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128 Cards in this Set

  • Front
  • Back
Classical Aplastic Anemia
- Destruction of pluripotent stem cells (CD34+ cells).
- 50% idiopathic cause; other causes include: radiation, chemo drugs, chloramphenicol (antibiotic for typhoid), benzene, infections (hep. c, parvo, epstein-barr, HIV), genetic (fanconi's aplastic anemia [dwarfism, other developmental disorders)
- Leads to a decrease in all blood lineages. (RBCs, platelets, neutrophils)
- BM biopsy shows dry bone marrow.
TREATMENT: whole blood/platelet transfusions, antibiotics, immunosuppressants (ALG, ATG, cycolsporin A) if autoimmune mechanism is causing stem cell destruction, growth factors (EPO, G-CSF, GM-CSF)
BM TRANSPLANT
Interleukin 3
- Source: T-cells
- Supports growth of stem cells and committed precursors
RGD sequence
- Tripeptide sequence which is recognized by RGD-receptors on immature cells.
- As the cells mature, the RGD receptors on the cells are downregulated, and the cells are released into the blood stream.
- Present on ahdesive BM proteins: fibronection, laminin, vitronectin, collagen, thrombospondin, fibrinogen, and vWF factor
Myelodysplastic syndrome
Clonal population of pluripotent stem cells (usually CFU-GEMM) arising from cytogenetically flawed and kinetically advantaged ancestor, takes over the BM
- SC numbers increase (due to proliferation of abnormal SC's)
- BM tap = normocellular, or hypercellular
- Neutropenia, thrombocytopenia
- Many result in Chronic Myeloid Leukemia
- TREATMENT: transfusion, recombinant GF's, antibiotics
Infiltrative Myelopathy
Tumor metastasis from other sites (breast, prostate, lung) infiltrate and take over the BM, crowing out the hematopoeitic cells.
- BM Tap = hypercellular w/ malignant cells.
- TREATMENT: treat the malignancy.
Chronic Myeloid Leukemia
Neoplastic pluripotent SC partially blocked in ability to differeniated into lymphocytes.
- Increase in myeloid cells (granulocytes, monocytes, basophils, eosinophils).
- Result of Philadelphia Chromosome (9q34 to 22q11).
EPO
EPO is synthesized in response to decreased oxygenation; it stimulated erythropoeisis
- New RBCs appear in the blood five days later.
- EPO stimulates the productio nof proerythroblasts from stem cells, and speeds up subsequent steps of differentiation
- When both kidneys fail, the patient becomes anemic b/c the 10% EPO production in the liver is not sufficient.
- Enhances CFU-GEMM to BFU-E step, BFU-E to CFU-E step, and CFU-E to proerythroblast step.
Ferrireductase
On the intestinal epithelium, reduces ferric to ferrous, which is then taken through the transporter protein -- DMT1.
DMT1 (divalent metal transporter 1)
Transports ferrous into the intestinal epithelium.
HCP1
Transports heme containing iron into intestinal epithelium.
Ferritin
Iron that is stored in tissues is complexed with apoferritin to form ferritin. When iron levels fall, it is released from ferritin.
Ferroportin
Iron is transported into the circulation from enterocytes through this transporter.
Hemosiderin
SMALL, insoluble quantities of stored iron.
Transferrin
Serum protein to which iron is bound in the blood. The Iron-Tf complex travels to tissues.
Antacids, phytates (from bran), tannins, antibiotics (tetracyclines, ciprofloxacin)
Reduce iron uptake
Total Iron Binding Capacity (TIBC)
- TIBC is larger in Iron deficiency
- TIBC is smaller in Iron overload.
Iron Deficiency Anemia
Under pressure from EPO (because of reduced oxygen uptake), the BM makes MICROCYTIC (small), HYPOCHROMIC (pale) RBCs.

CAUSES:
-Diet poor in Iron
-Pregnancy
-Growth spurts
-Chronic bleeding (peptic ulcer, menstruation)
-GI abnormalities (especially near duodenum)
-Hook worm (suck blood from the GI tract)
PRESENTS WITH:
-Fatigue
-Pallor
-Tachycardia
-Alterations in epithelial tissue (spoon-shaped nails, glossitis [sore or swollen tongue], gastric atrophy)
-Black stool (melena) in peptic ulcer patients

TESTS TO DIAGNOSE IDA:
-Blood smear to detect microcytic, hypochromic RBCs
-Mean Corpuscular Volume (normal is 80-96 femtoliters)
-MCH and MCHC are also decreased
-Hematocrit decreased
-Low serum iron (high TIBC)
-Free Erythrocyte protoporphyrin assay (FEP): In IDA, protoporphyrin IX, an intermediate in heme synthesis, accumulates in RBCs because there is insufficient iron to convert it to heme. FEP IS NORMAL IN THALASSEMIA -- A WAY TO DISTINGUISH IDA AND THALASSEMIA (especially because a blood smear will look similar for the two).
-Low ferritin, and low BM iron.
Mean Corpuscular Volume
Decreased in IDV

- MCV is the average volume of a RBC.
Mean Corpuscular Hemoglobin
Reduced in IDA

- Amount of hemoglobin in a single RBC.
Mean Corpuscular Hemoglobin Concentration (MCHC)
MCHC = MCH/MCV x100
Role of Vitamin B12 and Folic Acid in erythropoeisis
These two vitamins are essential for the final maturation of RBCs, because they are required for DNA synthesis (each in a different way being required for synthesis of thymidine triphosphate, a DNA building block)
- Lack of either vitamin causes diminished DNA synthesis, and consequently failure of nuclear maturation and division.
- As a result, erythroblasts, in addition to failing to proliferate, produce larger than normal, oval shapped "macrocytes."
- While these can carry oxygen normally, they are extremely fragile and fail to survive for extended periods of time.
- NEUTROPHILS AND PLATELETS ALSO SHOW MATURATION DEFECTS IN VIT. B12 and FOLIC ACID DEFICIENCY.
R Binder
Forms a stable complex with VitB12 in the stomach during digestion. Synthesized in the SALIVARY GLAND.
Intrinsic Factor
After the VitB12-R-binder complex is digested upon entering the duodenum, VitB12 forms a complex with IF -- a protein made by the PARIETAL CELLS of the stomach.
-This complex is resistant to proteolytic destruction and travels to the distal ileum where specific receptors on the mucosa bind the complex, which is taken into the mucosal cell. (IF is destroyed here)
Transcobalamin I and II
Transports VitB12 to the tissues. Cleared in ~1 hr.
TCII deficiency leads to sever megaloblastic anemia in early childhood.
Deoxyadenosylcobalamin
One of the two forms of Vitamin B12

-Converts Methylmalonyl CoA to Succinyl CoA (required for heme biosynthesis, and a TCA intermediate)
Methylcobalamin
Cofactor for methyltransferase in conversion of Homocysteine + N5-Methyltetrahydrofolate -> Methionine + THF. (The methyl from N5-MeTHF is transfered to homocysteine).
S-Adenosylmethionine (SAM)
Produced from ATP + Methionine.

-Methylates DNA, histidine residue in myelin basic protein of CNS, and phosphatidylethanolamine to form phosphatidylcholine (required for cell membrane formation).
Polyglutamate
Conjugates THF to create a form of folate that is retained in the tissues.
Vitamin B12 Deficiency (Cobalamin Deficiency)
- Homocysteine levels rise, which is an independent risk factor for heart disease and stroke.
- N5-MeTHF is not converted adequately to THF, the form that can be conjugated to polyglutamate. There is leakage of folate out of the cell, leading to deficiency of tissue folate.
- Methylmalonyl Coa levels rise.
- DEMYELINATION:
Decresed SAM leads to decreased methylation of a critical histidine residue in myelin basic protein (and also to decreased phosphatidylcholine); affects mostly proprioception and spasticity; PATIENTS COMPLAIN OF TINGLING and NUMBNESS IN FEET AND FINGERS, ALTERED GAIT, LOSS OF POSITION SENSE, BLINDNESS DUE TO OPTIC ATROPHY, PSYCHIATRIC DISTURBANCES.
- GI TRACT:
Nausea, weight loss, constipation, loss of appetite, beefy red tongue.
CAUSES
- Pernicious Anemia (most common cause of megaloblastic anemia in US): Autoimmune disease; anti-parietal autoantibodies (against proton pump) causes gastric atrophy; Consequently, IF production by the stomach is decreased. Lack of IF results in decreased absorption of cobalamin from BI tract.
- Gastric atrophy can also result from resection
- Fish Tapeworm: Accumulates cobalamin
- Failure of absorption from ileum: following resection, enteritis, strictures
- Poor Diet: Vegans (rare)

TREATMENT:
Parenteral administration of cobalamin -- correction of megaloblastic changes within 24-48 hours. Neurological manifestations may be partially corrected or not at all.
Folate adminstration -- may partially correct hematological abnormalities, because decreased tissue folate contributes to these changes. Folate will NOT stop neurological complications.
Megaloblastic Anemia
Results from deficiency of VitB12 and Folate.

- Leads to decreased thymidine synthesis, and consequently failure of nuclear maturation and division.

- RBCs:
Large RBCs (macrocytic anemia); increased MCV to 100-150 femtoliters.
- WBCs:
Hypersegmented neutrophils contain 5 or more lobes (usually 3-4)
- Platelets:
Decreased in number
- Marrow:
Packed with blood cell precursors.
Schilling Test
Medical investigation used to test for Vitamin B12 absorption deficiency.

- Radioactively labeled cobalamin given orally.
- Unlabeled cobalamin given intramuscularly to inhibit uptake of radioactive cobalamin by liver.
- Urinary secretion of radioactively labeled cobalamin measured to estimate whether orally administered cobalamin has been taken up.

- LOW SECRETION IN URINE SUGGESTS ABNORMALITY (eg. Pernicious Anemia) THAT PREVENTED UPTAKE OF COBALAMIN FROM GI TRACT.
Folic Acid Structure
Consists of three parts:
- Pteridine (nitrogen containing ring system)
- p-aminobenzoic acid (another ring)
- Glutamic Acid (aka glutamate) residues, which prevent the vitamin from leaking out of the cell

- The vitamin is inert

Active THF is produced by the reduction of DHF by DHF Reductase. THF has hydrogens on carbons 5-8 on the pteridine residue, while DHF has hydrogens on 7 and 8.
Methotrexate
A drug used in cancer therapy that inhibits DHF Reductase and causes a build up of DHF, and a reduction of THF (which, of course, is required for thymidine synthesis)

- LEUCOVORIN (5-formylTHF) can be administered to patients receiving methotrexate if they develop megaloblastic anemia.
Secondary Polycythemia
Secondary to CARDIAC FAILURE or HIGH AFFINITY HEMOGLOBINS

- EPO level is HIGH
- Red cell mass is high

(Physiologic Polycythemia: people living at altitudes of 14,000 to 17,000 feet - chronic tissue hypoxia leads to increased RBC levels)
Polycythemia Vera
MALIGNANT CONDITION

- Large number of RBCs are produced
- Hematocrit is high (65% compared to 45%)
- Red cell mass is HIGH
- Increased blood viscosity (leading to blindness and/or kidney failure)
- EPO level is LOW
- Increased platelets
NORMAL RANGE OF RED CELL VALUES IN ADULTS:

RBC Count
M - 4.5 to 5.9 (x 10^6/microliters)

F - 4.1 to 5.1
NORMAL RANGE OF RED CELL VALUES IN ADULTS:

Hemoglobin
M: 14.2 - 16.9 (g/dL)

F: 12.2 - 15
NORMAL RANGE OF RED CELL VALUES IN ADULTS

MCV
M: 83-99 (fL)

F: 83-99
NORMAL RANGE OF RED CELL VALUES IN ADULTS:

MCH
M: 28-32 (pg)

F: 28-32
NORMAL RANGE OF RED CELL VALUES IN ADULTS

MCHC
M: 32-36 (g/dL)

F: 32-36
NORMAL RANGE OF RED CELL VALUES IN ADULTS

Hematocrit
M: 42-49 %

f: 38-46 %
NORMAL RANGE OF RED CELL VALUES IN ADULTS

Reticulocyte
M: 0.5 - 1.8 %

F: 0.5 - 2.2 %
Howell-Jolly Bodies
Fragments of nuclei left in RBCs.

- The cells are unable to change shape, and are trapped in the spleen
- More Howell-Jolly bodies are found in blood cells following spleen removal.
Reticulocytes
Following denucleation, cells down-regulate RGD receptors and escape the BM.

- Reticulocytes stay in the blood for 1 day before maturing to RBCs.
- They contain cytoplasmic RNA and therefore stain with SUPRAVITAL DYES; the stained RNA precipitates give rise to a dark blue reticulin network (hence the name)

- NORMAL RETICULOCYTE COUNT:
0.5-1.8%
Reticulocyte Index
The rate of RBC production can be determined by the RETICULOCYTE INDEX:
= retic. % x (patient's hematocrit/ normal hematocrit)

RETICULOCYTOSIS: Increased reticulocytes as a result of increased hematopoeisis. Clinically important measure. Following a bleed or following increased destruction of RBCs, compensatory erythropoeisis in the BM results in increased reticulocytes in the blood.

DECREASED RETIC. COUNT:
Anemias associated with impaired hematopoeisis (aplastic anemia) are associated with decreased retic. count
RBC Migration through the circulation
- RBCs (the densest cells) enter the fast moving MID-AXIAL STREAM.
- Lighter lymphocytes move more peripherally
- Monocytes, neutrophils, platelets (lightest cells) tumble along the PLASMA-RICH MARGINAL STREAM
Rouleaux Formation
RBCs are forced into the mid-axial stream through a stacking mechanism called ROULEAUX FORMATION.

- In the presence of large linear molecules like FIBRINOGEN, RBCs stack up like coins, held together by weak surface-binding bonds.

- As the caps narrow, the stacked RBCs take on an umbrella shape, which allows for easier movement and a tight fit in the capillaries -- optimal for oxygen / CO2 exchange.
RBC Cytoskeleton
SPECTRIN: Most abundant protein; noodle shaped, comprised of alpha and beta chains.
- Interlinked by ANKYRIN, ACTIN, BAND 4.1

BAND 3: is an ANION CHANNEL deployed at frequent intervals in the membrane and is attached to ANKYRIN.
Hereditary Spherocytosis
Auto-hemolytic disease characterized by spherical RBCs (as opposed to biconcave RBCs)

- Abnormal RBC shape is a result of mutations in ANKYRIN (most common), SPECTRIN, or BAND 4.1

- The shape of change is so mild that the migration of RBCs through the circulation is impaired only in the SPLENIC SINUSES. In the spleen, the spherical RBCs breakdown and are engulfed by SPLENIC MACROPHAGES.

CLINICAL INDICATORS:
signs of chronic hemolytic anemia and signs of compensatory hematopoeisis.

TREATMENT: Splenectomy "cures" the disease.
Extravascular Destruction of RBCs
RBCs are estimated to live about 120 days. By about day 100, RBCs begin to show signs of senescence (including slowing down of glycolysis, decline in ATP levels, and marked change in shape - stiffening of cytoskeleton)

- Worn out RBCs are engulfed and destroyed by macrophages that line the liver sinusoids and splenic sinuses.
- 90% of RBCs are destroyed in this way.
- The globin chains and iron is stripped away from Hb; HEME is degraded to a toxic product called BILIRUBIN, which is excreted in the liver; IRON is complexed to ferritin or transferrin.
HAPTOGLOBIN and HEMOPEXIN
10% of RBCs die in the bloodstream.

HEMOGLOBIN released into the plasma is bound to a protein called HAPTOGLOBIN, and the HB-HAPTOGLOBIN complex is cleared by the the liver.

HEME released into the plasma is bound to a protein called HEMOPEXIN, and the HEME-HEMOPEXIN complex is cleared by the liver.
Hemolysis
Premature death, shortened survival of RBCs; leads to HEMOLYTIC ANEMIA.

- HEMOGLOBINEMIA: Free hemoglobin in the blood, once haptoglobin levels are depleted.
- HEMOGLOBINURIA: Free Hb is reabsorbed by the proximal tubule, but once this capacity is overwhelmed, free Hb appears in the urine. (You end up losing IRON)

JAUNICE: Increased extravascular breakdown of RBCs releases heme, which gets converted to bilirubin, which is cleared by the liver. However, if hemolysis is severe and prolonged, bilirubin accumulates in tissues resulting in jaundice (and/or gall stones).

LIVER and SPLEEN can enlarge in chronic hemolytic anemia because of increased extravascular destruction of RBCs and ALSO because th liver and spleen become sites of hematopoeisis (outside the BM).

BONES: In young children, extra hematopoeisis in the BM causes the long bones to become soft -- bow-leggedness and skull enlargement can result.
Osmotic Fragility test for Hereditary Spherocytosis
- RBCs are incubated in HYPOTONIC solutions (lower concentration of solute in relation to the inside of the cell) at various concentrations.

- RBCs imbibe water, swell and then burst

- Because of their biconcave shape, normal RBCs are able to handle lower osmolarities than the spherical RBCs of hereditary spherocytosis.

HS RBCs EXHIBIT INCREASED OSMOTIC FRAGILITY.
Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency
- Most common enzyme deficiency in RBCs.

- Main function of G6PD is to provide NADPH to protect RBCs from OXIDATIVE STRESS (H2O2).

- G6PD is on the X-chromosome, and is an x-linked disease.

- Hemolysis precipitated by oxidative stress during:
INFECTIONS, FAVISM (eating fava beans), ANTI-MALARIAL DRUGS (soldiers in Vietnam), SULFONAMIDES.

HEINZ BODIES: Intracellular precipitates of denatured Hb become visible when RBCs are stained with crystal violet -- seen in G6PD deficiency.
HEMOGLOBIN:

alpha chain family genes
zeta, alpha1, alpha2

on CHROMOSOME 16
HEMOGLOBIN:

beta chain family genes
epsilon, gammaG, gammaA, delta, beta

on CHROMOSOME 11
Embryonic Hemoglobin
[zeta]2, [epsilon]2
Fetal Hemoglobin
[alpha]2, [gamma]2

alpha can be 1 or 2
gamma can be G or A
Adult Hemoglobin

AND Adult Hemoglobin2
[alpha]2, [beta]2
* - commonest form (can be alpha 1 or 2)

[alpha]2, [delta]2
Hb Barts
[gamma]4

- Abnormal Hb due to loss of alpha genes in alpha-thalassemia.
- Instead of forming fetal Hb in utero, the absence of alpha chains results in free gamma chains that form a gamma-chain tetramer.
- [gamma]4 BINDS OXYGEN TIGHTLY AND DOESN'T RELEASE IT EASILY IN TISSUES - contributes to HYPOXIA
HbH
[beta]4

- Abnormal Hb due to loss of alpha genes in alpha-thalassemia.
- Instead of forming adult hemoglobin, the absence of alpha chains results in free beta chains that form a beta-tetramer.
- Susceptible to OXYGEN DENATURATION.
Hb Lepore
[alpha]2, [delta-beta fusion protein]2.

- Abnormal Hb due to loss of delta/beta genes in beta thalassemia. Cross-over function of delta and beta genes results in a NON-FUNCTIONAL hybrid protein in which the amino end of the delta chain is fused to the carboxyl end of the beta chain.
Intramedullary Hemolysis
Normally, both globin chains are synthesized coordinately; but, if synthesis is unbalanced, the total hemoglobin in RBCs may decrease to concentrations inadequate for survival. THEREFORE, MANY ERYTHROBLASTS DIE in the BM in THALASSEMIA - a process called INTRAMEDULLARY HEMOLYSIS.

Cells that escape the BM are destroyed by macrophages in the spleen.
Beta Thalassemia Major
- Anemia is severe, which places a severe burden on the heart that patients may develop congestive heart failure.

- The fetus is fine during the stages when embryonic and fetal Hb are the main oxygen carriers. (As HbF disappears, the first signs of anemia appear)
- THE RBCs are VERY SMALL and PALE - MICROCYTIC HYPOCHROMIC and low MCV - similar to what is seen in iron deficient anemia.

- To compensate for inefficient erythropoeisis, additional sites of BM are stimulated. The extensively hyperplastic BM fills and expands. (This is strikingly evident on x-rays, which show distended bones criss-crossed with hyperplastic trabaculae) The long bones become filled with BM (and they become bowed when the infant begins to walk), and the BM in the skull gives the child a chipmunk-like face.
- LIVER and SPLEEN enlarge.
- FATAL IF LEFT UNTREATED

Look at Page 13 of blood lecture 2 for quick rundown of symptoms.
Beta Thalassemia Minor
HETEROZYGOTE, one normal beta gene and one defective beta gene.

- Mild to moderate anemia with microcytic hypochromic RBCs, and low MCV.
- The anemia does not respond to iron therapy (unlike IDA).
- Furthermore, free erythrocyte protoporphyrin (FEP) is normal in thalassemia, whereas in IDA it accumulates in RBCs because there is insufficient iron to convert protoporphyrin to heme.
- Bilirubin might be increased because of extravascular destruction and the conversion of heme to bilirubin.
Alpha Thalassemia Minor
Deletion of 2 of 4 genes.
- In utero, HbF is reduced, and there is HB Barts ([gamma]4) in 2-10% of patients.
- After birth, HbA and HbA2 is reduced.
- MILD HEMOLYTIC ANEMIA
HbH Disease
An alpha thalassemia disease

DELETION OF 3 of 4 genes.

- In utero, the fetus is normal for the first 10 weeks since embryonic Hb production is fine.
- When the switch is made to to HbF, the lack of alpha chains cause gamma chains to tetramerize (causing Hb Barts), which has high oxygen affinity and therefore does not release oxygen to tissues.
- When the switch is made to adult Hb, the lack of alpha chains cause beta chains to tetramerize (HbH).
- HbH is sensitive to oxidative denaturation, which causes precipitates to be seen in RBCs as fine granular inclusions following staining with brilliant cresyl blue or cresyl violet.
- Moderate to severe microcytic hypochromic anemia, signs of hemolysis, signs of compensatory hematopoiesis.
-MEASURED BY ELECTROPHORESIS.
- TREATMENT:
BM Transplant
Hydrops Fetalis
Absence of all four alpha genes leads to fatal anemia in utero.
Sickle Cell Mutation
Point mutation in BETA GLOBIN gene.

- Negatively charged GLUTAMIC ACID is replaced by hydrophobic VALINE.
- Mutant Hb is sticky and forms polymers (deforming RBC and causing sickling).
Sickle Cell Pathophysiology
- The beta6 GLU>VAL mutation permits intermolecular bonding between adjacent HbS molecules in the deoxy state.
- The HbS molecules polyermize in the deoxy state and thereby cause the RBCs to take on a sickled state.
- LOSS OF POTASSIUM IONS through the KCa3.1 channel and the CORRESPONDING LOSS OF WATER causes the RBC to shrink, further aggravating sickling.

- THE SICKLED CELLS IMPEDE THE BLOOD FLOW THROUGH CAPILLARIES LEADING TO TISSUE INFARCTION, PAIN AND SEVERE TISSUE DAMAGE.
-In the late stages, the spleen shrinks due to repeated infarctions (autosplenectomy).

TREATMENT:
- Hydroxyurea is given to patients to PROMOTE THE FORMATION OF HbF (which inhibits the formation of HbS polymers).
- KCa3.1 channel blockers prevent erythrocyte dehydration.
Sickle Cell Trait
- Describes condition where only one of the beta genes contains the mutation:
IN OTHER WORDS, EACH RBC CONTAINS HbA and HbS, which is called HbAS.
- HbA = 60%, HbS = 40% (because alpha chains bind more effectively to beta chains without the mutation).

- RBC number and RBC morphology is normal EXCEPT when oxygen saturation falls below 40%.
- HbAS RBCs sickle only when oxygen saturation falls below 40%. Strenuous exercise or low pressurized planes or hiking at high altitudes can cause sickling of HbAS RBCs.

-SCREENING TEST:
Sodium Metabisulfite (a powerful reducing agent) is added to the patient's RBCs in saline - and HbAS RBCs will sickle.
- HEMOGLOBIN ELECTROPHORESIS can also confirm mutated beta chains.
Sodium Metabisulfite
Powerful reducing agent that is used to test for Sickle Cell trait.
Sickle Cell Disease
AUTOSOMAL RECESSIVE
- HbSS

- Sickled RBCs have shortened survival time mainly due to clearance by macrophages in the spleen -- resulting in CHRONIC HEMOLYTIC ANEMIA.
- Results in clinical signs of hemolysis and compensatory hematopoiesis.

- MICROVASCULAR OCCLUSIONS:
Responsible for most of the clinical features. Can affect any organ.
- Acute epsiodes of pain in the abdomen occur.
- Blood vessels to bones are blocked resulting in their destruction.
- Chronic osteomyelitis (INFECTION OF THE BONE) secondary to the poor blood flow to bones develops.
- Microvascular occlusion in the brain causes strokes, in the retina causes blindness, in the placenta causes fetal loss.
HEME BIOSYNTHESIS
Step 1: THE BUILDING BLOCK
- Glycine and succinyl CoA are catalyzed to form aminolevulinic acid (ALA). Pyridoxal phosphate (VITAMIN B6) is required. This OCCURS IN THE MITOCHONDRIA.

Step 2: THE FIRST TETRAPYRROLE
- This is synthesized in two steps.
- A) Condensation of 2 molecules of ALA to form MONOPYRROLE PORPHOBILINOGEN (PBG), catalyzed by PBG Synthetase.
B) 4 PBGs are condensed into a TETRAPYRROLE catalyzed by the enzyme PBG Deaminase. This OCCURS IN THE CYTOSOL.

Step 3: FORMATION OF HEME
- Heme is produced by three successive modifications of the tetrapyrrole followed by insertion of an IRON atam.
- This OCCURS IN THE MITOCHONDRIA, and Urogen Decarboxylase is the key enzyme in this stage.
- 4 HEMES combine with four GLOBIN CHAINS to form HEMOGLOBIN.
Porphyria:

EARLY STAGE BLOCK
(aka Acute Intermittent Porphyria)
Any block downstream of PBG causes an elevation of PBG and ALA; PBG acts like a NEUROTRANSMITTER, and ALA is NEUROTOXIC

- Stress in such patients (barbituates, menstrual period, infections, acidosis) causes PBG and ALA to accumulate. OFTEN A RESULT OF DECREASED PBG DEAMINASE.
- PSYCHOSIS and CONVULSIONS, INSTABILITY of TEMPERATURE CONTROL (brain stem), PERIPHERAL NEUROPATHY.
- ABDOMINAL and PELVIC PAIN, RESPIRATORY PARALYSIS.

- Diagnosis is made by detecting ALA and PBG levels in plasma and urine, and by the absence of tetrapyrroles in urine and stool.
TREATMENT:
- Avoid drugs that precipitate disease.
- Treat infections
- HIGH CARB DIET to PREVENT ACIDOSIS
- Intravenous heme injection in acute disease
Porphyria:

LATE STAGE BLOCK
(aka Porphyria Cutaneous Tarda)
Dermatological symptoms predominate.

Any block downstream to tetrapyrroles causes and elevation in URO III and other tetrapyrroles. OFTEN THE RESULT OF DECREASED UROGEN DECARBOXYLASE.
- These are deposited in the skin and cause photosensitivity and dermatitis.
- Painful "bullae" in the skin, scarring, irregular pigmentation.
- When urine is left to stand, the tetrapyrroles in the urine are oxidized and give rise to a DEEP RED COLOR.
Percent composition in blood:

Mature neutrophils
40 - 70%
Percent composition in blood:

Basophils
0.4 - 0.5%
Percent composition in blood:

Monocytes
4 - 5%
Percent composition in blood:

Lymphocytes
25 - 45%
Percent composition in blood:

Eosinophils
2 - 3%
Percent composition in blood:

Immature neutrophils (band forms)
~3%
Leukocytosis, Lymphocytosis, Monocytosis

Neutrophilia, Eosinophilia, Basophilia
Increased _______ in the blood.
Neutropenia, Lymphopenia
Decreased _______ in the blood.
Leukemoid reaction
Persistent NEUTROPHILIA (30,000 to 50,000/microliter).

Appears like leukemia (in sheer number of cells), but the cells look normal on a smear (unlike leukemia).
Neutrophils
NUMBER: 60% of white blood cells (~4300 cells/microliter)
- Neutrophilia = >10,000
- Neutropenia = <1500
* - You should get a temporary neutrophilia when you get a bacterial infection.

PRODUCTION TIME: 5 to 7 days in the BM

STAYS IN BLOOD: for 4-5 hours and in tissues for 4-5 days
- CIRCULATING POOL: 50% migrate in the CENTER OF THE BLOODSTREAM (measured during venipuncture)
- MARGINAL POOL: 50% roll along vessels in the PERIPHERAL BLOODSTREAM.

PRIMARY DEFENSE AGAINST:
bacteria
- margination and diapedesis
- chemotaxis
- phagocytosis
- killing
- prevention of bystander damage

DISEASES: Chronic Granulomatous Disease (CGD),
alpha1 anti-trypsin deficiency

** - 90% of the neutrophils in the body are in a reservoir in the BM; 2-3% are in the bloodsteram; and, the rest are in the tissues.

RELEASE OF EPINEPHRINE DURING VIGOROUS EXERCISE OR STRESS CAUSES NEUTROPHILS FROM THE MARGINAL POOL TO TRANSFER TO THE CIRCULATING POOL, RESULTING IN AN INCREASE IN THE PERIPHERAL BLOOD WBC COUNT.

STEROIDS INCREASE THE RELEASE OF NEUTROPHILS FROM THE BM INTO THE BLOODSTREAM AND INCREASE THE WBC COUNT.
Margination
Neutrophil function against bacterial challenge.

- Within five minutes of infection, cytokines such as INTERLEUKIN-1 and TUMOR NECROSIS FACTOR-alpha mobilize neutrophils from the BM and deliver them to the circulation.
- These neutrophils enter the marginal pool, where they adhere to and crawl along the endothelial lining of blood vessels, a process called margination.
Rolling
- Rolling of neutrophils takes place when tight interactions between SELECTINS and their targets tightly attach neutrophils to the blood vessel wall.
- L-SELECTIN on neutrophils bind to GlyCAM1 and MadCAM1 on blood vessels.
- Cytokines such as IL-1 and TUMOR NECROSIS FACTOR-alpha from macrophages "activate" endothelial cells to express P-SELECTIN and E-SELECTIN, which bind to PSGL-1 and CLA-1 molecules on neutrophils
Spreading
- Macrophages release IL-8 which induces endothelial cells to express ICAM (intercellular adhesion molecule) which along with the selectins make neutrophils spread, acquire motility and release enzymes that INDUCE SEPARATION OF ENDOTHELIAL CELL JUNCTIONS.
Diapedesis
Neutrophils shed their L-SELECTINS and crawl between endothelial cells into the surrounding tissue in a directed manner.
Interleukin-1 and tumor necrosis factor-alpha
- Mobilize neutrophils from the BM and deliver them to the circulation (margination).
- Activate endothelial cells to express P-Selectin and E-Selectin which bind PSGL-1 and CLA-1 molecules on neutrophils (rolling).
L-Selectin
Protein on neutrophils that binds to GlyCAM1 and MadCAM1 on endothelial cells during "rolling."
P-Selectin and E-Selectin
- Endothelial cell proteins that are activated by IL-1 and TNF-alpha.
- These bind to PSGL-1 and CLA-1 on the neutrophils during "rolling."
ICAM (intercellular adhesion molecule)
- Endothelial cell molecule that is activated by macrophage-released IL-8.
- Along with selectins, ICAMs make neutrophils spread, acquire motility and release enzymes that induce separation of endothelial cell junctions.
Chemotaxis
- Once neutrophils enter tissues, they are drawn in a directed manner to sites of INFLAMMATION by CHEMOTACTIC FACTORS (which bind to chemotactic factor-receptors on neutrophil surfaces).

- The chemotactic gradient is effective up to 100 microns from the inflammation site.

- Neutrophils move along the chemotactic gradient to the site of inflammation. (Polarization - signal transduction [calcium entry, potassium efflux, glucose uptake, altered membrane potential, g-proteins, kinases, TFs], polarized contraction of cell in direction of chemotactic gradient)

- THIS IS DEPENDENT ON COORDINATED WAVES OF ASSEMBLY AND DISPERSAL of ACTIN and MYOSIN. ("Reorganization of cytoskeleton")
Chemotactic Factors
CLOTTING FACTORS: thrombin, kallikrein, fibrin degradation products.

COMPLEMENT CASCADE:
C3a*, C5a
* - predominant chemotactic factor in the blood

LEUKOTRIENES:
LTA4 - LTE4
Products of arachidonic acid generated by LIPO-OXYGENASE ENZYMES.

N-FORMYL-METHIONYL OLIGOPEPTIDE:
A product of bacterial debris.

INTERLEUKIN-8
Lazy Leukocyte Syndrome
Deficiency in adhesion proteins (CD11/CD18) causes neutrophils to migrate sluggishly to target. PATIENTS SUFFER FROM REPEATED BACTERIAL INFECTIONS, BUT THEIR ABCESSES ARE "COLD" TO TOUCH AND LACK LEUKOCYTE INFILTRATES.
Opsonization
- When a neutrophil encounters a bacterium, it attempts to tether the bacterium to itself.

- Tethering is facilitated by immunoglobulin molecules that are attached to the bacterium via the antigen binding site, and to the Fc receptors on neutrophils.

- Another way to tether the bacterium is via the CR1 RECEPTOR for complement component C3b.

- PHAGOCYTOSIS follows opsonization: once tethered, the neutrophil engulfs the bacterium by extending its actin-rich lamellipodia around the bacterium until they fuse and create a phagosome.
- The phagosome eventually fuses with the lysosome to form a phagolysosome "death chamber."

NEUTROPHILS USUALLY PHAGOCYTOSE 5-20 bacteria.
Neutrophil Killing:

Primary Granules
- Contain MYELOPEROXIDASE, ELASTASE, ACID HYDROLASES, and DEFENSINS

- MYELOPEROXIDASE catalyzes the reaction between H202 and choloride ion to form hypochlorite, which is BACTERICIDAL

- ELASTASE AND ACID HYDROLASES digest the bacterial wall.
Neutrophil killing:

Secondary Granules
- Contain LYSOZYME, COLLAGENASE, and LACTOFERRIN

- LYSOZYME dissolves lipid membranes of bacteria.

- COLLAGENASE digests collagen in bacteria.
Neutrophil Killing:

Peroxisome
- The wall of the peroxisome contains a group of proteins that form NADPH oxidase, which generates superoxide and H202 via the HEXOSE MONOPHOSPHATE SHUNT.

- These agents are lethal to bacteria even in minute amounts.

- Defects in the generation of superoxide and H2O2 causes CHRONIC GRANULOMATOUS DISEASE.
Myeloperoxidase
Catalyzes the reaction between H2O2 and chloride ion to form hypochlorite, which is bactericidal.

Contained in primary granules of the neutrophil.
Superoxide Dismutase
- Inhibitor of oxygen metabolites in the neutrophil which help to protect it during the killing process.

- CONVERTS SUPEROXIDE TO H2O2.

* - Deficiency of SUPEROXIDE DISMUTASE results in ALS (Lou Gehrig's Disease).
Catalase
An inhibitor of oxygen metabolites, which protects neutrophils during the killing process.

CONVERTS H2O2 to H2O and oxygen

* - MADE BY BACTERIA
Reduced glutathione
An inhibitor of oxygen metabolites, which protects neutrophils during the killing process.

Converts H202 to H20
Protease Inhibitors
Help to protect the neutrophil during killing process.

- alpha1-antitrypsin
- alpha2-macroglobulin
- elafin
- secretory leukoprotease inhibitor
Chronic Granulomatous Disease
Because of a deficiency of NADPH oxidase, neutrophils and macrophages are incapable of generating superoxide and H202 (or just produce lower levels of these ROS).

- Thus, CGD cells can ingest but not effectively kill bacteria. The ingested bacteria multiply within the phagocytic cells and cause severe infections -- osteomyelitis, pneumonia, granulomas, skin infections, draining sinuses.

- Age of onset ~ 2 years.

- There are four different forms of the disease -- all of which involve abnormalities in the proteins that comprise NADPH oxidase.

CATALASE PRODUCING BACTERIA AND CGD INFECTIONS:
- As shown in the previous slides, catalase converts H202 to H20 and oxygen.
- The enzyme IS NORMALLY PRODUCED BY THE BODY TO PROTECT PHAGOCYTES.
- Some microbes - ASPERGILLUS and SERRATIA MARCESCENS can also produce catalase. When ingested by the CGD neutrophil, these bacteria release catalase which NEUTRALIZES THE SMALL AMOUNT OF SUPEROXIDE and H2O2 made by the CGD neutrophil.
- THUS, THESE BACTERIA THUS FLUORISH IN CGD NEUTROPHILS AND CONTRIBUTE TO MANY INFECTIONS IN THESE PATIENTS.
Nitroblue Tetrazolium Test
(NBT Test)
Most commonly used diagnostic assay to test for CHRONIC GRANULOMATOUS DISEASE.

- In this test, phagocytes are mixed with a CLEAR, WATER-SOLUBLE, YELLOW DYE called NBT in the presence of LATEX PARTICLES (substitues for bacteria).

- Normal phagocytes will engulf the latex particles and generate SUPEROXIDE and H2O2, which converts NBT into a BLUE PRECIPITATE called FORMAZAN.

- CGD Neutrophils produce less superoxide and H2O2, and so the NBT is not converted to formazan and the cells remain yellow in color.

Blood from CARRIERS of CGD contain a mixture of normal and abnormal neutrophils.
alpha1 anti-trypsin deficiency
PRODUCED IN THE LIVER BY TWO ALLELIC GENES, M and Z.

MM: Normal levels (200-400 mg/dl)
MZ: Mild deficiency; congenital (20-160 mg/dl)
ZZ: Absent; congenital; UNCONTROLLED PROTEASE ACTIVITY.

- Cigarette smoke inactivates alpha1 anti-trypsin (WORSENS DEFICIENCY, if already present); NO INHIBITION OF ELASTASE ACTIVITY. DAMAGE TO TISSUES.

EMPHYSEMA: 60% of MZ develop emphysema (especially those who smoke); 100% of ZZ develop it.

LIVER DISEASE: 10-15% of MZ; 100% of ZZ children.

DIAGNOSIS:
- alpha1 anti-trypsin phenotyping: ISOELECTRIC FOCUSING
- Serum protein electrophoresis: alpha1 globulins.
- Liver biopsy: Hepatitis, cirrhosis.

TREATMENT:
Supportive, antibiotic therapy, liver transplant.
Monocytes
Immature cells that inefficiently phagocytose bacteria and ARE OF LIMITED VALUE in fighting infections.

They circulate at the periphery of the bloodstream for 10-20 HOURS and then enter tissues where they differentiate into macrophages.

- 4-5% of WBCs
- ~370 cells/microliter
Macrophages
The maturation process from monocyte to macrophage involves a 5-FOLD INCREASE IN CELL SIZE to 20-50 microns.
- The cytoplasm also fills with granules containing lysosomal enzymes.
- Macrophages can engulf LARGER PARTICLES THAN NEUTROPHILS, like malarial and leishmania, TB, leprae bacilli, silica dust, and even whole senescent RBCs.
- THEY CAN LIVE IN TISSUES FOR MANY YEARS.

- They also secrete IL-1 (endogenous pyrogen) and IL-8 (promotes rolling/spreading).
Alveolar Macrophages
FIRST LINE OF DEFENSE WHEN SOMETHING IS INGESTED.

- Engulf particles in the alveoli of the lung.
- If the particle is not digestible, they for giant cell capsules around the particle (SEEN IN TB LESIONS).
Kupffer Cells
Line liver sinuses and remove bacteria in the portal circulation, and prevent them from entering general circulation.

THESE DIE IN CIRRHOSIS.
Osteoclasts
Resorb bone.

- Malfunction or absence of osteoclasts causes CONGENITAL OSTEOPETROSIS (extremely rare inherited disorder, whereby bones harden, becoming denser -- treated by BM transplant).
Microglia
Macrophages of the CNS
Dendritic Cells
Present antigens to lymphocytes.
Basophils and Mast Cells
- 0.4% of WBCs (~30 cells/microliter)

- These cells circulate briefly before entering "barrier" tissues such as the MUCOSA and SKIN.

- The prominent cytoplasmic granules of basophils, and their distant cousins, THE MAST CELLS, contain: HISTAMINE, SEROTONIN, BRADYKININ, and LEUKOTRIENES.

- These cells have Fc receptors for a class of immunoglobulins called IgE.

- Basophils differentiate from CFU-Bas. There is some controversy as to whether mast cells differentiate from CFU-Bas or another precursor.
Basophils, Mast Cells, and Allergy
FIRST EXPOSURE TO ALLERGEN:
- The allergen is taken up by APC and presented to the allergen-specific t-cells.
- These cells in turn activate antigen-specific B cells which differentiate into IgE-producing plasma cells.
- Secreted IgE molecules attach via the Fc portion to Fc receptors on mast cells/basophils, and can remain attached for years.

SUBSEQUENT EXPOSURE TO ALLERGEN:
- Following re-exposure to allergen, the allergen cross-links IgE molecules attached to mast cells or basophils, which induces THE RELEASE OF HISTAMINE, SEROTONIN, BRADYKININ, and LEUKOTRIENES (a process called DEGRANULATION).
- The secreted compounds cause arteriolar dilation, bronchospasm, enhanced vascular permeability and clinical symptoms of allergy (runny nose, urticaria, sneezing, asthma).

PHARMACEUTICAL TARGETING:
-Anti-Histamines
-Leukotriene Inhibitors
-CROMOLYN SODIUM prevents degranulation.
-STEROIDS inhibit the whole process.
Eosinophils
PRIMARY DEFENSE AGAINST PARASITES

- 2-3% of all WBCs (~160 cell/microliter)

- Eosinophils are morphologically indistinguishable from neutrophils, but when stained with WRIGHT-GIEMSA stains, they are revealed to contain large, lozenge-shaped red-orange granules that are larger than neutrophils' granules. THEY ALSO HAVE A BIOLOBED NUCLEUS.
- THEY PROLIFERATE IN RESPONSE TO IL-5.

- During inflammation, mast cells, macrophages, and platelets release EOSINOPHILIC CHEMOTACTIC FACTOR of ANAPHYLAXIS and PROSTAGLANDIN D2 and E2, which lure eosinophils to sites of inflammation.

- Eosinophils express receptors for the Fc portion of IgE and IgG molecules, and they also express receptors for complement proteins.
(THEY THEREFORE PARTICIPATE IN THE OPSONIZATION REACTION, as THESE CELLS PHAGOCYTOSE MICROBES).

- In the case of large parasites, they flatten themselves against the parasite and fire a furious fusillade of granules containing HYDROLYTIC ENZYMES, which KILL THE PARASITE.
Eosinophilia
Seen in allergies, hay fever, allergies to drugs (penicillin), eczema, parasitic infections, and asthma.
Loeffler's Syndrome
EOSINOPHILIA (50,000 to 100,000/microliter) with benign, acute eosinophilic pnuemonia.
* - USUALLY CAUSED BY ASCARSIS INFESTATION (Roundworm)
Four Cardinal Signs of Inflammation
RUBOR:
Redness (vasodilation)

CALOR:
warmth (vasodilation, increase in local metabolism, endogenous pyrogens like IL-1)

TUMOR:
swelling (vasodilation, increaes in leakage of fluid in tissues)

DOLOR:
Pain (swelling, damaged nerve endings, substance P, prostaglandins)

R.C.T.D.
Corticosteroids
BLOCK THE CONVERSION OF PHOSPHOLIPID to ARACHIDONIC ACID (blocks phospholipase A2)
Aspirin
Blocks the Cyclo-oxygenase pathway (COX) and thereby prevents the formation of prostaglandins.
NSAIDs (non-steroidal anti-inflammatory drugs)
IBUPROFEN

- BLOCKS the COX pathway reversibly.
Zileuton
LIPO-OXYGENASE (LOX) inhibitor which decreases LEUKOTRIENE production.
Zakirlukast
Inhibits the leukotriene (LTB4) receptor.
Singulair
Leukotriene inhibitor.
(prevents vasodilation and chemotaxis)