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Assessment of the Immune System

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Overview of immune system   Microbes enter thru break in epithelium, encounter APCs (dendritic) in submucosa and are presented by MHCs; Antigens are transported via lymph to regional node; circulating naive lymphocytes migrate into lymph node and are activated to differentiate  
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Overview of immune system, cont'd   Differentiated effector and memory lymphocytes enter circulation  
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Effector T cells and antibodies   filter into tissues and elimante antigen  
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Memory lymphocytes   take up residence in normal tissue in preparation for future infections (eliciting a stronger, faster response)  
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Assessment of humoral immunity: tests that measure specific antibody levels in a pt's serum   RIA, ELISA, Competitive Inhibition Assay, Western Blot  
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ELISA (enzyme-linked immunosorbent asay): to detect antigen "A"   purified antibody specific for antigen-A is linked to enzyme; test samples coated onto plastic wells; residual sticky spots are blocked; labeled antibody added to wells; bound antibody is detected by enzyme-dep color change read by spectrophotometer  
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Antigen Capture/Sandwich ELISA   a known antigen is added to bottom of well at first step; after adding serum, an enzyme-linked Ab (anti-HGG) is added and serum Ab is sandwiched btw the known antigen and anti-HGG  
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Clinical Use of ELISA: Screening donated blood for evidence of viral contamination by   HIV-1, HIV-2 (presence of HIV Ab); Hepatitic C (presence of Ab); Hepatitis B (test for Ab and viral antigen); HTLV-1, HTLV-2 adult Tcell/hairy cell leukemia(presence of Ab)  
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Clinical Use of ELISA: Measureing Hormone Levels   HCG (pregnancy); LH (time of ovulation); TSH, T3, T4 (thyroid fxn); Hormones (ex: anabolic steroids, HGH) possibly used illicitly by athletes  
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Clinical Use of ELISA: Detecting Infections   sexually transmitted agents (HIV, syphilis, chlamydia); Hepatits B, C; Toxoplasma gondii  
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Clinical Use of ELISA: others   Detecting allergens in food/house dust; Measureing "Rheumatoid Factors" and other autoantibodies in autoimmune diseases; Measuring toxins in contaminated food; Detecting illicit drugs (cocain, opiates, delta-9-tetrahydrocannabinol in marijuana)  
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Competitive Inhibition Assay (CIA)   determines presence/amount of antigen in unknown sample via competing for labeled reference antigen in a well; Standard curve allows calculation against a negative control  
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Radioimmunoassay   mixture of radioactive antigen (Iodine 125-I or 131-I via tyrosine)and Ab against that antigen; Known "cold" antigen is added to compete for binding sites on Ab; "cold" displaces "hot"; determine ratio of bound:unbound on standard curve  
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Radioimmunoassay: Separating Bound from Free Antigen   1. precipitate Ab-Ag complexes w/"second Ab" against 1st (ex: rabbit IgG-Ag w/antirabbit-IgG antiserum); 2. Ag-specific Ab coupled to test tube walls measure heat of supernatant & bound; 3. Ag-specific Ab coupled to particles & centrifuged (pellet=bound)  
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Drawbacks of RIA (radioimmunoassay)   expensive; hazardous; emission of gamma radiation requires special equipment; the body concentrates iodine atoms (radioactive or not, in thyroid gland and are incorporated into T4 thyroxine)  
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Use of RIA in clinical settings   assay plasma levels of hormones, digitoxin/digoxin in treated pts, certain abused drugs; Presence of HBV surface Ag (HBsAg) in donated blood; anti-DNA antibodies in SLE  
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Western Blot or Immunoblot   identifies presence of given protein in cell lysate; Confirms ELISA findings if there are false-positives (ex: diagnosis of HIV); More laborious/expensive than ELISA and more specific b/c Ag are identified by: SIZE and REACTIVITY to ANTIGENS  
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Western Blot and HIV Diagnosis   virus dissociated into constituent ptns w/SDS and run on gel; transfer to nitrocellulose and overlay w/serum sample; Ab in serum binds to different size-separated Ag; Bound Ab will be revealed w/anti-immunoglobulin antibody labeled w/enzyme  
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Different Immunoblots in Use: Southern   separates ssDNA with cDNS or RNA probe  
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Different Immunoblots in Use: Northern   separates RNA with RNA or cDNA probe  
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Different Immunoblots in Use: Western   separates protein with antibodies  
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Coombs Test   detects antibody to RBC antigens esp useful in hemolytic disease of newborn (anti-immunoglobulin antibody); Rh- mom forms antibodies against Rh+ fetus; 2nd pregnancy Ig anti-Rh cross placenta&damage fetal RBCs; presence of Ab shown w/Ig-Ab on coated cells  
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Total Hemolytic Complement (CH50) Assay: Modified Radial Immunodiffusion   determine efficiency of complement system by testing dilution of serum required for lysis of 50% Ab-coated RBCs (EA); EA on agar gel & poured on slide; standard vol/concent. added to wells punched in agar; measure lysis zone in 24hrs for active complement  
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CH50 Assay cont'd   the size of zone is proportional to amt of complement in well; the assay measures total activity of Classical and Alternate paths (C1-C9), BUT lacks ability to specify absnece of a particular compenent!  
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Detecting Defects in Specific Complement Factors   mix sensitized red cells w/"compement reagent" so sensitized cells + reagent contain all complement components except the one being tested (ex: to test for C4, EA are placed in C4-deficient guinea-pig serum, cells will be lysed ONLY if C4 is in serum)  
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Efficiency of complement system is determined by:   testing the dilution of serum required for lysis of 50% of antibody-coated red blood cells (EA = erythrocytes coated w/antigen)  
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More about the CH50 Assay   screens most complement disorders; specific component deficiency tests aren't always available; tests serum, synovial fluid, etc; ice bath/antibody-sensitized sheep erythrocytes (SRBCs) as indicator system; serial dilutions + comp serum 1hr, spec, curve  
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Interpretation of CH50 Assay   Deficiencies of C1-C8 (value of 0 ); C9 Deficiency (25% to 50% of normal); doesn't detect properdin/factor D deficiency (needs APH50)  
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C1 inhibitor deficiency   a/w decreased C4 levels; detected by functional assay or quantification of specific ptn; 85% pts have dec ptn  
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Normal C3 and C4 levels in the face of undetectable CH50   strong evidence of congenital complement component deficiency  
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Decrease in C4 and/or C3 with undetectable CH50   suggests complement consumption  
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Normal values of Ig and Complement Components: IgG   600-1400 mg/dL  
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Normal values of Ig and Complement Components:IgM   40-345 mg/dL  
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Normal values of Ig and Complement Components:IgA   60-380 mg/dL  
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Normal values of Ig and Complement Components: IgE   0-200 IU/mL  
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Normal values of Ig and Complement Components: CH50   125-300 IU/mL  
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Assessment of Cellular Immune Response: Lymphocyte Subpopulation in Human Blood - Resting Lymphocytes   appear as small round cells w/dense nucleus and little cytoplasm; can be IDed by differential expression of cell surface ptns detectable by specific antigens  
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All T lymphocytes express:   CD3  
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All B lymphocytes express   immunoglobulin receptors  
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NK cells   don't have CD3 or Ig receptors  
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a-b Tcells are subdivided on basis of:   CD4 or CD8 expression  
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d-g Tcells are identified with antibodies against:   d-g Tcell receptor  
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A minority of Bcells express   CD5  
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CD4 helper Tlymphocytes   stimuli for bcell growth/differentiation (humoral immunity); MQ activation by secreted cytokines (cell-mediated immunity); a-b Ag-receptor; CD3+, CD4+; 55% of lymphocytes in blood, nodes, spleen  
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CD8 cytolytic Tlymphocytes   kill virus-infected and tumor cells; reject allografts (cell-mediated immunity); a-b Ag-receptor; CD3+, CD8+; 25% in blood, 20% in node, 15% in spleen  
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B lymphocytes   antibody production (humoral immunity); surface antibody (immunoglobulin) Ag-receptor; Fc receptors, MHC class II, CD19, CD21; 15% of blood; 25% of node; 45% of spleen  
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NK cells   kill virus-infected and tumor cells; antibody-dependent cellular toxicity; killer cell Ig-like Ag-receptor; Fc receptor for IgG (CD16); 10% of blood and spleen; rare in lymph node  
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Identification and Isolation of Individual Cells by Fluorescence-Activated Cell Sorter (FACS)   cells to be IDed are labeled w/fluorescent dye-coupled Ab specific for cell-surface Ag; single cells pass thru laser and size/granularity emission detected and analyzed by CPU; sorted cells can be counted by expression of specific molecules  
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Analysis of Data Obtained from FACS   ex: fluorescence-conjugated Ab against IgM and IgD; when expression of only one molecule is analyzed, the data displays a histogram; when 2 or more parameters are measured the data is displayed as dot plots  
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Lymphocyte Function: Tcell activity can be divided into 2 phases - Induction Phase   Tcells are activated to divide and differentiate  
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Lymphocyte Function: Tcell activity can be divided into 2 phases - Effector Phase   their functions are expressed (effector T cells can be helper or cytotoxic)  
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Assessment of Lymphocyte Function   prolif of Ag-specific lymphocytes is prereq for differentiation of effectors & analysis of induced prolif is important  
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Assessment of Lymphocyte Function: Prolif of normal lymphocytes in response to specific Ab   difficult because only a minute proportion of cells will be stimulated to divide; but Assessment of Lymphocyte Function: many or all of lymphocytes to certain substances are polyclonal mitogens  
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Lymphocytes from pts w/suspected immunodeficiency:   are examined for ability to proliferate in response to non-specific stimulus; lymphocyte proliferation is measured by incorporation of 3H-thymidine into DNA  
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Polyclonal Mitognes   used to test the ability of lymphocytes in human peripheral blood to proliferate  
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Polyclonal Mitogens: Phytohemagglutinin (PHA) red kidney bean   T cells respond  
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Polyclonal Mitogens: Concanavalin (ConA) jack bean   T cells respond  
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Polyclonal Mitogens: Pokeweek mitogen (PWM)   T and B cells respond  
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Polyclonal Mitogens: Lipopolysaccharide (LPS) e.coli   B cells (mouse) respond  
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Antigen-specific Tcell Proliferation   assays Tcell response; Tcells obtained from pt immunized w/antigen-A; normal lymphocytes proliferate w/exposure to Ag-A, but not when exposed to unrelated Ag-B; Presence of APCs in culture is essential; measure incorporation of 3H-thymidine in dividingDNA  
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Ag-specific Tcell proliferation of lymphocytes establishes   the existence of efficient CD4 Tcell immunity  
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Functional Assessment of CD4 Tcells cont'd: when CD4 Tcells recognize Ag presented by Bcells or MQ:   they release cytokines which activate the antigen-bearing cells  
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CD4 Tcell function is studied by   measuring the type and amount of cytokines  
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Functional Assessment of CD4 Tcells: Supernatants from culture are used to measure   cytokine production by sandwich ELISA technique  
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Tcell Cytokine Production can be measured by:   ELISPOT Assay; a variant of ELISA in which Ab bound to plastic surface are used to capture cytokines secreted by individual Tcells; each cell producing cytokine makes a color spot; the # of spots is counted & freq is determined by known # of cells in well  
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ELISPOT Assay also measures   antibody secretion by Bcells; the wells are coated with Ag instead of Ab; after Ab binds to plate-bound Ag, an enzyme-labeled second Ab (anti-Ig) is used to detect bound Ab  
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PCR   starts w/ds-DNA from which millions of copies of select region are expanded w/Taq, nucleotides and ss-DNA primers  
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RT-PCR   can measure Cytokine Production! mRNA is isolated from cells and cDNA copies are made w/reverse transcriptase; the desired cDNA is selectively amplified by PCR w/sequence-specific primers to see RNA expression  
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DNA bands after electrophoresis   the amt of amplified cDNA will be proportional to its representation in the mRNA; stimulated Tcells actively producing a particular cytokine will produce a larger amt of that mRNA giving a correspondingly large amt of selected cDNA on RT-PCR  
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Intracellular Cytokine Staining for Detecting Cytokine Secreting Cells   inhibitors of ptn export inhibit cytokine secretion; fluorochrome-labeled Ab binds cytokines in cell; cells are fixed; the cells are also labeled w/Ab for cell-surface ptns to determine the subset of Tcells a/w particular cytokines; sort by FACS  
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Measurement of cytotoxic T lymphocyte (CTL) activity   activated CD8 Tcells kill cells expressing specific peptide:MHC-1 complex that is recognized by Tcell receptors  
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The activity of CD8 Tcells can be detected by:   its ability to kill a target cell; target cells are labeled w/radioactive sodium chromate; when labeled cells are killed they release 51Cr; the amt of free 51Cr is measured in supernatant mixture of target cells + CTLs  
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Technique for CTL activity is similar to:   assay to measure tumor-specific CD8 Tcell activity; 3H-thymidine is used in lieu of 51Cr; fragmented DNA collected in filter is measured for radioactivity  
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Measurement of Apoptosis by TUNEL Assay   DNA fragments; enzyme terminal deoxynucleotidyl transferase (TdT) is able to add nucleotides (biotin-labeled) to ends of DNA frags; Biotinalated-DNA is detected by streptavidin-coupled to enzymes that convert colorless substrate to brown precipitate  
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Delayed Type of Hypersensitivity   local response to Ag can indicate presence of active immunity (ex: tuberculin skin test); Ag is injected into skin and if individual has been exposed to Ag before, he/she develops a positive red/thickend reaction w/in 24hrs  
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Delayed Type of Hypersensitivity: reaction is teh result of   the reaction of sensitized T cells with specific antigen, followed by the release of cytokines and the influx of MQs to the injection site  
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Assessment of Phagocytic Function   chemotaxis, phagocytosis, intracellular killing, measurement of oxidative burst  
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Nitro Blue Tetrazolium (NBT) Assay   in nml phagocytes (PMN, monocytes/MQs) reactive oxygen intermediates (ROIs) are activated by phagocytosis; NBT yellow dye is taken up w/phagocytosis; w/in phagocyte NBT acceps H and is reduces dt NADPH oxidation and precipitates purple  
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NBT Assay: Pts w/defective ROI systems (ex: chronic granulomatous disease)   reduction of NBT does not occur and dye remains yellow  
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Cell Components of the Immune System: B cells   ~0.3x10^9/L blood; In vivo (serum Ig and specific Ab levels); In vitro (induced Ab production in response to pokeweed mitogen)  
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Cell Components of the Immune System: T cells   Total: 1.0-2.5x10^9/L blood (CD4 = 0.5-1.6; CD8 = 0.3-0.9); In vivo (skin test); In vitro (Tcell proliferation in response to phytohemagglutinin or tetanus toxoid)  
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Cell Components of the Immune System: Phagocytes   Monocytes (0.15x10^9/L); PMNs: Neutrophils (3.0-5.5), Eosinophils (0.05-0.25), Basophils (0.02); no measurement in vivo; In vitro (phagocytosis nitro blue tetrazolium uptake intracellular killing of bacteria)  
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When do we need to assess the immune system?   immunodeficiency diseases, autoimmune diseases, bone marrow or solid organ graft, diagnosis of HIV infxn and eval of status for treatment, allergic diseases, infectious disease screening (TB) and evaluating prognosis (leprosy)  
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Immunodeficiency Diseases: Primary   immunodeficiency dt genetic defects in the different components of the immune system (ex: def in Tcell, Bcell, or any other components of immune system)  
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Immunodeficiency Diseases: Secondary   aquired immunodeficiency syndrome; immunodeficiency secondary to chemotherapy for malignancy or transplantation; immunodeficiency dt infxn and/or nutritional deficiency  
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Primary Immunodeficiency: defective antibody response   B cell defect  
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Primary Immunodeficiency: defective cell-mediated immunity   Tcell or Tcell-related defects  
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Primary Immunodeficiency: hereditary complement deficiencies or complement component defect   C1 inhibitor deficiency  
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Primary Immunodeficiency: Defects in phagocytosis   defects in O2 reduction pathway in phagocytes; leudocyte adhesion deficiency  
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Chronic bacterial infxn; no tonsil (organ composed of 90% Bcells); maternal uncles died of pneumonia; elevated monocytes; many pre-Bcells in peripheral blood, Tcells normal, low IgG and IgM, NO IgA   X-linked agammaglobulinemia  
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Which component of immune system plays role in protecting against bacterial infxn?   B cells - humoral immunity  
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Non-random X inactivation in B cells of female carrier   nonrandom X chromosome inactivation in a particular cell lineage is a clear indicator that the product of X-linked gene is required for the development of that lineage  
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X-linked agammaglobulinemia (XLA)   first immunodeficiency disease to be fully understood; "model B cell" deficiency disease  
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X-linked agammaglobulinemia (XLA): Mechanism   defect in XLA (cytoplasmic ptn Bruton's tyrosine kinase (Btk)); Btk is involved in intracellular signaling from B cell receptor and is necessary for growth/differentiation of pre-B cells; role of Btk in Bcell maturation isn't known; Btk is a src oncogene  
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XLA: Clinical Features   usu M>F; presents after maternal-fetal antibodies wear of (6-12mo); very few or no B cells in blood/lymphoid tisue; small nodes/no tonsils; usu no IgA, IgM, IgD, or IgE and only small amt of IgG (<100mg/dL)  
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XLA: due to lack of antibodies   pts are vulnerable to infxn w/extracellular pyogenic bacteria w/polysaccharide capsule resistant to phagocytosis (H. influenzae; S. pneumoniae; S. pyogenes; S. aureus; enteroviruses that are usu controlled by neutralizing Abs)  
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Management/Follow-up of XLA   initial prep of IgG to maintain min of 600mg/dL; later 10g IgG/wk  
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Detection of Autoantibody in Autoimmune Diseases   react pt serum with tissue sections; examine for bound-antibody by indirect immunofluorescence using anti-human Ig labeled dye; most autoimmune diseases a/w autoantibodies directed at self tissue (distinguishes from inflam dt infection)  
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Immunofluorescence Microscopy   Ab labeled w/fluorescein green dye used to reveal presence of corresponding Ags in cells/tissues; cells making a particular enzyme light up under a fluorescent scope  
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Evaluation of Allergic Diseases: allergic asthma   long-standing asthma and allergic rhinitis; allergic to cats/dog/rabbit and probably to pollens, but roles not confirmed; strong family Hx of allergic rhinitis/asthma  
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Basis of allergic immune responses   clinical condition when host immune system responds to innocuous substances in environment; usu a sencondary response to re-exposure of Ag (Ag binds IgE Ab formed from previous exposure and bound to receptors on mast cell via Fc region); degranulate cells  
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Degranulation of mast cells releases:   histamine, cytokines, leukotreins, bradykinins, etc; they give rise to severe life-threatening conditions as in acute bronchial asthma and systemic anaphylaxis  
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Antigen binding to IgE on mast cells leads to amplification if IgE production   IgE secreted by plasma cells upon first exposure to allergen binds high-affinity IgE receptors on mast cells/basophils; on 2nd exposure, allergen x-link the IgE on mast cell (express CD40L and secrete IL-4 which binds IL-4R on B cells)  
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Binding of IL-4 to IL-4R on activated B cells results in:   isotype switching by B cells and production of more IgE; these interactions can occur at the site of allergen-triggered inflammation, such as in bronchial-associated lymphoid tissue (BALT)  
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Allergic Response involves:   activation of helper CD4 Th2 cells (source of cytokine); IgE antibody production; Mast cell sensitization and release of vasoactive compounds (histamine/leukotrein/prostaglandin/bradykinin, etc); recruitment of eosinophils  
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Acute Response in Allergic Asthma   degranulation of mast cells following x-link of IgE on cell surface leads to release of inflam mediators; mediators cause bronchial smooth m. contraction & influx of inflam cells (incl eosinophils & Th2 cells)  
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Activated mast cells and Th2 cells secrete cytokines which:   augment eosinophil activation and degranulation resulting in further tissue injury and influx of more inflam cells (basis of chronic inflam)  
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Markers of Inflammation in Pts w/ Asthma   1. peripheral blood or nasal eosinophils; 2. nasal secretion may have inc eosinophil cationic ptn (ECP) and IL-8; 3. Inc NO gas in exhaled air; 4. dec pH; 5. opaque sinuses; 6. Mast cell/eosinophil product in airway; 7. coll deposit/remodeling dt inflamm  
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Acute episode of asthma is accompanied by:   increased eosinophils in peripheral blood smear (in addition to PMNs w/red cytoplasm in H&E stains)  
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Localization of Major Basic Protein (MBP) in lungs of severe asthmatic   infiltration of eosinophils in submucosa of bronchus; MBP in infiltrating eosinophil (immunofluorescent stain)  
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Mediators that can be measured and used as indicators of allergic responses   IgE level; eosinophil count; histamine levels; tryptase levels; Th2 cytokines (IL-5, IL-13) levels; Chemokine (CCL11, formerly eotaxin) levels  
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Skin Testing can detect allergy to specific allergens   characteristic positive response is wheal and flare; skin response takes 15min and persists for 30min or more  
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Skin test wheals are caused by:   extravasation of serum from capillaries in skin which results from direct effect of histamine  
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Erythematous flares on skin testing:   dt increased blood flow; accompany wheals and pruritus (caused by histamine)  
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Late and Delayed Response to Allergen Skin Testing   can occur following an immediate response to either skin or lung exposure; response is diffuse and edematous and appears 2-3hrs after immediate response; may last 24hrs  
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Immediate and Late Response to Allergen in the Lungs   allergic response in airways often trigger asthmatic attack dt narrowing of airways caused by bronchial smooth muscles; can be measured by peak flow meter indicated by fall in peak expiratory flow rate (PEFR)  
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Immediate lung response to allergen:   peaks w/in minutes after inhalation and then subsides; after 6-8hrs of antigen challenge, there is a late stage response when PEFR falls again  
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Immediate lung response is due to:   direct effect of inflammatory mediators on the blood vessel and bronchi smooth muscle  
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Late lung response is due to:   influx of inflammatory cells attracted by the chemokines and other mediators released by mast cells and T cells during and after the immediate response  
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Hyper-inflated lungs are characteristic of:   obstructive airway disease  
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Immunotherapy/hypersensitization for allergic asthma   uses allergen extract for regular injections over a period of months; the dose is increased progressively starting btw 1-10ng and inc to 10mg/dose  
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The response to hypersensitization therapy:   increase in serum IgG antibodies, striking decrease in T cell response to antigen in vitro; marked decrease in late reaction in skin; eventually there is a progressive decrease in IgE antibodies in the serum  
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