BL+LOs

toc =Preamble= (from James Rose, the author of these notes) Monday, December 22, 2008

What follows is the original preamble (same as the notes from M2M). Just a couple notes on B+L: it’s probably the first lecture-based class you’ve had that’s both relevant and interesting (as opposed to D+D, which is relevant but uninteresting, or M2M, which is neither). Part of the reason it works well is that there’s a couple of dedicated, repeat lecturers who know their stuff and are reasonably good teachers. However, this advantage is largely lost if you don’t bother going to class. So I’d consider it, were I you. But whatever your opinion of class-going, for the love of God go to every last one of JJ Cohen’s lectures. He’s a great lecturer and you’ll walk away feeling like you know immunology inside and out. Dr. Ryder’s lectures can be hit or miss but it’s tough to know what to pay attention to in his (voluminous) notes unless you go. Finally, the B+L tests tend to be much more integrative (making you think) and less spit-out-the-memorized-facts—so study accordingly. –jcr

=Intro + Blood Cell Types=


 * [Notice that these aren't LOs- just food for thought and good background.]
 * 3 cell types: erythrocytes (red cells), platelets, and leukocytes (white cells).
 * All produced in bone marrow by pluripotent stem cells-- this process is called //hematopoiesis//.
 * __Erythrocytes__ are discs, thinner in the middle than at the edges (potentially to allow more surface area for oxygen exchange).
 * Latticework of proteins that covers the cell underneath the plasma membrane-- this gives both strength and flexibility to deform where necessary.
 * Mature red cells do not have a nucleus-- although they have one in the bone marrow, they get rid of it before they leave. Thus red cells can't make new proteins, repair its protein framework, etc.
 * Mature red cells also lack mitochondria-- thus they're dependent on anaerobic energy pathways to generate ATP.
 * What is does have is hemoglobin, and lots of it. Essentially it's crammed full of the stuff.
 * __Hemoglobin__: tetramer of globin chains (recall that normal adult hemoglobin is composed of two alpha and two beta globins). Each globin chain surrounds a porphyrin group with an iron atom (ie the __heme__ group- four of them per hemoglobin molecule). The iron atom is what actually binds the oxygen.
 * A mature red cell full of hemoglobin can carry about 1x10 9 oxygen atoms.
 * Anemia: lack of red cells, as discussed earlier. Potential reasons for this are (a) decreased production of RBC, and/or (b) increased destruction of RBC.
 * Reasons for decreased production:
 * low levels of enzymes or ions required for RBC synthesis (iron, folate, vitamin B12)
 * low levels of a hepatic hormone called __erythropoietin__ that stimulates RBC formation
 * Reasons for increased destruction:
 * mutations in cytoskeletal elements of RBC that make them more fragile, leading to RBC breakup (__hemolysis__)
 * defects in anaerobic pathway enzymes, or enzymes that exist to break down reactive oxygen species
 * autoimmune disorders where the immune system sees the RBCs as antigens and destroys them
 * hydrophobic point mutations in globin chains such that aggregates of hemoglobins form inside the cells and deform the cell structure (ie sickle-cell anemia)
 * partial thalassemias in which insufficiency of one type of globin makes homotetramers (ie four beta-globins together), which are unstable and prone to hemolysis
 * immune reactions in which cytokines produced by inflammation reactions shut down RBC production.
 * __Platelets__ are "itty-bitty fragments of a cell", derived in the bone marrow from cells called __megakaryocytes__ (cells with an enormous nucleus and lots of cytoplasm), which break off pieces of themselves-- these pieces are called platelets, have no nucleus, and function in hemostasis (coagulation/clotting).
 * Balance struck between having enough platelet-induced coagulation to avoid bleeding out, but not enough to cause thrombosis.
 * __Leukocytes__ are involved with fighting infection and inflammatory responses. There's all kinds of things that can go wrong with this plan-- it's like having circulating armed gangs in your bloodstream.

Blood Cell Types, 1/3/08
[Note: Dr. Ryder was going for a Guinness record for Explaining A Ton Of Shit Really Quickly, so some of the following is less complete than it might be.]


 * Understand the general principle of the Wright-Giemsa stain: describe how acidic components in the cell will stain and what the main acidic components of the cell are and how basic components in the cell will stain and what the main basic components of the cell are.
 * __Wright-Giemsa stain:__ uses two stains, eosin and methlyene blue, to stain basic and acidic cellular elements.
 * Uses eosin to stain basic cellular elements red.
 * Eosin-staining elements: hemoglobin, cytoplasmic granules
 * Uses methylene blue to stain acidic cellular elements blue.
 * Methylene blue-staining elements: primarily nucleic acids (DNA/RNA)
 * Generally, the nucleus in most cells are blue; cytoplasm is a mix.
 * In red blood cells, lots of basic hemoglobin-- thus lots of red.
 * Recognize the types of white cells that may normally be found in the peripheral blood.
 * Notice that white cells are **nucleated**, unlike the RBCs and the platelets.
 * Types:
 * **Granulocytes** : contain granules, which in turn contain various unpleasant substances to destroy antigens.
 * 3 types: neutrophils, eosinophils, and basophils.
 * **Neutrophils** : neutralize infectious bacteria by phagocytosis (engulfing). Contain toxin granules that break down and destroy the engulfed bacteria.
 * 3-5 segments or lobes under microscope
 * Myeloperoxidase: substance in these granules whose presence or absence helps to determine myelogenous leukemia vs. non-.
 * Absolute neutrophil count: 1.8-7.8 x 10 9 cells/L
 * **Eosinophils** : release granules that contain lots of basic stuff (which can be stained red with eosin, thus the name).
 * 2-3 segments or lobes under microscope
 * Eosinophils mediate type I immunopathology (hypersensitivity reactions; see next section, "Overview").
 * Absolute eosinophil count: 0-0.4 x 10 9 cells/L
 * No age- or sex-dependent variety in count.
 * Normal to have no count (zero value).
 * **Basophils** : release granules that contain lots of acidic stuff, counter-intuitively enough (stain blue with methylene blue).
 * Play a central role in type I hypersensitivity reactions (initiates the reaction; eosinophil mediates reaction).
 * No age- or sex-dependent variety in count
 * Absolute basophil count: 0-0.2 x 10 9 cells/L
 * Again, normal to have no count.
 * **Bands** : immature neutrophils. Discussed later, but tend to increase in number in response to infection.
 * **Monocytes** : phagocytic (engulfing) cells.
 * Big structure with vacuoles and some granules
 * Effectively macrophages in transit-- they're circulating potential macrophages (transport into structures to mature into macrophages)
 * Mainly involved in phagocytosis (engulfing foreign bodies).
 * Absolute count fluctuates through childhood and adolescence: adult ref range is 0.2-0.9 x 10 9 /L
 * **Lymphocytes** : T and B cells (also NK cells, which we haven't discussed yet).
 * Have a nucleus about the size of a RBC (very large); may or may not also have a lot of cytoplasm with granules in them.
 * "Natural killer" cells- function to be discussed.
 * Absolute count : 1.0-4.8 x 10 9 /L
 * Describe the difference between the white blood cell differential and the absolute count of a particular white cell type; given the white blood cell differential and the total white blood cell count, be able to calculate the absolute count of a particular white blood cell type.
 * Units: billions of cells per liter (10 9 /L).
 * Absolute or total white blood cell count: fluctuates through childhood, stays in the vicinity of 4.5-11.0 after that.
 * __WBC differential__: what percent of WBCs are lymphocytes, granulocytes, monocytes.
 * Notice that differentials are best expressed in actual counts, or number of cells per liter, rather than abstract percentages (ie "neutrophils are 50% of total count of 5.6," not "neutrophils are 50% of total count.").
 * Give a range for the absolute counts of the various white blood cells in a normal adult. Describe how the white cell counts fluctuate with age.
 * Absolute counts are discussed above for the various WBCs. The variability during childhood and adolescence is different for each WBC; generally, neutrophil counts go up during adolescence, lymphocyte and monocyte counts go down during adolescence, and eosinophil, basophil, and band counts don't vary at all.
 * Describe the shape of a red blood cell and the significance of this shape.
 * "Biconcave disc"- thinner in middle than at edges. Shape facilitates gas exchange.
 * Explain the difference between a red blood cell and a reticulocyte. Describe how a reticulocyte count is performed.
 * Reticulocytes: immature RBCs in the peripheral blood which still retain organelles (ribosomes, Golgi apparatus, etc).
 * After a day or two in the peripheral blood, these mature into RBCs-
 * Can stain the peripheral smear to highlight residual organelles- you can then make an easy count from that.
 * Stain essentially checks to see if your bone marrow is working properly-- an elevated reticulocyte count in the face of anemia means that the marrow is trying to compensate for the low RBC levels (thus anemia is due to RBCs being destroyed). If it's low, then there's likely a problem in the synthesis process of new RBCs.
 * 1-2% of blood cells in a normal adult are reticulocytes to keep a constant RBC level.
 * Give a range for the red blood cell count in a normal adult man and a normal adult woman. Describe how the red blood cell count fluctuates with age.
 * Range: measured in millions (10 6 ) of cells per microliter, or __10 12 per liter__.
 * Typical range varies throughout childhood and adolescence-- adult value is about **4.6-6.2** in men and **4.2-5.4** in women, at least in Denver.
 * Notice that this is influenced by altitude-- high altitude leads to larger numbers of RBCs, low altitude leads to low numbers of RBCs.
 * Be able to recognize a platelet in a peripheral blood smear.
 * 2-4 microns across; granular; 'purplish'.
 * Give a range for the platelet count in a normal adult. Describe how the platelet count fluctuates with age.
 * Platelet count: **150,000-400,000** cells per microliter.
 * Count does not fluctuate with age.
 * Platelet "granules"-- contain substances that play a role in the initiation of the coagulation cascade.
 * **Terms** :
 * Polycythemia: condition in which a patient has too many red cells (generally neoplastic or cancerous)
 * Erythrocytosis: condition in which a patient has too many red cells (generally benign)
 * Thrombocytopenia: too few platelets
 * Thrombocytosis: too many platelets.
 * Thrombocythemia: neoplastic condition in which there are too many platelets.
 * Leukemia: abnormal neoplastic proliferation of granulocytes and monocytes
 * Lymphoma: abnormal neoplastic proliferation of lymphocytes.
 * Neutropenia: too few neutrophils
 * Neutrophilia: too many neutrophils
 * Eosinophilia: too many eosinophils
 * Basophilia: too many basophils
 * Monocytopenia: too few monocytes
 * Monocytosis: too many monocytes (tuberculosis or neoplastic problems)
 * Lymphopenia: too few lymphocytes (can be caused by corticosteroids)
 * Lymphocytosis: too many lymphocytes (neoplastic causes, mainly)

=Hematopoiesis, Hematopoietic Precursors, and Bone Marrow=


 * (Hematopoiesis: process of making the cells that are found in the blood.)
 * Describe where hematopoiesis occurs before birth, in childhood, and in adulthood.
 * Before birth:
 * From 0-2 months, occurs in the embryo's yolk sac.
 * From 2-7 months, occurs primarily in the spleen and liver.
 * From 7 months til birth, and for the duration of the adult life, the bone marrow is the primary site of hematopoiesis.
 * As a person ages, the hematopoiesis tends to occurs more along the axial skeleton: ribs, skull, pelvis, sternum, and vertebrae.
 * List the types of myeloid cells found in the bone marrow.
 * __Myeloid__: Technically, means "derived from bone marrow," which isn't actually very useful here. Here, refers to __blood cells that aren't lymphocytes__ that are derived from the bone marrow (red cells, granulocytes, monocytes, platelets).
 * In the context of the "myeloid to erythroid ratio" (M:E), myeloid refers to granulocytes alone (erythroid is, obviously, the red cells).
 * Discuss maturation and differentiation as they relate to hematopoiesis.
 * CFU: "colony-forming unit." Refers to stem or progenitor cells that give rise to cells of different types (CFU-erythroid or CFU-E forms red cells, CFU-neutrophil or CFU-N forms neutrophils, etc).
 * BFU: burst-forming unit. Just a precursor of a CFU-E (erythroid colony-forming unit).
 * __Differentiation__: Here, the process of turning on some genes and turning off others to direct the development of the cell to a particular cell fate (erythrocyte, neutrophil, etc).
 * __Maturation__: Here, the process of accumulating the protein products that are the result of differentiation; directly results in phenotypic changes in the cell.
 * Define stem cell, progenitor cell, and precursor cell.
 * __Stem cells__: For the purposes of this discussion, self-renewing cells that are capable of differentiating into different types of blood cells.
 * Stem/progenitor cells are denoted by what follows the "CFU"-- CFU-GM means "CFU stem cell, can differentiate into granulocytes or monocytes".
 * Code for what follows the "CFU":
 * L: lymphocytes
 * M: in the context of CFU-LM, means "myeloid cells"
 * M: outside the context of CFU-LM, means monocytes
 * Meg: megakaryocytes (platelet-forming cells)
 * G: granulocytes
 * E: erythroid cells
 * __Multipotential stem cells__: CFU-LM, can give rise to any of the lymphoid or the erythroid cells
 * __Pluripotential stem cells__: CFU-GEMM, can give rise to any of the myeloid cells, or CFU-L, which can give rise to any lymphocytes.
 * __Progenitor cell:__ A cell with limited self-renewal capability; can only differentiate into one or two myeloid cell types.
 * Ie: CFU-GM (can give rise to granulocytes or monocytes)
 * __Precursor cells__: the immature forms of the mature blood cell forms seen in the peripheral blood. Notice that reticulocytes are technically mature cells, although they're not fully developed.
 * Describe how the phenomenon of self-renewal prevents the bone marrow from rapidly becoming depleted.
 * Stem cells, as described by Dr. Hooper last fall, can divide and keep one daughter cell as a self-renewing replacement and use one for differentiation into a non-dividing blood cell.
 * Discuss the role of hematopoietic growth factors in hematopoiesis. Include the names of the major hematopoietic growth factors, where hematopoietic growth factors are produced, and how they work.
 * Basically there needs to be a lot of other molecular factors in play before effective hematopoiesis can happen- can't just dump some stem cells in a dish and watch it go.
 * Hematopoietic growth factors (HGFs) are some of these:
 * CSFs: **C** olony- **S** timulating **F** actors. What most HGFs are called.
 * Multilineage growth factors (promote growth of a variety of cell types):
 * IL-3 (influences growth of all blood cell types, come from activated T and NK lymphocytes)
 * GM-CSF (influences growth of granulocytes and monocytes, come from activated T cells, macrophages, fibroblasts, endothelial cells)
 * G-CSF (influences growth of granulocytes, come from macrophages, fibroblasts, endothelial cells)
 * M-CSF (influences growth of monocytes, come from same as GM-CSF)
 * Erythropoietin or "Epo" (influence growth of RBCs and platelets, comes from kidneys)
 * Notice that erythropoietin, G-CSF, and GM-CSF are currently used in clinical practice for chemotherapy patients.
 * HGFs stimulate self-renewal and proliferation of stem/progenitor cells; also keep the mature blood cells working properly.
 * Using stem cells, progenitor cells, and precursor cells, draw the hierarchical scheme of hematopoiesis as it is understood today.
 * Stem cells give rise to progenitor cells give rise to precursor cells give rise to mature differentiated cells.
 * List and be able to recognize the granulocytic and erythroid precursors. Be able to recognize a megakaryocyte.
 * Neutrophil precursors:
 * Myeloblast: lots of blue stain- RNA/DNA. Large, unsegmented nucleus.
 * Large blast count = indicative of acute leukemias.
 * Promyelocyte: still have a lot of blue, but have a bunch of granules.
 * Myelocyte: more granules still. Slightly more muddy as the accumulation of basic proteins changes color away from blue.
 * Metamyelocyte: nucleus has indented less than half the nucleus.
 * Bands: nucleus has indented more than halfway (horseshoe shape).
 * Segmented neutrophil: fully segmented nucleus, ready to leave the marrow.
 * Eosinophils and basophils are similar.
 * Essentially: granulocytes accumulate granules and develop lobular nuclei as they mature.
 * Erythroid precursors:
 * Pronormoblast: lots of blue stain, large round nucleus and nucleoli.
 * Basophilic normoblast: Picking up more red color from accumulation of hemoglobin; nucleus has gotten slightly smaller.
 * Polychromatophilic normoblast: Still getting red color from hemoglobin but also have a lot of blue from RNA: muddy color. More condensed nucleus.
 * Orthochromatic normoblast: Full of hemoglobin; more or less a single color, small compact nucleus ("eight-ball-like structure").
 * Reticulocyte: the nucleus is lost; the organelles remaining inside are usually stained blue.
 * Mature erythrocyte: no nucleus, no organelles- red stain from hemoglobin.
 * Essentially: erythrocytes turn redder from accumulation of hemoglobin and compact/expel their nuclei once they differentiate to reticulocytes.
 * Megakaryocyte precursors:
 * Generally not enormously important, at least at the moment.
 * Mature megakaryocytes are very large cells with truly massive nuclei surrounded by granulocytic cytoplasm.
 * Megakaryocytes extend processes into sinuses and break off piece of their cytoplasm to form platelets.
 * Note that monocytes have big nuclei with a bunch of non-staining vacuoles in a lot of cytoplasm.
 * Define cellularity as it relates to the bone marrow.
 * Cellularity: the percent of the bone marrow that's hematopoietically active (producing blood cells). If it's not active, it's occupied by adipose tissue (fat).
 * Describe how bone marrow cellularity changes with age.
 * It decreases with age. Rule is that after the age of 50 your cellularity is roughly 100 minus your age. Take with a grain of salt.
 * Define the M:E ratio and what this ratio should be in a normal person.
 * Myeloid (here, means granulocytic) to erythroid cell ratio
 * Should be about 3:1.


 * [Bone marrow: has specialized vasculature designed to keep immature cells in and let mature ones out-- out, specifically, into the venous sinuses that run through the bone marrow.]
 * Notice that you want to see lots of different types of cells in bone marrow- this indicates that there are lots of different types of precursors and that the hematopoietic process is proceeding okay. If every cell looks the same on a bone marrow smear, it can be indicative of leukemia, etc.

=Hemoglobin: Structure, 02 Binding, Methemoglobinemia, Carbon Monoxide Poisoning=


 * Describe the overall structure of hemoglobin, including the number and type of subunits that make up its tetrameric structure, the heme prosthetic group, the placement of the iron molecule, and where oxygen or carbon dioxide binds.
 * Four globin proteins, generally two alpha and two beta, each of which are bound to a heme group (a porphyrin ring and an iron atom inside it).
 * Describe the function of heme in hemoglobin.
 * Heme: primarily, binds oxygen and CO2.
 * The iron needs to be in the ferrous form (Fe2+) to bind oxygen; if it's in the ferric form (Fe3+), it can't bind oxygen (more on this later).
 * It's reduced from ferric to ferrous by cytochrome b5 reductase.
 * Explain the contribution of quaternary structure to the function of hemoglobin and the phenomenon of allostery.
 * The globin subunits influence each other's oxygen binding capabilities: when one globin group binds to oxygen, it changes shape so as to make the other globins more apt to bind oxygen. Conversely, if a globin chain releases oxygen, it makes the other globins more likely to release theirs as well. This phenomenon is called 'cooperativity.' You can think of globins as herd animals- when one leads, the others tend to follow.
 * Allostery is just the word for a conformational change in a molecule changing the binding affinities of that molecule.
 * Compare the oxygenated and deoxygenated states of hemoglobin.
 * Globin with bound oxygen is in a "R" or relaxed form; globin without bound oxygen is in a "T" or taut form.
 * Describe how Hemoglobin A2 and Hemoglobin F differ from hemoglobin A. Indicate how these hemoglobins differ from Hemoglobin A in their oxygen affinity. Estimate what the normal percentages of these variants are and what diseases may cause elevations in those percentages.
 * Hemoglobin A2 is the other adult hemoglobin variant, formed of two alpha subunits and two delta subunits. Slightly higher affinity for oxygen. Normally found at only 2% of adult hemoglobin. Notice that if an adult has a problem making beta globins, the delta levels may go up to compensate.
 * Hemoglobin F (fetal hemoglobin, with two alpha chains and two gamma chains) has a higher affinity to oxygen; also a more highly pronounced Bohr effect (see below, but basically the effects of pH on oxygen saturation curve is much more pronounced) and 2,3-DPG has no effect (again, see below). Normally shouldn't find this form at all in adults (recall that gamma hemoglobin is replaced by beta hemoglobin after birth), but in some beta-globin thalassemias, gamma hemoglobin production continues into adulthood.
 * Describe what forms of hemoglobin are expressed during fetal development and hypothesize why fetuses produce a different form of hemoglobin from that seen after birth.
 * During fetal development, the zeta, epsilon, and gamma forms are seen; zeta is an immature alpha globin; epsilon and gamma forms are immature beta globins.
 * Fetuses produce a different combination of forms of hemoglobin primarily because they're not pulling oxygen from the ambient air-- they're pulling it from maternal circulation in the placenta. This means they need to bind oxygen more tightly to be able to pull it from the maternal hemoglobin. Again with the babies-are-vampires theme we're developing here.
 * Draw a typical oxygen dissociation curve. Explain why it is sigmoidal in shape. Roughly estimate % oxygen saturation with pO2s of 100 mmHg, 60 mmHg, 40 mmHg, and 30 mmHg.
 * It looks more like a J-curve than an S-curve to me, but whatever. Basically this is a consequence of allostery: as oxygen binds, the faster more oxygen binds; as oxygen unbinds, the faster the other oxygen atoms unbind.
 * Useful measurements of % oxygen saturation of normal hemoglobin at various ambient partial pressures of oxygen:
 * 100 mmHg: roughly 100% saturated.
 * 30 mmHg: 60% saturated.
 * 45 mmHg: 75% saturated.
 * 60 mmHg: 90% saturated.
 * Notice that these latter three are in a zone of the dissociation curve in which the partial pressure of oxygen is directly related to the percent of hemoglobin's oxygen saturation- for every 15 mmHg increase, another 15% of oxygen is bound. So 30-45-60 mmHg = 60-75-90%.
 * P-50, the oxygen pressure at which half of the oxygen binding sites on hemoglobin are bound and half are unbound, is 27 mmHg for a normal human adult.
 * oxygen partial pressure to saturation: 30-60, 40-75, 60-90
 * Remember that __lower__ oxygen saturation of hemoglobin means __more__ oxygen delivered to the tissues.
 * Explain effects of low pH, temperature, and 2,3-DPG on the shape of the oxygen dissociation curve.
 * They shift the curve to the __right__-- that is to say, at a given partial pressure of oxygen, there's less oxygen bound to hemoglobin under any of these conditions.
 * This is called the __Bohr effect__.
 * Another way to say this is that these factors __decrease__ the affinity of hemoglobin for oxygen.
 * Remember that this means that more oxygen is delivered to the tissues.
 * Notice that as levels of CO2 go up (as when muscles are working hard), the blood pH drops, thus shifting the curve to the right and increasing the flow of oxygen to tissues.
 * 2,3-BPG (also called 2,3-DPG): enzyme that binds to hemoglobin and decreases its affinity for hemoglobin (again, shifts curve to the right), Can occur as a result of altitude shift, exercise, etc. Remember that 2,3-DPG doesn't affect fetal hemoglobin.
 * Temperature: high temperature promotes flow of oxygen to the tissues in the same way as low pH or 2,3-DPG, by lowering the affinity of oxygen for hemoglobin.
 * Describe how a pulse oximeter works and what it measures. Describe situations where a pulse oximeter reading may inaccurately reflect a patient’s true oxygenation status.
 * It's just a device used for measuring the relative absorption of light at two different wavelengths (one absorbed by deoxyhemoglobin, one absorbed by oxyhemoglobin)-- this process measures the percentage of hemoglobin that's bound to oxygen.
 * If you've never seen one before, it's a little light-emitting clip that fits around your finger.
 * The normal percentage tends to be around 100% at sea level and goes down as altitude increases.
 * Notice that inaccurate placement, excessive movement of the patient, nail polish or deep pigmentation, a patient in shock or severely anemic, or high CO (not CO2) levels in the blood can all cause inaccurate readings.
 * Describe what methemoglobinemia is, what causes it, how to diagnose it, and how to treat it.
 * A anemic condition in which an iron atom of hemoglobin is stuck in the ferric state (Fe3+)- this means that the hemoglobin can't bind oxygen properly. Due to conformational changes, the other globins of the hemoglobin also bind more tightly to oxygen, pushing the dissociation curve to the left.
 * Cause: certain drugs, or anything that reduces levels of cytochrome B5 reductase, such as certain topical anesthetics, well-water contaminated with nitrites, etc.
 * Generally babies are most susceptible.
 * Can cause cyanosis (blueing) in tissues. Notice that carbon monoxide poisoning causes a reddening instead, despite having similar causes. Go figure.
 * Treatment: methylene blue, which reduces the ferric hemoglobin to ferrous.
 * Explain the pathophysiology of carbon monoxide poisoning and its treatment.
 * CO binds to hemoglobin much, much better than oxygen; hard to get rid of, and while it's bound to globin, that globin can't carry oxygen. It also shifts the dissociation curve to the left, as in methemoglobinemia (for the same reason).
 * However, CO poisoning causes cherry-red coloring in tissues rather than cyanosis.
 * Primarily found in heavy smokers, but also in anyone spending a lot of time in poorly-ventilated areas around hydrocarbon sources (heaters, gasoline, paint removers).
 * Treated with 100% O2 administration or a hyperbaric oxygen chamber.
 * Describe how unstable hemoglobins or hemoglobins with altered affinity can affect oxygen delivery to the tissues.
 * Some hemoglobins (ie "Chesapeake" globins) can have higher oxygen affinity (which leads to erythrocytosis, or lots of blood cells, to compensate) and a corresponding left-shift to the dissociation curve.
 * Other hemoglobins (fewer) have lower oxygen affinities- can seem cyanotic (low hemoglobin counts)
 * Neither of these two conditions are usually very severe.
 * Hemoglobins can also be unstable; a condition ranging from mild to very severe.
 * Effectively the hemoglobin breaks down earlier than its normal 120 day life span; can lead to hemolytic anemia (see later lecture on this topic).
 * Notice that this leads to jaundice, or a yellowish tint to the skin, due to the liver's inability to process so many heme breakdown products.
 * Notice also that this can lead to splenomegaly, as the spleen picks up way too many lysed hemoglobin cells from the blood.
 * "Heinz bodies:" precipitated, dense, crumpled-up hemoglobin clumps in a peripheral blood smear in a person with hemolytic anemia. (these are what's picked up by the spleen).
 * One important therapy in hemolytic anemia is the administration of folic acid-- need to be able to produce enough DNA to keep up an adequate red cell count.

=Anemia: Overview of the Approach to a Patient=


 * Define anemia and discuss the laboratory tests used to determine its existence in an individual. Explain the influence of age and gender on the definition.
 * **Anemia** : Insufficient red cell mass to deliver adequate levels of oxygen to peripheral tissues.
 * Tests:
 * Hemoglobin concentration in blood
 * Hematocrit: percentage red cells in blood.
 * Red blood cell count (cells x 10 12 /L)
 * A **CBC** (complete blood count) includes all of these, the mean corpuscular volume, mean corpuscular hemoglobin count, white count, platelet count, and white cell differential. More on these under the "CBC" lecture.
 * To diagnose anemia: look also at red cell morphology and reticulocyte count.
 * Varies by age, but not in a neat line. Starts large, gets smaller, gets larger again, but not as large as it started.
 * Varies by gender: men tend to have proportionally more red blood cells.
 * Varies by altitude: higher up, have more red cells.
 * Menstruating women and pregnant women tend to have lower hematocrits. No surprises there-- both bleeding and vampire babies lower hematocrit.
 * [Notice that the porphyrin rings of hemoglobin are produced in the mitochondria of the developing erythrocyte, joined to iron to form heme groups, then tacked onto globins.]
 * Define reticulocyte count, absolute reticulocyte count, and reticulocyte index and discuss how these measurements are used in assessing the rate of RBC production.
 * __Reticulocyte count__: the percent of 1000 red cells counted that are reticulocytes (recall that reticulocytes retain organelles, as opposed to mature RBCs). Multiplying this percentage by the total red cell count gives the absolute reticulocyte count.
 * Normal range of reticulocyte count: **0.4-1.7%** of total red cell count.
 * Reticulocytes are immature red cells- thus the number of reticulocytes in the peripheral blood gives a handy reference for new red blood cell production, as in the case of some leukemias. Effectively can use to quantity erythropoiesis rates.
 * The __reticulocyte index__ can be a tricky concept. Check this out.
 * During anemia, you want to see if the reticulocyte count is up-- that would mean that the body's responding to anemia by pumping up RBC production, like it should.
 * But given that the reticulocyte count is usually expressed as a percentage of the total RBCs, and the RBC count is likely to be artificially low, it's not always accurate to accept the ret. count on face value.
 * The reticulocyte index effectively is just a corrected reticulocyte count that figures in a stress factor-- the idea is to see whether the reticulocyte count is elevated relative to the __baseline__ red blood cell count, not relative to the __depressed__ red blood cell count that you can see in anemic situations.
 * Also notice that reticulocytes can hang around for longer before fully maturing in the periphery if the body is under stress- not always a neat 1-day turnover.
 * Draw a general classification scheme of anemias based on MCV and reticulocyte count and list the more common causes for anemia with low, normal, and high MCVs.
 * If anemia is one of a number of hematological symptoms, look into cancer. If it's just anemia, check reticulocyte count.
 * If reticulocyte count is high, look for evidence of hemolysis-- if positive, look for causes of hemolysis; if negative, look for causes of hemorrhage.
 * If reticulocyte count is normal, look at MCVs-- if very large (> 100), look for macrocytic anemias; if normal (80-100), look for normocytic anemia; if low (< 80), look for microcytic anemia (most commonly caused by iron deficiency).
 * Notice that thus far he hasn't discussed common normocytic or macrocytic anemias.
 * Recognize critical findings in the history and physical examination important in determining the cause of anemia.
 * Signs and symptoms [note: __symptoms__ are patient complaints; __signs__ are clinically measured parameters]:
 * These are predominantly concerned with cardio/pulmonary stress
 * Symptoms: shortness of breath, fatigue, rapid heart rate, dizziness, angina, pallor, claudication (pain while walking).
 * Signs: tachycardia, tachypnea, dyspnea, pallor.
 * Describe iron metabolism and the iron cycle, and describe where iron is distributed in the body.
 * Iron has two valence states, Fe2+ and Fe3+ (ferrous and ferric, respectively).
 * In aqueous solution, iron form insoluble hydroxides unless bound.
 * At low pH, iron is more soluble.
 * Note that the level of iron in the body is controlled only by iron absorption-- iron is __not__ actively excreted except for very small amounts in urine and skin (menstruation is a partial exception).
 * Distribution:
 * Most (60%) of iron in body is in hemoglobin.
 * 6% is in myoglobin.
 * 0.1% is in transferrin (in transit)
 * about 25% is in intracellular iron stores (ferritin and hemosiderin).
 * Iron is picked up as Fe3+ in the duodenum, converted to ferrous form (Fe2+), and transported into cells; there it can either be kept in storage or put into the bloodstream to be carried by transferrin to where it needs to go. If it's going into the bloodstream, the iron gets converted back to ferric (Fe3+) form to be bound to transferrin.
 * Iron cycle: it's in the plasma, bound to transferrin; it gets transported to the bone marrow and used to make red cells; when the red cells die, it's picked up by specialized macrophages (the reticuloendothelial macrophages in the spleen); from there, it's put in intracellular stores, bound to ferritin or hemosiderin; when needed, it's bound to transferrin and put back in the plasma.
 * List factors that increase or decrease iron absorption.
 * [Notice that food can contains both elemental iron and heme-bound iron (the latter is generally only in meats).]
 * Increase iron absorption:
 * Low pH: keeps iron soluble, more readily absorbable.
 * Iron is better transported in the presence of protein and amino acids.
 * Vitamin C keeps iron in ferrous state, thus more easily absorbed.
 * Higher quantity of iron ingested.
 * If erythropoiesis rates are up, iron is more actively picked up from the duodenum.
 * Decrease iron absorption:
 * Certain chelators (phytates, oxalates).
 * In many illnesses and during inflammatory progression, iron release from stores is halted, limiting hematopoiesis by reducing iron incorporation into heme.
 * The mechanism for this cessation is __hepcidin__, a small peptide produced in the liver that causes iron to be sequestered in intracellular storage forms. Hepcidin is actively produced during inflammation, infection, and abnormally high iron intake.
 * Explain the relevant facts related to production, function, and turnover of red blood cells.
 * I'm not sure what he's getting at here that we haven't already covered. Red blood cells need iron to be synthesized and to function properly. There's normally destroyed mainly in the spleen and produced mainly in the bone marrow of the axial skeleton. See "Hemolytic Anemia" for further notes.
 * List and describe some of the causes for iron deficiency and iron overload.
 * Iron overload: too much dietary iron, overactivity of iron transporters in duodenum. Can also be caused by multiple blood transfusions.
 * Iron deficiency: not enough dietary iron to keep up with demand.
 * Most common nutritional deficiency (other, presumably, than starvation).
 * During periods of rapid growth (fetal, adolescent) the demand for iron is higher- thus need more in diet.
 * Can also be caused by anemia of chronic inflammation or disease (hepcidin-mediated), or thalassemia.
 * Normal treatment is to give iron salts orally until serum iron, red cells, and iron stores are all normal.
 * Categorize the findings on a CBC and peripheral smear with iron deficiency and interpret iron lab tests (ferritin, iron, iron binding capacity, and % transferrin saturation) in patients with iron deficiency and disorders of iron metabolism.
 * Microcytosis (low MCV) on the CBC; hypochromia and wide range in the sizes of red cells in the peripheral smear.
 * CBC: Look for low serum iron, low ferritin and ferritin saturation, high transferrin saturation.
 * Describe the effects of over accumulation of iron in the body.
 * Causes organ damage: heart, liver, endocrine system (mainly pancreas)
 * Cardiac or liver failure
 * CBC: Look for increase in ferritin and serum iron.
 * Describe two treatments for iron overload.
 * Therapeutic phlebotomy (blood donations)
 * Iron chelators (desferal or exjade)

=Anatomy and Physiology of the Immune System=

[__Toleragen__]: An antigen that not only does not provoke an immune response, but prevents that stimulus from ever provoking an immune response. At this point the only toleragens we have are the various compounds of our own body. o __Antigenic determinant__ or __epitope__ (synonyms): The portion of an antigen that actually fits into and binds with an antigen receptor on the lymphocyte. Notice that this portion can be discontinuous in the primary sequence of the antigen.
 * Define:
 * __Leukocytes__: Nucleated blood cells (as opposed to anuclear erythrocytes and platelets), also called white blood cells (from Greek //leukos//, white). Sediment right above RBCs, but below plasma, in centrifuged whole blood.
 * __Mononuclear cells__: Leukocytes with a smooth nucleus. Types:
 * Monocytes
 * Lymphocytes
 * __Polymorphonuclear cells__: Leukocytes with a lobular nucleus (granulocytes).
 * __Granulocytes__: A synonym for polymorphonuclear cells: lobular-nucleated leukocytes. Called granulocytes on account of they've got lots and lots of granules in them, with a variety of contents. Types (see "Blood Cell Types" for further detail):
 * Neutrophils
 * Eosinophils
 * Basophils
 * [Notice that "__lymphoblasts__" are not inactive precursors to lymphocytes (as the word 'blast' is used elsewhere); lymphoblasts are activated, differentiated lymphocytes.]
 * Also notice that monocytes and lymphocytes have some granules in them as well, just not nearly as many as granulocytes.
 * Dendritic cells: See "Overview"-- the "alarm" cells, particularly in skin or mucosa, that bring antigens to the attention of lymphocytes.
 * Mast cells: More or less basophils, but in the tissues, not the plasma. Involved with type I immunopathology (hypersensitivity or 'allergic' reactions).
 * Plasma cells: Cells found in lymph nodes (not, actually, in plasma): enormous activated B lymphoblasts that are making and secreting millions of antibodies every minute. These die at a rapid rate, mainly due to exhaustion.
 * [Plasma: blood without the blood cells.]
 * [Serum: plasma without the clotting factors.]
 * Define antigen, and compare it to immunogen. Define antigenic determinant and epitope.
 * __Antigen__: Any substance which can be recognized by the immune system.
 * __Immunogen__: An antigen that actually activates an immune response (which can thus provide antibody-mediated future immunity to this antigen by means of the adaptive immune system).
 * Sketch schematically a dendritic cell; neutrophil; eosinophil; basophil; small lymphocyte; lymphoblast; plasma cell; monocyte. Indicate the characteristic features which distinguish each cell type.
 * You're going to have to sketch them your own damn self. But here's what they look like:
 * Round nucleus about the size of a RBC, with little cytoplasm: lymphocyte.
 * Same size nucleus but with bigger cytoplasm: lymphoblast.
 * Same size nucleus with a ton of protein production and ribosomes: plasma cell.
 * Segmented nucleus and lots of granules: neutrophil.
 * Lobular nucleus and lots of orange granules: eosinophil.
 * Lobular nucleus and lots of blue blobby granules: basophil.
 * Vacuoles, big nucleus, a lot of cytoplasm, and some granules: monocyte.
 * List the normal adult white cell count and differential percentage ranges. From these, calculate absolute counts for the different cell types.
 * Normal adult __total white cell count__: __4.5 - 11__ x 10 9 /L
 * Normal __neutrophil count__: __1.8 - 8.8__ x 10 9 /L (40-80%)
 * Normal __lymphocyte count__: __0.9 - 5.5__ x 10 9 /L (20-50%)
 * Normal __basophil count__: __0 - 0.22__ x 10 9 /L (0-2%)
 * Normal __eosinophil count__: __0 - 0.66__ x 10 9 /L (0-6%)
 * Normal __monocyte count__: __0.09 -1.21__ x 10 9 /L (2-11%)
 * Sketch a lymph node in cross section. Indicate the hilum, cortex, germinal centers, paracortex, medulla, afferent and efferent lymphatics.
 * Again, you're the artist, not me. Essentially the blood comes in (and goes out) one side, at the hilum. Moving away from that side, you get to the medulla, then to the paracortex, then to the cortex (containing **germinal centers** where lymphocytes are madly dividing) at the other side from the hilum. The afferent lymph (coming into this node from the last node in the chain) comes into the periphery near the cortex, and leaves (efferent lymph, going on to the next lymph node in the chain) at the other side, with the vessels at the hilum.
 * Cells in cortex: generally B cells.
 * Cells in paracortex: generally T cells.
 * Notice this means that incoming dendritic cells or antigens hit B cells first, then T cells- this prompts a collaborative B-T effort (talked about more later).
 * Name the major central and peripheral lymphoid organs.
 * __Central lymphoid organs__: organs in which lymphocytes develop.
 * Bone marrow
 * Thymus
 * __Peripheral lymphoid organs__: organs in which already-matured lymphocytes congregate to bushwhack antigens. Notice that the majority of mature lymphocytes in the body are found in the peripheral lymphoid organs.
 * Spleen
 * Lymph nodes (especially in the mesenteric gut)
 * Peyer's patches - remember the internal surface of the small intestine where the jejeunum gave way to the ileum, which was smoother (no plicae folds) but had a lot of little bumps? Those were Peyer's patches: masses of lymphoid tissue in the gut. See below for a couple more notes on these.
 * Adenoids: lymphoid tissue behind the uvula (you do remember the uvula, right?).
 * Tonsils: lymphoid tissue in the throat, sort of to the sides of the uvula.
 * Describe the recirculation of lymphocytes from blood to lymph and back; include in your discussion the specialized features of lymph node endothelium that permit recirculation.
 * General: Lymphocytes cycle from blood circulation to lymph circulation and back again. The entry points for lymphocytes to go into the lymphatic system from the blood are the lymph nodes themselves; the entry point for them to go back into the circulatory system is the lymphatic drainage into central veins.
 * Specifics: Lymph nodes have tall (for whatever reason, called "high"), cuboidal cells in their post-capillary venous endothelia. These allow lymphocytes in the circulation to pass between them, and into the node, via specialized transporters between the endothelial cells. From there they can migrate to the next node along the lymphatic flow, and so on until the lymph channel empties into the caval venous system (ie. thoracic duct empties into left BCV) and the cycle begins again.
 * This system allows antibody-mediated immunity to spread throughout the entire body from the lymph node nearest to the original site of infection.
 * Discuss lymphocyte activation by antigen with respect to: role of dendritic cells, receptor binding, proliferation, differentiation.
 * As discussed before, dendritic cells carry portions of ingested materials near an infection or wound site back to the lymphocytes in the lymph nodes to see if anything triggers an immune response.
 * Receptor binding: notice that several, preferably many, receptors on a lymphocyte must be simultaneously bound by antigen for proliferation to begin (more on this later in the course).
 * Proliferation: multiplication of identical cells to the lymphocyte whose antigen receptors the antigen triggered.
 * Differentiation: Resting lymphocytes look much different from activated lymphocytes-- resting cells are mostly nucleus, but activated lymphocytes get much larger and develop Golgi and ER organelles. These latter characteristics are particularly well-defined in plasma cells.
 * Again, activated and differentiated lymphocytes are called __lymphoblasts__.
 * [In gut epithelial lining: cells called "M cells." These are cells that are permeable to nutrients (and pathogens). Underneath these there's an area extremely rich in dendritic cells, then underneath that there's a big well of B and T cells. This is the **Peyer's patch** : the first decision-making cells about what kind of response to take to antigens in the gut.
 * [Notice that the brain and central NS have no lymphocytes in them-- the immune system hasn't "seen" them (thus can get immune response to brain material?]

=CBC/Peripheral Smear and Bone Marrow=


 * Understand the basic principles of flow cytometry.
 * You have a big expensive instrument. It pushes cells in a blood sample one at a time in front of a probe. The way the individual cells scatter light indicates some things about their size and properties- this information is collated and presented to you, the owner of the expensive instrument.
 * (Irrelevant note: when I was very young and starting college in 1996, my chemistry lab professor made a particular point of fixing us with his icy blue stare and barking: "Did I hear you call this a //machine// ? No, boy. This is an //instrument// ." Then, after holding the stare for a minute longer, he'd let it drop and smile slightly and add, "It's only a //machine// when it's not working." For whatever reason, I've been superstitious about calling instruments machines ever since.)
 * Describe the things that should be scrutinized when a peripheral smear is examined.
 * Things that I caught: hematocrit, red blood cell count, mean corpuscular volume, reticulocyte count. There's probably more.
 * Notice that hematocrits and hemoglobin measurements can be __artificially high__ if the patient is __dehydrated__, since they're both expressed as percentages of the total volume.
 * Another good parameter: **RDW** = red cell distribution width, ie. the width of the red cell impedance curve. Generally the wider, the more abnormal-- often, indicative of iron- or folate-deficient anemia. Essentially it's the inverse of the bone marrow: whereas in the bone marrow, a wide variety of sizes of red cells should be seen, in the peripheral blood, a very small variety of red cell sizes should be seen. You want red cells to mature in the marrow-- if they're released immature, at various sizes, it's a good sign that there's something wrong with red cell production.
 * Define and give the units for the hemoglobin (HGB) measurement.
 * Concentration of hemoglobin in blood, measured in grams per deciliter (g/dL).
 * Define and give the units for the hematocrit (HCT) measurement.
 * Percentage of blood made up of red cells. No units.
 * Define the MCV, the MCH, and the MCHC and relate these definitions to the units for these indices.
 * Mean corpuscular volume (MCV): average __volume__ of a given red blood cell in this smear.
 * Mean content of hemoglobin (MCH): average __mass__ of hemoglobin inside a given red blood cell in this smear.
 * Mean cell hemoglobin concentration (MCHC): average __concentration__ of hemoglobin inside a given red blood cell in this smear.
 * Note that concentration is mass over volume; thus MCHC is MCH/MCV.
 * Given the red blood cell count (RBC), the HGB and the HCT be able to calculate the MCV, MCH, and the MCHC.
 * How to calculate the MCV: the hematocrit divided by the red blood cell count, multiplied by 10 **(MCV = [HCT/RBC] x 10** )
 * Notice this doesn't always work (other factors can affect MCV).
 * How to calculate MCH: hemoglobin measurement divided by the red blood cell count, multiplied by 10 (**MCH = HGB/RBC x 10** ).
 * How to calculate MCHC: hemoglobin measurement divided by the hematocrit (**MCHC = HGB/HCT** ). Alternatively, **MCHC = MCH/MCV** (it's the same thing).
 * Describe how the MCV, the MCH, and the MCHC are used (or not used) in evaluating a patient’s red blood cells.
 * MCV: used to look at the size of the RBCs. A low MCV (microcytosis) can be indicative of thalassemia or deficiency anemia, while a high MCV can indicate macrocytic disorders.
 * MCH is rarely used- largely parallels MCV.
 * MCHC is also rarely used, for the same reason. The only times it's actually handy is either in case of __hereditary spherocytosis__ (causes the MCHC to be elevated) or in case of suspected hemolysis (error in drawing).
 * Describe the cause of platelet clumping and what should be done when platelet clumping is suspected as the cause of a spuriously low platelet count.
 * EDTA (anti-coagulant in blood draw tubes) sometimes forms big clumps of platelets, screwing up your count. (You would think anti-coagulants would be designed not to do this.)
 * Solution: draw another one with sodium citrate instead of EDTA as an anti-coagulant.
 * [Notice that if the patient has an abnormally low MCV, tiny little red blood cells can also be mistakenly counted as platelets on the platelet impedance graph.]
 * Describe the advantages and disadvantages of a manual white blood cell differential.
 * Advantages: You get to scrutinize the stuff what don't fit as expected.
 * Disadvantages: Your patient may have died of old age by the time you finish (lower volume counted, more subjective counting method)
 * Describe the advantages and disadvantages of an automated white blood cell differential.
 * Advantages: Really quick at counting and sorting normal white cells (larger volume counted, more objective counting method)
 * Disadvantages: Not nearly as smart as you and doesn't know how to classify unexpected shapes or how to recognize them well.
 * Be able to identify normal white blood cells in a peripheral smear.
 * See the powerpoint, the last three or four slides, for this. If it's got vacuoles, it's a monocyte. If it's got a horseshoe-shaped nucleus, it's probably a band. If it's got a distinctly segmented nucleus, it's probably a neutrophil. If it's round and its nucleus is pretty much the only thing you see, it's probably a nonactivated lymphocyte. If it's got a big round nucleus and irregular bunches of cytoplasm around it, it's probably an activated lymphocyte (lymphoblast, remember?). If it's orangish, it's probably an eosinophil. If it's dark blue with big lumpy blue granules, it's probably a basophil.

=Anemia due to Hemolysis=


 * [Stuff he mentioned that doesn't fit in anywhere in the LOs:]
 * [Under stress, the maturation time of RBCs is decreased and reticulocytes can be released early, and the bone marrow can also kick into overdrive and increase its red blood cell production by 6-8 fold.]
 * [Note that the normal red blood cell turnover is about 1% per day.]
 * Reasons for destroying red cells:
 * because they lose function over time;
 * because they accumulate oxidative injuries on account of they're playing with oxygen species all the time;
 * because their membrane composition changes;
 * because their calcium balance changes;
 * Red cells are normally covered with a layer of antigen, which in turn is covered by sialic acid- as the cells age, the sialic acid flakes off, allowing antibodies to bind the antigenic surface and signal the spleen to destroy the cell.
 * Note that a key antioxidant that prevents abnormal oxidation of red cells is __glutathione__. Without sufficient levels of active (reduced) glutathione, red cells accumulate oxidative damage much faster.
 * Just below the lipid bilayer (plasma membrane) of the red cell, there's a cytoskeleton that's interwoven with the transmembrane proteins embedded in the membrane. Recall that this cytoskeleton is what flexes to allow red cells to pass through capillaries. In the spleen, there are tight capillary gateways through which red cells have to be able to pass, with splenic macrophages waiting in the wings to haul off and destroy any erythrocyte that's having trouble making it through (a brutal system, really). Damage to the cytoskeleton is a sure way to mark a cell for lysis.
 * Is it just me or does this bring to mind a line of red blood cells all stepping forward in front of their splenic overseers and claiming, "I am Spartacus"?


 * Provide a definition for hemolysis and describe two main mechanisms of increased destruction of RBCs.
 * __Hemolysis__: a decrease in red cell survival or an increase in red cell turnover beyond normal ranges (normal RBC lifetime = 120 days plus or minus 20).
 * Keep in mind: we lyse red cells all the time. (yes, even you.) It's only called hemolysis when this lysis gets out of hand.
 * __Extravascular destruction__: responsible for most of the destruction of red cells, takes place in spleen.
 * __Process__: Macrophages pull aging or damaged RBCs out of the blood stream. Their globins are torn apart, their iron stored, and their porphyrin ring converted to __bilirubin__. Bilirubin is conjugated in the liver by glucuronide conjugation, then excreted into the bile duct to the small intestine, where it is further converted into __urobilinogen__ (which can be excreted in feces or filtered and excreted by the kidneys) by enzymes in the gut.
 * Notice that the enzymes that conjugate bilirubin in the liver can easily get saturated; unconjugated bilirubin can accumulate, causing a __jaundiced__ (sallow or yellow) appearance due to buildup of this bilirubin under the skin.
 * Notice also that high levels of conjugated bilirubin can crystallize and form gallstones in the bile duct.
 * Indications of extravascular hemolysis: increased unconjugated bilirubin or increased excretion of urobilinogen.
 * __Intravascular destruction__: responsible for less (10%) of the destruction of red cells; takes place within the blood vessels.
 * __Process__: Essentially, the red cell explodes in the vessel; this releases loads of hemoglobin, which dissociate into aB dimers. These dimers are bound by serum haptoglobin, but only a limited amount of haptoglobin is available. If haptoglobins are overwhelmed, the globin dimers are filtered through the kidney and are not reabsorbed (causing red urine). Also, the iron in globin dimer can be converted to the ferric state (Fe3+), which can cause metheme complexes to form; portions of these can bind to albumin in the plasma as well.
 * Indications of intravascular hemolysis: low serum levels of haptoglobin (the rest is bound to globins and excreted), the presence of metheme or methemalbumin, globin in urine.
 * Notice that the //rate// of hemolysis is important in all of this. There is a marrow response (increase RBC production) to attempt to compensate, but the response is innately limited (6-8 fold is as fast as the marrow can go), and may not be able to keep up, particularly in the face of rapid-onset acute hemolysis.
 * Describe the biochemical pathways of breakdown of hemoglobin and the relevant clinical lab tests for hemolysis.
 * I more or less went through how hemoglobin is broken down, both inside and outside the spleen. Here are some lab tests:
 * Morphology test: look for a change from biconcave disc to a different shape- this is indicative of a cell that will be lysed in the spleen.
 * Reticulocyte count: usually increased in the face of hemolysis.
 * Total and conjugated bilirubin: both are usually increased in extravascular hemolysis.
 * Hemoglobin in urine: associated with intravascular hemolysis.
 * Low levels of unbound haptoglobin: reflects intravascular hemolysis.
 * Elevated methemalbumin or metheme levels: reflects intravascular hemolysis.
 * Housekeeping enzymes (LDH, SGOT): increased levels may indicate lysed red cells, though the test isn't specific to hemolysis.
 * Describe the major constituents of the RBC membrane and cytoskeleton; identify the major defects in hereditary spherocytosis. Relate these to the clinical and laboratory findings of the disorder (CBCs and chemistries). Recognize other erythrocyte membrane disorders (elliptocytosis, pyropoikilocytosis).
 * The major constituents of the cytoskeleton are actin and spectrin, either of which can go wonky here. The major relevant constituents of the membrane are certain transmembrane proteins that have irritatingly unremarkable names.
 * **Hereditary spherocytosis** : genetic disease involving RBC cytoskeleton, characterized by anemia, intermittent jaundice, splenomegaly. Responds to splenectomy.
 * Presents in a variety of ways, but its hallmark is the loss of the red cell's plasma membrane due to cytoskeletal defects that cause the cell to assume a spherical shape.
 * Spherical shape causes the red cell to get stuck in the spleen and pulled out of the bloodstream by macrophages to be destroyed.
 * This condition is treated with folic acid administration (folate is required to synthesize new cells and DNA-- higher new of red cell production necessitates it).
 * Can also be treated well by splenectomy.
 * Usual clinical symptoms of hered. spherocytosis: anemia and jaundice.
 * Lab features: Variability in hematocrit/hemoglobin counts; high reticulocyte counts; high MCHC, low MCV relative to that of the reticulocytes, distinctive spheroid appearance; unconjugated hyperbilirubinemia.
 * Note most hereditary spherocytosis is dominantly inherited.
 * Other erythrocyte membrane disorders can present in similar ways.
 * Interpret an osmotic fragility test for diagnosis of spherocytosis.
 * In hypotonic solution, spherical red cells will lyse more quickly (weaker membrane).
 * Describe the clinical characteristics of hereditary spherocytosis. Explain when splenectomy is indicated.
 * Particular problem: in a viral infection, the bone marrow can stop RBC production for the duration; in a chronic hemolysis patient, this can have a severe effect, since the body relies on increased production to keep up with increased turnover.
 * Another problem associated with hereditary spherocytosis: gallstones formed from the crystallization of bilirubin in the bile pathways.
 * Splenectomy only when absolutely necessary, particularly in children.
 * Describe the major energy and antioxidant pathways in the RBC and explain how G6PD deficiency and pyruvate kinase deficiency cause hemolysis. Discuss the clinical and laboratory findings in patients with these syndromes. Recognize other enzyme deficiencies and their resultant hemolytic anemia.
 * Red cells are reliant on nonoxidative glycolysis (producing lactate) for energy. This glycolysis pathway takes a few detours along other reaction pathways that produce active glutathione (antioxidant pathway), 2,3-DPG, etc.
 * Prototypic disorder in enzyme pathways: glucose-6-phosphate dehydrogenase (G6PD) deficiency. Results from defect in G6PD enzyme (this defect may survive in the population by conferring a partial resistance to malaria). The lack of this enzyme causes the active (reduced) glutathione levels in the body to drop precipitously-- this means the hemoglobin tetramers get damaged by oxidation and stick to the red cell membrane, decreasing the deformability of the cytoskeleton. This causes the red cells to be trapped in the spleen and destroyed extravascularly.
 * Notice that this has clinical effects mainly under oxidative stress of some kind-- often shows up clinically as intermittent acute hemolytic anemia, depending on the oxidative stresses the person has undergone recently.
 * Can result in hyperbilirubinemia (jaundice), particularly in infants.
 * Managed by avoiding oxidant foods and drugs, and supplementation with folate; can also require transfusion (watch iron levels).
 * Good to know: aspirin is associated with hemolytic episodes in this disease.
 * Other disorder: pyruvate kinase deficiency. Briefly, the cell has trouble making ATP, becomes rigid, and is destroyed in the spleen. Anemia and high reticulocyte counts follow; folate, transfusions, and splenectomy are used to treat.
 * Identify other acquired causes of RBC destruction.
 * Not sure what he's getting at. Toxins can be 'acquired' that will lyse red cells.
 * Define autoimmune hemolytic anemia, explain the pathophysiology, distinguish “warm” and “cold” antibodies, and describe the testing used to identify antibodies causing hemolysis.
 * Autoimmune hemolytic anemia: hemolytic anemia caused by abnormal immune response to antigens on the surface of red cells.
 * Two basic types of antibodies involved:
 * 'Cold' antibodies: mainly IgM, with a little IgG. These transiently bind red cells, then activate complement to lyse them.
 * Notice that 'cold' hemolysis takes place intravascularly.
 * 'Warm' antibodies: IgG; these bind strongly to RBC. Once they get to the spleen, the antibody-RBC complex causes macrophagic activation to destroy the bound red cells.
 * Notice that 'warm' hemolysis takes place extravascularly.
 * 'Cold' antibodies are called cold because they bind in cooler areas of the body and fall off in warmer ones. 'Warm' antibodies bind optimally all throughout the body and don't fall off.
 * Testing for autoimmune hemolysis mainly involves the __Coombs test__. Two ways of doing this:
 * Direct (also called DAT): looks for the presence of antibodies or complement fragments on the surface of the patient's red cells. A positive reaction (agglutination) to the Coombs reagent indicates an autoimmune reaction.
 * Indirect: test patient's serum against normal red cells to see if antibodies or complement will bind to them.
 * Define the major syndromes of autoimmune hemolytic anemia and describe associated lab data (DAT, antibody and complement reactivity, antigen specificity, thermal range) and relate this to management options and prognosis.
 * This is a whole helluva lot more detail than he gave it in lecture. Here's my effort at a condensed version:
 * Clinical symptoms: acute/chronic anemia, jaundice, dark urine, pallor, decrease in oxygen carrying capacity, high reticulocyte count, high bilirubin levels.
 * To determine which flavor of autoimmune hemolysis it is, determine whether the hemolysis is intravascular or extravascular; if extravascular (warm), can treat with enzymes that block splenic macrophage activity.
 * Outline management options for inherited and acquired hemolytic anemia including autoimmune hemolytic anemia.
 * See under the various subtypes themselves above.
 * List some of the major foods, drugs, or other chemicals which can induce hemolytic anemia in patients with G6PD deficiency.
 * Aspirin, as noted. Also fava beans, methylene blue (for methemoglobinemia- remember?), and vitamins C and K.
 * Describe the distribution of G6PD deficiency among ethnic groups.
 * Essentially it's higher in areas with more brown people-- these are, more or less, the areas with higher rates of malaria. (Us crackers will stick with cystic fibrosis and no dancing skills for our ethnic plagues.)
 * List some of the risks and benefits of splenectomy. Describe when prophylactic antibiotics are indicated post-splenectomy, what antibiotics are used, and the role of vaccination.
 * Spleen: immune organ; important in development of immune response in children.
 * If removed, can lead to partially compromised immune system, which in turn can lead to bacterial sepsis if the kid gets infected with //Streptococcus pneumoniae// . If a splenectomy is unavoidable, vaccinate against influenza, //strep pneum.//, meningococcus, and administer prophylactic penicillin daily throughout childhood. Also make sure child is taken to get medical attention if he/she develops a fever over 101.3 degrees.
 * On the other hand, it's better than dying from uncontrolled splenic hemolysis.

=Sickle Cell Disease=


 * Explain the molecular bases for sickle cell disease and how specific mutation leads to the phenotype. Describe its mode of inheritance.
 * Sickle cell disease is an autosomal recessive illness caused by mutations in both copies of the beta-globin gene. At least one of these mutations has to be a characteristic single-amino-acid substitution on the first exon in the gene (which leads to the production of hemoglobin S); the other can also be the same substitution mutation (which combination is specifically called __sickle-cell anemia__) or a variety of other mutations, such as beta thalassemia or hemoglobin C (see below). See below under 'sickle cell trait' for normal-hemoglobin heterozygotes.
 * Hemoglobin S, when deoxygenated, polymerizes into helical fibers which dissolve again upon oxygenation. This polymerization of its internal hemoglobin damages the cellular cytoskeleton and forces the cell into a distorted sickle shape, which after a few cycles of polymerization and depolymerization it retains irreversibly. Once the cell is stuck in that shape, it is invariably caught and lysed by the spleen.
 * Describe the geographic distribution of sickle cell disease. Describe a situation where people heterozygous for sickle cell disease may have a survival advantage.
 * As described last year, sickle-cell disease appears in mainly equatorial regions, but is particularly widespread where malaria is endemic. Heterozygotes with sickle-cell trait (see below) seem less susceptible to malaria.
 * Describe the findings on the CBC and peripheral blood smear in patients with sickle cell disease.
 * Increase in reticulocyte count to attempt to compensate for anemia.
 * In some patients, increased WBC and platelet count. This is a byproduct of the hyperactivation of bone marrow progenitor cells.
 * Notice that a high WBC level also predicts for earlier mortality, since the constant damage to endothelial surfaces (see below) results in more inflammation and cell death.
 * Increased distribution of width of red cells, due to sickling effect on RBC size and immature RBC release from the marrow.
 * On peripheral smear: sickled/irregular/varied forms of RBCs, reticulocytes
 * Patients without a spleen: Howell-Jolly bodies (purple dots) within RBCs
 * Patients with hemoglobin C: hemoglobin C crystals (red rods) within RBCs, target cells (cells that look like a bullseye)
 * Patients with beta-thalassemia: microcytosis, target cells
 * Describe what “sickle trait” is. Describe the consequences of having sickle cell trait.
 * Sickle trait describes the genetic condition of being a heterozygote for the sickle-cell beta-globin gene when the other beta-globin gene is completely normal.
 * Persons with sickle-cell trait will not get sickle-cell disease-- they produce enough normal beta-globin to be asymptomatic. Notice that they still seem to benefit from increased malarial resistance.
 * Describe major variants of sickle cell disease, including sickle beta-thalassemia and SC disease.
 * Sickle beta-thalassemia: One beta-globin gene is S (sickle), one is Bo (major) or B+ (minor) thalassemic. Minor sickle beta-thalassemia is clinically mild, with a slightly elevated reticulocyte count and slight microcytosis. Major sickle beta-thalassemia is more severe, and shows full-blown sickle-cell disease, because there is virtually no normal beta globin production.
 * SC disease: One beta-globin gene is S (sickle), one is C (a beta globin variant in which the globin is unstable and easily broken down). SC disease is an intermediate- severity sickle-cell disease.
 * Describe:
 * __Hand-foot syndrome__: Acute swelling of the hands and feet in sickle-cell patients. Generally seen in infants with severe sickle-cell disease.
 * __Vaso-occlusive crisis__: The red cell membranes of sickle-cell patients are characteristically damaged and adherent to other surfaces. This means that the microvasculature (mainly post-capillary venules) of vessels that supply major organs can become occluded by 'stuck' RBCs. In addition, the walls of the endothelia can become damaged and more adherent as more damaged RBCs flow through them. This process is worsened under conditions of hypoxia, dehydration, inflammation, and infection.
 * A vaso-occlusive crisis is an event precipitated by one or more of those stimuli in which severe pain develops suddenly in a variety of places in the body, presumably because those regions are vaso-occluded and ischemic.
 * This can be fairly transient (corrects itself), or can be a chronic process that leads to major organ injury.
 * Chronically, the cause is usually red cells that lyse in the vessels and damage the endothelium, causing it to stenose over time.
 * Major stroke rate for sickle-cell patients is up to 10% by age 20.
 * __Aplastic crisis__: An aplastic crisis is simply anything that leads to a sudden drop in hemoglobin levels. Sickle-cell patients are much more susceptible to sudden changes in hematopoiesis because they rely so heavily on increased RBC production to keep up their red cell counts in the face of increased lysis. Infections, inflammations, or vitamin deficiencies (iron, folate) can precipitate aplastic crises in sickle-cell patients.
 * __Splenic sequestration crisis__: A type of vaso-occlusive crisis in the spleen, which due to its extensive microcirculation is particularly vulnerable to blockage. Massive blockage in the spleen can lead to severe anemia and circulatory shock (inadequate blood flow throughout the body). The spleens of nearly all sickle-cell disease patients are chronically occluded, develop splenomegaly, and are usually destroyed by age 5. Notice that splenic destruction can result in a lack of ability to destroy certain encapsulated microorganisms, and childhood sepsis can result. (prophylactic drops of penicillin past the age of 5 is usually recommended.)
 * __Acute chest syndrome__: Another occlusive crisis, this one occurring in the lung circulation. This damages the lung endothelium and allows fluid to leak into the alveoli, decreasing lung capacity. Chest pain, fever, and low O2 saturation result. Acute chest syndrome is a common cause of death among sickle-cell patients and is often triggered by pneumonia. Pulmonary hypertension is a common consequence of increased resistance in lung circulation.
 * In patients with sickle cell disease, explain other complications of sickle cell disease, including stroke, priapism, cardiovascular disease, kidney disease, retinopathy, and delays in growth and development.
 * Generally, these have to do with the adherence of altered RBCs to the microcirculatory pathways. Vaso-occlusion, total or partial, can result in all kinds of very unpleasant results depending on where it occurs.
 * Note that heart and liver are relatively unaffected.
 * Explain the relationship between aplastic crisis and parvovirus B19.
 * Parvovirus B19 infects red blood cell precursors in the bone marrow and causes arrest of erythropoiesis, which can lead to aplastic crisis due to the body's reliance on fresh blood cells.
 * Describe some of the therapies to treat patients with sickle cell disease and the rationale for therapies such as exchange transfusion, hydroxyurea, and bone marrow transplantation.
 * Exchange transfusion: The idea is to get around the problem with chronic blood transfusions, namely iron overload, by taking out the same amount of blood that you're putting in. To do this, you need IV access in both arms, apheresis machines, very well-matched blood to avoid immune response, and lots of money.
 * Bone marrow transplantation: The gold standard, since in principle you can completely cure a patient this way. The problem, as with any marrow transplantation, is that you're killing off the patient's immune system and replacing it wholesale, so if your match isn't A-1 perfect, your patient is dead. So if the sickle-cell patient has a sibling who's a great HLA-match (organ transplantation compatibility), this works great, but otherwise it's dangerous. Note that in principle there's no reason you couldn't use non-sibling marrow as long as it HLA-matches well.
 * Hydroxyurea: An inventive approach to the problem of abnormal beta-globin, hydroxyurea is an oral agent which induces the production of fetal hemoglobin (ie gamma globin) to make up the beta-globin deficit and lessen polymerization of hemoglobin S. This seems to help with the acute crises of sickle-cell disease and extends life expectancy, though long-term benefits are still under study.
 * Explain iron chelation therapy, its indications, and its drawbacks.
 * Iron chelation: If you're in a situation in which you need to repeatedly transfuse a patient, you need a way to take all the excess iron out of their system. Chelating agents bind to iron and allow it to be excreted. *Drawbacks? Infection potential?*
 * Explain how newborn screens can be used to diagnose sickle cell disease.
 * Can look at hemoglobin concentrations in the blood and look for hemoglobin S traces.
 * Interpret a labeled photograph showing a hemoglobin electrophoresis to make a diagnosis of sickle cell disease.
 * See notes. Effectively you look for differently-sized electrophoresis bands to determine whether you have hemoglobins A, C, S, F, etc.

=Thalassemia=


 * Review the normal structure of hemoglobin and indicate the globin chains that typically make it up. Describe how the composition of globin chains in hemoglobin changes during fetal development and after birth.
 * You should have seen this enough to know it by now. Two alpha, two beta globins is the predominant adult (A) form with a minor alpha2-delta2 (A2) form. Fetal hemoglobin is predominantly alpha2-gamma2 with early excursions in zeta and epsilon globins.
 * Describe what thalassemia is and explain in a general way the molecular basis for it.
 * Thalassemia- from Greek //thalassa//, sea, + //hema// , blood. Essentially a problem with globin production or stability. Again, you should know this by now. Recall that there are two alpha globin genes on each chromosome, so clinically severe alpha-thalassemias are relatively rare.
 * Explain the meaning of the terms thalassemia major, thalassemia intermedia, and thalassemia minor.
 * Thalassemia major, or Cooley's Anemia- a beta thalassemic disorder in which the body is making very little or no beta globin due to two completely silenced beta globin genes. The body compensates by trying to use fetal and A2 hemoglobin.
 * This means you wind up massively overproducing blood cells, which leads to bone marrow expansion, bone and joint damage, and hepato- and splenomegaly.
 * So you need pretty regular blood transfusions for your patients to survive past the age of 2 or so. You need iron chelation to avoid killing them at 10 from iron overload.
 * Thalassemia intermedia- 2 mildly to moderately silenced beta globin genes.
 * Thalassemia trait- 1 silenced beta globin gene, 1 normal.
 * Compare alpha-thalassemia and beta-thalassemia. Explain the genetic basis of both.
 * Genetic basis of both is that you're missing adequate globin production.
 * Alpha-thalassemias: Generally only clinically severe in hemoglobin H disorders (one chromosome with no alpha globin genes active, the other with only one alpha globin gene active).
 * Beta-thalassemias: There seems to be more clinical variation-- some genes are 'mildly silenced,' some are 'moderate,' some are 'severe.' Essentially, you need at least one well-functioning beta globin gene to be clinically normal.
 * List the ethnic groups who are more likely to have alpha- or beta-thalassemia.
 * Mediterranean, some African, Southeast Asian
 * Describe findings on the CBC and peripheral blood smear in patients with thalassemia. Sketch front and side views of a target cell.
 * Note that even in mild thalassemia, can see microcytosis/hypochromia.
 * In moderate or severe thalassemia, you can partly make up for microcytosis (Dr. Hassell: "wussy red blood cells") by making way more of them. Look for elevated reticulocyte counts.
 * Describe the geographic distribution of thalassemia. Describe a situation where people heterozygous for thalassemia may have a survival advantage.
 * Evidently it helps against diseases that get into the blood-- I don't really see it.
 * Explain why Southeast Asians with alpha-thalassemia are more likely than Africans with alpha-thalassemia to have a child with hydrops fetalis.
 * Hydrops fetalis: no alpha globins at all. Generally this is fatal in utero or shortly after birth.
 * It's more common in Southeast Asia because the more common alpha-thal allele there is one in which both alpha globin genes are silenced (vs. Africa, in which the common allele is one in which one alpha globin is silences and one is active).
 * Explain how newborn screens can be used to diagnose thalassemia.
 * Look at the hemoglobin breakdown- look for persistent hemoglobin F, abnormally high levels of hemoglobin A2 in beta-thalassemia. Also look for tetramers of gamma globin ("Bart's hemoglobin") or tetramers of beta globin (hemoglobin H) in alpha-thalassemia.
 * Interpret a labeled photograph showing a hemoglobin electrophoresis to make a diagnosis of alpha- or beta-thalassemia.
 * Look for decreased levels of hemoglobin or compensatory alternative hemoglobin forms.

=Disorders of granulocyte/monocyte number=


 * Identify the basic morphologic features of neutrophils, eosinophils, basophils, monocytes, and macrophages and explain their production, distribution, and turnover.
 * I think you should probably know this by now. Neutrophils have segmented nuclei; eosinophils (bi-lobed) stain red or orange due to their Major Basic Protein reserves vs. helminths; basophils stain blue due to their acidic granules; monocytes and macrophages are kind of amorphic and tend to show vacuoles.
 * [Recall that in folate or B12 deficiency you can see not only neutropenia (in very severe cases) but also hypersegmented neutrophils with 6+ lobes.]
 * All of them (recall macrophages are just monocytes that have left the vascular system) are produced by CFU-GM cells in the bone marrow.
 * Neutrophil distribution: Recall that neutrophils live a very short period of time in the peripheral circulation (we've been told values ranging from 7 to 24 hours, but pretty short in any case). After they've matured in the marrow, they're kept for a few days as a 'storage pool' to be released in case of infections; then they're released, circulate in the bloodstream for about 6 hours, and spend the rest of their brief lives in tissues.
 * Monocytes: no retention (storage pool) in marrow; developed in marrow for about 7 days, move to the peripheral blood for 3-5 days, then go into tissues and live there a long time- days to months.
 * Eosinophils and basophils: mature, go into the peripheral blood, go into tissues, then stay and survive for weeks. Notice that basophils have receptors for IgE-- probably the main actor in type I hypersensitivity.
 * Define neutropenia and describe the clinical consequences of neutropenia.
 * Neutropenia: a low absolute count of **neutrophils and bands** (evidently, not knowing the bands part of it is indicative of hematological Philistinism). Get the percentage of segs and bands in the smear and multiply it by the total white count to check for neutropenia.
 * Newborn: < 3000
 * Infant: < 1100
 * Child-Adult: < 1500
 * Can also vary by ethnicity.
 * Another thing is also to try and figure out if there's still a storage pool of neutrophils in the marrow. Can't check it unless you do a bone marrow biopsy.
 * Consequences of neutropenia: mainly, you get recurrent infections on account of you need the segs to beat away the bugs in your tissues; notice that your inflammatory response is also blunted.
 * Note that, for an adult, there's not a serious risk until you get below an absolute count of about 500 (cells/microliter) or so. Below 250 you're at risk for sepsis.
 * Remember that neutropenia is not a magical measure of all the neutrophils everywhere in the body; just of the neutrophils in a small sample of blood taken to be representative of the rest of the blood. Thus you can have lots and lots of neutrophils at an infection site in the tissues and still have 'neutropenia' because they've all moved out of your bloodstream.
 * Diagram the major causes and differentiate the major acquired or congenital/genetic disorders of neutropenia.
 * __Look for infection at teeth and gums__-- people with neutropenia will often have progressing gingivitis.
 * Most common cause of acquired neutropenia: __acute infection__. Can be due to:
 * Increased utilization of neutrophils
 * Increased margination of neutrophils (can be complement-mediated by C5a or chemokine-mediated in overwhelming infections)
 * Marrow suppression
 * Most often due to viral infections: EBV, CMV, influenza, HIV, etc.
 * Sometimes also due to bacterial infections (Gram neg. sepsis, TB, etc), fungal, etc.
 * Antibody reactions (where the antibodies released against some antigen bind to the neutrophils)
 * Antibodies can be against the infectious agent, or some toxin, or against various drugs (penicillin, etc) taken to alleviate illness.
 * Classification of neutropenia (by decreased production vs increased destruction):
 * Due to decreased production (decreased bone marrow reserve):
 * Kostmann syndrome (severe congenital neutropenia)
 * Apoptosis of myeloid precursors associated with elastase gene mutations. Can be AR or AD with incomplete penetrance.
 * Results in very severe neutropenia; marrow contains a very low M:E ratio with severe maturation arrest.
 * Babies: recurrent staph, E. coli, pseudomonas infections; usually die before 1 year of age. Can lead to acute myelogenous leukemia.
 * Treat with G-CSF; consider bone marrow transplant.
 * Schwachman-Diamond syndrome:
 * Apoptosis of marrow precursors; usually AR.
 * Multisystem problem: pancreatic insufficiency, dysmorphic features, recurrent infections
 * Treat with pancreatic enzyme replacement, G-CSF.
 * Many patients develop aplastic anemia or leukemia
 * Cyclic neutropenia (cyclic hematopoiesis):
 * Apoptosis of marrow precursors; recurrent fever, gingivitis, etc
 * Shows a highly individual cycle of low-then-normal neutrophil levels: during low phases, count gets down to less than 200.
 * Chemotherapy, folate/B12 deficiency, and aplastic anemia can also suppress stem cell activity in the marrow.
 * Due to increased destruction (mostly intact bone marrow reserve):
 * Immune neutropenia:
 * Chronic benign neutropenia of childhood: antibodies cross-react with neutrophils. Marrow production normal to increased, storage pool normal to decreased; turnover increased, thus low levels of neuts in peripheral blood. Generally asymptomatic and self-resolving.
 * Autoimmune neutropenia: antibodies made specifically against neutrophils. Can come along with SLE, immunodeficiency states.
 * Alloimmune neutropenia: immunization of the mother to fetal neutrophils. maternal IgG crosses placenta vs. fetal neutrophils. Frequently presents as severe neutropenia prolonged into early infancy (up to 3-4 months), easily mistaken for sepsis-associated neutropenia.
 * Hypersplenism: excessive sequestration/destruction of neuts in spleen.
 * Discuss major treatment strategies including growth factors for treating neutropenia.
 * Notice that you want CBCs twice a week for 6 weeks to follow up on neutrophil count-- determine if it's acute or chronic. Can also get bone marrow aspirate to check neutrophil storage pool.
 * For neutropenia induced by chemotherapy, congenital syndromes, or accompanied by a 101+ fever: culture blood and look for organisms; treat with broad-spectrum antibiotics (change to specific antibiotics when organisms are identified).
 * G-CSF (granulocyte colony-stimulating growth factor) can be given to normalize production and prevent infection. Note that in chronic conditions this may need to be given on a constant basis.
 * Define leukocytosis and provide reasons for a high white blood cell count. Describe the term “left shift” and what it indicates.
 * Leukocytosis: elevated total number of white cells.
 * Causes: infection, inflammation, physiological stress, leukemia.
 * Left shift: increased number of neutrophils and bands in the white count differential. Generally seen in infections (in which the bone marrow is pumping out neutrophils faster than they can mature-- think reticulocytes in anemia).
 * Define eosinophilia, basophilia, and monocytosis, and point out major causes for each.
 * Each of these is an abnormally elevated level of the appropriate type of cell.
 * Basophilia: seen primarily in drug or food hypersensitivity type I reactions, sometimes in infection/inflammation/myeloproliferative disorders.
 * Eosinophilia: seen primarily in parasite infection and drug/allergic reactions.

=Under-Production Anemias=

diminished: neutropenia, thrombocytopenia. o Elevated levels of unconjugated bilirubin or LDH o Low levels of reticulocytes (again, all underproduction anemias show low retic levels)
 * [Vitamin B12 = //cobalamin// if it comes up.]
 * Compare and contrast the clinical and lab features of anemia with the following syndromes:
 * Chronic inflammation and infection (and malignancy/acute sepsis):
 * Clinical: Features go along with whatever the underlying pathology is.
 * Lab: The severity of the anemia tends to follow the severity of the underlying disease but is generally mild to moderate. Can be microcytic with some hypochromia. Low serum iron, low TIBC (iron binding capacity) due to low number of iron receptors, some high ferritin (ferritin is an acute-phase reactant to inflammation or infection), low levels of erythropoietin (Epo), low reticulocyte count
 * In malignancy/acute sepsis: increased tumor necrosis factor (TNF) decreases the ability to mobilize iron and make erythropoietin; interferon beta (INF-beta) acts on marrow and decreases its ability to make new red cells.
 * In chronic inflammation/infection: same thing, but with IL-1 and INF-gamma respectively.
 * Renal disease/failure:
 * Clinical: Similar to normal ol' renal dysfunction (fatigue, pallor, dyspnea, tachypnea).
 * Lab: Generally don't find anemia until you progress to severe renal dysfunction. Moderate to severe anemia, low reticulocyte count, low hemoglobin, very low levels of erythropoietin (recall that it's made in the kidneys).
 * Lead intoxication:
 * Clinical: personality changes, headache, weakness, weight loss, abdominal pain, vomiting.
 * Lab: mild to moderate anemia, low reticulocyte count, microcytosis and hypochromia, basophilic stippling (blue dots in red cells on smear), high zinc protoporphyrin (zinc is being used in place of iron), high lead levels (durr).
 * In lead poisoning: lead inhibits the synthesis of protoporphyrin and the enzyme that links iron to the porphyrin.
 * It seems strange that lead should inhibit the synthesis of protoporphyrin, given that one observes levels of zinc-protoporphyrin as a lab sign. Maybe it just doesn't inhibit it very much.
 * [Note that low reticulocyte count is universal for all under-production anemias.]
 * Identify other underproduction anemia syndromes, including sideroblastic anemia, protein malnutrition, hypothyroidism, hypopituitarism, and decreased affinity hemoglobin mutants.
 * Sideroblastic anemia is a generic name for a lot of disorders in which iron starts piling up in your red precursor cells since it can't be put into heme (either because it can't be joined to porphyrin or because porphyrin production itself is impaired).
 * Hemoglobinopathies in which the O2-dissocation curve has shifted to the left can cause mild anemia or hemolysis.
 * Protein, calorie, or vitamin/mineral deficiency can also lead to underproduction anemias.
 * [He may just want us to be able to recognize the list above as possible causes of underproduction anemias.]
 * Differentiate management schemes for the major syndromes including those in the first objective:
 * First rule: correct underlying condition (sepsis, inflammation, malignancy, lead poisoning, etc) first where possible.
 * Lead: chelate.
 * Renal insufficiency: often can't correct except with transplant. However, can give infusions of erythropoietin as a transient fix.
 * Endocrine disorders: replace hormones.
 * Generally, don't transfuse except in case of extreme cardiovascular insufficiency.
 * Note can also give Epo at any point to correct for an RBC deficiency.
 * In sideroblastic anemia, treat with chelation therapy and B6 supplementation.
 * Describe the rationale for the use of erythropoietin and transfusion in the management of underproduction anemia.
 * Underproduction anemia: inadequate production of red cells. Erythropoietin: hematopoietic growth factor stimulating erythropoiesis. One understands why the latter might be used to treat the former.
 * Transfusion: replacing blood with a low red cell count with blood with a normal red cell count. Again, it makes sense.
 * Explain how vitamin B12 or folate deficiency can lead to anemia.
 * Folate and B12 deficiency arrest red cell precursors in S phase mitosis, after which they get destroyed in the bone marrow. The anemia is **megaloblastic**.
 * Basically the problem is that they're co-factors to methylate homocysteine into methionine (a common methyl donor, very important for proper DNA methylation, among other things). __Folate, in addition, is extremely important for making DNA bases__ (purines, pyrimidines) __and making sure you convert uracil to thymine__ (without which process there's no thymine to incorporate into DNA).
 * __B12 is also important to methylmalonyl-CoA conversion to succinyl-CoA. MM-CoA comes from amino acid degradation; succinyl-CoA gets dumped into the Krebs cycle to be recycled. B12 deficient patients wind up with a backup of methylmalonyl CoA.__
 * The reason this is important is that you can tell the difference between folate deficiency and B12 deficiency by looking at methylmalonic acid levels in serum (folate has nothing to do with MM pathway). If the levels of MM are high, it's B12 deficiency.
 * Describe how vitamin B12 deficiency can be distinguished from folate deficiency clinically. List which effects of B12 deficiency are non-reversible.
 * Effects of B12 deficiency are seen much more slower than folate deficiency (see below for why).
 * One clinical difference between the two is that B12 can cause neurologic deficiencies due to inadequately myelinated neurons.
 * Symptoms include sensory losses, loss of __proprioception__ (sense of location of body parts), cognitive/emotional changes.
 * Neurological changes can be non-reversible depending on how long they went on before the B12 deficiency was corrected.
 * Describe how vitamin B12 is absorbed from the gut and explain the rationale and procedure for the Schilling test.
 * B12 is released from food by stomach acid and binds to Intrinsic Factor (IF) secreted by the stomach; it's thus protected from digestion until it gets to the terminal ileum, at which point the complex gets taken up and separated. The B12 itself gets bound to a transport protein (TcII) to travel through the blood, and can either be taken to the liver to be stored or to the tissue (eg marrow) for use.
 * __Note that B12 is conserved in the body fairly well; thus it takes longer to show the effects of B12 deficiency than it does for folate.__
 * [Folate: the recycling system for folate is much less effective than that for B12- need a fairly high dietary intake of folate.]
 * Folate absorbed straight-up in duodenum and goes to the liver; can enter into enterohepatic recirculation.
 * Schillings: have patient eat radiolabeled B12. If all pathways intact, B12 absorbed into bloodstream. Patient given a "flushing" dose of B12 intramuscularly to prompt excretion and you look for radiolabeled B12 in the urine to evaluate the rate of B12 uptake.
 * Describe the findings on the CBC and peripheral blood smear of a patient with B12 or folate deficiency.
 * Megaloblastic changes: large, immature nuclei (thus larger cells and larger nuclei).
 * Lots of red cells in an attempt to compensate (RBC up).
 * Macrocytosis/ovalocytes
 * __Hypersegmented neutrophils__ (4+ lobes of nucleus)
 * In severe cases of folate/B12 deficiency, can also see other cell types beside red cells
 * List the major causes for B12 deficiency. Describe what elements of the patient’s history are important in determining the cause.
 * Dietary intake: generally found in animal products, thus watch out for all-vegan diets.
 * [Folate: generally found in leafy greens, some citrus.]
 * Also: autoimmune disease against Intrinsic Factor-producing cells or IF itself, gastrectomy (which means no more IF produced), ileal resections or problems with ileal absorption, and TcII deficiency.
 * [Problems with folate tend to be mostly dietary, since you need so much of it per day (can, in principle, also have metabolism enzyme deficiencies). Increased demand for folate during pregnancy or hemolysis.]
 * [Treatments: inject B12, orally replace folate. Anemia reverses quickly; retics elevate over a couple of days, Hgb rises over 1-2 weeks, WBC and platelet count get better rapidly, wait for normal turnover of abnormal red cells. Neurological improvement slow if present at all. Corrected B12 or folate deficiency that still doesn't correct anemia is probably predictive of a different cause of anemia.]

=Transfusion Medicine=


 * Describe the basic process of donor qualification and blood collection relating specific steps to blood safety.
 * Screening, ie weight and age restrictions-- are you bigger than a breadbox?
 * Donor interview and questions-- ever been to a Turkish prison?
 * Review of high risk behavior-- how come and how long?
 * Instructions for donor to call back with symptoms or questions
 * Abbreviated physical exam- general appearance, vitals, skin, extremities
 * Hematocrit count
 * Skin prep and phlebotamy technique (no bacterial introduction)
 * Some of these have to do with donor safety, others with recipient safety. It's pretty simple which is which.
 * Identify the basic components derived from blood donation, explain the biological characteristics of each component, and compare the optimal storage environment and storage time for each component.
 * Whole blood: 1 unit = about 450-500 mL + 67 mL anticoagulant/preservative. Can be divided into packed red blood cells (PRBCs) and fresh frozen plasma (FFP), or with an additional filtration step or two, you can also get some platelets out of the bargain.
 * Note that with particular types of apheresis equipment, you can take out just the RBCs or just the FFP or just the platelets without taking the rest (remove blood, take out components you want, return the rest to the patient).
 * Not all blood components are stored the same. They also have different properties that are important to know.
 * __Whole blood__:
 * Volume: **500-575 mL, HCT 36-40%**
 * Store at **4-6 degrees C**, expires on average in **35 days**.
 * Loses platelet and neutrophil function quickly (24 hrs)
 * Loses clotting factors more slowly but quicker than expiration of unit
 * __Packed red blood cells__:
 * Volume: **200-250 mL, HCT 70%**
 * Store at 4-6 degrees C, expires on average in 35 days (can be stored in special preservative for about a week longer).
 * Can specifically filter out any remaining white blood cells.
 * Can also glycerolize and freeze PRBCs. Good for at least 10 years.
 * __Fresh frozen plasma__:
 * Contains no cells, but anti-coagulation and clotting proteins.
 * Store at **-18 degrees C**, expires in about **a year**.
 * Cryoprecipitate: (frozen plasma, partially thawed at 4 degrees for 18 hours, centrifuge and remove the supernatant; what's left is your cryoprecipitate). You do this to get particular clotting factors (which are in what's left) at a particularly high concentration.
 * Store at -18 degrees C, expires in about a year.
 * __Platelets__:
 * Two types: donated from whole blood, or apheresed. The latter tends to be more concentrated.
 * Store at **room temperature away from light**, under gentle agitation (to avoid clotting), expires about 5 days later. Note the clotting factors degrade throughout storage.
 * As with PRBCs, can leukoreduce (filter out WBCs) to stop adverse reactions.
 * __Granulocyte__ concentrate: either apheresis or buffy coat from centrifuged blood.
 * **Room temperature storage**, probably because the durn things become ineffective so quickly that you'd waste too many of them in trying to preserve them.
 * Note that there's a 3-5% hematocrit that comes along with these, as well as a bunch of platelets.
 * Note that the criterion of an expiration (or "outdate") time is that 70% of the solution has to still have normal function. And it's not 100%, 100%, 100%, oh crap, 69%: function decreases slowly over time. "Valid" blood products can still be suboptimal.
 * Discuss the basic blood groups (ABO and Rh) and contrast the different compatibility requirements of basic blood components.
 * This was mainly done already in Cohen's lecture today. Remember that people have antibodies against any or either of the sugars that they don't make (O have A and B antibodies, AB people have none, Bombay people have A, B, and O antibodies).
 * Notice that all blood products should be microfiltered (evidently this means to filter against little microclumps of dead cells or fibrin in blood products).
 * Whole blood: Has to be typed and crossmatched.
 * PRBCs: Have to be crossmatched as whole blood.
 * FFP: Have to be ABO type specific, but not crossmatched.
 * Cryoprecipitate: "Consider giving ABO type specific or compatible".
 * Platelets: Again, "consider" matching ABO.
 * Granulocytes: Have to be typed and crossmatched.
 * Notice that for whole blood and PRBCs, matching (ABO, Rh), screening (vs known antigens), and cross-matching should take about __45 minutes__.
 * If you haven't got 45 minutes, just match and screen.
 * If you haven't got any time at all, use O-negative blood (if male or "non-child-bearing woman," can use O-positive).
 * Notice that typing and crossmatching are both required whenever you're giving someone a whole lot of either red cells or granulocytes.
 * Differentiate the specific indications for each of the basic blood components: packed red cells, fresh frozen plasma, and platelets.
 * PRBCs are used to give patient added oxygen transfer capacity.
 * FFP is used to treat coagulopathy related to procoagulant deficiencies; if it's a specific clotting factor deficiency, it's treated with the appropriate concentrated factor.
 * Platelets are used specifically to treat bleeding associated with platelet dysfunction or underproduction.
 * Explain the basic rules of blood administration.
 * This covers pretty much everything in this lecture. I'm going to go, preliminarily, with his "Practical Tips" section.
 * Never add anything to your transfusant, IV solutions or Ringer's or whatever.
 * Don't warm your transfusant over 37 degrees C.
 * After 4 hours at room temperature an open blood bag shouldn't be used. If it's kept at 1-4 degrees (cold fridge), extend its life to 24 hours but no more.
 * PRBCs should be transfused within 4 hours.
 * All products should be administered through a standard filter.
 * When transfusing RBCs in a patient with autoimmune hemolysis, only give 10% of the cells as a test dose. If there's an immune response (back pain, dyspnea, anxiety, blood in urine), don't give any more of that unit; open up a fresh one and test that.
 * You can cross-match a neonate with either the baby's or the mother's serum (I'm not sure why this is the case-- no response from Ambruso on it thus far).
 * Common-sense stuff that isn't as common as it should be:
 * Make sure you're giving the right stuff. Look at it.
 * Make sure you're transfusing the right patient. Ask them their name.
 * Make sure your setup is good to go.
 * Keep an eye on the patient as the transfusions go on.
 * Explain the infectious risks of blood transfusion and describe testing strategies to reduce risks of specific agents.
 * There are some risks. Some of these are syphilis, Hep A/B/C, HIV, and West Nile.
 * There are some tests to run to avoid them. Some of these are based on donor history.
 * Classify the non-infectious adverse events of transfusion, assess a constellation of symptoms and signs of the prototypic reactions, and describe the clinical management.
 * __Acute hemolytic transfusion reactions__: the ones you really want to avoid wherever possible. Generally caused by mistyped (ABO) blood. Activation of complement triggers intravascular hemolysis and, depending on volume, can lead to shock, acute renal failure, and DIC (disseminated intravascular coagulation) all of which are excellent ways to kill your patient. Management includes diuretics and heparin.
 * __Febrile non-hemolytic transfusion reactions and mild allergic reactions__: most common adverse event. Manage with antipyretics and/or antihistamines, or by transfusing with leukoreduced blood products.
 * __Delayed hemolytic reactions__: seem to represent the slow building of antibodies against something in the transfused blood. This should be noted in the patient's medical record, since the next time they're transfused they could have a much quicker and more violent reaction.
 * __Anaphylactic reactions__: rare, but unclear etiology. Airway closes up-- manage with epinephrine (bronchodilator), antihistamines, and steroids (anti-inflammatory/immune).
 * __Transfusion related lung injury__: Indistinguishable from ARDS (acute respiratory distress syndrome). Something - cytokines from donated blood, perhaps - reacts to make the lungs extremely inflamed, which leaks fluid into the alveoli and inhibits proper gas exchange. Presents as tachypnea and hypoxia. Manage supportively by sedation, analgesia, and intubation, and limit administration of fluids.
 * __Transfusion associated circulatory overload__: fluid overload. Treat with diuretics.

=Disorders of granulocyte/monocyte function=


 * [Note that the mitochondria of neutrophils generate no ATP-- get energy through glycolysis.]
 * Diagram the basic categories of neutrophil function and indicate, if possible, the defects expressed by leukocyte adhesion deficiency (LAD) I and II, actin dysfunction, specific granule deficiency, Chediak-Higashi syndrome, and chronic granulomatous disease (CGD).
 * __Adherence__ - to stick to endothelial surfaces prior to extravasation (defects cause LAD I and II).
 * __Chemotaxis and ingestion__ - to follow signals to the site of an infection and engulf the offending compounds (defects due to actin dysfunction or abnormal Fc receptors).
 * __Granule release__ - to merge granules with engulfed phagosome to kill microbes (defects due to Chediak-Higashi syndrome or a specific granule deficiency)
 * __Bacteriocidal (oxidative) activity__ - to use reactive oxygen species to kill ingested bacteria (defects result from chronic granulatomous disease).
 * Note that LAD, Chediak-Higashi, and CGD are all described under "Chronic Inflammation," in the Disease and Defense notes.
 * Characterize the types of infections you might expect to see with defects of phagocyte function or complement.
 * Phagocyte deficiency:
 * High rate and severity of bacterial and fungal infections, particularly with organisms that normally are not causative for pathophysiology.
 * In CGD: particularly high rate of catalase positive organisms (//Staph. aureus//, //Staph. epidermidis// ).
 * Early periodontal disease (bacteria at gumline is normally kept in check by neutrophils).
 * Complement deficiency:
 * Specifically have problems with //Neisseria// microbes (a genus particularly susceptible to complement lysis).
 * More generally, see bacterial infections similar to those seen in antibody deficiencies (pyogenic microbes like //Strep. pyogenes//, or //H. influenzae// or //Strep. pneumoniae// ).
 * Discuss tests which would characterize a phagocyte or complement problem. Differentiate between screening or confirmatory tests.
 * Phagocyte deficiency tests:
 * Screening:
 * CBC and white cell differential/morphology
 * Bacteriocidal assay
 * Chemotaxis assay
 * NBT test for CGD (NADPH deficiency)
 * Confirmatory:
 * Adherence tests, production of superoxide, essentially watching the neutrophil's entire process to see if something's out of whack.
 * Basically more complicated and/or expensive tests to confirm anything caught in the screening.
 * Complement:
 * Screening:
 * Measurement of C3 levels
 * CH50 (test of lysing activity of complement)
 * Confirmatory:
 * Measurement of all complement components
 * Evaluation of alternative vs. classical (vs. lectin?) pathway activity.
 * Describe the NADPH oxidase enzyme system, techniques used to determine its activity, and the consequences of a defect in one of its components.
 * NADPH oxidase enzyme system: the idea is to use NADPH on the phagolysosome's surface to react with oxygen and form superoxide (O2-) inside the phagolysosome (thus flooding the microbes with ROS and killing them).
 * Defects result in the absence of ROS production (respiratory burst)- see CGD description under "Chronic Inflammation" (D+D notes). Note that CGD is "granulatomous" because it forms granulomas-- the neutrophils can't destroy the bacteria they've engulfed, and wind up being lysed and destroyed by them (contributing to the pus and necrosis at the granuloma center and the ongoing nature of the infection).
 * Defect detection techniques: NBT test (**n** itro **b** lue **t** etrazolium) tests for superoxide free radicals.
 * Discuss the major neutrophil adhesion glycoprotein and clinical consequences of its pathologic mutation.
 * Beta-2 integrins: heterodimers with alpha and beta subunits. Consequences- see LAD-1 and LAD-2 (under "Chronic Inflammation" in D+D).
 * Describe the features of granules and their contribution to microbiocidal activity.
 * Lots of little vesicular packages with various unpleasant things as their contents.
 * Degranulation occurs, in neutrophils, mainly intracellularly after ingestion of a phagosome-- the granules merge with the phagosome to release their contents into it, presumably destroying the microbes inside.
 * [Note that neutrophils do not normally granulate extracellularly, except a little bit to get through the basolateral membrane after extravasation. But if they're stimulated in the wrong way at the wrong time they can basically blow up and spew their granule contents all over the nearest cell surfaces-- this phenomenon, at the endothelial surface, seems to be primarily responsible for causing Acute Respiratory Distress Syndrome.]
 * Discuss management strategies for patients with innate immune disorders.
 * Assume they're going to get sick all the time; try to figure out what's causing the disorder and fix it where possible (hematopoietic stem cell transplant or gene therapy if needed).
 * Surgery is OK for infected sites (both therapeutically and diagnostically).
 * Broad-spec antibiotics initially, switch to specifics once organism is known.
 * For neutropenia can give G-CSF, 3 mcg/kg/day.
 * Prophylactic antibiotics can, here, actually help. Also cytokine therapy (like INF-gamma therapy for CGD- attract and activate macrophages to make up for a dearth of effective neutrophils).

=Histology of the Thymus and Peripheral Lymphoid Organs=


 * Describe the basic structure and general movement of lymph and lymphocytes through a lymph node. What differentiates activated nodules from non-activated?
 * [Honestly I've tried my best here but it's easier just to go look it up in his notes.]
 * Dr. Bendiak: "It looks like a bean" (or a kidney). Got a big efferent (outgoing) lymphatic vessel on one side (medullary side) and a bunch of smaller afferent (incoming) lymphatic vessels on the other side (cortical side). The region between the cortex and the medulla is called the //paracortex//.
 * There's a whole bunch of nodules on the cortical side; these are stuffed full of B cells (cortical side is mainly B cells).
 * Normally these stain uniformly dark; when a nodule has been 'activated,' it shows up as a darker ring surrounding a lighter middle (rapid cell division). I'm guessing here, but I think when a nodule is 'activated,' one of its B cells has been selected for by an antigen and it's rapidly generating more of that clonal cell line.
 * Inside activated nodules you've got __germinal centers__ (the dark-surrounding-light pattern) that contain __follicular center cells__: mainly B cells, a few T helper cells, and a bunch of rapidly dividing cells called centroblasts (or centrocytes). Also contains dendritic cells/macrophages (the macrophages are eating dead B cells).
 * In the medullary area, you have a bunch of cords radiating outward from the hilum; these cords contain mainly plasma cells.
 * Between the medullary cords and the efferent lymphatic vessel you've got a collecting area called the medullary sinus; this is full primarily of macrophages.
 * The paracortex is mainly full of T cells.
 * Flow of lymph: in through afferent vessels to the cortex (B cells), the paracortex (T cells), the medullary cords (plasma cells), the medullary sinus (macrophages), and out through efferent vessels. Not entirely sure what this implies.
 * Outline the vasculature of lymph nodes. Know the importance of the high endothelial venule.
 * You get blood vessels (arteries/veins) running along with the efferent lymphatic vessel through the medullary sinus to vascularize the entire lymph node. Note that the efferent lymphatic vessel and the blood vessels are called the hilum of the node (on the medullary side, near the sinus and cords). Note also that no vessels accompany the efferent vessels (incoming lymphatic fluid).
 * __High endothelial venules__: have endothelial cells that bulge a bit into their lumens. This allows the lymphocytes in the bloodstream to diapedese into the lymph nodes via special receptors.
 * Describe the blood flow through the thymus. Be able to recognize the differences between a lymph node, thymic lymphoid tissue, and that of the spleen.
 * [Thymus: bilobed organ separated into many regions by thick trabeculae. Each internal region is divided into a central node (medulla) and the surrounding space (cortex). __Trabeculae are a defining pathological feature of thymus tissue__.]
 * Blood flow: Both arteries and veins enter/leave the thymus through the outer capsule and __run through inner trabeculae__. Note that there's no exposure of blood in vessels to the cortical areas of the thymus regions (just the medullary areas).
 * Arteries come mainly off the internal thoracic and inferior thyroid arteries.
 * [Maturation of T cells:]
 * Note that immature T cells, or thymocytes, progress as they mature from the cortex of a given thymal region to its medulla.
 * Thymocytes are accompanied by "nurse cells" as they progress-- these produce various cytokines that help the thymocytes grow and mature.
 * Be able to recognize the nuclei and cell bodies of reticuloendothelial cells in the thymus, Hassall’s corpuscles, and know how they are relevant to lymphocyte selection.
 * Reticuloendothelial cells: involved in selection process (positive and negative) for thymocytes as thymocytes progress towards the medulla.
 * Hassall's corpuscles: cells that thickly populate medulla-- produce lymphokines that promote thymocyte maturation into adult T cells.
 * Note that there's a thick layer of endothelial cells surrounding the medulla- form a 'blood-thymus barrier' that prevents developing T cells from being exposed to antigens in the bloodstream.
 * Notice that you get a population that's heavier on nurse cells (which seem to be more transparent) and less heavy on thymocytes (which seem to be denser) as you get closer to the medulla (many thymocytes have been selected out).
 * Describe the blood flow through the spleen. How does it differ markedly from that of the thymus and lymph node?
 * It's particularly different because it's got an open blood circulation through leaky sinuses. In the other organs you wanted to control blood interaction with the cells inside the tissue-- thus have tissue perfusion only at particular capillary sites. In the spleen you want the blood to run all around in there, to interact with the reticular fibers, lymphocytes, and macrophages. The spleen is your blood's filtration system.
 * Basically the arterioles have discontinuous endothelia-- they dump the blood into open sinus spaces around them. Small, trabecular veins run throughout the spleen to pick up the blood (also through open, discontinuous endothelia) and return it to the splenic vein.
 * There's a sheath of lymphoid tissue surrounding the leaky arterioles, called the __periarteriolar sheath__; these contain germinal centers, as in the lymph nodes, that are responsible for mounting immune responses to antigens in the blood.
 * Be able to recognize the cellular components of white pulp and red pulp.
 * White pulp: seemingly random distribution of lymphatic tissue throughout spleen. Looks white, grossly, or similar to lymphatic nodules on slides.
 * Red pulp: everything else. Looks red, grossly, or not-like-nodules on slides.
 * Note that macrophages are found in both red and white pulp. They're particularly important to remove senescent red cells and platelets that circulate through reticular fibers in the red pulp of the spleen.
 * Be able to recognize and describe the regions of mucosal-associated lymphoid tissue.
 * Mucosal-associated lymphoid tissue (MALT)- found primarily in gut and respiratory system (GALT and BALT respectively for Gut- and Bronchial-).
 * It's a specialized internal lining beneath the mucosa/epithelia of those tissues; mainly T cells with a significant fraction of B cells. Primarily, these make IgA for transport to the other side of the membrane (as dimers joined with a J chain, through secretory component receptors-- remember?).
 * Notice that in gut, you have specialized __M cells__-- generally macrophages that transcytose antigen from the lumenal to the basolateral side of the epithelia in order to display fragments of that antigen to the surrounding lymphocytes. There seems to be some decision-making involved in which antigens to transcytose and present and which to leave alone.
 * [Common theme: you've got reticular connective tissue (essentially fibrous strands that catch cells in fluid) inside the tissues that want to filter fluid passing through them. In this case, these are the lymph nodes and the spleen, but not the thymus.]

=Histology of the Thymus and Peripheral Lymph Organs, Part II=


 * Describe the functions and distribution of the lymph system.
 * To move potential antigens into contact with large numbers of cached lymphocytes, then to release the lymphocytes those antigens activate into the bloodstream.
 * It's all over the place. Go get your Netter's out of storage if you want specifics.
 * Describe the types of lymphoid cells. Understand the functions and interactions between these cells.
 * …so basically, immunology.
 * Explain the functions of all major lymph organs (spleen, thymus, lymph nodes, MALT).
 * Spleen: select out senescent red cells, defend against encapsulated organisms
 * Thymus: select against strongly self-reacting red cells and for slightly self-reacting red cells (see Cohen's notes).
 * Lymph nodes: store T and B cells in large numbers to expose to antigens and provide space for them to proliferate when activated.
 * MALT (mucosa-associated lymphoid tissue): to produce immune response to selectively transcytosed antigens across mucous membranes.
 * Delineate the differences between primary and secondary lymph organs.
 * Primary: where lymphocytes are made and mature. Secondary: where they go and are stored after they mature.
 * Thymus is primary; spleen and lymph nodes are secondary.
 * Be able to describe to describe the structure of all major lymph organs. Explain the function of the main lymph organ cell types and/or parts.
 * Take the quiz; read the notes; read the LO's from part I.
 * Describe the blood flow through the different encapsulated lymphoid organs, and the general flow of lymph through the lymph node.
 * Flow of lymph in lymph node: cortex to paracortex to medulla.
 * Flow of blood in lymph node: medulla to paracortex to cortex (& back again).
 * Flow of blood in thymus: through trabeculae to medullary regions (& back again).
 * Flow of blood in spleen: all throughout, out of the discontinuous endothelia; picked up by pervasive veins and returned to splenic vein.

=Overview: From Innate To Adaptive Immunity=


 * [You know, the immune system seems less like a monolithic "system" - less of a centralized monarchy - and more like a feudal system; lots of different "immune systems" that work together, or not, as the situation requires.]


 * Define (and recognize abbreviations):
 * Pattern-recognition receptor (PRRs): These are receptors for molecular patterns that are present in things that might cause us harm, but which are not present in our own bodies (ie. antibodies). Mostly these are the Toll receptors.
 * Pathogen-associated molecular pattern (PAMPs): The patterns just mentioned- ie lipopolysaccharides (LPS) on Gram-negative bacteria, double-stranded RNA, etc. Notice also you have DAMPs (damage-associated molecular patterns)- these are signals our cells send to their surface when they have been severely damaged or invaded.
 * Toll-like receptor (TLRs): specific PRRs. How the innate immune system recognizes antigens. Humans have 11 of them.
 * Note there's a lot of overlap between these. TLRs are PRRs; PRRs are, mostly, TLRs. TLRs - and thus PRRs - have PAMPs that they use to recognize antigens.
 * Identify some common foreign patterns recognized by TLR.
 * See above- LPS, double-stranded RNA, cell walls/peptidoglycans.
 * Identify the final transcription factor that is most commonly activated in inflammation.
 * This is NF-kB (NF-kappa-B), "the mother of all inflammatory processes."
 * Define cytokine and chemokine.
 * Genes expressed in nearby cells when TLRs detect antigens on the surface of another cell. (The affected cell can also produce cyto/chemokines all on its own.)
 * Cytokines: do a bunch of ill-defined stuff involved in inflammation. Seem to cause movement of cells of various types towards the cytokines.
 * Chemokines: cause inflammation; this is because they're very strongly attractive to white cells (which go through __chemotaxis__: chemically attracted movement. To observe in action, place a twenty-eight year-old man in the next room to an open container of good beer and the LSU-OSU game on Monday night).
 * Describe the function of the innate immune response, and compare and contrast it with adaptive immunity.
 * **Innate immune response** : in response to chemokines and cytokines, blood vessels dilate; the endothelial cells in vessels upregulate the expression of adhesion proteins that 'stick onto' WBCs in the circulation; the endothelial cells separate from each other and allow fluid and WBCs to leak out of the blood and into the tissue. WBCs either eat the foreign bodies or wall it off. Problem solved.
 * Trouble is that the innate immune system has limited resources-- there are only so many WBCs in the circulation.
 * **Adaptive immune system** is a later-evolved, 'backup' system in vertebrates with jawbones (which seems fairly arbitrary). It swings into action after the innate system has exhausted its resources but can't contain the antigens.
 * In the skin and mucosa, tissues are very rich in //dendritic cells// -- these function to phagocytose compounds in the vicinity of a wound site. Once it has ingested them and is activated by chemokines, it moves into the lymphatic system and is taken up to the nearest lymph node. En route it transforms into an antigen-presenting cell-- effectively it takes what it has engulfed and expresses it on its surface.
 * The lymph nodes are packed with lymphocytes, which are extraordinarily variable cells which express different patterns of receptors. These are capable of recognizing and binding to antigens, but not to native cells and molecules, the reason being that there are lymphocytes that express patterns that recognize just about any foreign antigen in the world, but //no// lymphocytes that express patterns that recognize our own tissues.
 * Once a lymphocyte recognizes a foreign pattern on the dendritic antigen-presenting cell, it becomes "activated"-- it begins to duplicate in those lymph nodes at the fastest rate of any cell division in the human body (division every 6 hours or so). Essentially your body mass-produces specific lymphocytes tailored to the specific antigen that was presented by the dendritic cell.
 * Name the cell that forms the bridge between innate and adaptive immunity.
 * This is the dendritic cell, as described above.
 * Discuss briefly the function of T cells.
 * Lymphocytes that recognize, with their surface receptors, antigens presented by other cells (either dendritic or antigen-presenting peptides on normal cells). They activate and proliferate as described above, and wind up circulating through the body and accumulating in places where their specific antigen is present on __cell surfaces__. Once there, they can use cytokines called //lymphokines// to attract macrophages to do their work ('helper-1 T cells'), or they can directly target the antigen-showing cells for destruction ('killer T cells').
 * Called T cells because they proliferate in the thymus (though they're made in the bone marrow).
 * Notice that some T cells ('helper-2 T cells') can activate B cells for antibody production.
 * Discuss briefly the function of B cells.
 * Lymphocytes that also recognize antigens (though these don't have to be presented by a dendritic cell), activate, and proliferate. However, instead of going out into circulation themselves, these secrete //antibodies// (soluble versions of their antigen receptors) to circulate into the __extracellular spaces__ (tissue fluids, blood, secretions) instead. See below for more on antibodies.
 * Notice that antibodies can't cross cell membranes.
 * [Here's the thing: whenever a cell creates a protein, the cell takes bits of that protein and expresses it on its surface-- these are __antigen-presenting peptides__.]
 * [If the antigen is free-floating, it's can be targeted by antibodies (thus B cell system). If the antigen is inside cells, it's a target - via antigen-presenting peptides - for the T cell system.]
 * Describe briefly the chief properties of the 5 immunoglobulin classes.
 * **IgE** (immunoglobulin E): attaches to mast cells in tissues-- this causes them to make and/or release all kinds of stuff (prostaglandins, leukotrienes, cytokines, granules with histamine, etc) that effectively cause an allergic reaction. This is evidently targeted against invading parasitic organisms.
 * **IgG** : most abundant antibody in blood. Two IgG molecules that bind an antigen trigger an inflammative process called //complement//, about which more later (basics: kicks the crap out of antigen cells).
 * Notice that maternal IgG is the only antibody to cross the placental barrier-- thus it's the fetus's only adaptive immune defense for a while.
 * **IgA** : most abundant antibody in secretions (saliva, tears, etc). This means it's first up against membrane-penetrating microorganisms.
 * **IgD** : antigen receptor on the surface of B cells.
 * **IgM** : sort of a rapid-response IgG. Also activates the //complement// process, and more quickly. It's the first antibody to appear in the blood after exposure to a new antigen, and is replaced by IgG after a couple of weeks.
 * Mnemonics (kind of hokey, use at own risk):
 * Types: **EGAD, M** an!
 * Ig**E** = **Explody** or **Extrovert** (cause mast cells to empty out their contents)
 * Ig**G** = **General** or **Gory** (all-purpose antibody in blood)
 * Ig**A** = **Aww** (contained in tears and other secretions)
 * Ig**D** = **Don't I Know You?** (recognition antibody on B-cell surface)
 * Ig**M** = **Mobile** or **Maroon** (rapid-reaction antibody in blood, gets there first)
 * Give examples of immunopathology.
 * **Type I immunopathology** : **immediate hypersensitivity** . Your run-of-the-mill allergic reactions to ordinarily harmless things like pollen, gluten, etc. Caused by an abundance of IgE (makes sense, given the allergy implies mast cell triggering).
 * **Type II immunopathology** : **autoimmune reactions** . Caused by antibodies that react against components of the body.
 * **Type III immunopathology** : caused by immune reactions to soluble antigens caught in capillaries. The resultant inflammation causes rash, arthritis, all kinds of bad mojo.
 * **Type IV immunopathology** : Not caused by antibodies, but by "collateral damage" wreaked by T cell-activated macrophages in the process of blowing the living shit out of nearby antigens. Think Rambo with a flamethrower in a crowded subway. This is a significant problem in tuberculosis and viral hepatitis.

=Antibody Structure=


 * Define:
 * [Ig: shorthand for immunoglobulin, or antibody class.]
 * H chains: 'Heavy,' larger proteins. Hooked to each other by disulfide bonds. H chains are where J chains and SC chains bind (see below).
 * L chains: 'Light', smaller proteins. Hooked to H chains by disulfide bonds. The L-H region is where the antigen-binding sequences are.
 * kappa and lambda chains: Kappa and lambda chains are the two genetic types of light chains. They don't seem to be terribly different from each other (though notice that when a B cell begins making a new type of Ig, it changes the heavy chain type but not the light chain type).
 * hinge region: Unsurprisingly, the region where the antibody hinges, between the region where the two heavy chains are bound to each other (Fc) and the region where the heavy chains are bound to the light chains (Fab).
 * Fab: A **F** ragment of an antibody above the hinge region (contains one light chain bound to one heavy chain, with an epitope-binding region in the middle). Note that the Fab fragment contains the variable, antigen-binding region.
 * F(ab)2: Two Fab fragments of an antibody whose heavy chain portions are still joined together. Comprise two heavy chains (joined), two light chains, and two variable, antigen-binding regions included in them.
 * Fc: The **F** ragment of an antibody below the hinge region, consisting only of two constant-region heavy chains. The Fc region does not bind antigen, but does signal inflammatory and phagocytic cells once the Fab region has bound antigen.
 * Complementarity-determining regions: Where the antigen-binding regions of particular immunoglobulins are. Notice that the antigen-binding region is formed by the combination of both heavy and light variable regions.
 * Variable (V) and constant (C) regions: parts of the light or heavy chains that either vary (the antigen-binding region) or stay constant between antibody molecules.
 * VL and CL: variable and constant regions of the light chain.
 * VH: Variable region of the heavy chain.
 * CH1, CH2, CH3, (CH4) : Constant-heavy-chain segment; can have a varying number of these depending on what immunoglobulin type you're talking about. CH1 is the first constant region of the heavy chain next to the variable region, CH2 is the second, etc.
 * Name the 5 antibody classes, and their characteristic heavy chains.
 * IgG, IgM, IgA, IgE, IgB. Differentiated by constant regions of heavy chains:
 * IgG: two light chains, two 'gamma' heavy chains (gamma refers to a particular type of constant-region of heavy chain)
 * IgE: two light chains, two 'epsilon' heavy chains
 * IgD: two light chains, two 'delta' heavy chains
 * IgA: immunoglobulin dimer, joined end-to-end; four light chains, four 'alpha' heavy chains joined at their other ends by a "J" chain and wrapped around by a chain called "Secretory component" (helps get secreted and protects from digestion).
 * IgM: immunoglobulin pentamer; ten light chains, ten 'mu' heavy chains joined in a radial fashion at their other ends by a J chain.
 * Notice that a single IgM antibody can bind lots of times to proximal antigens (ie. its valence is more than two- see below). This has consequences for activating complement (also see below).
 * Notice that a given activated, proliferating B cell will always produce the same variable chains on its immunoglobulin, no matter if it makes A, E, M, or whatever type of Ig.
 * "__Valence__": number of antigen molecules that a given immunoglobulin can bind (IgG: 2, IgA: 4, IgM: 10, etc).
 * Recall that your body makes IgM as the first-response antibody in the bloodstream. The reason you don't just keep making it - despite its superior complement-activating properties - is that IgM is too big and it makes your blood too viscous. IgM is a rapid-response antibody; IgG is its smaller, safer, steady-state replacement in the bloodstream.
 * Draw a diagram of the structure of typical molecules of each class. Do not bother with the exact number of CH domains. Label the heavy and light chains; Fc and Fab parts; J chains; antibody combining sites; main interchain disulfide bonds; secretory component.
 * This is getting ridiculous. Go look it up in the lecture notes, you lazy bastards.
 * Discuss the significance of the fact that in any one antibody molecule, both H and both L chains are identical.
 * Generally, each side of the immunoglobulin is going to bind to a different molecule of the same antigen. I think we get to the significance of //that// later on.
 * Distinguish the 5 immunoglobulin classes in terms of their approximate molecular weight and approximate concentration in serum.
 * By increasing molecular weight:
 * Hokey mnemonic: **G** reat **D** anes **E** at **A** **M** oiety
 * IgG: 150,000 daltons (or 150 kDa).
 * IgD: 180 kDa.
 * IgE: 190 kDa.
 * IbA: 400 kDa, fully assembled.
 * IbM: 900 kDa, fully assembled.
 * By decreasing serum concentration:
 * Hokey mnemonic: **G** reen **A** pples **M** ake **D** entists **E** nraged
 * IgG: 1000 mg/dL
 * IgA: 200 mg/dL
 * IgM: 100 mg/dL
 * IgD: 5 mg/dL
 * IgE: 0.02 mg/dL
 * Describe the structure of antibody combining sites.
 * Have "immunoglobulin folds"-- overlapping beta-sheets with variable regions poking out towards the surface to recognize and bind antigen.
 * Explain why complementarity-determining regions are also called hypervariable regions.
 * Well, that's where you find the variability, Alice. That's what you use to bind them thar antigens.
 * Give an example of a subclass, an allotype, an idiotype.
 * Subclass: Evidently, minor genetic differences between the constant regions of heavy chains. Ie: IgG1, IgG2, etc.
 * Allotypic differences: Notice that any given B cell only expresses the maternal or paternal heavy chain genes. Thus can have allotypic differences between B cells in the same individual depending on whether they're expressing paternal or maternal heavy chain genes.
 * Idiotypes: An idiotype is the antigen-binding site of an immunoglobulin, __considered as an antigen itself__. Thus can make immunity antibodies against specific antibodies: "anti-idiotypes."
 * Diagram an electrophoretic separation of human serum. Label the anode and cathode. Identify the albumin, alpha1, alpha2, beta and gamma peaks.
 * Damn, he likes pictures. Anode one end, cathode the other; from the cathode (+) to the anode (-) direction, you have five bands: in order, a big band, three small bands, and a really really big band.
 * Big band closest to cathode: albumin.
 * Bigger band closest to anode: gamma peak.
 * Little bands in the middle: alpha1, alpha2, and beta peaks, in that order.
 * Notice that from cathode to anode the bands go in alphabetical order: albumin, alpha1, alpha2, beta, gamma.
 * Define Fc-gamma receptors. Name the inflammatory cells which have them.
 * Fc-gamma receptors are receptors that respond to alterations in the constant heavy-chain region of IgG antibodies; these are found primarily on phagocytic or engulfing immune cells (like macrophages, neutrophils, etc). Thus IgGs that bind antibodies also bind immune cells to that location.
 * Notice: antibody-bound viruses can't get into cells (thus no longer a threat). But bacteria, even if bound to many antibody molecules, can still be pathogenic. Need to rip that sucker a new one.
 * So once you have at least __two__ antibodies bound to an antigen close together, a molecule found ubiquitously in the blood is activated: the complement compound C1q, which in turn attracts neutrophils, etc. We'll go into this more next week.
 * Notice that the IgM antibody, and probably IgA as well, can activate complement all by its lonesome on account of its multimeric structure.

=Antibody Genes=


 * Define:
 * Toxoid: A toxin with the 'toxic' part removed; antibodies can form to its delivery portion which then lend full immunity to the full toxin. This is good because bacterial toxins are often so virulent that an 'immunizing' dose of the active form would be lethal.
 * DNA recombination: Well, it's mainly recombination of DNA, or bits of DNA combining with other bits of DNA (or other bits of itself), to make a sequence of DNA that's not linearly found in either of the bits.
 * RNA splicing: Once you have a string of transcribed RNA, it can splice itself such that only parts of itself are translated. This becomes important where variable regions are joined with constant regions; see below.
 * Somatic mutation: A genetic mutation that doesn't affect the germ-line.
 * Define cross-reactivity. Give an example of a non-self antigen which cross-reacts with a self antigen. Explain, in terms of lymphocyte activation, how a self antigen might not itself elicit antibody, but might react with antibody elicited by a cross- reacting antigen.
 * Cross-reactivity: when antibodies bind both to the antigen they were raised against and also some other substance, usually endogenous, that has the bad luck to bind in a similar manner to the first antigen to the antibody. An example would be rheumatic fever after strep throat infections: the antibodies raised against streptococcus can sometimes cross-react with tissue in the joints and heart valves. So even though heart valve is an antigen that doesn't provoke antibody production, if antibodies are made by something else that happen to fit heart valve tissue, your valves are shit outa luck.
 * Discuss the Clonal Selection Theory in term of: the number of different receptor specificities it postulates per cell; the role antigen plays in the initial expression of receptors; the role of antigen in clonal selection; one experiment which provides strong evidence for the theory; how it differs from an instructional theory; whether it is Darwinian or Lamarckian.
 * Yeah, first rule of high-school biology: no accepted theory is ever Lamarckian.
 * Essentially, the body doesn't rearrange its DNA to make a B cell receptor given a particular antigen structure; it makes a whole ton of types of receptors in the hopes that a couple of them will bind to anything you could conceivably throw at them.
 * Define allotypic exclusion. Demonstrate your knowledge of the concept by first stating the number of chromosomes in a cell which bear H or L genes, and then the number that actually contribute to a particular B cell's antibody product.
 * Allotypic exclusion: you only express B cell immune genes from either your mother's or your father's chromosomes, not both.
 * Each chromosome set can produce one heavy chain and two light chains (kappa and lambda, effectively the same). But only one chromosome set actually gets those chains made- one heavy chain, one light chain, from one set of chromosomes.
 * The reason for this is that you really want to minimize cross-reaction; the way you accomplish this is to limit antibody-producing cells to each make only one type of antibody binding region.
 * Draw a diagram of the heavy and light chain gene regions of human DNA. Indicate V, D, J and C subregions. Show how a heavy or light chain gene is assembled out of these subregions during the differentiation of a B cell.
 * I don't have Dr. Cohen's magical pen arrangement, so I can't show you. But effectively the idea is that you have a bunch of different genetic variable regions (V, D, J in heavy chains; or V, J in light chains) that get schlepped together by enzymes called RAGs, thus creating a new recombination. It's basically like having three jars of paper clips of various colors. You pick up one paper clip out of the first jar and link it onto one from the second, then pick up a paper clip from the third and link it to the other end. If you have a lot of different colors of paper clips (ie genetic sequence variability in V, D, and J regions), you can have a whole lot of different possible color and order combinations.
 * When I say 'schlepping,' one of the things it's doing here is actually cutting off and discarding genetic material-- so once those paper clips have been put together, you throw out the jars containing all the rest. This means that even if the cell was inclined to go back and make a different variable region (paper-clip chain), there's scanty raw material to go back to. This is one reason why receptor editing (see below) rarely works.
 * Notice that the heavy chain is made first, then the light chain.
 * Notice that while the V and D and J regions are being brought together, their ends get some nucleotides randomly slapped on and have others chewed off-- this further randomizes the process and the shape of the variable region.
 * Here's the problem with that: two times out of three, you get a frame shift mutation that will likely result in a nonsense (premature stop codon) mutation. This means the antibody won't work well.
 * Here's how the cell deals with it: if the variable region from one parent's chromosome winds up with a prematurely truncated, useless antibody sequence, the cell will try the other chromosome instead and inactivate the first one. If neither of them works, generally the cell dies. Remember, though, that if the first chromosome's variable region comes out OK, the cell doesn't try the second one.
 * Once the variable region sequence is linked together, it's transcribed and spliced with a particular constant region (either mu or delta) to make either an IgM or an IgD, respectively. IgMs are the first antibodies made; IgDs are put on the B cell's surface to act as receptors a little later.
 * The reason for this order is to cull out self-reactive B cells. See "Ontogeny of T and B Cells."
 * Notice also that T cells effectively do this same VDJ process to come up with their antigen receptors.
 * Describe the somatic recombination model which explains how antibodies of the same specificity (idiotype) can be found in two or more different classes (“class switching”).
 * At a certain point, if the cell figures it wants to make a different class of antibody, the DNA itself is cut and spliced so that the constant region is joined to the variable region.
 * Notice a difference here: as opposed to switching between mu and delta constant heavy chains, which is done by mRNA transcript splicing, for other classes of Fc (gamma, epsilon, alpha) the mu and delta and whatever other regions are physically before the region it wants are cut out of the DNA itself and degraded.
 * This means that if the cell wants to make a class of antibody in the DNA it's just cut, so sorry, no luck. So a cell can make gamma after mu heavy chains, but can't go back to mu again.
 * Calculate the minimal number of genes required to code for a million different antibody molecules, based on the (outdated) concept of "one gene, one H or L chain". Show how breaking the variable region gene up into V, D and J subregions requires fewer genes.
 * If you really had the one gene-one H/L chain, you'd need 2000, 1000 of each (10002 = 1 million), total genes to get a million different combinations. On the other hand, if you have 5 variable regions (V+D+J heavy chain, V+J light chain), you can have a grand total of only 100 genes, 20 in each cluster, and still have 205 = 3.2 million combinations.
 * Describe the mechanisms by which more diversity is created by nucleotide insertion and removal during V(D)J recombination.
 * Described above.
 * Discuss somatic hypermutation.
 * There's an enzyme activated once a B cell is activated that slightly changes the variable region of a B cell's produced antibodies every time the B cell divides. This effectively attempts to make the antibody's affinity even higher for the antigen. This works because there's a kind of Darwinian selection going on- only the antibodies that fit really well to the antigen continue to bind.
 * Define receptor editing.
 * In some B cells, when a given genetic recombination in the variable region is faulty (nonsense mutation codes an early stop), the cell can try again to produce a viable variable region on the same chromosome. That's called receptor editing.


 * Somatic variation**


 * Order of C region genes:**
 * M D G E A**

Don't have D regions in light chains

=Antibody Function and Complement=


 * Define:
 * Valence: The number of antigen molecules a given antibody can bind to.
 * Affinity: How strongly antigens bind to antibodies.
 * Precipitation: A matrix of crosslinked molecular antigens and antibodies that fall out of solution. Generally happens when you have about as many antigens as you do antibody binding sites.
 * Agglutination: Precipitation without purified antigen (antigens are bigger and usually found in large complexes).
 * Hapten: Purified antigenic determinant molecules.
 * Distinguish the five classes of immunoglobulins in terms of:
 * Passage across the placenta: IgG only. Actively transported across the barrier.
 * Ability to activate complement by the classical pathway: IgG and IgM only.
 * Ability to activate complement by the alternative pathway: IgA only.
 * Involvement in allergic diseases: IgE only.
 * "First line of defense:” IgA (in secretions, gut, etc) only.
 * Most resistant to enzymatic digestion: Still IgA (it's in the gut).
 * Sketch the lattices obtained in antigen or antibody excess and at equivalence using an antigen with 4 different epitopes and IgG against them. Discuss the size of the complexes and their solubility in each zone.
 * All you.
 * Discuss why a line of precipitate may form in agar gel when antigen and antibody diffuse towards each other.
 * The concentrations of each of them lessen as they spread; at the line at which the concentrations of antigen and antibody are optimal, precipitations of matrix form out of the solution.
 * Basically if you’ve got too many antibodies or too many antigens, there isn't enough cross-linking to form precipitate.
 * Compare and contrast precipitation and agglutination in terms of the nature of the antigens involved, and sensitivity of the tests.
 * It's the same idea, but precipitation involves antigens that are molecule-sized; agglutination involves antigens that are cell-sized. Agglutination is easier to detect (larger complexes) and requires less sensitive tests.
 * List the components of complement in the order in which they become activated in the classical pathway. Name those that are also activated in the alternative pathway.
 * Classical: C1q activates C4, then C2, C3, C5-6-7-8-9
 * 1-4-2-3-5-6-7-8-9: just make 4 the 2nd number out of a normal order.
 * Alternative: C3, C5-6-7-8-9
 * Notice that C3 always has to be activated to activate C5 to form the membrane attack complex.
 * Discuss the different ways in which complement is activated by: IgG; IgM; IgA; polysaccharides.
 * IgA: acts via the alternative pathway. IgA, bound to microbes, provides a surface on which C3 and other factors can bind and activate C5 and the membrane attack complex.
 * IgG and IgM: activated classical pathway.
 * Polysaccharides on bacterial surface (eg. mannose-containing compounds): lectin-mediated pathway.
 * Identify the complement components which are:
 * Opsonizing: C3b. This fragment sticks to the nearest membrane. Phagocytic cells have a C3 receptor that make it easier for them to phagocytise the stuck-to cell. Remember that phagocytic cells can also attach to activated Fc regions of IgG antibodies.
 * Think of certain bacteria as greased pigs and opsons as, well, things that attach to greased pigs-- greased-pig handles, if you will.. Phagocytic cells are much better at holding onto the opson "handles," when present, than the greased pigs themselves.
 * Lytic: C3b, 5, 6, 7, 8, and 9, particularly 9. Effectively 3b-8 are there to position and attract 9, which actually bores a hole through the bacterial membrane and lyses it.
 * Anaphylatoxic: C3a and C5a fragments can attach to mast cells and release their granules (histamines, etc).
 * The point of this is to increase blood and immune supply to the area- blood vessel dilate and endothelial cells get leaky.
 * Chemotactic: C3a and C5a are chemotactic for a variety of immune cells, but most particularly neutrophils. Once complement is activated, neutrophils flock to that location.
 * Discuss how complement is important in immunity to bacteria even if the bacteria are resistant to lysis by C9. Identify the family of bacteria most susceptible to lysis.
 * //Neisseria// (ie gonorrhea, a Gram-negative coccus) is the family most susceptible to lysis. Even if a bacterium can't be lysed, however, complement has opsonizing, anaphylatoxic, and chemotactic elements that can help destroy it.
 * [Note that chronic //Neisseria// infections may indicate a defect in the C3b-C8 pathway.]
 * Discuss how complement plays roles in both innate and adaptive immunity.
 * The adaptive immune system (antibodies) can trigger complement; however, there are substances on the surfaces of pathogens that can activate complement without any antibody mediation (innate).
 * Classical complement activation: Adaptive
 * Alternative activation: Adaptive or Innate
 * Lectin activation: Innate
 * Discuss the lectin-mediated pathway of complement activation.
 * Essentially lectin proteins bind to mannose-containing saccharides on the surfaces of bacteria (we don't have mannose in our outer structures) and activates complement.

=Immunopathology Type 3: Immune Complex Disease=


 * Arthus reaction and serum sickness are local and general manifestations of immune complex disease; describe the mechanism of tissue damage. Discuss why this is sometimes called “innocent bystander injury”.
 * Effectively, lots of antigen-antibody complexes form, complement is activated, inflammation results, and local tissue is often damaged in the crossfire.
 * The point is that although the antibodies don't target the nearby membranes and structures, the neutrophils that show up discharge lysosomal enzymes that are toxic for those tissues. (complement can also activate mast cells, at which point you can also get a pseudo-allergic reaction like hives). It's friendly fire- you see your target and call in the strike, but he's sitting right next to a shopping mall. Or an orphanage. Or a nunnery. Or baby ducks. Your immune system doesn't really care what else gets hit; it's kind of myopic that way.
 * Indicate the critical size at which immune complexes get stuck in basement membranes.
 * There's an unfortunate presumed typo in the notes here, in which it describes both complexes that are too small to get stuck and complexes that aren't as " less than 1000000 MW." I've emailed Dr. Claman and hopefully clarification will be forthcoming.
 * Describe “one-shot” serum sickness. Make a chart showing antigen, antibody and immune complex levels in relation to time and to symptoms.
 * Serum sickness: inflammation within the vasculature (vasculitis) in which immune complexes get stuck on the walls of the endothelia. One-shot serum sickness comes from the large dose of antigen given.
 * Causes fever, joint pain, kidney pain, pericarditis.
 * Transient-- once most antigen-antibody complexes are excreted out of the system, the symptoms subside.
 * Note that upon hepatitis B infection, the host can never seem to fully clear the virus; get a serum sickness disease that never really goes away.
 * Notice that "antigen" can refer to anything you form an immune reaction to-- can be antibiotics, vaccines, bacteria, etc.
 * Notice also that this can refer to endogenous compounds in autoimmune disorders-- DNA in lupus, the Fc regions of your own IgG proteins in rheumatoid arthritis.
 * Notice that upon initial exposure to an antigen, this reaction takes about a week to start to manifest (need to make antibodies to that antigen); subsequent exposures occur almost immediately (because antibodies are already circulating).
 * Re his 'chart': antigen exposure occurs, antibodies are made, complexes form, serum sickness symptoms begin, the antigens are cleared from the body, symptoms subside, free antibodies are high for a while and then go away.
 * Discuss the types of tissues in which damage is most likely to occur from deposition of immune complexes. What do these tissues have in common.
 * Generally, immune complexes get stuck in basement (basolateral) membranes of places where the blood gets filtered.
 * Glomerulus (nephritis)
 * Pleura (plasma is filtered to provide pleural fluid)
 * Pericardium (for pericardial fluid, cause pericarditis)
 * Peritoneum (for peritoneal fluid)
 * Synovium of the joints (plasma filtered to make synovial fluid)
 * CSF (cerebritis, plasma filtered to make CSF)
 * Discuss the immunological mechanism of a typical Type III disease involving exogenous antigen.
 * Lots of antigen (eg. antitoxin) given; antibodies begin to be made; about 8 days later, the antibodies are released into the circulation, where they bind to the remnant of the original large dose, activating systemic complement, which produces serum sickness. After some time, enough antibody-antigen complexes are cleared out of the body that the serum sickness clears up.
 * Notice that antibody E can potentially be made to the original antigen; this means that any future exposure to the antigen will result in anaphylaxis.
 * Discuss how urticaria could result from interaction of antigen with either IgE or IgG antibody.
 * [Urticaria are hives.]
 * IgG antibodies activate classical complement fragments C3a and C5a, which induce mast cells to release their histamine contents.
 * IgE antibodies directly interact with mast cells to achieve the same thing faster.
 * Discuss the meaning of finding a fluffy white precipitate in a patient’s serum after a day in the refrigerator. Include the name used for such precipitates, the most likely composition, and the interpretation of the phenomenon.
 * The name is __cryoglobulins__; generally they are immune complexes of antigens and antibodies. Large numbers of them indicate that some level of serum sickness is going on.
 * Discuss the pathogenesis of post-streptococcal glomerulonephritis. Describe the diagnosis of this condition by fluorescent antibody technique and name the pattern of resulting fluorescence.
 * Effectively just a basic serum sickness-- streptococci + antibodies can get lodged in the glomerular basolateral membranes.
 * Discuss the pathogenesis of Farmer's Lung.
 * Caused by inhaled antigens from agricultural products (grain) that form immune complexes from the antibodies from a previous exposure, getting lodged in the lung.

=Ontogeny of T and B Cells=


 * Define:
 * stem cell: A cell that can self-renew or differentiate into one or more kinds of non-dividing, functional cells.
 * B cell: Lymphocytes that make antibodies.
 * T cell: Lymphocytes that either directly attack antigenic cells (killer T cells) or influence the development of B cells or attract inflammatory cells (helper T cells).
 * pre-B cell: An immature form of B cell in the bone marrow with cytoplasmic IgM but no surface IgM (or IgD).
 * pre-T cell: An immature form of T cell in the one marrow with no surface antigen receptors; to be transferred to the thymus.
 * self-tolerance: The normal cells of the body don't, generally, bind
 * Draw an outline diagram which shows bone marrow, thymus and spleen or lymph node. Indicate the development and movement of cells of the B and T lines, starting with the hematopoietic stem cell and ending with mature T and B cells.
 * T cells start out in the bone marrow, mature in the thymus, and move to the lymph nodes and spleen. B cells start out in the bone marrow, mature there as well, and then move to the lymph nodes and spleen.
 * Define the Bursa of Fabricius, and discuss where its functions take place in mammals.
 * Bursa of Fabricus is an organ in birds where B cells mature (which is why they're called B cells). B cells in humans mature in the bone marrow.
 * Describe the sequence of appearance of cytoplasmic and surface immunoglobulins in developing B cells. Using these data, derive a model that could explain self-tolerance at the B cell level (“clonal abortion”).
 * B cells develop IgM antibodies (antibody mRNA transcripts spliced with mu constant regions); later on it makes IgD to put on its cell surface.
 * Essentially, they put the IgM out on their surface and wait for something to react. If something reacts, the B cell attempts to reprogram itself with receptor editing, but if it fails (which, presumably, it most of the time), it immediately apoptoses and dies (clonal abortion).
 * The point is to make sure that any B cells that would react to anything they would normally be exposed to (that is, your own body) never make it to maturity. Thus your mature immune system never makes antibodies directly in response to your own tissues.
 * Notice, though, that anything that doesn't actually get into your bone marrow to be exposed to nascent B cells (like, I don't know, nose hair or something) can still potentially be an antibody-producing antigen.
 * Draw a graph showing the antibody response to a typical antigen in a primary and in a secondary response. Show both IgM and IgG antibody levels.
 * Primary response: Takes about eight days to produce the IgG antibodies used in the adaptive response, although IgM is produced fairly quickly.
 * Secondary response: The IgG response is much stronger and faster than before (notice that the timing of the IgM reaction is more or less unchanged).
 * This is a result of something significant: since IgG production is dependent on helper T cells, this implies that the 'memory' function that produces a faster IgG response is mediated by helper T cells as well. More on this later.
 * Draw a graph which shows relative IgG and IgM levels in a normal infant from conception to one year of age. Distinguish maternal from infant's antibodies.
 * In utero, the fetus can't make IgG, but it's getting it just fine across the placenta from its mother. This mother-derived IgG ramps up a few weeks before birth.
 * At birth, a newborn has roughly normal adult levels of IgG.
 * The baby doesn't start to be able to make IgG until about 3-6 months later.
 * Notice that the half-life of IgG is about **3 weeks**.
 * This means that there can be a big lag period between the time when the mother's IgG in the fetus is getting low and the time the kid can start defending itself with its own IgG.
 * This also means that prematurely born babies are particularly at risk, since they have low levels of maternal IgG at birth to begin with (didn't get that last surge before birth).
 * IgM is made by the fetus/baby at a steadily increasing rate throughout this whole period.
 * Note that IgA, imbibed from breast milk, gets into the baby's mucus membranes and serves as a barrier against infection there (notice that the antibodies don't get taken up into the bloodstream or anything-- they just sit in mucus membranes).
 * This is why colostrum is important- has a particularly high level of IgA antibodies.
 * Given a newborn's antibody titer, interpret its significance if the antibody is IgG or IgM. If IgG, calculate what the titer will be at 4 months of age, and state the assumptions that you made when you did the calculations.
 * If you have a low level of IgM at birth compared to an adult, that's normal (you're still making the stuff). If you have a low level of IgG at birth compared to an adult, you can be in trouble (it's going to become inactive long before you can make your own).
 * Use the half-life calculations: half-life of 3 weeks, 4 months is about 18 weeks (6 half-lives) give or take, thus the fraction left over should be 1/26 = 1/64th of the newborn titer.
 * Discuss the decrease in diversity seen in the immune repertoire of older people.
 * The thymus starts out, at birth, pretty gargantuan, but gets a lot smaller and replaced by fat as we age. Correspondingly, the number of new T cells made after age 40 starts to decline (B cells do this too, a little later on)-- the old ones keep getting made, but less so the novel ones. If you're not making a lot of new T cells, you're not able to make new antibodies against antigens you haven't already seen, though you're fine at responding to ones you've seen before.

=T Cells, Parts I + II=


 * List the main types of T cells, and define their functions. Discuss the positive and negative interactions between Th1, Th2, and Treg cells.
 * **Th1** : Also called __delayed-hypersensitivity__ or __contact-hypersensitivity__ cells. Interact with antigen-presenting cells and secrete lymphokines to attract and activate macrophages.
 * How this works:
 * Dendritic or other phagocytic cells phagocytose antigen, partially degrade it, load it onto antigen-presenting molecules, and stick them on the engulfing cell surface.
 * With prior exposure to an antigen, have circulating Th1 cells that recognize that antigen; it finds the dendritic cell and becomes activated. Releases lymphokines, partic. INF-gamma, as above (1 Th1 cell attracts up to 1000 macrophages). Takes some time for macrophages to accumulate; effect is not often noticeable for 24-48 hours. Once macrophages show up, they're activated to (actual technical term) "angry" macrophages, which gives them really extremely good engulfing/destroying properties. Even microorganisms that ordinarily use phagocytic macrophages to disseminate (like mycoplasma tuberculosis) will be destroyed by angry macrophages.
 * The first time you get exposed to an antigen, the antigens have to be taken to lymph nodes to produce the primary response: antibodies and so on. The second time, you've got Th1 cells circulating in your blood that know to recognize the antigen, and can produce an immediate and local rather than delayed and lymph-node reaction.
 * Notice that dendritic cells in particular promote T cell activation by not only presenting peptides but releasing a flood of factors that help activate the T cells.
 * **Th2** : Also called __classical helper__ cells. Effectively promotes B cell activation and antibody secretion. When activated by antigen exposure, starts to make lymphokines (primarily IL-4), which activates B cells that are bound to antigen.
 * __Interactions__ between Th1 and Th2 cells:
 * Differentiating interactions:
 * Note that T helper cells start out in the lymph nodes in an undifferentiated ("Th0"). There is a slight preference for Th0 cells to develop into Th1 over Th2.
 * But Th1-released lymphokines (ie IL-2) promote Th0 differentiation into Th1 and inhibit differentiation into Th2, and Th2-released lymphokines (ie IL-4) promote differentiation into Th2 and block differentiation into Th1. That is: activation of each species of T cell promotes its own species' growth.
 * Notice that that can be a bad thing. In HIV infections, the response is primarily Th2-related (antibody), possibly due to viral interactions with T cells. However, antibodies do more or less no good against HIV.
 * Notice also that this isn't an absolute division. Th1 cells, as noted below, activate and promote Th2 cells through their lymphokines. So it's not like the two systems are mutually exclusive-- just that they promote their own functions more than the function of the other.
 * Functional interactions:
 * Th1 binds to antigen, activates, produces IL-2, which activates both itself and other Th1 cells (which in turn produce more IL-2, as a positive feedback loop), as well as activating Th2 and killer cells.
 * When activated by IL-2, Th2 binds to antigen and activates, which produces IL-4, which turns off Th1 production of IL-2 (finish positive feedback loop).
 * Basically Th1 cells are the master cells: it activates antigen-bound Th2s (which in turn activates B cells) and antigen-bound killer cells.
 * **Killer** : Also called __cytotoxic__ cells. Work by physical contact with virally infected cells; the killer cell signals the infected cell to initiate apoptosis.
 * The infected cell shows a peptide on its surface that is determined to be infected or dangerous; the killer cell detects this and either activates specific 'death receptors' on the infected cell's surface, or injects a mix of proteins into the other cell, then moves off. The infected cell will apoptose shortly thereafter.
 * Notice that killer T cells tend to only kill the infected cells of their own host or native body. This is all involved with MHC (**Major Histocompatibility Complex** ) proteins.
 * Stuff about histocompatibility (doesn't really fit, but he talked about it here):
 * The dendritic antigen-presenting elements we've been talking about are MHC proteins.
 * "Professional antigen-presenting cells" are dendritic cells, B cells, and macrophages.
 * These make antigen-presenting proteins of a class called **MHC type II** as well as those of the **MHC type I** class.
 * Notice that dendritic cells, specifically, use __both__ MHC type I and MHC type II proteins to present antigen fragments that it has ingested. Thus can activate both Th1/2 and also killer cells in the lymph node.
 * Non-professional-antigen-presenting cells - that is, every other cell is the entire body - also has __MHC type 1__ class proteins on their surface, but MHC type 2 proteins are reserved for the pros.
 * Specifically, every cell takes proteasomal protein fragments of all proteins they produce and show them on the surface of the cell
 * Th1 and Th2 cells look at specifically MHC type II presented antigens (generally presented by dendritic cells).
 * Note that helper cells don't look at MHC type I presented antigens.
 * Killer cells, however, have to be able to look at any cell (viruses can infect any type of cell), so they look at MHC type I presented antigens.
 * Note that killer cells don't look at MHC type II presented antigens.
 * Describe the surface markers that can be used to distinguish between T and B cells in humans.
 * "CD" markers: cluster designation markers. Effectively specific proteins found on individual immune cell surfaces to provide surface recognition.
 * How this is used: the CD4 cluster on Th2, say, specifically helps bind to MHC type II antigen-presenting proteins (found on the surface of professional antigen-presenting cells). CD8 clusters on killer cells, by contrast, specifically help bind to MHC type I antigen-presenting proteins, in accordance with what T cells bind to what antigen-presenters (see below).
 * This means that a killer T cell, say, needs to bind to both an antigen and the MHC type I protein that's presenting it. If it would potentially bind to the antigen but the presenting protein's a type II MHC, no luck, it won't get activated.
 * CD20: found on the surface of B cells.
 * CD4: found on the surface of helper T cells.
 * CD8: found on the surface of killer T cells.
 * CD3: found on the surface of __all__ T cells. It's the transducer of the antigen-binding signal to inside the cell.
 * Interesting thing: we 'learn who we are' immunologically effectively early in life (can't inherit immune responses or our father's germ line would attack our mother's germ line and vice versa).
 * Early T cells express only CD3 on their surface (to make their antigen-binding receptors work properly), then, a little later, CD4 and CD8 all at once. This CD4-CD8 immature hybrid is going to brush up, in the thymus, against lots and lots of 'body' cells, all of which are expressing MHC I (and some that are presenting MHC II) on their surfaces. The cell does this, wandering around, waiting to bind its antigen-binding region to something on another cell's surface.
 * Interesting to note that thymus cells actually make and present an amazingly wide variety of body proteins for the T cells to learn to recognize as self. Makes sense-- if the T cell is going to be circulating in the bloodstream, having it only recognize thymus cells as self is a bad idea.
 * This stops when it simultaneously binds to some presented antigen and also its presenting MHC protein. If the presenting protein is MHC type II, it's the CD4 that will help bind to it; if it binds to a MHC type I protein, it'll be the CD8 that helps bind.
 * If the T cell has bound its CD4 region, it stops making CD8; if it's bound its CD8 region, it stops making CD4. Essentially your T cells stay with whatever CD region first bound something, sort of the immunologic equivalent of marrying the first person you kiss. No offense to anyone who's done that, I guess. De gustibus non est disputandum.
 * The point of this entire exercise, aside from determining the killer-vs-helper fate of your T cells, is to make sure your antigen-binding regions can adequately bind to antigen peptides within the confines of the antigen-presenting cleft of the MHC presenting protein. T cells whose antigen-binding bits can't fit with MHC proteins aren't much good, as all the antigens that T cells interact with are all presented by MHC proteins (recall that it's B cells that interact with free antigen). I guess another reason is to make sure your CD regions can bind to their respective MHCs as well.
 * There's a selection process similar to B cell selection that follows: If the T cells bind MHC + antigen strong enough to spontaneously activate, they die immediately (don't want T cells activated by self). However, if they don't bind well to any MHC complex + antigen at all, then they won't be able to bind to MHC complexes well enough to activate immune response sufficiently, and thus also die off.
 * Big important but nuanced point: T cells are raised specifically for their ability to bind to a particular individual's MHC complexes. Allotypic differences between individuals prevent you from being able to use one person's T cells to scan the surfaces of another person's cells (to shore up the faltering Th responses of a patient with AIDS, for example). BUT there's a great big honking exception to this, which is graft rejection and graft-versus-host disease. We'll get into this in exhaustive detail in the next section.
 * Describe markers that Th1, Th2, and killer T cell subpopulations in humans have on their surfaces.
 * As mentioned: Th1 and Th2 have CD4. Killer have CD8.
 * Define lymphokine, chemokine, and cytokine, and give an example of each.
 * Cytokines: This seems pretty vague, but his definition is short-range mediator compounds that affect the behavior of the same or another cell. Examples: IL-1, TNF-alpha, IL-12.
 * Chemokines: Small short-range mediators that primarily cause inflammation. Examples: MIP-1-MIP-4, RANTES, etc.
 * Lymphokine: Cytokines secreted by activated lymphocytes. Examples: IL-2, interferon-gamma (IFN-gamma).
 * Describe an activity of interferon-gamma (IFN-gamma).
 * Secreted by Th1 (helper-1) T cells. __Chemotactic__ for blood monocytes and/or tissue macrophages. Also helps to activate the monocytes/macrophages when they show up, causing them to step up phagocytosis and also to release pro-inflammatory cytokines (__TNF-alpha__ and __IL-1__).
 * Define mitogen, and name two T cell mitogens. Name a mitogen that stimulates both B and T cells in humans.
 * Mitogen: a substance that causes T cell mitosis (ie. T cell clonal division/proliferation, or "activation").
 * Examples: two plant lectins called //PHA// and //ConA//.
 * Essentially they 'fool' every T cell they contact into thinking they're being triggered by antigen.
 * This can be great if you want to look at T cell division; all you need to do is dump some in a plate with some ConA and off they go.
 * This can be a problem if you pick up that ConA and shoot up with it. Activated T cells release lymphokines, some of which cause capillaries to leak profusely (increases fluid/macrophage flow to area). If lots of T cells in the body are activated at once, the plasma fluid leaks out from every capillary simultaneously and you die from intractable shock very quickly (healthy to dead in less than 24 hours). This massive release of cytokine is called a __cytokine storm__.
 * Incidentally, this seems to be why the 1918 flu was so deadly to young people with a healthy, strong T cell system. More T cells = greater severity of the cytokine storm.
 * Distinguish between the effects of a mitogen and an antigen (and, when you learn about it later, a superantigen), when added to normal blood leukocytes.
 * Mitogen: doesn't actually bind to antigen-binding site on T cell, like an antigen does. What it does it to bind to is the CD3 domain that controls signal transduction from the antigen-binding chains. This is like bypassing the light switch in your house- the lights are going to always stay on because there's no longer a mechanism for turning them off.
 * Superantigens (mainly produced by dismaying common microbes, //Staph aureus// and //Strep pyogenes// ) are bacterial exotoxins that bind MCH type II presenting proteins on the one hand and T helper cell receptors on the other. Effectively they form an artificial "enzyme" that plugs the T cell into the MCH-presented antigen, even if the fit with the specific antigen isn't so hot with that T cell. This sets off a cytokine storm also, but by a different mechanism (artificially linking up T cells and MHC proteins rather than directly binding to the CD3 receptor of the T cells).
 * Compare and contrast the antigen receptors of T and B cells.
 * B cells' antigen receptors can interact directly with antigen molecules.
 * T cells' antigen receptors can only interact with antigens through an antigen-presenting intermediary (like a macrophage for type II or any cell for type I).
 * Discuss the structures recognized by T cell receptors. Distinguish between what is recognized by helper and cytotoxic T cells. Explain the special role of dendritic cells in this process.
 * This has to do with the next point. T cell receptors can only recognize antigens which are presented to them by MHC complexes (this is called "MHC restriction").
 * Dendritic cells are particularly handy because they not only present antigen fragments on MHC IIs, but also release factors that encourage T cell activation.
 * Discuss what is meant by “MHC-restriction”. Name the classes of MHC molecules by which CTL, Th1 and Th2 are restricted.
 * Types of T cells can only bind certain kinds of antigen-presenting MHC proteins.
 * Killer cells: type I only (on every nucleated cell)
 * Helper cells: type II only (on macrophages, dendritic cells, and B cells only)
 * Describe the role of T cells in ridding the body of a viral infection.
 * Killer T cells, remember, scour the body looking at the surfaces of cells. If a cell's been infected by a virus, then it's expressing viral proteins (though not true for latent retroviruses), and some of those viral proteins will be misfolded, or what have you, and wind up being chopped up by the proteasome. These fragments will be displayed on the cell surface by MHC type I proteins. Killer T cells can thus attack the infected cells.
 * Describe the characteristics of T-independent antigens.
 * See below. Basically they need to have a repeating motif that can bind to the B cell's receptors many times over. The only things that usually fit this bill are bacterial polysaccharides.
 * Outline an experiment that shows that an antibody response can be “T-dependent”.
 * I'm not sure if he has a particular thing in mind here, but I would test two leukocyte populations' ability to make antibodies to the same antigen-- one population with the full complement of T and B cells, the other with the T cells killed off by radiation (which selectively destroys T cells).
 * Discuss the mechanism by which T cells help B cells.
 * General B mechanism: the B cell's receptors bind to antigen and endocytose both the antigen and the receptor that bound to it. The antigen is broken down by lysosomes into little peptide chains. The antigen fragments are bound by MHC type II proteins and brought back to the surface.
 * Note that the fragments of the antigen may no longer have the same epitope that activated the B cell (may have been cleaved). The T cells that come activate them can be activated by completely different segments of the original antigen than the B cell.
 * Th2 cells come along, looking for MHC type II presentations. Any that recognize one of the antigen fragments begin to flood the B cell with helper factors (most importantly, IL-4) to activate the B cell. This 'switch' both activates massive antibody production and prompts the B cell to stop making IgM and start making IgG, IgA, or IgE. __Without this T-cell mediated switch, the B cell can't make any other immune antibodies than IgM__.
 * Note that although the T cell may have recognized a different fragment of the antigen than the B cell did originally, the activated B cell begins making antibodies to the antigen that __it__ saw, not the antigen that the __T cell__ saw.
 * Notice that there's one way you can activate the B cell without T cell help: namely if the antigen binds to a whole lot of the antibodies on the B cell at the same time. This is rarely the case with protein antibodies but is frequently the case with carbohydrates on the surface of bacteria. Take-home: bacterial carbohydrate capsules can stimulate B cells to activate without T cell help.
 * Note: this means in AIDS, when you're missing your T helper cells, you can still make antibodies to extracellular bacteria with carbohydrate patterns recognizable by B cells. AIDS patients really get hit by either viruses or intracellular bacteria.
 * Also note: B cell activation independent of T cells means that the B cell can only make IgM ('switch' not activated by T cells).
 * Describe the cellular and molecular events following intradermal injection of tuberculin antigen into a person who have cell-mediated immunity to it. Justify calling the process “delayed hypersensitivity”. Characterize the cells that would be seen in a 48-hour biopsy of the site with regard to whether T cells or macrophages predominate.
 * TB antigen comes in; it gets picked up by dendritic cells and broken up, then displayed; circulating Th1 cells bind to dendritic cell surfaces, causing their activation; they begin to release IL-2, which activates any nearby Th1 or Th2 cells that can bind to TB as well, and also release IFN-gamma, that cause the capillaries to open up and macrophages to be attracted to the site over 48 hours or so. Given that 1 activated T cell can attract 1000 macrophages with its chemotactic signals, I think it's a fair bet that the macrophages would outnumber the T cells.
 * The process is called delayed - I think - because the first time you're exposed to the antigen, it doesn't happen. This has to do with T cell timing-- if you're exposed for the first time, your dendritic cells have to bring antigen fragments to the lymph nodes, you have to make antibodies, etc. If you're exposed the second time, you already have circulating T antibodies that recognize those antigen fragments at the site of infection, and can prompt a quick, local, and painful reaction.

=Immunogenetics and Transplantation=


 * Define the Major Histocompatibility Complex (MHC). Distinguish between HLA-A and HLA-B antigens on the one hand, and HLA-D on the other, in terms of: which associate with foreign antigens for recognition by helper T cells; which, in association with foreign antigens, are the targets for killer T cells.
 * **MHC** : genetic basis for the MHC (antigen-presenting) proteins shown on cell surfaces. Also the main loci that determine whether or not transplanted tissues will be rejected.
 * **HLA** : Just the name for the human MHC group genes ( **H** uman **L** eukocyte **A** ntigen).
 * MHC segment of human genome: massively variable. Has three distinct regions: D, B, and A.
 * Note that the D region has several duplicated loci (these are the MHC class II loci) and the B/A regions are distinct duplicates of each other (these are the MHC class I loci).
 * Loci of the D region: DR, DP, DQ.
 * We describe different general patterns of genetic sequence - that is, alleles - at these loci by the letter of the HLA region and a number, eg.: A6, B12.
 * Notice that we have two different copies of the MHC chromosome-- thus we have two distinct sets of HLA alleles. See below under "haplotype."
 * The number of HLA locus matches between one individual and another determines how well transplanted organs or tissues are tolerated in the host. More on this later.
 * This means you always try matching first from siblings (your parents only have half your MHC phenotype-- again, more on this just below).
 * Define: alloantigen, haplotype
 * Haplotype- The set of HLA alleles that you acquire from one parent or the other. Every individual has two distinct haplotypes, one from each parent. Combined these form the "phenotype."
 * For example, an individual's haplotypes could be DR17, DP9, DQ10, B47, A16 on the one hand, and DR3, DP1, DQ5, B2, A7 on the other. But the phenotype for that individual (both haplotypes) would be DR17,3 / DP9,1 / DQ10,5 / B2,47 / A7,16.
 * Remember that D = MHC II (recognized by helper T cells) and A/B = MHC I (recognized by killer T cells).
 * Notice that there is very little crossing-over recombination during meiosis in this region of the genome-- so we don't really need to worry about haplotype variation coming from a parent to a child.
 * Alloantigen: NIH defines this as "antigens that exist in alternative (allelic) forms in a single species." I gather that this refers, in this context, to HLA variability and the MHC complex proteins.
 * Distinguish Class I and Class II histocompatibility antigens.
 * I: Made from genes found in A and B loci. Made in all nucleated cells. Used by killer T cells to recognize antigens on cell surfaces.
 * II: Made from genes found in DR, DQ, and DP loci. Used by helper T cells to recognize antigens presented by professional antigen-presenting cells.
 * Identify the chromosome on which the MHC is found in humans.
 * Chromosome 6.
 * Discuss HLA-A and B typing in terms of how many antigens a person has at each locus. Given two unrelated parents' haplotypes, predict their children's phenotypes.
 * At each locus (eg. HLA-A), a person has two antigens: one from the father and one from the mother.
 * Think of haplotypes as alleles. If your parents have haplotypes X and Y on the maternal hand, and Q and Z on the paternal, a quarter of their offspring will get XQ, a quarter XZ, a quarter YQ, and a quarter YZ. Just simple Mendelian crap. X, Y, Q, and Z can be any combination of HLA alleles; the point is that due to the lack of meiotic recombination events, each parent's haplotypes are inherited as whole units; there's no mixing and matching with one of your chromosomes having HLA-A from your mom and the HLA-B from your dad. One of your chromosomes will contain, verbatim, the HLA sequences of one of your mom's haplotypes. The other will contain, verbatim, the HLA sequences of one of your dad's.
 * Discuss the results of exchanging skin grafts P to F1 and F1 to P in inbred animals, and in humans.
 * Here, "inbred animals" means that all the loci on all the chromosomes are exactly identical in each parent, but the parents are genetically distinct (from two different inbred lines of animals).
 * Note that mice don't, sort of by definition, have HLAs (recall __human__ leukocyte antigens?). They have something similar enough for discussion.
 * P (parent) to F1 (first generation of offspring): yes, graft will work (all the HLA-type alleles in the parent are in the child). F1 to P: no, it won't (there are HLA-type alleles in the child that aren't in the parent).
 * Note that F1 to F1 would also work (they all have the same HLA complement).
 * In humans: not this simple. Parents are chromosomally variable. Even from siblings to siblings, the chance of both of them getting the same set of two #6 chromosomes is 1 in 4.
 * Describe the one-way mixed leukocyte reaction (MLR) and discuss its use.
 * I think the general concept is that when you put leukocytes from a donor next to leukocytes from a recipient, they will each start proliferating as T cells (mainly Th1 cells) from each population recognize D HLA regions (MHC type II proteins on professional antigen-presenting cells like B cells) from the other as antigens and activate each other and B cells. A "one-way MLR," in particular, is one in which the leukocytes of the 'donor' cells have been treated to prevent their activation; that way if you have a proliferation response, you can be sure that it's the potential host's T cells activating against the donor cells and not vice versa (though you could certainly reverse the test to look specifically for graft cells against host if you wanted).
 * Distinguish between “HLA-D” and HLA-DR, -DP, -DQ.
 * "HLA-D" is sort of a general term for the group of loci that give rise to MHC type II antigen-presenting proteins. It can also refer to a specific locus, HLA-DR.
 * HLA-DR, HLA-DP, and HLA-DQ are the individual loci within the D region of chromosome 6.
 * Describe the cellular and molecular events which go on during graft rejection, both of the usual type and hyperacute rejection. Include: killer T cells, Th1 cells, angry macrophages, antibody + complement.
 * Before I get into this I want to take a minute and lay out some thoughts on T cell activation by foreign human tissue (applies to both graft rejection and graft-versus-host disease).
 * Recall that T cell activation is predicated on two things: one, the recognition of an antigen (CD3 complex activation); and two, the binding of the T cell to the specific MHC proteins on the surface of the cell (killer T cells look for MHC I's on the surface of all cells, helper T cells look for MHC II's on the surface of professional presenting cells like B cells and dendritic cells). If you have one but not the other, the activation doesn't happen.
 * So the question is, how can your T cells recognize your buddy's kidney tissue in the first place to attack it? If his MHCs are different from yours (and they are, thanks to allotypic variation-- not such a useless concept after all, apparently), your T cells shouldn't be able to grab onto his cells at all, even if your T cells recognize them with antigen-binding domains.
 * The answer, if I understand this correctly, is that your T cells are trying to recognize the combination of antigen + HMC proteins together-- that is, they're trying to bind to a //slightly altered native MHC complex with the antigen inside it// . It's slightly altered, presumably, because of the particular shape and binding properties of whatever antigen it's presenting.
 * Given this much wiggle room (T cells are looking to bind to "almost me"), the problems begin when your T cells recognize the MHC complexes of your buddy's cells - sometimes with but especially without the antigens inside them - as "almost me." This means that your T cells activate an immune response, not just when they bind both foreign MHC and a specific antigen, but when they bind any foreign MHC at all (which are on, oops, all of your buddy's cells). Cohen sez about 5-10% of your T cells are likely to figure out that any particular bunch of foreign MHCs are "almost me" and get activated-- which is enough to make things bad for your buddy's kidney.
 * In a nutshell, that's how I think this is working. Evidently if you get one skin graft from a horse and one from a human, your body will reject the one from the human faster-- because your MHC complexes and the ones in the human graft are more similar (and thus have a higher rate of "almost me" recognition by your T cells) than yours and the horse's. Unless you're from the South, of course.
 * One more note on this: the idea behind HLA-matching in organ donations is that you want the MHCs (both type I and II if possible) to be //exactly identical// between host and graft. That way, the "almost me" factor gets taken out because the foreign MHCs are, by definition, "precisely me" and don't set off T cell reactions. More on different HLA matching below.
 * Okay, now we can get back to the topic at hand.
 * Plain old garden-variety graft rejection is more or less what I just talked about. You get Th1 cells that get activated by "almost-me" MHC type IIs (D region HLAs); they activate Th2 cells, which activate B cells to produce antibody against the foreign tissue, and killer T cells, which start attacking (once they bind to MHC type Is, or A/B region HLAs) the foreign tissue directly. In addition, the Th1s also bring in a whole mess of activated macrophages through their inflammatory response. The activated macrophages produce pro-inflammatory cytokines of their own, like TNF-alpha (**T** umor **N** ecrosis **F** actor). Poof, no more graft. Cohen mentioned in an email that the T cell response is frequently so strong that by the time the antibodies are made, they're irrelevant.
 * A particularly nasty version is called __hyperacute rejection__, in which the graft tissue is rejected almost immediately. Having watched a kidney transplant, I can attest that normally when you reconnect the vasculature and reperfuse the graft with host blood, it turns nice and pink within seconds. Apparently, with hyperacute rejection, it stays white and more or less bloodless even after reperfusion.
 * The reason for this is that there was circulating antibody against the graft's tissue (from a previous, failed graft) or against the graft's residual blood in the tissue's endothelium. Either way, the antibodies attach to the endothelium, activate lots and lots of complement, and thus set off anaphylatoxin release (C3a, C4a, and C5a, remember?) from mast cells that leads to a vasospasm, constricting the vessels and resulting in tissue ischemia. Evidently this can also lead to systemic inflammation unless the tissue is removed pronto.
 * Couple of lessons from this. One is to remember that T-cell-mediated rejection is slower but that complement-mediated rejection is a lot faster. The other is to always cross-type the ABO blood antigens from the donor and the recipient.
 * Notice that, specifically to avoid these types of reactions, immunosuppressants are usually given for the lifetime of a transplant recipient.
 * Explain the interaction of T cells recognizing HLA-D and T cells recognizing HLA-A or B in the generation of killer T cells. Include the roles of cytokines in your discussion.
 * Recall that Th1 and Th2 cells (with CD4 receptors) recognize Type II MHC proteins (professionally presented), while killer T cell (with CS8 receptors) recognize Type I MHC proteins (universally presented). Recall also that A and B HLA regions code for Type I MHCs and the D regions code for Type II MHCs.
 * This means that if the host 'sees' the graft's HLA-A and HLA-B MHCs as antigens, the host's killer T cells will bind to them and be activated, while if the host 'sees' the graft's HLA-D MHCs as antigens, the host's helper T cells will bind to them and become active.
 * The kicker here is to remember that Th1 cells really run the whole adaptive-immune show. So if the host thinks the graft's D regions are 'self' (thus no Th1 cells are activated), but doesn't like the A and B regions, a few killer cells will bind to the A/B MHCs and be activated, sure, but without the Th1 cells jump-starting T cell activation, by and large the reaction will be pretty slight and the graft has a chance of surviving.
 * However, if the host thinks the graft's A and B regions are 'self' (thus no killer cells are activated) but don't like the D regions, then a bunch of Th1 cells get activated, and even though they can't activate killer cells well if the killer cells aren't binding, they'll release a whole boatload of lymphokines (recall, interferon-gamma) to bring in and activate really pissed-off macrophages. Generally, having a bunch of activated macrophages right next to your new kidney is a bad idea, and remember that the Th1 cells will just keep getting stimulated by the kidney tissue until the whole damn thing is destroyed, in the process probably causing spectacular damage to the surrounding vasculature and soft tissue. You don't need killer cells activated with HLA A and B to make a mess of things.
 * The moral of the story is that HLA D compatibility from graft to host is the most important issue in transplant acceptance or rejection.
 * It should go without saying that if the host sees both A/B- and D-region MHCs (both types I and II) as antigens, the entire enterprise goes south a lot quicker.
 * Give an example of a disease whose incidence is tightly linked to a particular HLA allele. Speculate on the mechanism which might explain the linkage.
 * Example he's giving here is __ankylosing sponditis__, an unlikely-sounding disease involving the chronic inflammation and eventual calcification of the insertions of tendons into bones.
 * Evidently, 92% of people with this disease have a particular HLA-B allele (B27). If you put human B27 in rats they get the disease too. Strong correlation there.
 * The basic principle, I think, is that there's a price paid for the massive genetic variability of the HLA regions. The price is that, eventually, some variant is going to look similar to a reasonably common antigen (there's some suggestion that for ankylosing sponditis it's //Klebsiella// ), and in your immune response to the antigen you're going to develop an autoimmune response to your own tissues. Why specifically this disorder manifests in joints, I don't know, but the notes also cite HLA-linked cases of diabetes, lupus, and a kidney/lung degenerative disorder, so maybe it depends on something in the specific antigen.
 * Define graft-versus-host reaction, and discuss the immunologic requirements for its occurrence.
 * We’ve already discussed the problem of having the host's cells attack the newly transplanted donor tissue. The next question is, what about the donor tissue attacking the host cells?
 * T cells injected into another person will react against the new host's body. Normally this isn't a problem (the host's T cells will recognize the foreign cells and attack them back). But if the host is immunocompromised, it can't mount an effective defense and the T cells in the new organ can attack it with impunity. This is called graft-versus-host disease: the grafted tissue or organ is destroying the host's body.
 * Recall that about 5-10% of the graft T cells will see the host's MHC, by itself, as "MHC-plus-antigen" of self. This means they attack all the normal cells of the host. This is enough to do really nasty things in immunocompromised hosts.
 * [Important note: T cells are extremely sensitive to radiation and are killed off pretty easily with it while leaving the other cells intact. So when giving blood to an immunocompromised patient, you want to irradiate it to make sure there's no surviving T cells.]
 * So when you put an organ into an immunocompromised patient, if it has any significant amount of T cells on it, they can mount an attack on the host. This is what makes immunosuppressing transplant recipients prior to transplant such a gamble-- if graft-versus-host reactions start, you've just taken away their only shot at being able to fight it off.
 * Formal requirements for calling something a graft-versus-host reaction:
 * (1) Graft has to have immunocompetent T cells in it.
 * (2) Has to be something in the host that the T cells of the graft can recognize and become activated by (as mentioned above, this is the norm).
 * (3) Host must be immunoincompetent enough not to react against the graft's MHC proteins (otherwise graft is destroyed before it can react against the host).

=Immunohematology ABO/Rh=


 * [Note Rh problems comes into play mainly in repeat transfusion situations (since Rh isn't a ubiquitous substance like ABO antigens are, Rh-negative people don't generally make antibodies against it until they're exposed to an Rh-positive donor's blood. Thus, we're talking mainly about ABO typing here, with the notable exception of pregnancy (see below).]
 * That said: some more info on Rh. It's on a protein, not a carbohydrate, which has a couple of consequences: one is that it's not ubiquitous; thus with no prior exposure, there are no antibodies to it. (Note only about 15% of US is RH-negative; it's a recessive trait.) The other consequence is that the antibodies raised against it are IgG, not IgM (not a repeating carbohydrate pattern, so it's not independent B cell activation).
 * [**Notice also that ABO is on the surface of __all__ cells, not just blood cells** . So if you're doing a transplant, you better be sure you've matched not just HLA but also ABO from donor to recipient. **This in contrast to Rh, which is expressed only on the surface of red blood cells** .]
 * [Notice that you do __not__ use whole blood cells in place of red cells for oxygen transfer supplementation therapy, even if the blood is O. The whole blood contains patient plasma with antibodies (eg. for O blood, anti-A and anti-B) which can react against the recipient.]
 * [Sugar that O people have as their terminal blood antigen polysaccharide structure: L-fucose (evidently also called the "H antigen"). Sugar that A people add onto this fucose: N-acetylgalactosamine. Sugar that B people add onto the fucose: D-galactose. AB people add each of them on various ABO sugars. Bombay people don't have even the fucose.]
 * [A and B sugars are everywhere- people, hamburgers, plants, etc, etc. Thus, from an early age, we all have antibodies to any fucose-added sugar that we don't already have.]
 * For persons of the A, B, AB and O blood groups, give the following data: most and least common groups; red cell antigens; specificities of the ABO antibodies in their plasma; safe donors to that type; safe recipients of blood from that type; possible genotypes.
 * O:
 * Cell antigens: none (unless given to Bombay patients)
 * Antibodies: anti-A, anti-B
 * Safe donors: O
 * Safe recipients: all (not Bombay)
 * Possible genotypes: OO (recessive)
 * A:
 * Cell antigens: A
 * Antibodies: anti-B
 * Safe donors: O, A
 * Safe recipients: B, AB
 * Possible genotypes: AA, AO
 * B:
 * Cell antigens: B
 * Antibodies: anti-A
 * Safe donors: O, B
 * Safe recipients: B, AB
 * Possible genotypes: BB, BO
 * AB:
 * Cell antigens: A and B
 * Antibodies: none
 * Safe donors: all
 * Safe recipients: AB
 * Possible genotypes: AB
 * Frequencies: inadequate data here (no Latino or Asian US population), but O is about equally distributed between whites and blacks at 47% of each population, AB is likewise equal at about 4%, B is more common in blacks (21% vs 9% in whites), and A is more common in whites (42% vs 27% in blacks).
 * Name the class of most ABO isohemagglutinins.
 * Blood cell antigens are antigens because they have lots of repeating, foreign carbohydrate sequences. Hm.. sounds like a T-cell independent B-activating antigen, doesn't it? Which means, recall, that the antibodies produced are all IgM (to switch to IgG or other antibodies requires T cell activation). In the vast majority of cases, ABO isohemagglutinins (antibodies that react with blood) are, in fact, IgM.
 * Interesting side note here: I'm not sure why repeating carb sequences don't also activate T cells via dendritic cells and macrophages (which would result in IgG antibodies to ABO antibodies). I've emailed Dr. Cohen; I'll post the answer when I get it.
 * Notice that the IgM predominance is a good thing. If the antibodies produced in response to foreign red cells were IgG, they would cross the placental barrier and, in cases where the baby's blood type doesn't match the mother's, poof, no kid.
 * Notice that some women, in rare cases, do make IgG hemoagglutinins to ABO antigens. This results in refractory hemolytic disease of the newborn (see below, but can't be fixed with anti-ABO antibodies since mother is already making her own anti-ABO antibodies).
 * Explain the ABO antigen situation in a person of Bombay blood type, and the consequences of a transfusion of non-Bombay blood into such a patient.
 * In Bombay, you lack even the basic fucose sugar on your ABO antigen. So even if you have an A or B sugar-adding enzyme, they can't be added to the ABO antigen (no fucose). Bombay folks type as O (antibodies to both A and B), but actually have antibodies to O (thus will result in ABO-mismatch reaction, see "Transfusion Medicine." Probably best approach is for the individual to bank their own blood in case of emergency.
 * Note that this means, in principle, that a Bombay person (typing as O) can have an AB kid with an A or B spouse-- the other ABO sugar (B or A respectively) is coded for in the Bombay person but can't be expressed until the spouse's fucose gene is expressed with it.
 * Define the crossmatch, and explain why it is important. Explain how red cells are destroyed following a mismatched transfusion, and why this may be devastating to the recipient.
 * When blood's being donated: "type and screen": test blood against a wide variety of antigens. ABO, sure, but also testing for antibodies against pretty much everything else found in blood that might conceivably have caused antibodies to it to be formed in the recipient.
 * When blood's being used in a transfusion: you do the **crossmatch** (because you can never screen for everything and in every situation before it's there) by the following tests. From least to most sensitive (which is the order they're done):
 * (1) Take some donor red cells and add a drop of the patient's plasma. You're looking for agglutinating antibody reactions (the worst of which would be an IgM anti-ABO reacton, which would activate the complement system and either lyse or opsonize the red cells). If the cells agglutinate, then there's a really severe reaction going on and you don't use that blood in that patient.
 * (2) The next thing is to look for antibodies that are there but not expressing themselves under those conditions. So you repeat the donor red cells + recipient plasma test under low-ionic solutions (this minimizes red cell-red cell repulsion and allows the agglutination test more sensitivity).
 * (3) The last thing you do is the Coombs indirect test. This involves using __antibody to human antibodies__ (remember when we were talking about antibodies against antibodies?): what you do is apply some red cells and some recipient serum, wash off any unbound recipient antibody, and then use antibody against human-antibody to detect any bound recipient antibodies on the donor red cells. If agglutination occurs, it's a sign, again, of red cell antibodies. This test is extremely sensitive and it's the last way you crossmatch the red cells before giving them to the recipient.
 * Cells are destroyed in a mismatched transfusion the same way any other antigen would be in the presence of antibodies to it. Since the antibodies are IgM, this is mostly complement-mediated lysis, opsonization, anaphylaxis, and inflammation. There seems to be a competition among which complement result can kill your patient fastest, but lysis, with precipitation of hemoglobin in the blood, resulting in emergent kidney failure and death, is currently on top.
 * Compare and contrast the techniques of the direct and indirect antiglobulin tests and the question they are designed to answer.
 * Direct: Do these red cells __already__ have antibody on them? Tested by adding anti-antibody directly to washed cells.
 * Indirect: Are there antibodies in this plasma that __could__ react with these red cells I have? Test is described above, under (3).
 * Identify situations in which the direct and indirect antiglobulin tests would be of value in diagnosis.
 * Direct: autoimmune hemolytic disorders.
 * Indirect: crossmatching blood before transfusion.
 * Define heterophile antibody, and identify a common disease in which one type is increased enough to be useful diagnostically.
 * **Heterophile antibody: antibody that reacts with more than one antigen** . This fact is used to come up with easy ways to test if the antibodies a person is making are against particular organisms.
 * Eg.: antibodies against the Epstein-Barr virus (mononucleosis) in humans are the same antibodies, or as close as makes no difference, as antibodies against sheep red blood cells. If you have someone you suspect has mono, you can test their antibodies against sheep's blood cells (which are, for good or for ill, much easier to come by and store than a pocket full of EBV) and come up with your answer.
 * In Hemolytic Disease of the Newborn, explain: a. The consequences of severe hemolysis in the newborn. b. The way in which the mother becomes sensitized. c. The class of antibody to Rh(D) the mother makes. d. The consequences of sensitization to subsequent fetuses. e. The role of Rh-immunoglobulin.
 * Here's **the situation: you have a Rh-negative mother and a Rh-positive baby** (mother is dd; father is DD or Dd). At some point in the second trimester, blood cells from the fetus begin to trickle up back to the mother. Since the baby has the Rh antigen and the mother doesn't, the mother will begin, slowly, to make IgG antibodies (which will cross the placental barrier) against them.
 * Generally, **for the first pregnancy, there isn't a problem** (not enough blood and not enough pre-existing antibodies to cause hemolysis in the kid). For each successive pregnancy, the fetus gets more and more hemolysed by increasing levels of maternal anti-Rh IgG.
 * If a child is undergoing massive hemolysis, then heme's breakdown product, bilirubin, can spill across the blood-brain barrier, causing **cerebral palsy** in the fetus. To avoid this outcome in neonates you can put the newborn under bilirubin lights or do exchange transfusions.
 * Largest transfusion of blood cells from fetus to mother occurs during placental separation, at birth. Note that if you **infuse the mother at the point of delivery with anti-Rh antibodies**, Rh proteins never make it to the lymph nodes to be made into antibodies. This helps prevent the sequential worsening of the condition with subsequent pregnancies. The anti-Rh dose is low enough that the fetus is more or less unaffected.
 * Note you've got to get these Rh antibodies from putting Rh into Rh-negative men (no pregnancy complications).
 * Note also that there's a time window for this. You can give the shot up to about 3 days postpartum and still have it be reasonably effective.
 * Newer treatment: Put in one shot at 28 weeks, the other at birth. Can also give a shot after any kind of trauma that results in fetal blood leaking into the mother's circulation.
 * Notice that this is a prophylaxis only-- does not reverse the fact that there's maternal antibodies to the infant's blood. This is why the same principle doesn't work in women that express anti-ABO IgG for the ABO antigens they don't have-- they're already making the antibodies, so trying to intercept the offending ABO antigens before they make the mother sensitized is futile.
 * Explain the situation in which ABO hemolytic disease of the newborn can occur.
 * As mentioned: some women, rarely, make anti-ABO IgG instead of/in addition to IgM. So if an O, A, or B mother has an AB kid, the kid's blood is not long for this world. (I think I've always been waiting to say that.)

=Immunodeficiency=

can be useful replacement therapy. Compare and contrast intramuscular and intravenous therapy. o Originally (IM): full of aggregated immunoglobins, which activated complement and caused pain and inflammation at the site of injection. o Now (IV): purified, intact IgG, not clumped, doesn't cause inflammation. In fact it actually has anti-inflammatory uses; it's used primarily for that purpose, actually.
 * Draw a diagram of lymphocyte development. On the diagram, indicate possible locations of abnormalities of development in:
 * __Di George syndrome__: Basically you have a problem with the normal development of the thymus from the third and fourth pharyngeal pouches (remember those?) due to a huge deletion on chromosome 22. The thymus doesn't develop well, which means underdevelopment of T cell maturation and a __lack of effective T cells__.
 * Other regions affected by that deletion: parathyroid underdevelopment (__hypocalcemic seizures__) and great-vessel cardiac problems.
 * Results of T cell deficiencies: see next LO.
 * __Severe combined immunodeficiency (SCID)__: There's something wrong with the transition from the multipotential hematopoietic stem cell (CFU-ML) to the leukocytic stem cell (CFU-L). So there's __no leukocytes made at all, T or B__. This is a problem. Notice this diagnosis can be missed at birth due to the fact that the mother's antibodies are still pumping around in the kid's body.
 * Note that this can sometimes be a defect in the IL-2 receptor (recall that IL-2 is manufactured by Th1 cells to stimulate the growth of other T cells).
 * Also can be due to a defect in adenosine deamidase (actually, mostly the patients have both these deficiencies). The body accumulates adenosine, high levels of which are toxic to developing leukocytes. (see below for treatment)
 * In principle, can also have kids with problems in their RAG recombinases (therefore can't make V(D)J receptors, therefore can't make effective leukocytes).
 * Results of both T+B cell deficiencies: see next LO.
 * __X-linked (Bruton's) hypogammaglobulinemia__: Due to a developmental block between the pre-B cell (recall that it's expressing some IgM in the cytoplasm but nothing on the surface) and the mature B cell. Pre-B cells, recall, are in the bone marrow. Leads to __very little functioning antibody__ (<10% of normal IgG), meaning there's a problem with infections (no IgA, increased susceptibility to infections through skin or gut).
 * This condition is primarily why the oral (live) polio vaccine is no longer given.
 * __Common variable hypogammaglobinemia__: Normal B and pre-B cells, but some kind of a block with actually making and expressing the antibodies. Unknown etiology, sometimes resolves spontaneously; treat with other people's antibodies.
 * __X-linked hyperIgM syndrome__: Defect in the switch (the CD40 receptor and/or the CD40 ligand) in B cells from IgM to IgG production. B cells are activated and work normally except that they produce only IgM antibody.
 * IgM: doesn't penetrate well into tissues; no opsonizing activity; doesn't get out into surface membranes. Also no IgE, though that's probably the least of your worries in a developed country.
 * Characterize the infections you would expect in a pure B cell deficiency and in a pure T cell deficiency.
 * B cell deficiency: patient can't make antibodies. Antibodies are primarily used for defense against extracellular pathogens-- thus look for infections involving those. Killer T cells aren't activated by MHC II pathogen-presenting cells, so they won't get involved against extracellular bugs; Th1 cells can cause inflammatory responses and recruit macrophages, but the adaptive immune system won't come into play; Th2 cells can't activate B cells if the B cells aren't there.
 * T cell deficiency: similar reasoning-- it results in inadequate defense against intracellular organisms, particularly viral.
 * Notice you can break this down by specific type of T cell and it still works. A killer T cell deficiency would result in recurrent viral infections (can't read MHC I presentations). A Th2 deficiency would likely have less effect but would also result in viral infections (can't activate B cells effectively, but extracellular bacterial pathogens could still be picked up through T-independent B cell activation by bacterial carbohydrate moieties). A Th1 deficiency would result in de facto deficiencies in the other two, leaving only T-independent B activation as the body's immune response (effectively, AIDS, still only able to target extracellular bacteria). Kind of cool that all three types of T cells primarily target intracellular pathogens.
 * Note that T cells also form the main defense against yeasts and fungi- so you'd expect to see these in T-cell-compromised patients as well.
 * Describe the clinical features which, although not immunological, are associated with Di George syndrome.
 * Congenital heart disease
 * Cleft palate
 * Autism/developmental problems
 * Name the enzyme which is absent in some cases of SCID. Discuss possible approaches to replacing this enzyme.
 * As mentioned, adenosine deamidase (ADA). Can use purified ADA to treat; can also give transfusions of irradiated PRBCs (must be irradiated to avoid bringing T cells in that could begin a graft-versus-host problem); can also, in principle, use gene therapy to replace deficient gene (which has been done-- waiting to see how that comes out).
 * [Notice that the most common immunodeficiency is IgA deficiency. Generally asymptomatic.]
 * Discuss transplantation and gene therapy in immunodeficiency diseases. Include a consideration of side effects.
 * Can give thymus grafts to DiGeorge kids; can give bone marrow transplants to SCIDs.
 * [Interesting side note: with thymus grafts into kids with DiGeorge syndrome, you need to match at least one HLA-D allele and at least on HLA-A or B allele. The reason for this: remember that the thymus is where the T cells learn what their MHCs are supposed to look like. This gets screwed up with a completely foreign thymus graft, and the kid makes T cells that are completely useless to him (can't recognize his own MHC complexes). But note that you'll occasionally have endogenous macrophages wandering through, which would at least prevent the possibility of most self-reactive T cells.]
 * Given a child with recurrent infections, describe in principle tests which could be done to determine if there is a T, B or combined immunodeficiency, or a PMN, macrophage or complement problem.
 * Note change from earlier editions
 * Ok, I was wrong about this, it's right there in his notes.
 * B cells: quantitative IgG, IgA, IgM levels; look for specific antibodies against prior immunizations; look for ABO isohemagglutination. [In principle could also stimulate with T-cell-independent vectors like LPS.]
 * T cells: Total lymphocyte counts; CD3/4/8 counts; "skin test with recall Ag panel" (not clear what this is).
 * Macro/Neut: WBC count, differential, and morphology; NBT test/oxidative burst test. Also assays for phagocytosis and chemotaxis.
 * Complement: CH50 test (assay testing ability of complement to lyse antibody-coated sheep's blood cells), assay for C1 inhibitor or individual complement component levels.
 * Return to your diagram of a lymph node. Label T and B cell areas.
 * Go ahead on and do that. I'll wait here.
 * Describe the contents of commercial gamma globulin and indicate the conditions in which it
 * Name two viruses which are immunosuppressive in humans. Discuss a possible mechanism for the immunosuppression caused by one of these viruses.
 * Cytomegalovirus, measles, mononucleosis, HIV.
 * Mononucleosis: infects B cells; killer T cells go after B cells; susceptible during mono infection to secondary infections.
 * HIV infects Th cells (target CD4 cluster for infection site).
 * Describe the immunological problem of the Nude Mouse and name the immunodeficiency condition it resembles.
 * The thymus (and epithelial hair follicles) never develop, similar to full-on DiGeorge syndrome. This means pretty much no T cells, which means susceptibility to viruses (on the upside, also means no rejection of grafted tissue).

=Immunity and Vaccines=


 * [Note that here we're calling antibody-mediated immunity "humoral" and T cell-mediated immunity "cell-mediated" despite the considerable overlap of Th1 and Th2 cells with B activation. Go figure.]
 * Compare the roles of cell-mediated and humoral immunity in virus infections with regard to: preventing the infection; controlling spread of viruses in the body; which is responsible for recovery from disease; how each can cause immunopathology.
 * Preventing: mainly humoral (antibody-mediated)-- specifically, IgA (stop it from reaching the bloodstream).
 * Controlling spread of viruses: mainly cell-mediated (T cells), but can also be IgG (humoral) against free-floating virions.
 * Recovery from disease: Killer T cells are required for viral disease recovery (intracellular organisms can't be targeted by humoral mechanisms).
 * How they can cause immunopathology:
 * Antibodies can react with various body components (although the B cells don't make antibodies in response to endogenous compounds, the antibodies they make in response to exogenous compounds can cross-react, as in rheumatic fever after strep throat).
 * Th1 cells can cause collateral damage due to over-activation of macrophages (bring in granulocytes which release their toxic granules all over the place, also kick up systemic acute phase reaction).
 * Discuss the possible roles of Th1 and CTL in recovery from virus infection.
 * Natural killer cells, which have TLRs to bind viral DNA, activate dendritic cells when those TLRs are bound (recall that virus-infected cells release type I interferons and attract NK cells). The activated dendritic cells begin to scavenge for cellular debris and free viruses; at some point they'll pick up some virus, endocytose it, and go present it not only to Th1 cells on a MHC II complex but also killer T cells on a MHC I complex (remember that dendritic cells cross-present ingested antigens on MHC IIs and also MHC Is).
 * Th1s in turn activate killer T cells (cytotoxic leukocytes, or CTLs) to go after infected cells (which will be presenting viral fragments on their MHC I proteins).
 * Define “local immunity” and give an example.
 * It's pretty much what you think it is-- immunity to a given pathogen on a given tissue or surface (like the skin).
 * Generally, secretory IgA-mediated on skin and mucosa. The idea is to trap and destroy the pathogen before it gets inside the body.
 * Identify those organisms against which cell-mediated immunity is most effective.
 * Viruses, fungi, yeasts, intracellular bacteria.
 * Identify those organisms against which humoral immunity is most effective.
 * Extracellular bacteria and pathogens.
 * Identify the types of organisms against which IgE immunity may play an important role; discuss the roles played by IgE, mast cells, IgG, and eosinophils.
 * Mainly parasites, particularly worms.
 * The problem with parasites is that although they're bound by IgG, they're not affected by complement and can't be phagocytosed by neutrophils (too big). Parasites induce an IgE response (IgEs, when made, are bound to the surface of mast cells) by inducing Th2 cells to release lots and lots of IL-4. IgE binding causes mast cells to release their granules, some of which (prostaglandins and leukotrienes; they're also called ECFs for Eosinophil Attracting Factors) attract eosinophils; eosinophils (which have Fc receptors like neutrophils) contain granules with a substance (Major Basic Protein) that is particularly toxic to parasites (also called helminths, from the Greek word for worm).
 * Give an example of a human and an animal antitoxin; a toxoid; a killed virus vaccine; and a live virus vaccine. Identify the one which produces the longest-lasting immunity. Discuss possible hazards of each type of preparation.
 * Human antitoxin: IgG against tetanus. Animal antitoxin: also IgG against tetanus.
 * Practical difference, I think, is that IgG solutions (which is what anti-toxin is) tend to aggregate when they've been sitting around for a while in the bottle. With human IgG solutions, this causes lots of complement activation (pain, inflammation, etc) due to the proximity of multiple bound IgG antibodies. With animal IgG solutions, less complement is activated due to inter-species antibodies not activating each other's complement very well.
 * Killed virus vaccine: injected polio (Salk) vaccine.
 * Live virus vaccine: oral polio (Sabin) vaccine.
 * Longest immunity tends to be live virus vaccines (your body produces not just MHC II responses from professional ingester cells but also MHC I responses from your own, infected cells).
 * Hazards:
 * Antitoxin: as mentioned, possibility of complement activation.
 * Killed virus vaccine: I'm not sure, but could potentially cause pain and fever reactions if there was some kind of innate-immune response to the killed virions in solution. Also provides less thorough immunization that live virus vaccine.
 * Live virus vaccine: The obvious one here is that the virus isn't quite attenuated enough not to seriously infect the patient. More of a recurrent problem with immunocompromised people.
 * State the appropriate times for immunization of children against diphtheria, pertussis (whooping cough), tetanus, polio, and measles. Discuss why live viral vaccines tend to be ineffective in the very young.
 * Recall that he mentioned that the specific dates weren't actually all that important.
 * Diphtheria, pertussis, tetanus: 15-18 months
 * Polio: 2 months, 4 months, 6-18 months, and 4-6 years
 * Measles: 12-15 months, 4 years
 * Live viral vaccines tend to be ineffective in the very young because they tend to be destroyed by the mother's circulating IgG before the kid can make antibody to them.
 * Note, on sort of the same topic, that infants don't do T-cell independent B cell activation very well-- have some trouble building immunity to certain carbohydrate compounds. You can fix this by linking the carbohydrate you want to produce immune response against to a compound that the kid's already had a response to (like diphtheria); the B cells' receptors react with the carbohydrate and engulf both of them; then the B cells express the diphtheria fragments on its surface, they get recognized by Th2 cells, which kick the B cell into making antibodies against the carbohydrate. Good to go.
 * Discuss the use of IgG and IgM antibody titers in the diagnosis of intrauterine and neonatal infections.
 * What you want to look at to determine whether or not a kid has been exposed to an infection is either of:
 * (a) IgM: since it's made quickly and goes away quickly, it's a reasonably good touchstone for whether a kid's had a disease lately.
 * (b) IgG: if you measure it once and leave it at that, you don't know whether the level is getting bigger or staying pretty much the same (thus you don't know whether the kid is making a bunch of it due to a recent exposure or whether the kid's already had the disease and still has the antibodies floating around). Need to take a couple of time points and compare them (are they going up or down?).
 * Identify the oral and parenteral polio vaccines by the names of their developers. Discuss their relative advantages and disadvantages, and note which is currently used in the USA.
 * Currently the parenteral (injected) polio vaccine is used in the US.
 * The injected form is a killed (Salk) vaccine. The rationale for this is that some kids with impaired immune function might actually get sick from reversion of the attenuated live (Sabin) virus, which is given orally, to a virulent state. The rate of this occurring, says Wiki, is about 7,000 times higher in kids with agammaglobinemia than normal children.
 * Oral is, obviously, easier to distribute and administer, particularly in places in which access to healthcare is a problem (it's a four-part vaccine). It's also transmissable-- the immunized kids can infect their friends and family with the (attenuated) virus, which leads to greater spread of protection. It furthermore gives a better protection rate than killed virus (virus shows up on MHC I, not just MHC II proteins). On the other hand, it can also cause polio, mainly in immunocompromised kids.
 * Discuss the pros and cons and advances in pertussis (whooping cough) immunization.
 * Pros: you don't get pertussis, which can, and frequently does, kill you one time out of thirty-five.
 * Cons: there's about a 1 in 300,000 chance the pertussis vaccine causes brain damage.
 * Advances: now it's about a 1 in 3,000,000 chance.

=Autoimmunity: Immunopathology Type II=


 * Describe the molecular and cellular details of the immunologic mechanism by which tissue damage occurs in a Type II (“cytotoxic antibody”) reaction.
 * First off: Type II immunopathology is directly antibody-mediated (you're making antibodies that are reacting with self).
 * This differentiates it from Type I (innocuous substance triggers IgE activation),
 * Type III (antigen-antibody complexed get lodged in innocent membranes), and
 * Type IV (Th cells, responding to infection, call in and activate macrophages, which wreak havoc on surrounding innocent cells).
 * Three main mechanisms of Type II-brand problems:
 * (1) Complement: if you have normal cells that are bound by antibody, the first thing you want to think about is complement. Complement activation can lyse the cells (C6-9), opsonize them for phagocytosis (C3b), release histamine (C5a, C4a, C3a), and attract neutrophils (which can spill their granules and further damage tissue-- C5a) through the capillaries that the histamine has just opened up.
 * (2) __Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)__: this is, if you stop and look at it for a moment, a novel concept. Cytotoxicity is a word we've hitherto only applied to killer T cells, which are most definitely not antibody-dependent-- they depend only on MHC type I complexes on the surfaces of cells. But natural killer cells (sometimes called "K cells" in this role), as it turns out, can bind to the Fc portions of bound IgG and initiate apoptosis in the attached cell in the same kind of way.
 * Note natural killer cells, aka Large Granular Lymphocytes, make up about 10% of total lymphocytes. They're not T cells, they're not B cells. Alone among the lymphocytes, they have Fc receptors; alone among cells with Fc receptors, they initiate apoptosis in their targets instead of engulfing them.
 * Cohen sez: NK cells also have receptors for molecules that are expressed on the surface of "stressed" cells. These receptors are some of the elusive DAMPs (damage-associated molecular patterns) we heard about earlier. These are expressed in physically damaged cells, but also (notably) in cancer cells and in virally-infected cells (as per D+D, "Host responses to viral infections").
 * (3) Receptor-ligand mimicking. Recall that an antibody is just something that wants to attach to a particular pattern. We have another name for that: a ligand. So if your antibodies just happen to look a lot like the ligand for a particular receptor, you get activation of the receptor __all the time__ but without the normal control mechanisms you had over the actual ligand.
 * Example: IgG antibodies that look just like TSH (thyroid stimulating hormone). These bind to TSH receptors on the thyroid and cause thyroid hormones to be secreted constantly, a hyperthyroidism called Graves' Disease.
 * Give an example of a Type II mechanism disease of muscle, kidney, heart, red cells, platelets, lung, thyroid, pancreatic islets.
 * Pancreatic islets: Juvenile diabetes (ie. Type I diabetes).
 * Thyroid: Graves' Disease.
 * Muscle: Myasthenia gravis (though note the actual clinical pathophysiology seems to come from antibodies against acetylcholine receptors, not myosin).
 * Red cells: Autoimmune hemolytic anemia.
 * Platelets: Thrombocytopenic purpura (called either Idiopathic or Autoimmune).
 * Lung and Kidney: Goodpasture's disease.
 * Heart: Rheumatic fever (or rheumatic heart disease) after strep throat infection.
 * Describe the fluorescent antibody tests which would allow you to make the diagnosis of Goodpasture's Syndrome, given: patient's kidney biopsy, normal kidney biopsy, patient's serum, and fluoresceinated goat antisera to human IgG and complement.
 * Direct antibody test: use antisera to detect presence of attached IgG on patient's kidney and the absence of attached IgG on normal kidney (where present, IgG should look like nice lines of fluorescence attached to all exposed kidney surfaces).
 * Indirect antibody test: expose normal kidney to patient's serum, wash off unbound antibody, then use antisera to detect the IgG now bound to the normal kidney surface (again, in lines).
 * [Something that came up in discussion: notice that you can't use indirect antibody tests to look for Type III immunopathologies in tissue sections because the clumping of immunocomplexes in basolateral membranes of filtration tissues (like the kidney) only occurs when filtration is actively taking place-- that is, when the tissue is alive and actively using ATP to filter the fluid. Dead tissue isn't filtering jack, and thus you won't see immunocomplexes getting stuck in it after you wash it with the patient's serum.]
 * Distinguish between the “lumpy-bumpy” and linear immunofluorescent patterns in terms of the most probable immunopathologies they represent.
 * Lumps: probably clusters of antigen-antibody complexes stuck in the basement membrane (ie type III immunopathology).
 * Linear: probably antibodies that directly attack the basement membrane, all lined up in a row on the membrane (ie type II immunopathology).
 * Describe how you could tell, using fluorescent antibodies and biopsies of patient's kidney, if Type II or Type III immunopathology was involved. Name the antibodies you would use and the fluorescent patterns you would see.
 * This is what we just mentioned with lumps vs linear. Lumps: III, linear: II.
 * Antibodies: vs human IgG. He sort of gives it away in the next LO.
 * Given patient's serum, fluorescent antibody to human immunoglobulins, and slices of normal kidney, describe how you could tell if the patient's glomerulonephritis was due to Goodpasture's disease or SLE.
 * Goodpasture's: antibodies are specifically against glomerular capillaries (thus linear streaks on immunofluorescence).
 * SLE: serum-sickness (type III) accumulation in glomerular basolateral membranes will look lumpy under immunofluorescence.
 * Again, this is basically what we just mentioned with lumps vs linear fluorescence.
 * Describe how autoimmune disease could result from:
 * The innocent bystander phenomenon (hepatitis or TB): damage results due to normal cells' proximity or attachment to a genuine foreign antigen.
 * Cross-reaction of a foreign antigen with self (rheumatic fever, thromb. purpura): damage results from antibodies binding to self cells.
 * Coupling self antigen with a foreign antigenic “carrier:” if normally quiescent B cells react with self (engulf, fragment, and present self and foreign carrier fragments) and T cells react to foreign carrier fragments and stimulate B cell activation, the B cell will produce antibodies against self.
 * This is called the 'illicit help' model and can be responsible for all kinds of bad mojo. Cohen noted that if penicillin happens to bind to red cells, say, and you have a B cell against red cells that isn't normally expressed (lack of T cell stimulation) but have some T cells against penicillin, a B cell's engulfment of the RBC-penicillin complex can wind up getting triggered by the anti-penicillin T cell and producing anti-RBCs. Voila, autoimmune hemolytic anemia.
 * Notice also that the T cells have a much more extensive system for negatively selecting against self-reactive lymphocytes than the B cells (ie. the variegation in the thymal stroma via the //Aire// gene)-- this means that there are likely lots of potentially self-reactive B cells out there that just don't normally get activated by T helpers. The illicit help model shows that that's probably not a good thing.
 * Emergence of a forbidden clone: "Forbidden clone" is an overly dramatic phrase for a B (or, more likely, T) cell with reactivity against self that doesn't get clonally aborted. Ie: myasthenia gravis.
 * Exposure of a sequestered antigen: there are self-antigens that the immune system don't generally see (brain stuff, testicular stuff). If they get out into the bloodstream, for whatever reason, they can trigger immune responses because there's been no negative selection against those self antigens.
 * Failure of regulatory T cells: Treg cells repress T cell response (good in locations, such as Peyer's patches, where you don't always want to be making immune responses to new antigens). Sometimes the Th-Treg balance seems to get screwed up (possibly in response to soluble inflammatory mediators like IL-6), Treg isn't expressed adequately, and thus Th cells and cell actions aren't repressed adequately.
 * [Passive antibody: maternal anti-self IgGs can cross placenta and react with fetus; classic example is infant myasthenia.]
 * Identify “Rheumatoid Factor” and describe its molecular nature.
 * Rheumatoid factor: the stuff that shows up in the joints of patients with rheumatoid arthritis.
 * Molecular nature: it's IgM antibodies made against IgG antibodies. Etiology unknown.
 * Name the condition in which antibody stimulates rather than inhibits or harms its target cell. State the common name of the antibody.
 * As discussed above, Graves' Disease. The common name for the IgG that mimics TSH is long-acting thyroid stimulator or LATS.
 * Note also that some antibodies can stimulate the beta-adrenergic receptors in heart muscle (thus prompting inappropriate tachycardia).
 * Describe the role of the Aire gene in preventing autoimmune disease.
 * Aire (auto-immune regulator) gene: thymal transcription factor responsible for expressing various really out-of-place proteins in the thymus. This ensures that maturing T cells are exposed to lots and lots of different types of cells so that you can have negative selection against T cells that are self-reactive to things that you would otherwise never see in the thymus where they're maturing (though you'd certainly see it once you got out into the tissues).
 * Problems with either Aire or the genes it regulates, so that one doesn't interact well with the other, can cause the thymus to not express certain tissue types (and thus not negatively select against T cells that react with those tissue types). This can lead to problems (eg. myasthenia gravis).

=Immunology of AIDS=

cancer, and traffic accidents, and was killing about 2,700,000 people per year.
 * Explain the difference between “HIV-seropositive” and “AIDS”.
 * HIV-seropositive: you have antibodies against HIV. AIDS: you get major opportunistic infections and/or you have an absolute CD4 count below 200.
 * It should be fairly obvious by this point that having antibodies against HIV does very little to actually prevent the progression of the disease-- more on why below under pathogenesis info.
 * More to the point: HIV+ can be clinically apathological. AIDS patients are, by definition, not.
 * On average, at least from transfusion-acquired HIV, it's about a 9.5-year span between infection and AIDS.
 * Notice also that the rise in HIV antibody takes a while-- it's about a 7-9 week span after infection to make enough antibody to be detectably infected.
 * Name the virus that causes AIDS, and its classification.
 * Um, I'm going with influenza. No, wait, wait, HIV.
 * It's a "nontransforming retrovirus"-- that is, a RNA virus capable of retrotransposition that does not carry oncogenes (that's the nontransforming part).
 * Discuss the origin of the AIDS virus and the origins of the current epidemic.
 * Go look it up in the notes. Showed up first as a Pneumocystis outbreak in Los Angeles and then as a Kaposi's sarcoma outbreak in California and New York.
 * Identify the approximate number of cases in the U.S. and in the world, and discuss the rate of change in incidence.
 * Probably about 1 million infected people in the US (maybe a quarter of them are unaware of this fact). About 33 million infected worldwide. Incidence in the US has been falling since 1993; expanding in other places. By 2003 AIDS was the 4th leading cause of death worldwide, ahead of diarrheal diseases, tuberculosis, malaria, lung
 * Discuss the pathogenesis of AIDS, including target cell types, mode of entry of the virus into a cell, mode of exit, latency versus productive infection.
 * Seems possible it's taken up by dendritic cells at first, into some sort of special compartment that prevents its cleavage; once the dendritic cell gets to the lymph node, it gets out and infects its primary target, the T helper cells.
 * **Gp120** protein on the HIV virion surface attaches to CD4 regions. Once it binds, a stretch of the virion is exposed that also binds to a chemokine receptor (CCR5).
 * Once bound, the virus flashes out a kind of hydrophobic "switchblade" that opens up the cell's membrane, allowing viral entrance.
 * Structure of the virus: has an envelope consisting of the plasma membrane from the last cell that it infected, studded with gp120 proteins.
 * Note that the fact that it's enveloped means it's fairly physically fragile-- it doesn't resist UV light or environmental stress very well.
 * Notice that HIV only has about 11 genes-- the fact that it can do all this with so little is both astounding and terrible.
 * Really unpleasant thing: the reverse-transcribed viral genome carries with it a promoter that is activated precisely by the same transcription factor that activates IL-2 in Th1 cells. Activation of Th1 cells therefore also triggers hypertranscription of the viral genes.
 * The mode of exit is one of the most irritating things about it. The infected cell will fuse with other nearby Th cells, allowing the virus to spread without moving out into the extracellular space. This makes all that lovely antibody completely useless, since antibody doesn't get into cells and the virus never goes outside them.
 * Latency: partly this is the mechanism of pandemic. Because it doesn't cause rapid death but the host is still infectious for years, it doesn't 'burn out'-- it can build its reservoir of infected hosts over a long period of time.
 * Distinguish between the roles of Th1 and Th2 in the progression of HIV infections.
 * Th2 seems to be preferentially activated against HIV. This is unfortunate. Not only are the B cells that the Th2 cells activate useless (antibodies don't target HIV well, for reasons discussed above), the lack of Th1 activation means that the killer T cell response (which you'd expect to limit the extent of the infection) is weak and ineffective.
 * Note that this preferential Th2 stimulus seems to have something to do with how it's presented by the dendritic cell.
 * Also note that there's a problem with just stimulating Th1 cell response (thus producing lots of IL-2) due to the fact, as noted earlier, that the same factor that makes IL-2 in Th1 cells also transcribes the viral genome-- so if you kick up Th1 response you also kick up HIV transcription.
 * Discuss the types of infections seen in AIDS patients, and provide an immunological basis for this spectrum.
 * Largely opportunistic viral and fungal infections-- mainly herpes, CMV, and hepatitis for the viruses, and //Candida// and //Pneumocystis// for the fungi. Also protozoa (mainly //Toxoplasma// ).
 * More or less what you'd expect to see with a suppressed cell-mediated immune response.
 * Discuss possible reasons for which the total number of CD4 cells in AIDS patients decline.
 * Well, they're being infected and lysed, that's a good mechanism.
 * As noted, if Th1 cells start expressing IL-2, the virus begins to replicate much faster.
 * Discuss reasons for which antibody seems to be ineffective in HIV infection.
 * Discussed above.
 * Describe the laboratory diagnosis of AIDS.
 * ELISA is the screening test for HIV antibodies in patient's blood-- you put some HIV+ T cells (not from your patient) in a well; you add the patient's blood, wash it off, and look for bound antibody with immunohistologically-labeled, goat-derived anti-IgG (there's an enzyme bound to the goat anti-IgG which can be detected by the addition of another substrate).
 * Cheap and sensitive (few false negatives), but not necessarily specific-- get false positives. So you run a Western blot to make sure that the patient has the right antibody.
 * Notice that this is technically not a diagnostic tool for AIDS-- it's a diagnostic tool for HIV. Once HIV+ people get major opportunistic infections, then it's AIDS.
 * Discuss risk groups and risky behavior in AIDS.
 * Lots of sex: risky. Anywhere where you're exposing torn mucous membranes to other people's blood: risky. Being a health care worker: risky (particularly because most infections that arise from accidental needle sticks come from patients who haven't yet been ID'd as HIV+).
 * Discuss the prospects and problems of AIDS vaccine development
 * One problem: vaccine would, presumably, make you seropositive for HIV (problems for insurance, employment, marriage, etc).
 * Another problem: given long latency period, hard to statistically prove that a vaccine is actually working (not about to stick HIV in vaccinated volunteers).
 * Obviously can't do vaccine work in animal models very well (__human__ immunodeficiency virus).
 * Latest trial in 2007 wound up as a failure.
 * [Recall that CCR-5 is necessary for the virus to bind-- humans who don't have CCR5 genes (no phenotype) seem to be immune to HIV progression (can get HIV in macrophages, etc, but can't get into T cells). Heterozygotes get a much milder, slower form of AIDS.]

=Type I Immunopathology: Allergies=


 * [Dr. Claman is great. His notes are not as great. Take with one grain of salt (= about 65 milligrams, see "Prescription Writing" under D+D notes).]
 * [Recall that there's a certain kind of balance between Th1 and Th2 cells. If the balance is tipped in favor of Th1, you see mainly cell-mediated immune responses; if in favor of Th2, you see mainly humoral immune responses. I think the idea is that allergic reactions, because they require so much IL-4 production, are more Th2-driven.]
 * Define:
 * Atopic: Atypical; that is, in the entire population it is rare. Seems to be used to refer to allergic reactions.
 * Immediate hypersensitivity: (as opposed to delayed hypersensitivity) Aka Type I immunopathology, an immune reaction against a sensitized allergen by pre-existing IgE, characterized by activation of mast cells.
 * Allergy: same as immediate hypersensitivity.
 * Allergen: A nonparasitic antigen capable of stimulating Type I immunopathology in atopic individuals.
 * Anaphylaxis: release of histamine from mast cells, causing vasodilation in arterioles and bronchoconstriction in the lungs. Wiki sez "true" anaphylaxis is IgE-mediated, while "pseudo" anaphylaxis is due to anything else (see below on anaphylactoid rxns).
 * Asthma: Chronic condition in which the bronchial passages constrict, become inflamed, and secrete los of mucus. Can be triggered by allergen contact.
 * Immune deviation: the shift in Th2 to Th1 dominance (or vice versa) in immune response.
 * Urticaria/Hives: Skin condition caused by Type I hypersensitivity reactions-- vasodilation, edema, redness, etc.
 * Wheal-and-flare reaction: Rash, swelling, and inflammation associated with skin allergies.
 * State the approximate incidence of atopic diseases in the general population, and in individuals with allergic parents.
 * About 10% of population has some kind of atopic Type I hypersensitivity (ie genetically influenced tendency to get IgE-mediated allergic reactions).
 * Tends to run in families; tend to occur in younger patients.
 * Describe the mechanism of IgE-mediated hypersensitivity in terms of: IgE attachment to basophils or mast cells; reaction with allergens; mediator release; effects of mediators on target tissues and cells.
 * Interaction of allergens with IgE: allergens cross-link two IgE molecules on the surface of a mast cell, degranulation of histamine follows.
 * [Note that in allergic people, IgE plasma levels are higher than normal.]
 * Histamine: Vasodilation, edema, itching (pruritis), chemotaxis for eosinophils.
 * Activated mast cell begins to synthesize and release leukotrienes and cytokines.
 * Discuss the features that the various atopic diseases have in common which justify lumping them together.
 * IgE-mediated response to various antigens; IgE sensitization to antigen occurs 'silently' at some prior point to pathophysiology.
 * State the principles of the RAST and basophil degranulation tests. Compare these tests to intradermal skin tests with reference to safety and specificity.
 * RAST: in vitro test for reactivity of an allergy to a patient's IgE.
 * Skin tests: in vivo tests against particular allergens.
 * Problem is here that they don't prove that the exposure to the allergen produces clinical symptoms (high false-positive rate) and needs to be correlated with clinical history.
 * [Basophil degranulation: generally not done (hard to do, hard to measure).]
 * Neither said anything nor wrote anything about comparative safety and specificity. Wiki sez not only was the RAST superseded in 1989 but that skin tests are usually better when possible-- more specific and sensitive.
 * Discuss specific hyposensitization therapy of allergic disease, considering duration of effect, risk of anaphylaxis, percent of patients obtaining significant relief.
 * See below. Can have a long duration, low risk, 75% percent obtaining relief.
 * Describe the immediate allergic reaction and the late-phase reaction in terms of: time course of the reaction, mediators involved, and histology.
 * Two phases of allergic reactions: immediate and 6-12 hours later.
 * Early symptoms: due to histamine release.
 * Late symptoms: due to mast-cell-synthesized cytokines (attracting neutrophils?).
 * Discuss the similarities and differences between anaphylactic and anaphylactoid reactions.
 * Some compounds (anaphylactoid-producing) do not go through IgE mediators but can directly trigger mast cells to degranulate.
 * Indicate which tissues are most rich in mast cells.
 * Upper respiratory mucosa, bronchial mucosa, gut mucosa, skin.
 * Note that these are the "shock organs" (maybe the organs susceptible to hypoxic injury in large-scale histamine release?).
 * Outline the "3-fold pathway" for treating allergies.
 * Identify and avoid specific triggers
 * Treat symptoms
 * Hyposensitization ("allergy shots"): trying to induce specific immunulogical tolerance in in a sensitized patient. Not an easy affair.
 * Promotes switch from Th2 to Th1 (away from IL-4 and antibody production).
 * Decreases IgE and mast cells.
 * Works in about three-quarters of patients.
 * Low risk of systemic anaphylaxis.
 * Can have a "persistent benefit," whatever that means.
 * Note that Dr. Claman feels strongly that anti-allergy shots given to young patients with allergic rhinitis (hay fever, etc) can prevent development of asthma.
 * Discuss the asthma pandemic in terms of the hygiene hypothesis.
 * Claman: "Germs are good" (and evidently prevent asthma). He should, obviously, have a talk with our "Vaccines in Colorado" coordinator. I'm betting Claman in three rounds.

Even though Type I rxns are called "immediate hypersensitivity," remember that they have a secondary, delayed phase (6-12 hours later) due to cytokines released by mast cells.

Immune deviation: shift in response from Th1 to Th2 or vice versa.

10% of population has atopic disease of some kind.

Anaphylactoid: activate mast cells directly (no IgE mediators)

Tissues rich in mast cells: upper respiratory and bronchial mucosa, GI mucosa, skin.

Hyposensitization involves switching to Th1 response. Low risk of anaphylaxis. 75% success.

=Immunomodulators=


 * Distinguish between NSAIDs and DMARDs in terms of: mechanisms of action, use in autoimmune and rheumatic diseases, effects on those diseases.
 * NSAIDs: non-steroidal anti-inflammatory drugs.
 * MOA: COX-1, -2 inhibitors (plus whatever acetaminophen acts on)
 * Use: First-line auto-immune relief.
 * Effects: well, anti-inflammatory. They __don't prevent progression of disease__-- symptomatic relief only. Often given while the specific autoimmune diagnosis is being made.
 * DMARDs: disease-modifying anti-rheumatoid drugs.
 * MOA: Lots of them. Cohen sez, when pressed about whether we need to know specific MOAs for these:
 * "NSAIDS: Anti-inflammatory, make you **__feel__** better. DMARDS: anything that makes you **__get__** better."
 * Use: Nearly all RA patients get DMARDs. Also used in SLE and other autoimmune disorders, not just rheumatism. Generally preceded/accompanied by NSAIDs. Can be given concomitantly with other DMARDs.
 * Effects: __Will prevent progression of the disease__, unlike NSAIDs. Reasonably safe at DMARD levels (as opposed to chemo dosages).
 * Outline an approach to the treatment of rheumatic or autoimmune disease based on the use of NSAIDs, DMARDs, and biological response modifiers.
 * DMARDs should be given as first-line drugs. They decelerate the rate of accumulation of rheumatoid injury.
 * NSAIDs should also be given as first-drugs, but are effective to relieve symptoms only; they do nothing to change the rate of bone degeneration.
 * Biological response modifiers: something that’s more or less endogenous (biological) that modifies an immune response (response modifiers). Here, refers mainly to antibodies (see below for more detail). I think these are supposed to be used acutely or if the others aren't doing so hot.
 * Discuss the mode of action of Cyclosporine A. Indicate its most severe toxicity.
 * Mainly target helper T cells. Bind to a cellular binding protein (cyclophilin), which inhibits __calcineurin__-- this prevents transcription factor **NFAT** from activating **IL-2** (which in turn is necessary for activating Th1 and Th2 cells, thus nipping both cell-mediated and humoral immunity in the bud).
 * Nice specificity to it- inhibits mainly a T-cell-specific pathway (calcineurin to NFAT).
 * Most severe problem is renal toxicity (unfortunately, the __other__ calcineurin-similar pathway is in the kidneys), which is a bitch if you're getting immunosuppression for a kidney transplant.
 * Here's an important question: with renal dysfunction in a transplant situation, are you looking at kidney rejection (thus increase cyclosporine admin) or are you looking at renal toxicity (thus decrease cyclosporine admin)?
 * Discuss the use of monoclonal antibodies as anti-inflammatory agents.
 * MABs: product of a single B cell. Hybridize the activated B cell desired (from the mouse) with a B-cell lymphoma (also from the mouse)-- and you get immortal, monoclonal antibody-producing cells.
 * The problem is that if you use mouse antibody in humans, the host becomes immune-sensitive to the mouse Ig. Thus if you use some more of it, (a) it won't work, and (b) you'll get immune complex disease. So you can use murine (mouse)-derived antibodies exactly once (or, rather, you have about a week's window while the host is building antibodies) before they're useless.
 * Antibody against mouse antibodies: __HAMA__: Human anti-mouse antibody.
 * __Anti-CD3__ antibodies: used to wipe out existing T cells without wiping out entire T cell production system (acutely suppress T cells).
 * __Anti-CD20__ antibodies: vs. B cells/lymphomas.
 * **Ibritumomab** : You can use radiolabeled (111I) murine (mouse) anti-CD20 to visualize/locate the CD20+ tumor, then follow it up with 90Y radiolabeled anti-CD20 to destroy not only the CD20+ cells, but the cells nearby to it (which are probably tumor cells that aren't expressing CD20). This is called a "crossfire" irradiation approach.
 * Note that this is a case in which you can use murine antibodies, since it's effectively a single-dose therapy that ends before the immune response to the mouse Fcs is mounted.
 * __Anti-TNF-alpha antibodies__: as you would expect given TNF-a's role in immune response. These are antibodies you would probably want to use chronically-- thus the immune response to murine antibodies pose a problem.
 * Distinguish between mouse monoclonals, chimeric, human, and humanized monoclonals, and indicate in overview how each is made.
 * [For more detail on how monoclonal ABs are made, see FAQs on Blackboard.]
 * Essentially these different sources of MABs constitute a progression of how much of the antibody is made of human protein and how much is mouse protein. This correlates to how quickly the body makes antibodies against them.
 * __Mouse monoclonals__: monoclonal antibodies made in mice, using mouse B cells, resulting in mouse antibody. See details above for how to make immortalized B cells. The problem with these is the rapid rejection described above by HAMA-- only get one or two rapid uses out of it.
 * __Chimeric monoclonals__: take out only the bits of the mouse antibodies north of the hinge region (which contain the epitope-binding regions) and stick them onto human heavy-chain Fc 'stalks,' thus avoiding some of the IgG vs. mouse antibody response in hosts.
 * Notice that this buys you a few more uses, but the hosts still eventually develop antibody (now called __HACA__, for human anti-chimeric antibody) against the therapy.
 * __Humanized monoclonals__: take __only__ the epitope-binding regions from MABs and graft them onto human 'stalks' (thus only about 2% mouse). Buys more immune-response-free time (host antibody is now called __HAHA__, for human anti-humanized antibody).
 * __Human monoclonals__: Inject human bone marrow, lymph node, and thymus tissue into a SCID mouse. T cells (which mature in the human thymus) and B cells (stored in the human lymph node) from the human bone marrow are the only immune response the mouse makes to various antigens. Injected into a human, these won't prompt an immune response (custom-made, fully human antibodies). They cost more than the collective organs of our class sold on the black market to ailing hedge fund managers.
 * Discuss the use of growth factors in bone marrow transplantation.
 * Donor is often treated with G-CSF/GM-CSF to promote stem cell activity prior to donation. Recovery in recipient also is hastened by the use of G-CSF or GM-CSF.
 * [Glucocorticoids:]
 * Used in most autoimmune disorders.
 * Glucocorticoids: induce apoptosis in eosinophils, prevent apoptosis in neutrophils.
 * Thus steroids help more with eosinophil-mediated disorders and help less (or harm) with neutrophil-mediated disorders.

=Tumor Immunology=


 * State the concept of the Immunosurveillance Hypothesis and the updated concept of "immunoediting." Discuss whether data from immunosuppressed and immunodeficient patients support the theory.
 * Immunosurveillance Hypothesis: The immune system **should** be able to recognize and eliminate developing tumors based on recognition of neoantigens from the mutations of the transformation process.
 * The idea is that tumors are occurring to some extent all the time, but normally the immune system's surveillance keeps the process under control.
 * Evidence: virus-associated, and some non-virus-associated, tumors increase with immune suppression. Tumor-infiltrating Lymphocytes (__TIL__s) are found in many tumors and seem to be acting to try and keep them in check.
 * T cells, NK cells, and the innate immune system seem to be most central to this process.
 * Immunoediting: I think what she's talking about is the idea that the immune system constantly 'edits' the body's cells to eliminate anything that's out of place. There are three main outcomes associated with this:
 * __Elimination__: Tumor is destroyed. Acute cytokines/chemokines trigger innate and adaptive immunity to target and destroy the tumor.
 * __Equilibrium__: Tumor is not destroyed but is kept in check. T cells infiltrate the tumor but don't completely destroy it. This is tricky because the tumor is now (a) under tremendous selective pressure to adapt to avoid or inhibit the immune system, and (b) increasingly genetically unstable, meaning that it's racking up all kinds of mutations. Eventually it'll find a way to proliferate in a way that's hard for the immune system to deal with, leading to the following:
 * __Escape__: Immune system is not able to destroy or contain the tumor, mainly because the tumors have hit on some mechanism to inhibit or destroy the immune cells that are trying to keep it in check.
 * Evidence for immunoediting: woman with melanoma has it excised with no visible recurrence; 16 years later she dies and donates her kidneys; the immunosuppressed woman into which one is placed develops metastasizing melanoma, which kills her. The idea is that the melanoma had been kept in check by the donor's immune system but proliferated in the immunoincompetent host.
 * Describe tumor-associated antigens (TAA), and tumor-specific antigens (TSA). Compare and contrast these tumor antigens: viral, mutant, and normal gene products.
 * **TSA** : tumor cell antigens not found on corresponding normal cells. Easier for the immune system to target.
 * **TAA** : tumor cell antigens found on corresponding normal cells; they're just more common on tumor cells. Harder for the immune system to target (it's 'self' to T cells).
 * Viral tumor antigens: generally TSAs (viral antigens not found in uninfected cells).
 * Mutant gene product antigens: novel gene products made by tumor cells. Also generally TSAs since normal cells don't make them.
 * Normal gene product antigens: normal gene products made __to excess__ by tumor cells. Generally TAAs since normal cells do make them, albeit at lower quantities. Types:
 * Oncofetal: antigens made in fetus but not in normal adult cells.
 * Differentiation: antigens involved in, yeah, differentiation.
 * Oncospermatogonal/testis: antigens usually found only in germ cell development. Can be targeted well (since immune responses don't generally travel into the testes).
 * Clonal: antigens only on the clone of cells that the tumor came from.
 * Describe the mechanisms by which the body may kill tumor cells: Killer T cells (CTL), Th1 and “angry” macrophages, natural killer (NK) cells.
 * __Killer T cells__: as you'd expect, through either Fas- or granule (perforin)-mediated induction of apoptosis. Activated in lymph nodes by dendritic cells, the standard drill. Produce IFN-gamma when bound to their target cells.
 * However, note that she mentions that "unmanipulated" killer T cells, even ones that recognize the TAA antigen, don't actually kill the tumor cells very well-- they expand, but don't stop the tumor's growth.
 * __Th1 cells__: secrete TNF and lymphotoxin, which seem to preferentially target tumor cells. In testing.
 * __Macrophages__: Can, obviously, target tumor cells in the area TNF-a is being secreted. But notice that angry macrophages also secrete factors that promote angiogenesis and cell growth, both of which can cause tumor growth.
 * __NK cells__: Preferentially target cells which have downregulated their MHC I complexes. Tumor cells like to downregulate MHC I, because then they can hide their irregular proteins from killer T cells. NK cells catch these cells and kill them.
 * Note that endogenous antibody and complement mechanisms seem particularly weak against tumors, mainly since you wouldn't expect it to work well against TAAs anyway.
 * In the context of the above list of mechanisms, discuss the low incidence of spontaneous tumors in nude mice and Di George syndrome patients.
 * Despite not having working T cells, these mice/patients still have macrophages, NK cells, and a working innate immune system.
 * Discuss the principles underlying antibody or T cell methods that might be used as treatments of tumors.
 * According to Dr. Slansky, what she's getting at in this LO is "the mechanisms used by T cells and antibodies that makes them effective in immunotherapies. For example, specific antibodies may kill the target cells by antibody-dependent cell-mediated cytotoxicity [NK cells--jcr], and specific CTL may be stimulated to kill the tumor using perforin, Fas, and TNF pathways when the patient is treated with specific vaccines (say antigenic peptide plus adjuvant [like the illicit help mechanism--jcr])."
 * Describe two mechanisms by which BCG treatment may cause tumor regression.
 * BCG: vaccine designed to prevent TB.
 * Injecting it straight into tumors can cause non-specific killing of tumor cells by T cells (nobody seems entirely sure why this is).
 * It also induces a delayed-hypersensitivity reaction to BCG (angry macrophages) which can engulf and destroy the surrounding tumor cells.
 * Discuss prospects and problems concerning the use of monoclonal antibodies in the diagnosis or treatment of cancer.
 * Prospects: Can use passive antibodies to target TAAs the body wouldn't otherwise make antibody against. In the news lately: Herceptin (anti-HER2 surface growth stimulatory molecule) and antibodies against VEGF (angiogenesis factor).
 * Problems: The passive use of monoclonal antibodies against TAAs (like melanin) will probably affect normal cells expressing the TAA as well.
 * List four actions of interferons that might be important in the response to tumors.
 * IFN-gamma:
 * (1) Activates NK
 * (2) Activates macrophages
 * (3) Stimulate preferential Th1 development (over Th2)-- thus more killer cells
 * (4) Induce cells to express more MHC I antigens to increase immune surveillance
 * (5?) She also mentioned that it can induce anti-proliferative and apoptotic effects on the tumor itself (inhibits angiogenesis).
 * Describe the therapeutic use of tumor-infiltrating lymphocytes, TIL, in adoptive cellular transfer therapy.
 * The idea is to take out TILs from tumors (since you figure that these are the ones doing the most good against the tumor), culture them to grow lots of them, then ablate the patient's native immune system and re-'sow' the patient with the TILs. The more-variegated T cell response does, slowly, regrow, but in the meantime there's lots of anti-tumor T cells in the patient's marrow and an absence of Treg cells to downregulate their growth.

Tumors generally only poorly targeted by endogenous antibodies/Th2 responses.

=Hemostasis, Normal Mechanisms=


 * [Hemostasis: injury to blood vessel triggers certain reactions, leading to a fibrin plug at the bleed site.]
 * [Thrombosis: uncontrolled pathologic clotting; can occlude vessels.]
 * Identify the elements that compose the hemostatic system. Understand the basic paradigm for coagulation factor activation.
 * Components:
 * Coagulation factors (13 known)
 * Platelets
 * Endothelial wall (site of injury)
 * General idea: Endothelial wall becomes damaged, exposing collagen, tissue factor, and von Willebrand factor. (vWF, if you're interested, is produced constitutively in endothelia and binds platelets once the endothelium is damaged. Also binds Factor VIII and keeps it viable.)
 * Platelets bind to the site of injury and change configuration, exposing certain proteins that attract fibrinogen and vWF (thus binding more platelets).
 * They also release granules containing thromboxane (leading to vasoconstriction of damaged vessel), vWF and ADP (to bind and activate other platelets), various clotting factors, etc.
 * They also serve as a ground on which clotting factors can easily activate.
 * Note that the endothelium is normally actively preventing coagulation; prevents platelet aggregation, promotes clot breakdown. As with so many other things, it's a balancing act. When the endothelium is breached, the balance shifts (towards pro-coagulation) until it's resolved.
 * Describe the blood clotting pathway. List which components of the system are vitamin K dependent factors. Distinguish the extrinsic and intrinsic pathways and describe the screening tests to measure both (PT and APTT, respectively).
 * A few notes about the components of the clotting pathway:
 * Several clotting factors are called by name, most are called by numeral. An "a" after the numeral indicates that the factor has been activated and is enzymatic.
 * By name: **Prothrombin** (factor II), **Fibrinogen** (factor I). These, when activated, are called thrombin and fibrin respectively.
 * By numeral: Factors III-XIII.
 * Several clotting factors are large enzymes that serve to __orient__ the others.
 * These are: **Factors V and VIII** . Tissue factor also serves this role.
 * Most of the rest of the clotting factors are proenzyme __serine proteases__ which, when cleaved and activated, cleave and activate each other.
 * These are: **Factors II, VII, IX, X, XI, and XII.**
 * The others seem to have particular, difficult-to-generalize-about roles. For example, Factor XIII cross-links fibrin fibers to make a 'hard clot.'
 * Some clotting factors require carboxylation in the liver by vitamin K (recall from the MCAT that it's a fat-soluble vitamin stored in the liver). If there's no vitamin K available or there's a lot of liver damage, these factors may not function correctly. Note that she emphasized this a lot in lecture.
 * Vitamin K-dependent factors: **Factors II, VII, IX, and X**.
 * Note that several important anti-clotting factors are also vitamin K-dependent: **proteins C and S**.
 * __So the end goal__ of clotting is to produce, cross-link, and harden a mat of fibrin fibers to plug the endothelial damage. There are two pathways, the **extrinsic** and the **intrinsic**, to get things set up to do this; both of them end by activating **Factor X** to feed into the same clot formation mechanism, the so-called " **common pathway** ."
 * **Extrinsic pathway** (Factor VII-mediated, triggered by release of tissue factor from damaged endothelium):
 * Tissue factor serves as a co-factor with Factor VIIa (it's not clear what activates VII) to activate Factor X to Xa.
 * Note VIIa can activate elements in the intrinsic pathway as well (XI, IX).
 * **Intrinsic pathway** (Factor VIII-mediated, Factor VII and tissue factor independent):
 * Factor XII is activated (unclear what does this).
 * XIIa activates XI.
 * XIa activates IX.
 * IXa, with Factor VIII as its co-factor, activates Factor X to Xa.
 * **Common pathway** (Factor V-mediated):
 * **Factor Xa**, oriented by **Factor V** , cleaves **prothrombin** (Factor II) to **thrombin** (Factor IIa).
 * **Thrombin** cleaves **fibrinogen** (I) to **fibrin** (Ia), a soluble fiber that begins to polymerize.
 * **Factor XIII** cross-links fibrin polymers to form a hard, insoluble clot.
 * And that's clotting. To reiterate:
 * Extrinsic: tissue factor + VIIa -> Xa.
 * Intrinsic: XIIa -> XIa -> IXa + VIII -> Xa.
 * Common: Xa -> IIa -> Ia + XIII -> clot.
 * Several notes:
 * Notice that many steps in these pathways require Ca2+. No calcium, no (or little) clotting.
 * Fibrin clots binds the platelets at the site together and attaches them more firmly to the vessel wall.
 * Understand that thrombin is the central regulation point for coagulation. Understand normal homeostasis in the coagulation system.
 * Thrombin (IIa) is the central regulation point for coagulation. Activates platelets, cleaves fibrinogen to form fibrin. It also __activates Factors VIII and V__, which further accelerates the clotting cascade (recall that these are the two large orientation co-factors).
 * Normal hemostasis involves a certain amount of regulation of thrombin. There are anti-thrombin proteins that normally circulate to bind free thrombin to prevent its being activated. Thrombin also activates another anti-coagulation protein, protein C, which cleaves Factors Va and VIIIa to slow down clotting. There's also a protein called tissue factor pathway inhibitor that inhibits Xa and the tissue factor-factor VIIa complex. Point is that there's a balance going on.
 * Understand the function of platelets in hemostasis. Describe the process of platelet aggregation.
 * As mentioned, adhere to site of endothelial injury, partly mediated by vWF. Activated by thrombin (IIa) to release vasoconstrictors (TXA2), calcium, and ADP, as well as changing conformation, exposing proteins (factors IIb and IIIa, if I'm reading my notes right) to bind more vWF and fibrinogen, thus binding and activating more platelets.
 * Platelets also have receptors for Factor V, which (recall) is the orienting co-factor for the conversion of prothrombin to thrombin. This speeds up that conversion.
 * Platelets also expose the __phospholipid complex__, which provides a greatly pro-clotting enzymatic effect on the coagulation cascade.
 * Explain what factors influence platelet activation and platelet aggregation. List some of the drugs that can inhibit platelet function and describe their mechanism of action.
 * As mentioned just now, platelets __adhere__ due mainly to vWF but are __activated__ largely by thrombin (from clotting factor activity). Once activated, platelets expose receptors which bind vWF and thus bind other platelets to the site, as well as releasing vWF, ADP, etc in granules to recruit and activate them.
 * Note epinephrine, ADP, and collagen also activate platelets.
 * Drugs: see "Pharmacology of Anticoagulation Therapy." Essentially aspirin, ADP receptor blockers, and adhesion glycoprotein inhibitors.
 * Understand the contribution and influence of endothelial cells on coagulation.
 * As mentioned, when damaged, they expose collagen (mainly type II), vWF, and tissue factor.
 * Collagen and vWF bind platelets.
 * Tissue factor activates the extrinsic clotting pathway.
 * Understand the regulatory mechanisms of coagulation, i.e., protein C pathway, antithrombin, fibrinolytic pathway.
 * Fibrinolytics:
 * Fibrin (clot material) is degraded by __plasmin__, which is cleaved and activated by __tissue plasminogen activator__ (TPA). After degradation of fibrin, fibrin split products (also called fibrin degradation products) are formed, which are also anti-coagulation factors.
 * Protein C pathway:
 * Activated thrombin (IIa) activates Protein C, with __thrombomodulin__ as a cofactor.
 * Protein C, with Protein S as co-factor, inactivates Factors Va and VIIIa.
 * Note that a mutation in Va can cause resistance to its inactivation.
 * Antithrombin:
 * __Antithrombin III__ forms a complex with thrombin and other serine proteases to block their activity.
 * It's activated by heparin (endogenously produced anti-clotting substance).
 * Note that, in the absence of endothelial damage, the balance is generally in favor of anti-clotting activity.


 * Once endothelium is broken, vessel constricts to divert blood flow away from it; in addition, three elements below the basement membrane are exposed: collagen, tissue factor, and von Willebrand factor.
 * These cause platelet adhesion and aggregation; this surface of platelets are what starts all the rest of the clotting pathway going. No platelets, no intravascular clotting. (*)
 * Platelets, bound (ie activated), expose certain membrane glycoproteins (IIb and IIIa), which bind fibrinogen and von williebrand factor, which recruit and bind (activate*) more platelets.
 * Platelets agonists: thrombin, ADP, collagen, AAcid, epinephrine-- these cause platelets to form spiny processes and degranulate.
 * Granules: Dense granules contain serotonin, ATP, ADP, Ca2+ / non-dense*: fibrinogen, vWF, factor V, platelet factor 4, etc. Also released are lysosomes.
 * Clotting factors: 13 of them; mostly referred to by number except for prothrombin (II) and fibrinogen (I).
 * Prozymogens (cleaved to become serine proteases, which act on each other): XII, XI, X, IX, VII, II
 * Cofactors: tissue factor, VIII, V (orient serine proteases)
 * Fibrinogen: when cleaved, during into polymeric fibrin strands (forms clot proper).


 * Coagulation cascade: series of enzymatic reactions activated when collagen, tissue factor, or negatively charged surfaces (ie membrane of activated platelet or blood draw test tube) are exposed-- 'point'* is to produce __thrombin__.
 * Extrinsic system (extrinsic to blood, starts in tissue):
 * Exposure of tissue factor to blood: acts as a cofactor for VIIa. Phospholipid, tissue tactor and VIIa cleaves X to Xa
 * Intrinsic system (independent of factor VII): contact activation of XII activated XI; Xia activated IX; IXa with VIII (orienting protein to align with Ca2+ and phospholipids) can activate X.
 * Common pathway:
 * Once X is activated, Xa reacts with (oriented by V) Ca2+, converts prothombin (II) to thrombin (IIa).
 * Once you have thrombin, it binds to fibrinogen and liberates fibrinopeptides A and B; these fibrin monomers link together to form polymers.
 * Factor XIII cross-links polymers to form a hard clot.
 * Fibrin mesh binds platelets together; increases attachment to vessel wall.


 * Vitamin K: Factors II, VII, IX, and X undergo vitamin K dependent carboxylation in the liver. Without either Vit K or a functioning liver, these elements don't work.
 * Notice anticoagulants C and S are also carboxylated in liver by vitamin K


 * Thrombin also activates VIII and V, which accelerates clotting cascade.


 * Overview: Fibrinogen cleaved by activated thrombin to form fibrin (which is what ultimately stops blood flow).
 * Have to activate thrombin with Xa (Ca2+ and Va as cofactors).
 * Extrinsic pathway: exposed tissue factor to blood and activated VIIa (not known what activates VII) form a complex and activate X.
 * Intrinsic pathway: XIIa is activated by something unknown; XIIa activates XI which activates IX which activates VIII which activates X.
 * After fibrin's been formed, need XIII to form a hard fibrin clot instead of a soft clot.
 * Activated thrombin, as said, activates V and VIII and XIII to accelerate process.
 * Note VII can also activate IX and maybe XI- link between intrinsic and extrinsic.
 * Note can trap red and white cells in fibrin polymers - danger of occlusion.


 * Platelets: have receptors for factor V (orients prothrombin complex). When they change shape after binding to damaged surface, activates X with enzymes on surface.


 * Anticoagulants
 * Fibrinolytic pathway:
 * Fibrin degraded by plasmin; plasminogen (proplasmin) activated by tissue plasminogen activator (TPA). (Fibronolysis inhibited by plasminogen activator inhibitors and alpha-2 antiplasmin.)
 * After degradation of fibrin by plasmin, have certain fibrin degradation products.
 * Antithombin III
 * Proteins C and S
 * Thrombomodulin
 * Generally, balance seems to be towards anticoagulation during steady-state.
 * Process:
 * Activated thrombin activates protein C with thrombomodulin as a cofactor.
 * Protein C (with protein S cofactor) __inactivates__ VIIIa and Va. Note mutation in Va can cause resistance to inactivation by protein C.
 * Antithrombin III forms a complex with thrombin and other serine proteases; activated by heparin to block protease (clotting) activity.

=Hemostasis, Defects=


 * [General coagulation screening mechanisms, used to pinpoint specific factor deficiencies:]
 * (1) Prothrombin time, international normalized ratio. aka "__protime__" or __PT/INR__
 * (2) Activated partial thromboplastin time, aka __PTT__
 * (3) Thrombin time
 * (4) Bleeding time/Platelet function analyzer
 * [Important note I wish she'd mentioned: Tissue factor is Factor III is thromboplastin.]
 * [Recall that warfarin (coumadin) inhibits vitamin K utilization.]


 * Be aware of both congenital or acquired disease states causing bleeding and/or clotting.
 * Classification of hemophilia severity:
 * Severe hemophilia: less than 1% clotting activity (sometimes can make antibodies against the factor you don't have)-- tend to die early, need to take clotting factor supplementation every week to avoid becoming crippled.
 * Mild hemophilia: less than 10% clotting activities-- bleed with trauma and surgery. Tend to only be found after surgery or trauma.
 * Carrier females (hemophilia A and B): 15-80% clotting activity depending on how many of which X chromosomes are activated (lyonisation).
 * **Hemophilia A and B** :
 * X linked (predominantly males) deficiency of factors VIII or IX, respectively.
 * Screen males with unexplained bruises or post-surgical/trauma bleeds
 * Can assay specific factor activity to distinguish between A and B
 * Carrier females are often symptomatic.
 * __Abnormal PTT is the only abnormal screening test seen__ (bleeding time ok due to okay platelets and vWF).
 * A is about 1:5,000 live births
 * B is about 1:50,000 live births
 * Lots of new-mutation causes
 * Symptoms:
 * Soft-tissue hematomas
 * Joint bleeding (blood in a joint begins inflammatory processes and ankylosis/arthritis)
 * CNS bleeds (frequent cause of death)
 * Retroperitoneal or psoas bleeds (can't straighten knee due to femoral nerve compression with expansion of psoas muscle)
 * With falls, can get ecchymoses (bruises) all over legs and back that can stay for months. Can bleed a large proportion (1/3-1/2) of total blood volume into large muscles.
 * Notice severe hemophiliacs can take regular doses of prophylactic clotting factors.
 * **Hemophilia C/factor XI deficiency** :
 * Usually have > 10% clotting activity, usually not picked up until trauma or surgery.
 * Autosomal recessive; variable incidence in various populations.
 * __Abnormal PTT is the only abnormal test observed__.
 * **Factor VII deficiency** :
 * Rare; __abnormal PT is the only abnormal test observed__.
 * **Von Willebrand disease** :
 * Common, mild; deficiency in vWF, with associated difficulties aggregating and binding platelets.
 * __Shows an abnormal bleeding time__; also can show __an increased PTT__ due to vWF's effects to prolong the half-life of Factor VIII.
 * Acquired factor inhibitors: extremely rare, but can get acquired clotting factors inhibitors (most commonly Factor VIII), which will cause a prolonged PTT that's refractory to resolution by mixing with standard plasma.
 * Can acquire autoimmune hemophilia.
 * Understand the PT or the APTT. Understand the differential diagnosis of an abnormal PT or APTT. Be aware of other tests used to evaluate patients with thrombotic or bleeding disorders.
 * __Activated Partial Thromboplastin Time__ (aPTT or PTT): A surface activating agent (proclotting), calcium, and platelet phospholipid are added to citrated (anticoagulant) plasma-- measure how many seconds until a clot forms. Generally about 30 seconds is normal.
 * **Mainly measures intrinsic/common pathway** ; thus measures activity of the entire clotting cascade __except__ Factor VII (VII is extrinsic pathway).
 * Sensitive to inhibition by heparin and fibrin split products (which are also anticoagulants).
 * **Prolonged PTT primarily indicates deficiency of Factors VIII, IX, X, and XI.**
 * __Differential for prolonged aPTT__:
 * Heparin in sample (contamination)
 * Hemophilia A/B (Factor VIII or IX deficiency)
 * Factor XI deficiency (Hemophilia C)
 * Factor XII deficiency
 * Acquired hemophilia (autoimmune)
 * Von Willebrand Disease
 * Lupus Anticoagulant
 * (see under Prothrombin Time for others)
 * If long PTT is observed: mix patient's plasma with control plasma. If it corrects the long PTT, you have a factor deficiency; if it doesn't, you have either lupus anticoagulant or a specific factor inhibitor.
 * __Prothrombin Time__ (PT): Ca2+ and thromboplastin (ie tissue factor) are added to citrated plasma and time to clotting is measured.
 * **PT/INR measures the extrinsic/common pathway** : used mainly to measure II, V, VII, and X (the factors in that pathway).
 * "Normal" PT depends on how good thromboplastin is (by manufacturer); normalized by adjusting for the known potency of given drugs.
 * "INR" -- 1 is a normal, normalized PT.
 * **Prolonged PT indicates deficiency of Factors II, VII, V, and X.**
 * Prolonged also with Vitamin K deficiency and liver disease (need liver-enzyme-mediated carboxylation from K).
 * __Differential for prolonged PT__:
 * If protime is more prolonged than aPTT:
 * Liver disease
 * Vitamin K deficiency
 * Warfarin or rat poison ingestion
 * If PTT is more prolonged than protime:
 * Disseminated intravascular coagulation (DIC)
 * [Other tests she mentioned:
 * __Thrombin time__: Excess thrombin added to plasma. Fibrinogen cleaves, forms fibrin, clots; measure time in seconds.
 * Measures common pathway's conversion of fibrinogen to soluble fibrin.
 * **Detects low or abnormal fibrinogen** ; also sensitive to the inhibitory effects of fibrin split products and/or heparin.
 * __Bleeding time__: measures platelet function, vessel wall, von Willebrand's factor activity, and skin integrity. __Does not measure clotting factors in blood__. Small device makes a cut in forearm; time to clot in measured (2-9 minutes).
 * Rare. Mostly use PFAs (below) instead now.
 * __Platelet function analyzer__: can determine an in vitro bleeding time with agonists. Faster.
 * Understand the hypercoagulable state and causes for inherited thrombophilia.
 * Hypercoagulable state: abnormal propensity to develop thromboses.
 * Acquired: Prolonged impairment of blood flow (cast on leg, prolonged sitting/lying down), chronic damage to vessel endothelia, or imbalance of clotting vs anticlotting factors can lead to a hypercoagulable state. The lupus anticoagulant (see below) is another common culprit.
 * Inherited: mainly deficiencies in anticlotting factors (antithrombin III, proteins C and S). Can also occur due to the aforementioned mutation in Factor Va that resists inactivation by protein C.
 * Describe the role of liver disease in coagulopathy.
 * **Liver disease** :
 * Decreased __synthesis__ of most factors aside from VIII and IX; can't use vitamin K to __carboxylate__ II, VII, IX, and X.
 * Can also stop making fibrinogen (I) and also increased consumption of platelets by spleen due to venous backup in portal hypertension.
 * [For diagnosis: both PT and aPTT prolonged, but PT much more so than aPTT (no carboxylation of Factor VII).]
 * [Treatment:]
 * give plasma infusion; give vitamin K; can use recombinant factor VII (VII has shortest half-life, dies out of FFP quickly); platelet transfusions.
 * List some of the causes for both quantitative and qualitative platelet abnormalities.
 * (she doesn't have a particular section about this that I can see-- this is what I've gleaned thus far. Feel free to contribute others.)
 * Quantitative: lupus anticoagulant (platelets destroyed), DIC (platelets used up).
 * Qualitative: von Willebrand's disease (no platelet aggregation)
 * Good note from Jeff Dunn: "From BRS Pathology (really good review book) I'd just add that qualitative means that you have bleeding disorders in spite of a normal platelet count--aspirin induced inactivation of COX can also be a cause. Quantitative means you have decreased platelets (thrombocytopenia) so you could throw in Idiopathic Thrombocytopenic Purpura."
 * Describe disseminated intravascular coagulation (DIC) with its associated conditions. Understand diagnostic testing for DIC and associated conditions.
 * **DIC** (disseminated intravascular coagulation):
 * Systemic activation of coagulation.
 * Starts forming small fibrin clots all over, mainly in arterioles but also in larger vessels.
 * Leads to organ failure from hypoxia and infarction of tissues.
 * Also depletes clotting factors and platelets-- can bleed out easily.
 * Lab: PTT and protime both abnormal, but PTT much higher than protime (reverse of liver disease).
 * On smear: looking for schistocytes (helmet cells, have sharp points) and an abnormally low number of platelets.
 * Caused by:
 * Sepsis (most common)
 * Trauma with fat embolism (bone, head trauma)
 * Cancer
 * Obstetric complication (amniotic fluid embolism)
 * Dissecting aortic aneurysm or other vascular disorders
 * Some snake venoms
 * Mismatched blood transfusion (immediate hemolytic reaction)
 * Long PTT usually reflects nonspecific inhibition by fibrin split products
 * Low fibrinogen is the key indicator-- low is bad, higher is better.
 * Note you don't generally treat DIC directly- treat the underlying disorder. Trying to stop progression with anticoagulants is sometimes tried but can be dangerous due to bleeding.
 * Replace blood products and factor if bleeding out.
 * Explain what a lupus anticoagulant is, how it affects coagulation, and ways to test for it.
 * Lupus anticoagulant: antibodies to phospholipid in platelet wall.
 * Named incorrectly. In vitro it causes a prolonged PTT, __but__ in vivo it causes thrombotic disorders.
 * Test: absorb out anti-phospholipid antibodies by mixing with normal platelets; repeat PTT test. Can also use a test called the Russell's Viper Venom Test (that's so the name of my next band).
 * Look for nonspecific inhibition: assay factors VIII, IX, XI, XII. If they're all inhibited to some extent, probable lupus anticoagulant.
 * Note most of these patients don't actually have lupus proper. Who named this thing?
 * Can be treated with heparin. Note that it's really important to make sure it's this disease, since treating most of the other prolonged PTT disease states will not react kindly to heparin (anticoagulant + bleeding disorder = bad news).

=Pharmacology of Anticoagulation Therapy=


 * [Classes:]
 * Heparin/oral anticoagulants (interfere with coag. cascade)
 * Fibrinolytic agents (+plasmin)
 * Platelet inhibitors (-aggregation/activation of platelets).
 * Describe the mechanism of action and pharmacokinetics of heparin and low molecular weight heparins, and differences in management of patients on these therapies.
 * __Unfractionated__ heparin **binds to antithrombin III** (recall ATIII is an endogenous anti-clotting agent that binds to and inactivates IIa, IXa, Xa, XIIa) and greatly **increases the rate of its thrombin inactivation** . Note that once ATIII is bound to thrombin/heparin, heparin dissociates from ATIII and can bind another ATIII molecule.
 * Not absorbed from GI tract- given IV for immediate effect or SC for delayed effect. Has a very short half-life and unpredictable dosing response.
 * **Inpatient therapy only**, for the above reasons.
 * __Doesn't cross placental barrier__, unlike warfarin.
 * Used for:
 * Post-orthopedic surgery
 * Venous thromboembolism (with warfarin)
 * Acute MI/angioplasty/cardiopulmonary bypass surgery management
 * __Low-molecular weight__ heparins [LMWH] can't bind to ATIII/thrombin complexes; instead they selectively **bind to Factor Xa** / **ATIII complexes** and increases the rate of Xa inactivation.
 * Given SC; have longer half-lives and more predictable dosing response.
 * Thus these are **outpatient therapy**.
 * [Note a third type of heparin: __Fondaparinux__, a synthetic peptide consisting of the minimal sequence of the heparin sequence necessary to bind antithrombin and Factor Xa. Seems to act like LMW heparin.]
 * The difference here is that only unfractionated heparin has enough saccharide units to effectively bind ATIII-thrombin complex; low-MW heparins can still bind ATIII-Xa complexes.
 * Notice that they're both effective anticoagulants (Factor Xa is needed to activate thrombin anyway). Used for:
 * Venous thrombosis (with warfarin)
 * Managing unstable angina/acute MIs
 * During surgeries requiring cardio bypass or involving angioplasty/stents.
 * Describe the complications associated with heparin therapy, including excessive bleeding and heparin-induced thrombocytopenia with associated thrombosis.
 * Excessive bleeding is exactly what it sounds like, and for the reasons you would expect (interferes with thrombin activation). Can be treated by administration of anti-heparin agents (**protamine sulfate** ).
 * __Heparin-induced thrombocytopenia__: Unintuitively, this is a **pro-clotting disease** caused by heparin.
 * Patients' platelets decreased by greater than 50%.
 * In 3-5% of patients taking unfractionated heparin. Occurs about 5-10 days after heparin administration. Less common with low-MW heparin.
 * Antibody formation to platelet factor-heparin complexes; the antibodies bind to and activate platelets, leading to prothrombotic state.
 * Describe the alternative anticoagulant therapies used for patients with heparin-induced thrombocytopenia.
 * Direct thrombin inhibitors: //Argatroban// (small molecule inhibitor), //Lepirudin// (recombinant leech anticoagulant).
 * Describe the mechanism of action, pharmacokinetics and uses of oral anticoagulant warfarin.
 * Warfarin: **Vitamin K analog** . As such, it's an antagonist for an enzyme (vitamin K reductase) in the pathway that uses vitamin K to carboxylate Factors II, VII, IX, X. If those clotting factors don't get carboxylated, no clotting is possible (either pathway).
 * (remember, from "Hemostasis, Defects,", that the protime will be relatively more increased than the PTT in vitamin K deficiency/liver disease. Protime is thus also used in examining warfarin's effect on patients.)
 * Rapidly absorbed, good bioavailability, long half-life (36-48 hours). Doesn't reach full anticoagulant activity for 2-3 days (til existing coagulation factors expire)- __slow onset__. This means as an acute treatment it's not so hot-- often used as a prophylactic in people with pro-thrombotic conditions.
 * Used for:
 * Venous thromboembolism (with heparin)
 * To prevent embolism in patients with prosthetic valves/atrial fibrillation
 * To prevent stroke or recurrent infarctions
 * Note that it __does cross the placental barrier__.
 * Describe the adverse effects and potential complications associated with use of warfarin.
 * Hemorrhage and bleeding, as above (fixed with administration of vitamin K long-term and replacement of plasma short-term).
 * Can't be used during pregnancy (teratogenic).
 * Lots of drug interactions described, some of which follow.
 * Increased action: aspirin and antibiotics
 * Decreased action: barbiturates and rifampin
 * Describe the relationship between mechanisms of action and speed of onset of action of heparin and oral anticoagulants.
 * __Heparin actively inactivates clotting factors; warfarin inactivates the synthesis of new clotting factors__.
 * Makes sense, then, that heparin is fast-acting and warfarin takes a while to ramp up-- the old clotting factors are still rattling around in the blood for a few days.
 * Describe the mechanisms of action and uses of fibrinolytic agents.
 * Dissolve already-formed clots by breaking down fibrin.
 * **Tissue plasminogen activator (t-PA)** : endogenous compound that converts plasminogen to plasmin, which degrades fibrin and lyses existing clots. It converts plasminogen by binding fibrin first.
 * given at 100x physiological concentration. Can be given as a bolus injection with long half-life.
 * **Urokinase** ; endogenous compound that converts plasminogen to plasmin as above, but doesn't bind fibrin itself.
 * **Streptokinase** : substance isolated from beta-hemolytic streptococci that forms a complex with plasminogen and activates it (to cleave fibrin).
 * Note that tissue plasminogen activator is about 30-40 times more expensive that streptokinase (which is about $100/dose).
 * On the other hand, you can form antibodies and allergic reactions to streptokinase more easily.
 * Fibrinolytics used for:
 * Emergent/acute MI, with aspirin
 * Acute ischemic stroke (effective if administered less than 3 hours after stroke)
 * Deep vein thrombosis (with heparin/warfarin)
 * Side effects: systemic fibrinolytic state. Plasmin degrades not just fibrin but other coagulation factors.
 * Intracranial hemorrhage = most severe side effect.
 * Describe the mechanisms of action and uses of antiplatelet agents.
 * Antiplatelets: __aspirin__, __ADP receptor blockers__ (ticlopidine/clopidogrel), __glycoprotein IIb/IIIa inhibitors__.
 * MOA: depends on agent.
 * __Aspirin__: irreversibly inactivates COX-1 on platelets (platelets don't have COX-2), which inhibits TXA2 synthesis (preventing platelets from releasing it to activate other platelets).
 * __ADP receptor blockers__: Irreversibly bind to ADP receptors; this blocks platelet activation and also blocks alpha granule release (which prevents release of adhesion proteins - glycoproteins IIb/IIIa - that would otherwise recruit more platelets).
 * Both aspirin and ADP receptor binders:
 * Used to prevent acute MIs and stroke (with aspirin).
 * Due to irreversibility, both leave a lasting effect-- it'll be about a week til the platelets are replaced with fresh, unbound replacements.
 * __Glycoprotein IIb/IIIa inhibitors__: block binding of fibrinogen to adhesion proteins (glycoproteins IIb/IIIa). Fibrinogen normally binds to GIIb/IIIa and begins to bind other platelets to the site and activate them.
 * Used after angioplasty, to treat acute MI, for unstable angina.
 * Side effects: bleeding and/or another kind of thrombocytopenia from heparin-induced. Can be reversed by platelet infusions.

Inpatient: unfractionated heparin Outpatient: LMWH

Anti-heparin agent: protamine sulfate

Heparin-induced thrombocytopenia results from antibody formation to heparin-platelet complexes; activated platelets, induces pro-thrombotic state; shows as platelet count below half of normal.

Lepirudin- leech anti-clotting extract

Warfarin: vit. K-mediated clotting factor carboxylation inhibitor. Note it's a vitamin K analog itself.

For venous thromboembolism: use heparin + warfarin

Can't use warfarin during pregnancy. Watch out for drug interactions, especially with aspirin (decreased platelet function anyway) and antibiotics (kill vitamin K-producing bacteria in gut).

=Hemostasis: Approach to Patient=


 * [Good diagnostics:]
 * __Brisk bleeding__ from obvious trauma suggests a localized vascular defect.
 * __Prolonged or recurrent bleeding__ is more likely a generalized hemostatic disorder.
 * __Sudden resumption of bleeding__ from the injured site is possibly excessive fibrinolysis or abnormal clot formation.
 * __Multiple site bleeding__ is often a more severe generalized hemostatic disorder.
 * __Mucocutaneous bleeding__ suggests a von Willebrand factor or platelet defect.
 * __Soft tissue/joint/deep bleeding__ implies coagulopathy.
 * [Also good stuff: causes of abnormal bleeding.]
 * Platelet abnormalities (too few or not working):
 * Generally due to immune thrombocytopenia or von Willebrand disease.
 * Coagulation factor abnormalities (too few or not working):
 * Generally due to hemophilia or antibodies against clotting factors.
 * Fibrinolysis abnormalities (too much or working overtime):
 * Generally due to liver disease.
 * [Reason for this seems to be decreased clearance of plasminogen activators in the blood with impaired liver function.]
 * Review events occurring during hemostasis. Compare primary and secondary hemostasis.
 * Platelets interact with the damaged vessel; thrombin (IIa) leads to fibrin (Ia) to form a clot; this is regulated by fibrinolytic system and clotting factor inhibitors.
 * **Primary hemostasis** : interaction of damaged vessel with platelets.
 * **Secondary hemostasis** : formation of clot through clotting cascade.
 * List important questions to ask when obtaining a bleeding history in a patient with excessive bleeding.
 * (1) Excessive, prolonged, recurrent, or delayed bleeding (too much or too little)?
 * Particularly after surgery, tooth extraction, childbirth, etc?
 * (2) Abnormal bruising/petechiae?
 * (3) Nosebleeds or excessive menstrual bleeding (mucocutaneous bleeding)? Soft tissue/joint hemorrhage?
 * (4) Hematemesis (vomiting blood) or hemoptysis (coughing up blood), melena (black, tarry stool), hematuria (blood in urine) ("blood coming out of any orifice it shouldn't?")
 * (5) Family history of bleeding?
 * (6) Recent meds (vitamin K antagonists or antibiotics that kill K-producing microbes)?
 * (7) Anemia or iron deficiencies?
 * (8) Recent transfusions?
 * List important laboratory studies to obtain when evaluating a patient with excessive bleeding.
 * CBC and peripheral smear (the ubiquitous answer to everything)
 * Bleeding time (with or without platelet function analyzer)
 * Prothrombin time (PT-INR) (measures extrinsic + common pathways)
 * Activated partial thromboplastin time (aPTT) (measures intrinsic + common pathways)
 * Fibrinogen level (measures fibrinogen, no kidding)
 * Thrombin time (TT) (measures fibrinogen activity)
 * Describe the molecular defect, typical clinical course, and general approach to treatment for a patient with Hemophilia A, Hemophilia B, or Von Willebrand's Disease.
 * [Also see notes on this under "Hemostasis, Defects.")
 * Hemophilia A and Hemophilia B:
 * Defect: Deficiency of Factor VIII or IX, respectively; both are X-linked recessive.
 * Clinical:
 * Bleeding into the joints, muscles, and GI tract are the most common manifestations.
 * Note a highly variable presentation depending on how much activity of the particular factor is still retained.
 * Treatment:
 * Replace Factor VIII or IX, as needed; severe patients may need lifelong supplementation.
 * Von Willebrand Disease:
 * Defect: Deficiency of von Willebrand's factor. Inherited autosomally (some forms recessive and some dominant, although our notes just say dominant).
 * Clinical:
 * Basically, mucosal bleeding. Look for the following:
 * Nose bleeds
 * GI bleeds
 * Menorrhagia
 * Bleeding after surgery
 * Treatment:
 * Wiki sez patients can be given prophylactic vWF + Factor VIII (remember that vWF also maintains Factor VIII in the blood) before surgery but generally require no other treatment.
 * List five acquired bleeding disorders due to defects in secondary hemostasis.
 * Liver disease
 * Vitamin K deficiency
 * Warfarin/heparin use
 * DIC (disseminated intravascular coagulation)
 * Acquired clotting factor inhibitors (autoimmune, etc)
 * Describe the process of platelet plug formation.
 * (1) Platelets adhere to damaged vessel wall by collagen or vWF binding.
 * (2) Platelets become activated (by collagen, TXA2, etc) to expose adhesion proteins for more platelets and releases granules that activate them.
 * (3) Platelets become loosely laced together by fibrinogen; once fibrinogen is cleaved to fibrin by thrombin, this firms and stabilizes the platelet plug.
 * List some causes for thrombocytopenia.
 * [Platelets under 50,000: Generally don't see spontaneous hemorrhage, but see increased risk of hemorrhage for trauma or surgery.]
 * [Platelet under 10,000-20,000: Can see life-threatening, spontaneous hemorrhage.]
 * Decreased production of platelets:
 * Nutritional deficiencies (B12), diseases of bone marrow (either from external sources, cancers, or chemotherapy agents).
 * Increased destruction of platelets:
 * Mainly immune-mediated (idiopathic thrombocytopenic purpura, see below).
 * Increased sequestration of platelets in spleen.
 * Due to splenomegaly secondary to another condition (hemolytic disorders, severe thalassemia, parasite infection, etc).
 * Describe an approach to the treatment of idiopathic thrombocytopenic purpura.
 * Acutely (mostly in children or young adults), generally it resolves on its own. Can administer steroids if severe.
 * Chronically (mostly in adults), can be treated with steroids, IVIg, and splenectomy.
 * List some risk factors for development of venous thrombosis and some important questions to ask when taking a history from a patient with suspected venous thrombosis.
 * Risk factors for __venous thrombosis__ (big old list in the notes):
 * Age
 * Previous thrombosis
 * Immobilization (bed rest or post-surgery or both, long plane trips)
 * Surgery
 * Pregnancy
 * Oral contraceptives
 * Myeloproliferative diseases
 * etc.
 * Questions:
 * Pain and swelling in affected leg?
 * Previous deep vein thrombosis or a family history?
 * Recent surgery or immobilization or long plane flights?
 * Got cancer?
 * Smoke?
 * Use oral contraceptives?
 * Pregnant much?
 * Discuss the management of a patient with suspected thromboembolism.
 * Immediate subcutaneous low-molecular-weight heparin (pricey but outpatient) or IV unfractionated heparin (cheaper but inpatient). Administer warfarin as well; stop heparin administration once warfarin reaches therapeutic levels in blood.
 * Aspirin does no good. See why?
 * Recall that aspirin is used to deactivate TXA2 production in platelets. But intravascular clot formation in veins (as opposed to arteries) doesn't generally involve many platelets-- it's clotting factor-heavy.
 * List some of the genetic causes for thrombophilia and describe situations when testing for genetic thrombophilia is indicated.
 * Increased prothrombotic proteins:
 * Mutant Factor V (V Leiden)- reduced Protein C inhibition
 * Prothrombin gene mutation
 * Hyperhomocysteinemia
 * Increased Factor VIII (maybe- mixed evidence).
 * Decreased antithrombotic proteins:
 * Antithrombin, Protein C, or Protein S deficiencies can lead to thrombophilia.


 * Liver disease: too much fibrinolysis due to decreased clearance of plasminogen activators.

Primary/secondary hemostasis: platelets first, clotting cascade second.


 * Hemophilias: bleeding into joints and muscles


 * vWD: muscosal bleeding (types I/II/III; desmopressin for I)

Acquired secondary hemostatic disorders

Can also be caused by B12 defic, bone marrow suppression, ITP, splenomegaly
 * Platelets < 50k risk after trauma/surgery; < 10-20k risk of spontaneous bleeding

Chronic ITP: treat with steroids, IVIg, splectomy. Acute: few steroids.

Thromboembolism: immed LMWH + warfarin, no aspirin

=Rheumatology and Osteoarthritis Overview=


 * [Recall: type II collagen, the strongest collagen in the body, is found mainly in joint cartilage, along with water, absorbent proteoglycans, chondrocytes, and matrix proteins.]
 * Describe the symptoms and signs, synovial fluid analysis, and x-ray features of osteoarthritis. (note some of these are taken verbatim from notes, thus are a little wooden-- not to say incomprehensible.)
 * Osteoarthritis [OA] symptoms:
 * **Joint pain with use that's improved by rest** (doesn't hurt as much in the mornings, hurts more as the day progresses).
 * **Localized** - no systemic effects (patient doesn't "feel sick"). **Rarely symmetrical** (vs. rheumatoid arthritis).
 * Relatively good retention of joint function.
 * "Stiffness"-- generally for less than 30 minutes.
 * Rarely significant effects before age 40.
 * Minimal inflammation- no demineralization of bone.
 * OA signs:
 * Localized joint tenderness, bony enlargement, crepitus, restricted movement, and sometimes some swelling or instability (__inflammation is not in the cartilage__ - as in rheumatoid arthritis - but __in the synovium__).
 * OS synovial fluid analysis:
 * Type I fluid (minimally inflammatory), __200-2000 WBC__ (more than normal, less than RA), 25% PMNs (inflammation of synovium).
 * Negative 'crystal exam' (crystals are found in gout, etc).
 * Normal glucose.
 * OS X-ray features:
 * "Gull wing" patterns in the interphalangeal joints.
 * Medial compartment disease of the knee.
 * Horizontal **osteophytes** (bony outgrowths) of the vertebrae.
 * Decreased joint space superiorly with relative medial preservation in the hip.
 * Hallux valgus (bending of the great toe towards the second toe).
 * Discuss the risk factors for getting OA.
 * Some genetic factors
 * Metabolic abnormalities of cartilage (iron overload deposition in cartilage, etc)
 * Trauma to joints
 * Inflammatory joint disease (if you get gout, that joint's at a higher risk of getting OA)
 * Obesity (risk factor for hands as well as knees and hips)
 * Age (three out of four people over 70 have OA)
 * There are some postulates that repetitive motion injuries can also contribute to OA.
 * Explain the various theories on the pathogenesis of OA.
 * **Focused on cartilage** -- effectively the chondrocytes overproduce joint-breakdown factors ( **catabolic** factors) relative to joint-reconstruction factors.
 * Injury (as in trauma) can damage chondrocytes and cause them to release predominantly catabolic factors.
 * Reparative mechanisms fail; cartilage loses its ability to retain water; **joint increases its water content** as less is taken up by cartilage.
 * Catabolic factors in specific:
 * __IL-1__: sustains inflammation and cartilage degradation.
 * __TNF-alpha__: similar to IL-1.
 * Various other interleukins (IL-6, -17, -18).
 * __Nitric oxide__ (NO): induces chondrocyte apoptosis (inhibiting repair).
 * __Prostaglandins__
 * Anti-catabolic factors: inhibitory cytokines IL-4, -10, -13, -1RA (decrease breakdown/inflammation).
 * Compare and contrast the aged joint with the OA joint.
 * Point here is to know that aged joints are very different from OA joints.
 * __Aged__ joints have __decreased__ water content; __OA__ joints have __increased__ water content.
 * __Aged__ joints have __increased__ GAG (glycoaminoglycans, from Pfenniger's lectures in M2M) content; __OA__ joints have __decreased__ GAG content.
 * __Aged__ joints have __more__ type II collagen; __OA__ joints have __loose, smaller__ type II collagen.
 * Discuss the treatment of OA as it relates to the pathophysiology.
 * He seems to be saying to fit the treatment to the specific case. Lose weight if obesity is the problem, modify activities if repetitive motion is the problem, take weight off knees or ankles with canes/crutches, etc.
 * Medicinally, treat with topical agents, NSAIDs, pain meds (analgesics), intra-articular injections (corticosteroids or hyaluronic acid), glucosamine, or potentially surgery (from arthroscopy to joint replacement).

=Rheumatoid Arthritis=


 * Describe the general clinical, laboratory, and x-ray features of rheumatoid arthritis (RA), including joint distribution, synovial fluid analysis, serologies, and extra-articular manifestations.
 * Clinical features: swollen, warm joints with limited motion. **Joint pain with rest, extensive morning stiffness** (as opposed to OA).
 * X-rays: Bone degeneration, deviated joints, 'swan neck' patterns in the fingers. Soft tissue swelling.
 * Joint distribution: generally symmetrical, peripheral. The upper cervical spine is often involved.
 * Synovial fluid analysis:
 * Synovium: Increase in Type A and B synovial cells. A are like macrophages; B are like fibroblasts. Shows lots of lymphocytes (B, T, NK) and mast cells, also plasma cells (producing RF).
 * This inflamed, abnormal synovium is called **pannus** and is the main agent responsible for the pathology (bone and cartilage degeneration) in RA.
 * Synovial fluid: __Early on__: lots of mononuclear leukocytes. __Later on__: lots of neutrophils. RF leaks into the fluid, as well, causing complement activation within the fluid (which is presumably what attracts neutrophils-- C5a, recall?).
 * Serologies: RF often present; more than 2000 WBC count; citrullinated peptide antibodies often found; anemia and hypogammaglobulinemia often found.
 * Extra-articular manifestations: RF complexes can cause vasculitis; **Rheumatoid nodules** can occur in skin, lung, heart, and eye. In general, patient can present with feelings of malaise, low-grade fever, fatigue, and weight loss.
 * Discuss the genetic factors that may determine the severity of RA.
 * A particular gene of the DR-4 HLA antigen, **DRB1**, is linked to increased incidence of RA. In particular, the **QKRAA** sequence within that gene is located around the antigen-binding groove of the MHC I proteins and seems to be partly responsible.
 * Note that if it was that simple we could probably treat it better. It's not.
 * Explain the pathogenesis of RA in the synovial tissue and synovial fluid, including cell types, cytokines, and proteolytic enzymes.
 * Thing to recall here is that RA is primarily a synovial disease and only secondarily a bone and cartilage disease-- the synovium becomes inflamed, turns into pannus, and begins to invade and break down adjacent bone and cartilage.
 * First thing that seems to happen is microvascular injury in the synovium, causing edema, which leads to infiltration of mononuclear leukocytes (not neutrophils at this stage). T cells go after some collagen that's been altered by collagenases. The RF produced by the plasma cells infiltrates the synovial fluid, triggering complement, bringing in neutrophils. Fibroblast-like cells in the pannus lay down lots of new tissues and blood vessels, expanding the synovium into neighboring structures.
 * The macrophages in the synovium produce cytokines, stimulating collagenase and protease production as well as promoting osteoclasts and chondrocytic catabolism. The neutrophils in the synovial fluid may also contribute by leaking proteolytic enzymes and reactive oxygen species into the joint space.
 * IL-1, TNF-alpha, and IL-6 (produced largely by the macrophages) seem primarily responsible for the systemic ill effects, as well as primary pannus development. As with OA, the thing is that the balance between pro- and anti-inflammatory cytokines seems to get thrown out of whack.
 * Note that we're still not entirely sure why RA happens other than it seems to be partly autoimmune.
 * Discuss the treatment of RA as it relates to pathophysiology.
 * Anti-inflammatory and analgesic drug treatment (NSAIDs and prednisone).
 * Disease-modifying anti-rheumatoid drugs (DMARDs-- seriously?) are used to inhibit macrophage/lymphocyte functions and limit tissue damage.
 * Physical therapy and surgery also options.
 * Contrast OA and RA on a clinical basis and discuss their differences in pathophysiology.
 * OA: asymmetrical, little inflammation, bone outgrowth (osteophytes). A cartilage disease first and a bone disease second. Better in the mornings, worse as the day goes on.
 * RA: symmetrical, lots of inflammation, bone degeneration. A synovium disease first and a bone disease second. Worse in the mornings, extensive morning stiffness.

=Rheumatology, Part II: Crystal Arthritis and Axial Arthropathies=


 * [generally found in men over 40, esp. with a history of drinking and/or hypertension.]
 * Review normal uric acid metabolism and identify secondary causes of hyperuricemia.
 * It's part of normal amino acid metabolism pathways. Note that the reaction producing uric acid is irreversible; thus humans, lacking an enzyme to convert uric acid to some other compound, have to excrete it.
 * Causes of hyperuricemia: overproduction or underexcretion of uric acid.
 * Underexcretion:
 * Alcohol: dehydrator, resulting in relative increased concentration and decreased solubility of crystals.
 * Hypertension inhibits kidney filtration of uric acid. Note other kinds of renal failure can also promote gout.
 * Diuretics: again, dehydrates.
 * Overproduction:
 * Rare. If you have wonky levels of a couple particular enzymes, you can wind up with an abnormal shunt down the uric acid synthesis pathways.
 * Specifics: The enzymes in question are HGPRT (hypoxanthine-guanine phosphoribosyltransferase) and PRPP (phosphoribosyl-pyrophosphate) synthetase.
 * Note a complete absence of HGPRT also leads to Lesch-Nyhan syndrome (self-mutilating disease, found usually in young boys, from M2M notes).
 * Describe the general clinical and synovial fluid analysis features of gout and calcium pyrophosphate dihydrate deposition disease (CPDD), including crystal morphology and birefringence.
 * Gout:
 * Clinical: Sudden onset; red, inflamed, warm/hot joint with excrutiating pain. Particularly found in the MP joint of the hallux, since it's a large, distal (cool) joint susceptible to trauma.
 * Classic symptom (from cases): "Even the weight of the bedding hurts my toe."
 * Synovial fluid: negatively birefringent uric acid crystals (they look yellow when parallel to red compensator). Elevated WBCs, predominance of neutrophils.
 * The precipitated uric acids are recognized as antigens (by TLRs) and PMNs are released on site.
 * Note there's also a buildup of antibodies against uric crystals if it goes on long enough.
 * Pseudogout (aka calcium pyrophosphate dihydrate deposition or CPPD):
 * Clinical: Sudden onset of severe pain, swelling, redness, warmth, like gout, but usually in a large joint, particularly the knees, sometimes the wrists and ankles. Rare involvement of MP joints. Often found in the elderly along with RA or OA.
 * Synovial fluid: no uric acid crystals; instead . Generally found in large joints like the knee, found in elderly people often along with OA or RA. Crystals are blue in parallel to the red compensator (as vs. yellow).
 * Get an inflammatory response, similar to that found in gout.
 * Contrast the differences in the pathophysiology of gout and pseudogout.
 * Gout: Precipitation of uric acid crystals cause the inflammation. Generally found in distal small joints, particularly MP of big toes.
 * Pseudogout (CPDD): Get high levels of pyrophosphate incorporated into the cartilage, leading to calcium crystals shedding out of the cartilage and into the synovial fluids, which causes inflammation.
 * Discuss the treatments for acute crystal-related arthritis and chronic symptomatic hyperuricemia.
 * Check for sepsis in synovial fluid by culture and Gram stain.
 * Acute: NSAIDs or steroids to reduce local inflammation. Can use colchicine to diminish PMN infiltration.
 * Chronic: For gout, promoting renal excretion (uricosurics) or preventing synthesis (xanthine oxidase inhibitors) of uric acid should serve as treatment and prophylaxis. For CPDD, there is no good way to remove crystals from the joints.
 * Describe the clinical, laboratory, and x-ray features of Ankylosing Spondylitis, including any extra-articular manifestations.
 * Clinical: history (> 3 mo.) of __inflammatory back pain, prolonged morning stiffness, improvement of pain with exercise__. Physical examination of back shows sacroiliac joint tenderness and loss of spinal range of motion (can also find back deformities and reduced chest expansion in late disease).
 * Some patients (25%) also have peripheral arthritis in joints close to spine.
 * Unlike rheumatoid arthritis, AS frequently affects the manubriosternal joint, costovertebral joints, and pubic ramus (areas of lots of cartilage in direct contact with bone).
 * Characteristic AS symptom: pain and inflammation where tendons insert into bones (heel with calcaneal/Achilles tendon, sole with planter fascia).
 * Lab: Elevated sedimentation rate, negative RF test (rhematoid factor, IgM anti-IgG), negative ANA test.
 * The ANA test is a test for a particular type of autoantibody (__a__nti-__n__uclear __a__ntibody), usually found in lupus.
 * The fact that the RF and ANA tests are negative is why these disorders are called "seronegative spondyloarthropathies."
 * X-rays: Sacroiliitis with bone erosion and sclerosis in all patients by age 45.
 * "Radiographic spondylitis" (thin cartilaginous junctions between adjacent vertebrae) in two-thirds of patients; complete spinal fusion in only about one of ten.
 * About a quarter of patients show inflammatory hip disease that can lead to bone fusion.
 * Extraarticular:
 * Acute anterior uveitis (inflammation and scarring of iris and surrounding tissue)
 * Osteoporosis (common)
 * Colitis/Crohn's-similar lesions (common)
 * Bunch of other, rarer crap. See notes.
 * [__Note in contrast to RA, Ankylosing Sp involves thoracic and lumbar spine and large joints.__]
 * [__Note in contrast to OA, Anklyosing Sp involves morning pain that gets better with exercise.__]
 * Discuss the epidemiology and genetics of the seronegative spondyloarthropathies.
 * 90% of Caucasian patients with ankylosing spondylitis have the HLA-B27 gene.
 * 6-9% of Caucasian population have HLA-B27 gene.
 * Of Caucasian people with HLA-B27 with no family history, have only 1-2% chance of developing disorder; with family history in first degree relative, have more like a 10-20% chance.
 * Explain the theories of the pathogenesis of the seronegative spondyloarthropathies.
 * "Molecular mimicry": HLA-B27 looks like a common bacterial pathogen, cross-reaction of antibodies with (for whatever reason) tissue in joints.
 * Or: don't make a response to a particular infectious agent because it looks so much like HLA-B27, which results in chronic infection/inflammation (in joints?).
 * "Arthritogenic peptide": HLA-B27 presents particular peptides in such a way as to prompt an immune response.
 * Homodimers: HLA-B27 proteins may form homodimers or misfold enough to trigger pro-inflammatory factors.
 * The other main seronegative spondyloarthropathy: **reactive arthritis** . This is generally secondary to //Chlamydia//, //Salmonella// , //Yersinia// etc infections which reach the joint. Note HLA-B27 not strongly correlated with reactive arthritis.
 * Discuss the treatment of the seronegative spondyloarthropathies as it relates to the pathogenesis.
 * Specific physical therapy, sleep with small or no pillow; no smoking; NSAIDs and steroid injections.
 * Sulfasalazine sometimes used, as is tetracycline (for reactive arthritis due to //Chlamydia// ) and anti-TNF-alpha (TNF-a evidently partially drives inflammation).

ANK gene involved with abnormal PPi processing Often found in larger joints than gout.


 * Ankylosing Spondylitis = seronegative spondyloarthropathy (no RF, no ANA)**
 * Reactive Arthritis (reactive to joint infections) = other seronegative SPARpathy.**

=Rheumatology, Part III: Autoimmunity/SLE/Vasculitis=


 * Describe the clinical, laboratory, and X-ray features of systemic lupus erythematosis (SLE), including the organs involved and serologies.
 * Clinical:
 * Malar rash (butterfly rash, on face)
 * Discoid rashes (oval plaques, anywhere on body)
 * Serositis (pericarditis/pleuritis- chest pain, dyspnea)
 * Oral ulcers
 * Arthritis
 * Psychosis, seizures
 * Photosensitivity
 * Lab:
 * ANA test positive
 * Pancytopenia: white cells, anemia, platelets, etc, all low.
 * Antibodies vs. DNA and phospholipids
 * X-ray:
 * Patient in case study's X-ray was normal. No info in notes that I can see.
 * Explain the difference between organ specific autoimmunity and systemic autoimmunity.
 * This is more or less what you think it is.
 * Organ specific autoimmunity: Immune response directed against one or more specific autoantigens within a given organ; results in destruction of only those organs expressing the autoantigens.
 * Examples: myasthenia gravis (vs. acetylcholine receptors), Goodpasture's syndrome (vs. type IV collagen), autoimmune thyroiditis, type I diabetes.
 * Systemic autoimmunity: Immune response against multiple autoantigens not limited to a specific organ system. Resulting disease affects multiple organ systems, both because of immune complexes in basement membranes (type III immunopathology) and also because of direct autoimmune attack on organs (type II immunopathology).
 * Examples: systemic lupus erythematosus (classic), rheumatoid arthritis, polymyositis.
 * Discuss the epidemiology and genetics of SLE including the predisposing factors and environmental factors that can modulate disease.
 * By a 9:1 ratio, found more in post-pubertal women than men, particularly young women. Notice that pre-pubertal ratios are closer to 1:1, implicating sex hormones.
 * Also implicated: UV exposure, which can exacerbate SLE and trigger a flare.
 * Prevalence: 0.5-5 per 1,000, more common in African-Americans, Asians, Hispanics.
 * **SLE is associated with HLA allele DR3, C4A null allele** (complement deficiency).
 * C4a: one way of thinking about this is that classical complement is the body's way of clearing IgG that's attached to something. No C4 activation, no classical complement activity, less clearance of IgG that can be stuck in immune complexes, generating type III immunopathology.
 * Risk goes up with affected relatives.
 * IFN-a and IFN-b activation upregulates certain gene expression patterns.
 * Explain the pathophysiology of SLE and the various theories used to explain autoimmunity.
 * Type II manifestations (direct antibodies against self): Hemolytic anemias, anti-phospholipid antibodies, CNS stuff.
 * Type III manifestations (immune complex damage): Lupus nephritis, antinuclear antibodies.
 * Theories: Essentially see under "Autoimmunity: Immunopathology Type II."
 * Loss of T cell tolerance (emergence of autoimmune T cells)
 * Polyclonal B cell activation (kind of a mitogen B cell effect by certain agents)
 * Cross-reaction of antibodies with 'self' tissues
 * "Illicit help" model (self-antigen coupled to foreign-antigen)
 * Sequestered antigen
 * Immunodeficiency (complement deficiencies or Fc deficiencies)
 * Note also a great article in the NEJM this week that Scott Koski mailed out earlier that postulates that some kind of macrophage dysfunction means that apoptotic cells - instead of being properly engulfed before they can spill their contents - spill out some DNA into the extracellular space, thus potentially sensitizing the immune system to dsDNA.
 * Discuss the treatment of SLE as it relates to the pathophysiology.
 * Decrease exposure to triggers (wear lots of clothing + sunblock)
 * Decrease inflammatory response (NSAIDs, steroids)
 * Decrease cell-mediated (T) immune response (anti-malarials, immunosuppressive)
 * Also now using anti-B cell drugs (rituximab: CD20 antibody)
 * Administer IVIg (no clear mechanism of action)

Signs of SLE: 4 out of 11 of the following:
 * MD SOAPBRAIN**


 * M** alar rash (butterfly rash, on face)
 * D** iscoid rash (oval plaques, anywhere on body)
 * S** erositis (pericarditis/pleuritis- chest pain, dyspnea)
 * O** ral ulcers
 * A** NA test positive (found 95-98% in SLE patients)
 * P** hotosensitivity
 * B** lood tests: pancytopenia, Coombs test often positive
 * R** enal dysfunction
 * A** rthritis
 * I** mmune antibodies vs. DNA and phospholipids
 * N** eurological: psychosis and seizures

=Rheumatology Review and Vasculitis Case=


 * [Review:
 * OA:
 * Large bony nodes in hands
 * DIP, PIP, 1st carpal-metacarpal (CMC) joints
 * Cervical/lumbar spine
 * Great toe
 * [See slide for summary]
 * Predisposing: age, obesity, trauma, inflammatory (anything producing synovitis), metabolic (eg. iron overload). No risk from sports/exercise (may be protective). Some repetitive-motion stuff (miners, weavers).
 * Once cartilage damaged, it doesn't repair itself.
 * Cartilage: type II collagen, chondrocytes, hyaluronic acid, proteoglycans, water, and matrix metalloproteases and their tissue inhibitors.
 * In early OA: higher water content and chondrocyte content, no systemic features, type I noninflammatory synovial fluid (200-2000 WBCs)
 * Note that there are some, minor, inflammatory processes going on. Cytokines: IL-1 (+MMP production), nitric oxide (NO), prostaglandins.
 * Clinical: joint space loss on medial and lateral sides. Sclerosis: thickening of bones. Subchondral cysts, osteophytes.
 * RA:
 * Misshapen joints, digits pointing in the wrong directions.
 * Unknown etiology; peripheral, bilateral symmetric synuvitis.
 * Classically spares DIP, unlike OA.
 * Disease severity associated with the __shared epitope__ (QKRAA) in epitope binding groove of HLA-DR4 and HLA-DR1 proteins.
 * RF: Not specific for RA, but largely present in RA patients. Generally IgM, vs Fc regions of IgG.
 * Anti-cyclic citrullinated peptide antibody: found more specifically with RA patients, though not all RA patients have it (low sensitivity). Detect modified arginine residues (to citrulline) found in RA. Both RF + anti-CCP: specificity nearly 100%, though both are only found in about half of RA patients.
 * Hypertrophy of synovium (pannus) invading into bone and cartilage ("marginal erosions").
 * Maybe: APCs activate T cells, activate B cells and macrophages, make RF and pro-inflammatory cytokines that induce MMP production.
 * In synovium: most are helper (CD4+) T cells. Some B and plasma cells. Lots of the T cells are memory cells.
 * Nodules outside joints: caused by RF.
 * The Gout + CPDD
 * Podagra: in 1st MTP
 * In cool, peripheral joints of lower/upper extremities
 * Caused by either over-production (occasionally) or under-excretion (mostly).
 * 2 X-linked causes of over-production:
 * PRPP synthetase overactivity
 * HGPRT deficiency (complete deficiency = Lesch-Nyhan)
 * Uric acid = product of purine metabolism. Don't have the enzyme to break down uric acid.
 * Long, needle-shaped crystals, negatively birefringent.
 * Use allopurinol to block xanthine oxidase activity to avoid making uric acid.
 * Driven by TLR activation by MSU crystals. IL-8 produced (chemotactic to neutrophils). IL-1 produced (+infiltration, vasodilation).
 * [MSU crystals: monosodium urate.]
 * IgG binds to MSU crystals (not epitope specific, just H-binding, but activated neutrophils).
 * Ultimately self-limiting: releases factors that inhibits its own processes. Apolipoprotein B inhibits phagocytosis and inflammation, ACTH release (+Na+ retention, more water volume in blood, thus higher solubility of crystals), etc.
 * CPDD: psuedogout due to abnormal pyrophosphate metabolism.
 * Pyrophosphates: generated by metabolism of NTPs (ATP, GTP, etc). Chondrocytes expel lots of pyrophosphate, joins with calcium to form crystals.
 * Calcified cartilage seen on X-ray, released into synovial fluid.
 * Crystals: short, rhomboid, positively birefringent.
 * Ankylosing Spondylitis:
 * Axial arthritis: SI joint, spine; morning stiffness; usually joint involvement close to spine (hips, shoulders).
 * __Enthesitis__ (inflammation where ligaments, tendons, and fibrous tissues insert into bone)
 * Uveitis
 * HLA-B27 association (low % of HLA-B27 people get AS but very high % of AS patients have HLA-B27).
 * **If you have HLA-B27, your risk of getting AS:**
 * **2% if no AS relatives**
 * **20% if first-degree AS relative**
 * Synovium inflammation
 * Unknown etiology (genetic susceptibility, environmental trigger)
 * Reactive arthritis:
 * After GI infection or urethritis, dead antigens go to joints (inside macrophages) and trigger inflammation
 * Lower extremity arthritis
 * Chlamydia can remain latent in joints
 * Reiter's syndrome: can't see (conjunctivitis), pee (urethritis), or climb a tree (arthritis)
 * Systemic lupus erythematosus:
 * Chronic, systemic autoimmune disease, affects lots of stuff. See "Rheumatology, Part III."
 * Type II autoimmune disease with type III complications.
 * Both a T cell and a B cell process (autoimmune responses driven by Th2s)
 * Increased risk among relatives
 * Association with HLA-DR3 and especially complement deficiencies (C4A null allele)
 * Increased with UV exposure (expose more DNA from damaged cells)
 * ANA not specific for SLE but found in almost all SLE patients, also anti-dsDNA antibodies.
 * Type II rxns: anti-red cells, -white cells, -phospholipids (Lupus anticoagulant)
 * Note type II does not cause vasculitis.
 * Type III rxns: anti-dsDNA; find glomerulonephritis (lumpy-bumpy)
 * Vasculitis: (see more below)
 * High sed rate, fever, rash, malaise, immune dysfunction, weight loss, glomerular inflammation
 * Inflammation within/through vessel wall-- damage to vessel integrity, decreased flow. Anything distal to inflamed region will have decreased flow.
 * Can get ischemic neuropathy in distal organs/extremities.
 * Varying degree of various immune cells invading damaged vessel wall,
 * Focal, segmental: never entire vessel at once, but a spotty infiltration.
 * Name the different types of vasculitis according to the Chapel Hill Consensus Conference classification.
 * Classified based on the size of the involved vessel and the clinical presentation.
 * (Probably don't need to know all the names but for completeness' sake:)
 * Large-vessel vasculitis:
 * Giant-cell arteritis (anything off coronary arteries or carotids outside the brain)
 * Takayasu's arteritis (anything off the aortic arch)
 * Medium-vessel vasculitis:
 * Polyarteritis nodosa
 * Kawasaki's disease
 * Small-vessel vasculitis:
 * Antineutrophil cytoplasmic antibody (ANCA)-positive vasculitides:
 * Wegener's
 * Churg-Strauss
 * ANCA-negative:
 * Henoch-Schonlein Purpura
 * Essential cryoglobulinemic vasculitis
 * Cutaneous leukocytoclastic angiitis
 * Describe the clinical features and the laboratory abnormalities suggestive of vasculitis.
 * In a nutshell: clinically you feel crappy. Fever, pain. Lab values suggest lots of inflammation. That's about it. Think Type III immunopath with an option on focal ischemia.
 * Clinical features:
 * Skin lesions
 * "Constitutional:" Fever, anorexia/weight loss, weakness/fatigue.
 * Musculoskeletal: arthralgias (joint pains), arthritis, myalgias, peripheral neuropathy.
 * Lab features:
 * Indicative of systemic inflammation.
 * Anemia
 * Thrombocytosis
 * Low albumin
 * Elevated sedimentation rate + C-reactive protein
 * Low complement levels
 * &c.
 * Keep an eye out for stuff that's indicative of Type III immunopathology-- cryoglobulins (immune complex precipitate in serum samples stored in the fridge overnight), renal failure, etc.
 * Discuss the different immunopathogenic mechanisms that mediate vasculitis.
 * (1) Immune complexes:
 * Note that the immune complexes do not magically (or stochastically if you prefer) get stuck in random endothelia (recall that they get preferentially stuck in serous filters). It takes some manner of pre-existing inflammation of the endothelium first-- this activates Platelet Activating Factor (PAF), resulting in vascular permeability, which is what permits the immune complexes to attach and cause complement.
 * Again that immune complexes are not the precipitating event-- it's inflammation activating PAF.
 * (2) T cell-mediated: I think he's trying to say you can have antigens in your endothelia that piss off your T cells. Beyond that and some scrambled discussion on giant cell arteritis and association with HLA-DR4, I got nothing.
 * (3) Antineutrophil cytoplasmic antibodies (ANCAs) (see below)
 * (4) Anti-endothelial antibodies (type II immunopathology)
 * (5) Infection of vascular endothelial cells: promote immune complex binding and PMN adhesion (also probably ANCA-related problems), drive inflammatory response.
 * **Note that these seem to break up into two categories:**
 * Root causes of endothelial inflammation (type II immunopath, infection, T cells)
 * Exacerbating factors of endothelial inflammation (ANCAs, immune complexes)
 * Distinguish the different types of ANCAs and discuss their role in the pathogenesis of vasculitis.
 * This is a little tricky. Just to keep this straight: ANCAs are __endogenous antibodies__ that __bind to certain granulatory enzymes__ that are released to the surface of neutrophils when activated by certain inflammatory cytokines (like those at the site of vascular inflammation). Antibody binding further activates the neutrophil to induce greater inflammation. So if your neutrophils aren't activated, ANCA isn't going to do much. Once they're activated, though, ANCA is going to take the inflammation up a notch.
 * Thus: __ANCAs do not seem to precipitate inflammation, but they exacerbate the inflammation that is already extant__.
 * The two types of ANCAs are named for where they stain in the lab; this has nothing to do with their involvement in the inflammatory process.
 * Anti-neutrophilic cytoplasmic antibodies (types of ANCAs):
 * (1) Cytoplasmic (c-ANCA): entire cytoplasm stains in the lab.
 * Antigen = Proteinase-3 (PR3) in primary granules in PMNs.
 * c-ANCA is associated with Wegener's granulomatosis.
 * (2) Perinuclear (p-ANCA): only stains around the nucleus in the lab.
 * Antigen = myeloperoxidase (MPO) in primary granules in PMNs.
 * p-ANCA is associated with microscopic polyangiitis.
 * These amplify the inflammatory response in endothelium-- once the granule contents are released onto the endothelial surface, circulating ANCAs can bind to their targets and cause further damage.
 * Discuss the treatment of vasculitis as it relates to the extent of organ involvement.
 * Treat the antigen that's either causing the underlying inflammation or forming the immune complexes: Drugs, bugs, connective tissue disease, malignancies.
 * Manage inflammation with steroids.
 * Treat rapidly progressing disease with high-dose steroids and chemo drugs. Plasmapheresis can be helpful with immune complexes (partic. vs. hepatitis C) and ANCA problems.

TLR activation by MSU crystals

Lower extremity arthritis in reactive arth

Mechanisms of vasculitis:
 * Primary causes:
 * Type II immunopath (B-cell mediated)
 * T-cell mediated
 * Infection
 * Secondary to inflammation
 * Immune complexes
 * ANCAs

=Chronic Myeloproliferative Diseases and Myelodysplastic Diseases=


 * Chronic Myeloproliferative Diseases and Myelodysplastic Diseases, 2/21/08:**
 * Note on leukemia lectures: I ran all this stuff by Dr. Ryder, who actually sat down with a pen and ran through them and corrected anything he didn't like. He said this should represent pretty close to exactly what he wants us to learn. So it may still be confusing as f*ck, but at least it's relatively complete.
 * That said, he also sped through it like a doctor whose time is worth more than my student loans. So he may have missed on some specifics-- so if you see anything that doesn't look right, email me.
 * [Side notes: ]
 * On the classification of these diseases: done by four criteria, some of which are more significant in some leukemias than others.
 * By morphology (toned-down version):
 * Malignant cell = blast-- classified as __acute__ (rapid onset).
 * Malignant cell = more mature cells-- classified as __chronic__.
 * By immunophenotype:
 * Classified by the type of over-proliferating immune cell (as defined by immuno/CD markers)
 * By genetic features:
 * Eg. the Philadelphia chromosome (9-22) -> CML.
 * By clinical features:
 * How it presents.
 * Recall: myeloid cells = hematopoietic cells that ain't lymphocytes.


 * Discuss the similarities and differences between the chronic myeloproliferative disorders and the myelodysplastic syndromes. Include the appearance of the peripheral blood, the appearance of the bone marrow, disease course in the absence of successful clinical intervention or death from some other cause, and who gets these diseases.
 * **Similarities** : Both are clonal, neoplastic disorders that can progress to marrow failure or acute leukemia. They can both be traced back to problems with pluri/multipotential stem cells. Both show hypercellular marrow (almost all of the cells in the marrow are hematopoietic). They both progress from chronic (low blast-cell content) to actute (high blast-cell content).
 * **Differences** :
 * CMPD: increased proliferation of myeloid cells (one or more types). See __elevated counts__ of these types in the peripheral smear and on the CBC.
 * MDS: "Perturbed" maturation of one or more types of myeloid precursors, leading to increased apoptosis of those precursors in the marrow. See __cytopenias__ of those types in the peripheral smear/CBC.
 * CMPD: Dysplastic changes not seen in the marrow.
 * MDS: Dysplasia in the marrow (thus the name).
 * CMPD starts out with a problem in a CFU-LM cell and can therefore can progress to __myeloblastic as well as lymphoblastic leukemias__.
 * MDS: starts out with a problem in a CFU-GEMM cell-- thus can progress to __myeloblastic leukemias only__.
 * List the four most common myeloproliferative disorders using WHO terminology.
 * **Chronic myelogenous leukemia** (granulocyte proliferation)
 * **Polycythemia vera** (red cell proliferation)
 * **Essential thrombocythemia** (megakaryocyte/platelet proliferation)
 * **Chronic idiopathic myelofibrosis** (mainly also megakaryocyte proliferation-- these involve stimulation of fibroblasts that scar up the bone marrow)
 * Discuss the diagnostic cytogenetic abnormality in chronic myelogenous leukemia (CML). Discuss why cytogenetic studies should be performed when making a diagnosis of any of the chronic myeloproliferative disorders.
 * Philadelphia chromosome (9-22). 95% of patients with CML have the Philadelphia chromosome. It's also the most common chromosomal abnormality in adult acute lymphoblastic leukemias, and is a marker of poor prognosis. So a good thing to look for.
 * Describe the three phases of chronic CML.
 * (1) __Chronic phase__: patient may be asymptomatic, but usually have splenomegaly. Have **granulocytosis**, __always__ have **basophilia** , often have high platelet counts. Hypercellular marrow aspirate.
 * (2) __Accelerated phase__: with increasing number of blasts in peripheral blood; basophilia over 20%; persistent thrombocytosis. Essentially, the neoplastic clone is becoming more active and aggressive.
 * (3) __Blast phase__: More than 20% blasts in the peripheral blood (20% is the somewhat arbitrary cutoff between chronic and acute leukemias); extramedullary blast proliferation.
 * Describe complications that are common to both polycythemia vera (PV) and essential thrombocythemia (ET).
 * Lab: Bone marrow is nearly identical between the two. Both show some degree of thrombocytosis, though ET's is usually higher.
 * Clinical: Both share a predisposition to arterial/venous thrombosis, though ET patients can also show predilection to hemorrhage (possibly due to malfunctioning platelets).
 * Discuss what needs to be considered when evaluating a patient with erythrocytosis and entertaining a diagnosis of PV.
 * Mainly:
 * (1) Too many red cells (RBC mass > 25% over mean normal predicted values).
 * (2) Without evident secondary causes (left-shifted oxygen dissociation curve, etc).
 * Secondarily:
 * Splenomegaly.
 * Most patients will have basophilia.
 * Thrombocytosis, high WBC, marrow biopsy with panmyelosis (general sign of CMPDs)
 * Almost always have no iron stores in the bone marrow.
 * Note that these abnormal cells don't need Epo to grow //in vitro// -- good diagnostic test. Look also for low serum Epo levels with high red cell counts in patients.
 * Note you don't need to know these lists off your head. But they should make sense.
 * Discuss what needs to be considered when evaluating a patient with thrombocytosis and entertaining a diagnosis of ET.
 * Again, some or all of the following:
 * Sustained platelet count > 600 x 109/L
 * Generally, no basophilia.
 * Marrow with predominantly megakaryocyte proliferation.
 * No evidence of PV (unelevated red count, existing iron stores in marrow), CML (no Philadelphia chromosome), CIMF (fibrosis in marrow), or myelodysplastic syndrome (no dysplasia in marrow).
 * No evidence of secondary causes: inflammation, infection, neoplasm, or splenectomy.
 * Describe what is typically found in the peripheral blood of a patient with chronic idiopathic myelofibrosis (CIMF). What other conditions cause this peripheral blood picture?
 * Smear: teardrop red cells (dacrocytes), nucleated red cells, immature lymphocytes. This is called a "leukoerythroblastic" smear if you want to get fancy.
 * Other conditions that can cause this: Same stuff that causes marrow fibrosis (next LO)-- metastatic carcinoma, infection, lymphoma, Hodgkin's disease, etc.
 * Discuss what needs to be considered when evaluating a patient with marrow fibrosis and entertaining a diagnosis of CIMF. Which other chronic myeloproliferative disorders progress to a stage that is essentially identical to CIMF?
 * Needs to be considered for CIMF:
 * Widespread splenomegaly and/or hepatomegaly.
 * Megakaryocytic hyperplasia in the marrow.
 * Extramedullary hematopoiesis.
 * Occasionally, basophilia/eosinophilia.
 * Fibrosis can also be caused by: metastatic carcinoma, infection, lymphoma, Hodgkin's disease, etc.
 * Discuss why chronic myelomonocytic leukemia (CMML) is defined as a myelodysplastic/myeloproliferative disease under the WHO classification system.
 * You see both dysplasia of non-monocyte precursors in the marrow (thus MDS) and monocytosis in the peripheral smear (thus MPD).
 * Discuss dysplasia in relation to hematopoiesis in the myelodysplastic syndromes.
 * In MDS:
 * **Dysplastic erythrocytes** :
 * Megaloblastic cells with weird-looking nuclei, **ring sideroblasts** (blue-staining iron accumulation in mitochondria in erythroid precursors/cells), misshapen/variously sized or chromic cells.
 * **Dysplastic granulocytes** :
 * Too few granules, too many or too few segments in nucleus, decreased blasts.
 * **Dysplastic megakaryocytes** :
 * Micromegakaryocytes (single and/or nonlobular nucleus) or megakaryocytes containing disconnected nuclei, too few granules in platelets in periphery.
 * Describe the evaluation of patient in whom myelodysplasia is suspected. What should be evaluated before a patient with anemia, neutropenia, and/or thrombocytopenia is subjected to a bone marrow biopsy? Does dysplasia of hematopoietic precursors and mature blood cells necessarily mean that the patient has a myelodysplastic syndrome? What special study or studies should be considered if a bone marrow biopsy is deemed necessary?
 * Evaluate, essentially, what other marrow-suppressive etiologies might be present:
 * Folate/B12 deficiencies (also megaloblastic, hypersegmented neutrophils)
 * Current meds (ie chemo drugs)
 * Infections (parvovirus, probably mononucleosis/CMV)
 * Toxins (arsenic)
 * Invasion of marrow by tumor
 * G-CSF (granulocyte colony-stimulating factor) administration
 * Also look at **cytogenetics** : specifically the deletion of chromosome 5q, but the more chromosomal abnormalities, generally the poorer the prognoses.
 * Look at blasts: the more blasts in blood or bone marrow, the more advanced the MDS. Under old system: 30% blast cutoff between MDS (< 30) and acute leukemia (> 30). Under new system: 20% blast cutoff between MDS and acute leukemia.
 * Look at different types of dysplasia: with more types of cell lineage involved in the dysplasia, the worse the prognosis.
 * Dysplasia does not necessarily mean anyone's got MDS.
 * Prognosis: look at #blasts, karyotypic abnormalities, #cell lineages involved in cytopenias. More, in all of the above, is bad.
 * Special studies: cytogenetic analysis, as mentioned above.
 * [Note that smoking, chemo, radiation, and various genetic syndromes can cause MDS.]
 * [The clinical symptoms of MDS are generally due to the underlying cytopenias. Eg.: ecchymoses from depressed thrombocytes levels.]
 * **Auer rods** : seen in smear: __abnormally fused primary granules__. Sign of acute myeloid leukemia or advanced MDS (see "Acute Leukemia").

20% blast content = cutoff between acute and chronic.

Basophilia: CML: Always (100%) PV: Mostly (66%) ET: Rarely (~0%) CIMF: Sometimes (30%)

Morphology of dysplasia in MDS

=Acute Leukemia=


 * Acute Leukemia, 2/26/08:**


 * [Notice, germane to this discussion, that a B cell is no longer classified as a "blast" once it's expressed surface antibody.]
 * Compare and contrast the way in which the WHO and FAB systems classify acute myelogenous leukemia (AML).
 * FAB:
 * Classification of AML:
 * Done by morphology and cytochemistry:
 * Morphology: if **Auer rods** are seen in the blasts, it's **AML** (granulated cells).
 * Cytochemistry:
 * Looking mainly with __stains__:
 * do the blasts contain **myeloperoxidase** ( **AML** indicator)?
 * do the blasts contain **non-specific esterase** (monocytic cell, thus **AML**, indicator)?
 * do the blasts contain chloracetate esterase (neutrophilic cell, thus AML, indicator)?
 * Can use the Periodic Acid-Schiff test: results in AML are diffuse and granular, results in ALL are clumpy.
 * (remember, old FAB "acute" cutoff is > 30% blasts, unlike WHO "acute," which is > 20% blasts)
 * Essentially there's a system of naming: "M" for myeloid, and a number from 0 to 7 that indicated what kind of cell it is. Without too much detail:
 * M0: undifferentiated myeloleukemias (found with anti-CD34)
 * M1, M2, M3: granulocyte blasts
 * M4: granulocyte and monocyte blasts
 * M5: mainly monocyte blasts
 * M6: erythrocyte blasts
 * M7: megakaryocyte blasts
 * WHO:
 * Based on morphology, immunophenotyping, clinical presentation, and genetic typing.
 * Immunophenotypying-- Antibodies vs:
 * Myeloperoxidase (AML)
 * CD34 (progenitor cell antigen, AML/ALL)
 * CD45 (AML/ALL)
 * CD14 (monocytic)
 * CD13 and CD33 (granculocytes and monocytes)
 * CD41, CD61, and CD42b (megkaryocytic antigens)
 * Classification of AML:
 * AML with recurrent cytogenetic findings (abnormalities)
 * AML with multilineage dysplasia (frequently preceded by MDS)
 * AML and myelodysplastic syndromes, therapy-related (caused by therapeutic agent)
 * "Uncategorized" AML (essentially FAB system)
 * Compare and contrast the way in which the WHO and FAB systems classify acute lymphocytic leukemia (ALL). Under the WHO system which type of ALL is most common in the United States?
 * Can have pre-T, pre-B, or pre-NK (last is very rare and undiscussed here) lymphomas.
 * FAB:
 * Classification of ALL (using mainly morphology):
 * Again the system is "L" for lymphocytic and a number that designates something about the blasts in question, in this case the morphology.
 * To remember here: L1 and L2 are pre-T and pre-B cells, while L3 is Burkitt's lymphoma cells (which are mature B cell lymphomas).
 * Note that, consistent with the idea that acute leukemias should really consist of blasts, Burkitt's lympomas isn't technically acute, it's a non-Hodgkin lymphoblastic leukemia (at least according to First Aid). That said, it's still worse than many acute leukemias.
 * WHO:
 * Classification of ALL:
 * ALLs arise from either precursor T-cells or precursor B-cells. The remaining question is how to discriminate between them.
 * Immunophenotypic markers:
 * CD34, CD45: any acute leukemia (blasts)
 * CDs 1, 2, 3, 5, 7: T cell antigens
 * CDs 19, 20, 22, 79a: B cell antigens
 * Note that cytogenetic analysis in ALL is often useful for prognosis.
 * WHO sez __pre-B lymphomas__ are by far the most common ALL (80-85%).
 * Explain the significance of cytogenetic findings in classifying AML under the WHO system. Be familiar with the four recurring cytogenetic abnormalities that define leukemia types.
 * Four recurring cytogenetic abnormalities:
 * Note that spe cifics are not that important here except as noted in the following LO.
 * t(8,21)(q22;q22): generally gives rise to AML-M2, often with eosinophilia.
 * inv(16)(p13;q22) or t(16;16)(p13;q22): always indicative of AML-M4, often with abnormally formed eosinophils, even if < 20% blasts.
 * t(15;17)(q22;q12): APL (acute promyelocytic leukemia) = AML-M3.
 * Often get DIC as complication.
 * Can treat with ATRA (trans-retinoic acid) and chemo.
 * 11q23 abnormalities: often found in AML-M4 and AML-M5.
 * Essentially, from the cytogenetic findings, you should be able to make some manner of prognosis or even start thinking about treatment options. Note that the first three listed above are associated with good prognoses.
 * [Multi-lineage myelodysplasias tend to evolve into poorer prognoses.]
 * Discuss acute promyelocytic leukemia in terms of its cytogenetic abnormality, special complications associated with the disease, its treatment, and its prognosis.
 * As mentioned, arises from t(15;17) as an abnormal retinoic acid receptor, is associated with DIC, is treated with ATRA (all-trans-retinoic acid), and generally has a favorable prognosis.
 * This is the one he mentioned that he wanted us to know in detail.
 * Describe the difference between precursor B or T-lymphoblastic leukemia and precursor B or T-lymphoblastic lymphoma.
 * As mentioned in "Chronic Lymphoid Leukemia," a lymphoblastic leukemia is one that's primarily found in the bone marrow or peripheral blood. A lymphoblastic lymphoma is one that's primarily found in the lymph nodes. Notice that there really doesn't seem to be much difference between them.
 * Define sanctuary site and the anatomic location of such sites.
 * Site at which leukemia can get into to avoid getting hit by chemotherapeutic agents. Mainly in CNS and testicles (blood-testes/brain barrier).
 * Be familiar with which type of acute leukemia is likely to affect children and which type is likely to affect adults.
 * AML is mainly found in adults.
 * ALL is mainly found in children:
 * Risk factors:
 * Between age 1 and 10
 * Nonwhite race
 * Male
 * CNS disease
 * Discuss CD34, CD10 (CALLA) and TdT in terms of their expression by normal cells and its expression by acute leukemia cells.
 * CD34: primitive stem-cell antigen (only found in blasts, but found in both myeloblasts and lymphoblasts).
 * TdT: expressed by T + B lymphoblasts.
 * CD10: usually made by B cell lymphomas. Also called CALLA.
 * Essentially you can use these to try and figure out what kind of leukemia you're looking at. TdT and CD10 positive, probably pre-B cell; TdT positive but CD10 negative, probably pre-T cell, etc.
 * Explain the significance of a leukemic blast being positive for myeloperoxidase. Explain the significance of a leukemic blast being positive for non-specific esterase.
 * Myeloperoxidase is only found in myeloblasts and some myelocytes. Thus a positive myeloperoxidase test unequivocally indicates **AML** and not ALL.
 * Non-specific esterases are found in monocytes. Thus a positive non-specific esterase test indicates monocytic cells in acute leukemia (thus **AML**, not ALL).

=Chronic Lymphoid Leukemia, Plasma Cell Neoplasms, and Hodgkin and Non-Hodgkin Lymphomas=


 * Chronic Lymphoid Leukemia, Plasma Cell Neoplasms, and Hodgkin and Non-Hodgkin Lymphomas, 2/28/08:**


 * [Note that Ryder seemed to use 'leukemia' and 'lymphoma' more or less interchangeably throughout his lecture. Possibly this is to make the point that they're more or less the same.]
 * [Notice genetic analysis is increasingly common to determine specific cancer type.]


 * Without attempting to memorize the entire classification system, be familiar with the types of data used by the WHO Classification system to define different types of lymphoma.
 * When I asked Ryder about this, he said, "indolent--aggressive--highly aggressive. Highly aggressive, it's Burkitt's or lymphoblastic lymphoma; aggressive, it's follicular large-cell lymphoma; indolent, it's everything else." Take with a grain of salt but there it is.
 * When I asked again, he clarified: "WHO can be broken into three easily remembered clinical categories: highly aggressive, aggressive, and indolent." Have to love it. One can only speculate that if I'd asked a third time he would have just grunted.
 * Define lymphadenopathy and give general disease categories that are associated with lymphadenopathy.
 * Lymphadenopathy- enlarged lymph nodes.
 * Associated with, basically, a mess of other junk: infectious diseases (viral, bacterial, fungal, parasitic, etc), immunologic diseases, malignancies (cancers), lipid storage diseases, hyperthyroidism, sarcoidosis, and a host of others, more or less none of which we've seen thus far.
 * Describe what is meant by “effacement of lymph node architecture”.
 * Effacement: basically it looks all messed up.
 * Note on follicular vs diffuse patterns: in follicular lymphomas, you see abnormal clusters of neoplastic lymphocytes inside the lymph node; in diffuse lymphomas, the neoplastic cells are found more or less throughout the entire space of the node. Either one can be called an "effaced" node.
 * Be able to define non-Hodgkin lymphoma in terms of the types of neoplastic cells involved.
 * **Types of non-Hodgkin lymphoma: T or B lymphocytes.**
 * Describe the type of pattern or patterns that may be observed in lymphomas of B-cell type and lymphomas of T-cell type. Relate the ability of T and B-cells to form pattern(s) to normal lymphoid architecture.
 * This has to do with the fact that lymphoma cells tend to mimic the distribution of their 'normal' counterparts, and gets back to the division between follicular and diffuse node patterns. B cells, recall, are found primarily in cortical follicles within lymph nodes; B-cell lymphomas are, similarly, found mainly in follicular patterns (though they can also show diffuse patterns, as described next) in nodes. T cells, by contrast, are generally not found in concentrated patterns within the node, and thus T-cell lymphomas are __never__ found in follicular patterns and always diffuse within the node.
 * Be able define indolent, aggressive and highly aggressive as these terms relate to lymphoma. Be familiar with the most common types of indolent and aggressive lymphomas and the three types of highly aggressive lymphoma.
 * Indolent: slowly growing, slowly spreading. Most lymphomas. Survival measured in years if untreated.
 * Aggressive: growing more rapidly. Mostly follicular large-cell lymphomas. Survival measured in months if untreated.
 * Highly aggressive: growing like a son of a bitch. Burkitt's and lymphoblastic lymphomas. Survival measured in weeks if untreated.
 * [Small cells are generally better-behaved than big cells. Exception is Burkitt's lymphoma, which has medium-sized cells and behaves worse than either of them.]
 * Be familiar with the common types of lymphoma in adults and children.
 * B-cell Chronic Lymphocytic Leukemia or (B-CLL):
 * By far __the most common chronic lymphoid leukemia__.
 * The affected cells are evidently related to naïve, but mature, B cells. They show up as small and round (as opposed to prolymphocytes, see below).
 * Presents primarily in older age (> 50).
 * Increased absolute lymphocyte counts (10,000-100,000/mcL), often mild anemia, sometimes thrombocytopenia.
 * Can progress (generally to prolymphocytic leukemia, below), but is well treatable before that.
 * Prolymphocytic Leukemia:
 * Prolymphocytes: larger than B-CLL cells and ovaloid.
 * Mostly a more severe, progressed form of B-CLL; occasionally //de novo// . More aggressive; median survival time of 3 years.
 * Hairy-Cell Leukemia:
 * Rarer; have cells that look "hairy" in the peripheral blood.
 * Is well treatable.
 * Be able to describe the difference (or lack thereof) between a leukemia and lymphoma when the same neoplastic lymphoid cell is involved.
 * If the lymphoproliferative disorder presents in the blood or marrow, you call it a leukemia. If it presents in the lymph nodes, you call it a lymphoma. They are effectively the same thing named two different ways.
 * Be able to describe the difference between nodular lymphocyte predominant Hodgkin lymphoma and classical Hodgkin lymphoma.
 * Classical Hodgkin lymphoma cells have a distinctive immunophenotype: CD15+, CD30+, CD45-, negative for T- and B-cell antigens. These __are__ Reed-Sternberg cells.
 * Nodular lymphocyte predominant Hodgkin leukemia cells don't have as specific an immunophenotype. Positive for B-cell antigen (CD20), positive for leukocyte common antigen CD45, negative for CD15 and CD30. These __are not__ Reed-Sternberg cells (they're "L-H cells" or "popcorns cells").
 * One way of thinking about this:
 * Classic = Reed-Sternberg: CD15+, CD30+, CD45-
 * Nodular = Popcorn cells: CD15-, CD30-, CD45+
 * Be able to describe and recognize a classic Reed-Sternberg cell.
 * Reed-Sternberg cells are the malignant cells in classic Hodgkin lymphomas.
 * They're actually fairly rare in the involved lymph nodes. They tend to be surrounded by inflammatory cells.
 * Ryder: "..the Reed-Sternberg cell is some sort of spazzed-out B cell."
 * Diagnostically: has a __bilobed-nucleus__ (classically the nucleus looks like "owl's eyes") with large eosinophilic nucleoli.
 * Describe the features of people who are at greatest risk of being diagnosed with Hodgkin lymphoma.
 * Young adults; men more than women, some association with mononucleosis and EBV.
 * Present with painless lymphadenopathy of the cervical nodes; sometimes also mediastinal, axillary, or para-aortic lymphadenopathy.
 * More advanced symptoms ("**B symptoms** "): __fever, night sweats, weight loss__.
 * Sometimes: pruritis, alcohol-induced pain in the lymph nodes.
 * [Generally good cure rate for Hodgkin lymphoma (50% 5-year survival even when there's metastasis outside the lymph nodes).]
 * Describe the usual location of tumor in plasma cell myeloma, and features of the neoplastic cell in plasma cell myeloma and how these relate to presenting laboratory and radiographic findings.
 * Note plasma cell myeloma is often called __multiple myeloma__ (old terminology).
 * Usually found in the bone marrow.
 * The neoplastic cells are plasma cells; they're producing monoclonal proteins, often monoclonal antibodies but also antibody fragments (free light chain proteins are called **Bence Jones proteins** ).
 * Describe typical laboratory and radiographic findings in a patient presenting with plasma cell myeloma.
 * Labs:
 * Nearly all plasma cell myeloma patients have monoclonal protein in the serum/urine.
 * Note that most patients will have Bence Jones proteins (light chains) in the urine.
 * As mentioned, mostly you also find antibodies, but in about 15% of cases, it's Bence Jones only.
 * Of the remaining 85%, 50% is IgG, 20% is IgA, 2% is IgD. Rarely (1%), it's two immunoproteins instead of one (not monoclonal but biclonal).
 * Normal serum antibodies are depressed.
 * Most plasma cell myeloma patients show moderate or severe normochromic, normocytic anemia.
 * Increased serum calcium, renal dysfunction (secondary to increased serum calcium and Bence Jones protein, which is nephrotoxic).
 * "Varying atypia." They look wonky. The plasma cell, not (necessarily) the patients.
 * Radiographic:
 * Osteoclasts are abnormally activated, leading to bone breakdown and the increased serum calcium.
 * Explain what is meant by the terms MGUS, smoldering myeloma and indolent myeloma.
 * **MGUS** : Monoclonal gammopathy of unknown significance. Asymptomatic patient with monoclonal antibody but can't find the causative cells.
 * Smoldering/indolent myeloma: Asymptomatic patient with lab findings characteristic of, but not dire enough to diagnose, plasma-cell myeloma.
 * [Note on Burkitt's from First Aid: shows a classic "starry night" pathology presentation with monocytes surrounded by tons of B cells; results from translocation of c-myc oncogene right next to the heavy chain gene.]

Highly aggressive: Burkitt's, lymphoblastic Aggressive: large-cell follicular Indolent: everything else.

Prolymphocytic: more advanced B-CLL

=New Directions In Immunology=


 * Describe an experiment that shows that immune responses might be “conditioned” in a Pavlovian sense.
 * Ader:
 * Take mice genetically predisposed to get lupus (100% of females will die of glomerulonephritis in first year). If given an immunosuppressive drug every month, they will all survive their first year. If treated only every other month, all of them die in a year.
 * First month, saccharine in drinking water and immunosuppressive; second month, just saccharine; alternate like that for a year. Thus were getting effectively a once every other month treatment, which normally results in all of them dying in a year.
 * The mice all survived as if they were being treated monthly.
 * Describe in principle the procedure for making a monoclonal antibody. Discuss advantages and disadvantages compared to a conventional antiserum.
 * Describe in principle the way in which a person could be induced to make an auto-antiidiotypic response. Suggest a use for such a procedure.
 * Culture a one-way MLR between two people's blood (allow host's T cells to react against donor's cells). The cells that proliferate are the host T cells which react against the donor cells. Harvest activated cells (which should be larger than unactivated cells). Now we have a bunch of host anti-donor cells. Stop their growth, add a good immune-stimulating adjuvant; immunize host with these host anti-donor cells. Donor makes an antibody response against these-- but these antibodies should also kill the cells that recognize the donor as foreign.
 * Ok, I still don't understand this.
 * Define superantigens, and show diagrammatically how they work to stimulate T cells.
 * Bind to certain families of T cell TCRs and also class II MHC proteins; this brings the two into such close proximity that the T cell becomes activated, thinking it's recognizing antigen.
 * This is bad news because if you activate even 3% of your given Th cells are activated at once (and superantigens tend to vary between 2-20%), IL-2 and IFN-g release ensues all over; coordinated IL-2 release causes intractable shock due to enormously increased vascular permeability.
 * This is bad news because if you activate even 3% of your given Th cells are activated at once (and superantigens tend to vary between 2-20%), IL-2 and IFN-g release ensues all over; coordinated IL-2 release causes intractable shock due to enormously increased vascular permeability.