Microbiology

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Microbiology Bio 205

• Test 3
• Dr. Rhoads
• I will NOT test you on text in GREEN.

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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)

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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?

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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

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• 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

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• 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

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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

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• 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.

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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???

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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

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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
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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
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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

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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
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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.

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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.

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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.

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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

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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 ???

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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

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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

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Edible vaccines
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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

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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

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

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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.

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Serology tests

• Compliment fixation indirect
• Fixation phase Indicator phase

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Continued

• Fluorescent antibody a Fl precipitin test -
  direct

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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

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Continued

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

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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

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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?)

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ABO blood typing

• type O universal donor type AB universal
  acceptor
• foreign antigen is the problem, NOT foreign
  antibody

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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
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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

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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

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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.

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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

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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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