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MedMicro-Y2S1B2
Assessment of the Immune System
| Question | Answer |
|---|---|
| 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 |
| Overview of immune system, cont'd | Differentiated effector and memory lymphocytes enter circulation |
| Effector T cells and antibodies | filter into tissues and elimante antigen |
| Memory lymphocytes | take up residence in normal tissue in preparation for future infections (eliciting a stronger, faster response) |
| Assessment of humoral immunity: tests that measure specific antibody levels in a pt's serum | RIA, ELISA, Competitive Inhibition Assay, Western Blot |
| 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 |
| 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 |
| 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) |
| 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 |
| Clinical Use of ELISA: Detecting Infections | sexually transmitted agents (HIV, syphilis, chlamydia); Hepatits B, C; Toxoplasma gondii |
| 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) |
| 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 |
| 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 |
| 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) |
| 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) |
| 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 |
| 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 |
| 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 |
| Different Immunoblots in Use: Southern | separates ssDNA with cDNS or RNA probe |
| Different Immunoblots in Use: Northern | separates RNA with RNA or cDNA probe |
| Different Immunoblots in Use: Western | separates protein with antibodies |
| 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 |
| 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 |
| 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! |
| 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) |
| 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) |
| 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 |
| 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) |
| C1 inhibitor deficiency | a/w decreased C4 levels; detected by functional assay or quantification of specific ptn; 85% pts have dec ptn |
| Normal C3 and C4 levels in the face of undetectable CH50 | strong evidence of congenital complement component deficiency |
| Decrease in C4 and/or C3 with undetectable CH50 | suggests complement consumption |
| Normal values of Ig and Complement Components: IgG | 600-1400 mg/dL |
| Normal values of Ig and Complement Components:IgM | 40-345 mg/dL |
| Normal values of Ig and Complement Components:IgA | 60-380 mg/dL |
| Normal values of Ig and Complement Components: IgE | 0-200 IU/mL |
| Normal values of Ig and Complement Components: CH50 | 125-300 IU/mL |
| 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 |
| All T lymphocytes express: | CD3 |
| All B lymphocytes express | immunoglobulin receptors |
| NK cells | don't have CD3 or Ig receptors |
| a-b Tcells are subdivided on basis of: | CD4 or CD8 expression |
| d-g Tcells are identified with antibodies against: | d-g Tcell receptor |
| A minority of Bcells express | CD5 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| Lymphocyte Function: Tcell activity can be divided into 2 phases - Induction Phase | Tcells are activated to divide and differentiate |
| Lymphocyte Function: Tcell activity can be divided into 2 phases - Effector Phase | their functions are expressed (effector T cells can be helper or cytotoxic) |
| Assessment of Lymphocyte Function | prolif of Ag-specific lymphocytes is prereq for differentiation of effectors & analysis of induced prolif is important |
| 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 |
| 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 |
| Polyclonal Mitognes | used to test the ability of lymphocytes in human peripheral blood to proliferate |
| Polyclonal Mitogens: Phytohemagglutinin (PHA) red kidney bean | T cells respond |
| Polyclonal Mitogens: Concanavalin (ConA) jack bean | T cells respond |
| Polyclonal Mitogens: Pokeweek mitogen (PWM) | T and B cells respond |
| Polyclonal Mitogens: Lipopolysaccharide (LPS) e.coli | B cells (mouse) respond |
| 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 |
| Ag-specific Tcell proliferation of lymphocytes establishes | the existence of efficient CD4 Tcell immunity |
| 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 |
| CD4 Tcell function is studied by | measuring the type and amount of cytokines |
| Functional Assessment of CD4 Tcells: Supernatants from culture are used to measure | cytokine production by sandwich ELISA technique |
| 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 |
| 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 |
| PCR | starts w/ds-DNA from which millions of copies of select region are expanded w/Taq, nucleotides and ss-DNA primers |
| 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 |
| 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 |
| 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 |
| Measurement of cytotoxic T lymphocyte (CTL) activity | activated CD8 Tcells kill cells expressing specific peptide:MHC-1 complex that is recognized by Tcell receptors |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| Assessment of Phagocytic Function | chemotaxis, phagocytosis, intracellular killing, measurement of oxidative burst |
| 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 |
| NBT Assay: Pts w/defective ROI systems (ex: chronic granulomatous disease) | reduction of NBT does not occur and dye remains yellow |
| 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) |
| 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) |
| 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) |
| 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) |
| 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) |
| Immunodeficiency Diseases: Secondary | aquired immunodeficiency syndrome; immunodeficiency secondary to chemotherapy for malignancy or transplantation; immunodeficiency dt infxn and/or nutritional deficiency |
| Primary Immunodeficiency: defective antibody response | B cell defect |
| Primary Immunodeficiency: defective cell-mediated immunity | Tcell or Tcell-related defects |
| Primary Immunodeficiency: hereditary complement deficiencies or complement component defect | C1 inhibitor deficiency |
| Primary Immunodeficiency: Defects in phagocytosis | defects in O2 reduction pathway in phagocytes; leudocyte adhesion deficiency |
| 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 |
| Which component of immune system plays role in protecting against bacterial infxn? | B cells - humoral immunity |
| 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 |
| X-linked agammaglobulinemia (XLA) | first immunodeficiency disease to be fully understood; "model B cell" deficiency disease |
| 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 |
| 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) |
| 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) |
| Management/Follow-up of XLA | initial prep of IgG to maintain min of 600mg/dL; later 10g IgG/wk |
| 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) |
| 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 |
| 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 |
| 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 |
| 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 |
| 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) |
| 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) |
| 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 |
| 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) |
| 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) |
| 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 |
| Acute episode of asthma is accompanied by: | increased eosinophils in peripheral blood smear (in addition to PMNs w/red cytoplasm in H&E stains) |
| Localization of Major Basic Protein (MBP) in lungs of severe asthmatic | infiltration of eosinophils in submucosa of bronchus; MBP in infiltrating eosinophil (immunofluorescent stain) |
| 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 |
| 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 |
| Skin test wheals are caused by: | extravasation of serum from capillaries in skin which results from direct effect of histamine |
| Erythematous flares on skin testing: | dt increased blood flow; accompany wheals and pruritus (caused by histamine) |
| 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 |
| 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) |
| 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 |
| Immediate lung response is due to: | direct effect of inflammatory mediators on the blood vessel and bronchi smooth muscle |
| 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 |
| Hyper-inflated lungs are characteristic of: | obstructive airway disease |
| 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 |
| 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 |
Created by:
bscaryp