Micro FINAL: UNIT 4 **KEY TERMS**

• -INNATE IMMUNITY!
• -complement: proteins in serum; work with or w/o antibodies to destroy cells to get rid of cellular microbes & opsonization
• -Interferons: made by virally infected cells; help protect other cells from infection by making warning proteins (non-specific; for any virus)
• -Phagocytosis by Macrophages (myeloid mononuclear) & Neutrophils (polymorpho granulocyte): engulfment & digestion of something by a cell; produce cytokines/chemokines

• a. Opsonization: microbe is coated for recognization by complement proteins -->Lysis of pathogen: poking holes in membrane “Membrane Attack Complex”
• b. Two pathways: Alternative & Classical

• Alternative pathway: active all the time; doesn’t require antibodies. C3B (activated protein) binds to ANY cells and assembles another complement proteins (C35,etc): host cells inactivate this BUT in “foreign” cells it leads to Opsonization & MAC formation
• Classical pathway: requires antibodies in specific immunity

• Produce by neutrophils and macrophages;chemical signals used by immune cells; combined actions lead to inflammation at infection site
• --> 4 signs of inflamation: Heat & Redness (increased blood flow), pain, and swelling (increased permeability of blood vessels).
• a. Chemokine IL-8: recruits more phagocytes.
• b. Cytokines IL-1, IL-6 &TNF-alpha: cause fever (WBC work better since bacteria dislike heat) and allow immune cells to work better.

complement proteins (C3B-activated) are on the antigens; recognized by macrophages/neutrophils that have "complement receptors" that bind C3B--> then CD14 proteins on macrophages then bind to LPS cells (gram neg) --> works with TLR4 receptor on macrophage (to start transcription/translation of genes to start making inflammatory cytokine).

Then macrophage performs phagocytosis by joining a phagosome (vesicle/vehicle for antigen) and a lysosome (digestive enzymes) to make a phaglosome.Remaining bits become MHC2 peptide used on surface of APC cells. MHC2 antigen (a peptide from original gram neg cell) which later communicate with specific immunity

**Note: Listeria monocytogenes: escape the phagosome & Mycobacterium tuberculosis adapted to evade this step**

• "Major histocompatibility complex": proteins located on surface of host cells and present antigenic peptides to T Cells; two types w/ 6 diff complexes: half are MHC 1 and rest are MHC 2.
• -Polymorphisms :MHC vary in alleles, reason why blood transfusions trigger immune responses.
• -->MHC 1: displays intracellular pathogen peptides on all host cells; recognized by cytotoxic T Lymphocytes ( CTL/ CD8+ T cells); HLA-A,B&C

-->MHC 2: surface of APC ONLY; displays peptides from phagocytosed pathogens like bacteria; Recognized by T Helper Cell (CD4+ T cells= first to be alerted)

• -based on the constant region;
• -these isotypes can switch (retain same specificity but use a diff constant region thru alterations in DNA by editing cutting/splicing out variable region genes in RNA)

a. IgM: membrane bound; 1st antibody secreted; works w/ classical complement pathway (specific); works in agglutination

b. IgG: Blood/tissues; most common in blood; type 2& 3 Hypersensitive reaction (see later notes)

c. IgA: mucosa/secretions (breastmilk)

d. IgD: membrane bound

e. IgE: works w/ mast cells to respond to allergies & parasitic functions; involved in Type I Hypersens. Reaction

• Naïve B cells has membrane bound antibody which encounters antigen==> differentiate into plasma cell and secretes antibody which binds to antigen.
• a. Naïve B cell have membrane bound antibody which allows to recognize antigen (IgM or IgD)
• b. Secreted antibody: after activation and differentiation, naïve B cells become plasma cells (clones)-secrete same/monoclonal antibodies into plasma

• THREE examples of how antibodies specifically fight infections:
• i. Bind & neutralize exotoxins/viruses by surrounding membrane so that virus can’t use surface proteins to activate at target site after traveling in blood stream.
• ii. Opsonization: bind to bacteria and help phagocytose small cellular pathogens.
• iii. Activate complement (innate immunity)classical pathway to poke holes in pathogen cells; example of how innate and specific defense work together.

-“BOSS”; has CD4 & T Cell Receptor; has two roles:

i. antigen recognition: “Sounds alarm”, APC stimulates T Helper cell (CD4+) by 2 signals including MHC2/peptide; occurs in lymph nodes, spleen, tonsils, salivary glands, etc

• ii. activating other WBC’s: Naïve T helper cell produces cytokines (chem messengers) so that:
• (1)CD8+ cytotoxic T Cells are ready to kill infected host cells
• (2) B cell-->plasma cells which secrete antibodies
• (3) activate macrophages (phagocytosis); one of the cytokines is IL-2 (a cell growth factor; stimulates proliferation) *know it*

-CD14: bind to LPS of antigens

-TLR4 receptors: tells nucelus to start translation/scription to create cytokines

-Phagocytosis: Lysosome + phagosome

-makes the MHC2 angtigen/peptide complex to communicate w/ specific immunity

• -has CD8 and T Helper Cell
• -activated by CD4+ cells; divide and kill infected cells: Apoptosis (cell suicide)
• -CTL’s recognize MHC 1/antigen complex on host cells; perforins (pokes holes in cell membrane) and granzymes (proteases) allow programmed cell death/suicide; neat death so viruses are stuck inside
• -BETTER than necrosis: water balloon pops-->messy.

helps w/ cellular and humoral immunity and maintain tolerance (CD25)

1. ANTIGEN RECOGNITION (“Sounding the Alarm”): Antigen Presenting cells stimulate CD4+ (helper T cells) w/ at least 2 signals such as MHCII/Peptide which interacts with T-Cell Receptor on the T Helper cell. Once activated, CD4+/T Helper Cell produces IL-2 cytokine (growth factor) so that T-Cells can divide and proliferate.

• 2. ACTIVATION (“Prepare to Fight”): Activated CD4+ makes cytokines that will activate other T-Cells such as CTL’s to become ready to kill infected host cells, B Cells to become plasma cells and macrophages activated.
• -->B & T cells undergo Clonal expansion (division and duplication of many identical daughter cells) and Differentiation (cytokines produce development changes in the cells to express new genes when encountering the antigen)
• -->Note: “Clonal Selection hypothesis”: T & B cells already have receptor specificity before exposure to the antigen; so clonal expansion and differentiation is choosing from one of these “pre-made” cells (one billion variations in receptor specificity) and cloning it.
• -->“Gene Rearrangement”: The molecular reason for the diversity of receptor specificity; DNA of T/B Cells is shorter due to rearrangement or changes during cell development. Occurs by splicing DNA segments and ligating remaining pieces (and mistakes just add to diversity)

3. EFFECTOR PHASE: Pathogens/infected host cells are destroyed; B Cells make antibodies; Macrophages phagocytose & kill pathogens BETTER; CTLs kills infected cells (apoptosis)

• Type I ( IgE mediated, soluble antigen): response to harmless antigen so that Mast Cells (in tissues; release cytokines, histamine, leukotrienes which cause the symptoms) bind to IgE and IgE antbodies bind antigen when it is encountered again.
• --> Systemic symptoms: reacts to ingest/injected allergens and release of mast cell chemicals-->drop in BP and anaphylactic shock.
• --> Local symptoms: caused by asthma, hay fever symptoms & hives; very sensitive to small amts of antigen.

Type II (IgG): penicillin allergies & RH incompatibility

Type III (IgG): Immune complexes like Lupus; antibodies grab onto each other and stick to an antigen to create clumps where large clumps are phagocytosed but intermediate sized get stuck in tissue and cause pain.

Type IV (delayed): Based on T Cell reactions; memory T cells respond quickly to macrophage that present antigen and active macrophages to cause inflammation; TB skin test

• -->Virion features
• • Linear dsDNA genome: different from previous viruses
• • Very large genome
• • Icosahedral capsid 20 small triangles
• • Envelope with glycoproteins (sugars + protein)

-->Who gets infected? Humans, Primates, Reptiles, Elephants, etc.

--> Common feature of herpes viruses: Ability to establish latency as closed circular DNA; dna can sit in infected cell and wait

• - Majority of adults in U.S. have virus by age 40
• -Most people have no symptoms; establishes latency
• -->• Possible signs and symptoms: fever, sore throat, fatigue, swollen glands

• -->CMV transmission
• Most bodily fluids including: saliva, urine, tears, breastmilk and blood & Also sexually transmitted

-->CMV disease in babies: CMV passed to fetus from mother; 1 out of 5 children with CMV have severe developmental disabilities

--> HIV patients can become blind

1. Attachment: HIV binds using gp120 that attaches CD4 & CCR5 on the T Helper cell.

2. Fusion: Uses gp41 and entry into Host cell

3. Viral RNA is now sitting is host cytoplasm, gets copied by Reverse Transcription enzyme and makes DNA copy from the RNA (opposite of transcription) to eventually make dsDNA (ProViral DNA)

4. Integration: Proviral dna ends up in nucleus; Integrase enzyme intergrates proviral dna into chromosome (explains why virus is hard to remove)

5. Biosynthesis: making more RNA/viral proteins, RT enzymes to make more virions

6. Protease: enzyme that cuts polyproteins (three proteins in a row)

7. Assembly

8. Release of virions

• how do they work:
• • Block action of viral proteins; like enzyme inhibitors (enzymees are proteins)
• • Reverse transcriptase inhibitors
• DRUGS:

1. Nucleoside analog RT inhibitor: nucleotide before the phosphate is added to it; "fake nucleotide", looks like a normal nucelotide but it's not, it's a terminator rather than ATGC, stop RT from making viral RNA when the RT runs into this fake nucleotide terminator ex: zidovudine (AZT) first drug used in 1980’s; strict regimen

2. Non-Nucleoside RT inhibitors: act like allosteric inhibitors; Ex: Nevirapine: shown to stop transmission of HIV from preggie mom to child

3. More antiretroviral drugs:(targets) Protease Inhibitors, Fusion inhibitors-gp41, Integrase inhibitors: inhibits proviral dna integrated into chromosome, CCR5 inhibitor: unusual b/c it blocks OUR proteins that HIV uses to get into host cells- new in 2007

• • Highly active anti-retroviral therapy; triple drug threrapy to avoid resistance
• • Use a combination of several antiretrovirals at the same time ( e.g. 2 RT inhibitors and a protease inhibitor)

enzyme-linked immunosorbent assay

• --> uses specificity of antibodies to captures and stick to antigen
• --> detection method: using enzyme
• -->Limitations of test: people can be poor produces of antibodies and won't be detected, false positives are possible: unrelated antibody reacts w/ antigent nospecifically
• -->AVOID SOURCES of error: use replicates, wash properly, use positive & neg controls and follow instructions carefully.

A first antibody is adhered to the well, then the solution that may contain antigen (pentagons) is added, and finally an enzyme-labeled antibody against different parts of the antigen. Substrate is again converted to colored product.

• 
• 1. Antibody is captured with specificity for the protein that is used (pretreated)

2. Human blood Sample then added to well and incubated, washed off.

3. Detection of antibody: secondary antibodies which is attached to an enzyme ( ex: HRP) is added that will specifically bind the antigen in sample if antigen is present

4. Substrate for HRP added and color change is observed if HRP is prsent in wells.

• Antigen (pentagons) is bound to the bottom of a well, and patient's serum that may contain antibodies to that antigen is added. Next, enzyme-labeled second antibody (made in a diff species)to the first antibody is added. A colorless enzyme substrate (S) is converted into a visible
• product (P)
• 

• 1: antigen is coated onto plasatic wells.
• 2. Serum from patient is added to wells; plate is washed
• 3. detection antibody w/ enzyme attached is added. Wash.
• 4. Enzyme substrate is added: color change indicates patient has antibodies

Enveloped, ssRNA; segmented type A genome pandemics (Influenza)

enveloped, dsRNA; latency; large genome; ex: VZV, HSV1&2, EBV,CMV,HHV8

enveloped,2copies +ssRNA; latency, proviral integration, reverse transctipion; ex: lentivirus ( HIV &SIV) and oncoviruses

icoshadedral; +ssRNA; smallest-22 to 20 nm; ex: Rhinoviruses/cold and Polio

enveloped, -ssRNA; cause of several childhood diseases;ex: RSV, measles-rubeola, and mumbs

complex shaped; dsDNA; VERY LARGE-250 nm; ex: smallpox-size: 20-250 Nm)

• -Common Causes of viral respiratory infections
• -Trasmitted: Respiratory droplets
• -Characteristics: enveloped negative ssRNA (complementary mRNA) virus, easily killed by soap/water/disinfectants
• -Intials symptoms: wheezing, cough, runny nose, croup in babies (inflammation of larynx/trachea which lead to difficulty in breathing, a barking cough and noisy breathing)
• -complications: the most common cause of bronchiolitis especially in babies (Inflammation of the bronchioles) and pneumonia in children <12 months

-Diagnosis: look for viral RNA, viral antigens/proteins, viral antibodies against RSV, rapid test is also possible.

-Treatment: For severe infections, oxygen is given or a ventilator may be used.

-Prevention: No available vaccine; there is a "Palivizumab" monocloncal (all variable regions attack RSV) antibody for children at high risk for severe disease; circulate in baby and give protection by neutralizing RSV

*syncytium*: when a several viruses infect several host cells and then virus causes infected cells to merge into one giant multinucleated cell.

• -->family: orthomyxovirus
• -->Three types: A (associated with pandemics; mutates quickly), B and C
• -->structure of Type A:
• 1. 8 nuclear capsids: ss-RNA helical capsids; haploid genes (only 1 copy of every gene; not repeated anywhere else)
• 2. Envelope w/ layer under called M1 proteins and embedded M2 protein (ion channel)
• 3: ON the surface:
• - Hemaglutinin(HA): glycoprotiein; "glutinin"(clumping); sticks to cell it attacks, binding sialic acid which is on host cell (used in attachment)
• -Neuraminidase (NA): enzyme; cuts sialic acid so that the virions can release after infecting the host cell.

• Antigenic variation: change the outside of virus; can change in two ways.
• 1. Antigenic drift: slight change in either HA or NA; from a spontaneous mutation, might not get too protected from flu vaccines
• 2. Antigenic Shift: Big change in protein; completely new and different HA or NA; occurs by genetic reassortment (the virus actually gains a new HA or NA gene; done when 2 viruses infect same cell and same time)ex: H2N2 became H3N2

Quadruple reassortment: swin flu origin pandemic 2009; new H1V1 (HA & NA subtypes)

A: antibodies: Anti-B; Receives: A & O

B: antibodies: Anti-A ; Receives: B & O

AB: antibodies: none ; Receives: ALL (universal receiver)

O: antibodies: Anti-A & Anti B (both) ; Receives: ONLY O (UNIVERSAL DONOR)

RH Incompatiblility: RH- mom & RH + baby

Hemolytic disease in newborn: RH antibodies of mom attack the babies RBC's

visible clumping of solids in a liquid; two kinds: direct and indirect

A.direct agglutination: blood typing lab; soluble antibody protein dissolves in liquid interacts with antigen like "A" or "B" on cell ex: hemagglutination of RBC to detect blood type (lab)

• B.indirect/passive agglutination: solube antigen or antibody is coated onto a latex bead;(take a bead and attach constant region of antibody specific to a microbe, mix beads with sample and if you see clumping then the microbe is there)
• ex: "Rapid Strep Test" use small latex beads coated with antibody against Staph aureus & Strep pyogenes plus patient sample

• antibody-antigen interaction will inhibit a biological activity; cells to detox: antibodies may inhibit the ability of a virus to infect a cell; antibody bind to antigens and stop them from infectiing other cells.
• --> Neutralization assay: control: virus and cells--> cells are dead. To see if someone has antibodies against something, you add antibodies to the control and if the cells are surviving then you know that antibodies are working and neutralizing the virus.

fluorescently labeled monoclonal antibodies are incubated with cells to detect microbial antigen or normal proteins; usually fluorescent red/yellow-green molecules are used; detected by microscopy using a special microscoby (ex: Giardia lamblia intestinalis); detected by flow cytometry using a FACS machine -flow cytometer (ex: detection of CD4+ T cells and CD8+ T cells in HIV patients)

(monoclonal antibody w/ attached flourescent molecule to bind to specific antigen; then use flurescent molecule to observe it; can also be used to differentiate T and B cells)

• --> direct method: primary antibody is attached to a fluroescent molecule
• --> indirect method: secondary antibody is attached to a fluorescent molecule

proteins (antigens) are separated by size or electrophoresis gel and detected by antibody "sandwich" using radioactivity or visible light detection (ex: used to confirm a positive HIV antibody ELISA test); present antibodies will stick... color response. End result: bands. Does the patient respond to one, two or three proteins from microbe. ex: HIV has three proteins..

(other notes: The basic technique of a Western blot involves sorting proteins by length on a gel and then probing the gel with antibodies that react to the proteins that are being searched for. However, when Western blots are used for HIV testing, the process is actually performed in reverse. Instead of unknown proteins being tested for with known antibodies, labs work with prepared protein samples, and look to see if there are any antibodies in a person's blood that stick to them.)

• **STEPS**
• 1.Proteins from a known bacterium or virus are separated by an electric current in electrophoresis
• 2. The proteins are then transferred to a filter by blotting.
• 3. Patients serum is washed over the filter. IF the patient has antibodies to one of the proteins in the filter then antiboidies and protein will combine. Anti human serum linked to an enzyme is then washed over the filter.
• 4. THis will be made visible as a colored band on the filter after addition of the enzyme's substrate

use monoclonal antibodies; sample interacts with labeled antibody, then with capture antibody after moving through a lateral flow membrane (ex: canine parvovirus test, RDT or Rapid TB test, Oraquick test for HIV (in video), west nile virus, Treponema and pregnancy test is similiar...ehh? (http://www.rapid-diagnostics.org/tech-lateral.htm)

The bacteria is killed (Bordetella pertussis: bacterium in old DPT vaccine) or the virus is inactivated so the microbe can't cause disease Ex:Bordetella pertusis (causes whooping cough) old DPT vaccine, Salk pollio vaccine-injected;also standard flu shot.

typically used for viruses; weakened virus but still alive so it can REPRODUCE, weakened-should not cause serious illness, may be missing some genes

• ex: Sabrine oral polio vaccine
• MMR-measels, mumps, rubella
• varicella-chicken pox virus, Rotateque :diahrrheal rota virus

treated exotoxin inactivated by fomaldehyde

• EX: Diphtheria and Tetanus toxoids in DPT-not used anymore,
• DTap -inital baby shots; more antigen in them
• Tdap vaccines-booster shot,less antigen since less capitals; for teens.

Uses antigen from a piece of the microbe OR dna by using restriction enzymes and placing it in something else like yeast and making more proteins ; may be genetically engineered protein

• Ex:
• new pertussin vaccine
• DTaP-babies; Tdap for older children and adults
• Hepatitis B vaccine: using yeast, and malaria vaccine-children (clinical trials)

• capsule is linked to a carrier protein--stimulates a better response in young children since babies respond better to proteins but anyone can take it.
• ex1: Hib children's vaccine for Heamopilus influenzae-see meningitis
• ex 2: MCV4 vaccine effective against 4 strains of N. meningitidis
• ex3: PCB: Pneumococcus conjugated vaccine (aslo on meningitidis handout)

• experimental; take patience cells treat em w/ cytokines and develop dendritic cells and give antigens to dendritic cells in lab to train so that in the future the dendritic cells will present antigens better; self dendritic cells are grown in the lab and pulsed w/ antigen from a tumor/microbe and given back to the patient..
• ex: tried against cancer w/ DC+ tumor anitgens

-live attenuated: Oral "sabine" vaccine; swallow it, administered the same way you get the disease; but you can get disease if you have weak immune respnse and can transmit it to others /:

-inactivated/injected: "Salk" vaccine; IPV

-live attenuated: vaccinia (skin scraping); live viral, the only one we have and military still get it; ppl w/ eczema or skin infections can't get this vaccine b/c it reacts badly

-inactivated/injected: none

Virology: Part of flavivirus family (includes Encephalitis and dengue fever); 40-60 nm in size, enveloped, icosahedral, +ssRNA

Transmission:-vector: female culex mosquitor carries virus and infects bird-crows/jays (host: life cycle happens) and humans are dead end.

• signs/symptoms: Usually appears up to 15 days after a bite from an infected mosquitos)
• -Most ppl are asymptomatic
• -20% of infected get "West Nile Fever": Including fever, headache, bodyaches, swollen lymph nodes, and occassional rash.
• -1 in 50 ppl exposed develop severe disease "neuroinvasive": including headache, high fever, neck stiffness, convulsions, confusion, and other symptoms of meningitis or encephalitis (45% of population that developed disease had severe form in 2004 U.S)
• -Risk factors: older age(illness and death)
• - Younger patients may end up with long term neurological problems

• Diagnosis:
• 1. RT-PCR: genetic test to detect viral RNA; covert RNA to dna (PCR)

2. MAC-ELISA: used for initial detection of IgM antibodies against West Nile virus in either serum/CSF. False Positive is possible if person has been immunized against/exposed to related virus (yellow fever). Serum IgM antibodies can exist for about a year after virus exposure.

3. PRNT (Plague reduction neutralization test): type of neutralization assay; take virus incubated w/antibodies, do neutralization assay and then spread on a dish to look for reduction of plaques in order to test for antibodies; confirm cases of West Nile virus; takes a week or more b/c involved incubating patient serum w/ a virus from lab then infecting tissue culture cells; tissue cult cells checked to see if serum was able to reduce plaque formation

**Treatment: None; IV fluids and respiratory support are provided as support in severe cases.

• **Prevention:
• -Use mosquito repellent with DEET, get rid of standing water, wear loose fitting, long sleeved shirts and paths when mosquitos are active (dawn and dusk).
• -A plant based lemon eucalyptus insect repellent (repel) is CDC's alt to DEET.
• - Picaridin: different, newly approved insect repllent

**Vaccine: No human vaccine exxists; 2 dose vaccine for horses.