Prologue Ii

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Benefits of normal bacteria

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- assist in digestion & absorption of nutrients (bifidobacteria)
- stimulate host immune system and prevent colonization by pathogens (bifidobacteria, lactobacillus)
- acidify the vagina to prevent infection
- occupy space to prevent overgrowth/colonization of pathogens

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2 Mechanisms by which microorganisms cause disease

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Microbial factors:
- secreted toxins (tetanus, diptheria, cholera), host cell lysis (viral), cell death
Host response:
- inflammation, abnormal coagulation, vascular changes

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Frank vs. Opportunistic pathogen

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Frank pathogen: always causes disease when infected. Ex: HIV
Opportunistic pathogen: infection must coincide with other circumstances to get disease (antibiotic use, loss of beneficial bacteria, compromised immune system). Ex: toxoplasmosis, candida albicans, pneumocystis jerovicii

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Koch's postulates

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= a way to identify the etiologic agents of disease (often not able to fulfill with certain disease processes)
1. take a patient with a disease and isolate the potential pathogen from them in a pure culture
2. inoculate a susceptible animal with the pathogen and see if disease develops
3. Isolate the pathogen from the infected animal in pure culture

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Routes by which microbes enter the body

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- areosol/fomites: the primary route of most human infection is the respiratory tract; a fomite is any inanimate particle/object capable of carrying an infectious agent (ex: dust, ties, stethoscope)
- fecal/oral: usually via food/water contaminated with human waste
- sex
- penetrating trauma (wounds)
- bites

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Host and tissue specificity of infecting microorganisms

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- infecting pathogens recognize and bind specific cell surface molecules and so can only colonize certain hosts/cells
- Bacteria express adhesins (recognition proteins); different bacteria/strains express different adhesins

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Define virulence factors

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=substances secreted by pathogens that help cause infection in the host
- may be involved in: avoiding the host immune system, tissue colonization, entry/exit from host cells, deriving nutrients from host, etc
EX:
-bacterial toxins: endotoxins (structural component of the bacteria with toxic properties, lipopolysaccharide), exotoxins (secreted compounds, tentanus toxin)
- immunoglobulin proteases: secreted by some pathogens (strep pyogenes), break down host immunoglobulins, interfere with immune resonse
- capsules: carbohydrates of exterior of bacteria that prevent phagocytosis

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Define virulence

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= a quantitative estimate of pathogenicity.
- relates the transmission rate, infectious dose, and severity and duration of symptoms for a particular pathogen.
- usually measured by case/fatality ratio

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Mechanical and chemical immune defenses

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Mechanical: physical barriers to pathogens
- skin, tears in eyes, fluid flow in the respiratory tract and GI tract
Chemical: chemical/environmental barriers
- salt (sweat on skin), pH (low in stomach, vagina), fatty acids, secretions (lysozyme in tears), defensins (antimicrobial peptides secreted by microphages and other immune cells in the skin)

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3 genetic conditions that influence the ability of a pathogen to infect the host

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1. Sickle-cell disease: autosomal recessive mutations of hemoglobin. Cells lyse prematurely, conferring resistance to malaria b/c infected cells lyse before organism completes life cycle
2. G6P-DH deficiency are also resistant to malaria due to impaired parasite growth in RBCs due to poor metabolism
3. Deletion/mutation of CCR5 chemokine receptor confers resistance to HIV, because is normally required for the virus to enter cells

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Importance of the innate immune system in combating infection

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- first line of defense: physical/chemical barrier, generalized immune system
- required for activation of the adaptive immune system: T cells have to be activated by antigen-presenting cells (B cell require T cell activation)
- Studies in mice show that absence of innate immune system results in total failure to control infection, while loss of adaptive system still give some measure of infection control

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Activation and outcomes of the complement system of antibody presentation

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- A complement is a proteolytic cascade that activates zymogens
Triggers:
- Alternative pathway: presence of a microbe
- Classical pathway: recognition of antibody on pathogen surface
- Lectin pathway: presence of lectins on microbe surface
Activation: inactive C3 is cleaved into C3a and C3b.
Effect:
- Eat: C3b is deposited on the microbe surface (or an allograft bound by antibody) marking for phagocytosis or degranulation (if too big)
- Destroy: deposition of C3b triggers the formation of the membrane attack complex (MAC)--a giant pore in the membrane
- Attack: C3a recruits and activates leukocytes triggering microbe destruction by leukocytes

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Mechanism of recognition of microbes by phagocytes

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- macrophages (tissues) and neutrophils (plasma) have toll like receptors (TRLs) that recognize pathogen associated molecular patterns (PAMPs) on the surface of pathogens, enducing phagocytosis of the pathogen
- once inside the phagocyte, the vesicle containing the pathogen (phagosome) fuses with a lysosome and the pathogen is killed.

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Define opsonization

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= the addition of a molecule to a given antigen which will target it for immune response.
- Ex: pathogens are coated with antibodies or complement molecules enabling recognition and response by phagocytes

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Importance of dendritic cells in immune response

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= antigen presenting cells
- dentritic cells are found in tissue that contact the external environment (skin, respiratory, GI)
- immature dentritic cells become activated by phagocytosing and degrading a pathogen. They then migrate to the lymph nodes and present remnants of degraded pathogen on their cell surface. T and B cells are activated by these antigens initiating the adaptive immune response

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Systemic effects of inflammation

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IL-1/IL-6/TNF-α trigger different responses in different systems:
- Liver: activating of acute phase proteins leading to activation of complement opsonization
- Bone-marrow endothelium: neutrophil mobilization leading to phagocytosis
- Hypothalamus (fat, muscle): increased body temperature (fat and muscle increase energy mobilization to generate heat) leading to decreased viral and bacterial replication, increased antigen processing, facilitates adaptive immune response
- Dendritic cells: TNF-α stimulates migration to the lymph nodes and maturation leading to initiation of the adaptive immune response

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Differentiating between Staph. aureus, strep. pyogenes, E. coli, Shigella dysenteriae

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1. S. aureus and S. pyogenes are gram positive cocci:
- Test for catalase activity: add H2O2 (S.A. positive (bubbles, aerobic), S.P. negative)
2. E. Coli and Shigella are both gram negative rods:
--use differential media with a pH indicator: shigella cannot ferment lactose, will use peptone instead producing ammonia raising pH, forming white/colorless colonies. E. coli can ferment lactose and will form pink colonies.

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Surface structures of gram positive bacteria

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- have single cytoplasmic membrane of lipids with integral membrane proteins
- thick cell membrane of peptidoglycans (N-acetylglucosamine & N-acetylmuramic acid) associated with teichoic acid (lipoteichoic acids anchor structure to the membrane)
- Thick cell wall holds violet stain so remained stained on Gram stain
- cell wall is unique to bacteria so peptidoglycan synthesis is a target for antibiotics

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Surface structures of gram negative bacteria

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- inner layer of membrane
- single layer of peptidoglycans, no teichoic acids (anchored to membranes by lipoproteins)
- second outer membrane containing porin proteins (which allow the passage of small molecules) and lipopolysaccharides (LPS) (lipid A (endotoxin, in outer membrane) + O antigen polysaccharides; together block diffusion))
- Thin cell wall does not hold stain so appear clear (pink) on Gram stain
- outer membrane acts as a selective permeability barrier making gram negative bacteria resistant to antibiotics that attack the cell wall (target for gram positives)

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Surface structures of mycobacteria

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- single membrane
- outer peptidoglycan cell wall
- outer waxy coat composed of mycolic acid and lipoarabinomannan (LAM), has porins to facilitate diffusion
- hydrophobic outer coat takes up acid fast stains
- Different from mycoplasma which lack the cell wall (so resistant to beta-lactam antibiotics)

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Encapsulation and bacterial virulence

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- many bacteria secrete a polysaccharide or protein capsule that makes them resistant to phagocytosis.
- Capsules hide PAMPs (including peptidoglycans, LPS, other antigens)
- Capsules prevent antibodies from binding and coating the bacteria
- Capsules are essential for bacterial virulence (unencapsulated Pneumococci are not virulent)

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Baltimore classification of viruses

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- classifies viruses based on nucleic acid structure and how they process that nucleic acid to produce active/infectious viral mRNA (+RNA)
- +RNA is infectious: it can be transferred into a cell and directly translated (while a negative complement of the strand is not itself infectious--requires viral proteins to copy into active complement--so vRNApolymerase usually is transmitted with it)
Classes:
- Single stranded DNA: Parvovirus
- Double stranded DNA: pox virus, herpesvirus, adenovirus, papovirus, hepadnavirus
- + RNA (converted to double stranded DNA): retroviruses (HIV, human T cell lymphotrophic viruses)
- -RNA (converted to mRNA): othromyxovirus, paramyxovirus, arenavirus, rhabdovirus, bunyavirus
- Double stranded RNA (to mRNA): Reovirus
- +RNA: picornavirus, togavirus, flavivirus, coronavirus, calcivirus

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Virus classification based on presence of envelope

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DNA viruses:
- enveloped: Pox, herpes, hepadna
- Naked capsid: polyoma, papiloma, adeno, parvo
RNA viruses:
+RNA: N (picorna, noro), E (toga, flavi, corona)
-RNA: E (rhabdo, filo, orthamyxo, paramyxo, bunya, arena)
+/-RNA: double capsid (Reo)
+RNA via DNA: E (retro)

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Influence of viral structure on replication strategy

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1. Entry in to the cell (or injection of nucleic acids): can occur through receptor mediated endocytosis, receptor docking, or membrane fusion depending on whether the virus is encapsillated
2. Exposure of the nucleic acids for use: may require uncoating or modification of the capsid
3. Replication and transcription: determined by genomic content and structure--DNA viruses generally replicate in the nucleus (except Pox virus) while RNA viruses stay in the cytoplasm
4. Viron assembly and maturation: may occur in the cytoplasm or ER/golgi or at the plasma membrane to acquire a viral envelope
5. Exit from the host cell: rupture of the cell, budding, exocytosis

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Hemaglutinin (HA) mediated fusion of membranes

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HA induces fusion of viral membrane with the endosome and release of influenza ribonucleoproteins
1. HA binds monosaccharide sialic acid on target cells, causing the viral particles to adhere to the surface and inducing endocytosis
2. The viral material in the endosome begins to denature at pH 6.0. HA partially unfolds exposing a hydrophobic region (fusion protein) that inserts into the endosomal membrane. The rest of the HA molecule refolds into a low-pH stable form and pulls the endosomal membrane into contact with the viral membrane, inducing fusion and release of the nuclei acids into the cytoplasm
- A homolog of HA, gp41 is found in HIV and mediates fusion of the viral envelope at the cell membrane (rather than the endosome)

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Unique microbial structures and functions exploited in treatment

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- goal selective toxicity (affect microbes only) with a large therapeutic index (ratio between good and harm)
Targets:
- peptidoglycans (cell wall): beta-lactams inhibit biosynthesis
- Bacterial ribosomes: inihibit protein synthesis, clydamycin
- nucleic acid synthesis
- folic acid metabolism: bactrim
- sterols in fungi

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Mechanisms of antibacterial resistance

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Genetic changes in the microbes (spontaneous mutation, genetic exchange...) may result in:
- decreased permeability to the drug
- secretion of drug-inactivating enzymes (especially in genes acquired by lateral transfer)
- increased efflux (increased removal of toxic metabolic intermediates from cells)
-- Must use combination therapy for treatment. Monotherapy results in fast resistance and may have effect of other treatments
-

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Emperic vs. Definitive antibacterial therapy

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Empiric is based on:
1. likely pathogens given clinical situation (don't know exact)
2. site of infection
3. review of specimens (including Gram's Stain)
--use broader range of antibiotics, more common in practice
Definitive is based on:
1. isolation by culture of the bacteria
2 .resistance testing of the bacteria
--takes longer before a result is available so less common although preferred

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9 main principles governing antibiotic use

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1. Is an antibiotic indicated? (significant infection/suspected infection/can't delay treatment/prophylatic use)
2. What is the likely pathogen? (site of infection, age, context)
3. Obtaining appropriate specimens for examination and culture (Gram stain, cultures/timing)
4. Best drug for the pathogen? (activity of drug, antimicrobial spectrum broad/narrow, cost, toxicity, resistance patterns, site of infection)
5. Risk to the patient? (consequences of uncontrolled infection, site, context)
6. Need for bacteriostatic vs. bactericidal agent
7. Important host factors? (allergies, renal/hepatic insufficiency, age, pregnancy, immunosupression, storage disorders)
9. Is combination therapy important? (prevent resistance)

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Bacteriostatic agents

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- prevents growth but does not kill bacteria
- includes most protein synthesis inhibitors (except aminoglycosides)
- Good for most infections, allows the body's immune processes to eradicate the infection
- Not sufficient in cases of immediate danger to the patient, if the immune system cannot access the infected site (immunologically protected area), if the host is immuno-compromised. Ex: Meningitis, endocarditis, any immunodeficiency)

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Bactericidal agents

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- include cell wall inhibitors (usually), quinolones, aminoglycosides, and rifampin
- Usually not required except:
1. Meningitis: in an immunologically protected area
2. Endocarditis: avascular, so little immune response
3. Immuno-compromised host

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MIC

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= Minimum inhibitory concentration
- lowest concentration of an antibiotic that produces no visible growth after overnight incubation of a standard inoculum

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MBC

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= Minimum bactericidal concentration
- minimum concentration of drug that reduces the colony count of viable organisms to less that 99.9% of initial value overnight incubation
- ineffective against bacteria that express beta lactamase

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Penicillins/beta lactams

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Ex: methicillin, amoxicillin, cephalosporins (ceftrioxone), carbapenems (meropenem)
- Bacteriocidal (bacteria lyse under osmotic pressure)
- inhibit synthesis of the peptidoglycans in cell walls by binding to/inhibiting the transpeptidases which form cross links to add rigidity to the cell wall

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Glycopeptides

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Ex: vancomycin
- bacteroicdal against enterococci only (lyse due to inability to grow cell wall)
- binds terminal dAla-dAla residues on pentapetide so that it cannot interact with other cell wall peptides, preventig cross linking and inhibiting cell wall growth

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Macrolides

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Ex: azithromycin
- for gram positives, mainly bacteriostatic (bacteriocidal in high enough concentrations)
- inhibit protein synthesis: bind to 50S ribsomal subunit preventing binding between successive amino acids, no polypeptide made

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Tetracycline antibiotics

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Eg: tetracycline, doxycline
- effective against gram postives, bacterostatic
- inhibits protein synthesis via the 30S ribosome: prevents tRNA from binding and resulting in the wrong amino acid being incorporated in a misfolded/non-functional protein
- resistance is due to enzymatic inactivation, efflux, and protection of the ribosome

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Aminoglycosides

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Ex: streptomycin, kanamycin, gentamycin
- usually bacterostatic
- inhibits 30S ribosomal subunit so that an incorrect codon is read and the wrong amino acid is incorporated resulting in a non-functional protein

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Quinolones

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Ex: ciprofloxacin, levofloxacin
- bactericidal, effective on gram negatives
- prevents replication by inhibiting topoisomerases (involved in DNA uncoiling) and DNA gyrase (required for correct coiling)

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Rifamycins

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Ex: rifampin
- bacteriostatic, effective against mycobacteria (TB, leprosy, MAC)
- Binds RNA polymerase preventing mRNA synthesis
- resistance conferred by modified forms of RNA polymerase

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Lipopeptide

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Ex: daptomycin, surfactin
- bactericidal, effective against gram positives
- binds to and inserts lipid tail through bacterial cell membrane: causes calcium-dependent rapid efflux of potassium and depolarization of the cell/disruption of electrical gradients causing cell lysis and death

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Sulfonamides

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Ex: Bactrim/Septra
- bacteriodstatic, effective against strep, staph aureus, E. coli, H. flu, oral anaerobes, PCP/toxo
- inhibits folic acid synthesis by competitive inhibition of pteroate synthase (key step in pathway). Folic acid needed for nucleic acid synthesis

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Dihydrofolate reductase inhibitors

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Ex: trimethoprim
- bacteriostatic, effective against PCP prophylaxis
- inhibits folate synthesis by inhibiting dihydrofolate reductase

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Clinical significance of antibiotic resistance

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- increased resistance from over prescription and monotherapy use. Now recommend multi-drug/combination therapies to treat (especially resistant strains)
- factors that increase risk for antibiotic resistance: especially in hospitals there is a lot of antibiotic pressure from antibiotic use and competition from other microbes, leading for resistant, aggressive species
- development of resistance can result in failure of regimens and relapse and the need to use more costly and aggressive antibiotics.

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3 forms of genetic exchange in bacteria

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- genetic exchange is a mechanism by which by which bacteria can acquire resistant mutations from other strains. This is exceedingly common
1. Conjugation: plasmid transferred through physical contact between bacteria (pili)
2. Transformation: bacterium picks up free plasmid DNA for the extracellular environment
3. Transduction: bacteriophages or viruses acquire DNA and infect other bacteria, incorporating it into the bacterial genome.

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4 Primary mechanisms of antibiotic resistance (and antiobotics/bugs that it effects)

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1. Altered target site: antibiotic binding site is changed so that it cannot be bound.
- Beta lactams (MRSA, new penicillin binding protein), vacomycin (VRE, changed end residues of crossing-linking protiens), rifampin, daptomycin, erythromycin
2. Decreased entry: altered membrane to reduce drug entry
- beta lactam (pseudomonas: altered permeability by deleting D2 porin protein from outer membrane), tetracycline (bacterial efflux pump)
3. Bypassing effector pathway: bacteria develops alternate pathways around the target pathway
- sulfonamide (development of a antibiotic-resistant enzyme)
4. Enzymatic degradation: bacteria develops way to degrade/inactivate the antibiotic
- beta lactam (beta lactamases hydrolizes bond in drug, inactivating it, can be transferred by plasmid or chromosomes; carbapenemase more versatile beta lactamase, found on klebsiella), aminoglycosides (inactivated by a number of enzymes)

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The innate immune system

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Characteristics: non-specific, limited diversity, no memory, non-reactivity to self
Components: physical/chemical barriers (skin, mucosal epithelia, intimicrobial chemicals), blood proteins (defensins, complement), cells (phagocytes (macrophages, neutrophils), NKs)
- Timeline: Ubiquitous response 0-4 hrs non-specific affectors; induced response 4-96 hrs, recruited effector cells (NKs, neutrophils, macrophages)

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The adaptive immune system

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Characteristics: specific for antigens (microbial and otherwise), large diversity (receptors produced by somatic recombination), memory (B cells), non-reactivity to self
Components: barriers (lymphocytes in epithelia, antibodies secreted from epithelia), blood proteins (antibodies), cells (lymphocytes: T and B cells)
Timeline: Initial exposure >96, requires antigen transport, presentation, clonal expansion and maturation of effector cells; protective immunity (re-infection) immediate, removal by preformed antibody and effector cells; immunological memory ~hrs, reinfection triggering rapid expansion and differentiation of effector cells

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Neutrophils

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= polymorphonuclear leukocytes
- effector cells of the innate immune system
- short lived (form pus when dead), stored in bone marrow (also some in circulation) and released upon infection (attracted to site by chemotaxis and deposited on vessel walls by integrins)
- contain granules (lysosomes) of degradative enzymes and can produce oxidative metabolites. Release is good for antimicrobial activity but also can damage cells.
- Have complement receptors, CR3/4, allowing them to phagocytose bacteria
- Does not express MHC so does not present antigens to T cells
- Deficienct or ineffective neutrophils can cause chronic granulomatous disease

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Macrophages

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- effector cells of the innate immune system and antigen presentor cells of the adaptive immune system and help with tissue repair
- derived from bone marrow monocytes. Found in circulation and in localized in tissues. Attracted to sites of inflammation by chemotaxis.
- Have surface receptors for IgG and C3b which mediates phagocytosis
- Presents bacterial antigen and MHC II on surface for antigen presentation to CD4 helper T cells
- Secrete cytokines: IL-1 (helps activate helper T cells), TNF (mediates inflammation), IL-8 (attracts neutrophils and T cells)
- activated by bacterial proteins/components: LPS (endotoxin), bacterial DNA or peptidoglycan, which interact with TLRs and signal cytokine production. Also activated by gamma interferon (increases MHC II production)
- are good for killing intra/extracellulr pathogens, and infected or altered host cells

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Natural killer cells

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- effector cells of the innate immune system
- kill virus infected cells and tumor cells by secreting cytotoxins
- not part of the inflammatory response
- circulate in the blood in a partially active state (activity regulated by activating/inhibitory receptors on surface)
- are active without prior exposure to virus, not enhanced by exposure, not specific
- Derived from same lymphocytic precursor cells at T and B, but have no memory function or response to MHC II
- Activated by IL-12, response enhanced by IgG
- secrete cytokines that activates macrophages and T cells

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5 Main functions of the innate immune system

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Without the innate immune system, organisms are not able to control infection at all (critical to survival)
1. recruit immune cells to infection via production of cytokines
2. activate complment cascade to identify bacteria, activate cells and promote clearance of dead cells and antibody complexes
3. identify and remove foreign substances from the body
4. activate the adaptive immune system via antigen presentation
5. act as a physical and chemical barrier to infection

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3 Functions of the complement system

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1. recruitment of inflammatory cells: C3a (anaphylatoxins) promote inflammation by increasing vascular permeability to allow other cells/proteins/components to reach the tissue
2. Opsonization: C3b mark pathogen for ingestion and destruction by phagocytes
3. Perforate pathogen cell membrane: C3b aggregate in membrane attack complexes creating pores in the membrane, destroying the pathogen

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Alternative pathway of activation for the complement system

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Pathway: thioester bond in intact C3 (usually protected) is attacked by water forming iC3 which complexes with other subunits to form C3 convertase which attacks intact C3 to form C3a/b. Once deposited on the membrane C3b can also recruit the same subunits to become a C3 convertase which can activate deposition of more C3
Regulators: properdin (stabilizes C3 convertase on pathogen surface), Factor H (enables binding of Factor I to C3), Factor I (inactivates C3b as iC3b to prevent C3 depletion), DAF/MCP (disrupts C3 convertase on host cells)

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3 Pathways of complement activation (in order of action)

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- Activation consists of cleaving C3 (releasing C3a/b) exposing thioester bond which undergoes nucleophilic attack by phospholipids to bind C3b to membrane surface
1. Alternative: C3 hydrolized by H2O and converted to iC3 then C3 convertase, which cleaves C3
2. Lectin Pathway: mannose-binding lectin binds to pathogen surface
3. C-reactive protein or antibody binds to specific antigen on pathogen surface

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Consequences of inflammation

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Pros: (if localized)
- isolation of damaged site
- mobilization of effector cells (phagocytic) and molecules to the site
- initiates healing
- Systemic response: cytokine secretion, activation of complement system, neutrophil migration, fever (neutrophils on hypothalamus increased energy production in muscle/fat), migration of dendritic cells to lymph nodes (via TNFα) to activate adaptive immune system
Cons: (if uncontrolled --> shock/death)
- lipopolysaccharide release in the blood stream increases vascular permeability ---> hypotension, shock
- effects on liver --> hypglycemia
- activation of clotting factors --> disseminated intervacular coagulation and thrombosis

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Detection of pathogens by the innate immune system

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Pattern recognition receptors: these are present on immune cells and recognize pathogen associated molecular patterns (PAMPS: structural motifs that are highly conserved on microbes but generally absent in the host). 3 types:
1. Lectins: (ex: mannose receptor, glucan receptor) present on macrophages and scavenger receptors. Bind carbohydrates on microbes
2. scavenger receptors: highly positively charged, so affinity for negative charge of molecules on pathogens (ex: sulfated polysaccarides). Also recognize nucleic acids, lipotechoic acids (gram postive cell walls). Receptors recruit macrophages
3. Toll like receptors: transmembrane proteins (PM or endosome) that recognize PAMPs inside and outside cells

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Toll-like receptors

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- transmembrane proteins that a pathogen pattern recognition domain (multiple subdomains, leucine rich repeats) and in intracellular domain for initiation of signalling casade
- can be found as dimers (hetero or homo) or as monomers
- 10 major TLRs in the human immune system which recognize different ligands (ex: LPS)
- found on plasma membranes to recognize extracellular PAMPS, and in endosomes to recognize phagocytosed microbes
- signaling domain activates cascade leading to NF-kappa Beta activation, and translocation to the nucleus for proinflammatory cytokine synthesis

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Link between innate and adapative immunity

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- dendritic cells: recruited to infection by cytokines, immature cells internalize and process antigens and present pieces of microbial peptides on class II MHC molecules. They then migrate to lymphoid tissue to present the antigen to CD4 T cells.
Macrophages can do this too.

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MHC class I

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- expressed on nearly every cell in the body (except RBCs). Key to recognizing self (expression will resist degradation by NK cells). Important to match in transplants
- Structure: light chain + heavy chain supporting length-restricted peptide binding groove
- 3 antigen presenting varieties: HLA A, B, C (codominant expression from parents, so each person has a unique set)
- Activates CD8 killer T cells
- Used to fight intercellular pathogens (results in destruction of infected cell)
- Peptide binding region can hold peptides of 8-10 aa's (binding groove pinches at end, restricting length)

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MHC class II

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- expressed mainly on cells of the immune system (ex: B cells, dentritic cells, macrophages)
- Structure: two heavy chains with non-length restricted peptide binding groove
- 3 varieties: HLA - DR, DP, DQ
- Activates CD4 helper T cells
- used to fight extracellular pathogens
- Can hold long peptides in groove (ends do not pinch off)

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Define MHC restriction

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T cells are specific for foreign peptides (antigens) and the host-MHC molecule. Without either the T-cell will not be stimulated

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3 Ways MHC diversity is maintained

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- Polymorphism: lots of different versions, both withing the individual (multiple HLA-As/Bs/etc and each can bind multiple polypetides as long as the binding residue is conserved) and across the population (most diverse set of human genes). Mate selection linked to pheromones.
- Polygenicism: multiple different genes involved. Different combinations of sequences can be combined.
- Codominant expression: both copies from parents are expressed at the same time--diversity from mixing (alpha from one parent, beta chain from the other)

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MHC Class I processing/activation

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Handles mostly intercellular agents.
- proteins in cytosol are degraded by the proteosome into peptide fragments
- peptide fragments are transported to the ER via TAP (transporter associated with antigen processing; uses ATP binding cassette; cannot differentiate between self/non-self, always on)
- Chaperone proteins stabilize MHC until it binds a peptide (MHC unstable without a peptide or chaperone)
- MHC+Peptide is released to the surface of the cell
-

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MHC Class II Antigen Processing

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Mostly extracellular antigens
- Antigen is phagocytosed and proteins brokend down
-MHC II leaves ER in vesicle (stabilized by invariant chain that jams in the binding groove to prevent early binding)
- 2 vessicles fuse and MHC releases stabilizing protein loads target protein
- Loaded MHC transported to the surface to serve as antigen for CD4 cells

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CD8 T lymphocytes

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- Naive CD8+ T lymphocytes bind and are activated by MHC class I molecules on antigen presenting cells (dendritic cells)
- Active CD8+ T lymophocytes then either mature into memory cells or cytotoxic T lymphoctes.
- CTL's kills cells with internal pathogens (especially viruses) by introducing holes (perforins/MHC) and releasing enzymes that activate apoptosis.

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CD4 T lymphocytes

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- naive cells are activated by MHC class II on antigen presenter cells. (good for extracellular pathogens)
- Mature into CD4+ T helper cells which secrete cytokines to support cell and antibody mediate immune systems
- produce cytokines that promote differentiation of B cells into plasma cells (produce antibodies), activate microphages to become phagocytic, activate cytotoxic T cells, induce inflammatory response

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Superantigens

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- bind directly to conserved regions of MHC ptoteins and T-cell receptor
- can activate whole families of T cells resulting in a cytokine storm
- produced mostly by bacteria, may result in food poisoning or toxic shock syndrome

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T vs. B lymphocytes

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T cells:
- originate in the bone marrow but mature in the thymus
- conducts the cell-mediated immune system
- mature into cytotoxic, helper or regulatory T cells and migrate to peripheral lymphoid system
- found in the thymus, the periarterial lymphatic sheath (PALS) of the spleen white pulp, the paracortex of lymph nodules, mucosa-associate lymph tissue (MALT)
- recognize a linear sequence of amino acids (linear epitope) only when bound to MHC
B cells:
- originate and mature in the bone marrow
- mediate the humoral/antibody immune system
- mature into plasma cells (secrete immunoglobulins) and memory cells and migrate to the peripheral lymphoid tissue
- found in the cortex and medullary cords of the lymph nodes, in the lympoid nodules/follicles of the PALS, and throughout the MALT
- recognize soluble 3D/conformatioal epitopes

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Primary and secondary lymphoid organs

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Primary:
- bone marrow: where lymphoid precursors originate and B cells mature
- thymus: where T cells differentiate and mature
Secondary: tissues that maintain mature lymphocyte and allow for antigen presentation to occur (mobilize adaptive immune response)
- lymph nodes
- mucosa associated lymphoid tissue
- spleen

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Thymus

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- bilateral organ in the mediastinum
- peak development in childhood during T cell maturation, atrophies with age and T cell migration to the rest of the body
- much of the organ is immune-protected to prevent premature activation of T cells
Functions:
- generation of diverse T cell antigen receptors
- selection of function T cells and deletion of autoreactive T cells
- differentiation of T cell subpopulations
Structure:
- consists mostly of fibrous connective tissue and adipose
- connective tissue capsule segments the organ into lobules (portion of cortex + medulla)
- reticular epithelia cells extend processes into the cortex and medulla surrounding lymphocytes to isolate them from blood (connected by tight junctions and desmosomes to prevent antigen passage)
- Cortex: higher concentration of T cells at different maturation stages, so stains darker
- Medulla: ligher stain, contains differentiated CD4/8, Hassall's corpuscles (concentric layers of cells, help in continued maturation of T cells)

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Bone marrow in immune cell generation

Back 72

- consists of flexible tissue found within the interior of flat bones.
- source of lymphocyte progenitors (for B, T, and NK cells)
- T cells progenitors and mature B cells leave the center of bone via sinusoids and enter the blood stream.

Front 73

Spleen

Back 73

- biggest lymphoid organ, storage/filter/processing site for aged RBCs
Structure
- connective tissue capusle (easily ruptured) with trabeculae partitioning interior
- splenic artery enters and branches several times, converges and leaves as the splenic vein
- Red pulp: rich in RBCs; consists of splenic cords containing macrophages, lymphocytes, plasma cells surrounded by reticular fibers; sinuses with fenestrated endothelia (allows for open circulation and passage of all blood contents)
- white pulp: lymphoid tissue surrounding the artery; 3 parts: periarterial lymphatic sheath (PALS, T-cells), marginal zone (sinuses, loose lymph tissue), follicles (B-cells)
- has smooth muscle to contract and eject erythrocytes

Front 74

Lymph nodes

Back 74

- 1000 distributed throughout the body
- responsible for filtering the lymph and allowing lymphocytes to interact with antigens and initiate immune response
- concentrated on organs with epithelia (mucosa-associated lymphoid tissue, MALT) or where the limbs join the body
Structure:
- 2-10mm in diameter, bean shaped, connective tissue capsule with trabeculae in interior, subcapsular sinuses
- Functional regions: cortex, paracortex, medulla
- afferent lymph vessels send lymph to the subcapsular sinuses which are continuous with cortical and medullary sinuses, where it is filtered by lymphocytes, then sinuses region in efferent lymphatic vessel
- Cortex: contains lympoid nodules (clonal expansion of activated B cells if germinal centers)
- paracortex: mostly T cells, no precise boundaries
- medulla: cords (branched extensions of the paracortex, mostly B cells), sinuses (dilated spaces between cords, contain lymph, join at the hilum to for efferent vessel)

Front 75

Mucosa-associated lymphoid tissue

Back 75

- system of small concentrations of lymphoid tissue found in mucosal linings of various body tissues
- components include gut, bronchus, nose, and vulvovaginal -associated lymphoid tissue

Front 76

T cell maturation

Back 76

- precursor cells migrate from bone marrow to the thymus in the blood stream during fetal/postnatal life
- precursors enter the cortex and undergo massive proliferation
- As they mature, the surface proteins change (TCR, CD4/CD8/CD25, markers for maturation) and they move toward the medulla
- mature t cells undergo thymic selection: positive selection: bind to specific antigen or else destroyed; negative selection: don't bind to self antigen or else destroyed
- Cells differentiate into CD4/8 cells and migrate to the medulla
- from the medulla they enter venules and migrate to the secondary lymphoid organs

Front 77

Thymocyte selection

Back 77

- purpose: identify T-cells that will interact with MHC/antigen complexes but not MHC/host protein
- Each T cell has a different TCR, initially express both CD4/8
- 99% are killed (not selected)
- Positive selection: T cell exposed to self MHC on cortical epithelial cells, if not response --> apoptosis. Either CD4 or CD8 is also down regulated so only one expressed
- Negative selection/clonal deletion: occurs in the medulla. Dendritic cells present with self-antigen, if bound too tightly--> apoptosis. AIRE Tx factor (autoimmune regulator) control process by randomly activating expression of self proteins for presentation

Front 78

2 Step activation of a T-cell

Back 78

1. Costimulation:
- T cells binds with MHC receptor + antigen. This is the specific antigen for the T cell plus MHCII (CD4) or MHC (CD8)
- Then CD28 on T cell must interact with B7 on the APC (upregulated when the cell is infected)
- When both of these occur, T-cell secretes IL-2 to initiate cloning
2. Anergy: if the TCR binds to the MHC+antigen complex but not B7 the T-cell will be deactivated to prevent autoreactivity

Front 79

CD4 function, activation, and regulation

Back 79

Function:
- signals B cells and initiates cell cycle
- stimulates B cell proliferation and antibody production (mostly Th2)
- activates macrophages (Th1) to promote inflammatory cellular immunity
- Th1 is better for mycobacteria, Th2 dominates allergic and anti-parasitic response
Activation: IL-2 and IFN-g activate macropoages; IL-4 activate B-cells
Regulation:
- Treg cells inhibit Th1 and Th2 via production of inhibitory cytokines (IL-10, TGF-b)
- Treg cells are activated by IL-2 (produced by CD4's)-->negative feedback loop

Front 80

CD8 function and activation

Back 80

Function:
- attack and kill other cells
- responds to the presentation of foreign antigen on MHCI
- uses porforins and granzymes to form a pore in the target cell and insert proteases to initiate apoptosis
Activation:
- activated by TCR binding to MHC+specific antigen in conjunction with CD28 binding with B7 on an APC. Can then kill the cells presenting the specific antigen

Front 81

Allelic exclusion and tolerance in B cell development

Back 81

Allelic exclusion: the gene expression producing the multiple combinations of heavy and light chains. Each B-cell expresses only one combination (so only 1 antibody produced) ensuring mono-specificity and avoiding autoimmunity
Tolerance: B cells in the bone marrow are trained to recognize only foreign antibodies--> immature B's expressing IgM are exposed to cell bound and soluble self antigens. Those that bind cell antigen are eleminated (clonal deletion), those that bind soluble antigen become anergic (only can express IgD, unable to respond to antigen)

Front 82

One-cell, one-specificity

Back 82

each B cell carries antibodies that are specific to only one antigen. Only the presentation of that specific antigen will result in B cell proliferation and immune response

Front 83

Heavy chain class switching in B cells

Back 83

= when the constant region of antigen binding receptor is changed between one domain to another (variable region is unchanged). This allows the cell to change the immunoglobulins they express while still responding to the one-specific antigen.
- Changing the Ig class changes the effect of antigen binding (so change be diverse effects for each antigen)
- 5 major Ig classes (G, D, E, A, M)

Front 84

B cell differentiation and memory generation

Back 84

Differentiation: B cells can respond to 10^7 antigens, once activated B cells can:
-- secrete antibodies as a short lived plasma cell in the lymph nodes and and spleen
--become a germinal B cell. Clones of the germinal cell can then become memory cells (bone marrow) or or long-lived plasma cells (life long antibody production)
Affinity maturation: the process of hypermutation and selection
-- in the germinal center a range of B cell mutants are generated and the antigen selects the high affinity cells to become memory cells ( these have better affinity than the original cell--> faster, greater immune response if reinfected)

Front 85

Function of antibodies

Back 85

- recognize antigen
- non-covalent interaction occurs between antibodies and a variable region of antibodies, requires a very specific fit
- once bound the antigen can neutralize and interact with effector cells to destroy the pathogen: neutralization of microbes and toxins, opsonization and phagocytosis of microbes, activation of the complement system (lysis or phagocytosis of microbes, initiation of inflammation)

Front 86

Creation and function of monoclonal antibodies

Back 86

- monoclonal antibodies are exact clonal copies the the antibody produce by a single unique B cell or its clones.
- they are grown in lab by fusion with myeloma cells
- they are used in therapy for cancer, autoimmune disease, and after organ transplants.

Front 87

4 types of abnormal immune response

Back 87

1. Hypersensitivity
- when repeat exposures trigger a pathogenic response
- may be elicited by both endogenous and exogenous antigens
- often associated with inherited susceptibility genes (like HLA genes)
- reflects an imbalance between the effector mechanisms of immune response
2. Autoimmunity
- when immune system mounts reactions against self-antigens
3. Immunodeficiencies
- conditions in which certain components of the immune system are impaired (either humoral, cell-mediated, or both)
- primary (genetic) secondary (environmental)
4. Rejection of tissue transplants
- both cell-mediated (T cell mediated direct and indirect pathways) and humoral (antibody-mediated can result in hyperacute rejection)
- both graft v. host and host.v graft

Front 88

Type I hypersensitivity

Back 88

Immune reactant: IgE
Antigen: soluble antigen
Effector Mechanism: Mast-cell activation
Speed: immediate (antibody-mediate)
Ex: allergic rhinitis, asthma, systemic anaphylaxis

Front 89

Type II hypersensitivity

Back 89

2 mechanisms:
1. IgG mediated; cell- or matrix-associated antigen; immediate response; mechanism: complement, FcR+ cells (phagocytes, NK cells)
- Ex: some drug allergies (penicillin)
2. IgG mediated; cell-surface receptors; immediate reaction; mechanism: antibodies alter signaling
- Ex: chronic uticaria

Front 90

Type IIII hypersensitivity

Back 90

- IgG mediated
- against soluble antigen
- mechanism: complement, phagocytes
- immediate reaction
- ex: serum sickness, Arthus reaction

Front 91

Type IV hypersensitivity

Back 91

3 type:
1. Th1 cell mediated; soluble antigen; delayed reaction; mechanism: macrophage activation
- Ex: contact dermatitis, tuberculin reaction
2. Th2 cell mediated; soluble antigen; delayed reaction; mechanism: IgE production, eosinophil activation; mastocytosis
- ex: chronic asthma, chronic allergic rhinitis
3. cytotoxic T cell mediated; cell-associated antigen; delayed reaction; mechanism: cytotoxicity
- Ex: contact dermatitis

Front 92

Mechanisms and site of action of 6 types of B cell tolerance

Back 92

1. Central tolerance: clonal deletion/editing to elimiate self-reactivity; occurs in thymus, bone marrow
2. Antigen segregation: physical barrier to self-antigen access to the lymphoid system; occurs in peripheral organs (thyroid, pancreas)
3. Peripheral anergy: cellular inactivation by weak signaling without co-stimulus; occurs in secondary lymphoid tissue
4. Regulatory cells: suppression by cytokines, intercellular signals; occurs in secondary lymphoid tissue and sites of inflammation
5. Cytokine deviation: differentiation to Th2 cells limiting inflammatory cytokine secretion; occurs in secondary lymph tissue and sites of inflammation
6. Clonal exhaustion: apoptosis post-activation; occurs in secondary lymph and site of inflammation

Front 93

Main processes of autoimmune disease

Back 93

- breakdown of T cell tolerance
- combination of genetic factors (like MHC), environmental factors (infection), random events, and gender (predominantly in females)
- infections potentially lead to molecular mimicry (bacteria have antigens with similar sequence to self-antigens) so antibodies against bacteria target host (ex: rheumatic fever)
- normally some areas of the body are immune-privileged (brain, eye, testis, placenta), when these barriers break antigens from these areas are identified as foreign so the cells are attacked.

Front 94

Basis for primary vs. secondary immunodeficiencies

Back 94

Primary
- genetically determined and affect the humoral and/or cell-mediated adaptive immunity
- recessive gene defects (many x-linked in males)
Secondary:
- arise as a result of external insults such as cancers, infections, malnutrition, or side effects of immunosupression, irradiation or chemotherapy
- classic example is AIDS

Front 95

Immune response during transplantation

Back 95

T-cell mediated rejection:
- general: APCs and antigen exit the graft and travel to the host lymph nodes where they encounter T cells, which identify them as foreign and activate T and B cell proliferation. Effector T cells then travel back to the graft and destroy the tissue.
- direct T-cell pathway: T-cells from the recipient recognize donor MHCs and differentiate into CTLs (which attack the graft) and Th1 (which release cytokines triggering a delayed hypersensitivity reaction: increased vascular permeability, local accumulation of mononuclear cells cells, graft injury by activated macrophages, activation of antibiody production by B lymphocytes)
- Indirect: graft releases antibodies that are presented by the host MHCs. CTLs produced cannot destroy graft cells (don't recognize graft MHC), so damage caused by Th1 cytokine production and the delayed hypersensitivity pathway
Humoral rejection:
- hyperacute: preformed anti-donor antibodies are present in circulation of the patient (ex: from someone who previously rejected a transplant)
- exposure to class I and II HLA antigens of the donor graft results in antibody formation against them

Front 96

Features of HIV virus

Back 96

- enveloped RNA virus with 2 copies of the genome in each viron.
- RNA genome encodes for 3 major proteins: the envelope, GAG (needed to form viral particles), and polymerase
- reverse transcriptase will be found inside the envelope
- When the virus binds, the envelope is uncoated, the core enters the cell then is uncoated, releasing RNA. Reverse transcriptase converts genome to RNA/DNA hybrid then double stranded DNA which gets integrated (half-life of many years) and replicated
- Reverse transcriptase is highly error prone, leading to many variants and rapid evolution

Front 97

HIV cell tropism

Back 97

- HIV targets cells expressing CD4 and chemokine coreceptors CCR5 or CXCR4 so: helper T cells, macrophages, and dendritic cells
- People with homozygous CCR5 mutation are relatively resistant to HIV and ligands for CCR5 and CXCR4 can inhibit HIV entry
- Immunodeficiency is caused by destruction of T-helper cells
- HIV primarily targets immune activated CD4+ T cells for high level replication, which express CCR5, transport RNA to the nucleus and have transcription factors that drive viral promotors

Front 98

HIV interactions with macrophages and dendritic cells: influence on viral transmission and persistance

Back 98

- HIV can cross the epithelial barrier by binding and infecting dendritic cells and then spreading to CD4s and other cells. Macrophages can also similarly be vectors for infection (especially in tissues where there are no T cells normally--vagina)
- Infection may initially be confined to the portal of entry but then spread to the draining lymph nodes and beyond.
- DCs and macrophages can serve as a reservoir of ongoing viral replication and lead to infection of CD4 cells leading to HIV persistence.

Front 99

Typical clinical course for HIV (if untreated)

Back 99

3 Major phases:
Acute infection (2-6 weeks): rapid rise in viremia and dip in CD4s then recovery as immune system activates (Gut CD4s decline completely and do not recover). May be characterized by flu-like symptoms (often goes unnoticed)
Asymptomatic phase (variable length): consistent viral load, slow decline in CD4s
Symptompatic phases: increasing viral load and faster decline of CD4s usually bringon onset of symptoms (usually around CD4<500). At CD4<200, clinically described as AIDS, and high susceptibility to opportunistic infections

Front 100

Immune responses to HIV and implications for vaccine development

Back 100

- after initial infection the host generates CTLs and antibodies against viral envelope and GAG proteins. The antibodies can clear free virus and virus infected cells (CTLs also do this), but neither can help latently infected cells. (this is a problem for vaccine)
- Virus can escape immune responses (making vaccine difficult) by: developing envelope structural features that reduce antibody access (occulsion, variable loops, glycosylation); antigenic escape (modification of viral antigen to avoid recognition); exhaustion of CTLs (loss of function due to porforin expression, cytokine production, and proliferative capacity mediated by inhibitory receptors); down regulation of MHCs; integration and latency in the host genome

Front 101

HIV-induced chronic immune activation leading to immunodeficiency (as opposed to direct effects of viral replication)

Back 101

- humans there is chronic immune activation in response to HIV leading to eventual depletion of overall CD4+ T cell pool (increased activation of naive and memory cells and increased death of mature cells leading to loss of T-cell reserve)
- In contrast monkeys remain healthy despite high viral loads due to how immune activation
-

Front 102

Important dates in HIV history

Back 102

1981: first cases of PCP present in 5 previously healthy homosexual males, also first Kaposi sarcoma in same population.
1982: AIDS syndrome defined
1983: HIV virus isolated in France
1984: serological CD4 antibody test developed
1987: AZT developed as first antiretroviral
1995-7: HAART introduced
2000: Recognition of HIV as world-wide problem
2008: 30mil cases worldwide, HIV largely a chronic condition

Front 103

Mechanism of HIV infection

Back 103

- glycoproteins on the viral envelope recognize the target cell (gp120-->CD4, gp41-->CCR5) which binds and forces a fusion event, allowing the virus to inject its DNA
- Inside the cell viral reverse transcriptase is activated and synthesized linear DNA for viral RNA. dsDNA is integrated into the host genome and host replication machinery is used to generate viral mRNA and proteins (host proteins may be neglected-->cell death)
- New synthesized virions bud from the host cell. The protease that cleaves the envelope from the host cell causes membrane damage-->cell death

Front 104

Pneumocystis pneumonia

Back 104

- opportunistic infection of Pneumocystis jiroveci
- unicellular fungus that causes lung infection
- previously occurred in 60-85% of patients with HIV
- patient experiences shortness of breath, cough, fever
- disease only occurs when humoral and cellular immunity are suppressed (most common upon reactivation of latent infection)

Front 105

Toxoplasmosis

Back 105

- opportunistic infection of Toxoplasma gondii
- bacteria may normally colonize the serum/GI tract (from cats) but are supressed by immune system
- past incidence: 20-33% of seropositive HIV cases
- causes lesions in the brain resulting in headache, focal neurological seizures, swelling of the brain
- diagnosed via CT scan or MRI in conjunction with serology

Front 106

Kaposi Sarcoma

Back 106

- tumor associated with compromised immune function, particularly HIV
- cancerous tumor of connective tissue (manifests on skin)
- caused by interactions between weakened immune system, human herpes virus, and HIV
- tumors appear as bluish-red or purple bumps on the skin (aggressive form of African Kaposi Sarcoma can spread to bones)

Front 107

CMV Retinitis

Back 107

- opportunistic infection of cytomegalovirus
- infection occurs in 60-100% of AIDS cases, late in onset (CD4 <5)
- results in visual loss, biliary disease, pneumonia, diarrhea, encephalitis, blindness
- diagnosed via histology

Front 108

Mycobacterium avium complex

Back 108

- opportunistic infection of Mycobacteria avium (commonly found in water and soil)
- occurs in 20-40% of patients with AIDS (systemic effects when CD4 <100)
- symptoms include fever, weight loss, sweating
- diagnosis via acid-fast blood cultures and assays of bone marrow

Front 109

Cryptococcal meningitis

Back 109

- opportunistic infection of Cyptococcus neoformans (fungi present in soil worldwide)
- found in 6-10% of patients with AIDS
- patient experiences fever, headache, nausea, dizziness, and systemic disease or in connective tissue
- diagnosed via lumbar puncture, culture, and antigen tests
- Usually CD4 ~100

Front 110

4 Main mechanisms of antiretrovirals

Back 110

1. CCR5 blockers: prevent virus from infecting new cells by inhibiting the ability of the virus to recognize the receptor
2. NRTIs/NNRTIs: inhibit reverse transcriptase (by introducing mutated nucleotides or by direct binding); easy to develop resistance to (only 1 AA change is sufficient)
3. Protease inhibitors: interfere with the virus's ability to splice proteins and assemble envelope to exit the host cell
4. Integrase inhibitors: block integration of viral DNA into the genome

Typical regimen consists of a reverse transcriptase inhibitor + protease or integrase inhibitor. Ideal is 3 drug therapy

Front 111

Current problems with HIV drugs

Back 111

Toxicity & side effects, including:
- immediate: headaches, GI intolerance, hypersensitivity
- short term: anemia, peripheral neuropathy, renal or hepatic insufficiency
- Long term: hyper lipidemia, fat redistribution, possible thrombotic events
Adherence issues: complex regimens (different times of day, food requirements, injection/oral)
Cross-resistance & cross-toxicity:
- different drugs may interact with each other and cause adverse side effects
- resistance to one drug may confer resistance to the entire class (ex: AZT --> NNRTIs)

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Prostoglandin biochem abbreviation and structure

Back 112

- abbreviated (EX: PGF₂α) PG + letter (ring structure, 7 naturally occuring) + subscript # (# unsat bonds outside the ring) + α/β (IF a 9-hydroxyl is present on the ring)
- found in nearly all (except RBC's), named b/c originally isolated from prostate tissue
- PGG and PGH are unstable and used to synthesize other PGs. PGI (prostocyclin) is the most stable circulating hormone and the only one w/ 2 rings.
- produced from 20C chains with 3+ double bonds (2 are destroyed to make the ring)
- class-specific substitutions are critical for differential recognition of these hormones by receptors
- synthesis: constant supply of linoleic (omega 6) is required. Stable precursors and enzymes are tissue specific. Rate limiting step is the freeing of arachadonic acid from phospholipid membrane

Front 113

Thromboxanes biochem abbreviation and structure

Back 113

- abbreviation: (ex: TXB₂) TX + letter (type) + subcript (# db outside ring)
- are potent thrombus forming agents
- derived from arachidonic acid (20C + 4db)

Front 114

Leukotrienes biochem abbreviation and structure

Back 114

- abbreviation: (ex: LTA₄) LT + letter (class of substitutions) + subscript (# dbs)
- lack a ring structure
- first found in leukocytes (hence name)
- derived from arachidonic acid (20 C + 4db)

Front 115

Hydroxyeicosatetraenoic acids biochem abbreviation and structure

Back 115

- abbreviation: (ex: 5-HETE) #- (position of hydroxyl group) + HETE
- no ring structure
- derived from arachidonic acid (20C + 4db)

Front 116

Biosynthetic pathways of eicosanoids

Back 116

- dietary precursors: linoleic acid (omega 6), essential for all except some PGs
- linoleic acid --> arachidonic acid, esterified in phospholipids
- RL step: release of precursor from phospholipids by phospholipase A2 (main regulation of pathways)
- Precursors then proceed in Cycolooxygenase pathway (PGs and TXs) or Lypoxygenase pathway (HETEs and LTs)

Front 117

Functions of eicosanoids

Back 117

PGs: locally regulate smooth muscle contraction/relaxation (vasodilation) and other inflammatory responses; involved in temp control, bronchial tone, cytoprotection of stomach mucosa, BP, intestinal motility, myometrial tone, semen viability, vasodilation
LTs: trigger SM lining trachea and can lead to asthma and allergic rhinitis during over expression; made in cells of myeloid lineage in response to cytokines
- TXs: thrombus formation stimulating agents
- HETEs: affect neutrophils and eosinophils, involved in chemotaxis, stimulate adenylate cyclase, induce PMN leukocytes to degranulate and release hydrolytic enzymes

Front 118

Drugs that inhibit eicosanoids

Back 118

Aspirin: irreversible inhibitor of COX enzyme; potent anti-inflammatory
Indomethacin (and other NSAIDS): revesibly bind COX enzyme; anti-inflammatory; can be used to hasten closure of ductus arteriosus after birth
Steriods: block production of all LT and PG as well as the ability to recruit immune cells to inflammation (reduction in response to infection)
- Zyflo: 5-lipoxygenase inhibitor; inhibits production of LTs and LTB4 and 5-HETE (for asthma/allergies)
- CysLT: receptor agonists; inhibit CysLT receptor and used to avoid steroids in severe allergic/asthmatic responses

Front 119

Hypoxia

Back 119

= a reduction in blood oxygen levels below normal physiologic levels (90-100%). It deprives cells of adequate aerobic oxidative respiration making them ATP deficient (some cellular systems may fail, anaerobic energy production induced)
- Can be caused by obstructed breathing/O2 absorption (ex: chronic bronchitis due to smoking/damaged cilia), or by obstructed flow to a tissue (ischemia)

Front 120

Ischemia

Back 120

= sudden reduction in blood supply to a tissue or part of the body resulting in insufficient O2 for normal aerobic respiration.
- Triggers utilization anaerobic energy sources (glycogen) until exhausted and causes decreased clearance of metabolic products (toxicities), if prolonged may cause significant tissue damage and death
- can be caused by impeded arterial flow (from atherosclerosis or thrombosis) or by reduced venous drainage (less common).
- tissues more vulnerable if: ↓PaO2 (smoking), ↓Hb, ↓BP, high energy dependence/few stores (cardiac muscle, brain)

Front 121

Atherosclerosis

Back 121

- condition in which there is progressive accumulation of lipoproteins (and cholesterols), monocytes, and collagen in walls of vessels. Plaques may occlude the lumen resulting in ischemia, or rupture and thrombose resulting in obstruction at the site or embolism
- risk factors: high lipids, low LDL, diabetes, hypertension. Symptoms may be exasperated by conditions that limit perfusion normally (↓PaO2, smoking, ↓BP, ↓Hb)
- if obstructing the coronary arteries may cause exertional cardiopectus (heart shifts of anerobic metabolism, causing lactate accumulation); treat with nitroglycerin for vasodilation

Front 122

Thrombosis

Back 122

- abnormal activation of the clotting cascade, where a thrombus forms in a blood vessel and obstructs flow.
- often caused after rupture of atherosclerotic plaque: leakage of lipid into the lumen induces the clotting cascade (recruits platelets and fibrin). The thrombus may then occlude the vessel locally or embolize distally causing ischemia (may cause CVA, MI, or PE)

Front 123

Events occurring immediately following a coronary artery occlusion

Back 123

↓↓O2 to the myocardium resulting in (cessation of ischemic fiber is 1 min, death of heart in 30min):
- aerobic respiration in the mitochondria stops-->↓ATP --> cellular systems that require constant ATP supply begin to fail.
- ion pumps in plasma membrane fail: ↑↑Na, ↓K, ↑Ca, ↑↑H20 → cell swell
- ↑AMP, anaerobic metabolism (glycolysis) begins and cellular glycogen is consumed→ ↑lactate, ↑phosphates→ ↓pH (also ↑osmotic load → ↑swelling)
- cell swelling damages cytoskelton, reduces membrane integrity
- ↑↑Ca in cytosol activates proteases (degrade cytoskeleton) & phospholipases (degrade membrane), forms conductance channels in mito (Ca kills mito)
- impaired membrane integrity: ER dilates, ribosomes detach, PM forms blebs, mitochondria swell (cristae detach, Ca aggregates--irreversible), lysosomes deteriorate (release enzymes with degrade cellular proteins), cellular proteins and enzymes leak out of cell (CPK, troponin 4-6hrs)
- Massive influx of Ca + ↓pH denatures and coagulates cytoplasmic and lysosomal proteins → coagulative necrosis (loss of nuclei & glycogen (slick look), very eosinophilic, neutrophil infiltration) (visible 12-36 hrs after MI)

Front 124

Coagulative necrosis

Back 124

- characteristic pattern of ischemic cell death (except in CNS)
Macroscopic:
- in tissues without collateral circulation: pale segment of tissue, usually wedge shaped away from obstruction
- in tissues with collateral circulation: dark, red tissue to do accumulation of blood
Microscopic:
- lighter staining with no nuclei, little apparent structural damage "ghost cells" due to coagulation of proteins
- coagulated cell structure is preserved for several days

Front 125

Tissue susceptibility to ischemic necrosis

Back 125

neurons: 3-5 min
renal tubule epithelia (proximal): 30 min
cardiac myocytes: 30 min
hepatocytes: 1-2 hrs
skeletal muscle: many hours (because it can rest)

Front 126

Free radicals and mechanism of protection against them

Back 126

3 types of free radicals
- exogenous chemicals which are enzymatically metabolized, especially by P450. Ex: CCl4 (metabolized to •CCl3 which attacks lipids, producing peroxides-->more damage)
- reactive oxygen species from aerobic respiration (can attack lipids and proteins (changes structure) and DNA (break, link strands)
- ionizing radiation (UV): attacks skin, esp DNA --> cancer
Mechanisms for removing free radicals:
- antioxidants: block initiation or scavenge (VIt E, A, C)
- restriction of iron and copper levels (catalyze ROS formation)
- enzymatic neutralization: catalase, superoxide dismutase, glutathione peroxidase

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Steosis and cell swelling

Back 127

- major patterns of reversible cell injury
Steaosis: fatty change and accumulation of triglycerides withing parenchymal cells (esp. liver)
- tissue develops mottled yellow color, large fat vaccoles visible microscopically
- caused by acetominophen OD or alcohol due to excessive entry/synthesis or defective metabolism/export of lipids (more in acute injury). Protein malnutrition or CCl4 injury also can cause this

Front 128

Apoptosis, morphological characteristics, importance in neoplasia and autoimmune injury

Back 128

Apoptosis: regulated cell death in which activated enzymes degrade the cell, breaking it into apoptotic bodies that are phagocytosed. No inflammatory response.
Appearance: cell shrinkage, chromatin condensation, cytoplasmic bleb formation, apoptotic bodies (usually with macrophages)
- Ensures removal of malignantly mutated cells in neoplasia; involved in T-cell negative selection, allows for pruning of tissue during development (esp. reproductive tissue)

Front 129

Liquefactive necrosis

Back 129

- characterized by digestion of dead cells resulting in the transformation of tissue into a liquid viscous mass
- seen in focal bacterial/fungal infections (due to accumulation of leukocytes and liberation of enzymes from these cells
- necrotic tissue is cream/yellow colored (pus) due to presence of dead leukocytes
- occurs in ischemia of the brain

Front 130

Caseous necrosis

Back 130

- occurs most often in foci of TB infection
- necrotic tissue is white ("cheesy"), granuloma (lysed cells and granular debris with distinctive inflammatory border)

Front 131

Gangrenous necrosis

Back 131

- not a specific pattern of death, used clinically
- appears with a limb has lost blood supply and undergone necrosis (typically coagulative) involving multiple tissue planes
- if bacterial infection is superimposed there is more liquefative necrosis--"wet grangrene"

Front 132

Fat necrosis

Back 132

- not a specific pattern of necrosis
- focal areas of fat destruction resulting from the release of activated pancreatic lipase (occurs in acute pancreatitis: enzymes leak out of acinar cells and liquefy membranes of fat cells in the peritoneum)
- appearance: macro (chalky-white areas of fat saponification), micro (shadowy outlines of necrotic fat cells with basophilic calcium deposits surrounded by inflammatory cells)

Front 133

Thombus

Back 133

= pathologic extension of then normal hemostatic mechanism which occludes blood flow
- typical homeostatic process: injury to the epithelium exposes vWF allowing platelets to bind using GPiB receptors. Then thromboxane A2 and ADP encourage platelet adhesion and expand the plug. Exposed factor XII (in connective tissue) initiates thrombin formation from prothombin and then fibrinogen stabilization of the clot.
Thrombosis: proliferation of the clot continues, not degraded, and occludes the vessel

Front 134

Principle functions of thrombin

Back 134

1. Conversion of fibrinogen to fibrin, stabilizing the clot
2. Activation of Factor XIII which initiates cross-linking of fibrin molecules, further stabilizing the clot
3. Stimulation of platelet aggregation and TxA2 production
4. Activates endothelial cells to produce other adhesion molecules
5. Activates endothelial cells to produce vasoactive molecules (NO and PGI2, as well as the fibrinolytic molecule t-PA
6. Thrombin is also a cofactor in several of the clotting cascade reactions leading to positive feedback loops

Front 135

6 Agents or mechanisms that function as inhibitors of coagulation and help prevent thrombus formation

Back 135

1. Antithrombin-III (AT-III): protease that cleaves thrombin, inactivating it
2. Tissue Plasminogen Activator (t-PA): coverts Plasminogen to plasmin which degrades fibrin and leads to breakdown of a thrombus
3. Thrombomodulin: binds thrombin irreversibly, this thrombin-thrombomodulin complex then activates Protein C to cleave and thus inhibit Factor V and VIII
4. Heparin: Increases the ability of AT-III to bind thrombin sigificantly
5. NO: acts as an inhibitor of platelet aggregation
6. pGI2: inhibitor of prostacyclin and platelet aggregation

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Mechanisms by which heparin and warfarin inhibit coagulation

Back 136

Heparin: increases the ability of AT-II to act on thrombin by protein cleavage, thus removing thombin from the blood and inhibiting coagulation
Warfarin: disrupts the vitamin K pathway by inhibiting the body's ability to synthesize vitamin K so Factors II, VII, IX, X, thrombin, protein C and protein S cannot be formed. Reduction in these proteins inhibits the ability to clot

Front 137

Virchow's Triad

Back 137

Factors which predispose thrombosis:
1. alterations in vessel wall: primarily endothelial injury
2. alterations in blood flow: turbulence and stasis
3. alterations in blood constituents: "hypercoaguable state"

Front 138

Anatomical lesions that initiate thrombosis: aterial vs. venous

Back 138

Venous lesion: damage from needle, catheter, static blood (prolonged lack of movement--plane, car, post-op)
Aterial lesion (usually turbulent flow): aneurism, atherosclerotic plaque

Front 139

Mechanism by which turbulence and stasis predispose thrombosis

Back 139

Turbulence:
- causes thrombus formation by reducing shear stress which reduces the amount of NO produced (therefore reducing the ability to inhibit platelet aggregation)
- Occurs where arteries bifurcate and branch, which disrupts laminar flow and increases platelet aggregation.
Stasis:
- lack of movement for a prolonged period of time, particularly post-OP. Muscle contractions of normal movement typically prevent stasis from occurring.
- allows for platelet aggregation due to slow flow, especially in older patients

Front 140

7 Causes of the :hypercoagulable state"

Back 140

(modes of inheritance include primary/genetic and secondary/acquired)
1. Deficiency in anti-thrombin III: (rare) patients at risk for thrombo-embolism, heparin less effective
2. Deficiency of protein C or S: (rare) greater risk for thrombo-embolism, since protein is part of inactivation pathway
3. Release of thrombogenic factors by neoplasm: tumors (lung/pancreatic esp.) secrete thrombus inducing factors--Trousseau's sign (multiple clots)
4. Alterations in the fibrinolytic system: rare, risk for embolus
5. Increased blood viscosity: (due to chronic hypoxia (increased RBCs) so will have high HCT also)
6. Exogenous estrogen, esp in smokers: smoking makes platelets more reactive and increases fibrinogen in the blood.
7. Factor V Leiden mutation: pt. mutation common among caucasions, alters protein conformation so can't be cleaved by protein C, slowing inactivation cascade

Front 141

Mechanisms by which neoplasm predispose hypercoagulable state

Back 141

- tumors secrete mucin, which enters the blood and can activate Factor X triggering thrombin activation and frequent coagulation
- Trousseau's sign: recurrent thrombophlebitis occurring in veins (simultaneous or random), is a sign of malignancy (especially lung or pancreatic)

Front 142

Embolism

Back 142

- fragment of a thrombus or plaque (or air bubble or amniotic fluid, etc) that has detached and occluded a distal vessel
3 Types:
- Venous embolism: originates in deep peripheral veins (due to statis or injury) end up in pulmonary arteries
- Arterial embolism: originates in left ventricular wall/atrial wall/any systemic artery, end up in distal arteries, capillary beds or organs (brain, kidney, toe, heart, etc)
- Paradoxical embolism: originates in a deep vein but crosses from the right ventricle to left atria (due to congenital heart defect) occludes systemic artery/capillary bed/organ

Front 143

3 principle factors that determining clinical and pathological effect of pulmonary embolism

Back 143

1. number of emboli:
- small PE showers may occlude flow to certain alveoli, resulting in dyspnea, coughing, but no large scale ischemia
2. Size of embolism
- large PE occluding >60% of the major pulmonary artery will cause acute right ventricular failure and sudden death.
- obstruction of small pulmonary branches may cause hemorrhagic infarction of that region. Obstruction of mid-sized arteries can cause greater hemorrhage, but supplemental flow from collateral vessels may prevent total ischemia (patient may experience hemoptysis and pleuritic chest pain)
3. Pulmonary and cardiovascular status of the patient:
- hypercoagulable state, chronic hypoxia, smoking, etc can increase risk and severity of thrombus in general

Front 144

Role of the bronchial arteries in preventing ischemia (PE)

Back 144

- bronchial arteries branch from the thoracic aorta and help supplement the lungs with blood and nutrients
- they are separate from the pulmonary arteries so they may be secondary source of blood if the pulmonary arteries are obstructed--keeps lungs from dying/infarction during a PE

Front 145

Amniotic fluid embolism

Back 145

- occlusion of an artery or vein by segments of amniotic fluid
- can occur during childbirth if the intrauterine vessels rupture and are exposed to fluid in the canal
- can lead to DIC (disseminated intravascular coagulation--small clots form everywhere sequestering platelets away from major bleeds) in the mother, resulting in organ failure in some cases

Front 146

Fat embolism

Back 146

- when an artery or vein is occluded by a fat droplet which can enter the blood stream after bone fracture, plaque disruption, or excess cholesterol in the blood.
- Patients with FE may experience pulmonary insufficiency, neurological symptoms, anemia, and thrombocytopenia
- 1-3 days after embolism, pt. may experience sudden onset of dyspnea, tachypnea, and tachycardia
- usually not fatal (only 5-15%)

Front 147

Air Embolism

Back 147

- obstruction of an artery or vein by a volume of air (100cc minimum) that is introduced in to the blood stream; large volumes make blood frothy, impeding perfusion
- complication of coronary bypass or neurosurgeries; diving induce decompression sickness (N2 bubbles)
- severe embolisms can lead to hemorrhage of capillaries and microvessels, also edema in the lungs and severe chest pain

Front 148

Sudden death due to pulmonary embolism

Back 148

- an obstruction of one or both (saddle embolus) of the pulmonary arteries (impeding >60% of circulation) results in impeded flow to the right ventricle, increasing pressure during diastole, causing actute right ventricular failure and death

Front 149

Hemoptysis due to pulmonary embolism

Back 149

= productive cough with bloody sputum
- can correlate to a middle lung embolus, in which obstruction of a middle-sized or end arteriole causes hemorrhage and blood in the alveoli
- Collateral circulation of the lungs from the bronchial arteries helps prevent greater infarction of the lung (and death)

Front 150

Pleuritic chest pain due to pulmonary embolism

Back 150

- caused by friction of the alveolar wall with the lung pleura as the lung moves during respiration
- often associated with hemorrhaging in the distal pulmonary lobes due to end-arteriolar pulmonary embolism (alveoli fill with blood instead of air, and have increased adhesion to the pleura, causing friction)

Front 151

Bloody pleural effusion due to pulmonary embolism

Back 151

- accumulation of blood in the pleural fluid
- increased pressure due to hemorrhage from PE cause increased capillary pressure, resulting in damage to membranes and leakage of blood and fluid into the pleural space.

Front 152

3 major disorders the predispose systemic embolism

Back 152

1. Atherosclerosis
2. Atrial fibrillation: develop thrombi (due to tissue damage) on the left atrial wall which then dislodge)
3. Left ventricular thrombosis (accounts for 20% of systemic emboli)

Front 153

Clinical and morphological consequences of emboli in: brain, spleen, liver, superior mesenteric artery, upper vs lower limb

Back 153

Brain: collateral circulation, infarct tissue undergoes liquefactive necrosis
Spleen: solid organ (light infarct tissue), no collateral circulation but not critical organ
Liver & kidney: hemorrhagic/white infarct tissue (coag. necrosis), collateral circulation, have to lose a lot of tissue to have pathologic effect
Superior mesenteric artery: infarcted small intestine looks dark, undergoes gangrenous changes (fatal), no collateral circulation (unlike inferior mes. art)
Upper limb (vs lower): upper extremities have collateral circulation, but lower do not so greater risk for ischemia/necrosis

Front 154

Blood compartments and components

Back 154

Plasma: 55% of total blood volume
- 91% water, 7% blood proteins (including clotting factors, if removed-->serum), 2% nutrients
Cellular components: 45% of blood volume
- buffy coat: less that 1% of total volume (middle density), contains WBCs and platelets
- RBCs: most dense

Front 155

Clinical relevance of CBC

Back 155

- can indicate stresses (including infection, anemia)
- gives absolute counts (WBC, RBC) and relative information (HCT)
- All values are relative to the patient's status, time, other conditions etc. (ex: value of pulseOx also depends on Hb)
- Can't diagnose w/ CBC but can corroborate clinical indications (dehydration)

Front 156

Cellular components of blood and their functions

Back 156

WBCs:
- not usually isolated except for genetic testing (or for certain viruses, Epstein-Barr). Also important for transplant, immunocompromised patients
- Buffy coat typically <1% of volume, elevated count could indicate infection (extreme elevation leukemia)
- 5-6 different types of WBCs in peripheral circulation, function in immune response
Platelets:
- small cell segments used in clotting. Platelet count below 20,000 is a moderate risk for spontaneous CNS/GI bleeds
RBCs:
- make up 25% of the body. Lack nucleus and organelles, uniform in shape and size with central pallor
- usually survive ~120 days, with turnover rate of 0.8%/day
- CBC values: MCV (mean corpuscular volume), RDW (red cell distribution width--is cell size uniform or bimodal?)

Front 157

Differential for hemolysis

Back 157

- CBC comes back with low Hb, retculoycte count is high (compensating for blood loss):
1.Autoimmune Hemolytic Anemia (AIHA): perform direct coombs test or direct antibody test
- IgG mediated: "warm," occurs that the normal body temperature, more common in hospitals. Look for antibody
- IgM mediated: "cold," occurs when body temp drops (often with mycoplasma infection)
2. Inherited: dysfunction/destruction of 3 parts of RBCs: hemoglobin (ex: sickle cell), membrane, or enzymes
3. Acquired: look at smear to diagnose. Could include microangiopathic, iatrogenic, hypersplenism, or direct injury.

Front 158

Differential for low RBC/Hb

Back 158

After CBC, look at reticulocyte count (production of new RBCs)
Normal retic--> acute hemorrhage (external/internal, retics have not caught up (2-3 days))
High retic (increased destruction):
-chronic bleed
- Autoimmune disease (IgG/IgM, perform direct coombs or antibody test)
- inherited RBC dysfunction/destruction
- Aquired RBC destruction
Low retic (decreased synthesis)-->perform MCV:
- Low MCV: hemoglobin problem (inborn or aquired)
- Normal MCV: exogenous problem (hormonal, environmental)
- High MCV: cell synthesis problem (DNA, membrane, hypothyriodism)

Front 159

Differential for decreased RBC synthesis

Back 159

- Low RBC/Hb on CBC + low retic count indicates synthesis problem-->perform MCV (average cell size)
1. Low MCV: indicates problem producing hemoglobin molecule
- inborn: problems with iron metabolism (sideroblastic anemia), heme synthesis (porphyria), hemoglobin synthesis (thalassemia, hemoglobinopathy)
- Acquired: from prolonged inflammation (acts like iron deficiency), iron deficiency, lead poisoning
2. Normal MCV: problem is exogenous to the cell
- renal failure, hormone deficiency (cortisol), early inflammation, pregnancy, marrow failure, marrow replacement
3. High MCV: problem in cell synthesis
- impaired DNA synthesis (B-12/folate deficiency, myelodysplastic syndrome, marrow failure), excessive membrane and target cells (liver failure, jaundice, post splenectomy), hypothyroidism

Front 160

Differential for high/low WBC

Back 160

1. High WBC:
- stress (trauma and acute issues), infection, steroids, inflammation, malignancy (huge)
2. Low WBC
- Increased destruction or use: sepsis or injury, autoimmune disease, hypersplenism/sequestration
- Decreased production: replacement, viral infection (like CMV), aplasia (from chemo or iatrogenic)
- marginalization: adherence to vessel wall to migrate to tissues (artificial lowering of WBC)

Front 161

Differential for High/low neurtrophils

Back 161

High neutrophils and bands (immature)
- bacterial infections, acute inflammation, stress, congenital
Low Neutrophils/bands (neutropenia is <1500, moderate bacterial infection at 500)
- increased destruction: consumption, autoimmune
- decreased production: marrow failure, viral infection

Front 162

Differential for high/low lymphocytes

Back 162

High lymphocytes:
- intracellular infections (pertussis, EBV), malignancy
Low lymphocytes
- increased destruction: viral infections (HIV), autoimmune disease, excess steroids (endogenous or exogenous)
- decreased production: immunodeficiency, marrow failure

Front 163

Differential for elevated eosinophils, monocytes, high/low platelets (each individually)

Back 163

High eosinophils:
- parasitic infection, allergy
High monocytes:
- intracellular infections
High platelets:
- acute phase reactant-->inflammation/infection
Low platelets:
- increased destruction: microangiopathic process/bleeding, large volume transfusion, autoimmune
- decreased production: marrow failure

Front 164

Ways inflammation affects the blood

Back 164

- acute plasma reactants are activated: immunoglobulins, platelets, complement proteins, ferritin, C-reactive protein, and others
- hemoglobin production falls (usually) due to iron sequestration (iron is growth factor for bacteria, so inhibited to fight infection)
- increased white cell count
- ESR will increase: measure of who much RBCs will settle in an hour

Front 165

Normal pathways of Acetaminophen metabolism by the liver

Back 165

- 95% coverted to APAP glucuronide or APAP-sulfate by glucuronidase + sulfination pathways
- 5% coverted to NAPQI by P450 (N-acetyl-p-benzoquinone imine)
- with a normal dose NAPQI is reduce by glutathione and excreted as mercaptopuric acid. If glutathione is unavailable (overdose) then cell death
Other factors:
- drugs (alcohol) metabolized by the sER/P450 system can cause induction of that pathway, causing more toxic metabolism

Front 166

Consequences of acetaminophen overdoes

Back 166

- toxic dose: ~8g (16 500mg tablets). Not necessarily lethal dose (but adverse effects seen)
- with more drug in the liver, more is metabolized by p450 system--> NAPQI saturates the glutathione metabolism causing cell death.

Front 167

Treatments for acetaminophen overdose

Back 167

- Charcoal: (standard for most overdoses), absorbs drug in GI and prevents absorption
- Acetylcysteine ("Mucomyst"): source of extra glutathione (normally used for CF to loosen mucous)
- Oral lactulose: synthetic disaccharide that helps remove ammonia by causing osmotic diarrhea (prevent protein absorption, acidifies gut to prevent ammonium ion reabsorption)

Front 168

Pattern of liver injury in patients with acetominophen overdose

Back 168

- takes several days to die from liver failure due to acetaminophen overdose. Most patients treated early can recover
Early:
- jaundice
- high bilirubin, AST, ammonia (indicative of liver damage)
- normal total protein (indicates acute rather than chronic damage)
Middle:
- jaundice, hepatomegaly (cell swelling/fat accumulation), asterixis (hand flapping, due to ammonia in CNS)
Late:
- hepatic encephalopathy (disordered CNS due to failed detoxification by liver, esp ammonia), hypotension, bradycardia, anuria, seizures, death

Front 169

Define asterixis

Back 169

= abnormal tremor/flapping of the hands when extended at the wrist.
- Due to interruption of the neural impulses needed to sustain muscle contrations
- Indicates (but not diagnostic) CNS toxicity occurring in liver failure (from ammonia accumulation)

Front 170

Define hepatic encephalopathy

Back 170

- a state of disordered CNS functioning resulting from failure o the liver to detoxify noxious agents from the intestine (particularly ammonia).
- Due to hepatocellular dysfunction and port-systemic shunting of ammonia
- Symptoms: initially subtle changes in logical thinking, personality, and behavior; then increasing drowsiness, confusion, and eventually loss of consciousness and coma.

Front 171

Michaelis-menton Equation

Back 171

V₀= Vmax[S]/Km+[S]

Vmax: max rate of catalysis for enzyme concentration (when saturated)
Km: affinity of enzyme for substrate (lower is better)

Front 172

Kcat

Back 172

Kcat = Vmax/[Enz]

measure of the catalytic efficiency of the enzyme

Front 173

Competitive inhibition

Back 173

- inhibitor binds at the active site, preventing substrate binding (generally similar structure). Depending on the nature of the binding, may be overcome by adding more substrate or new enzyme synthesis.
- Will increase Km, but not effect Vmax (or Kcat)

Front 174

Non-competitive inhibitors

Back 174

- bind an allosteric site, changing binding site conformation
- Can overcome by mutating the allosteric site to prevent binding.
- Do not effect Km, but will decrease Vmax (and Kcat)

Front 175

Reversible vs. Irreversible inhibitors

Back 175

Reversible:
- not covalently bound, can be removed
- good for drug administration because results can be removed.
Irreversibly
- covalently bound to enzyme. New enzyme must be synthesized to restore function.

Front 176

Pharmacokinetics vs pharmacodynamics

Back 176

Pharmacokinetics: the body's treatment of a drug--factors which influence drug dosage and delivery.
- ADME interactions (absorption, distribution, metabolism, elimination)
Pharmacodynamics: the biochemical and physical effects of a drug on the body, including mechanism of effect

Front 177

describe EC50

Back 177

= Effective concentration in plasma at which 50% of maximal drug effect is observed
- found on a dose/response curve at half the dose (Y) of the asymptote

Also:
ED50 (effective dose for 50% response)
IC50 (inhibitory concentration)
TD50 (toxic concentration)
LD50 (lethal does)

Front 178

Drug efficacy vs potency

Back 178

Efficacy: the level of effect that a drug elicits (a response, mostly Y axis)
Potency: the amount of drug responsible for a response (a dose, mostly X axis)

Front 179

Define drug side effects and therapeutic index

Back 179

Side effects are undesired effects of drug administration and are usually due to:
- therapeutic mechanism of action (target has multiple functions)
- off-target mechanism of action: (drug interacts with a different system from target)
- interaction/interference from other drugs/foods/substances
Therapeutic index = ED50 of side effect/ED50 of therapeutic effect
- the greater the space (larger TI) between the effects, the less side-effects in general

Front 180

Common mechanisms of drug action

Back 180

Directly/indirectly (metabolized) and reversibly/irreversibly on
- receptors
- channels
- transporters
- enzymes
- free molecules (small or macro)
They often inhibit these (mimic endogenous materials) but may be them themselves (especially enzymes)

Front 181

Origin of the concept of receptors

Back 181

- receptors were first inferred from observations of chemical and physiological specificity of drug effects. Interpreted as side chains on tissues that the drug bound to--> "lock and key" hypothesis
- Drugs today still classified by what lock/receptor they target. Normal hormones are keys while drugs are lock picks (agonists) or dud-keys which block all use (antagonists)

Front 182

define generalized or drug receptor

Back 182

anything that a drug binds to (enzyme, channel, transporter, cytoskeleton, etc) is the receptor for that drug

Front 183

Define specialized or biological receptor

Back 183

proteins that mediate the transfer of information from outside the cell to inside (not other function). This is a large receptor gene family, with expansions in multicellular organisms

Front 184

Describe Kd

Back 184

= the dissociation constant
- describes the affinity of a drug for its receptor (high means less affinity); reflects the chemical binding forces, is unique for each receptor-drug pair
Kd = [D][R]/[DR] ; [DR]/[Rtot] = [D]/[D]+Kd (use to predict receptor occupancy)
- when 50% of the receptors are occupied, Kd = [D]

Front 185

Difference between agonists, partial agonists, antagonists, and inverse agonists

Back 185

Agonists: bind to a receptor and fully activate it
Partial agonist: bind to receptor but are not as effective as a full agonist (considered competitive antagonist if full agonists present)
Neutral antagonist: has no activity, but can competitively block activity of agonists
Inverse agonist: induce opposite response as agonists

Front 186

4 major receptor superfamilies

Back 186

1. Ligand-gated channels
2. G-protein couple receptors
3. Enzyme/cytokine receptors
4. Ligand-activated transcription factors

Similarities: first 3 found on cell surface (4 is in the nucleus), require ligand binding to change function which leads to some downstream change within the cell.

Front 187

Ligand-gated channels (receptor functions)

Back 187

- activated by certain neurotransmitters, providing a direct link between agonist binding and conductance
- act by opening ion-selective pores in the membrane, allowing specific ions to move
- composed of multiple subunits and have multiple agonist binding site and allosteric regulatory sites

Front 188

G-protein linked receptors (receptor functions)

Back 188

- activated by neurotransmitters and hormones (and lots of durgs)
- when activated they associated with heterotrimeric G proteins, which activate effectors to generate small second messengers (cAMP, cGMP, Ca2+)
- Usually operate as monomers (with exceptions) but still have multiple transmembrane domains.
- Ex: HIV uses CCR5/CXCR$ (GPCRs) to gain access to cells

Front 189

Enzyme/cytokine receptors (receptor functions)

Back 189

- typically include receptor tyrosine kinases (also guanylate cyclases)
- single polypeptides with either intrinsic enzymatic activity (auto-phosphorylation) or act act as docking sites for other enzymes (ex: JAKs)
- receptors often dimerize on activation to cross phosphorylate and initiate intracellular signals (including recruitment and phosphorylation of other proteins)
-ex: EGF receptor (inhibited in treatment for breast cancer with HER2 mutation)

Front 190

Nuclear receptors (receptors properties)

Back 190

- aka. ligand-activated transcription factors, usually on nuclear envelope
- usually activated by steroids or other hydrophobic molecules that can enter the cell (fairly common drug targets)
- dimerize upon binding, which induces DNA binding or recruitment of other transcription factors to bind DNA, leading to gene transcription
- ex: progestin receptor (binds estrogen then dimerizes to bind DNA)

Front 191

Receptor-effector coupling and spare receptors

Back 191

- in many cases not all receptors must be activated to cause maximal response: leaves "spare receptors", usually occurs in more potent drugs
- presence of spare receptors increases the efficacy of partial agonists
- as spare receptors are lost, potency decreases to a point, until maximal effect of the agonist is reduced--> EC50 depends on the amount of free receptors available.

Front 192

Genetic basis for varying drug responses between individuals

Back 192

- polymorphisms in genes that encode enzymes involved in drug metabolism or receptors/signaling pathways
- SNPS or Indels (insertions/deletions)
- pharmacodynamic polymorphisms are much rarer than pharmacokinetic ones (less evolutionary reason to select against exogenous chemicals)
- the combinations of polymorphism in the individual haplotype determine response (may be multiple enzymes involved)

Front 193

Polymorphic responses to drugs (pharmacodynamic vs. pharmacokinetic polymorphisms)

Back 193

Pharmacodynamic:
- genetically determined change in a response to drug (ex: changed/absent target enzyme)
- less common and/or subtle phenotype: less evolutionary pressure to select against non-essential/exogenous pathways
Pharmacokinetic polymorphisms:
- genetically determined change in drug disposition, usually metabolism
- Many examples, low selection pressure because phenotype only seen with drug challenge

Front 194

Consequences of altered drug metabolism

Back 194

Effect depends on therpeutic index and whether a drug is inactivated or activated by metabolism:
Inactivated by metabolism:
- decreased metabolism: ↓clearance so ↑half-life, plasma concentration, clinical effect. Possible toxicity
- increased metabolism: ↑clearance so ↓half-life, [plasma], clinical effect
Activated by metabolism
- decreased metabolism: ↓ active metabolite available so ↓therapeutic effect
- increased metabolism: ↑active metabolite available so ↑clinical effect, possible toxicity

Front 195

Ways to detect pharmacogenetic traits

Back 195

Phenotyping:
- ex: pharmacokinetics, urinary metabolite ratios, enzyme activities in blood
- Can detect all mutant alleles, but not specific mutations
- affected by epigenetic factors, can change with time
Genotyping:
- ex: allele-specific PCR, gene array, sequencing
- Does not always reflect phenotype (epigenetics); can only detect known alleles
- Can give exact mutation and heritability

Front 196

CYP2D6 polymorphism

Back 196

- mutations of P450 (CYP) enzyme superfamily that metabolizes many drugs (including codeine). Can result in therapeutic failure at standard doses
- function of enzyme is additive depending on amplification of the gene (more gene copies more effect) (appears autosomal recessive inheritance)
- 3 polymorphisms in humans: "normal" activity (most common), low activity (no codeine effect), very high activity (exaggerated effect)

Front 197

Frequenciees of polymorphisms with respect to race, geographical location and/or gender

Back 197

- certain polymorphisms occur more frequently in certain populations than others. All the mentioned characteristics can effect PM frequency (and then there is variation within those populations)
- population/race differences (ex: CY2D6); inherited disorders (ex: G6PD deficiency)
- can test for these PMs or use populations stats/family history to tailor treatment to expected haplotype

Front 198

Drug bioavailability

Back 198

= the fraction (0-1 scale) of administered dose that reaches systemic circulation
- Affected by dissolution in GI fluids, absorption, first pass metabolism by the liver.
- Used to adjust dose when given orally (IV has 100%)

Front 199

Volume of distribution (pharmacokinetics)

Back 199

= the amount of drug (mg)/plasma concentration (mg/ml)
- relates the amount of drug in the body to plasma concentration. Given in mL but not necessarily a physical dose
- Vd = dose (mg)/C₀(mg/mL); where C₀ is the [ ] if distribution was instantaneous (extrapolate to time zero when drug amount is known)
- used to determine dose required for desired plasma concentration (single or loading dose)
- affects biological half-life

Front 200

Biological half-life (of a drug)

Back 200

= the time required to reduce the plasma drug concentration by 50%
- determines the duration of action of a single dose (5-6 half lives for effective total elimination)
- In chronic dosing schedule determines time to reach a new steady state (for 1st order drugs) when rate of administration changes
- together with therapeutic index determines choice of dosage interval.
- Determines Elimination rate constant: fraction of drug eliminated per unit of time.
T1/2 = 0.693(Vd)/CLp

Front 201

Drug Clearance (pharmacokinetics)

Back 201

= the volume of plasma from which the drug is removed per unit time (plasma clearance = sum of clearance from all organs)
- Clearance = rate of elimination/plasma concentration = hepatic extraction ratio (E) X hepatic flow (Q, ~1.5L/min normally)
- determines rate of drug administration required to reach desired steady state level (rate of elimination = rate of administration); rate of admin = [SS]xCL/F* *if oral factor in bioavailability
- Clearance is inversely proportional to half-life, directly proportional to elimination rate constant (Ke). It is biologically independent of volume distribution (Vd). CL = Ke x Vd = 0.693 x Vd/ T₁₍₂

Front 202

Two-compartment model of pharmacokinetics

Back 202

- two compartments in which drug is located in the body: central (Vc, plasma) and peripheral (Vp, organs). Drug enters/exits the central compartment via administration, distribution, excretion (Vd = Vc + Vp)

Front 203

Plasma concentration vs. Time curve for single dose administration

Back 203

Distribution phase: initial peak during which drug is moving from the central to peripheral compartment. Shows exponential decline as the two compartments equilibrate
Elimination phase: elimination is the predominant determining concentration after distribution. If first order metabolism, will show linear decline.
- C₀ = the initial concentration if there was no distribution phase (extrapolate back from elimination curve, used to calculate Vp from Vd)
Concurrent absorption/elimination:
- absorption dominates initially, decreases and peaks when absorption = elimination
- after peak, elimination dominates so rate of [ ] decline increases as absorption slows.

Front 204

First order drug elimination

Back 204

- constant fraction of drug eliminated per time (concentration dependent)
- Rate constant Ke; T₁₍₂ = 0.693/Ke
- Preferred for clinical drugs because easier to control plasma concentrations

Front 205

Zero order drug elimination

Back 205

- constant, absolute amount of drug eliminated per unit time (concentration independent)
- can occur when the excretion mechanism is saturate or a required cofactor is limiting
- More difficult to control clinically (easier to overdose). Ex: opiods

Front 206

Effect of rate and route of drug absorption on onset of action, peak plasma levels and duration of action

Back 206

Faster absorption/IV/bolus
- high peak of plasma drug levels, high immediate bioavailability--> rapid onset, but metabolized fast (due to high concentration) so short duration
- Depending on therapeutic index could have adverse effects
Slow absorption/Oral/slow infusion
- smaller peak, slower onset of action, longer duration.
- Depending on the MEC could not have effect

Front 207

Plateau principle for steady-state plasma concentration

Back 207

- for drugs eliminated by 1st order kinetics (0 order administration), giving a dose at fixed intervals will result in a increase in plasma concentration until the dose=the amount eliminated in the interval
- takes 5-6 half lives to reach steady state. Can be reached earlier by giving loading dose (usually double in usual interval)
- half-life determines the amount of time to reach the steady state b/c it determines rate of elimination (dose is constant)
- dose determines the concentration at steady state

Front 208

Rate of administration needed to reach effective drug level

Back 208

rate of administration = dose/dose interval = [SS]xCL /F* (*F = bioavailability for oral administration)
- choice of dose rate depends on drug half-life, therapeutic window, frequency/severity of adverse effects, convenience/compliance of the patient
- Loading doses are used for drugs with a long half life or in emergencies to reach [SS] faster. Loading dose = [SS]Vd/F
- optimal dose rate is usually around 1 half-life, and a loading dose is usually 2x the maintenance dose

Front 209

Effect of physical barriers on drug absorption

Back 209

Drug must cross physical barriers to reach target tissue. The drug properties and transport mechanisms at the tissue determine how this is accomplished
2 mechanisms to cross barriers:
- passive diffusion: through lipid bilayers (lipid soluble) or paracellularly (water soluble)
- transport by specific transporters: usually requires energy (ATP or co-transport), drugs can compete for the transporter
Major barriers
- gut epithelium: tight junctions between cells, many receptors/carriers (though often not usable by drugs). Generally drugs must be lipophilic and non-ionized to be absorbed
- plasma: lipophilic drugs must bind to proteins, but then dissociate to be active/absorbed by target
- capillaries/BBB: most are fenestrated (allow most to cross), BBB has tight junctions so only lipophilic drugs can cross but then has transporters that actively pump stuff out.

Front 210

Factors effecting drug absorption through oral administration

Back 210

- solubility: must be lipid soluble to cross membrane but water-soluble to pass in gut environment/reach the epithelium
- stability in low pH (stomach acid)
- First pass metabolism: all absorbed products first travel to the liver (via portal vein) not too much should be lost
- pH range and ionization: ions cannot cross membrane, sp drug must have appropriate pKa to not be ionized in the intestine

Front 211

Ion trapping

Back 211

- drugs will tend to concentrate in the medium in which it is most ionized.
- good drugs will be non-ionized in the gut (so absorbed) and more ionized in the plasma so concentrated (less needs to be administered) and can be distributed
- most drugs are weak electrolytes and so will be partially ionized at some pH
- Ex: bactrim is a weak base so gets ionized in urine and concentrated so good for UTIs

Front 212

Plasma protein binding of drugs

Back 212

- Occurs for most drugs (lipophilic, use albumin), drugs are not active when bound
- binding is reversible and saturable: the higher the drug concentration the greater volume of free (active) drug
Consequences:
- only free drug can cross membranes/be active
- drugs compete for binding sites on protein, possible site for interactions (can displace each other, increasing the concentration/effect of one). Ex: warfarin: normal 99% bound/1% free, with other drugs 96% bound/4% free
- not usually that large concentration in the plasma because of peripheral distribution (unless %bound is very high)
- binding slows the onset of drug action: slows the rate of equilibration with extracellular fluids (keeps concentration of free drug low, which slows movement by mass action)

Front 213

Sites of drug biotransformation

Back 213

Liver: main site of metabolism as enzymes are concentrated there and receives first pass from portal blood
GI track: enzymes and bacteria metabolize drug so that it can or can't be absorbed
Kidney: metabolized to help excretion
Other tissues: most often have toxicological concerns, not part of normal excretion

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Phases of drug metabolism

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Phase I: non-conjugative
- redox, hydrolysis
- adds or unmasks a functional group
- usually inactivating, sometimes activating
Phase II: conjugative (synthetic)
- covalent coupling of drugs to endogenous molecule
- increases polarity, adds bulky moiety (recognized by transporters into the bile)
- usually inactivating
- may follow phase I activation
- ex: glucuronidation, acetylation, sulfation, methylation, glutathione conjugation (not the major route for drug clearance, more protective)

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Causes of altered drug metabolism resulting in altered therapeutic effect

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Increase effect/reduced metabolism:
- occurs in neonates, liver failure, genetic deficiencies, enzyme inhibition (from drugs/ diet/herbal; competitive or mechanism based (inhibitor suicide binding))
Decreased effect/increased metabolism
- occurs due to enzyme induction (from drugs/diet/herbs), gene amplification

These factors differentially affect individuals, especially their P450 family

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Renal excretion of drugs

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- only free (non-protein bound) is filter and can be excreted (also influenced by BP and flow)
- drug reabsorption in the tubule occurs by passive diffusion, so more lipid soluble/less polar are reabsorbed while ionized drugs are excreted
- pH modulation of the urine can have toxic effects because it changes the elimination of drugs normally ionized and excreted
- there are specific acid and base transporters, drugs can compete for those transporters

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DNA damaging agents

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Exogenous:
- UV radiation: can produce covalent linkage between adjacent pyrimidine bases resulting in deletion upon replication. UV-C most dangerous but filtered. UV-B is most ubiquitous carcinogen
- Ionizing radiation: introduces elections creating radicals on DNA or on water creating and ROS that interacts with DNA (more common-->more O-H bonds)
- chemicals: potency determined by structure/reactivity w/ DNA, often require metabolic activation. Ex: benzopyrene (combustion-->lung/bladder), aflatoxin B1 (fungus on crops-->liver), 2-actylaminofluorine & β-napthyline (bladder), Nitrogen mustard (anti-cancer, cross-links DNA to kill)
Endogenous
- ROS: electrophilic agents from normal respiration
- Alkylation damage: adds CH3 to DNA
- Spontaneous hydrolysis (deamination, depurination): links to doxyribose hydrolize (no base) or C-->U; most common spontaneous damage
- Errors in replication: mismatches, insertions, deletions, single--> double strand breaks by DNA polymerase
Radiation, chemotherapy, free radicals, replisome stalling all are sources of strand break

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Mechanisms of DNA repair

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- Base excision repair: primarily responsible for removing small, non-helix-distorting base lesions form the genome (ex: modified or incorrectly incorporated bases).
- Nucleotide excision repair: repairs bulky helix-distorting lesions (longer/larger modifications or errors). Primary pathway for UV damage repair (defective in xeroderma pigmentosum)
- transcription-coupled repair (occupies in tandem with transcription): RNApolymerase stalls at lesion recruiting CSB to recruit other proteins (highest priority). Defective in Cockayne syndrome (accelerated aging, death; no cancer--> dead cells from failed transcription)

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DNA damage Tolerance

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- translesion synthesis: allows replication machinery to replicate past lesions. Low fidelity, high mutation rate, destabilizes genome
- Recombination: strand exchange reaction with sister chromosome; damaged DNA section exchanged with other stand; mutations and inappropriate recombination may occur; increases mutation rate, destabilizes genome

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Relationship between genetic instability and cancer

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- mutations in the genome increase the rate of mutated proteins causing functional errors resulting in further damage (ex: replication errors), instability (ex: failure to repair) and cancer
- Chromosomal instability (CIN): large sections or whole chromosomes are gained or lost in rearrangements, deletions, insertions, inversion, amplifications. Characteristic in 85% of cancers, correlates to more aggressive tumors w/ metastatic potential and resistance. Ex: xeroderma pigmentosum, Ataxia telangiectasia (defective DNA damage sensor), Boom's syndrome (defect in DNA helicase)

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Mutator hypothesis of cancer

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the frequency with which a cell would normally accumulate mutations is so slow that it would improbably for anyone to independently accumulate enough mutations for cancer (4-7 critical genes). Instead early mutations must increase the frequency of subsequent mutations (ex: mutations in repair mechanisms). This takes many years (disease of old age)

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Evolution in cancer cells

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- Development of cancer over time is an evolutionary processes because the host environment exhibits selective pressure for the fittest cells
- Tumor-genesis is viewed as successful clonal expansion of a aberrant cell population (progressive mutation increases ability to replicate and avoid destruction by the host).
- 3 stages of carcinogenesis: initiation, promotion, progression
- expanding populations of genetically unstable cells increase the statistical probability of mutations in additional genes giving rise to growth or survival advantage

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Top 6 acquired capabilities of cancers

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1. sustained angiogenesis (blood flow)
2. tissue invasion & metastasis
3. Evaded apoptosis
4. self-sufficiency in growth signals
5. insensitivity to anti-growth signals
6. unlimited replicative potential
(order of obtaining abilities is variable among cancers, may involve multiple events for one ability)

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Synthetic lethality and application in cancer treatment

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- arises when a combination of mutations/deletions in two or more genes results in cell death but mutation in only one is still viable.
- In cancer if a mutation is in one of the genes, attacking the complement will kill cancer cells without killing host cells (drug or mutation alone cannot kill the cell, but together they do).

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Mechanisms for converting pro-growth signals into oncogenes (especially receptor tyrosine kinases)

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Point mutations and deletions: make the receptor constitutively active (signal without ligand binding)
- deletions: loss of ligand binding domain due truncation of the protein
- point mutations: can cause truncation or linkages between receptors to activate them independently of ligand
Over-expression: (increase in pro-growth/anti-apoptotic signals)
- ↑ receptor→ ↑ opportunity for ligand binding→ ↑ total growth signaling→ cancerous growth
- often associated with gene amplification (ex: Her2/neu) or mutations that allow for RNA amplification
Autocrine:
- cell produces it own ligand→ ↑receptor binding → ↑growth signaling

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Phorbol esters as tumor promotors

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- they increase the potency of carcinogens by activating the inflammation cascade (and other cellular proliferative mechanisms)
- TPA binds protein kinase C (PKC) (usually binds DAG and Ca2+) and activate it for a prolonged period of time (no way to turn it off unlike DAG).Activation increases inflammation, cell proliferation, and anti-apoptosis signaling.
PKC → (+) IKK → (+)NF-κβ → TNF-α (inflammation), COX-2 (inflammation, proliferation, Ø contact inhibition), cyclin D (proliferation), Bcl-X & IAP-1 (protection from apoptosis)

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Diseases associated with defective hemostatic mechanism

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Bleeding:
- Hemophilias (A, B)
- Idiopathic thrombocytopenic purpura (decreased platelets)
- Disseminated intravascular coagulation (rampant thrombin action)
Thrombosis
- MI, CVA, PE, Peripheral artery disease, Trousseau syndrome

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Components of the hemostatic mechanism

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1. Vessel wall: endothelial cells and subendothelial constituents
2. Platelets: have no nucleus, alpha granules contain proteins (fibrinogen, vWF, Factor V), dense granules contain non-protein components (ADP, serotonin)
- Coagulation factors
- Coagulation factor inhibitors
- Fibrinolytic system

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Primary hemostasis, formation of platelet plug

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Vascular damage:
- produces transient vasoconstriction by smooth muscle contraction (slows blood flow)
- exposes subendothelial components (collagen) which bind Von Willenbrand Factor which binds platelets at GP1B
- Platelets are activated by binding thombin, thromboxane A2 (TXA2, inhibited by NSAIDS), ADP (secreted by platelet dense granules), collagen at different extracellular receptors
- Activated platelets extend pseudopodia (increase surface area), release alpha and dense granule contents (recruit more platelets, induce intracellular signaling, create negative PM to aggregate more platelets

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von Willebrand Factor (VWF)

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- plasma protein that binds to exposed collagen in subepithelium. It then binds GP1B receptors on platelet surface, initiating primary hemostasis
- Structure: very large multimer of repeating subunits linked by disulfide bonds
- carries VIII in plasma (would degrade otherwise
- Von Willebrand Disease occurs in individuals who have reduced or abnormal VWF synthesis. Severe form is rare (1/1mil) mild form is common (1/100)

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Platelet Activators

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Thrombin: Product of coagulation cascade
Thromboxane A2: a prostoglandin, inhibited by aspirin and NSAIDs (COX inhibitors)
ADP: Secreted from platelet dense granules, promotes further activation of platelets
Collagen: exposed in vessel damange, binds VWF, which then binds platelets through GP1b

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Platelet activation events

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1. Pseudopod formation: Leads to an increase in surface area
2. Release of alpha and dense granule contents: ADP, Serotonin, Fibrinogen, VWF, factor V
3. Induction of receptor-mediated signaling cascades:
4. Exposure of phosphatidylserine on the exterior plasma membrane: Provides a negatively-charged surface for platelet adhesion
5. Aggregation: Bridging of nearby platelets by fibrinogen and GPIIb-IIIa

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Thrombin

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- Is the key enzyme in hemostasis
- Is a serine protease (cleaves at arginine residues)
- is activated from prothrombin by prothrombinase complex (Xa+Va) on platelet surface. Inactivated by antithrombin
- Catalyzes fibrin formation from fibrinogen
- Activates platelets
- Activates procoagulant factors V, VIII, XI and XIII
- Activates protein C, an anticoagulant factor

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Fibrin

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- trinodular structure (2D, 1 E).
- fibrinongen activated at E domain by thrombin (cleaves peptide chains making them adhesive for D subunits)
- polymerized fibrin forms a gel like mesh. Secondary stabilization from Factor XIIIa (which cross-links D domains)
-

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Vitamin K dependent Coagulation factors

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- 10a, 9a, 7a, 2a
- all contain Ca2+ binding Gla domains
- Ca2+ and a protein cofactor (increase catalytic efficiency) are required for all reactions
- synthesis is inhibited by Warfarin because it inhibits Vit K reductase which is necessary for γ-carboxyglutamic acid synthesis (in these proteins)

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Lab tests for clotting function

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PT: Prothrombin time
- measure of the time to clot via the extrinsic pathway (add tissue factor/thromboplastin and excess Ca2+)
- normal is 11-13 sec. If prolonged Factors 7, 5, 10, 2, and/or 1 are substantially decreased
- Warfarin will prolong PT
aPTT: activated partial thromboplastin time
- measure time to clot via intrinsic pathway (add activated partial thromboplastin (includes silica) and excess calcium)
- normal is 25-35s. If prolonged, prekallikrien, HMWK, factors 12, 11, 8, 9, 5, 10, 2, and/or 1 are substantially decreased
- Heparin will prolong aPTT

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3 Mechanism for terminating the hemostatic process

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Antithrombin III: serine protease inhibitor
- inhibits thrombin, factors 10a, 7a, 9a (covalently binds active site of enzyme)
- catalyzed by Heparin (binds both thrombin and antithrombin), changes AT to better recognize 10a
Activated protein C:
- inactivates factor 5a and 8a via proteolysis
- activated by protein S (cofactor), thrombin/thrombomodulin (fibrin in this form cannot promote coagulation).
- vitamin K dependent (as is S), so inhibited by warfarin
Tissue factor pathway inhibition (TFPI)
- inactivates Factor 7a and 10a

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Heparin

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- CATALYST that promotes anticoagulation via antithrombin
- structure: repeating suflated disaccharides
- binds target factor and AT, increasing the reaction rate (facilitates diffusion)
- induced conformational change in AT that makes it better able to recognize Xa.
-LMWHep acts mainly to help AT inhibit Xa though it does target other factors
- Must be given subQ
- produces elongation of aPTT

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Fibrinolysis

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- Thrombin induces the release of tPAand uPA from endothelial cells
- tPAand uPA (don't need activation) catalyze the conversion of plasminogen (plasma protein) to plasmin
- PIasmin is a serine protease that cleaves fibrin between D and E domains and solubilizes it
- In contrast to uPA, tPA has a higher affinity for pIasminogen bound to the fibrin clot and is said to be clot- specific
- tPA is used therapeutically (eg. in the treatment of acute myocardial infarction and stroke)

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Define signal transduction

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the conversion of extracellular signals into intracellular signals

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Define hormones

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- signal molecules secreted by endocrine cells
- travel through the blood stream to communicate with cells nearly anywhere in the body (long distance)

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Define receptors

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the protein on which a signal molecule binds to effect change in the target cell (intracellular or extracellular)

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Define Agonist

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molecules which bind to receptors and activate them

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Define antagonist

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molecules that bind to receptors but do not activate and by binding prevent agonists from binding and activating

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5 types of cell communication

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Contact dependent: signals remain bound to the surface of the signalling cell to it must be indirect contact with target cell to have an effect
Autocrine: cells produce signals they (or adjacent cells of the same type) respond to. Can produce positive feed back quickly
Paracrine: cells produce local mediators that affect only the cells in the nearby environment
Synaptic: signals (neurotransmitters) are transmitted across a tight synpase; release and uptake is couple to ion channels/electric potential
Endocrine: cells secrete hormones into the blood stream to effect cells that are far away

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Effect of physiochemical properties on hormone function

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- hydrophobic nature of ligands determines whether they can diffuse into/across a membrane and target intercellular receptors (lipophilic) or travel in the blood stream and target extracellular receptors (hydrophilic). Some small molecules are still able diffuse across the membrane and target intercellular or nucleic receptors

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SH2 domains in RTK signaling

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- SH2 domains are found in different intercellular signaling proteins and are specific for phosphorylated (activated) tyrosine residues adjacent to a specific 3AA region on the protein kinase domains of TKRs.
- each has 2 binding domains: 1 for phosphorylated TK, one for a 3AA sequence specific to the SH2 domain.
- Allows for high specificity and regulation (requires ligand binding) in post-receptor signal transduction: SH2 domains binding is first level of post-receptor signaling
- when RTK binds ligand, the receptor dimerizes and autophosphorylates Tyr residues, allowing recruitment of intracellular proteins with SH2 domains that recognize and bind specific phosphorylated Tyr motifs.

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Domains involved in post-receptor signaling of RTKs

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- SH2: recognize activated/phosphorylated Tyr residues in a specific 3AA motif
- PH (Pleckstin homology domain): on proteins recruited to the surface to bind phosphorylated inositol phospholipids (Phos'd by activated PI3 kinase and moved to membrane)
PTB (phosphotyrosine binding domain): binds phosphotyrosine residues (Y-Phos)
SH3 (Src homology 3 domain) : binds proline rich motifs (PPP) (ex: SH3 on GRB2 binds SOS in the Ras-MAP kinase pathway)

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Ras-MAP kinase pathway

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- EGFR receptors bind EGF and dimerize, autophosphorylating and recruiting proteins with SH2 domains (PLC-γ, PI3 kinase, GRB2)
- GRB2 is bound to SOS (via SH3), which is a guanine nucleotide exchange factor (GNE). When recruited GRB2-SOS converts Ras-GDP (membrane bound) to Ras-GTP (active)
- Activation cascade: Ras-GTP → Raf-1 → MAPKinaseKinase (MAPKK) → MAPKinase → transcription factors

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Cellular Effects of EGFR binding

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- Activation of Ras-MAP pathway: activation of transcription factors to promote growth
- Activation of PI3-Kinase (by Phos'd Tyr): phosphorylates inositol moieties on the inner PM to create PIP2
- Activation of PLC-γ: cleaves PIP2 (membrane bound) → IP3 (which raises Ca2+ levels, activating Ca-sensitive proteins) + DAG (can be cleaved to arachidonic acid or can activate Protein Kinase C (Ca2+ dependent))
***PLC-β activated by GPCR, does the same thing (different regulation)

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Regulation and termination of TRK signaling

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- receptor sequestration (excess agonist can do this: cells produce receptors slower than they take them in. Would initially get signaling peak but then gradual loss)
- receptor down regulation
- receptor inactivation
- inactivation of signaling protein
- production of inhibitory protein (to the signaling cascade)

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Post receptor signaling in RTKs vs GPCRs

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- both activate an intermediate protein that leads to downstream activation of proteins and transcription factors (gene expression)
- GPCRs: activated G-proteins in close proximity to the receptor on the membrane. The Gp then activates downstream signaling
- RTKs: recruit docking proteins to the receptor (via high affinity bonding). These proteins then activate downstream signaling.

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Post receptor signaling in RTKs and TKARs (tyrosine kinase associated receptors)

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- Both phosphorylate Tyr residues in order to recruit docking proteins
- RTKs: auto-phosphorylate, in which each half of the receptor dimer phosphorylates the counterpart.
- TKARs: employ a separate kinase (ex: JAK in α-interferon pathway) for Tyr phosphorylation. Upon ligand binding JAKs get recruited and activated to cross-phosphorylate each other then the Tyr residues on the receptor (this allows STAT2 to bind, get phosphorylated, dissociate and dimerize and act on DNA + regulatory proteins)

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TGF-β Receptor signaling pathway

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- TGF-β R is a receptor serine/threonine kinase; binding causes formation & activation of a heteromeric complex (Type I + II)
- TGF-β binding on receptor causes it to recruit and phos a receptor of a different type, which then recruits and phos's a Smad2/3. Phos'd Smad dissociates and binds Smad4 and migrates to the nucleus (binds other proteins) to activate transcription of specific genes (incl: p15 (cdk inhibitor), PAI-1 (ETC matrix protease inhibitor))
- TGF-β Receptors are important in development (differentiation, morphogenesis, proliferation, migration)
- Loss of function mutations found in cancer, thought to cause deregulation of the cell cycle and increase metastasis.

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Development of "smart drugs"

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- target cancer cells based on mechanism that is specific to the mutated mechanisms in a tumor (target different steps in the pathways of growth signals). Ex: tyrosine kinase inhibitors

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Protein kinase inhibitors as anti-cancer agents

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- developed to inhibit PTK receptor and non-receptor oncogenes that are involved in cancer
- goal is to find inhibitors that can selectively inhibit specific cancer-relevant PTKs without inhibiting normally functioning ones
- EX: Bcr-Abl inhibitor: inhibits PTK resulting from fused Abl gene (normal PTK) that only occurs in cancerous cells (90% of CML). Resistance develops (from surviving mutants): mutations, over-expression, const. active downstream signalling
Ex: EGFR inhibitor, Iressa, is effective in some Non-small cell lung carcinomas. Can inhibit a number of activated EGFRs (more possible uses) but other mutations confer resistance

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Prostate Cancer treatments and side effects

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**most prostate cancers are testosterone dependent, so strategy is to limit T -- slows growth, not cured**
- GnRH receptor agonists: block GnRH receptors→ ↓FSH, ↓LH production→ ↓testosterone (downstream)
- GnRH agonists: utilize negative feedback mechanism of steroid hormones→ androgens inhibit GnRH (at hypothalamus), LH/FSH (at pituitary); also agonists cause down-regulation of receptors at the cell. Initially helps growth, but after down regulation causes inhibition
- Orchiectomy (castration): removes main source of androgens (but others still produced in adrenal glands)
- Estrogen: negative feedback on androgen production, not good side effects
- CYP-450 inhibitors: shut down T synthesis by P-450. Multifunctional enzyme though so side effects (including loss of cortisol production, can supplement)
-5α-reductase inhibitors: prevent conversion of T→ DHT (more active form)
- Antiandrogens (steroidal and not): act as receptor antagonists for androgen receptors on prostate cells. Can block hormones from multiple sources since downstream in pathway
**major side effects: loss of sex drive, sexual dysfunction, psych disturbances, loss of muscle mass. May develop hormone-independent tumor

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Breast Cancer treatments and side effects in hormone dependent tumors

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- Estrogen receptor antagonists: block estrogen signaling directly on cancer cell.
- Estrogen biosynthesis inhibitors (aromatase inhibitors): prevent the conversion of androgens into estrogen/estradiol. Can be used both for hormone dependent breast cancer and endometriosis. Build up of androgens can cause side effects: fatigue, hot flashes, loss of bone density