VALDOSTA STATE UNIVERSITY

Biology 2900--Spring 2005

PART IV. HOST-MICROBE INTERACTIONS IN DISEASE

1. Defense systems
   
1. innate defenses (covered previously)
   
2. adaptive responses -- target specific invaders/foreign molecules; leads to acquired immunity (chapter 16)
   
1. key players
1. antigen -- substances that lead to the production of antibodies
1. epitope -- particular portion of antigen leading to response
2. haptens -- small molecules that can elicit a specific response, but only if bound to a larger molecule first
2. antibodies -- complex proteins consisting of several subunits
1. structure
1. two heavy chains composed of four domains; three of the domains are relatively constant, fourth is variable; constant regions responsible for antibody function, variable for antigen recognition
2. two light chains composed of two domains; like the heavy chains one domain is constant, the other variable; variable chain involved in antigen recognition
   
2. types
1. IgG -- monomer; binds to phagocytes, activates complement system; helps with opsonization, agglutination, immobilization
2. IgM -- pentamer; first antibody produced in primary response; helps with agglutination, complement activation
3. IgA -- dimer; in bodily secretions; helps with protection of mucous membranes
4. IgD -- monomer; helps with immune responses somehow
5. IgE -- monomer; attaches to mast cells and basophils; causes allergic (histamine) responses
3. MHC proteins
1. MHC-I proteins -- on almost all cells of the body; help display antigens produced within the cell, these may be either normal antigens or antigens caused by viruses or other problems; recognized by CD8 receptor proteins on the surface of cytotoxic (killer) T cells
2. MHC-II proteins -- on antigen presenting cells only (B-cells, dendrocytes, macrophages, some T-cells; recognized by CD4 receptors on helper T-cells (CD4 has been implicated in HIV infections)
4. lymphocytes -- each recognizes only one antigen
1. B cells, plasma cells -- antibody production;
2. T cells, cytotoxic T cells, helper T cells, suppressor T cells
3. all lymphocytes go through phases: immature, naive, activated, effector, memory
1. clonal selection theory and specificity of responses
2. responses
1. antibody-mediated (humoral) response
1. T-dependent antigens -- require assistance of T-helper (type Th2) cells
1. immunocompetent (naive) B-cells bind antigens to their surface; when sufficient numbers are bound they are lumped together, swallowed, and digested; left over bits are mixed with MHC-II proteins and displayed on the surface
2. helper T-cell with T-cell receptor (TCR) matching the displayed antigen binds to B-cell; helper T-cell is activated
3. activated helper T-cell secretes interleukin-2 and other lymphokines; these cause the B-cell to reproduce rapidly, forming a clone of activated B-cells specific to the antigen; some of the cloned cells continue to divide, others convert to plasma cells and begin to secrete antibodies, IgM at first, later IgG or IgA, depending on where the cells are located; in any case the concentration (titer) of antibodies specific to the antigen rises
4. antibodies bind to antigen leading to antigen inactivation, invader cell death, etc.
5. some of the cells convert to memory B-cells; speed up secondary response--almost instantaneous (hours, peak in 2 days)  and much stronger the second time
2. T-indepedent antigens -- uncommon, include several polysaccharides, LPS; response more or less the same as with T-dependent antigens, except activation by T-cells not required
2. cell-mediated response
1. naive (immunocompetent) CD4 helper T-cell (type Th1) encounters antigen in conjunction with MHC-II on surface of antigen-presenting cell (dendritic cell, macrophage) and is activated; a co-stimulatory molecule may be required
2. activated helper cell begins to both secrete interleukin-2 and the receptor for it; interleukin-2 causes clonal reproduction of lymphocytes, including the activated helper T-cell
3. some activated helper-T cells secrete additional cytokines and interleukins (macrophage activating factor) that activate non-specific and specific responses
4. killer T-cells with proper TCR recognize cells with MHC-I and antigen; can poke holes in membrane (perforin), secrete chemicals causing DNA to degrade, secrete tumor necrosis factors that kill cells, secrete migration inhibition factor that keeps macrophages in the area
5. both helper-T cells and killer T-cells form clones of memory cells
3. applications of adaptive responses -- acquired immunity (chapter 17)
   
1. passive immunity -- uses someone else's antibodies; no memory
1. naturally acquired -- IgG from the mother crosses the placenta and remains active in the bloodstream for several months after birth; IgA in mother's milk can prevent infections in the alimentary tract
2. artificially acquired -- injections of antibodies (gamma-globulin, anti-serum); often given during the incubation period after exposure to the disease
1. examples:  tetanus, rabies, hepatitis A and B
2. active immunity -- an adaptive response creates a bank of memory cells
1. naturally acquired -- exposure to disease with recovery
2. artificially acquired -- exposure to the antigens without risks associated with disease
1. attenuated vs inactivated, inactivated can be whole agents or fractions (protein subunits and polysaccharide capsules; sometimes the polysaccharides are linked to proteins making them more effective); adjuvant are sometimes added to increase the immune response
2. how long does immunologic memory last
3. examples:
1. Sabin (attenuated) vs Salk (inactiviated) vaccines for polio; both contain a mixture of viruses; both cause antibodies in blood; Sabin leads to mucosal immunity and helps prevent the spread; Sabin also leads to disease in small number of cases; currently, Salk-type (inactivated) is the vaccine of choice, Sabin was until the 1990's
2. smallpox
3. MMR -- risk of neurological damage (1/1,000,000), etc.
4. DPT -- improved when switched to acellular subunit of pertussis instead of whole cell vaccine
4. prospects
1. more stable vaccines from peptides, produced by plants
2. DNA-based
3. directed against new targets
4. monoclonal antibodies
   
4. applications -- testing and identification
1. rise in titer of antibodies indicator of active infections; titer monitored using variety of tests in conjunction with serial dilutions
1. precipitation tests in tubes or agar (immunodiffusion tests)
2. agglutination (direct or indirect) tests
1.  hemagglutination inhibition (for diseases where virus causes agglutination, antibody prevents)
2. latex beads (usually direct)
   
3. complement fixation tests -- serum to be tested is heated to destroy the complement system, but not antibodies; antigen plus complement components added; if antibodies were present the complement system is fixed;  testing for fixation involves adding red blood cells and antibodies to red blood cells, if complement not fixed before, it will be now, lysing the red blood cell, if it was fixed the red blood cells will remain intact
4. immunofluorescence -- attach fluorescent dye to constant end of antibody
1. direct (tests for antigens) immunofluorescence -- antibodies to antigen bound to surface; serum with possible antigen added, then washed off; antibodies with attached fluorochrome added and washed off; if antigen in the serum, the surface will fluoresce
   
2. indirect (tests for antibodies) immunofluorescence -- antigen is bound to slide or sides of a chamber; serum with possible antibodies placed on slide then washed off; antibodies that react to the constant portion of human antibodies, which themselves have fluorescent dyes attached, are added then washed off; if antibodies in the serum then the surface will fluoresce
5. enzyme-linked immunosorbant assay (ELISA) -- similar to indirect immunofluorescence except that instead of fluorescent marker an enzyme is linked to the antibody; after rinsing the sustrate for the enzyme is added; positive results indicated by color change as the enzyme acts on the substrate; quick test for HIV; some false positives
6. sol particle immunoassays (SPIA) -- similar to immunofluorescent except this time the second antibody is linked to colloidal gold and the complex appears colored; pregnancy tests are usually direct versions of this procedure
   
7. western blots -- similar to ELISA; antigens separated by electrophoresis, transferred to membrane, then exposed to serum purported to contain antibodies; after period of incubation, antibodies to the constant portion of human antibodies with marker added; used as second, more definitive test for HIV
8. RIA -- bind antibodies to substrate; hit with mix of purported antigen, followed by known, radioactive antigen; used to find hormones
2. cell-mediated system harder to monitor
1. fluorescent markers for CD4 and CD8 help distinguish the size of the help-T population
5. disorders of the immune system (chapter 18)
   
1. Type I hypersensitivity -- IgE mediated responses leading to release of granules from basophils and mast cells (note that IgE antibodies bind to mast cells and basophils first, then bind to antigen-- vascular permeability and inflammation
1. localized anaphylaxis -- hives and hay fever (histamine); asthma -- based on eosinophils and mast cells, histamine not involved in asthma attacks, leading to bronchial constriction and mucus production
2. generalized -- anaphylactic shock: systemic vascular permeability, vasodilation
3. treatment usually involves building up IgG response (desensitize); new methods involve monoclonal antibodies developed against the constant portion of the IgE antibodies
2. Type II hypersensitivity -- antibody-mediated cell lysis; confusion about what to attack
   
1. transfusion reactions -- IgM antibodies present for antigens not on red blood cells
2. Rh problems with newborns -- IgG attack on red blood cells with Rh antigen in the uterus; not problem in uterus because breakdown products can be destroyed by enzymes from mother; this is not the case in newborns
3. Type III hypersensitivity -- antigen-antibody complexes not removed fast enough, cause reactions including skin rashes, joint pain, kidney problems
4. Type IV hypersensitivity -- helper T-cell mediated; delayed 2 to 3 days
1. contact dermatitis
5. transplant immunity -- blocked by cyclosporins (inhibit T-cell reproduction) and monoclonal antibodies to IL-2 receptors
6. autoimmune diseases -- tissue destruction by immune system
1. examples:  insulin-dependent diabetes mellitus (T-cell); Grave's disease (antibodies to thyroid hormone); myasthenia gravis (antibodies to acetylcholine receptors); etc.
2. treatment with immunosuppressors (cyclosporin) and anti-inflammatories; oral tolerance?
7. immunodeficiencies -- system not working properly; genetic absence of components or loss of components through infections (measles, syphilis, leprosy, malaria, HIV)
8. superantigens -- bind to TCR's outside of the normal binding site; this means that they can bind to TCR's on different clones of T-cells, activating more types of cells than normal; result is a very large response that can be damaging to the body
   
2. Medications (chapter 21)
   
1. terminology -- see Table 21.1
1. minimum inhibitory and bacteriocidal concentrations; Kirby-Bauer disc diffusion
2. adverse effects
1. allergic reactions
2. toxic effects on kidneys, peripheral nervous system; shut-down of blood cell production possible with chloramphenicol; some of the more toxic forms used topically
3. resistance mechanisms
1. drug-inactivating enzymes
2. mutational changes in the target
3. decreased drug uptake
4. increased elimination through efflux pumps
4. classes of antibacterials and their mechanisms
1. interfere with peptidoglycan synthesis
1. beta-lactams -- block cross-linking in peptidoglycan (competitive inhibitor of the enzyme; some bacteria produce beta-lactamases that digest beta-lactam
1. penicillins -- penicillin, amoxicillin, ampicillin, etc.
2. cephalosporins -- cefaclor, cefixime, etc.
3. carbapenems
4. monobactams
2. vancomycin -- blocks cross-linking by binding to the linker peptide; can't pass through outer membrane of Gram negatives; resistance by mutations in the linker peptides
3. bacitracin -- blocks transport of precursors from cytoplasm
2. interfere with protein synthesis
1. aminoglycosides (streptomycin, gentamycin, neomycin) -- bind to small subunit of ribosome and blocks initiation; enter cell through active transport involving the proton motive force and respiratory chains so more effective against aerobes
2. tetracyclines bind to small subunit and block tRNA attachment; resistence through efflux pumps
3. macrolides (erythromycin) bind to large subunit; resistance through mutational changes in binding site
4. chloramphenicol binds to large subunit and prevents peptide bond formation
5. lincosamides bind to large subunit
6. oxazolidinones bind to large subunit and prevent initiation
7. streptogramins bind to two sites on teh large subunit and inhibit two independent steps in protein synthesis
3. interfere with DNA and RNA formation
1. fluoroquinalones (ciprofloxacin) inhibit proteins involved in the twisting of DNA
2. rifamycins (rifampin) block RNA polymerase and transcription
4. block general metabolic pathways
1. sulfonamides competitively inhibit enzyme that uses para-aminobenzoic acid in the folic acid pathway (not found in humans); resistance through alternative enzyme
2. trimethoprim inhibits the next step in the pathway; resistance through an alternative enzyme
5. interfere with cell membrane structure
1. polymyxin B makes cell membranes leaky
5. classes of anti-viral medications
1. block uncoating of the virus (influenza only)
2. block nucleic acid synthesis
1. nucleoside analogues (acyclovir, zidovudine, didanosine, lamuvidine) -- competitive inhibitors; must be converted to nucleotide analogue (phosphates added)
2. polymerase inhibitors and reverse transcriptase inhibitors that bind away from the binding site
3. protease inhibitors -- block cutting of some viral proteins into smaller, active subunits
4. neuraminidase inhibitors -- block neuraminidase in influenza
6. classes of anti-fungal agents
1. cell membrane formation -- target production/function of ergosterol, found in fungi, but not humans
1. polyenes (nystatin, amphotericin B) bind to ergosterol
2. azoles (imidazole) block the formation of ergosterol
2. griseofulvin seems to block spindle formation
3. flucytosine converted by yeast cells to a form that blocks DNA synthesis