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Microbiology

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Title: Microbiology


1
Microbiology Bio 205
  • Test 3
  • Dr. Rhoads
  • I will NOT test you on text in GREEN.

2
Immunology
  • Very complex we are ignorant a frontier
    science
  • Includes more than often given credit for
    microbes, etc.
  • Our great friend? Most of the time, yes.
  • Non-specific (innate) vs Specific (adaptive)

3
Non-specific immunity
  • Non-specific immunity involves 3 lines of
    defense
  • The most basic line of defense involves simple
    things
  • age Toxoplasma, S. agalactiae ? ?
    Klebsiella, VRE
  • diet
  • stress
  • sleep
  • exercise
  • exposure to pathogens your job?
  • compromised barriers your job?

4
Non-specific (innate) immunity
  • The next level of defense involves 3 things
  • 1. Physical / mechanical
  • skin keratin acid salt ?AW
  • tracheal cilia
  • urinary tract natural flushing action
  • lacrimal apparatus glands ducts
  • 2. Chemical
  • low pH on skin, in GI tract, in female urethra
  • lysozyme in tears, sweat, kidneys, lysosomes, etc
  • beta lysin vs Gram positives in blood (from
    platelets)
  • siderophores bio-chelators
  • many other examples here

5
  • 3. Microbial Dont kill your friends!
    Enemies or allies?
  • Skin 1 Propionibacterium (acnes), S.
    epidermidis, LOTS more 182 species!
  • Oral cavity
  • pre-teeth aerotollerants S. mutans, S.
    sobrinus
  • post-teeth obligate anaerobes
    Actinobacillus
  • consider plaque and tooth decay
    Streps perio Actinobacillus, Porphyrimonas,
    Bacteroides, Fusobacterium, Campylobacter,
    Treponema

6
  • 3. Microbial continued
  • GI tract pH? as approach stomach, pH? as
    depart - stomach pH 2.5
    Lactobacillus, S. lactis, other? - colon
    microbes 1/3 1/2 the weight of feces!!!
    facultatives enterics 1 E. coli
  • obl. anaerobes x 103 1st is
    Bifidobacterium
  • then Bacteroides (1) Clostridium
  • Female genitourinary tract your friend
    Lactobacillus

7
Non-specific immunityyour 3rd level of defense
cellular
  • Leukocytes are WBCs ALL of them (specific
    non-specific)
  • Leukocytes include Lymphocytes (specific immune
    cells)
  • Non-specific immune cells granulocytes
    agranulocytes
  • 1. Granulocytes polymorphonuclear leukocytes
  • neutrophils abundant non-specific phagocytes
  • eosinophils eukaryote (esp. helminth)
    phagocytes
  • basophils secrete cytokines (cell signaling
    proteins) involved in regulating the immune
    response
  • a) histamine vasodilation (mainly
    arterioles)
  • increased venous permeability
  • b) heparin anti-coagulant blood
    thinner

8
  • 2. Agranulocytes mononuclear phagocytes
  • a) macrophages
  • functions
  • 1. highly efficient phagocytes
  • 2. wound repair
  • 3. antigen presenting cells (macrophages are not
    the only APCs. Others include Dendritic cells
    and certain B cells). They present antigen to
    naïve B cells
  • 4. fever interleukin ? hypothalamus
  • 5. Inflamation how? Many ways
  • Leukotrienes group of cytokines involved in
    sustaining inflamation induced by a non-specific
    immune response.

9
Specific (adaptive) immunity
  • 4 characteristics
  • Antigen (Ag) specificity targets a particular
    bad guy
  • Antigen memory carries (remembers) antigen shape
  • Self-tollerance (???)
  • It is about antigen re-exposure, not the 1st
    exposure
  • Our main area of ignorance in medical science???

10
Specific (adaptive) immunity
  • Ag specificity from various mechanisms. Just a
    few are
  • 1. APCs Macrophage APCs present Ag to B cells,
    dendritic cell APCs present Ag to naïve T cells,
    and more.
  • 2. Mucosal antigen sampling M-cells over Peyers
    patches transfer intestinal contents to
    lymphocytes beneath
  • 3. Antigen sampling across other epithelial
    barriers
  • Major components 1. Humoral (next slide)
    2. Cellular

11
Specific immune system - Humoral
  • Humoral fluids NOT cells plasma proteins
  • Antibodies immunoglobulin
  • Complement
  • Cytokines proteins produced by a variety of
    cell types involved in cell communication roles
    including, but not limited to immuno-regulation.
  • Ex. Interferons immune proteins secreted in
    the presence of dsDNA (viral) who then activate
    macrohages and T cells. They interfere with
    viral success. Interferon also has activity
    against parasites cancer cells

Antigen binding site (Fab region of
heavy chain) Fc
12
Antibody structure
Heavy chain H
Fab region antigen binding, highly variable
region
Light chain L
Constant region
Fc region interacts with cell surface
receptors
Fc end of heavy chain determines Ig type G M A
D E Fab region (or both chains) is highly
variable
13
Specific immune system Humoralcontinued
  • Prevalence
  • IgG 1. 1 most abundant type 80
  • 2. involved in complement fixation (and IgM)
  • 3. capable of placental transfer
  • IgM 1. largest antibody type pentameric
  • 2. 1st type produced in response to infection
    5-15
  • 3. involved in complement fixation (and IgG)
    4. primary RBC A,B Ab cant cross placenta!
  • IgA 1. 1st encountered in mucosal glandular
    5-15 secretions other epithelial
    surfaces
  • IgD little known but thought to fnc as
    Ag-receptor 0.2 on ( expressed by) memory
    B-cell surface
  • -- IgE 1. involved in Type 1 hypersensitivity
    0.002
  • 2. with Eosinophils vs helminths, fungi, others

14
Specific immune system - Cellular
  • Cellular Lymphocytes B-cells T-cells
  • B-cells involved in humoral response
  • responsible for direct antigen
    interaction
  • antigen memory
  • antibody production and
    lots more
  • plasma B-cells
  • APC (Ag) TH cell
  • memory B-cells
  • plasma cell make Ab (not D?) lots of cells
    short life
  • memory cell remember few cells long life
    IgD?
  • they differentiate upon Ag
    re-exposure

undifferentiated naïve B-cell
differentiation
15
Genetics of antibody production
  • Could the human genome contain all of the coding
    for the production of each discrete antibody?
    No way! 1. Not enough space for gt 10 million
    antibody genes 2. Variability from a duplicative
    process? replication
  • So, how is it done? Plasma cell DNA coding for
    amino acid sequences at variable regions exist in
    sections, at least 3 sections for both the L and
    H chains. For each section, up to 300 variations
    exist. Combinations of these variations result
    in the huge variability necessary to accommodate
    the plethora of antigens that exist.

16
T-cells (good things)
  • TH ie. CD4 Tcells stimulate (help) B-cells to
    differentiate.
  • TR regulate B-cell function the brakes
    formerly called TS cells
  • TC cytotoxic T-cells (or T killer cells)
    directly kill via perforins (vs membranes),
    proteases, etc.
  • 1. intracellular pathogens 2. cancer cells
  • 3. closely related foreign cells transplant
    rejection
  • TM rapid response to Ag re-exposure like memory
    B cells Fnc (among others) like TC cells in
    regard to direct attack of pathogens cancer
    cells
  • NK 3rd type of cell differentiated from common
    lymphoid cell also (like Tc) directly attack
    using perforins, etc., but they 1. are less
    specific bridge gap between adaptive and
    innate
  • 2. kill the host cell rather than just the
    intracellular pathogen
  • 3. are NOT B or T-cell type lymphocytes
    although they do come from lymphoblasts (like B
    T cells), and therefore are lymphocytes, they do
    not exhibit the Ag specificity of B cells, and do
    not mature as do T cells. They also do not carry
    surface markers common to B and T cells.

17
Continued - T-cells (bad things) NOT on the
test
  • T-cells cell-mediated? Yes and no
  • Some types of T-cells produce compounds
    (cytokines) that interact with the cells, whereas
    other types of T-cells directly interact with the
    bad guys or other cells.
  • T-cells produce secrete cytokines proteins
    that regulate cell function and control
    interactions / communication between cells.
    Among other things, the cytokines include the
    interleukins (made by virtually all leukocytes
    and many other cells), lymphokines (T cells make
    to hail macrophages) and cell signal molecules
    (such as tumor necrosis factor), and the
    interferons, which trigger inflammation and
    respond to infections.
  • Some T-cells / cytokines result in pathology.
    Examples include TC cell liver damage during
    Hepatitis B virus infection, global TD cell
    tissue damage in tertiary syphilis, perhaps
    multiple schlerosis and type 1 diabetes, and who
    knows what else (hog with a watch). This is not
    the first time that you have heard about your
    immune systems causing pathology.

18
Vaccination - Types of immunity
  • What is the point of vaccination?
  • Passive Active
  • Natural transplacental (IgG)
    infection
  • colostrum (G, M, AD? E?)
  • Artificial serum transfusion vaccination
  • Active
  • Advantages lasts longer (lifetime? VS weeks,
    months)
  • more specific more effective
  • Disadvantage not immediately effective

19
3 types of vaccines
  • 1. Whole agent / cellular the whole bacterium,
    virus, etc.
  • Killed / inactivated non-viable cell (or virus)
  • advantages safer? cheaper
  • BUT less effective cant multiply altered
  • ex. Salk injectable killed polio (IPV)
  • Attenuated viable but non-pathogenic
  • advantage more effective via can multiply
    less altered
  • ex. Albert Sabin oral attenuated polio (OPV)
  • disadvantages expense danger?
    iatrogenic (contact)
    polio ?Flumist is attenuated ???

20
Continued
  • 2. Sub-unit / sub-cellular Ag only?
    advantage safer!
  • ex. bacterial capsule (HIb) or fimbriae
    (Neisseria)
  • viral envelope (flu) or capsid (HBV)
  • 2 ways to make them 1. fragment purify
    antigen 2. recombinant methods
  • toxoid inactivated toxin ex. DPT DTaP
    Tdap etc.
  • conjugated combination of surface Ag and
    toxoid
  • adjuvant alum aluminum salts chelators
    others

21
continued
  • 3. Recombinant vaccines
  • advantages fastidious pathogen? No problem
  • safe to the max, but.who knows?
  • examples lets start simple progress to out
    there
  • Std. subunit like we have already discussed
  • edible vaccines E. coli, Cholera, HBV, other?
  • potato, banana, other? ? slide
  • Trojan horse vaccine HIV HBV coming soon! ?
  • DNA vaccine 1. inject naked DNA not
    persistent
  • 2. direct transfection of somatic cells
  • will it happen? malaria? rabies? HIV?
  • Monoclonal Ab technology not a vaccine, but ?
    slide

22
Edible vaccines
23
Monoclonal antibodies
Inject antigen of choice to stimulate naïve
B-cell
Cancerous lymphocyte Myeloma
  • Antigen stimulated B-cell Myeloma Hybridoma
  • Product of the Hybridoma monoclonal antibodies
  • Applications hormone regulation, birth control,
    vs cancer, etc. Could also use to make
    antibodies for diagnostic purposes

24
Serology as a diagnostic tool
  • Old days diagnose via symptoms ID pathogen
    (maybe)
  • Serology detecting Ag - Ab interaction
  • Indirect use Ag to look for Ab ex. old
    rapid Strep test
  • Direct use Ab to look for Ag where to get
    the Ab?
  • Advantages of direct 1. early detection
    (before self Ab titer has risen) 2. late
    detection (after self Ab titer has diminished) 3.
    immune suppressed individuals
  • Disadvantages of direct 1. greater
    expense 2. requires more expertise,
    facilities, special storage, etc

25
  • How to detect the Ab Ag reaction?
  • 1. precipitin test see the lattice at
    zone-of-equivalence
  • 2. agglutination test ex. blood typing

26
continued
  • Advantages of serology
  • 1. Sensitive diagnose early infection
  • 2. Specific hard to mis-diagnose
  • 3. Fast
  • 4. Quantitative zone-of-equivalence
  • Various tests have been developed that increase
    the sensitivity of serology testing. See some of
    the examples on the next 4 slides.

27
Serology tests
  • Compliment fixation indirect
  • Fixation phase Indicator phase

28
Continued
  • Fluorescent antibody a Fl precipitin test -
    direct

29
Continued
  • Radio-immuno assay another indirect
    method used to be called RIA
    now is called RAST
  • adsorb antigen to surface
  • add your serumantibody present?
  • add radioactively labelled antibody (MC)
  • rinse measure radioactivity
  • The current RAST test (similar to RIA) is used to
    measure allergen-specific IgE in type 1
    hypersensitivity

30
Continued
  • ELISA expensive! Uses 2 batches of monoclonal
    Ab - direct

31
Immune disorders 3 categories
  • Hypersensitivity autoimmunity immune
    deficiency
  • 1st two a) resemble infection immune
    response
  • tissue damage
  • b) treatment anti-histamines,
    steroids, epinephrine, intravenous
    fluids
  • -------------------------------------------------
    -----------------------
  • Hypersensitivity look at the name over react
    allergy allergen re-exposure
  • 4 types types 1-3 involve antibody and
    something else
  • type 4 involves T-cells, but NOT antibody

32
Hypersensitivity
  • Type 1 immediate Ab (IgE) histamine
    (basophils )
  • a) atopic local / mild pollen, etc.
    hay fever, etc.
  • b) anaphylactic systemic / severe bee
    sting, antibiotics, food
  • What is the difference? Antigen type? Amount?
    ???
  • Type 2 cytolytic, cytotoxic IgG, M
    complement
  • Mainly RBC related, but not exclusively
  • ABO incompatibility what determines blood
    type? ? slide
  • Rh incompatibility 85 of people carry rhesus
    monkey Ag
  • Rh() Rh Ag on RBCs but no anti-Rh Ab in
    serum
  • infant hemolysis Rh(-) mother bears Rh()
    baby ? slide
  • RhoGam covers Rh Ag on babys RBC (or
    destroys?)

33
ABO blood typing
  • type O universal donor type AB universal
    acceptor
  • foreign antigen is the problem, NOT foreign
    antibody

34
Rh incompatibility infant hemolysis (HDN)
Can lead to a) kernicterus jaundice leading to
brain damage b) hydrops fetalis effusion
resulting in anemia damage to various
tissues and organs
35
continued
  • Type 3 immune complex IgG, M Ag complex
  • complex precipitates ? basement membrane
  • tissue damage via neutrophils, etc.
  • inject horse serum vs tetanus serum
    sickness
  • infect post-Streptococcal glomerulonephritis
  • others Farmers lung arthritis? lupus?
    malaria?
  • Type 4 delayed or cellular T-cells TC,
    TM, NK
  • takes gt 12 hrs to develop, peaks lt 2-3 days
    later
  • tissues affected are mainly surface /
    epithelial
  • contact dermatitis, TB test, skin graft
    rejection,
  • necrosis leprosy, HBV liver damage, 3
    syphylis, etc

36
Autoimmunity
  • Attacking self-Ag with no apparent external
    impetus
  • self-destruction
  • low self-tollerance
  • MHC problem? The Major Histocompatibility
    Complex (MHC) is a set of molecules displayed on
    cell surfaces that are responsible for lymphocyte
    recognition and "antigen presentation". The MHC
    molecules control the immune response through
    recognition of self and non-self.
  • Why? Prevailing theories
  • a) sequestered Ag theory CNS, eyes,
    thyroid, other?
  • b) loss of TS function
  • Some may not really be autoimmune conditions
  • could pathogens (viruses) be changing our
    antigens?
  • could pathogens be mimicking our antigens?
  • consider rheumatic fever

37
A few autoimmune diseases
  • Rheumatoid arthritis attacking protein in
    joints called rheumatoid factor tissue
    destruction, inflammation
  • Systemic lupus erythematosus all connective
    tissue, malar rash other epidermal lesions , CV
    tissue heart, blood, vessels joint pain
    virtually all organs 91 women men
    Great Imitator non-European women
  • Diabetes mellitus sweet fountain glucose
    passes kidneys destroys ability of
    pancreas to produce insulin, but not
    to produce glucagon.
  • Graves disease 1 form of hyperthyroidism
    therefore effects metabolic fnc 81 women
    men
  • Crohns disease chronic progressive inflamation
    of the GI tract, especially the bowel. Jews 3-5
    X incidence
  • Diagnosis ANA, other autoantibodies CRP
    (inflammation) etc.

38
Immune deficiency
  • Congenital developmental usually thymus
    problem rare bubble boy
  • Acquired steroids other medications
  • radiation
  • heavy metals (Hg)
  • cancer leukemia, etc.
  • infection HIV, rubeola, mono-viruses,
  • syphilis, many more

39
Antimicrobial drugs
  • History
  • Quinine in Europe 1600s vs malaria protozoan
  • Alexander Flemming 1928 discovery of
    penicillin
  • Paul Ehrlich same time? Arsenic compounds
    vs syphilis
  • Many microbes synthesize antibiotics, but most of
    our naturals are derived from 4 genera
  • Streptomyces (1 most), Bacillus,
    Penicillium, Cephalosporium
  • Naturals vancomycin, polymyxin, penicillin,
    cephalosporin
  • Semi-synthetics ampicillin, amoxicillin
  • Synthetics ciprofloxacin, isoniazid,
    methicillin?
  • Range mechanism of action

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