Cellular & Molecular Basis of Medicine, Exam 3 - 2014

Expression of virulence genes can be upregulated by a change in cell density

Biofilms are important pathogenic factors Signal molecules do not bind to receptor w/ low population density ∙Will bind more receptors, causing allosteric change in regulator and may activate expression of genes

Erythromycin: Mechanism of Action, Pharmacokinetics & ADRs

*Primarily bacteriostatic, binds 50S of bacterial ribosomes, inhibits protein synthesis* Resistance - *change in ribosomal binding site (primary)*, inc. hydrolysis, dec. drug uptake / efflux pump Decomposed by gastric HCL to irritant, adm PO as enteric-coated or esterified preps to reduce this decomposition Primarily biliary\GI exc; urinary minor ADRs: GI disturbances, rash, allergy, cholestatic hepatitis (estolate ester), ototoxicity / tinnitus/ hearing loss @ IV/ high doses

Common Virulence Factors of Enterobacteriaceae

All have endotoxin ∙If they gain access to blood, they can cause endotoxic shock Capsule Antigenic & phase variation Type III secretion systems Sequestration of growth factors Resistance to serum killing Antimicrobial resistance (i.e. ESBL's of E. coli and Klebsiella pneumoniae)

Mediators of neutrophil extravasation

(A) Endothelial cells have pre-formed P selectin molecules, in the form of Weibel Palade bodies, that can be relocated to cell surface upon activation ∙Histamine or thrombin acts as the signal to express the P selectin on the surface ∙This allows for up-regulation of P selectin on the endothelium to occur in minutes to hours →The process would take much longer if it depended on changes in gene expression ∙Endothelial cells are ready to express surface selectins quickly if an inflammation event takes place (B) Neutrophils will rapidly up-regulate their surface molecules in response to pro-inflammatory TNF or IL1 Cells can have low affinity receptors that are always present on the cell surface ∙These can easily and rapidly be turned into high affinity receptors with the right pro-inflammatory stimuli ∙This allows for a rapid response of cells during inflammation ∙An example of this is capping the active site to prevent binding → Binding would only be needed during inflammation, in which the active site's cap would be removed (C) Chemokine stimulation changes the conformation of the leukocyte to go from expression of low affinity to high affinity receptors

Sequence of Events Following Acute Injury

(A) Normal cellular architecture with a limited number of white blood cells (C) Initial event in acute injury is edema formation and preformed mediators start to appear in tissue ∙Neutrophils are the first to respond to the leaked stimuli ∙If neutrophils can't resolve the injury, they will call in the monocytes, which will differentiate into macrophages (B) Now we start to see changes in the tissue, including a greater predominance of nucleated cells

Acute inflammation in lung

(A) represents normal lung tissue (B) represents vascular congestion and stasis with increased numbers of leukocytes and erythrocytes (C) shows rupture of alveoli due to massive leukocyte infiltration Fluid in lung impairs normal function Chronic stimulus results in damage to this tissue and a permanent loss of function Chronic inflammation results in a progressive condition (not shown)

Extrinsic Apoptotic Pathway

"Death receptor" ligand interaction ∙Contain cytoplasmic death domains that delivers the apoptotic signal →TNF-R - tumor necrosis factor receptor superfamily →Fas Fas ∙Three or more Fas molecules become cross-linked by the Fas ligand ∙Fas cytoplasmic death domains form a binding site for an adaptor protein (FADD) →It contains a death domain and an intercellular tail for caspase binding →In humans can be either caspase 8 or 10 ∙Complex of death receptors and FADD binds inactive caspase-8 (pro-caspase-8) via death domains →This is called the death inducing signaling complex (DISC) ∙Multiple pro-caspase-8 molecules are brought into proximity and cleave one another to generate active caspase-8 →This is autocatalytic activation ∙Caspase 8 then triggers a cascade of caspase activation ∙These other caspases mediate the execution phase of apoptosis ∙This pathway can be inhibited by FLIP →Binds to pro-caspase 8 but cannot cleave and activate it because it has no enzymatic activity →Produced by viruses and some normal cells →We only need one FLIP to cause inhibition TNF-R (Tumor Necrosis Factor) ∙"Death receptor" ligand interaction (TNF: TNF-R) ∙TNF is an important mediator of inflammatory processes and it also induces apoptosis ∙When TNF binds its receptor, cascade is very similar: →Association of the receptor with the adaptor protein TRADD →TRADD binds to FADD →Caspase activation ∙Signaling pathways that initiate apoptosis: →Major functions of TNF are not by induction of apoptosis →NF-kB is a potent transcriptional factor that causes activation →→→NF-kB and its inhibitor (IkB) →→→Important in regulating cell survival and inflammation ∙TNF induces apoptosis AND promotes cell survival with adaptor proteins that attach to the TNF-R →TRADD and FADD - apoptosis →TRAFs (TNF receptor associated factors) favor activation of NF-kB

Clinical indications of cephalosporins

Alternative to pens in beta-lactamase-producing bacteria Otitis, sinusitis, lower respiratory tract infections Surgical prophylaxis (e.g., cefazolin) Meningitis, resistant gonorrhea 3rd generation, (e.g., ceftriaxone) Common gram (-) infections

APP changes over an inflammatory event

0 is the starting point C Reactive Protein and Serum Amyloid A will both increase more than 1000-fold initially Later haptoglobin and fibrinogen are up regulated to 100-fold Albumin and transferrin (the negative APPs) can be seen to initially slightly down-regulate. ∙This provides ample amino acids for synthesizing positive APPs

Caenorhabditis elegans

131 of 1090 somatic cells generated during development of this organism are eliminated by apoptosis During development we grow a stump at the end of our arms and apoptosis removes cells to create digits ∙Incomplete apoptosis leads to webbing between digits Homologues play a role in development, differentiation, and programmed cell death ∙CED-3: caspase homologue ∙CED-4: Apaf-1 homologue ∙CED-9: bcl-2 homologue

CRP serves as a marker for inflammation in chronic conditions

10-50 fold increases, but it varies by lab and by patient It may eventually replace ESR

History of Macrolides

1952 - erythromycin is produced by Streptomyces erythreus Semi-synthetic derivatives: azithromycin, clarithromycin, dirithromycin Macrocyclic lactone ring is characteristic of drugs in this class Alternate to penicillin/cephalosporins Bacterial resistance when developed to one macrolide becomes common to other macrolides (i.e., alteration of binding site, methylation in gram-positive bacteria)

Fibrinogen

2 symmetric half molecules ∙Each consists of one set of three different polypeptide chains ∙It has an elongated shape and is abundant in plasma It is highly heterogenous ∙Alternative splicing ∙Extensive post-translation regulation →Allows high variability ∙Proteolytic degradation It is induced transcriptionally by IL-6 ∙Glucocorticoids and oncostatin-M also induce fibrinogen Made in the hepatocytes It is considered a moderate APP ∙100-2-fold up regulation ∙There are baselines for each range of individuals' ages because the rate increases with age

Aminopenicillins (2 - Generation Spectrum)

2nd generation or extended spectrum pens has advantage of reliable oral bioavailability vs pen G (e.g., amoxicillin/ ampicillin) Gm + spectrum same as Pen G Common gram( -) rods: E. coli, P. mirabilis, H. influenzae, Salmonella \ Shigella, anaerobes Not effective against gram negative Proteus vulgaris, Pseudomonas aeruginosa, Serratia, Gonococci *Not resistant to B - lactamases*

Bacterial morphology

3 basic shapes are round spheres (cocci), rods (bacilli), and spirals (spirillum) There are other organisms that are modifications of these basic shapes

Acute Inflammation: Immediate and early responses

3 major components ∙Alterations in vascular caliber leading to increased blood flow →Histamine and other vasoactive amines cause capillaries to dilate which allows the blood to slow down ∙Structural changes permitting plasma proteins and leukocytes to leave the circulation →Reorganization of the cytoskeleton of endothelial cells to allow space for small molecules to slip through →Eventually there will be enough room made for leukocytes to slip through the endothelium ∙Emigration of leukocytes from the circulation and accumulation at site of injury →This process moves along a chemical gradient and involves chemokines that attract the leukocytes to the site of infection

Structure of Tetracyclines

4-ringed structures

A-B subunit internalized exotoxins

A = active subunit ∙Possess toxic enzymatic activity ∙Mediates host cell damage B = binding subunit ∙Mediates binding ∙Mediates translocation of A subunit into cell ∙Often a multimer

Pathogenicity Islands

A contiguous set of genes that encode for virulence properties, surrounded by transposon-like elements These islands are not found in the non-pathogenic strains

Transudate

A fluid w/ low protein content (albumin) and a specific gravity less than 1.012 Alternative definition: An ultrafiltrate of blood plasma - results from hydrostatic imbalance across endothelia ∙the permeability of the endothelium is normal fluid and small molecules will cross the membrane as small openings are created b/w endothelial cells

Regulation of CRP

A lot of regulation makes this very important in inflammatory processes ∙It is more specific than erythrocyte sedimentation rate →There has been debate to making CRP counts to new gold standard for diagnosis ∙Massive up regulation occurs in short periods of time →Increases of 1000x are possible within 24-48 hours →→→Parallels neutrophil and macrophage up regulation →The body can return to normal levels of CRP within 4-5 days after onset of inflammation ∙Regulation occurs at the transcriptional level →IL-6 induces →→→Predominant regulator →Other cytokines then act synergistically to increase the response ∙It is persistently elevated in chronic inflammatory conditions and advanced cancer 20→50-fold increases are normally seen

What is a pathogen?

A pathogen is an organism that has the capacity to cause disease ∙Opportunistic ∙Primary Its ability to cause disease is its pathogenicity

Exudate

An inflammatory extravascular fluid w/ high protein content, cellular debris and specific gravity above 1.020 Exudate is typically formed later on in immune response Purulent exudate = pus, made up of neutrophils

Albumin

A small but very abundant protein in blood Accounts for 50-60% of plasma proteins Is responsible for 80% of the osmotic pressure of blood Represents 25%of total protein synthesized in the liver Is an important negative APP The rate of albumin synthesis depends on nutritional status It transports a variety of substances in the blood, notably copper and zinc Negative regulation is less than 50-fold ∙But you don't need a large decrease to make other APPs with the free amino acids ∙Very well regulated

Antigen

ANTIbody GENerator Any molecule capable of binding specifically to antigen receptors on B or T lymphocytes ∙Usually a protein →For T cells, must be processed and presented by major histocompatibility complex (MHC) ∙Can also be a carbohydrate, lipid, or nucleic acid →Particularly for B cells →Better if conjugated to protein ∙Bigger is generally better, for recognition purposes ∙More complex is usually better

Examples of intracellular accumulations

Abnormal Metabolism ∙This would be seen in a fatty liver after drinking alcohol ∙This is reversible early on Disruption of protein folding ∙The proteins build up in the cell Lack of enzyme ∙This is usually genetic ∙There is a complex substrate that can't be broken down ∙Good example of this is a lysosomal storage disease ∙This can initiate apoptosis Ingestion of indigestible materials ∙This occurs when the cell ingests material that cannot be digested, such as inorganic substances like silica or asbestos ∙Cell does not have the mechanism to breakdown the material so the material accumulates ∙If it is carbon, it won't kill you but something like asbestos can result in neoplasm Summary: Intracellular accumulations can cause apoptosis → Some of these accumulations include fat, cholesterol, proteins, glycogen, and pigments ∙Lipid accumulation →Steatosis is fat in the liver or heart →Cholesterol and cholesterol esters can cause atherosclerosis, xanthomas, inflammation and necrosis →It is reversible in the liver but you can die of it →In alcohol abuse, the liver appears greasy and this will kill you ∙Cholesterol Accumulation →Cholesterol accumulates in macrophages and smooth muscle cells →Can lead to atherosclerosis resulting in blockage of blood vessels →The buildup in the lumen narrows it and results in ischemia ∙Protein Accumulation →Reabsorption problem: droplets in the proximal renal tubules →Proteins leak out in renal disease →You can have excessive amounts of proteins →Accumulations will lead to Russell Bodies →Disease: Multiple Myeloma: immunoglobulin is produced abnormally and in abnormal amounts →If proteins are not folded appropriately, can't mature and go to golgi →→→Chaperone proteins attempt to properly fold proteins to prevent aggregation

Integrins

About 30 structurally homologous proteins that promote cell-cell or cell-matrix interactions The name of this family of proteins derives from the hypothesis that they coordinate or integrate signals from extracellular ligands w/ cytoskeleton-dependent motility, shape change and phagocytic responses The extracellular domains of the two chains bind to various ligands, including extracellular matrix proteins (ECM) glycoproteins ∙Activate complement components ∙Several integrins bind to arg-gly-asp (RGD) sequences in fibronectin and other ECM glycoproteins ∙Fibronectin: ECM protein involved in adhesion of cells to matrix ∙Also important in movement of cells during embryogenesis ∙Some integrins bind to other cell Cytoplasmic domains of the integrins interact w/ cytoskeletal components ∙Talin: intracellular anchorage protein ∙Actin: major constituent of actin filaments (major component of cytoskeleton) ∙Tropomyosin: protein involved in the stabilization of actin filaments ∙α-actinin: bundling protein ∙Vinculin: membrane-cytoskeletal protein in focal adhesion plaques

Prototype 1st Generation Drug: Penicillin-G/Pharmacokinetics

Absorption - low oral bioavailability, acid-labile, usually adminstered IV or IM Distribution - H2O soluble drugs, low penetration CNS ∙Increased CSF levels attained in meningitis with increased dosage Metabolism - very little, t 1/2: ~ 30-60 min Excretion - unchanged in urine via tubular secretion (90%), remainder thru GF Probenecid - slows/blocks tubular secretion of B-lactam antibiotics ∙Keeps drugs around longer to extend action Advantage: Penicillin V is reliable oral absorption, only used for minor infections

Pharmacokinetics of Cephalosporins

Absorption - many acid-labile and administered IM/IV; however each generation has some drugs that provide adequate oral absorption Distribution - 1&2 generations mostly H2O soluble; 3/4 generations increased lipid solubility and enters CNS without inflammation Metabolism - very little, some deacetylated to active metabolites Excretion - mostly unchanged in urine; exception cefoperazone & ceftriaxone with increased amounts via liver\biliary/GI Probenecid slows/blocks tubular secretion

Disorders associated with inhibited apoptosis and increased cell survival

Accumulated cells can give rise to cancer ∙p53 mutation ∙Hormone-dependent (breast, prostate, ovary) Autoimmunity ∙Individuals with complement component C1q defects have an increased risk of developing systemic lupus erythematosus (SLE) and glomerulonephritis

Pyroptosis - inflammatory form of programmed cell death

Activated after cellular insults such as bacterial infection or exposure to toxins, and mediated by caspase 1 and/or caspase 11 Triggers the release of IL-1 Results in plasma membrane lysis

Type of specific immunity

Active ∙Natural (via infection) ∙Artificial (by vaccination) Passive ∙Natural (by transplacental or colostral transfer of antibody) ∙Artificial (by administration of immune globulins) Adoptive: transfer of immune cells

General Features of Acute Inflammation

Acute inflammation is a normal, healthy process when it occurs over a short period of time Involves rxn of: ∙Blood vessels ∙Accumulation of fluid and leukocytes in extravascular tissues Acute inflammation is intertwined w/ process of repair (hopefully) ∙The two processes are occurring at the same time →We are trying to re-establish the cellular architecture, but there are still leukocytes present that are causing collateral damage ∙Hopefully down-regulating response by day 4-7 ∙Regeneration ∙Scarring Fundamentally a protective response ∙Rids the body of initial cause of injury and consequences of such ∙May be potentially harmful (chronic) Inflammation occurs in vascularized connective tissue ∙Need intact vascular network in order to provide necessary contents to site ∙The presence of insult or injury can cause inflammation →Repetitive motions of skin can separate the epidermis from the epithelial cell layer, creating a blister - this serves as a stimulus for inflammation →Inside the blister is serous inflammation fluid - It has a low protein content and is very watery ∙Plasma, circulating cells, blood vessels, cellular and extracellular constituents of connective tissue Organs that prevent inflammation? Organs w/o a lot of regenerative capacity ∙Eye - scarring would contribute to blindness ∙CNS and reproductive system are other examples

Outcomes of acute inflammation

Acute inflammation is associated with changes in vascular caliber If the acute inflammation is not resolved, we move into a primary immune response. If the inflammation is still not resolved, chronic inflammation results. ∙Chronic inflammation occurs when the process has been going on for 7-10 days Acute inflammation can be resolved. This would lead to restoration of normal cellular architecture Acute inflammation can result in pus formation, in which all cellular architecture is lost In the figure above, the Fibrosis (bottom right) picture indicates that the tissue is healing Resolution phases consist of pus formation and fibrosis (as seen in the figure above)

Non-Suppurative Complications (Sequelae) of S. pyogenes

Acute rheumatic Fever (ARF) ∙Multisystem disease resulting form an autoimmune rxn to infection w/ Group A strep ∙Cross-reactivity of streptococcal antigens and human heart, joint and nervous tissues ∙Follows respiratory, but not skin infections ∙Characterized by pancarditis and arthritis Post-Streptococcal Glomerulonephritis (PSGN) ∙An autoimmune complex disease ∙Antigen-antibody complexes deposited in the kidneys fix complement and damage glomeruli ∙May follow respiratory or skin infection ∙Acute inflammation of renal glomeruli w/ edema, dark urine, HTN

Adaptations of cellular growth and differentiation

Adaptations are reversible changes in size, number, phenotype, metabolic activity, or function of cells in response to changes in their environment Different types of adaptations (very important to know): ∙Hypertrophy ∙Hyperplasia ∙Atrophy ∙Metaplasia Causes of Cellular adaptations ∙Changes in cells and tissues ∙Prolonged stimulation (can be hormonal, physical, or a combination of stimuli) ∙Lack of stimulation ∙Lack of oxygen, lack of blood flow - this can lead to death of cells ∙Lack of nutrients that are necessary for a cell to maintain normal function ∙Chronic injury

Adaptive Immunity Overview

Adaptive immune responses can be humoral (antibody mediated) or cell-mediated ∙Whether either or both are activated depends on where the pathogen is B lymphocytes ∙When appropriately stimulated, make antibody ∙Different subsets of B cells respond preferentially to protein and non-protein antigens T lymphocytes ∙Centrally important in most, if not all, adaptive antigen-specific immune responses ∙Derived from thymus gland ∙2 major types: CD4+ and CD8+ ∙CD4+ T cells make cytokines →Most are helper/inducer →Some are Tregs →Regulate: →→→Afferent (generation) and efferent (effecting the response) arms of many different types of response →→→Production of antibody →→→Generation of cell mediated immune responses ∙CD8+ cytolytic T cells →Cytotoxic or cytolytic for cells bearing relevant antigen →Regulation of immune responses (suppression???) Important points ∙T and B lymphocytes are 'antigen specific' ∙The receptor structures on lymphocytes form the molecular basis of the antigen-specificity of the adaptive immune system ∙The antigen receptor on B cells is surface immunoglobulin ∙The antigen receptor on T cells is called the T cell receptor (an immunoglobulin-like structure)

Fluoroquinolones - 2nd Generation: Structure-activity relationship

Addition of fluoride and side rings significantly extends gram (-) spectrum ∙Adds some gram (+) activity, but its reliable Ciprofloxacin - PO, IV Ofloxacin - PO, IV

Ischemia Reperfusion Injury

After ischemia and perfusion to an organ, the blood flow is re-established ∙This is a good thing, but it carries some negative effects, such as the induction of free radicals ∙You can have mitochondrial changes, lipid peroxidation of membranes, DNA lesions, and cross linking of proteins ∙The complement pathway causes cell injury and inflammation →You are not responsible for all of the steps, just know that there are a variety different types of change

Biofilm

Aggregate of bacteria that bind to each other on a surface w/in a slime layer This particular process can be regulated

Microbial surface components recognizing adhesive matrix molecules

Aka MSCRAMMs A subfamily of surface adhesions that target host extracellular matrix proteins such as fibrinogen, fibronectin, and collagen for adhesion Have only been studied in gram (+) bacteria (i.e. lipoteichoic acid, clumping factor)

Fimbriae

Aka pili Hair-like structures composed of protein subunits called pilin that cover entire surface of bacterium Mediate specific binding to host cells and other bacteria (often required for infection) The specific adhesion is located that the tip of the pili ∙Good example is in UTI ∙When urine passes through urethra, the adhesion of the pili allows the bacteria to not be washed away F-pili (sex pili) are involved in interactions b/w bacteria for genetic exchange (conjugation)

Platelets

Aka thrombocytes Small, disk shaped clear cell fragments with no nucleus 2-3 micrometers in diameter Derived from fragmentation of precursor megakaryocytes ∙megakaryocytes live in bone marrow and break off into pieces to form platelets Normal lifespan is 5-9 days Source of growth factors Circulate in blood of mammals Involved in hemostasis, leading to formation of blood clots Platelet Microparticles (PMP) ∙Not super important ∙Submicron size ("dust") ∙Released mainly from platelets (approx. 90%) also from monocytes and other cells after activation or during apoptosis ∙Platelet-derived microparticles have procoagulant activity ∙Monocyte microparticles contain encrypted tissue factor →Tissue factor plays an important role in the blood coagulation cascade Platelets are the first responders ∙Become activated by outside signals and induce signal transduction to alert the cell ∙Platelet undergoes abrupt shape change ∙Granule contents spill into surrounding area →Factors and enzymes necessary for coagulation ∙Platelet adhesion and aggregation →Aggregation: adhere and bind to each other ∙Synthesis prostaglandins ∙Surface is negatively charged →Negatively charged surfaces are very important for coagulation Formed in bone marrow from megakaryocytes ∙Megakaryocytes fragment into the minute platelets either in bone marrow or soon after entering blood Normal concentration in blood is 150,000 - 300,000 per microliter

1st Generation Spectrum of Cephalosporins

All 1st-generation drugs generally have same antibacterial spectrum Gram (+) similar to pen G, but often effective against lactamase + staphylococci Gram (-) similar to 2nd-gen pens (Not effective against gram negative Proteus vulgaris, Pseudomonas aeruginosa, Serratia, Gonococci) Preferred use for surgical prophylaxis (cefazolin); has longer t ½ than others

Sulfonamide ADRs

Allergy - rash, fever, hives, photosensitivity and more serious (Stevens-Johnson syndrome, less than 1%) Sulfa allergy, cross-allergenic e.g. thiazide diuretics, sulfonylurea hypoglycemics Hematologic - *hemolytic anemia esp. with G-6-PD deficiency*, bone marrow suppression, newborn at the risk of devp. Kernicterus (*yellow pigment in brain*) Renal - nephrotoxicity/crystalluria in acidic urine, esp. N-acetylated metabolites (treated by NaHCO3) GI disturbances-n-v-d, headache May decrease absorption / drug levels of oral contraceptives and folic acid

How is complement activated?

Alternative pathway ∙Triggered with a hydrolysis product of C3 is deposited on a pathogen surface Lectin pathway ∙Initiated when mannose binding protein binds to mannose on the pathogen surface ∙MBP structurally similar to C1 in classical pathway ∙How is complement activated? Classical pathway ∙IgM bound to targets on cell/pathogen surface ∙IgG (two or more) bound to targets on cell/pathogen surface ∙C reactive protein →Pentameric protein similar to C1

There are three pathways for activating complement (in order of activation)

Alternative pathway: This pathway does not involve immunoglobulin deposition on a pathogen surface, *it is triggered at sites of microbial infection* Lectin pathway: This pathway also does not involve immunoglobulin deposition on a pathogen surface. *It is activated by binding of a plasma protein to mannose-containing peptidoglycans on microbial surfaces* Classical pathway: *Triggered as an effector mechanism when certain isotypes of immunoglobulin or C-reactive protein bind to a pathogen*

Degranulation: platelet granules expel their contents upon activation

Alpha-granules contain fibrinogen, thrombospondin, beta-thromboglobulin, platelet factor 4, PDGF and more vWF (van-Weibel Factor) is found in Weibel-palade bodes (in endothelial cells) and in alpha-granules (in platelets) Dense granules contain serotonin, ADP, ATP, calcium and more Prostaglandins and leukotrienes are produced during this process from arachidonic acid coming from cell membrane phospholipids ∙Arachidonic acid is released upon rupture of the cell membrane ∙focus on prostaglandin production, which is important for hemostasis ∙Cycloxygenase converts arachidonic acid to prostaglandin G2 allowing for the producing of prostacyclin and thromboxane A2 ∙Prostacyclin, produced in endothelial cells, causes vasodilation and inhibits platelet aggregation ∙Thromboxane A2, produced in platelets, causes vasoconstriction and promotes platelet aggregation ∙This process only occurs in the endothelial cells ∙This is where NSAIDS are important →Aspirin is an irreversible inhibitor of cyclooxygenase and has a stronger inhibitory effect than NSAIDS →NSAIDS are reversible, Aspirin is not ∙Prostaglandins cause circulatory adjustments by widening or narrowing the vessels →Vasoconstriction →→→Platelets secrete serotonin and thromboxane A2 →→→Endothelial cells release endothelins →Vasodilation →→→Endothelial cells produce NO and prostacyclin →→→High molecular weight kininogen produces bradykinin Procoagulant phospholipids (pro-coagulants) become exposed on platelet surface ∙Arachidonic acid is cleaved from membrane by phosphoipids by Phospholipase A2 ∙In activated platelets, phospholipids are "flipped" to the external side (exposing a negatively charge surface) ∙negative charges bind clotting factors via calcium ∙Surface bound reactions help to: →Concentrate the reactants (clotting factors) →Achieve 2D diffusion of factors usually within a lipid raft →Orient the reactants for the enzymatic reaction

β2 Integrins

Also called CD11a-cCD18 or LFA-1 family (lymphocyte function associated antigen) ∙a-c refers to a different α chains and CD18 to the common β2 chain associated w/ each LFA-1 (CD11aCD18) ∙Plays an important role in the adhesion of lymphocytes and other leukocytes w/ other cells such as APCs and vascular endothelium Other membranes of the family include: ∙CD11bCD18 (Mac-1 or CR3) ∙CD11cCd18 (p150, 85 or CR4) ∙Both mediate leukocyte attachment to endothelial cells and subsequent extravasation CD11bCD18 also functions as a fibrinogen receptor and as a complement receptor on phagocytic cells, binding particles opsonized w/ a by-product of complement activation called the inactivated C3b (iC3b) fragment

General properties of the proteins encoded by the human major histocompatibility complex (MHC)

Also called the human leukocyte antigens or HLA They are members of the immunoglobulin superfamily of proteins Unlike immunoglobulins (and the TCR), these molecules are encoded by conventional, stable genes that do not rearrange or undergo any other somatic or developmental change There are two classes of MHC proteins: Class I and Class II ∙Class I: HLA-A, B, C, E, F and G ∙Class II: HLA-DP, DQ, DR, DM and DO However, there is inherited diversity w/in the MHC which has two components ∙Gene families: consisting of multiple, similar genes encoding these proteins ∙Genetic polymorphism: the presence, w/in the population, of multiple, alternative forms or alleles of a gene When considering the diversity of MHC Class I and Class II molecules that can arise from the combination of multiple genes and multiple alleles, the term isoform is used to denote a particular MHC protein Certain MHC Class I and Class II genes have many different alleles and the proteins encoded by these are highly polymorphic ∙Others have no polymorphism - monomorphic ∙Others have only a few alleles - oligomorphic ∙Note: the products of the highly polymorphic genes (HLA-A, HLA-B, HLA-DR) are used to "match" donor and recipient for transplantation

Characteristics of Bacterial Toxins

Alter normal metabolism of host cells Typically, by themselves, cause disease Two categories: ∙Endotoxins ∙Exotoxins

Once-Daily Dosing of Aminoglycosides

Aminoglycoside bactericidal effect is concentration-dependent, with significant post-antibiotic effect Aminoglycoside toxicity both time- and concentration-dependent Once toxicity concentration threshold is reached, time above this threshold is critical for toxicity Multiple smaller daily doses of drug increase time above toxicity threshold more than single larger daily doses Graph shows that the time you are above the threshold for toxicity w/ multiple dosing much more than with once-daily dosing

Blood Coagulation: series of ordered reactions with a specificity of enzymes

Amplification of enzymes is important for thrombosis to be made at the end Initially, coagulation was believed to be set up in a "waterfall" cascade which was later revealed to include both an intrinsic and extrinsic pathway which is important because blood tests are still based on this Later, the cell based model was developed which describes the normal physiological coagulation cascade in the body

Staphylococcal food poisoning

An acute gastroenteritis (an intoxication) ∙Short incubation period (1-6 hrs) Caused by pre-formed staphylococcal enterotoxin serotypes A-E (SEA, SEB, SEC...) ∙*Heat stable, so active even if bug is dead* Characterized by intensive intestinal peristalsis, diarrhea and vomiting

Cholera

An acute secretory diarrheal disease that can be severely dehydrating A toxin mediated disease

Hyperplasia

An increase in the number of cells in an organ or tissue usually resulting in increase mass of the organ or tissue May be physiological or pathological Arises from stem cells Takes place in a cell population that is capable of dividing Hyperplasia and hypertrophy are two distinct entities but can occur together Physiologic Hyperplasia ∙Hormonal: increases the functional capacity of a tissue when needed ex: hyperplasia of the pregnant uterus or proliferation of the glandular epithelium of the breast during pregnancy ∙Compensatory: increase tissue mass after damage or partial resection →ex: partial hepatectomy →Hepatocytes are stable cells - they can reenter the cell cycle after leaving the cycle, pending the appropriate signal, and they can rebuild after damage occurs Pathologic hyperplasia - these are important because they can be pre-malignant ∙Excessive hormonal stimuli acting on target cells →ex: excessive estrogen leads to uterine bleeding or in benign prostatic hyperplasia (BPH) →It is NOT hypertrophy! ∙Generally the process is controlled if the hormone is eliminated →Sometimes this is possible, and sometimes it's not ∙May contribute to cancerous proliferation, as seen in endometrial hyperplasia. This hyperplasia can result in a neoplasm like endometrial carcinoma ∙May also be due to excessive growth factor production that have been linked to many viruses such as HPV (like genital warts or squamous cell carcinoma) Mechanism of hyperplasia ∙Increase local production of growth factors ∙Increase levels of growth factor receptors on cells ∙Activation of intracellular signaling pathways ∙Production of transcription factors ∙Turning on of genes encoding for growth factors or their receptors ∙Net result of all these actions is cellular proliferation Image is great to study concepts discussed, but we don't need to worry about growth factors and cytokines just yet

There are four types of cellular junctions

Anchoring junctions ∙Actin filament attachment sites →Cell-cell junctions (adherens junctions) →Cell-matrix junctions (actin-linked cell-matrix adhesions) ∙Intermediate filament attachment sites →Cell-cell junctions (desmosomes) →Cell-matrix junctions (hemidesmosomes) Occluding junctions ∙Tight junctions (in vertebraes) ∙Septate junctions (in invertebrates) Channel-Forming Junctions ∙Gap junctions (in animals) ∙Plasmodesmata (in plants) Signal-Relaying Junctions ∙Chemical synapses (in the nervous system) ∙Immunological synapses (in the immune system) ∙Transmembrane ligand-receptor cell-cell signaling contacts (Delta-notch, ephrin-Eph, etc.) →Anchoring, occluding, and channel-forming junctions can all have signaling functions in addition to their structural roles

Salmonella

Animal and human reservoirs (lots of reptiles) S. enterica - 2500 unique serotypes (or serovar) w/in a single species ∙i.e. S. enterica serovar. Typhi Capsular antigens are called Vi (for virulence) antigens instead of K antigens

S. aureus evasion of host response

Anti-phagocytic capsule Protein A: finds Fc portion of IgG, prevents opsonophagocytosis by PMNs Coagulase: fibrin clots protects bugs from white cells Intracellular survival of small colony variants in endothelial cells and macrophages Panton-Valentine Leukocidin (PVL) ∙Cytotoxin that's presence is associated w/ increased virulence of S. aureus ∙Particularly effective at damaging neutrophils ∙Bi-component exotoxin (lukF-PV & lukS-PV) ∙Carried by nearly every CA-MRSA strain

Treatment and Prevention of Streptococcus pneumoniae

Antibiotics will be discussed in systems Polyvalent vaccines for young children and for 55+ available

Epitope

Antigenic determinant ∙That part of an antigen that directly interacts w/ antigen receptor on lymphocytes Epitopes are usually linear for T lymphocytes (b/c of processing) Epitopes can be conformational or linear for B lymphocytes Usually 5-7 AA for antibody, longer for T cell epitopes

Sites of Microbial infection and shedding

Any place open to environment, including break in integrity in the skin

Immunogen

Any agent capable of inducing an immune response by T and/or B lymphocytes ∙All immunogens are antigens, but not all antigens are immunogens

Apoptosis in Physiological circumstances

Apoptosis plays a role in every stage in life: development, differentiation, and health maintenance in adult life Embryogenesis ∙Implantation ∙Organogenesis ∙Developmental involution ∙Metamorphosis Hormone dependent involution in the adult ∙Regression of lactating breast after weaning ∙Endometrial cell breakdown during menstrual cycle ∙Ovarian follicular atresia in the menopause Cell deletion in proliferating cell populations ∙Intestinal crypt epithelia ∙Maintenance of constant cell number Death of host cells that have served their purpose ∙Neutrophil death in acute inflammation →About 70% of the circulating WBC are neutrophils →They can persist in circulation for 24 hours →They have a huge turnover and maintain a constant cell number →→→Once they become effector cells they have a very short life at the site of injury ∙Lymphocyte death at the end of an immune response →Lymphocytes undergo a different and slower cell death compared to neutrophils →Antigens serve as pro-survival stimuli for lymphocytes →Apoptosis occurs as a result of deprivation of necessary survival signals Elimination of potentially harmful self reactive (autoimmune) lymphocytes ∙During or after maturation ∙There's positive and negative selection →In positive selection, the antigen has to be recognized in the context of the receptor molecule and it can't respond to itself Cell death induced by cytotoxic T lymphocytes ∙All T cells express CD3 on surface ∙Helper T cells express CD4 in addition to CD3 →These cells don't directly kill, but prime macrophages and assist B cells to produce cytokine ∙Direct effector cells express CD8 →They are antigen specific cells for virus or bacteria

Electron micrograph in apoptosis

Apoptotic cell ∙We can see cell shrinkage ∙Organelles are retracting but the plasma membrane stays intact ∙This process is energy dependent Late apoptotic cell ∙Blebs begin to form ∙Subcellular organelles and plasma membrane remains intact within each bleb Necrotic cell ∙Chromatin clumping ∙Organelle swelling will cause leakage of intercellular space, losing the ability to generate energy from the electron transport chain →If the cell loses the ability to make ATP, DNA repair mechanisms can't occur

Phagocytosis - Final stage

Apoptotic cells and their fragments have markers on their surface that facilitate early recognition by nearby phagocytic cells ∙Loss of phospholipid asymmetry in the plasma membrane ∙Translocation of phosphotidyl serine (PtdSer) and annexin I to the outer membrane of the lipid bilayer →Serves as an "eat me" trigger →Flipping the parts of the interior cell to the outside, triggers macrophage phagocytosis and uptake →This is very efficient with very little inflammation ∙Other cell surface associated molecules enhance uptake →Mannose binding lectins (MBL) →C1q (complement) →Deficiencies in complements can result in autoimmune diseases

Does organism cause disease?

Apply principles of Koch's postulates ∙Agent must be present in every case of disease ∙Agent must be isolated from host and grown in a lab dish ∙Disease must be reproduced when a pure culture of the agent is isolated into a healthy susceptible host ∙The same agent must be recovered again from the experimentally infected host

Trimethoprim ADRs

Approximately 75% of incidence of adverse effects involve increased skin effects/ rashes Increase risk life-threatening toxic epidermal necrolysis with TMP/SMX GI disturbances, nausea/vomiting/diarrhea Less renal effects; usually no urinary crystalluria; weak base Increased incidence of adverse effects in AIDS patients with TMP/SMX : fever, rash, decreased blood cells; *folate deficiency may cause macrocytic anemia*

Arachidonic acid metabolites

Arachidonic acid metabolites play a role in every step of inflammation The production of arachidonic acid starts with cell membrane phospholipids ∙Steroids inhibit pro-inflammatory events by blocking the action of phospholipases →This can leave a patient susceptible to infection →Inflammation is a normal, necessary process that occurs in the body Cyclooxygenase ∙Cyclooxygenase 1 (COX1) is constitutively active, it performs normal cellular activation events ∙Cyclooxygenase 2 (COX2) is the inducible form that is typically associated with inflammatory events →There are inhibitors for COX2 that block only this part of the pathway ∙Aspirin is taken for a headache to block some of the split products of the cyclooxygenase pathway ∙Cyclooxygenase leads to the production of prostaglandins, prostacyclin, and thromboxane A2 →Prostacyclin causes vasodilation →Thromboxane causes vasoconstriction →Since there are different effects depending on which metabolite is produced, the cell types and stimuli are important in determining whether or not there is vasodilation or vasoconstriction occurring →If we have different populations of cells producing different factors (prostacyclin and thromboxane A2), you can have balance and a net effect of no change on the vessel →If you have predominance of a particular cell type that produces either thromboxane or prostacyclin, you will have either vasoconstriction or vasodilation In general, the type of response and metabolites generated depends on: (1) The time of activation, as certain cell types are present at specific times in the inflammatory response (2) The cell types that are activated (3) The initial stimulus for activation

Local manifestations of acute inflammation

Arterioles (red) break off into capillary beds, and then merge with (blue) post capillary venules There is no smooth muscle around the capillaries When vasodilation of the arterioles occurs, it allows the same volume of blood to enter a larger space ∙A result of the dilation is that blood spends more time in this localized vascular network In acute inflammation, we are using the intact vascular network all around the injury/insult to deliver the cells we need to the site of the injury/insult There is a balancing act being performed in this vascular network normally ∙Erythrocytes need to be able to perform their job of nutrient exchange and flow through the capillaries, but we don't want leukocytes moving through (normally) ∙Normally, leukocytes are not exiting the capillaries, they continue to flow through the vessels ∙Leukocytes only exit the capillaries when they are activated - this results in collateral damage ∙The balance is maintained by "factors" that change the permeability of the vascular network →The further from the site of insult/injury, the less the amount of "factors" are activating and pulling the leukocytes into the injured tissues →Immediately around the area of insult/injury, there are many factors present that promote the leukocyte activation, leukocyte transmigration, and the entire process of an inflammatory event ∙The swelling from acute inflammation must be localized so that a cut on the finger, for example, does not lead to swelling of the entire arm

Interrelationships b/w the plasma mediators systems

As a result of the clotting cascade, kinins are produced The kinins are very potent; they alter the vascular caliber The kinin cascade is connected with the complement cascade

How a humoral immune response proceeds: Class switching

As immune response proceeds, B cells undergo somatic hypermutation "mutation that occurs at high frequency in the rearranged variable region DNA of immunoglobulin genes in activated N cells, resulting in the production of variant antibodies, some of which have a higher affinity for the antigen IgM declines, IgG and IgA produced

Drugs that regulate platelet function in thrombosis

Aspirin inhibits cyclooxygenase (COX) which inhibits TXA2 synthesis Ticlopidine (Ticlid) and clopidogrel: ADP receptor inhibitors which inhibit ADP-induced expresion of GpIIb/IIIa ∙Used in patients that cannot tolerate aspirin, or need dual anti platelet therapy Abciximab (reopro): directly inhibits GpIIb/IIIa

Teichoic Acids

Associated with gram positive bacteria Wall teichoic acid (WTA or TA): polymer of glycerol or ribitol phosphate covalently attached to peptidoglycan Lipoteichoic acid (LTA): polyglycerol phosphates covalently linked to glycolipids in the cytoplasmic membrane ∙LTA can initiate innate immune responses similar to lipopolysaccharide Major antigenic determinates Promote adherence b/w bacteria and mammalian cells

Factors that affect bacterial growth

Availability of major and minor essential elements ∙Carbon source - required by all organisms ∙Nitrogen needed for protein synthesis ∙Iron required for some enzymes (i.e. electron transport enzymes) Hydrogen ∙Most bacteria grow best at pH 6-8 ∙Some can grow and sometimes prefer lower pH conditions (i.e. lactobacillus) Osmotic conditions ∙If the concentration of solutes becomes higher in surroundings than cell (hypertonic solution), water tends to leave the cell and the growth of the cell is inhibited Temperature ∙Most clinically relevant bacteria grow at 37°C ∙However, bacteria can vary considerably in their ability to grow over the range of 25-40°C Oxygen ∙Bacteria exhibit different growth responses to presence of oxygen ∙Aerobic metabolism produces hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) →These can damage the cell →Aerobic growth is dependent on bacteria producing protective enzymes (i.e. SOD, catalase) →If they don't have these enzymes, they are sensitive to presence of O₂ and can be killed in presence of O₂

Lymphocytes

B lymphocytes ∙These lymphoid cells arise in bone marrow and are key players in adaptive immunity, particularly humoral immunity T lymphocytes ∙T cell precursors arise in bone marrow, but move to thymus to undergo proliferation, differentiation and selection processes ∙They are key players in both cell mediated and humoral adaptive immune responses NK (natural killer cells) ∙These cells arise in the bone marrow ∙They are larger than T or B lymphocytes and have abundant granular cytoplasm, hence the term "large granular lymphocyte," which is used somewhat synonymously w/ NK cell ∙These cells are prominent effectors of innate immunity, particularly against virally infected and possibly tumor cells

Bacterial genetics: So what?

Bacteria are haploid organisms that have the ability to sense and quickly adapt to their environment through regulated and coordinate regulation of genes → short term adaptation Bacteria can develop inheritable changes in their genome through mutations that they can pass on to daughter cells → vertical transfer Bacteria can acquire genes from other bacteria through transformation, transduction, or conjugation → horizontal gene transfer

Multiplication

Bacteria multiply before their presence is noted by symptoms (incubation period) Exceptions are those organisms that cause disease through the production of a toxin

Transformation

Bacteria take up exogenous, naked (foreign) DNA nd incorporates it into their genome ∙DNA can be a linear or circular (i.e. plasmid) Naturally competent bacteria are capable of taking up exogenous DNA w/o manipulation (i.e. Streptococcus pneumonia, Neisseria gonorrhoeae) Occurs in nature and can lead to more pathogenic bacteria

Quinolones/Fluoroquinolones

Bactericidal ∙inhibits bacterial DNA gyrase, bacterial enzyme more susceptible than human counterpart Resistance involves decreased binding to gyrase and uptake by bacteria, and increased efflux of drug (energy requiring drug pump)

Aminoglycosides: Mechanism of Action

Bacteriocidal, *irreversible binding to 30 S (& some 50S) to inhibit protein synthesis* Diffuses thru porin channels (outer wall) Active transport by anionic transporter thru cytoplasmic membrane; requires O2, not active against anaerobes Additional destructive membrane effect Most common resistance due to production of microbial enzymes, acetylases, phosphorylases, adenylases ∙also impaired entry into bacterial cell and altered binding receptor protein on ribosomal unit

EMB (eosin-methylene blue) agar

Bacteriostatic dyes inhibit gram (+) E. coli are green-black w/ a metallic sheen Klebsiella & Enterobacter are pink/purple Non-lactose fermenters are colorless

Chloramphenicol: Mechanism of Action, Clinical Indication and Pharmacokinetics

Bacteriostatic, binds 50S, inhibits protein synthesis Broad spectrum: gm+, gm-, anaerobes, esp. H. inf. meningitis, Salmonella typhi, Rickettsia ∙not effective for Pseud. aeruginosa or Chlamydia Good PO, wide dist. \ CNS, metabolism glucuronidation (usu deficient in infants) "Gray baby syndrome": depressed breathing, CV collapse, cyanosis, abdominal distension, loose green stools, related to inhibition mitochondria (70S) ∙extremely contraindicated in pregnancy Inhibits CYP450 enzymes, drug interactions

Clindamycin: Mechanism of Action, Clinical Indication and Adverse Effects

Bacteriostatic, binds 50S, site overlaps ERY site, antagonism for ERY binding Spectrum: most gm + & anaerobes, Bacteroides/Clostridium, but not C.difficile Main clinical use: Gm (+) strepto- and staphylococcal infections (e.g., bacterial endocarditis), anaerobic infections Gm (-) usu. resistant; & Clostridium difficile ADRs: GI / diarrhea, superinfections, esp. pseudomembranous colitis due to overgrowth of Clostridium difficile within GI tract

Sulfonamides: Mechanism of Action

Bacteriostatic, synthetic analogs of PABA essential for bacterial synthesis folate Inhibit dihydropteroate synthase, enzyme catalyzing conversion PABA to folic acid required for DNA synthesis Selective action, mammalian cells require preformed folic acid (diet) Resistance usually to increased or altered production of enzyme, mutation of PABA binding site, dec. permeability bacterial membrane No cross-reactivity with Pen allergic rxns

Structure of penicillins

Beta-lactam ring imparts activity to penicillins When this ring is cleaved, penicillin is inactivated → major form of resistance bacteria develop

Bacterial Resistance of Penicillins/Cephalosporins

Beta-lactamases, bacterial enzymes that break open/inactivate B-lactam ring inactivate penicillins (penicillinases) and cephalosporins (cephalosporinases); generally refer to them as just B-lactamases Resistance can be due to modified PBPs, reduced binding Can be due to decreased entry of drug thru outer membrane/porins Can be due to drug efflux pumps, transport mechanism in some bacterial membranes that actively pumps drug out

Overview of antibiotic classes

Beta-lactams ∙Bactericidal drugs ∙Prevent cell wall synthesis by blocking beta-lactamase ∙Penicillins and cephalosporins →Vancomycin effective for gram (+) bacteria, good for those w/ penicillin allergy →Aztreonam will fight gram (-) bacteria, also good for those w/ penicillin allergy →Both of these, for the most part, must be given parentally ∙Used for a wide variety of indications ∙Very effective, little side effects ∙Cannot use in patients w/ allergies Quinolones ∙In general, used to treat gram (-) negative bacteria, as well as pseudomonads ∙Block DNA replication ∙Bactericidal Sulfonamides ∙Antimetabolites ∙Block folic acid synthesis ∙By stopping folic acid production, you stop synthesis of DNA ∙Can be bactericidal if used in tandem w/ other drugs Protein synthesis inhibitors (30S) ∙Bacteriostatic drugs (except aminoglycosides) ∙Inhibit at 30S ribosome (unique to bacteria) Protein synthesis inhibitors (50S) ∙Large group of drugs ∙Inhibit at 50S ribosome (unique to bacteria)

MacConkey agar

Bile salts inhibit gram (+) bacteria Lactose fermenters grow pink colonies Non-lactose fermenters have colorless colonies

Mechanism of Action of Macrolides

Block transferase site Basically, stops transfer of tRNA from one site to another on ribosome during translation

Model for superantigen induction of toxic shock syndrome

Boxes contain main clinical features Other superantigen (SAg) effects include direct effects of superantigens on endothelial cells and the ability to synergize w/ agents such as Gram (-) endotoxin to release even greater amounts of TNF

Fluoroquinolones - 2nd Generation: Antibacterial Spectrum

Broad gram negative - most Klebsiella, Enterobacteriaceae, Pseudomonas (Cipro most active/potent), Hemophilius, Mycobacteria, Legionella, N. gonorrhoeae, C. trachomatis Selected gram positive - some Staph. aureus, active against penicillinase producers; minimal activity against streptococci and not generally indicated for these infections Minimal anaerobic activity, not used

Antipseudomonal (4 - Generation Spectrum)

Broad spectrum antibacterial activity (e.g., piperacillin) Requires parenteral administration Spectrum similar to 3-gen pens with greater potency/effectiveness against more gm (-) strains/species; Gm (+)similar to ampicillin,> 3rd generation Often used with an aminoglycoside (e.g. Streptomycin) for serious infections with enterococci e.g. endocarditis Not resistant to B-lactamases Really only want to use this as a last resort → do not want to make drugs that are resistant to this

Mechanism of Action of Quinolones/Fluoroquinolones

By inhibiting DNA gyrase, you will keep DNA in supercoiled positive form, limiting DNA replication and therefore bacterial growth

Disease/Tissue Injury

By-products of bacteria growth (acids, gas) Secretion of enzymes that breakdown cells and intracellular matrices of host tissues Secretion or elaboration of bacterial toxins Damage by immune response (host-mediated pathogenesis) Ex: Clostridial cellulitis ∙Clostridia can be introduced into tissue during surgery or by traumatic injury ∙This patient suffered compound fracture of tibia ∙5 days after injury, skin became discolored, and bullae and necrosis developed ∙a serosanguineous exudate and subcutaneous gas were present, but there was no evidence of muscle necrosis ∙the patient had an uneventful recovery

Classical pathway in detail

C1 complex consists of 3 proteins ∙C1q ∙C1r ∙C1s IgM has receptors for C1q ∙IgM in planar (soluble) configuration does not bind C1q ∙Upon binding to pathogen surface, IgM undergoes a conformational change to a 'staple form,' which can then bind C1q at multiple sites ∙*C reactive protein: another pentameric protein w/ multiple binding sites for C1q* IgG also has receptors for C1q ∙IgG1 and IgG3 fix complement via the classical pathway better than IgG2 Activation of C1 ∙Activated C1q activates C1r ∙Activated C1r activates C1s ∙Activated C1s has proteolytic activity on C4 and C2 Regulation of C1 ∙Proteolytic activity of C1r and C1s is inhibited by C1 INH ∙Deficiency of C1 INH causes hereditary angioneurotic edema →Autosomal dominant →Intermittent skin and mucosal edema →→→Abdominal pain, vomiting, diarrhea, airway obstruction →Caused by increased breakdown of C2 and C4 →→→Proteolytic fragment of C2 (C2 kinin) is responsible for edema C3 convertase ∙After cleavage of C4 and C2, C4b fragments that are covalently attached to the pathogen surface bind C2a ∙C4b2a is the classical C3 convertase ∙Important note: there is a discrepancy among textbooks as to what they call the C2 cleavage product that becomes part of the C3 convertase and which one is released →For historical reasons, the small cleavage product of C2 is called C2b and the larger one C2a →Whereas for other complement components, the smaller cleavage component is called the a fragment (i.e. C3a, C4a) and the larger one is the b fragment ∙C3 is activated by C3 convertase and is split into C3a and C3b →Important amplification step →C3b becomes attached to the target cell membrane ∙C3b also binds to C4b2a to form active C3b2a4b complex, which is called the C5 convertase C5 convertase ∙C5 convertase splits C5 into C5a and C5b ∙each have important biologic properties ∙C5a is an anaphylatoxin (so is C3a) →Smooth muscle contraction →degranulation of mast cells and basophils →release of histamine and other vasoactive amines →Increase vascular permeability ∙C5a is also a chemotaxin for neutrophils →substances that attract phagocytic cells and cause their migration from areas of lower concentration to areas of higher concentration ∙C5b initiates the membrane attack complex

Pro T cells

CD4-/CD8- (double negative) TCR genes still not rearranged at the beginning Most pro-T cells will give rise to CD4+ and CD8+ T cells, bearing the α/β TCR Express Rag-1 and Rag-2, showing that rearrangement will occur Rearrangement of the β chain of the TCR via mechanisms that involve Rag-1 and Rag-2 (like Ig gene rearrangement)

Complement receptors

CR1 ∙Expressed on many cell types, including →macrophages and neutrophils →erythrocytes (facilitates transport of immune complexes to the spleen for clearance) ∙Bind C3b or C4b on pathogen surface (high affinity) ∙Facilitate uptake and destruction of pathogen →C3b and, to a lesser degree, C4b are acting as opsonins ∙Markedly enhanced when Fc region of IgG also binds phagocyte via FcRg or in presence of IFN-g CR2 ∙Expressed on follicular dendritic cells and B cells ∙Component of the B cell receptor complex (co-receptor) ∙Binds to degradation products of C3b (C3d) on microbial surfaces →Delivers activation signals to B cell →Innate to acquired immune response bridge ∙Later: you will learn it is the surface receptor for Epstein-Barr virus CR3 and CR4 ∙b-integrins →CR3 (also called CD11b/CD18 and Mac-1) →CR4 (also called CD11c/CD18) ∙Bind iC3b (proteolytic product of C3b) ∙Both expressed on phagocytes →Promotes phagocytosis of microbes opsonized with iC3b →Also involved in adherence of leukocytes to endothelial cells at sites of inflammation via binding to ICAM-1 (CR3, maybe CR4)

Cyotoxic T Lymphocyte (CTL) Mediated Lysis

CTL recognizes foreign antigens presented by Class I MHC at the cell surface Upon recognition CTL secrete: ∙Perforin - transmembrane pore-forming molecule →This is more of a direct path than granzyme B *∙Granzyme B - serine protease* →Entry facilitated by perforin →Cleaves proteins at aspartate residues →Activates caspases -8, -3, -6, and -7 →By-passes up-stream signaling events and induces target cell cytochrome c release →Acts directly by inducing execution phase (3, 6, and 7) →CTL also express Fas-L on their surface and can induce cell death via this pathway

Serological Classification of Enterobacteriaceae

Can be classified based on their O, H, K antigens O (somatic) antigen ∙O side chains on LPS H antigens ∙Flagellar antigens K antigens - sugars that are making up capsule ∙Capsular antigens ∙Type specific polysaccharides Example: E. coli O157:H7 (refers to specific antigens) ∙Related - E. coli K1 is associated w/ neonatal meningitis

Growth responses to oxygen

Can classify bacteria this way Strict aerobes NEED oxygen ∙Ex: M. tuberculosis, B. subtilis Strict anaerobe can be killed in presence of O2 b/c they don't have necessary enzymes ∙Growth occurs only when there is no O2 ∙Cause mixed infections in body (have other organisms that can deal w/ O2 in body, allowing these anaerobes to grow ∙Ex: C. botulinum, B. fragilis Facultative anaerobes can growth w/ or w/o O2 ∙Respires w/ O2, ferments in absence of O2 ∙*Most bacteria that cause disease* ∙Ex: E. coli, S. aureus Aerotolerant anaerobes ferment regardless of presence of O2 ∙Will not utilize O2, but tolerate low levels of O2 ∙Ex: S. pnemoniae, S. pyogenes Micoaerophilic need O2, but grow best when O2 pressure is low (low [O2]) ∙Ex: C. jejuni Capnophilic: grow in an atmosphere enhanced w/ CO2

Virulence factors of Streptococcus pneumoniae

Capsule Surface protein adhesions Release of cell wall components → inflammatory response Pneumolysin ∙Exotoxin that creates pores in ciliated epithelial cells and phagocytes ∙Activates complement → migration of inflammatory cells → tissue damage Secretory IgA protease

Other B-lactam/glycopeptide antibiotics

Carbapenems (e.g. Imipenem-cilastatin); mechanism of action similar to Pens ∙Adm. I.V. ∙Broad spectrum: gram (+), gram( -), and anaerobes, doc for Enterobacter infections, not effective for MRSA ∙cross-reactivity with Pen allergic rxn. *Monobactams (e.g., Aztreonam)*; MA similar to pens; Adm. I.V.; high affinity for PBP of gram(-); poor affinity for PBPs of gram (+) or anaerobes; no cross-reactivity with Pen allergic rxn *Vancomycin*, a glycopeptide that inhibits transglycosylase (cell wall synthesis) ∙bactericidal for gram (+) bacteria (e.g. MRSA); ∙renal elimination ∙no cross-reactivity with Pen allergic rxn Demonstrates good resistance to beta - lactamases; used only parenterally (all three)

Primary pathogens

Cause disease in healthy individuals ∙Have virulent mechanisms to overcome mechanical, innate and adaptive immune responses Rarely associate w/ their host except in case of disease ∙i.e. Bacillus anthracis

Staphylococcal Scalded Skin Syndrome (SSSS)

Caused by exfoliative toxins A and B ∙Tissue specific serine proteases that cause separation of the layers of the epidermis at the desmosomes Characterized by extensive sloughing of the skin remote from infection site

Toxic Shock Syndrome (TSS)

Caused by exotoxins that are superantigens ∙Toxic Shock Syndrome Toxin-1 (TSST-1) ∙Staphylococcal enterotoxin serotypes B & C ∙IL-1 → fever ∙TNF-α and β → capillary leakage ∙Interferon-γ and IL-2 → rash Characterized by fever, skin rash, hypotension, and peeling of skin on recovery Originally associated w/ use of highly absorbent tampons ∙Oxygen stimulates TSS-inducing toxins Increasingly associated w/ lung infections, necrotizing pneumonia, and MRSA strains

Diseases caused by Staphylococcus aureus

Causes more varied diseases than any other pathogen

Apoptosis in pathologic states

Cell death by injurious stimuli Cell injury from viruses *Atrophy from duct obstruction ∙If a duct is obstructed by a tumor or a stone, the duct distal will undergo apoptosis* Cell death in tumors: even the most aggressive malignancy will have some apoptosis

Apoptosis in Pathological circumstances

Cell death induced by a variety of injurious stimuli ∙Radiation and anti-neoplastic drugs →Excess sun exposure can trigger a limited amount of cell death →This will cause DNA damage →→→Cell proliferation will be blocked via p53 →→→DNA repair mechanisms will be implemented →If repair mechanisms fail, cell undergoes apoptosis →If greater radiation damage has occur, there will be a massive number of cells that need to undergo apoptosis but only a limited amount of phagocytosis cells that can complete this process →→→The extra cells that have not undergone apoptosis will start to secrete intracellular components →→→Necrosis will cause inflammation, pain, and swelling ∙Mutation in caspase1 will predispose cells to particular tumors ∙Mild heat and hypoxia ∙Stress to the endoplasmic reticulum →Accumulation of unfolded proteins will trigger intercellular proteasome Cell injury in certain viral diseases ∙Viral hepatitis triggers massive apoptosis Pathologic changes in parenchymal organs after duct obstruction ∙Pancreas ∙Kidney ∙Parotid gland Cell death in tumors, primarily as a result of preventing new vascularization of the tumor itself

Apoptosis in physiologic states

Cell destruction during embryogenesis is seen because during development, you want parts to stop growing at certain points Hormone dependent involution: Such as during menstruation with different hormones waxing and waning Cell deletion in proliferating cell populations ∙This is important ∙In lymphocytes, you will want them to stop growing or else who will have a neoplasm, a tumor ∙Neoplasms are driven by a lack of apoptosis ∙Acute inflammation response is triggered in certain cells ∙Eliminating self-reactive lymphocytes ∙Cell death induced by cytotoxic T cells

Morphology of apoptosis

Cell shrinkage Chromatin will condense and shrink down The little fragments or blebs form apoptotic bodies that have receptors which are recognized by macrophages and broken down

Morphology of Apoptotic cells

Cell shrinkage ∙Dense cytoplasm ∙Relative maintenance of cell organelles, but more tightly packed Chromatin condensation ∙Highly characteristic of apoptosis ∙Chromatin aggregates peripherally around the nuclear membrane ∙Eventually nucleus may break up into two or more fragments Formation of cytoplasmic blebs and apoptotic bodies ∙Apoptotic bodies composed of cytoplasm, tightly packed organelles, and sometimes nuclear fragments ∙There is very little release of intracellular components, but these are usually anti-inflammatory Phagocytosis of apoptotic cells with NO INFLAMMATION ∙Apoptosis is a regulated process

The immune system is composed of cells and molecules

Cells include: ∙Lymphocytes ∙Granulocytes (polymorphonuclear leukocytes, PMNs) ∙Mononuclear phagocytes Molecules include: ∙Antibodies ∙Cytokines ∙complement

Cephalosporins

Cephalosporin-isolated from Cephalosporium acremonium in 1948 Beta-lactam antibiotics, larger ring attached to B-lactam ring, structure inherently more resistant to B - lactamases; often effective in penicillin-R (lactamase +) infections *Cross-allergenicity with pens; about 5-10%* ∙DO NOT USE W/ ANAPHYLACTIC RXN ∙Can use if its only a rash Cephs divided into 4 generations based on spectrum of activity against gm negative rods Effective against staphylococcal infections and typhoid fever

Significance of Mutations

Changes in amino acids of bacteria structures can result in resistance to antibiotics Frame shifts and inversions can cause antigenic and phase variation of major surface components ∙Antigenic variation: changes in the composition or structure of a molecule ∙Phase variation: the turning on or off of the expression of a molecule

Clinical relevance of APPs

Changes in certain APPs are general indicators that something is wrong with a patient ∙These values are not diagnostic of a specific disease ∙Tell us "an inflammatory event is happening" But if a test is performed incorrectly, then bad results will be obtained and an incorrect diagnosis could be made

Characteristics of APPs

Changes in concentration can be caused by: ∙Substantial changes →Infection, trauma, surgery, burns, infarction, inflammatory conditions, advanced cancer ∙Moderate changes →Strenuous exercise →Heatstroke →Childbirth →Any type of injurious stimuli ∙Minimal changes →Psychological stress →Possibly psychiatric illnesses

Changes in vascular permeability

Changes in vascular permeability can be due to a number of different stimuli *Image* (A) A normal, intact endothelium →Leukocytes are being bombarded by factors that "shut them off" and keep them moving along in vascular network (B) When retraction of endothelial cells occurs, small molecules and fluid can pass through the gaps in the endothelium →If this process continues, there will be large enough gaps to allow leukocytes to pass through the endothelium (C) A mechanism of endothelial injury: Leukocytes have been loitering longer in the vessel and the hydrostatic pressure imbalance causes them to migrate through efferent lymphatics →Moving through the lymphatics is a passive process →The lymphatic system moves fluids uni-directionally →It has valves that open and shut according to pressures in the vessels (D) There can be damage to the intact endothelium by leukocytes if they are not able to get out into the tissue quick enough →Essentially, the leukocytes attack the endothelial cells instead of a pathogen (E) Increased transcytosis can occur through an intact endothelial cell layer →Up to this point, all the processes described have been passive and based on the osmotic pressure of the vessel wall

Biochemical events in leukocyte activation

Changes that occur for the leukocyte upon activation: ∙Changes to the cell surface receptors ∙Cytoskeletal rearrangements, such as pseudopod formation →occur to allow the cell to squeeze through the endothelium and into the tissue →Rearrangements are dependent upon release of intracellular calcium ∙There is an increase in the number and affinity of adhesion molecules ∙Arachidonic acid split products are formed, which are factors that act locally to change microenvironment and activate other cells ∙Enhance the respiratory burst of the cells to enable them to phagocytize pathogens or damaged cellular components Neutrophils destroy damaged cells or pathogens ∙Neutrophils are producing and secreting cytokines Macrophages destroy pathogens but also can process and present antigens to lymphocytes All of these events are taking place and gearing up for a primary immune response, if necessary ∙Neutrophils predominate for 48 hours, then macrophages predominate until about day 5, then the T cells will begin to respond ∙Each wave has cells that perform a function →If the injury isn't under control, other factors are called into play

α1 antiprotease (AKA α1-antitrypsin)

Characteristics ∙It is a serine protease inhibitor (SERPIN) ∙It consists of a single chain protein with three oligosaccharide side chains ∙High polymorphism →Over 75 forms possible →MM is the normal genotype and makes PiM →ZZ genotype is associated with emphysema and makes PiZ →→→This is due to a missense mutation that affects folding and processing in the ER of the liver →→→→→Functionally deficient copies of the gene →→→Usually some type of liver cirrhosis and decreased levels of enzyme activity at distant sites are seen Regulation ∙Transcriptional level induction by IL-6 ∙Almost all is produced by hepatic cells, but also in monocytes (about 1%) ∙In acute phase levels increase 3→4-fold Protects tissue distally to limit collateral damage ∙Proteases will be active at the site of inflammation ∙Acts as a panprotease inhibitor →Trypsin, elastase, and other proteases are inhibited through formation of inactive complexes ∙Elastic fibers and ECM components in lung alveoli are protected from neutrophillic elastase Loss of lung function and cirrhosis occur with deficiency ∙Methionine in the active site of neutrophils is oxidized by cigarette smoke ∙In patients with ZZ genotype, mutant proteins are not secreted properly and form aggregates in the ER of hepatic cells

Macroscopic Distinctions in Bacteria

Characteristics of colonies on growth media: ∙Color ∙Size ∙Shape ∙Texture ∙Metabolism of sugars ∙Lysis of RBC's (hemolysis)

Structure of MHC molecules

Class I MHC molecules are composed of one peptide chain w/ three globular domains designated α1, α2, and α3 ∙All Class I MHC isoforms are complex w/ a molecule called β2 microglobulin Class II MHC molecules are composed of 2 peptide chains, designed α and β ∙Each chain has two globular domains, designated α1 and α2, and β1 and β2, respectively Overall, the structure of MHC Class I and Class II are very similar Both Class I and Class II MHC have peptide binding clefs ∙α helix 'walls' ∙β pleated sheet 'floor' ∙difference: peptide binding cleft of Class II MHC is not closed at both ends like Class I ∙peptides are less uniform in size for Class II MHC polymorphism is largely confined to peptide binding cleft

Tetracyclines: Mechanism of Action

Classified as "broad-spectrum" antibiotics Bacteriostatic, reversibly binds 30 S subunit of bacterial ribosomes, block protein synthesis ∙b/c of this, they aren't used frequently anymore Blocks binding of amino-acyl tRNA Resistance protein (TetA) involves Mg dependent drug efflux (drug pump) R-factor; also changes in ribosomal binding and production inactivating enzymes Passive diffusion thru outer cell wall Drugs actively transported thru bacterial cytoplasmic membrane

Quinupristine-Dalfopristine: Mechanism of Action, Clinical Indication and ADRs

Classified as a streptogramin, two drug synergistic combination inhibiting protein synthesis, binds 2 sites on 50S ribosomal subunit; IV adm Main indication resistant gram-positive infections; MRSA, drug resistant Strep. pneumoniae, vancomycin-resistant Enterococcus faecium (VREF), but not VRE faecalis ADRs: GI/N-D, pain/phlebitis @ inj site, arthralgia, myalgia Enzyme inhibitor (CYP3A4)

Classification of Sulfonamides: Clinical Indication

Classified by indications; systemic use vs. local and topical use Systemic use: sulfisoxazole and sulfamethoxazole, primarily for UTIs Local use: sulfasalazine, bowel lumen (ulcerative colitis) Topical use: sulfacetamide (ophthalmic infections)

Lipases

Clostridium perfringens alpha-toxin (phospholipase C, lecithinase) ∙Hydrolyzes phosphorylcholine in cell membranes → gas gangrene

Inheritance of the MHC

Co-dominant expression - protein products of both alleles at a given locus are expressed Image ∙For purposes of explanation, each haplotype is artificially designed by a color ∙Dad has haplotype blue and red, mom has yellow and green ∙During meiosis, each egg and sperm gets one chromosome 6 and hence, one haplotype ∙After fertilization, each offspring has 2 chromosomes 6, so there are 4 possible outcomes for offspring: →Green/blue →Red/yellow →Blue/yellow →Red/green Remember that each haplotype contains loci encoding all Class I molecules (HLA-A, B, C, E, F and G) as well as all Class II molecules (HLA-DP, DQ, DR, DM and DO) ∙So every individual expresses 2 HLA-A molecules or isotypes (encoded by the maternal and paternal alleles) and so on for each polymorphic gene

Streptococcus agalactiae (GBS)

Colonize the lower GI and GU tract, transient vaginal carriage A leading cause of neonatal sepsis, pneumonia and meningitis Early onset (endogenous): bacteremia, pneumonia or meningitis Late onset (exogenous): bactermia w/ meningitis Anti-phagocytic polysaccharide capsule is a major virulence factor

Clinical considerations of S. agalactiae

Colonizes female GU tract ∙All pregnant women screened for colonization ∙60% of infants born to colonized mothers become colonized Pregnant women ∙Postpartum endometritis, wound infections, UTIs Non-pregnant women and men ∙Opportunistic infections (HIV, diabetics, cancer) →Skin and soft tissue infecitons, bacteremia, urosepsis

Pre-thymic (T) stem cells

Come from the bone marrow Committed to the T lineage, but no markers of mature T cells Enter thymus at the subcapsular region Cannot recognize antigen T cell receptor genes are not rearranged

What is complement?

Complement is a group of approximately 30 proteins whose functions "complement" the antigen-binding function of antibodies Complement is essentially a proteolytic cascade Each complement component exists in the plasma in an inactive or zymogen form When activated (usually by proteolytic cleavage), each component cleaves the next component in the cascade This process leads to the deposition or 'fixing' of complement components to the pathogen surface

Function of secondary lymphoid organs

Concentrate antigens that are introduced through common portals of entry ∙Include skin, respiratory tract, GI tract Antigen is brought to secondary lymphoid tissue by dendritic cells ∙Processed and present to T lymphocytes Naïve lymphocytes that have not yet encountered antigen migrate through these peripheral organs ∙When encounter antigen, initiate immune responses ∙Anatomy of secondary lymphoid organ promotes cellular interactions that are required for activation phase of adaptive immune responses ∙Generation of memory and effector cell populations ∙Migrate into the periphery Important things to remember from figure ∙Naïve (non-activated) lymphocytes exit the primary lymphoid organs and migrate to secondary lymphoid organs where they may or may not encounter antigen that is brought from the sites of infection ∙Antigen-induced activation of lymphocytes, the generation of an adaptive immune response w/ production of effector cells and molecules, occurs in secondary lymphoid tissue ∙These effector cells and molecules then migrate back to site of infection to participate in eradication of the infection

Components of inflammatory response

Connective tissues Resident cells ∙Mast cells, macrophages, etc. ∙Both functional and nonfunctional Components of ECM Vascular smooth muscle (sometimes) ∙Histamine causes this to constrict ∙Part of initial response - vasoconstriction of larger vessels

Penicillin-G Spectrum

Considered narrow spectrum drug Staph (non-R); Strep species (ear/throat/sinus pneumonia) ∙Serious enterococcal infections → complexed with aminoglycosides Pen G not resistant to B-lactamases Difficulty penetrating gram (-) rods Cross-reactivity to other pens regarding the allergic reactions

Members of the immunoglobulin superfamily

Contain one or more of the extracellular globular domains that are characteristic of immunoglobulin molecules Include two endothelial adhesion molecules ∙ICAM-1 (intercellular adhesion molecule-1) ∙VCAM-1 (vascular cell adhesion molecule-1) ∙Both of these molecules serve as ligands for integrins found on leukocytes Also includes NCAM (expressed on multiple cell types including neural cells) Represent the major endothelial proteins recognized by the integrins

Lower levels of CRP have been associated with increased risk of Cardiovascular (CV) disease

Could be associated with plaque buildup 1-10mg/L CRP is not good indicator of atherosclerosis, though, just of chronic inflammatory events

Insertion Sequences (IS)

DNA capable of replicating itself into a new site in the genome by site-specific recombination

Edema

Swelling in tissues Edema is either a transudate or an exudate Greater pressure on one side of endothelium forces fluid to other side

Typical Structures of a Prokaryotic Cell

Cytoplasm ∙Contains DNA chromosome, mRNA, ribosomes, proteins and plasmids →DNA is not w/in nuclear envelope ∙Some cells will have granules Cytoplasmic membrane ∙Responsible for many functions that occur in organelles in eukaryotic cells ∙For those that carry out aerobic respiration, ETC is found in this membrane ∙Secretion of molecules occurs here, as well as absorption of molecules ∙Flagellum attached here, for movement ∙Structure →Lipid bilayer that resembles eukaryotic cytoplasmic membranes but no sterols (except mycoplasma) ∙Functions →Electron transport & ATP production →Uptake of and secretion of substances →Maintenance of membrane potential →Septum formation for cell division →Site of synthesis of DNA and cell wall components Cell Wall ∙Outermost component common to all bacteria (except mycoplasma) ∙Structural components & functions distinguish gram positive from gram negative bacteria ∙Prevents osmotic lysis of cytoplasmic membrane, determines and maintains cell shape →Interfering w/ integrity of cell wall will cause the cell to lyse ∙Components unique to bacteria and the repetitive structures elicit the innate immune response ∙Contains peptidoglycan →Polymers of sugars (glycan) cross-linked by short chains of amino acids (peptides) →Synthesis is target for antibiotic therapy b/c it is unique to bacteria

Subcellular Responses to Injury

Cytoskeletal abnormalities ∙The skeleton of the cell is comprised of a variety of filaments that allow for movement ∙If there is an impairment of the filament, locomotion is impaired ∙Example: Kartagener's syndrome: Abnormal microtubules results in problems with cilia, such as those in the respiratory tract →Patients are prone to respiratory infections →Men are sterile because the sperm flagella will also be altered ∙Intermediate filaments coalesce and form certain bodies →Mallory Bodies: occur in liver disease, mostly alcoholic liver disease →→→The liver is damaged and intermediate filaments breakdown and coalesce →Neurofibrillary tangles: Seen in Alzheimer's disease →→→This is a sign of the damage rather than the cause of the damage Lysosomal catabolism ∙Heterophagy is when there is phagocytosis or pinocytosis by another cell ∙Autophagy: the cell is broken down by itself →The enzymes will break down the cell and material is released →In autophagy, you have some organelle or problem in the cell and this portion breaks itself down by formation of lysosomes that connect to the abnormal part →This will then linger on as a pigment like lipofuscin Mitochondrial alterations ∙Hypertrophy ∙Atrophy ∙Abnormalities SER induction ∙Smooth ER is induced, which is the basis of tolerance to many types of medication ∙Metabolism of chemicals is done here ∙P450 is modified Heat shock proteins

Clostridium difficile

Cytotoxins A and B Enteroxin (Toxin A) - chemotactic for neutrophils, PMNs → release of cytokines, hypersecretion of fluid into ileum of gut Cytotoxin (Toxin B) - causes actin depolymerization → destruction of cellular cytoskeleton When symptomatic: mild diarrhea to life-threatening pseudomembranous colitis

Conditions that favor/trigger thrombosis

Damage to endothelial cells which promotes platelet activation and adhesion and exposes tissue factor (TF) ∙TF is also exposed to some degree on monocytes

Roles of the immune system

Defense against infections ∙Deficient immunity results in increased susceptibility to infections, exemplified by AIDS ∙Vaccination boosts immune defenses and protects against infections Defense against tumors ∙Potential for immunotherapy of cancer Immune systems recognizes and responds to tissue grafts and newly introduced molecules ∙Immune responses are barriers to transplantation and gene therapy Immune system can injure cells and induce pathologic inflammation ∙Immune responses are the cause of allergic, autoimmune and other inflammatory diseases

Complements in health and disease

Deficiencies in complement proteins or complement regulatory proteins have been associated with enhanced susceptibility to infection or immune abnormalities C3 deficiency ∙Predisposes individuals to many infections and is fatal if not treated ∙This is a critical step in promoting opsonization C2 and C4 deficiencies ∙These deficiencies are associated with autoimmune diseases, specifically systemic lupus erythematosus Inability to form the MAC (C5-C9) ∙ Enhanced susceptibility to Neisseria infections

Classical pathway: The membrane attack complex

Deficiency in terminal complement components is associated w/ an increased susceptibility to infection by Neisseria

Standard Precautions

Definition: ∙Designed to prevent transmission of HIV, HBV and other blood borne pathogens when providing health care ∙Standard precautions apply to blood, semen, vaginal secretions and body fluids such as cerebrospinal, synovial, pleural, peritoneal, pericardial and amniotic ∙Standard precautions do not apply to feces, nasal secretions, sputum, sweat, tears, urine, saliva and vomitus unless they contain visible blood Protective barriers: gloves, gowns, aprons, masks and eyewear Needle stick injuries: needles should not be recapped by hand, purposely bent or broken by hand or removed from disposable syringes or otherwise manipulated by hand ∙Sharp items should be placed in puncture-resistant containers, which should be located close to use area Hand washing is the single most effective means of minimizing health-care associated infections ∙Hands should be washed immediately after gloves are removed and in b/w patient contacts In addition, isolation procedure should be used when required (i.e. TB, MRSA, VRE, etc.)

Diagnosis of Toxin-Mediated Disease

Demonstration of toxin-production by bacteria isolated from clinical specimens ∙i.e. Corynebacterium diphtheriae demonstration of exotoxin in a clinical specimen ∙i.e. Clostridium difficile

Cross-Presentation

Dendritic cells: major antigen presenting cell involved in activation of naïve T cells (priming) Dendritic cells can present internalized (exogenous) antigen on either MHC Class I or Class II Phenomenon is called cross-presentation Involves transfer of peptide from the endocytic pathway to the cytosol Can then be picked up by a protesosome, processed, and transported to the ER for presentation on Class I MHC

Mechanisms of cell injury

Depletion of ATP ∙Activity of sodium pump reduced: cell swelling, dilation of ER →This is reversible early on ∙Cellular energy metabolism is altered: increased glycolysis, decreased glycogen, and decreased pH →When glycogen is depleted, the energy reserves are gone ∙Failure of calcium pump: leads to influx of calcium damaging cellular components →It can also enter the mitochondria →It can be very toxic when not controlled ∙Calcium is toxic to many intracellular structures even in small amounts ∙Reduction in protein synthesis ∙Unfolded protein response: decrease in oxygen or glucose leads to unfolded or misfolded proteins leading to cellular injury or death →Lots of misfolded proteins can interact and accumulate, and cause cell damage Mitochondrial damage ∙Mitochondrial injury through the formation of pores inhibiting oxidative phosphorylation ∙This leads to further depletion of ATP ∙Increased permeability ∙Leakage of cytochrome C with alteration of electron transport chain leading to cell death ∙Mitochondrial image shows formation of pores and loss of cytochrome C Calcium homeostasis ∙Cytosolic calcium usually lower than extracellular levels ∙Most intracellular levels are sequestered in the mitochondria and endoplasmic reticulum →Once mitochondria are damaged, the sequestered calcium can enter the cytoplasm and cause damage ∙Influx of intracellular calcium - this can activate a variety of enzymes in a cell, depleting energy, and initiating chain reactions that can be irreversible →Decreased ATP: compromises calcium gradient →Release of calcium from the mitochondria and ER →Decreased phospholipid: from activation of phospholipases causing membrane damage →Decrease ATP from activation of ATPases →Disruption of membrane and cytoskeletal proteins →Chromatin damage from endonucleases ∙Dr. McDonald suggests knowing this diagram! →Summary of influx of calcium into a cell Accumulation of oxygen derived free radicals-causes ∙Radiant energy: uv light (this is why you should wear sunscreen!), x-rays ∙Chemicals or drugs: CCL4 to CCL3 (This can cause massive damage) ∙Accumulation of oxygen derived free radicals-causes ∙Reduction-oxidation reactions: water generated from O2 forms toxic metabolites ∙Transition metals: iron, copper may donate or accept free electrons and generate free radicals ∙Nitric oxide (NO) is generated by endothelial cells & macrophages and acts as a free radical →NO is also a very potent vasodilator ∙Effects of free radicals →Lipid peroxidation of membranes: peroxides are reactive and unstable →Oxidative modification of proteins →Effects of free radicals →Lesions in DNA: free radicals react with thymine causing single strand breaks leading to cell death or malignant transformation →→→Sometimes it is better for a cell to die than to replicate →Cross linking of proteins: leads to increased degradation or loss of enzymatic activity ∙Removal of free radicals →Antioxidants: block the initiation of free radicals or inactivates them ex: vitamins A & B and glutathione →Removal of free radicals →Binding of metals to storage and transport proteins →Enzymes: catalase, Super Oxide Dismutase, glutathione peroxidase Defects in membrane permeability ∙Mitochondrial dysfunction ∙Loss of membrane phospholipids ∙Cytoskeletal abnormalities ∙Reactive oxygen species ∙Lipid breakdown products

Lactose fermentation properties of Enterobacteriaceae

Do not memorize!

E. coli associated w/ gastroenteritis

Different O & H serotypes are associated with different types of pathogenic E. coli ETEC = enterotoxigenic E. coli ∙Can cause traveler's diarrhea (buildup up in water/food sources in underdeveloped nations) ∙Water, no blood or WBCs EHEC = enterohemorrhagic E. coli ∙O157:H7 ∙Can cause bloody diarrhea, possibly from genes acquired from shigella (acquired Shiga toxin?) EIEC = enteroinvasive E. coli EPEC = enteropathogenic E. coli EAEC = enteroaggregative E. coli DAEC = diffusely adherent E. coli

Transmission

Direct ∙Sexual, kissing, animal bite, transplacental, droplet infection ∙Organisms transmitted by direct contact have minimal ability to survive stressful environmental conditions (changes in temperature, humidity, pH) Indirect ∙Vector borne, air borne (aerosol - i.e. TB, measles, chicken pox) and vehicle borne (water, food, blood, tissue, fomite, and surgical instruments) ∙Organisms multiplying in environment have unique adaptation for survival (spores, protozoan cysts, worm eggs)

Diagnosis of S. pyogene infection

Direct gram smear, culture on blood agar Sensitivity to bacitracin, presence of L-pyrrolidonyl arylamidase (PYR+, all other strep negative) Antibody to streptolysin-O (ASO test), DNase B (ADB test), streptokinase, hyaluronidase can be determined to monitor progress of disease

Leukocyte-induced injury

Disorders in the chart above involve the cellular mediators we have discussed Acute transplant rejection is due to lymphocytes, which will destroy tissue if HLA is not matched ∙HLA is not a topic we have covered yet Septic shock involves infection with a microorganism that spills into the bloodstream ∙This causes a massive change in the blood vessels over the entire body ∙Blood pressure drops significantly as the cells leave the circulation and enter the tissues ∙This global activation of inflammatory events is life threatening In a lung abscess, pus is generated ∙Pus has a high DNA content of all cells the neutrophils have destroyed

Defects in leukocyte function

Don't memorize the above chart ∙Realize that there are genetic and acquired defects that can occur in leukocyte function There can be a loss of leukocyte adhesion to the endothelium if there are defects in integrins There can be direct loss of function of leukocytes Defects in leukocyte function leave people with a predisposition to infection

Chloramphenical: Hematologic Toxicity

Dose-related bone marrow suppression and anemia due to inhibitory effect on protein synthesis and Fe incorporation into heme Anemia; genetic predisposition or hypersensitivity reaction ∙Mortality increases as time between treatment and onset aplastic anemia increases ∙Increased risk of leukemia in those who develop this and recover

Chemical injury

Dr. McDonald won't list all of the chemicals but know some of the concepts Chemical can have direct cytotoxic effects ∙If direct, it combines with molecular component or organelle Chemical may also undergo metabolic change and be a toxic metabolite ∙An example would be carbon tetrachloride Tylenol can be devastating to the liver ∙In prescribed doses, it isn't a problem ∙If taken in excess, it can cause liver problems and massive necrosis of the liver →This is often taken in overdoses as a suicide →Or accidentally, when parents overdose their children who have fevers or upper respiratory infections and the child will end up with liver necrosis

Droplet transmission

Droplet nuclei < 5um in diameter → measles, TB, varicella Droplet > 5um in diameter → need close mucosal contact (w/in 2 meters), i.e. SARS, influenza, pertussis, mumps

Mechanism of Action of Pencillins/Cephalosporins

Drugs bind/inhibit PBPs (penicillin-binding-proteins), the bacterial transpeptidase enzymes required for new bacterial cell wall synthesis Defective cell walls cause osmotic swelling/release of autolytic enzymes and bacterial lysis (bactericidal) Vancomycin ALSO inhibits cell wall synthesis, but DOES NOT WORK AT THE SAME ENZYME

Gangrenous Necrosis

Dry Gangrene has more coagulation type necrosis ∙Is called dry because occurs in the limbs and looks mummified and dried out Wet Gangrene has a lot more of the liquefactive component, the limbs look moist You can have a combination of the two Occurs especially in diabetes, which impairs the blood flow and is associated with neuropathy Risk factors result in gangrenous necrosis which could result in amputation

Pathologic Calcifications

Dystrophic Calcifications ∙This is when calcium is deposited in abnormal organ, calcium is deposited in abnormal structure ∙This adds to the pathology and dysfunction ∙You have a normal serum calcium level ∙You MIGHT have hypercalcemia but often it is normal ∙Does produce damage ∙Occurs in areas of necrosis ∙Can be intracellular or extracellular calcifications ∙Cause organ dysfunction ∙Accentuated by hypercalcemia Metastatic Calcifications ∙You have hypercalcemia, results in calcium deposition in otherwise normal organs ∙Metastatic does not produce damage to that organ with the accumulation, it is an incidental finding ∙Does not produce underlying damage ∙Occurs in normal tissues →E.g. Gastric mucosa, kidneys, lungs ∙Usually causes no clinical dysfunction ∙Seen in x-ray and autopsy

Transport proteins

During an acute phase response, iron is internalized in circulation over several days There is no physiological pathway for iron excretion Iron gets absorbed in the small intestine as heme or free ions In capillaries, transferrin binds iron to transport it to iron consuming tissues like bone marrow and liver ∙It is a negative APP ∙It has two binding sites for ferric ions (Fe3+) ∙Single chain glycoprotein ∙Transferrin is a β globulin that carries iron in the blood ∙Iron availability determines saturation changes in transferrin →Proper stimulation releases free iron back into extracellular environment which then induces transcription of more transferrin molecules in the hepatocytes →The presence of unbound sites protects against infection by iron-dependent pathogens →Transferrin iron binding capacity (TIBC) increases in iron deficiencies Ferritin ∙It is comprised of 24 subunits surrounding a core of 3-4000 ferric ions ∙There are two subunit isoforms, L and H ∙It is mostly found in tissues, but can also be see in blood in low concentration →In blood, is indicative of iron reserves ∙Most of the intracellular iron is stored, or sequestered, in ferritin ∙It regulates itself over 24 hours ∙Why don't we leave iron free in cytoplasm? →Iron is a very reactive oxygen species →This is a protective mechanism →Ferritin is an antioxidant

Macrolide Drug Interactions

ERY and clarithromycin inhibit cytochrome P-450s / isoenzyme CY3A4, and the metabolism of other drugs may be inhibited > to increase adverse effects Cardiac arrhythmias ( increase QT interval) leading to possible sudden death ∙Drugs that inhibit the metabolism of & increase blood levels of ERY may cause ERY-induced arrhythmias

B cell differentiation in the bone marrow: Pro B cells

Earliest bone marrow cell committed to the B cell lineage Marker expression ∙Do not produce immunoglobulin ∙Immunoglobulin heavy chain rearrangement occurs ∙Do express other B cell lineage-restricted markers (CD19 and CD10) At the early pro-B cell stage: D-J rearrangements At later pro-B cell stage: V-DJ rearrangements Mechanism of recombination ∙Protein products of the RAG-1 and RAG-2 genes mediate recombination ∙DNA loops out ∙Excision ∙Ligation ∙Involve the action of *recombinases*, exonucleases and DNA ligases (probably part of the RAG-1/2 complex) *B cell rearranged DNA contains V exons at the 5' end, "selected" VDJ with intervening sequences deleted, J exons at 3' end and C region exons at 3' end* Transcription yields a primary RNA transcript containing rearranged VDJ, some portion of the rest of the J region, and the C regions for m and d

Commitment to B and T cell lineages

Early B and T cell development is characterized by the proliferation of progenitors, stimulated mainly by the cytokine IL-7, particularly for human T cell progenitors ∙Results in marked increase in cell numbers →Important b/c the next step, the generation of 'useful' antigen receptors, is an inefficient process involving random genetic recombination events ∙*Ensures large enough pool of progenitors to generate highly diverse repertoire of antigen-specific lymphocytes* IL-7 made by stromal cells in the bone marrow and thymus ∙This is why knowing about IL-7 is important: *mutations in IL-7 receptor causes SCID (x-linked) in humans* Proliferation precedes antigen receptor rearrangement In order for student to understand how antigen receptor rearrangement proceeds and the vast array of antigen receptor specifities is generated, it is important to understand how the genes for antigen receptors are different than other genes and how they encode the antigen receptor proteins

Metronidazole: Clinical indication and ADRs

Effective against protozoa & anaerobic bacteria, undergoes nitroreduction Forms reactive intermediate (radical) > damages DNA, DNA strand breaks DOC Clostridium difficile pseudomembranous colitis Potent antiprotozoal and anaerobic activity GI disturbs/metallic taste, headache \ paresthesias, ataxia, liver dysfunction Disulfiram rx (alcohol) Contraindicated in pregnancy, mutagenic

Mature B cells

Emigration from the bone marrow Use of alternate splicing events to co-express both IgM and IgD Fully responsive to antigen

Encounter and Transmission

Endogenous: normal flora moved from normal niche Exogenous: encounter from an outside source Congenital: passed from mother to fetus

Thrombin and EC inhibit thrombosis by inactivating two cofactors

Endothelial cells (EC) produce Thrombomodulin (TM) which complexes with Thrombin (T) T-TM complex activates Protein C leading to Active protein C (APC) Factors Va and VIIIa are cleaved/inactivated by APC

Toxin-Mediated Disease & S. aureus

Enterotoxin and Toxin Shock Syndrome Toxin (TSST-1) ∙Both function as superantigens that can cause TSS ∙Note: a different region of the enterotoxin molecule mediates symptoms of food poisoning Exfoliative toxins (ETA and ETB) ∙Disrupt the desmosomes that link adjoining cells, causing superficial skin desquamation (SSSS, staphylococcal scalded skin syndrome)

Clonal selection theory

Every individual contains numerous clonally derived lymphocytes Each lymphocyte has an antigen receptor specific for a single antigen These are present prior to exposure to antigen Antigen selects a pre-existing clone and activates it

What's the clinical significance of these transfer mechanisms?

Evolution of new pathogens ∙Image shows a non-pathogenic strain of E. coli acquiring foreign DNA to produce E. coli strains that cause diarrheal disease or UTIs Transfer of Antibiotic resistance

IgA

Exists (mostly) as a monomer or dimer ∙Monomeric IgA functions w/ IgM and IgG to protect all tissues reached by blood ∙Dimeric IgA protects the mucosal surfaces of the body that communicate w/ the external environment →GI tract →Eyes, nose, throat →Genitourinary tract →Respiratory tract →Breast milk How dimeric IgA exits into lumen ∙Poly-Ig receptor on basolateral side of epithelial cells ∙Binds to J chain of IgA and IgM ∙Secreted on luminal side w/ remnant of the poly-Ig receptor called the secretory component ∙Secretory component binds mucins ∙Keeps IgA bound microbes from attaching to epithelia

IgG

Exists as a monomer 2 high affinity binding sites per molecule *easily moves into tissue b/c of specialized endothelial receptor (FcRn)* Functions: ∙Precipitation ∙Agglutination ∙Complement fixation ∙Neutralization

IgM

Exists as a pentamer 10 antigen binding sites per molecule *large size impairs penetration into tissues* protects internal tissues *first antibody class to be secreted* strongly binds to pathogens and particulate antigens *activates complement via classical cascade*

CD4+/CD8+ (double positive) thymocytes

Express both CD4+ and CD8+ Also express a complete TCR (α/β) w/ associated CD3 complex and ζ chains Stage at which positive and negative selection occur *Positive selection: name given to process whereby cells are signaled to mature further* ∙failure to undergo positive selection, cell undergoes apoptosis ∙takes place in thymic cortex ∙DP cells interacts w/ thymic epithelial cells (TEC), which express both Class I and Class II MHC ∙α/β TCR T cells is 'tested' ∙*If can interact w/ thymic epithelial cells ("self" peptide + MHC): psotive selection* →if TCR fails to 'see' peptide + MHC: apoptosis and phagocytic clearance →*this represents a failure of positive selection and NOT negative selection* ∙If see class I: cell stimulated to make only CD8 (single positive) ∙If see class II MHC: cell stimulated to make only CD4 (single positive) ∙Positive selection halts further α chain rearrangement *Negative selection occurs if α/β TCR T cell binds self peptide antigen + MHC very tightly* ∙Very tight binding suggests cells are self-reactive and could mediate autoimmune rxns Result of thymocyte interactions w/: ∙TEC ∙Dendritic cells ∙Macrophages Deletion via apopotosis or cell is rendered anergic (if manage to exit thymus)

Earlier model of coagulation

Extrinsic Pathway ∙Involves TF and VIIa producing factor Xa which makes prothrombinase ∙Prothrombinase makes thrombin Intrinsic Pathway ∙Includes Factor XII, which is not included in the cell-based model because if patients are deficient in it they do not have any clinical manifestations. ∙Factor XI makes Tenase which makes prothrombin ∙Relies on platelets and blood vessels Extrinsic and Intrinsic pathways merge around where prothrombin is converted thrombin (where factor X is being produced) Extrinsic pathway occurs first (similar to activation in cell-based model) Shortly, after TF pathway inhibitor (TFPI) inhibits the complex of VIIa-TF-Factor X Thrombin concentration increases due to extrinsic pathway and activates intrinsic side through feedback loops (by making more XIa, Xa and VIIa)

Mechanism of apoptosis

Extrinsic Pathway (left image): Cell surface death receptors, FAS and TNF Intrinsic Pathway (right image): Balance of apoptotic and protective molecules is changed, and increased pro-apoptotic and then death inducers are seen Execution phase: Proteolytic cascade from caspases Removal of dead cells by phagocytosis

Respiratory Diphtheria

Exudative pharyngitis: diphtheria toxin mediated necrosis of respiratory epithelial layer

Fragments of immunoglobulins

Fab = antigen binding portion Fc (crystallizable) ∙Biologic activity Cleaved into 2 Fab and 1 Fc fragment by papain Cleaved into one F(ab)2 and one Fc by pepsin

Enteric bacteria

Facultative, gram (-), rod-shaped bacteria ∙Facultative = growth in both presence and absence of oxygen Found as normal flora or pathogen in GI tract of humans/animals Ex: ∙Enterobacteriaceae ∙Other aerobic gram (-)'s, i.e. vibrios, pseudomonas

The Toll-Like Receptor (TLR) Family

Family of signaling receptors ∙each specific for a different set of microbial products ∙TLRs are expressed on different cell types associated with innate immunity ∙Allows the innate immune response to vary according to the type and site of infection

Contributing Factors to Antibiotic Resistance: Overuse of antibiotics

Fear of malpractice suits Patient volume, time and expectations Lack of knowledge of natural course of viral disease Diagnostic and prescribing habits Need to return to work, school or day care Release of enormous quantities of antibiotics into agriculture, fisheries and animal husbandry Inappropriate use - no infection is present, incorrect drug selected, excessive dose/duration, too frequently prescribed, loose drug control in some controls

Overview of Apoptosis

First described in 1972 From the Greek, meaning "falling off" Apoptosis is cell death that can occur under normal or abnormal conditions ∙Normal conditions: →Eliminates unwanted or potentially harmful cells →Cells that have outlived their usefulness ∙Abnormal conditions: →May be triggered during/as a result of pathologic event →Viruses can trigger or prevent apoptosis →Cells damaged beyond repair (particularly DNA and/or mitochondria) →Ex. Severe sun poisoning can cause hypoxic injury or morphological changes, where the cell can no longer function properly ∙In general, there should be very little or NO inflammation when apoptosis occurs properly Whereas, necrosis is an uncontrolled cell death ∙Cells inability to repair itself with loss of energy production ∙Dis-regulation of intercellular organelles and breakdown of nuclear material ∙Leakage of intracellular proteins and oxygen radicals ∙Triggers inflammatory responses

P-Selectin

First identified in the secretory granules of platelets Also found in secretory granules of endothelial cells (Weibel-Palade bodies) When platelets or endothelial cells are stimulated, P-selectin is translocated w/in minutes to the cell surface Where it mediates binding of neutrophils, T lymphocytes and monocytes Carbohydrate moieties that P-selectin binds appears similar to those that E-selectin binds

The alternate pathway: General points

First to act Initiated by hydrolysis of a thioester bond in C3 that occurs in the plasma at low level but is increased when pathogens are present ∙When C3 is first made in the liver, a thioester bond is sequestered in the hydrophilic innards of the protein In the aqueous environment of the plasma, conformational change occurs ∙Thioester bond is exposed and hydrolysed ∙Hydrolysis occurs at low rate ∙Yields a molecule called iC3 (C3H20)

What happens after a vessel injury?

First, blood is lost due to the injury of the blood vessel Vascular spasm: constriction of vessel, occurs immediately ∙First step to inhibit loss of blood ∙Regulated by reflex neurogenic mechanisms and by local secretion of factors (endothelium) Formation of platelet plug ∙Remains loose until coagulation which takes place after or simultaneously to develop a permanent clot ∙Temporarily blocks the flow of blood from the wound ∙Platelets attach to injured vessel and to each other ∙Aggregation Blood coagulation (fibrin > thrombin) ∙Clot forms in 3-6minutes ∙Clotting factors induce the production of a fibrin "mesh" around platelet plug ∙Fibrin cross-links the platelets ∙Forms a "hard clot" that completely blocks the loss of blood from the wound ∙Thrombin mediates the conversion of fibrinogen to fibrin and also activates other components of the blood coagulation cascade Growth of fibrous tissue into the clot to close hole permanently ∙Red and white blood cells Size of the clot must be restricted ∙Clot must not overgrow and restrict blood flow ∙Enzymes restrict the growth of the clot ∙Others dissolve the clot after tissue is repaired

Map of the human MHC - chromosome 6

Five Class II loci ∙DP, DQ, DR, DO and DM ∙Polymorphism varies w/ the gene Six Class I loci ∙HLA-A, B and C: highly polymorphic ∙HLA-E and G: oligomorphic ∙HLA-F: monomorphic ∙Inherited as a unit or haplotype Haplotype - in respect to a linked cluster of polymorphic genes, the set of alleles carried on a single chromosome = haplotype ∙Every person inherits two haplotypes, one from each parent ∙The term was first used in connection w/ genes of the MHC

Burkholderia cepacia

Found in moist environments & around roots of plants Low virulence and morbidity except in CF patients or patients w/ granulomatous disease ∙CF patients w/ B/ cepacia: associated w/ poor lung transplant outcome, risk of post-operative sepsis Clinical manifestations ∙Cepacia syndrome - respiratory distress and septicemia in CF patients ∙Ventilator associated pneumonia Treatment ∙Intrinsically resistant to many antibiotics

Epidemiology of P. aeruginosa

Found in moist environments: soil, plants, vegetables, tap water, countertops, etc. ∙When patients are infected, ask family to NOT bring flowers During 1950's and 60's (but now on decline) a major cause of morbidity and mortality in: ∙Burn patients ∙Patients undergoing cytotoxic chemotherapy Most common source of gram (-) bacteremia in neutropenic patients in Far East and parts of Latin America Most common contributing factor to respiratory failure in CF patients, and death among CF patients

Outer Membrane

Found only in gram negative bacteria Barrier to large and hydrophobic molecules Small hydrophilic molecules pass through PORINS ∙Have size limitations, i.e. can prevent vancomycin from entering ∙Add level of selective permeability to membrane

Cephalosporin Generations

From 1st (cefazolin) to 4th (Cefepime) generation, there is: ∙Gradual decrease in gram (+) coverage ∙Increase in gram (-) coverage ∙Increase in CNS penetration (3/4th) ∙increase in resistance to inactivation by B-lactamases

Tetracycline ADRs

GI distress, "heartburn", esophageal ulcers Phototoxicity - reactions in skin exposed to sunlight, esp. demeclocycline> doxycycline *Skeletal effects - chelates teeth and bones during calcification* Contraindications: not use in children (<8 yrs) or during pregnancy Neurotoxicity - vestibular: dizziness, vertigo, ataxia (mainly with minocycline) Hepatotoxicity - esp. in pregnancy - jaundice, fatty liver necrosis Nephrotoxicity - Fanconi syndrome (impairment in the proximal tubular function of the kidney): use of outdated/degraded drug Superinfections - development of resistant bacteria

Fluoroquinolone ADRs

GI: nausea-vomit-diarrhea (n-v-d); anorexia, taste disturbance; CNS: headache, dizziness, insomnia Skin: rash, allergy, photosensitivity (2 types: photoallergic (uncommon) and phototoxicity,) highest incidence with lomefloxacin, others less Nephrotoxicity, crystalluria may occur in alkaline urine Produced cartilage erosion and inhibition of juvenile cartilage development in animal studies; in humans: joint swelling, tendonitis, tendon rupture, especially involving weight-bearing joints; therefore, these drugs contraindicated: pregnancy/nursing/children Cardiovascular; tachycardia, QT prolongation, hypotension (sparfloxacin) Note: how does FQ adverse effects profile compare to penicillins/cephalosporins

Antibacterial Spectrum of Aminoglycosides

GM (+): limited, Staph > Strep; increased resistance, not used alone; used in combination with pens/cephs for serious staph, strep, enterococcal infections > bacteriocidal synergism *Aerobic Gm (-) is primary indication*, the big guns, indicated for serious infections ∙Affects almost all Gm (-) organisms ∙*Adm with 3rd/4th gen. pens for Pseudomonas*, and with cephs for Klebsiella and also with other drugs for serious Gm (-) infections

Clinical disease of Salmonella

Gastroenteritis - poultry, eggs, dairy ∙Inflammatory diarrhea ∙Remains localized Typhoid fever/Enteric fever ∙Systemic disease ∙Salmonella Typhi & Salmonella Paratyphi →Disease only in humans →Chronic carriage in gall bladder (typhoid Mary)

Capsule

Gelatinous, covering entire bacterium Composed of polysaccharide (sometimes peptides) Antigenic differences define serotypes *Protects bacteria from phagocytosis* Promotes non-specific adherence to tissues, prosthetic devices and catheters

Erythromycin Antibacterial Spectrum

Gm (+): alternative to pens/cephs for streptococcal and pneumococcal minor throat/ear infections, e.g. in pen/ceph allergy; resistant to penicillinases, has some staph activity but usually not an indication Gm (-): Legionella pneumophila, Mycoplasma pneumonia, Genital infections: Chlamydia, syphilis, gonorrhea, C. diphtheriae / carrier state Not useful against Gm (-) enteric organisms

Streptomycin: Clinical Indications

Generally used in combination with other drugs; deep IM injections With a penicillin for enterococcal endocarditis Tuberculosis, esp. with multi-resistant strains, in initial therapy, IM adm Tularemia and plague (Y. pestis) usually with PO tetracyclines Has less activity against gram - rods than other aminoglycosides

Function of primary lymphoid organs

Generative ∙T cells arise in thymus ∙B cells arise in bone marrow Support differentiation from progenitor cells to mature, naïve (non-activated) cells Selection processes

Organization of Immunoglobulin genes

Genes encoding κ and λ and the single locus containing the various heavy chain genes are located on different chromosomes ∙Heavy chain: chromosome 14 ∙Kappa light chain: chromosome 2 ∙Lambda light chain: chromosome 22 w/in each heavy chain V region, there are actually three separately encoded sections: ∙V = variable ∙D = diversity ∙J = joining w/in each light chain V region, there are actually two separately encoded sections ∙V and J (no D)

Bacterial Gene Expression

Genes required for basic survival are constitutively expressed Expression of non-essential genes often tightly regulated (positively or negatively) at the level of transcription mRNA's often (but not always) encode for more than one protein (polycistronic) ∙operons: sequence of DNA that contains multiple genes to produce multiple proteins for a single purpose ∙genes are under the control of one promoter and one terminator ∙Genes in eukaryotic cells are only monocistronic → no operons

Genetics and regulation of toxin expression

Genes that encode for exotoxins can be found on the bacterial chromosome, plasmids, or lysogenic (aka temperate) phage The expression of the toxin genes are often governed by environmental conditions ∙i.e. pH, temperature, oxygen, iron concentration, etc.

Integrons

Genetic elements that can promote the acquisition and expression of genes embedded in gene cassettes Gene cassettes are non-replicative mobile elements

S. saprophyticus

Genitourinary mucosa flora Catalase (+), coagulase (-), non-hemolytic, novobiocin resistance Virulence: epithelial adhesions Clinical disease: outpatient UTIs in women

Gentamicin/Tobramycin: Clinical Indications

Gentamicin most widely used systemic aminoglycoside ∙less expensive ∙active against Serratia species ∙better than tobramycin when used in combination with a penicillin against enterococci ∙However, streptomycin is preferred with Pen Tobramycin 2-4X more potent against Pseudomonas than gentamicin; ∙However, it is *less effective against enterococci* and ineffective against mycobacteria

Thrombasthenia

Glanzmann: platelet aggregation disorder with a lack of GpIIb/IIIa receptors on platelets to bind fibrinogen between 2 platelets Bernard Soulier: Platelet adhesion disorder with a lack of GpIb/IX/V receptors to bind vWF to subcellular surface (only know GpIb, not the others) Both bind via RGD 3 amino acid sequences (arginine, glycine, aspartate) found on fibrinogen and vWF

Lipopolysaccharide (LPS)

Glycolipid only in outer leaflet of gram negative outer membrane Aka endotoxin: major mediator of fever and inflammation caused by infections w/ gram negative bacteria In large doses can cause shock and even death Present permeability barrier to hydrophobic molecules (i.e. antibiotics)

Antibacterial Spectrum - Trimethoprim

Gm (+) activity against Staph (MRSA) and strep, but increase resistance and variability Gm (-) activity is main indication and includes most Gm - bacteria and Enterobacteriaceae, not Pseudomonas Indications: UTIs, GI, respiratory infections (doc for Pneumocystis jiroveci), prostatitis / vaginitis, otitis media

Sulfonamide Antibacterial Spectrum

Gm (+): strepto-, gono-, meningococci , but generally not used alone due to inc resistance and preference for bactericidal drugs Gm (-): E. coli, P. mirababilis for uncomplicated UTIs; this is main indication for systemic use of sulfonamides adm. alone H. influenzae/ H. ducreyi and Chlamydia trachomatis, effective but increase resistance often limits systemic use as single agents Nocardia asteroides infections (lung/brain infection/abscess)

Sulfonamide Pharmacokinetics: Systemic Drugs

Good PO bioavailability Wide distribution: plural, peritoneal, CNS Metabolism by acetylation Excretion: mainly urinary, both metabolites and unchanged (acidic urine decreased solubility and causes crystalline drug deposits; alkaline urine favors solubility and excretion

Spores (Endospores)

Good example of bacteria that forms spores: Anthrax Metabolically dormant forms of bacteria resistant to heat, cold, drying and chemicals Formed in response to environmental stress (i.e. nutrient depletion) Vegetative state returns when good conditions (nutrients) return The location of a spore w/in a cell is characteristic of the bacteria and can assist in identification Bacillus and Clostridium are medically important spore formers Spores NOT formed by gram (-)'s

Antipseudomonal (3 - Generation Spectrum)

Gram (+) activity less than 1/2-gen; not indicated for gram (+) infections; 1/2-gen preferred Main indication: Gram (-) ∙P. vulgaris, P. aeruginosa (often in combo with an aminoglycoside), Not resistant to B-lactamases (e.g., ticarcillin, poor oral bioavailability)

General characteristics of Staphylococci

Gram (+) cocci, arrange as grape-like clusters Facultative anaerobes Catalase (+) ∙2H₂O₂ → 2H₂O + O₂ pyogenic (pus forming) bacteria prolonged survival on inanimate objects under various conditions common cause of community acquired nosocomial infections antibiotic resistance is a problem w/ these bacteria

Enterococci

Gram (+) cocci, usually in pairs Variable hemolysis Normal intestinal flora (as name implies) Virulence: resistance to commonly used antibiotics One of the most common causes of nosocomial infections

Streptococcus pneumoniae

Gram (+) diplococcic, lancet shaped in short chains Alpha-hemolytic on blood agar Optochin sensitive, lysed by bile Encapsulated 90 serotypes → most serotypes included in polyvalent vaccine

Morphology and General Characteristics of Streptococci

Gram (+), occurs in chains or pairs Catalase (-) Usually encapsulated Normal flora: respiratory tract, genitourinary tract, GI tract

Antibacterial Spectrum: Tetracyclines

Gram (+): susceptible species of staph\ strep, however other drugs preferred due to bacteriostatic action / increased resistance Propionibacterium acnes (anaerobe) Gram (-): H. influenzae, K. pneumonia-3rd line drugs; ineffective against Pseudomonas B. burgdorferi (Lyme disease) Helicobacter pylori (GI ulcers) Main indications: Rickettsiae, Mycoplasma pneumonia, Chlamydia trachomatis, V. cholera Not effective against resistant Gonococci

Moraxella catarrhalis

Gram (-) diplococci Strictly aerobic Oxidase (+) Settings for infection = elderly patients w/ chronic pulmonary disease (i.e. COPD) Clinical manifestations ∙Bronchitis ∙Bronchopneumonia ∙Sinusitis ∙Otitis media (children) Treatment ∙Resistant to penicillins, but susceptible to other antibiotics

Lab Features of Pseudomonas aeruginosa

Gram (-) rods, in pairs, motile Non-fermentative, aerobic or anaerobic respiration Growth on agar plates ∙Diffusible pigments: →Pyocyanin (blue) →Pyoverdin (yellow-green) →Pyorubin (reddish brown) →Gun-metal appearance & fruity odor (grape-like) Does not ferment lactose on MacConkey agar Oxidase (+) ∙Presence of cytochrome oxidase differentiates it from Enterobacteriaceae and Stenotrophomonas Isolates from CF patients: mucoid, produces large amounts of the exopolysaccharide alginate

Bacterial cell wall structure

Gram negative bacteria can be difficult to attack b/c the only way into the cell is via porin proteins

Lab Diagnosis of S. aureus

Gram stain and microscopic examination of infected site/tissue Culture PCR assays

Diagnosis of S. agalactiae infection

Gram stain of CSF PCR-based amplification assay

Gram negative cell wall

Gram stain pink or red Have very thing peptidoglycan layer, which is still susceptible to cell wall antibiotics Have outer membrane ∙Outer leaflet has ability to stimulate immune response ∙Has porins which control what enters/exits cells

Leukocyte-endothelial cell interactions

Greatest changes to the vascular endothelium are found right after the capillary at the site of post capillary venules ∙This is the equivalent of a high endothelial venule in secondary lymph tissue ∙This site will have the greatest and earliest up-regulation of factors that will promote leukocyte extravasation

Medically Important Streptococci

Group A: Streptococcus pyogenes Group B: streptococcus agalactiae Group C and G: S. milleri, S. dysgalactiae Group D: ∙Enterococci: E. faecalis and E faecium ∙Nonenterococcal group D: S. bovis →Endocarditis associated w/ GI neoplasms Viridans streptococci ∙Hetergenous group ∙Several species: S. salivarius, S. mitis, S. sanquis, S. mutans Streptococcus pneumonia Anaerobic streptococci ∙Peptostreptococci Abiotrophia and Granulicatella species ∙Nutritionally variant streptococci ∙Will initially grow in broth but not when sub-cultured on agar ∙Bacteremia, endocarditis, eye infections Leuconostoc and Pediococcus ∙Resistant to vancomycin ∙Opportunistic infections

Initiators of Apoptosis

Growth factor deprivation DNA Damage TNF receptors Cytotoxic T lymphocyte mediation Intracellular accumulations ∙Normal cellular constituents ∙Abnormal substance ∙Pigments ∙Heat shock proteins →Proteins need to be processed into alpha helices or beta pleated sheets →If the proteins are not folded properly the proteins will accumulate →If proteins continue to aggregate cell death will ensue →This is seen in Alzheimer's, Parkinson's, and Huntington's diseases →Just know that if these proteins are not folded correctly they can aggregate and cause cell death →Ubiquitin helps regulate the destruction ∙Endogenous substance is seen in normal cells but for some reason begins to accumulate →This can be due to genetic or acquired defects in metabolism →Decreased transportation or secretion of this material out of cell →Increased amount of the substance produced ∙Hyaline Change →Homogenous, glassy, pink change in cells →Accumulation of material in small blood vessels (such as in HTN) ∙Glycogen →Present in most cells, is the storage form of glucose →Can accumulate in the PCT of the kidney in diabetes →Also seen in liver and pancreas →This is not the worst problem of diabetes →Also seen in glycogen storage diseases, inborn errors of metabolism ∙Pigments →Exogenous pigments: carbon with anthracosis and tattooing →Endogenous pigments: most have to do with hemoglobin, become red in appearance →Example: Hemosiderin →→→If someone has CHF, the heart is not pumping the way it should, will have a build up of blood and the pressure accumulates in the blood vessels →→→Extravasation of blood from the blood vessels is seen (this often happens in the lungs) →→→→→The blood leaves the blood vessels and floats to the alveoli →→→→→The alveolar macrophages will breakdown hemoglobin producing hemosiderin resulting in a golden brown pigment →→→Remember in CHF: you have Hemosiderin Laden Macrophages →→→Bruising: If you get punched, extravasation of blood cells from the vessels occurs →→→→→The blood enters tissue and the hemoglobin breaks down to hemosiderin, so it changes from red to golden brown →→→Lipofuscin can accumulate with aging →→→Melanin can accumulate with melanoma tumor

Laboratory identification of Enterococci

Growth in 6.5% salt Grows at 42°C Grows in presence of 40% bile salts PYR-test: produces L-pyrrolidonyl-arylamidase (only S. pyogenes is +)

Development of T cells

Has elements in common w/ development of B cells ∙Stem cells for both arise in the bone marrow ∙Both undergo antigen receptor gene arrangement ∙Both must undergo negative selection Differences ∙T cell precursors must emigrate from the bone marrow and go to thymus ∙Developing T cells must undergo positive selection to ensure that receptor can recognize either Class I or II MHC Structure of the thymus ∙Contains immature T cells (thymocytes) ∙Embedded in network of thymic epithelial cells called stroma ∙Together form an outer (closely packed) cortex ∙Inner (less dense) medulla

Free radical cell injury

Have a single unpaired electron Unstable, react with organic and inorganic chemicals. This leads to more cell damage. Lipid peroxidation of membranes DNA lesions Cross linking of proteins Image shows effects of ischemia and reactive oxygen species on a cell ∙Ischemia can also create free radicals Superoxide (O2-) are converted to hydrogen peroxide ∙The superoxide stimulates degradative enzymes in leukocytes, and may directly damage lipids, proteins and DNA ∙This is associated with inflammation, neutrophils are released and they release enzymes, resulting in the formation of free radicals Hydrogen peroxide can be converted to OH ∙OH is the most reactive oxygen derived free radical

Classification of Streptococcus by Hemolysis

Hemolysis exhibited on blood agar Alpha (α) - incomplete hemolysis, colony surrounded by a green halo Beta (β) - complete hemolysis, colony surrounded by a clear zone Gamma (γ) - no hemolysis (non-hemolytic)

Hemophilia vs. Thrombophilia

Hemophilia = tendency to bleed Thrombophilia = tendency to clot

Hemostasis

Hemostasis = stoppage of blood flow through a blood vessel In the instance of an injury - there is an injured blood vessel ∙i.e. cut on arm the goal is to produce a platelet and fibrin plug (thrombosis) in order to seal an injured blood vessel wall

Heparin

Heparin: mucopolysaccharide chain Antithrombin III (ATIII) is a 'serpin' Heparin can bind to antithrombin-III (ATIII) to enhance its inhibitory activity Causes a conformation changes in ATIII and assists in interactions ATIIII inhibits factor Xa, thrombin (IIa), IXa, XIa, and XIIa

Viridans Streptococci

Heterogenous collection of alpha and non-hemolytic strep Constitute main facultative oral flora Contribute to dental caries Bacteremia in neutropenic patients Sub-acute bacterial endocarditis in patients w/ abnormal heart valves, dental caries, and intra-abdominal infections

Purulent inflammation (subcutaneous bacterial abscess)

High concentration of neutrophils starts to destroy tissue Substantial tissue destruction-loss of normal architecture Edema - purulent exudate (pus) Destruction of normal architecture

Actions of the principal mediators of inflammation

Histamine and serotonin are early mediators Reactive oxygen species and reactive nitrogen species are anti-microbial Cytokines TNF and IL-1 are pro-inflammatory

Receptor-mediated adhesion defines:

Host-specificity ∙E. coli CFA/1 (Colonization factor antigen 1): mediates binding of enterotoxigenic E. coli to human intestinal cells → diarrhea in humans Tissue-specificity ∙E. coli P pili bind a digalactoside receptor on the surface of urinary tract epithelium

Shigella

Humans only natural reservoir S. sonnei (most common in US) ∙Causes non-inflammatory diarrhea (enterotoxin mediated) S. dysenteriae ∙Produce Shiga toxin (cytotoxin) ∙Dysentery, an inflammatory diarrhea w/ mucus, blood and WBCs in stool (due to invasion of gut mucosa)

Dissemination Facilitated by Spreading Factors

Hyaluronidase: breaks down hyaluronic acid the ground substance of connective tissue Deoxyribonuclease (DNase): thins pus made viscous by DNA released by dead WBCs Streptokinase: activates plasminogen and converts it to plasmin, which attacks fibrin clots, allowing spreading

Transudates vs. Exudates

Hydrostatic pressure gives the net fluid movement out, as seen in (A), the normal vessel in the figure above ∙Movement of fluid is called pinocytosis ∙Leukocytes are not normally moving out of a vessel ∙Erythrocytes, or red blood cells, can move to a limited degree through the endothelial layer under normal conditions →This is the function of the capillary beds- to allow exchange of nutrients by red blood cells between the vessel and the surrounding environment In (B), the net movement of fluid out of the intact vessel is what results in a transudate ∙There is an increase in hydrostatic pressure and a net movement of fluid out of the vessel In (C), there are gaps formed between the endothelial cells, allowing for the formation of an exudate ∙Cells can move out through the gaps in endothelial cells ∙This is caused by an alteration in the endothelial cell architecture

Selection of Antimicrobial Agents

Identification of organism(s)/educated guess bacterial statistics vs. Gram stain/culture Safety of drug vs. severity of infection Site of infection vs. pharmacokinetics; in-patient vs. out-patient Patient factors - age, *pregnancy*/lactation, liver/kidney function, immune status, allergy Initial empiric vs. definitive therapy for severe infections Incidence of bacterial resistance

Transposons (Tn)

IS elements that flank a structural gene(s) and carry this gene to a new genome sites and to other organisms A transposon can move from place to place in the genome, leaving a copy of itself behind at the previous site

Bacterial arrangements

If you looked at stained preparations of bacteria, some will have characteristic arrangements ∙Dependent on how they divide as cells Streptococci basically divide in one plane ∙When you see them together, they tend to form chains b/c sometimes daughter cells do not detach from mother cells Staphylococci tend to form clusters, b/c they form in 3 different planes Bacilli tend to divide in one plane ∙Sometimes separated in pairs, also chains Sometimes cocci can divide into diplococci ∙Streptococcus pneumoniae is an example

Negative selection in the bone marrow occurs at the immature B cell stage

Immature B cells express only IgM Still in bone marrow *Negative selection* in the bone marrow (immature B cell) If interact with antigen at this stage of differentiation, it is *SELF-ANTIGEN*. Cell is: ∙Deleted (clonal deletion) by apoptosis →Seems to occur with cell surface antigens ∙Cell is inactivated, but doesn't die (anergy) →Can't respond to further antigenic stimulation →Seems to occur more with soluble antigens ∙Undergoes receptor editing →Receptor editing appears to preferentally target light chains

Immunity/Prevention

Immune clearance is mediated by opsonizing IgG, which allows for more efficient phagocytosis Immunity short lived and incomplete so individuals can be re-infected Cutaneous infections can recur over many years Good hand washing is best prevention

Bacterial Chromosome

In general: one copy (haploid), circular, dsDNA (majority) ∙Some >1, some linear Encodes for essential and non-essential genes Looped and supercoiled to fit the cell ∙No nucleus No histones, no introns

Bacterial Catabolism

In increasing order of efficiency, bacteria can produce energy by: ∙Fermentation ∙Anaerobic respiration (nitrate, sulfate, CO2 used in place of O2 as terminal electron acceptor) ∙Aerobic respiration

Tetracycline Pharmacokinetics

Incomplete PO, chelated by di- & trivalent ions, i.e. antacids (Al, Mg, Ca, Zn, Fe), dairy products → decrease PO absorption Wide distribution; some CNS (25%), fetus and milk/nursing (50-60%) Excreted primarily unchanged in the urine via glomerular filtration, accumulates in renal failure

Hypertrophy

Increase in size of cell and organ Not due to swelling but *due to synthesis of more structural components* Seen in non-dividing cells, ie permanent cells such as nerves, cardiac myocytes and skeletal muscle ∙An example of this would be going to the gym and lifting weights If you have systemic hypertension, your heart needs to work harder to pump blood to the body because the venous return is low ∙This will cause the heart to hypertrophy, and this is not desirable, and can have deleterious effects Nuclei have higher DNA content Caused by increased functional demand on cells or hormonal stimulation Mechanism of hypertrophy ∙Signal transduction pathways induce genes that stimulate synthesis of cellular proteins which help enlarge the cytoskeleton ∙Increase production of transcription factors lead to induction of proteins and increased production of growth factors ∙Induce genes that encode transcription factors , growth factors and vasoactive agents (all of which will increase the size of the cell) ∙May be a switch of contractile proteins from adult to fetal or neonatal forms ∙In skeletal muscle the alpha form of the myosin heavy chain is replaced by the beta form which has a slower, more economical contraction ∙Some genes that are only seen in early development may be re-expressed in hypertrophic cells ∙Hypertrophy may eventually cause cell injury or death if the stressor is not eliminated Image of hypertrophy ∙A = Hypertrophied uterus, post giving birth, on the left. The normal uterus is the smaller one on the right. ∙B = Normal smooth muscle cells ∙C = Hypertrophied smooth muscle cells *Sub cellular hypertrophy* ∙Smooth endoplasmic reticulum (sER) may become hypertrophied in response to barbiturates which is an adaptive response to increase enzymes such as cytochrome P-450 to detoxify the drugs resulting in the concept of tolerance →The sER adapts to the medication, and you need larger doses to feel the effect ∙Mitochondria may also undergo hypertrophy

Fluoroquinolones - 3rd generation: Pharmokinetics and Clinical Indication

Increased PO absorption, inc Vd, tissue conc > serum concentrations Longer t1/2, may allow once/ day dosing Spectrum: increased Gm +, esp. for streptococci; Gm (-) similar to Cipro; however Cipro generally considered most active against Gm (-) bacteria Levofloxacin, t1/2- 7 hr Moxifloxacin, t ½ - 10 hr

Antistaphylococcal Penicillinase-R (Resistant) Drugs

Indicated only for staphylococcal (i.e., gram + cocci) infections that are resistant, i.e. B-lactamase-producers (e.g., *nafcillin*) ∙Basically, these drugs are resistant to B-lactamase Used in initial therapy when resistance with these organisms is suspected ∙If cultures are negative for B-lactamase, switch to regular penicillin if practical Serious staph infections combined with aminoglycosides Dicloxacillin is the only antistaphylococcal penicillinase-resistant penicillin currently available in US

Stenotrophomonas maltophilia

Infection established by *major* changes in normal flora Settings for infection ∙Debilitated patient w/ impaired host defenses ∙Broad spectrum antibiotic therapy Clinical manifestations ∙Pneumonia & bacteremia ∙High incidence of complications and death ∙Contamination can be traced to hospital equipment, ice, etc. Uniformly resistant to carbapenems Susceptible to trimethoprim-sulfamethoxazole

Functions of fibrinogen

Indirectly effects ESR ∙It is the APP with the greatest effect on ESR ∙ESR definition given: if inflammation is happening, then there is more rouleaux, or stacking, of red blood cells, and the red blood cells fall at a faster rate when a mix of blood and anticoagulants is left in a vertical tube to settle for long periods →It is an indirect measure of APP →Increased aggregation of red blood cells (rouleaux) causes them to fall faster →Increased by high immunoglobulin concentrations →Red blood cell size and shape are also influential →Will increase progressively with age

Clinical considerations of enterococci

Infections in patients: ∙Elderly or debilitated ∙Mucosal or epithelial layer has been disrupted (i.e. catheterization) ∙Treated w/ broad spectrum antibiotics UTIs Nosocomial bacteria ∙Associated w/ localized infection or endocarditis *Bacterial endocarditis* ∙10-20% of cases on both native and prosthetic valves Peritonitis ∙typically polymicrobial

Clinical manifestations of P. aeruginosa

Infections of immune competent ∙Eye ulcers from extended wear of contact lenses ∙Wounds from punctures through tennis shoes ∙External otitis →Swimmer's ear →Malignant external otitis (diabetics & elderly) ∙Hot tub folliculitis Infections in immune compromised ∙Burn victims: cellulitis w/ blue-green pus ∙Septicemia: symptoms may include ecthyma gangerenosum and may lead to gram (-) shock and death ∙Repeated pneumonias in CF patients →Often cause of death in CF ∙Septicemia and pneumonia common in leukemic, transplant or neutropenic patients ∙UTI's in patients on long, indwelling catheters ∙Diabetics at risk for malignant otitis externa

Definition of Infectious Disease

Infectious diseases result from an encounter of a potential pathogen w/ a susceptible host in conjunction w/ a suitable portal of entry They carry the traits of life that seeks to perpetuate itself, evolving and trying to achieve equilibrium

Inflammatory actions of eicosanoids

Inflammatory actions of the arachidonic acid split products are responsible for all the signs and symptoms of inflammation See the above table for the predominant eicosanoids and their actions

Types of hemostatic inhibitors

Inhibitors that block clotting: ATIII, C1inh, alpha1-Protease inhibitor Inhibitors that block fibrinolysis: plasminogen activator inhibitors (PAI), plasmin inhibitors Inhibitors that shift reactions: TFPI inhibits tissue factor (TF)

Overview of Innate and Adaptive Immunity

Innate immune system engages against microbial infection before the adaptive immune system Innate immune system triggers and regulates the adaptive immune response The key cells that are engaged in an adaptive response are lymphocytes (B and T) Elements of the innate immune system often serve as effector (clearance) mechanisms for adaptive immune responses

DNA-damaged Mediated Apoptosis

Involves p53: ∙Accumulates when DNA is damaged (ionizing radiation) ∙Arrests the cell cycle at the G1/S boundary to allow for repair →If repair fails, p53 triggers apoptosis →If p53 absent or mutated, favors survival ∙p53 may up-regulate Bax, Fas and APAF-1 →Activate caspases and cause apoptosis

Internationally Reportable Infectious Disease

Is the public health impact of the event serious? Is the even unusual or unexpected? Is there a significant risk of international trade or travel restriction? Is there a significant risk of international spread?

Other effects of ischemia

Ischemia can lead to increased calcium levels in the cell ∙If this were to happen, damage to DNA, ATP, lipids, and cytoskeleton ∙Then you would have reperfusion, memory damage can occur in these areas

Functions of CRP

It enhances innate immune response and phagocytosis It can bind nuclear components of apoptotic or necrotic cells ∙In limited apoptosis, there is no great degree of leakage, so CRP is not usually needed ∙In high apoptosis and necrosis, however CRP is necessary and elevated Calcium dependent binding of phosphocholine (PCh) ∙PCh is present on lipid membranes of bacterial and fungal pathogens ∙It is not accessible for binding on human cells, but becomes available due to membrane changes in apoptotic or necrotic cells It may be found functionally bound by c1q ∙Will activate the classical complement pathway May help recruit phagocytes By binding Fc receptors Augments neutrophil and macrophage function

Ceruloplasmin (Ferroxidase 1)

It is a protective antioxidant It is an α2 globulin with 6 copper binding sites It binds copper very tightly, so it is not readily exchangeable It transports 90% of the copper in blood ∙Albumin is the other transporter which donates Cu more easily to tissues (same as with Zinc) Use Ceruloplasmin to sequester copper in the blood to regulate copper levels ∙Copper accepts and donates electrons in reactions involving dismutation, hydroxylation, and oxygenation ∙Excess copper can oxidize proteins and lipids, bind nucleic acids and enhance production of free radicals But increased ceruloplasmin 1 levels are associated with CV risk ∙It just slows the rate of damage to tissues but does not prevent the damage ∙Both antioxidant and pro-oxidant properties have been demonstrated in vitro ∙We don't know the true cause of CV disease yet and it is unclear whether ceruloplasmin is a marker for inflammation or has a role in development of atherosclerotic plaques Deficiency in ceruloplasmin or copper leads to iron build up in tissues

Haptoglobin

It is an α2 globulin is a positive APP is an antioxidant because it binds extracorpuscular hemoglobin ∙This is to sequester and decrease generation of free radicals It stimulates angiogenesis

The most severe type of free radical formation is Carbon Tetrachloride formation

It is extremely damaging, and you will have a decrease in apoprotein synthesis ∙So there will also be an accumulation of fat, cellular swelling, and accumulation of calcium You will also have massive liver destruction

Example of Gram stain of Enterobacteriaceae

Klebsiella pneumoniae is a member of Enterobacteriaceae All members of this family tend to stain more intensely at their ends ("bipolar" staining) This characteristic staining property is very useful for making a preliminary identification of these enteric bacteria

Plasminogen and apolipoprotein A both have kringle domains

Kringle domains are autonomous protein domains that fold into large loops stabilized by 3 disulfide linkages Bind to the clot so plasminogen/plasmin can digest it

Defects in selectins

L-selectin-deficient mice have poorly formed, small lymph nodes w/ few T cells Mice deficient in P-selectin or E-selectin have mild defects in leukocyte dysfunction Double knockouts are more severely impaired leukocytes and an increased risk of infection Humans who lack one of the enzymes needed to express the carbohydrate ligands for E-selectin and P-selectin in neutrophils have similar problems ∙Leukocyte adhesion deficiency 2

TLR4 Activation by LPS

LBP = Lipid A binding protein MD2 = myeloid differentiation protein 2 CD14 = cluster of differentiation 14 MYD88 = myeloid differentiation primary response gene (88)

Hinge region of immunoglobulin

Links Fc and Fab portions of the immunoglobulin molecule Allows for flexibility Molecular ball and socket joint

Phases of Bacterial Growth

Lag phase - metabolic activity increased in preparation for cell division ∙Cells grow in size, not in number Exponential (log) phase - maximum growth rate ∙Not the same for all organisms Stationary phase - equilibrium b/w cell division and cell death ∙Nutrients exhausted, waste builds Decline (death) phase - number of deaths exceeds number of newly formed cells

Lab diagnosis of Streptococcus pneumoniae

Lancet-shaped diplococcus Optochin sensitive Bile soluble (dissolves in bile) Quellung rxn to detect capsule Pneumococcal C polysaccharide can be detected in urine (immunoassay detection)

Components of LPS

Lipid A ∙Mediates endotoxic activity ∙Relatively constant structure in gram (-)'s ∙FA chain anchors lipid A in the outer membrane Core polysaccharide ∙Short series of sugars nearly the same in most gram (-)'s O antigen ∙Linear, hydrophilic carbohydrate chain ∙Excludes hydrophobic compounds and antibiotics ∙Highly variable, reason for different antigenic specificities among gram (-)'s (i.e. E. coli O157)

Leading Infectious Diseases

Leading killers are AIDs, TB and malaria

Clarithromycin: Mechanism of Action, Clinical Indication & Pharmacokinetics

Less GI irritation than ERY, same MA Gm (+) spectrum ~= ERY, but greater potency, t ½ 3-7 hr, adm. BID *Increased Gm (-) activity over others for Leg. pneumophila, C. trachomatis, Mycobacterium avium complex (AIDS); Helicobacter pylori, Borrelia burgdorferi* Forms active metabolite,14-OH in 1-pass metabolism *Inhibits hepatic drug metabolizing enzymes* Excretion primarily by urinary tract

Defects in integrins

Leukocyte adhesion deficiency: ∙Failure to synthesize β2 chains ∙Repeated bacterial infections

LPS

Lipid A portion of LPS can induce endotoxic shock ∙Fever, shock, hypotension, disseminated intravascular coagulation (DIC) ∙This is what binds to CD14, TLR4 → leads to activation of genes, etc. Lipid A and core is very similar in all gram negative bacteria O antigen, unlike other portions, can under antigenic mutation ∙Recognized by antibodies ∙Defines different serotypes of bacteria

Endotoxins of Gram Negative Bacteria

Lipopolysaccharide in the cell wall Liberated when bacteria lyse and/or release as part of membrane fractions (i.e. Neisseria meningitidis

Encountering S. aureus

Live on people, inanimate objects, fomites Colonization of skin and mucus membranes (MSCRAMMs) ∙Fibronectin-binding proteins (FnbpA and FnbpB) →Invasion of epithelial and endothelial cells →Attachment to fibronectin in wounds ∙CNAs (collagen adhesion) mediates collagen binding →Collagen in connective tissue, bones and joints ∙Clumping factors A and B →Cell bound coagulase →Bind fibrinogen →Important for clot formation

Flagella

Long helical protein filaments that endow bacteria w/ motility (tumbling or swimming) Antigenic (H antigen) ∙Recognized by TLRs Number of flagella varies w/ species Located at one end, both ends, or completely surrounds bacteria Only bacilli cells make flagella ∙CILLI DO NOT

Role of cadherins in cancer

Loss of cadherins can lead to a malignant phenotype ∙Allows easy disaggregation of cells ∙Local invasion ∙Distant metastasis Reduced cell surface expression of E-cadherin noted in cancers of: ∙Esophagus ∙Breast ∙Colon ∙Ovary ∙Prostate Germ line mutations of E-cadherin can predispose to familial gastric carcinoma

Life Cycle of Bacteriophage

Lytic (Virulent) ∙After binding → rapid replication: 20 min from infection to lysis ∙Leads to cell death and production of new (progeny) phage Lysogenic (temperate) ∙Infection leads to cell lysis or integration of phage DNA into the chromosome (prophage)

Order of complement activation in classical pathway

MEMORIZE C1, C4, C2, *C3*, C5, C6, C7, C8, C9 C5b-C9 represents the common terminal pathway for all three pathways

Tissue distribution of MHC

MHC Class I → all nucleated cells in body Class II ∙Some activated T cells ∙B cells ∙Macrophages/APC ∙Thymic epithelial cells

Different MHC isoforms bind different peptides

MHC polymorphism is largely confined to peptide binding cleft Reason for polymorphisms and co-dominant expression pattern ∙Peptide binding is selective, not antigen specific like antigen receptors on B and T cells ∙One isoform can bind many different peptides w/ same overall shape Non-polymorphic areas bind CD4 (Class II) or CD8 (Class I) co-receptors

Mononuclear phagocytes

Macrophages/monocytes ∙Monocytes circulate in the blood →These cells are larger than PMNs and have a distinctive indented or 'kidney-shaped' nucleus →They are progenitors of the tissue macrophage ∙Macrophages are differentiated tissue phagocytes arising from monocytes →These are long-lived irregularly shaped cells w/ extensive cytoplasm and numerous vacuoles →They are general scavengers and can dispose of dead cells and debris →Importantly, these cells process and present antigen to T cells via molecules encoded by the major histocompatibility complex →They also secrete many different cytokines that regulate other cell types (neutrophils and other leukocytes) →b/c of their regulatory function and their ability to process and present antigen, they are important bridge b/w innate and acquired immunity Dendritic cells ∙These star-shaped cells also arise from myeloid differentiation and share many properties w/ macrophages ∙They have a unique function - to initiate adaptive immune responses →They pick up antigen in infected tissue and move to lymphoid tissue, where they can interact w/ T cells and present antigen

Classification of Bacteria

Macroscopic and microscopic appearance ∙Characteristics on special media ∙Gram staining morphology Growth and metabolic properties ∙Metabolism (i.e. aerobic growth, lactose fermentation) Antigenicity Genetic properties/makeup Special features (i.e. spore formation)

2nd Generation Spectrum of Cephalosporins

Main feature is increased gram (-) activity including against beta-lactamase-producing H influenzae or K pneumoniae and penicillin-resistant pneumococci Gram (+) activity = slightly less effective against Bacteroides fragilis & most anaerobes (cefoxitin) Not active against P. aeruginosa

L-selectin

Major role: L-selectin is a homing receptor for lymphocytes to enter lymph nodes by binding to high endothelial venules Minor role: it also serves to bind neutrophils to cytokine-activated endothelial cells at sites of inflammation L-selectin is located at the tips of microvillus projections of leukocytes facilitating its interaction w/ ligands on endothelium At least three endothelial ligands can bind L-selectin ∙GlyCAM-1 (glycan-bearing cell adhesion molecule 1): secreted proteoglycan found on HEVs of lymph node ∙MadCAM-1 (mucosal addressin cell adhesion molecule 1) expressed on endothelial cells in gut associate lymphoid tissue ∙CD34 is a proteoglycan found on endothelial cells and bone marrow cells ∙Protein backbones of all these ligands are modified by specific carbohydrates which are molecules actually recognized by selectin

Prostatitis

Males < 35 with infection often due to C. trachomatis - fluoroquinolone e.g. ofloxacin BID 7 days, 6 weeks if chronic infection Older males infection usually Enterobacteriaceae (E. coli) or Pseudomonas or enterococcus ∙FQ usually Cipro or ofloxacin or TMP/SMX BID 10 -14 days →6 weeks - 3 months for chronic infections

Genetic Disorders of clotting

Many defects are not viable, while some show no symptoms The outcome is often bleeding Hemophilia A and B ∙X-linked disorders of coagulation ∙Mutations of clotting factor VIII (A) or factor IX (B) ∙Factor VIII is a cofactor and factor IX is the enzyme in the reaction to activate Factor Xa (same reaction) ∙Deficiency of either results in decreased clotting because there is less factor X ∙Characterized by bleeding into soft tissue, muscle and weight-bearing joints, hours to days after trauma and continuing for days to weeks ∙Management: intravenous replacement of the deficient factor

Chemical mediators

Mediators are all derived from cells These are the factors that were mentioned previously that can move through the endothelium with the changes in osmotic pressure Many of the factors are formed in the liver and delivered via the plasma Clotting is one of the first events to occur with damage to the vascular network ∙With vessel damage, the first priority is to clot and stop the bleeding Complement proteins are released early with microbe infection to result in destruction of the pathogen Cells make chemical mediators ∙Some of the mediators are preformed, such as histamine and lysosomal enzymes ∙Other mediators are newly synthesized, such as prostaglandins and leukotrienes →Once the arachidonic acid pathway begins, the newly synthesized mediators can be active in the environment →Cytokines produced initially will call in neutrophils, and then later call in more specific cells

Hemolysins

Membrane active toxins Pore forming toxins that bind to host cell membranes and polymerize to form pores that allow water and ions to pass → cell lysis Ex: Staphylococcus aureus alpha toxin: hexamer forms pore

Complement activation

Microbes activate the alternative pathway if the body hasn't encountered the organism before If the body has encountered the organism prior, the classical pathway will be activated. Preformed antibodies will be able to bind to the microbe The mannose binding lectin pathway is activated only when certain organisms enter the body What happens when complement is activated? ∙C3a and C5a are cleavage products of certain steps in the complement cascade. These serve to promote leukocyte activation ∙Phagocytosis of microbes ∙Direct lysis of microbes using the membrane attack complex (MAC)

Bacterial Growth

Microbial cells growth by doubling cellular constituents and dividing into two cells Division is asexual (binary fission) and continues in an exponential fashion ∙The explosiveness of exponential growth means that even a small number of bacteria can initiate infection ∙However, bacteria are usually stressed by nutritional limitations or damage by the immune system Time required for doubling is called the generation time

Urinary Tract Infections

Microbiology: E. coli (80%), Staph saprophyticus, Proteus, Enterococci, Klebsiella, Pseudomonas, other Gm negatives Treatment (PO); FQ; TMP/SMX:- 3 days treatment. In pregnancy usually 7 days (no FQ, no sulfonamides near term) Kidney infections usually 14 days treatment, hospitalized usually get IV therapy

Normal Flora (Normal Microbiota)

Microorganisms frequently found in or on the body of health persons (usually bacteria) Inhibits the establishment of pathogens Stimulates local and systemic immune responses Aids in digestion and the production of vitamins (i.e. Vitamin K) Can become pathogenic in immunocompromised, or when removed from usual anatomic nice ∙Ex: E. coli in the gut are pathogens in the urinary tract

Result of Mutations

Missense mutation → changes in amino acid sequence of the protein product Frame shift mutation → shift in the reading frame of the gene Inversion mutation → segment of DNA is removed and reinserted in reverse direction

What properties of the organism are involved in the disease process?

Molecular Koch's postulates (newer) ∙Trait should be associated more often w/ pathogenic strains of a species than non-pathogenic strains ∙Inactivation of the gene(s) associated w/ trait should decrease or eliminate virulence ∙Virulence should be restored when mutated gene is replaced w/ wild-type gene

Antigen receptors on T lymphocytes

Most CD4+ and CD8+ T cells bear the 'α/β' TCR A minority of T cells bear the γ/δ TCR ∙Limited diversity in antigen specificity ∙Recognize non-peptide antigens w/o processing and presentation via MHC ∙Microbial antigens? ∙May be an important first line of defense in skin and gut

Treatment of S. aureus

Most S. aureus is resistant to penicillin due to β-lactamase Drain abscess and treat w/ β-lactamase resistant penicillins and cephalosporins (i.e. nafcillin) ∙Effective for methicillin sensitive strains (MSSA) Use vancomycin for MRSA ∙Treat VRSA w/ linezolid, synercid and daptomycin Anti-toxin antibodies protect against TSS, SSSS and staphylococcal food poisoning ∙Consider protein synthesis inhibiting antibiotics to treat toxin mediated disease

Inhibitors of proteases (including clotting enzymes)

Most are serine protease inhibitors - "serpins" Produced by liver Circulate in the blood Either inactivate or "trap" protease enzymes "suicide substrate"- complex of inhibitor and clotting factor tightly bound which is then cleared from circulation All serine proteases have a central triad ∙Includes Factor XIII, XI, IX, X, II, VII, plasmin, complement, C1r, C1s, elastase, trypsin, and many other ∙1/3 of all known proteases are serine proteases Serpins are irreversible inhibitors because they fit into the serine active site resulting in a complex which is stable and does not dissociate ("suicide substrate")

Clotting Factors

Most are synthesized in liver Most are enzymes that require Ca2+ for their function and some of them require Vitamin K ∙Ca2+ acts as a bridge between clotting factors and negatively charged surface ∙Certain clotting factors have extra carboxyl groups to aid in binding to surface and some others need a carboxyl group added to create a double negative charge (vit K) ∙Adding gamma-carboxyl to glutamate residues ('Gla') requires cofactor vitamin K →Factors II (thrombin), VII, IX, X, protein C and protein S are vitamin K dependent ∙Vitamin K is oxidized during the reaction and must be reduced before the next reaction →Normally occurs via a reductase in the liver →Warfarin (Coumadin) blocks the reductases and inhibit the recycling of vitamin K and consequently reducing clotting A few of them are cofactors (not all enzymes) Active form denoted with "a" (X—>Xa)

Minocycline: Pharmacokinetics and Clinical Indication

Most lipid soluble tetracycline, 100% PO bioavailability, wider distribution, esp. to CNS, longest t1/2 Undergoes significant metabolism, mostly excretion of metabolites in urine and feces Especially effective in treatment of acne (Propionibacterium acnes) due to increased penetration of skin Used to eradicate meningococcal carrier state, 200 mg/5 days /PO

Skin infections caused by S. aureus

Most localized staphylococcal infections lead to the formation of a collection of pus called an abscess Folliculitis - pyogenic infection in hair follicle ∙Stye if in base of eyelid Furuncle - extension of folliculitis ∙Large, painful, underlying dead and necrotic tissue (abscess) Carbuncles - multiple interconnected boils that extend into deeper tissues ∙Fever and chills point to systemic spread

Pseudomonas aeruginosa

Most medically important strain of Pseudomonas Ubiquitous, tolerates a wide range of temps Fast growing, requires minimal nutrients for growth Infections are primarily opportunistic ∙i.e. important pathogen in individuals w/ cancer, cystic fibrosis, and burns

Intrinsic (Mitochondrial) Pathway

Most of cytochrome c is sequestered in the intermembrane space ∙Blc-2 is the regulatory factor that prevents release of cytochrome c This pathway is not induced by a receptor ligand interaction, but by multiple stimuli to the nucleus or cytoplasm ∙Apoptosis inducing factors are up-regulated by intrinsic pathway damage (hypoxia or DNA damage) Bcl-2 is removed from the mitochondrial outer membrane of mitochondria to cause the release of cytochrome c and Apaf-1 (Apoptosis Protease Activating Factor 1) Cytochrome c and apaf-1 associate with pro caspase 9 ∙Pro caspase 9 will autocatalyze, active itself to caspase-9 ∙Executioner caspases are released Bcl-2 family ∙There are about 20 proteins in this family and they all regulate apoptosis ∙Pro-apoptotic members of the Bcl-2 family →Include Bax and Bak ∙Normally reside in mitochondrial membranes and in the cytosol →When cells are deprived of survival signals or are under stress, these proteins are lost from the mitochondrial membrane →They are replaced by Bax and Bak ∙Main ones that are anti-apoptotic →Bcl-2 →Bcl-x ∙Decrease in Bcl-2 and Bcl-x levels, increases the permeability of the mitochondrial membrane, causing leakage of proteins that can activate caspases →The best known protein is cytochrome c Cytochrome c ∙Important for role in mitochondrial respiration ∙Result of increased mitochondrial permeability and release of pro-apoptotic molecules into the cytosol →No known role for death receptors ∙Once out in the cytosol, cytochrome c binds to apaf-1 →Complex of apaf-1 and cytochrome c activates caspase 9 →Bcl-2 and Bcl-x may also directly inhibit apaf-1 activation ∙Other mitochondrial proteins such as apoptosis inducing factor (AIF) can enter the cytosol →Bind to and neutralize inhibitors of apoptosis ∙Net effect: initiation of the caspase cascade Growth factors stimulate the production of anti-apoptotic members of the Bcl-2 family ∙The intrinsic pathway balances pro- and anti-apoptotic molecules that: →Regulate mitochondrial permeability →Release of death inducers that are normally sequestered in the mitochondria ∙There is evidence that the intrinsic pathway can be triggered without the mitochondria →Not well-defined →May also be overlap between the extrinsic and intrinsic pathways (may not be distinct) →Ex: Hepatocytes Fas signaling activates pro-apoptotic bid which activates the mitochondrial pathway

Staphylococcus aureus

Most pathogenic species of Staph genus Unique lab characteristics ∙Coagulase (bond & free) → converts fibrinogen to fibrin →Bound aka clumping factor ∙Ferments mannitol →Yellow color on mannitol-salt agar indicates fermentation of mannitol ∙Blood agar → golden colonies, beta-hemolytic ∙Protein A → binds Fc portion of IgG Epidemiology ∙Humans are a major reservoir →Anterior nares of 30-90% of healthy →Transiently: skin, oropharynx, vagina, feces ∙Carriage rate higher in hospitals, persons w/ skin diseases, or those who use needles regularly (i.e. diabetics) ∙Leading cause of nosocomial infections →Surgical wound infections and primary bacteremia ∙Community acquired infections (CA) →Skin and soft tissue infections →Respiratory infections →Infective endocarditis (IVDU's) ∙CA-methicillin resistant S. aureus (CA-MRSA) →Groups = prisoners, athletes, drug users →Risk factors = poor hygiene, close contact, contaminated material, damaged skin

Processing of exogenous antigen for presentation on Class II MHC

Most peptides that are destined for presentation on MHC Class II molecules (to CD4+ cells) are capture and internalized into endosomes by antigen presenting cells (dendritic cells, mononuclear phagocytes, B cells) ∙Macrophages and dendritic cells use scavenger and related receptors to bind and internalize extracellular particles that recognize structures common to many microbes ∙Macrophages also can bind antigen via C3b or Fc receptor ∙B cells bind antigen via surface immunoglobulins, so binding is antigen-specific After internalization, proteins are localized into endosomes (intracellular vesicles with an acid pH that contain proteolytic enzymes) • Particulate matter is internalized via phagosomes. ∙Each of these structures can interact or fuse with lysosomes, which are more dense, enzyme containing vesicles ∙Many different enzymes participate in breakdown of antigen (cathepsins, thiol and aspartyl proteases) ∙Class II molecules are synthesized in the ER, where they couple with a protein called the invariant chain, which blocks peptide binding within the peptide binding cleft of MHC Class II →Importantly, binding of the invariant chain prevents binding of peptides to Class II MHC in the ER. ∙MHC Class II are exported from the ER into exocytic vesicles, where they are targeted to late endosomes and lysosomes →Fusion of the exocytic vesicles with these compartments brings together the MHC Class II proteins with processed peptides derived from extracellular proteins →These fused structures, called MHC Class II compartments (MIIC), contain proteolytic enzymes, Class II HMC, the invariant chain and HLA-DM ∙The invariant chain is acted upon by proteolytic enzymes leaving only a small remnant called the Class II-associated invariant chain peptide (CLIP) →CLIP sits in the peptide binding groove in the same way as a peptide would →→→The groove becomes vacant through the action of HLA-DM, which appears to catalyse the removal of CLIP →Once vacant, it appears that the peptide binding groove is filled with peptide and that HLA-DM may facilitate this process ∙Class II MHC are stabilized by the bound peptide and these stable complexes are delivered to the surface of the cell

Genetic analysis of bacteria for identification

Most precise method of classification Highly conserved sequences identify a genus and highly variable sequences identify species or subspecies ∙Ex: E. coli O157:H7 ∙Genus: Escherichia ∙Species: coli ∙Subspecies: O157:H7 Methods: DNA hybridization, plasmid analysis, ribotyping, analysis of chromosomal DNA fragments

Toxoid Immunization

Most toxins invariant (mono-polymorphic) Mild denaturation by treatment w/ formaldehyde (formalin) or heat inactivates toxin activity, but not immunogenicity → toxoid (used to raise immunity against toxin Current toxoids: diphtheria, tetanus

Mechanisms of Changes in Genome

Mutations can occur spontaneously as a result of errors in DNA replication (most common) or by mutagens ∙Point mutations ∙Deletion, replacement, insertion or inversion of many bases (may involve recombination)

Metaplasia

NEED TO KNOW EXAMPLES Reversible change in which one differentiated cell type is replaced by another cell type May represent an adaptive substitution of cells that are sensitive to stress by cell types better able to withstand the stress ∙This isn't always a good thing! Most common type involves the transformation of columnar to squamous cells as seen in the respiratory tract in smokers. This occurs of a period of years, but is reversible when the stimulus is removed. Elderly patients may not be able to reverse these processes. This allows for a more rugged cell to replace the more delicate cells However the replacement cells often lack the functional properties of the original cells, this can lead to pathologic in the long term. Barrett's Esophagus - The lower 1/3rd of the esophagus, which normally has stratified squamous epithelium, is replaced with columnar epithelium with goblet cells. This is due to chronic gastroesophageal reflux disease (GERD). The columnar cells are better equipped to handle the acid compared to the squamous cells. Mechanisms of metaplasia ∙Results from the reprogramming of stem cells which exist in normal tissues or of undifferentiated mesenchymal cells present in connective tissue ∙The precursor cells differentiate along a different line as a result of the signals generated by cytokines, growth factors and ECM components in the cells' environment ∙The stimuli promote expression of genes that drive cells toward a specific differentiation pathway Multiple images ∙Epithelial cartoon - The reserve cells are stimulated to change into squamous cells in a chronic smoker, for example. ∙Histological slide shows columnar cells on right side, squamous on left ∙Depending on stimuli, the mesenchymal cells can differentiate into a variety of other cells

Nitric Oxide

NO is a product of nitric oxide synthase (NOS) acting on L-arginine NO is released from endothelial cells to relax vascular smooth muscle and promote vasodilation NO prevents interactions between leukocytes and endothelial cells. It keeps leukocytes moving in the vessel There are 3 forms of NOS ∙Endothelial (eNOS)- This is a product of endothelial cells →It maintains normal homeostasis ∙Neuronal (nNOS)- Will not be discussed in this lecture ∙Inducible (iNOS)- This is active in the macrophage and creates RNS (reactive nitrogen species) NO function ∙NO is an endogenous compensatory mechanism that reduces inflammatory responses →Primarily through eNOS action ∙NO production is enhanced in response to microbial infection →Primarily through iNOS action and the production of RNS ∙NO and derivatives are microbicidal →Genetic inactivation of iNOS results in enhanced microbial replication (observed in experimental animal systems)

Quinolones - 1st Generation

Nalidixic acid and Cinoxacin (older drugs); limited usage ∙Used infrequently for lower Urinary Tract Infections (UTIs) for common Gram (-) organisms, e.g. E. coli Resistance can develop fairly rapidly to these drugs within a few days Rapidly absorbed, rapidly excreted ADRs: rash/GI disturbances

Natural vs. specific immunity

Natural immunity ∙Innate defense mechanisms present prior to exposure to infectious microbes or other foreign macromolecules ∙First lines of defense (w/in hours) ∙Not specific for any particular agent ∙Evolutionarily more primitive ∙No memory ∙Important point: innate immunity against microbes is very important in stimulating adaptive or specific immune responses Specific immunity ∙Evolutionarily more advanced ∙Usually responsible for resolving infection ∙Slower (days to weeks) ∙Often confers life long immunity (memory) ∙Usually specific for a particular organism Image shows recognition structures (receptor) on cells involved in innate and specific immune rxns

Apoptosis vs. Necrosis

Necrosis ∙Danger activated molecular patterns (DAMPs) stimulate different molecules intracellularly ∙Activation of inflammation and may lead to an apoptotic response Apoptosis ∙Trigger cascade activation, get blebs, and rapidly internalize ∙There is a fine line between apoptosis and uncontrolled cell death, which can lead to intercellular break down →In order to not trigger inflammatory response, DAMP is inactivated Limited Inflammation ∙Uptake of apoptotic cells has been shown to result in the release of: →IL-10, TGFb, and PGE2 →These are anti-inflammatory mediators Caspases coordinate the inactivation of DAMPs during apoptosis ∙Caspase activity, downstream of MOMP, directly and indirectly leads to the inactivation and/or sequestration of multiple endogenous DAMPs →IL-33 →Genomic DNA →ATP →HMGB1

Types of Cell Death

Necrosis (always pathologic): Cellular swelling, protein denaturation, and organellar breakdown, lysosomal enzymes enter cytoplasm and digest cell causing its contents to leak out ∙There is a risk of an inflammatory response Apoptosis (pathologic or physiologic): Programmed cell death, noxious stimuli damage DNA with nuclear damage without loss of membrane integrity!

Ischemic and hypoxic injury

Need to know difference b/w reversible and irreversible Reversible injury ∙Acute cellular swelling ∙Depleted glycogen ∙Reduced intracellular pH ∙Reduction in protein synthesis Irreversible injury ∙Increased calcium in mitochondria ∙Damage to plasma membranes ∙Swelling of lysosomes ∙Leakage of intracellular proteins ∙Mechanisms of irreversible injury →Loss of membrane phospholipids →Cytoskeletal abnormalities →Toxic oxygen radicals buildup →Lipid breakdown products MUST KNOW DIAGRAM ∙Ischemia leads to a lack of oxygen, thus a decrease in oxidative phosphorylation and ultimately a decrease in ATP ∙You would do a blood test in order to look for elevated levels of enzymes or other compounds that shouldn't normally be in the blood ∙After an irreversible injury, these enzymes leak out of the cell

Netilmicin/Amikacin: Clinical Indications

Netilmicin more resistant to microbial enzymes, used in gentamicin resistance, otherwise it is comparable to these drugs Amikacin exhibits the greatest resistance; used in gentamicin & tobramycin resistance ∙Amikacin less vulnerable to microbial enzymes because of protective side chain substitutions, more $$$

Granulocytes (polymorphonuclear leukocytes, PMNs)

Neutrophils ∙Can capture, engulf and kill microorganisms ∙Contain reactive substances that kill microorganisms and enhanced inflammation ∙Can be rapidly mobilized to enter sites of infection ∙Can work in anaerobic environments ∙Form pus Basophils ∙Least abundant of the granulocytes Eosinophils ∙Defends against helminthes and intestinal parasites

Fosfomycin: Mechanism of Action, Clinical Indication & ADRS

New bactericidal drug approved for UTIs due to common Gm( +)/GM (-) urinary pathogens Administer PO, single dose Excreted unchanged in urine Blocks peptidoglycan synthesis ADRs = GI → diarrhea, headache, vaginitis

Linezolid: Mechanism of action, clinical indication and ADRs

New class: oxazolidinones PO 100% (advantage) and IV Blocks early stage of protein synthesis, binds ribosomal RNA of 50S subunit *Indicated for MRSA, Vancomycin resistant Enterococcus faecium / E. faecalis (VREF), bacteriostatic* Bacteriocidal for streptococci, including those resistant to penicillins/cephalosporins Adverse: n/v/diarrhea, headache, rash, dec platelets/anemia with prolonged treatment MAO inhibitor, caution with sympathomimetics

Escherichia coli

Nonpathogenic strains are normal flora in the colon Most common gram (-) isolated from patients w/ sepsis Uropathogenic strains are leading cause of UTI ∙Have pathogenicity islands that encode for special features that allow them to survive well in urinary tract to cause UTI K1 capsule (along w/ Group B strep) is associated w/ neonatal meningitis ESBL in some strains ∙ESBL = extended-spectrum beta lactamase, an enzyme produced by beta-lactam resistant bacteria

Cellular responses to stress and injury

Normal cells are in a state of homeostasis If the cell is disturbed by various influences the cell can undergo biochemical and morphologic changes. The change can be biochemical, or observable through microscopy or grossly. Cell injury may be reversible or irreversible. The injury can begin as a reversible change and become an irreversible change. This depends on the cell type and the extent of the damage. Irreversible changes usually lead to cell death. The cell may undergo various adaptations to compensate for disturbed homeostasis Cell death is the end result of progressive cell injury and has many causes both physiologic and pathologic. Stressors may induce cellular changes resulting in intracellular accumulations. There are a variety of stressors, and we'll talk about them. Some of them are due to an accumulation of pigments, fats, other lipids, etc.

Epidemiology/Pathogenesis of Streptococcus pneumoniae

Normal flora of throat and nasopharynx, infection can be endogenous or exogenous Highest incidence in children and the elderly b/c of low levels of protective antibodies

S. epidermis

Normal flora, most abundant species on skin Catalase (+), coagulase (-), non-hemolytic, novobiocin sensitive Virulence: slime/biofilm formation allowing adhesion to plastic surfaces Clinical disease: nosocomial infections associated w/ plastic prosthetic devices, shunts, grafts and catheters

Regulation of APPs

Not coordinated Changes in concentration are highly variable among different APPs ∙Some APP concentrations go up/down by only a little while others increase/decrease by thousands Inflammatory cytokines such as IL-6, IL-1β, and TNFα play important roles in APP changes ∙Transcription is the primary mode of regulation ∙Post-transcription and translational changes are seen also but to a lesser extent than transcriptional level changes

The genes that encode the immunoglobulin heavy and light chains in their 'germline' or urrearranged state

Note that each heavy chain locus, in its germline configuration, contains multiple 'V' gene segments (~100, but estimates vary), about 23 "D" segments and about 6 'J' segments ∙On the 3' end, there are various gene segments that encode various the constant domains associated w/ the various isotypes or classes of immunoglobulin In the genes encoding the kappa and lambda light chains, you will note that there are again multiple 'V' and 'J' gene segments, but no 'D' gene segments ∙You will also note that the 'J' and 'C' segments are ordered differently b/w kappa and lambda gene loci

Virulence factors of S. aureus

Note: S. aureus possess an antiphagocytic microcapsule

Review: Structure of antigen receptor proteins

Now notice that the heavy chain protein contains only 1 V region, only one D region and only one J region in the variable domain This means that only one gene segment for each is actually transcribed ∙How does this happen? ∙When during development do those rearrangements occur in DNA?

B cell differentiation in bone marrow: Pre B cells

Occurs following productive rearrangement of one heavy chain locus ∙Productive rearrangement leads to synthesis of m heavy chains and halts rearrangement of the second locus (*allelic exclusion*) ∙Marks the transition to the pre-B cell stage of differentiation There are two phases of pre-B cell development ∙Large pre-B cells ∙Small pre-B cells Large pre-B cells are less mature, ∙m is complexed with a surrogate light chain ∙Some go to the cell surface ∙Most remain in the ER ∙Surrogate light chains composed of: →VpreB (like the variable region) →l5 (like a constant domain) ∙Expression of the pre-B cell receptor is an important checkpoint in B cell differentiation ∙Confirms productive rearrangement of the heavy chain gene ∙Presence of a pre-B cell receptor halts rearrangement of the other heavy chain locus and synthesis of surrogate light chains ∙Large pre-B cell proliferates and yields more mature small pre-B cells *Small pre-B cells*: ∙No pre-B cell receptor at the cell surface (instead m is restricted to the cytoplasm) ∙Rearrangement of the light chain proceeds, rearrangement of the k chain gene is attempted first ∙If rearrangements of both k chain genes fail, then the l chain genes are rearranged (this is why most immunoglobulins in the body contain k light chains) →Molecular mechanisms of rearrangement are very similar to those used for heavy chain rearrangements →Once there is successful rearrangment of a light chain gene, IgM can be made

Coagulase Negative Staphylococci

Often referred to as CoNS Strains: ∙Staphylococcus epidermis ∙Staphylococcus saprophyticus ∙S. lugdunensis & S. schleiferi

Endothelial molecules and their roles

P and E selectin expressed on the endothelium interact with Sialyl-Lewis X protein on the leukocytes ∙These selectins are expressed on endothelial cells to promote rolling of the leukocytes along the endothelium GlyCams interact with selectins ICAMs and VCAMs interact with integrins for more permanent binding The point of this table is to illustrate that both the endothelial cell and the leukocyte must be activated to allow for extravasation to occur

Serum Amyloid A (SAA)

One of the largest APPs It is part of a family of apoplipoproteins ∙Some are acute phase reactants and some are constitutive SAA contains an α helix with β pleated sheets SAA is elevated during inflammation and may be increased higher than CRP ∙Like CRP, it is regulated transcriptionally by induction by cytokines →IL-1 and IL-6 are the predominant regulators It serves as a functional global marker of inflammatory events ∙It is a precursor to amyloid A protein, a component of secondary amyloid plaques →Note that secondary plaques are not the same as those in Alzheimer's disease →SAA does not have a direct effect, but will influence mediators and sequester microorganisms ∙When present, SAA causes altered properties of acute HDL →Enhancing uptake of HDL by macrophages →→→This would seem to be protective because this increased uptake decreases the foam cells indicative of the plaques, but over time atherosclerotic plaques still will build up and cause issues →Cleared from the circulation more rapidly →5x increased binding to macrophages →There may be a possible role for SAA in reverse cholesterol transport from macrophages during acute inflammation →→→SAA reduces storage of cholesterol esters in macrophages →→→Decreases number of foam cells

Gram negative cell wall

One thin layer in periplasmic space Cross-linking different and less than gram positive cells Resistant to lysozyme and less susceptible to cell wall antibiotics than gram positive b/c of outer membrane

Minor pathogens

Opportunistic & nosocomial pathogens Antibiotic resistance is a problem Citrobacter koseri ∙Similar to Salmonella ∙Meningitis and brain abscesses in neonates Enterobacter cloacae & Serratia marcescens ∙similar to Klebsiella ∙normal flora in the GI tract; also in soil ∙have been associated with UTI & pneumonia (Serratia) in hospitalized patients Providencia rettgeri & Morganella morganii ∙similar to Proteus ∙produce urease; cause UTIs in patients in LTCF's

Atrophy

Opposite of hypertrophy Can also be physiological or pathologic Shrinkage of the size of a cell due to loss of cell substance May culminate in cell death, not always, but it may. Net result is the decrease in size of an organ Physiologic: during fetal development some embryonic structures undergo atrophy, uterus atrophies after child birth - you don't want it to be that large after giving birth Pathologic: local or generalized from decrease workload, loss of innervation, decreased blood supply , inadequate nutrition, pressure on cells or tissue ∙An example is a cervical/spinal cord injury ∙The nerves will not regenerate, so the muscles that were innervated/stimulated by those damaged nerves, will rapidly atrophy Mechanism of atrophy ∙Autophagy: cell digests it's own contents by using lysosomes ∙Ubiquitin-proteosome pathway: ubiquitin attaches to cellular proteins targeting them for destruction in proteosomes which degrade the cytoskeleton (the skeleton of the cell that gives it its structure) Image ∙The brain on the left is atrophic, while the one on the right is the control ∙Can see that on the left there is a decrease in the size of the gyri and an increase in the size of the sulci ∙This is indicative of something like Alzheimer's disease ∙Upon autopsy, a brain less than 1000 grams, may have undergone an atrophic process

Major functions of complement

Opsonization Direct lysis of target cells/pathogens Chemoattraction and activation of inflammatory cells

Contact w/ an organism does not always lead to disease

Organism may be eliminated via defense mechanisms Can become a carrier of organism, as it becomes part of the normal flora

Toxicity of Aminoglycosides

Ototoxicity - cochlear (esp. amikacin) and vestibular (esp. streptomycin and gentamicin), hair cell damage, degeneration 8th nerve, esp. in combo with loop diuretics/other ototoxic drugs; *contraindicated in pregnancy* Nephrotoxicity - increased BUN, proteinuria, increased serum creatinine, inability to concentration urine, adjust dose to creatinine clearance *Common cause of drug-induced renal failure* Neuromuscular blockade - respiratory difficulties, decreased Ach release & receptor blockade, with anesthetics/neuromuscular blockers, but can be attenuated with calcium salts Superinfections Usually low incidence of allergy, immediate reactions rare

Metabolic signature to identify bacteria

Oxygen requirements ∙Some bacteria have large O2 requirements, some cannot live in presence of O2 Substrate utilization ∙Ex: lactose or non-lactose fermenter Fermentation end products: acid, gas, alcohols, etc.

Pathogenesis/Clinical Considerations of P. aeruginosa

P. aeruginosa can infect almost any body tissue ∙Wound infections ∙UTI ∙Sepsis ∙Pneumonia Usually requires alteration in host defense ∙Trauma/surgery ∙Urinary tract catheterization ∙Leukemia/neutropenia ∙Chronic lung disease (i.e. CF, patients needing ventillary assistance) Encounter and Entry ∙Ubiquitous, found mostly in moist environments ∙Infections of immune compromised initiate at the site of compromise →i.e. catheter associated UTI → bacteremia ∙Large numbers of organisms needed to cause infection in immune-competent Adherence ∙Pili - adheres best to injured cell surfaces ∙Flagella - flagellin binds to cells →Flagellar cap attaches to mucin ∙LPS - outer core binds to cystic fibrosis transmembrane conductance regulator (CFTR) ∙Alginate coat of mucoid strains - enhances adhesion to cells and mucins Evasion of host defenses ∙Inhibition of phagocytosis - attached bacteria inject toxins via a type III secretion system into phagocytic cells →Exo S and T: ADP-ribosylate target proteins →ExoT - interferes w/ internalization by epithelial cells and macrophages →Exo-U - phospholipase that lyses host cells (associated w/ severe disease) ∙Killing of phagocytes: Exotoxin A & leukocidin ∙Degradation of host effector molecules: multiple elastases Tissue Injury ∙Type III secretion toxins: probably acts locally ∙Exotoxin A: Inactivates EF2 thru ADP-ribosylation ∙Elastases: degrades elastins →Lung damage and ecthyma gangrenosum ∙Phospholipases: hydrolyzes phospholipids ∙Pyoverdin: siderophore that binds iron ∙Pyocyanin: catalyzes production of superoxide and hydrogen peroxide Inflammatory components ∙Inflammatory response to components most important factor in disease causation ∙Sepsis syndrome and septic shock due to inflammatory response to Lipid A (TLR4) and flagellin (TLR5) ∙Cell death during disease → release of cellular components (e.g. heat shock proteins) → activation of TLR system

Treatment of P. aeruginosa

P. aeruginosa is highly resistant to most antibiotics ∙Sensitivity testing required Can mutate to more resistant strain during therapy Monotherapy ineffective and can select for resistant strains Mutation of porin proteins the major mechanism of resistance Produces beta-lactamases

Lipid A is a Pathogen-Associated Molecule Pattern (PAMP)

PAMP's are molecular motifs conserved w/in a class of mirobes that are recognized by pattern recognition receptors (PRR's) of the innate immune system PAMP's of bacteria ∙Lipopolysaccharide (LPS), lipoteichoic acid, lipoproteins, mycobacterial lipoarabinomannan ∙Peptidoglycan, flagella ∙DNA (has unmethylated CpG motifs not in mammalian DNA, toxins

Doxycycline: Pharmacokinetics and Clinical Indications

PO bioavailability almost complete, food/dairy does not significantly dec bioavailability, metal ions do, esp. iron The most widely used tetracycline, long t1/2, usually once/day or BID administration Excreted mostly by nonrenal mechanisms, via bile > feces as inactive conjugate or chelate, the only tetracycline that does not accumulate in renal insufficiency, therefore dosage adjustment not required Activity against Chlamydia trachomatis, anthrax, cholera, Lyme disease, Rocky Mountain Spotted fever (Rickettisiae) Doxycycline + Cephalosporin against anaerobic bacteria

Whether or not disease results depends on:

Pathogenicity (virulence) of pathogen Number of infectious bacteria (# needed for infection not the same for all bacteria) Host factors (i.e. immune status, genetic defects, medications, etc.)

Eukaryotic vs. Prokaryotic Cells

Peptidoglycan is an important target in antibiotic therapy ∙Least toxic b/c they do not have target in eukaryotes Antibiotic therapy is based on differences b/w prokaryotes and eukaryotes

Periplasmic space

Peptidoglycan w/ little to no cross-linking Contains: ∙Hydrolytic enzymes to break down large macromolecules for metabolism ∙Binding proteins for uptake of metabolites ∙Virulence (collagenases, proteases) and antibiotic inactivating enzymes

Peptidoglycans as target for antibiotic therapy

Peptidoglycans are unique to bacteria, so their synthesis is a good target for antibiotics Transpeptidases involved in crosslinking of peptidoglycan bind penicillin so they are called penicillin binding proteins (PBPs)

Suppurative Manifestations of S. pyogenes

Pharyngitis - most common bacterial cause of exudative pharyngitis in children ∙Nonsuppurative complications: ARF and PSGN ∙Scarlet fever exanthema associated w/ expression of Spe A, B, or C Impetigo (pyoderma) - superficial infection of skin Erysipelas - form of cellulitis ∙Involves skin and subcutaneous tissue ∙Includes systemic signs (usually preceded by respiratory or skin infection w/ S. pyogenes) Necrotizing fasciitis - destruction of muscle and fat ∙Streptococcal gangre (necrosis and obstructed blood flow) ∙Hallmarks - toxicity, multiorgan failure, death ∙Strep implicated in 60% of necrotizing fasciitis cases Streptococcal Toxic Shock Syndrome ∙Associated w/ strains producing Spe A or C, M serotypes 1 or 3, prominent capsule (mucoid strains) ∙Frequently associated w/ necrotizing fasciitis and bacteremia ∙Criteria available for diagnosis ∙Aggressive medical intervention indicated →>30% of cases are fatal

Elements of natural immunity

Physical barriers ∙Skin • Breached by physical damage (wounds, burns, surgery) ∙Mucosal surfaces • Line respiratory, GI and urogenital tracts • Bathed in mucus Chemical barriers ∙pH of stomach, vagina, skin ∙Degradative enzymes ∙Tears (lysozyme) ∙Saliva ∙Digestive enzymes and bile in the small intestine Normal flora ∙Skin ∙GI ∙Vagina Mechanical barriers ∙Flushing ∙Tears, urination ∙Coughing Fever ∙Elevation in body temperature that exceeds the normal daily variation and occurs in conjunction with an increase in the hypothalamic set point →> 37.2oC (98.9oF) in AM (oral) →> 37.7oC (99.9oF) in PM (oral) ∙Caused by pyrogens →Exogenous →→→Microbial toxins, products or the organisms themselves →→→LPS →Endogenous (pyrogenic cytokines) →→→IL-1 →→→IL-6 →→→IFN-α →→→TNF-α ∙These cytokines act on the immune system, but also on many other tissues including: →vascular endothelium →hypothalamus →muscle →fat ∙Alter metabolic processes to generate heat →Decreases bacterial and viral replication →Alter leukocyte/lymphocyte migration patterns →Antigen processing enhanced →Specific immune processes become more potent Cellular elements of natural immunity ∙Phagocytic cells ∙Mononuclear phagocytes ∙Neutrophils

Apoptosis vs. Necrosis

Picture refers to the differences between apoptosis and necrosis Necrosis ∙The cell membrane slowly starts to break apart ∙Then the intracellular accumulations and enzymes have access to the blood ∙The cell has enzymatic breakdown of the cellular membrane ∙Always pathologic Apoptosis ∙The cell membrane remains intact ∙Fragments start to break off "like leaves off of a tree" and forms these little apoptotic bodies, each with an intact cell membrane ∙These are targeted by macrophages/phagocytes and is destroyed ∙Pathologic or Physiologic

Main Virulence Factors of P. aeruginosa

Pili and flagella can mediate motility as well as adhesion in this particular organism

How to identify changes in APPs?

Plasma electrophoresis ∙Serum is taken from a patient and placed in a gel electrophoresis chamber to separate the proteins in the sample ∙Based on size and charge, we get a distribution of globulin bands ∙Height of the bands indicates amount of the protein within the sample ∙The farther the bands travel from the start point, the smaller and more positively the molecule Image: Distribution can be seen as the series of bands or a graph showing relative amounts of globulins present ∙Albumin has the largest spike and thus is in the largest quantity in this sample →Provides the most protein and osmotic pressure to the plasma

Thrombolytic therapy

Plasminogen activators (PAs) are approved by FDA for thrombotic diseases ∙Streptokinase (SK) ∙Urokinase ∙Alteplase (tissue plasminogen activator (t-PA)) ∙Recombinant t-PA They are usually delivered directly to the clot via catheters (not used in pulmonary embolism, PE)

Proteolysis of the clot

Plasminogen is cleaved by plasminogen activator (PA) to plasmin (a protease) Plasmin breaks down the clot Fragments of the clot are removed through the blood

Superantigens

Powerful polyclonal T-cell mitogens Stimulates non-specific T-cell responses ∙20% of APC's stimulated vs. 1/10^5-10^6 (normal clonal response) Binds to MHC class II molecule and T-cell receptor outside of antigen binding sites Leads to improper immune response and a toxic cascade of cytokines

Variable Domain/Region of Immunoglobulin

Present in heavy and light chains Antigen binding Contain highly variable hypervariable regions, and less variable framework regions

Antigen processing and presentation: General rules

Preference of MHC classes for different subpopulation of T cells ∙CTL (CD8+) recognize antigen in context of Class I MHC ∙Helper T cells (CD4+) recognize antigen in context of Class II MHC Size of peptide ∙8-25 AA ∙Class I appear to associate w/ shorter peptides (8-10 AA) b/c they are anchored Preference for endogenous vs. exogenous peptide ∙MHC Class II - exogenous (for presentation to CD4+) ∙MHC Class I - endogenous (for presentation to CD8+) MHC can bind self peptides

Colonization

Presence at a site in the body Establishment at site can be mediated by: ∙Specific receptor mediated interactions ∙Nonspecific interactions

von Willebrand Disease (vWD)

Presentation: nose bleeds, skin bruises, hematomas Most common hereditary coagulation disorder vWF required for adhesion and to protect Deficiency of vWF means that Factor VIII is also deficient because it is involved in the regulation of Factor VIII vWF: a multimeric protein produced by endothelial cells Because many clotting factors are produced in the liver, having a clotting disorder may be indicative of a liver disorder ADAMTS-13 is produced by hepatocytes (liver) ∙A Disintegrin And Metalloproteinase with ThromboSpondin-1-like domains ∙Cleave vWF under high shear stress ∙Major regulator of vWF's final size in plasma ∙Protects against uncontrolled platelet adhesion ∙Congenital deficiency found in patients with TTP (thrombotic thrombocytopenia purpura) →Due to lack of ADAMTS-13 →Without ADAMTS-13 equilibrium and size of the von Willebrand clumps are not maintained causing thrombosis

E-selectin

Previously known as endothelial leukocyte adhesion molecule 1 (ELAM-1) Expressed on cytokine activated endothelial cells Recognizes complex sialylated carbohydrates groups ∙Related to the Lewis A or Lewis X family Ligands are found on surface proteins of: ∙Granulocytes ∙Monocytes ∙Previously activated effector and memory T cells E-selection is important in the homing of effector and memory T cells to some peripheral sites of inflammation, particularly in the skin Endothelial expression of E-selections is the hallmark of acute cytokine mediated inflammation

Lymphoid organs can be primary or secondary

Primary ∙Thymus ∙Bone marrow Secondary (peripheral) ∙Tonsils, adenoids ∙Spleen ∙Lymph node ∙Mucosal surfaces

Three critical arms of prevention

Primary - education and disease prevention Secondary - early identification of disease in patients at risk Tertiary - reduction of illnesses in patients w/ diseases

Tumor Necrosis Factor and Interleukin-1

Primary mediators of endotoxemia Produced by macrophages when stimulated by endotoxin Also referred to as endogenous pyrogens (fever) Systemic release leads to adverse effects ∙Local release → containment and removal of infection ∙Systemic release → hypotension, DIC, systemic shock

Host response to S. aureus

Primary responses is recruitment of PMN's Anti-capsular and anti-MSCRAMMS facilitate opsonization and protect against infection (in vitro and animal studies)

Cytokines in inflammation

Pro-inflammatory cytokines are IL-1 and TNF Many chemokines are produced in acute inflammation, mainly by neutrophils and resident macrophages ∙Chemokines will recruit leukocytes to the site of inflammation Chronic inflammation is associated with lymphocytes

IL-1 and TNF

Pro-inflammatory factors you need to know: IL-1 and TNF Microbial products or cytokines activate macrophages. This results in production of IL-1 and TNF TNF and IL-1 can have local or systemic effects ∙Systemic effects include fever and leukocytosis ∙Local effects occur on the vascular endothelium, leukocytes, and fibroblasts →Examples of local effects are expression of leukocyte adhesion molecules, up-regulation of production of IL-1 and chemokines (chemotactic cytokines), and an alteration in coagulation events

Cadherins

Probably the most important class of proteins that mediate cell-to-cell attachment, at adherens junctions (which are anchoring sites for actin filaments) and at desmosomes (which are anchorage sites for intermediate filaments) Cadherins take their name from the calcium dependence of binding Two classes of cadherins: ∙Classical cadherins (all closely related to each other) →N-cadherin (expressed on nerve, muscle and lens cells) →P-cadherin (cells in the placenta and epidermis) →E-cadherin (many types of epithelial cells) ∙Non-classical cadherins →More distantly related in terms of sequence →More than 50 expressed in the brain alone →Include proteins w/ adhesive properties →→→Protocadherins (brain) →→→Desmocollins (form desmosome junctions) →→→Desmogleins (form desmosome junctions) →→→Proteins w/ signaling function (T-cadherin is expressed in nerve and muscle cells) Cadherins mediate hemophilic (like-to-like) adhesion ∙Specific cadherins on one cell bind to same (or closely related) cadherins on the second call ∙Binding probably occurs at the N-terminal tips ∙Protein knob and pocket structures →Cadherins have b/w 5-30 repeated extracellular domains called 'cadherin domains' →This is where the next knobs are →Each cadherin domain is joined to the next cadherin domain by a flexible hinge region →Calcium binds near the hinge and prevents it from flexing, so that the whole chain of cadherin domains behaves as a slightly curved, rigid rod →*When calcium is absent, the hinge flexes and the structure becomes floppy* ∙Knob from one fits into pocket of other, and vice-versa ∙Binding is of relatively low affinity ∙Spacing b/w cell membranes at an anchoring junction very tightly defined →Depends on which cadherins are expressed ∙Strength of interaction increases b/c of multiple molecular interactions →Think 'velcro' Intracellular domains of cadherins provide anchorage for filaments of the cytoskeleton ∙Anchorage to actin at adherens junctions ∙Intermediate filaments at desmosome junctions ∙Linkage to cytoskeleton is indirect ∙Depends on intracellular anchorage proteins that assemble on the tail of the cadherin ∙In general, a central part is played by a protein called β-catenin

S. lugdunensis & S. schleiferi

Produce more serious infections than other CoNS Clinical disease: native-valve endocarditis and osteomyelitis (but more commonly associated w/ endocarditis of artificial valves)

Cholera toxin (ctx)

Produced by Vibro cholerase An enterotoxin encoded on a pathogenicity island, acquired by horixontal gene transfer through a bacteriophage A2-5B ∙B: binds GM1 in small intestine ∙A: ADP ribosylates, activates adenylate cyclase Increased cAMP levels → secretion of fluid and electrolytes into intestine → very watery diarrhea

Diphtheria toxin

Produced by toxigenic Corynebacterium diphtheria Prophage encoded One A subunit, one B subunit ∙A subunit: ADP-ribosylase that transfers ADP-ribose from NAD to elongation factor (EF-2), and inhibits protein synthesis

Proteus mirabilis

Produces large quantities of urease ∙So pronounced it can change pH of urine ∙Very clinically relevant Swarming motility → VERY MOTILE Community & hospital-acquired UTIs (about 70% of UTIs) ∙Common cause of UTIs behind E. coli ∙Urease produces ammonia →Alkalinization & precipitation of Ca2+ and Mg2+ salts contributes to formation of kidney stones

Necroptosis

Proinflammatory form of cell death initiated by receptor-interacting protein kinases (RIPK1 and RIPK3) May be inhibited by caspase-8

Klebsiella pneumoniae

Prominent capsule - anti-phagocytic Community and hospital acquired infections Clinical disease ∙Necrotic pneumonia (primary lobar) ∙Pneumonia produces a thick, bloody sputum referred to as "current jelly" sputum ∙Alcoholics can be particularly vulnerable ∙Multidrug resistant strains a problem (ESBL)

Regulation of C3 in the alternative pathway

Properdin ∙Increases the speed and power of complement activation by binding the C3 convertase C3bBb on the pathogen surface ∙Prevents its' degradation by proteases Factor H counteracts properdin ∙Facilitates cleavage of C3b to iC3b by plasma factor I ∙iC3b cannot form the C3 convertase Factor I deficiency ∙Formation of the C3bBb (C3 convertase) is unchecked until depletes reservoir of C3 ∙When faced with bacterial infection: Less C3b deposited ∙Individuals are more susceptible to ear infections and abscesses caused by encapsulated bacteria →These infections cleared more efficiently when opsonized by C3b Another type of regulatory proteins prevent complement activation on human cell surfaces ∙Decay accelerating factor: DAF binds to C3b in the alternative C3 convertase, causing dissociation and inactivation ∙Membrane co-factor protein: MCP binding to C3b makes it susceptible to action of Factor I (so it's similar to Factor H)

Acute inflammation

Protective, normal response Short duration → minutes to several hours to a few days ∙It takes time for the endothelial cell layer to reorganize and change surface marker expression →Permitting fluid from the endothelial layer to the tissues requires minimal changes →Promoting transmigration of leukocytes requires more modifications to the endothelial layer to make room for the large cells to pass through If it lasts longer than a week, we are moving into primary immune response ∙If adaptive immunity cannot resolve insult/injury, chronic inflammation will occur Associated with: ∙Exudation of fluid and plasma proteins (edema) ∙Emigration of leukocytes (in particular neutrophils)

Biochemical features of apoptosis

Protein cleavage by caspases which drive a lot of the portions of the apoptotic cascade Blebs recognized by phagocytes DNA breakage

Biochemical features of apoptotic mediators

Protein cleavage through activation of caspases targeting: ∙Lamins ∙Nuclear scaffold ∙Cytoskeleton ∙DNAses DNA breakdown in a specific pattern Phagocytic recognition at plasma membrane

Antibodies

Protein that binds specifically to an antigen Produced by plasma cells (terminally differentiated B cell) in response to infection or immunization Binds to and neutralizes pathogen or prepares for destruction by phagocytes or by complement

IgE

Provides a mechanism for rapid *Ejection* of pathogens from body Little circulates in plasma Most binds via FcR (FcRe1) on: ∙Mast cells (connective tissue) ∙Basophils (blood) ∙Eosinophils (mucosal tissue) Cross-linking of receptor leads to release of mediators ∙Acts on smooth muscle to cause 'violent' expulsion of pathogen ∙Sneezing, vomiting, diarrhea Same mechanisms work in allergies and asthma ∙b/c your body mistakes Fluffy's dander for a parasite

Pathogenesis of S. aureus: General considerations

Pyogenic disease: abscess formation at local and/or metastatic sites Immune response: intense ∙PMNs w/ influx of macrophages and fibroblasts Immune response will contain, or infection can spread to adjoining tissue or blood Toxigenic disease: may or may not involve viable bacteria

Opportunistic pathogens

Rarely cause disease in healthy hosts Regularly cause disease in compromised hosts ∙Burn victims susceptible to infection w/ Pseudomonas aeruginosa ∙HIV patients more susceptible to intracellular bacteria (i.e. mycobacteria) ∙Hospitalized patients on broad-spectrum antibiotics (i.e. Clostridium difficile)

How do phagocytes recognize pathogens?

Recognition can be direct or indirect Direct recognition involves surface receptors on the phagocyte surface that interact directly with the pathogen ∙Toll-like receptors ∙C-type lectins →Mannose receptors ∙Scavenger receptors →Pathologic role in the generation of cholesterol-laden foam cells in atherosclerosis →Recognize and mediate uptake of microbes into phagocytes Indirect recognition involves phagocytic binding to host proteins that have bound to the pathogen ∙Antibody (via phagocyte Fc receptor) ∙Complement (via phagocyte C3b receptor) ∙others

4 Cardinal Signs of Inflammation

Redness (from release of RBCs) Swelling (edema) Heat ∙Activation of many cells at local environment ∙Also produce factors that have global influence, so may influence body temp Pain

Role of β-catenin in cancer

Related to germline mutations in another protein called APC (adenomatous polyposis coli): ∙APC represents a class of tumor suppressor genes whose main function is to down-regulate growth promoting signals ∙Germline mutations in APC are associated w/ familial adenomatous polyposis: individuals born w/ one mutant allele develop thousands of polyps in the colon in the late teens/early 20's ∙One or more these polyps undergoes malignant transformation (colon cancer), which is associated w/ loss of both APC copies ∙70-80% of non-familial colorectal carcinomas and adenomas also have loss of both APC copies ∙mutations in β-catenin also present in 50% of hepatoblastomas and 20% of hepatocellular carcinomas Molecular mechanism ∙APC is an important component of the WNT signaling pathway, which has a major role in controlling fate, adhesion and polarity of cells during development and is also important in hematopoiesis ∙An important function of APC is to down-regulate β-catenin ∙In absence of WNT signaling, APC forms a complex w/ other proteins and degrades β-catenin, thus preventing its accumulation in the cytosol ∙When WNT binds to its receptor, this destruction complex is blocked ∙This allows β-catenin to move to the nucleus, where it complexes to a transcriptional factor that up-regulates cell proliferation ∙If APC is deleted, there is no degradation of β-catenin and cell proliferation is unchecked

Apoptosis and Alzheimer's disease research

Research shows unaffected cells can be triggered to apoptosis Affected cells protect the host cell and won't trigger apoptosis

Acute phase proteins are involved w/ major functions of the blood

Respiration ∙Transport of oxygen from the lungs to the tissues and of CO2 from the tissues to the lungs Nutrition ∙Transport of absorbed food materials Excretion ∙Transport of metabolic waste to the kidneys, lungs, skin and intestines for removal Maintenance of the normal acid-base balance in the body Regulation of water balance through the effects of blood on the exchange of water between the circulating fluid and tissue Regulation of body temperature by the distribution of body heat Defense against infection by the white blood cells and circulating antibodies Transport of hormones and regulation of metabolism Transport of metabolites Coagulation During apoptosis, there are changes to the phospholipid membrane and a number of these factors will associate with those changes and promote internalization

Patterns of Acute Cell Injury

Reversible cell injury ∙Cellular swelling is seen ∙Fatty change can be seen, such as a fatty liver after alcohol ingestion Necrosis ∙Loss of the cellular wall integrity, which would result in leakage of material into serum, such as enzymes ∙This elicits an acute inflammatory response with neutrophils ∙There would be lipid breakdown and enzymatic digestion of the cell ∙Intracellular materials leave the cell and what is left is digested enzymatically *Morphology of Necrosis* ∙There are different types of necrosis, each type with a different morphology ∙Increased eosinophilia: Refers to a pinkish change to the cell's cytoplasm, not a leakage of eosinophils ∙Myelin figures: Some accumulations of phospholipids are found in the cells themselves ∙There are three types of nuclear changes - KNOW THESE →Karyolysis is where the nuclei are broken down into small fragments →Pyknosis refers to the nucleus being shrunken down, and it looks like a raisin →Karyorrhexis refers to the nucleus breaking into large fragments →The nucleus only undergoes one of these changes, not all three

Cell Injury and Death

Reversible cell injury: injury leaves the cell with functional and morphologic features that are reversible, causes decreased oxidative phosphorylation and decreased ATP Irreversible injury: cell exhibits morphologic changes in response to a noxious stimulus that are recognized as cell death, functional changes are prominent at this point Causes of cell injury ∙Hypoxia - leads to decreased aerobic respiration, cell may adapt or die. Ischemia is a lack of blood flow (which also stops oxygen delivery to tissues). The effects are similar. ∙Physical agents: mechanical trauma, extremes of temperature, radiation, electric shock ∙Chemicals and Drugs: legal and illicit drugs, poisons, & ETOH →Alcohol can be a result of death due to liver damage, driving accidents, etc; it is very common ∙Microbiolgic agents: virus, bacteria, parasites, protozoans ∙Immunologic Rxns: anaphylactic reactions, autoimmune diseases. Over many years of this, real damage can occur. ∙Genetic Defects: chromosomal abnormalities, inborn errors of metabolism ∙Nutritional imbalances: malnutrition, obesity, hyperlipidemia →This is common in the United States now →For the first time in our country's history, people being born now may not have the same life expectancy as their parents ∙Aging: cellular senescence. Mechanisms of Cell Injury ∙Cellular responses to injurious stimuli depends on type of injury, its duration and severity ∙Consequences of cell injury depend on the type, state and adaptability of the injured cell. The brain is more sensitive to lack of blood flow than skeletal muscles, for example. ∙Cell injury results from functional and biochemical abnormalities of cellular components

Execution of apoptosis

Role of caspases ∙Nuclear targets with proteins involved in: →Transcription (c-Myc and NF-kB-inhibited) →DNA replication →DNA repair poly ADP-ribose polymerase (PARP) ∙Disassembly of cell structure: →Nuclear lamina (keratins 18, 19 and vimentin) →Elements of the cellular cytoskeleton →→→B-catenin-disrupting cell-cell interactions →→→Fodrin and gelsolin-disrupting the actin filament network ∙Caspase 3 activates endonuclease: →DNA fragmentation factor 45 (DFF45) →DFF45 in turn activates DFF40, which plays a critical role in the internucleosomal DNA degradation →Mitochondrial substrates - disrupts electron transport →→→Loss of mitochondrial transmembrane potential after 4-8hrs Role of Endonuclease ∙DNA breakdown ∙Cleaves in internucleosomal space regions →First into large fragments (50-300kb), then into multiples of 180 base pairs →This is a regulated breakdown of the nuclear material ∙Forms a ladder on agarose gels

Selectins mediate transient cell-to-cell adhesions in the bloodstream

Selections are cell surface carbohydrate-binding proteins (lectins) ∙All selections are single chain transmembrane glycoproteins w/ an amino terminus that is related to carbohydrate-binding proteins called C-type lectins ∙Binding is calcium dependent Family of three closely related proteins that differ in their cellular distribution ∙L-selection (CD62L): white blood cells (lymphocytes and other leukocytes) ∙P-selection: platelets and activated endothelial cells in an inflammatory response ∙E-selection (CD62E): activated endothelial cells

Azithromycin: Mechanism of Action, Clinical Indication & Pharmacokinetics

Same MA, less GI irritation / HCL destruction Gm (+) strep activity ~ same as ERY, alternates to B-lactams for pharyngitis, sinusitis, community-acquired pneumonia Increased Gm (-) H. influenzae, N. gonorrhoeae, Mycoplasma pneumoniae, Legionella pneumophila, B. burgdorferi, Chlamydia Long t1\2 (2-3 days), initial loading doses may be indicated, adm once/day Primarily biliary/GI excretion unchanged; urinary minor, similar to erythromycin

Triple sugar iron agar (TSI Slants)

Screening test for gram (-) enteric organisms ∙Ferment glucose and/or lactose or sucrose ∙Produce gas as a by product of metabolism ∙Reduce sulfur to form hydrogen sulfide gas →H2S reacts w/ iron to form iron sulfide (black) →Requires acid environment, only occurs if fermentation has occur Slant itself ∙Oxygen-poor butt ∙Oxygen-rich slant ∙Glucose, lactose, & sucrose →[glu] < [lac & suc] ∙Phenol red (pH indicator) →Red = alkaline →Yellow = acid

Tissue factor pathway inhibitor

Secreted from endothelial cells TFPI acts as a serine protease inhibitor (Serpin) TFPI causes shifts so thrombin formed from extrinsic coagulation will feedback to activate the intrinsic coagulation side

Characteristics of Exotoxins

Secreted or membrane bound and released upon lysis Specifically destroy or inhibit cellular functions or tissue components Vary in specificity (neurotoxin, cytotoxin, enterotoxin) Many (but not all) possess enzymatic activity

Lancefield Grouping

Serological (A-W), based on major cell wall carbohydrates Originally developed to differentiate beta-hemolytic strains System inadequate Currently used to identify few medically important strains (A, B, C, D, F, G)

Conjugation

Transfer of DNA from a donor to a recipient cell Mediated by conjugative plasmid (F plasmid, fertility plasmid) that encodes for transfer genes and sex pili Requires initiate cell contact Plasmid transfers from F+ (male) to F- (female) cell ∙Result = 2 F+ cells

Antigenicity to identify bacteria

Serotyping: strains of bacteria can be distinguished using antibodies that detect characteristic antigens on their surface Good example - throat swab for strep throat

Patterns in acute and chronic cell injury

Serous inflammation will result in serous fluid accumulation ∙Serous fluid is straw colored, clear fluid that is protein poor ∙Seen in blisters from grabbing something hot, fluid accumulates under epithelium Fibrinous: You will have vascular permeability and accumulation of fibrin, associated with acute inflammation ∙Appears string-like Suppurative: accumulation of neutrophils, most commonly seen in bacterial infections ∙This occurs when you haven't really beaten the infection but it is walled off and the accumulation of dead and dying neutrophils is really an abscess Ulceration: Refers to the erosion of epithelial surface, exposure of underlying blood vessels ∙Example is a stomach ulcer, exposure to too much gastric acid, or maybe not enough mucous, so you have damage and erosion ∙Also seen in bed sores

Acquisition of Iron for Multiplication

Siderophores: compounds produced by bacteria that bind ferric ions for transport into the cell Some bacteria have membrane proteins that bind lactoferrin and/or transferrin to scavenge iron from host

Membrane-active, other mechanisms

Staphylococcus aureus exfoliative exotoxin ∙Protease that causes erythema and intradermal desquamation by disrupting epidermal cell-cell junctions ETEC ST toxin (plasmid encoded) ∙Heat stable, bind membrane bound guanylate cyclease → increase cGMP → watery diarrhea

Slime layer

Similar to capsule, but can cover other organisms around it An extracellular polymeric substance (EPS) composed of extracellular DNA, protein and polysaccharide

Examples of Non-Specific Adherence

Slime layer/glycocalyx promotes biofilm formation on teeth, prosthetic devices, damaged heart valves, etc. Copious capsule of Klebsiella facilitates retention in the urinary tract

Plasmids

Small (1.5-120kbp) circular supercoiled DNA 1-5 copies/cell, large plasmids ∙10-20 copies/cell, small plasmids replicate independently of chromosome ∙inherited by daughter cells conjugative plasmids encode transfer enzymes and sex pili for transfer to other cells nonconjugative plasmids-lack transfer genes but can transfer if in cell w/ conjugative plasmid Plasmids may carry genes that provide resistance to antibiotics (R-plasmids) or make proteins that act as toxins or other pathogenic factors

Cytokines

Soluble, hormone-like molecules Secreted by cells of the immune system Act on cells by binding to specific receptors Influence cellular activity Proliferation Activation of a particular function

Targets of Injurious Stimuli

Some are seen microscopically, some are seen functionally Mitochondrial aerobic respiration (thus a decrease in ATP) The integrity of the genetic apparatus ∙This can be the basis of neoplasms/cancer if one cell has the right mutation and replicates The integrity of the cell membranes ∙When compromised, the contents will be lost to the ECM Protein synthesis, a very complicated process. The cytoskeleton, can lead to compromise of the membrane

Invasion

Some bacteria bind and then enter host cells Facultative intracellular bacteria: invasion of cells is part of pathogenesis Obligate intracellular bacteria: invasion of cells is required for growth/survival

Coagulation necrosis

Sometimes referred to as coagulative Early on in coagulation, you have preservation of the outline of the cell You will lose that eventually The intracellular components and proteins are denatured and broken down If you look microscopically, you will see a "ghost of the former cell" An example of this would be a myocardial infarct (IMAGE) ∙On the left, the cells are intact, you can see the cell outline intact and you can see the striations ∙On the right, you can see increased eosinophilia, cell becomes dark and the outline is still intact →You can no longer see the nucleus or striations →You can see neutrophils if you look closely ∙This is seen antemortum, and this helps show how long it has been since someone had an MI →If someone died within minutes of an MI, you wouldn't see much of a change in the tissue →If someone dies after a lingering MI, you can see the MI evolving in the tissue

Ultrastructure of neutrophil granules

Specific granules are useful in cleaving the basement membrane and in combating microorganisms ∙Some of these granules are released into the tissue and others are contained in phago-lysosomes →Azurophilic granules are typically not released into the tissues

Cardinal Features of an Adaptive Immune Response

Specificity ∙An immune response is specific for distinct antigens ∙Function of distinct antigen receptors on the lymphocyte surface ∙Antigen receptors present before exposure to antigen ∙Basis on the CLONAL SELECTION THEORY Diversity ∙Total number of antigenic specificities of lymphocytes in an individual (lymphocyte repertoire) is LARGE ∙Individual can discriminate at least 1 billion different antigens ∙Result of incredibly variability in the antigen receptors among lymphocytes Adaptability ∙Ability to respond to antigens not previously encountered in nature Memory ∙Exposure of the immune system to foreign antigen enhances its ability to respond again ∙Secondary immune responses are: →More rapid →Bigger →Qualitatively different Clonal expansion ∙Increases number of antigen-specific lymphocytes to keep pace w/ microbes during an infection Specialization ∙Generates responses that are optimal for defense against different types of microbes Self-limitation (contraction and homeostasis) ∙Immune responses wanes w/ time ∙Elimination of antigen ∙Effector cells short lived ∙Regulatory feedback elements of the immune response Discrimination of self from non-self

Medially important superantigens

Staphylococcus aureus Toxic Shock Syndrome Toxin (TSST-1) Staphlyococcus enterotoxins (SEA, SEB, SEF) Streptococcus pyogenes pyrogenic exotoxins (SPE-A, B, C) ∙Toxic shock syndrome-like ∙Responsible for rash seen in scarlet fever

New coagulation model is a cell based model which recognizes that all clotting factors are interrelated and cell surfaces are important for clotting factor activation

Step 1: initiation (activation) ∙TF bearing cells are exposed to blood ∙Once TF is exposed, active factor VIIa will bind to TF ∙Active factor VIIa/TF complex will activate factor X which is in the blood stream in its inactive form ∙Small levels of active factor V (activated by small levels of thrombin) is present in blood and binds to factor X →Factor V also converts of pro-thrombin to thrombin via prothrombinase →→→Only very small levels of thrombin made during initiation ∙TF and VIIa binding also activates a significant amount of factor VIIII which makes a significant amount of factor IX ∙Role of TF in initiation of clot formation: →Tissue factor is found both outside and inside the blood vessels →→→TF is an integral membrane protein, normally expressed by extravascular cells (especially heart, lungs, testis, uterus, placenta) →→→Separated by primary hemostatic barrier around all blood vessels, but following injury, rapidly activates blood coagulation →Also present in blood in encrypted form, circulating on monocyte-derived micro particles ('dust') →→→Not clear how TF becomes 'unencrypted' →→→Encrypted = unactivated Step 2: amplification: amplification of clotting factor activation ∙Thrombin produced on tissue factor-bearing cells activates platelets and other clotting factors ∙Enough thrombin is made to hit the threshold to activate factor VIII ∙vWF also is protective to enhance the half life of factor VII ∙Once factor VIII is activated it can no longer bind to vWF ∙Activates factors XI and V ∙Feed forward affect Step 3: propagation ∙Occurs on the surface of the platelets ∙During the amplification step there is an increase in activated platelets and clotting factors with high levels of Tenase (VIIIa-IXa) →Tenase is when active factores VIIIa and IXa come together in a complex →Tenase activates Xa →This causes a large burst of thrombin formation and eventually the thrombin clot ∙Surge of active factor X and V which complex together and form prothrombinase which converts prothrombin to thrombin →Enzyme is factor X and the cofactor is factor V →Factor Xa cuts the prothrombin to activate it →Factors V is a cofactor, not an enzyme and is activated by thrombin to Va →It binds to receptors on the platelet surface and helps to properly align all of the components and is inactivated (Vi) by activated protein C (APC) Thrombin cleaves fibrinogen to form fibrin monomer forming the soft clot Thrombin also activates factor XIII (fibrin stabilizing factor) which stabilizes the mesh for a period of time allowing it to cross-link

Leukocyte activation

Stimulation of leukocytes can occur in response to: ∙Bacterial peptides, lipid mediators, chemokines, microbes, phagocytic signals The stimulation of the leukocyte stimulates intracellular responses such as: ∙Production of cytokines ∙Production of reactive oxygen species Toll like receptors respond to basic microbial patterns such as LPS

Cellular Aging

Structural and biochemical changes with aging include oxidative damage, abnormally folded proteins If your antioxidants decrease then your defense mechanisms breakdown In replicative senescence, there is a limited amount of dividing for every cell Accumulation of genetic damage Exposure to carcinogens DNA repair mechanisms and apoptotic genes don't function as well Damage accumulates over time

Virulence factors of S. pyogenes

Structural components ∙M protein: blocks C3b binding ∙M-like proteins: bind Fc portion of antibody ∙Hyaluronic acid capsule: weak immunogen, antiphagocytic ∙Forms biofilms ∙Adhesion proteins: LTA, M protein, F protein ∙Invasion of epithelial cells: M and F protein Extracellular products ∙Spreading factors: streptokinase, DNase, protease ∙Streptolysin S and O: damage cell membranes and accounts for hemolysis ∙Streptococcal pyrogenic exotoxins (SpeA, SpeB, SpeC) →Produced by lysogenic strains; diphtheria toxin →Act as superantigens →Responsible for rash associated w/ scarlet fever →Linked to severe invasive infections

Spectinomycin: Clinical indications

Structurally related to aminoglycosides *Only used as alternative to treat gonorrhea in patients allergic to pens/cephs or where gonococci are resistant to other drugs (i.e., fluroquinolones)* *May also be used during pregnancy* Single IM injection/2 grams, pain @ injection site

C Reactive Protein (CRP)

Structure = homopentameric molecule ∙It is five non-covalently associated proteins arranged symmetrically around a central pore

Biochemical Classification of Enterobacteriaceae

Substrates used or metabolites they secrete ∙Lactose fermentation →Preliminary step in the identification of Gram negative enteric rods ∙Resistance to bile salts →Separates Shigella & Salmonella (more resistant) from commensals Bacteria can be identified by acid or alcohol fermentation products

Pre T cells

Successful rearrangement of the TCR β chain gene marks the transition to pre T cells Also CD4-/CD8- (double negative) β chain is expressed at the cell surface complexed w/ an invariant protein called pre-Tα (this is called the pre-T cell receptor) pre T cell receptor now expressed w/ associated signaling proteins (CD3 complex and ζ chains Successful expression of pre-T cell receptor stimulate rearrangement of the α chain (accompanied by expression of RAG-1 and RAG-2

B-Lactamase Inhibitors

Suicide molecules, bind / inhibit lactamase enzymes (e.g., clavulanic acid, sulbactam, tazobactum) Different inhibitors combined with extended spectrum penicillins, see handout (e.g., piperacillin-tazobactam) Basic antibacterial spectrum usually the same or slightly increased, hopefully the inhibitor ties up the lactamase so that the antibiotic is not inactivated

Clinical diseases of S. pyogenes

Suppurative infections = produces pus Non-suppurative infections

Treatment of Enterococci

Synergistic combination of antibiotic therapy (aminoglycoside and a cell wall active antibiotic) Can use vancomycin, however, VRE exist

Trimethoprim/Sulfamethoxazole

Synergistic combo inhibits sequential steps in folic acid synthesis Drugs have similar spectrum and t1/2, trimethoprim 20 times more potent Resistance develops quickly when either drug is used alone Enhanced efficacy, combination often bacteriocidal with increased effectiveness against bacteria Not effective for enterococci, Pseudomonas aeruginosa, or anaerobes

MHC Restriction

T cells cognize processed peptide antigen present by products of the HC locus w/in a given individual, each clone of T cells recognizes peptides ONLY in the context of a particular MHC isoform this is called MHC restriction

Identification of medically important species

THIS IS FOR GRAM (+) COCCI Catalase test differentiates staph from strep Among staph, coagulase (+) vs. coagulase (-) (aka CoNS) ∙Most important coagulase (+) = S. aureus ∙For CoNS, differentiates using novobiocin sensitivity test

Thrombopoietein (TPO) targets megakaryocytic for platelet synthesis

TPO = Hematopoietic growth factor ∙a cytokine and a hormone which specifically targets megakaryocytic Made in liver, some in kidney Proposed 50 years ago, but wasn't purified until 1994 Synthetic Recombinant human thrombopoietin (rHu-TPO) may be way to increase the number of platelets in cancer patients receiving chemotherapy ∙Alternative to harvesting platelets from other patients or donors Potential regulation: platelets can remove TPO from circulation

Gram staining procedure

Take a sample, smear it on a slide, and then heat fix it Stain w/ a crystal violet solution ∙All of the bacteria on that slide will take up that stain Flood the slide w/ iodine solution that complexes w/ stain to help it stay on slide Rinse off excess Decolorize w/ acetone ∙Gram positive cells, due to structure of cell wall, will retain crystal violet ∙Gram negative cells will release stain and become colorless Apply counterstain (safranin, red stain) to be able to see gram negatives In the end, the gram positives will be purple and the gram negatives will be red

Aminoglycosides: Microbial Resistance

Take home: tobramycin is most vulnerable, amikacin has the most resilency

Formation of platelet plug

Temporarily blocks blood flow out of injured vessel Concentration of platelets at the site of injury is necessary for coagulation Begins first but continues through the rest of the "coagulation cascade" ∙Thrombin activates platelets ∙Platelets accumulating at site of injury continues to occur as the rest of the clotting cascade continues platelet recruitment to site of injury ∙Collagen is exposed and platelets adhere directly to collage via glycoprotein IV ∙Platelets adhere to damaged vessel and undergo degranulation and release cytoplasmic granules which contain serotonin, a vasoconstrictor, ADP and thromboxane A2 →ADP attracts MORE platelets to be attracted to the site →Thromboxane A2 promotes platelet aggregation, degranulation and vasoconstriction →Positive feedback promotes the formation of a platelet plug ∙Von Willebrand Factor (vWF): produced by endothelial cells which is normally folded, but in the presence of vessel injury (sheer stress) it exposes collagen in vessels when it unfolds and binds to glycoprotein Ib receptor on platelet ∙Phophatidylserine (PS): exposed on dying cells and activated platelets which are able to directly bind to phosphatidylserine allowing them to start binding to each other ∙Bind to Each other: platelets bind to each other via glycoprotein IIb/IIIa receptor (with fibrinogen as an intermediate) →Fibrinogen molecule spans two platelets and holds it together

What is an antibody titer?

Term used to denote the relative amount of specific antibody in a preparation (such as serum) Serum is serially diluted, usually in 2-fold, increments until some measurable property of the antibody is no longer detectable Titer is usually expressed as a reciprocal of the last dilution where the effect is seen (1:4, 1:256, et.) Acute vs. convalescent titer ∙Acute: serum taken while illness is symptomatic ∙Convalescent: serum taken after symptoms subside ∙Significant of the 4-fold increase in titer (convalescent compared to acute) ∙Significance of IgG vs. IgM

Assessing function of clotting factor pathway (secondary hemostasis)

Tests intrinsic and extrinsic pathways Partial thromboplastin time (PTT) ∙Tests intrinsic and common pathways: factors XII, XI, IX, VIII, X, V, Thrombin and fibrinogen ∙In tube, put: patient's plasma, phospholipid, kaolin and calcium →Kaolin acts as a very good surface to activate Factor XII ∙Normal: 25-27 sec, used to test effect of heparin (goal: 2-2.5x normal) ∙Used to analyze severe deficiencies ∙Heparin greatly enhances inhibitory activity of antithrombin II →Antithrombin III primarily inhibits Factor Xa and thrombin (IIa) →Also inhibits factors IXa, XIa, and XIIa Prothrombin Time (PT) ∙Tests extrinsic and common pathways: Factors VII, X, thrombin, V and fibrinogen ∙In tube put: patient's plasma, phospholipid, tissue factor and calcium ∙International normalized ration (INR) standardizes PT nationwide →INR is the ratio of (Pt patient/ PT normal mean) →If INR=1 it means that the patient is normal (10-12 sec), if it is 2 it means the patient is 2x normal (20-24 seconds) ∙Normal: 10-12 seconds used to test effect of coumadin (warfarin)- goal is 2-2.5x normal ∙Also used to analyze deficiencies ∙Coumadin (warfinin) blocks action of vitamin K in liver →Post-translational modification →Gamma-carboxylation of factors II (prothrombin), VII, IX, X, protein S and protein C →Actions of coumadin takes time →→→When you first administer there are plenty of normal proteins →→→It takes time to recycle with new proteins being made in the liver

The alternative C5 convertase

The C5 convertase is assembled when a C3b fragment created by C3bBb is added C3bBb: C3 convertase C3bC3bBb: C5 convertase The alternative pathway:membrane attack complex works just like the classical one!

What is virulence?

The capacity or power of an organism to cause disease

The central role of C3 in all complement cascades

The central event in the complement activation is proteolysis of the complement protein C3 to generate biologically active products and the subsequent covalent attachment of a product of C3, called C3b, to microbial cell surfaces or to antibody bound to antigen Why? ∙Patients lacking C3 are prone to successive severe infections ∙C3 represents an important amplification step in the complement cascade ∙When complement is activated by infection: leads to the cleavage of C3 into C3a and C3b →C3b becomes covalently bound ("fixed") to the pathogen surface →→→Tags pathogen for destruction by phagocytes (opsonization) →→→Organizes the formation of protein complexes that damage the pathogen's membrane (MAC) →Soluble C3a fragment →→→Chemoattractant to recruit effector cells (including phagocytes)

Exudate vs. transudate clinically

The exudate appears cloudy (left) due to the high protein content The transudate (right) appears clear, and contains a low amount of cellular components

The Endocytic Pathway

The feedback loop ∙Decreasing iron then increases protein binding to mRNA leads to less ferritin being made and more transferrin receptors ∙In an acute response, iron is increased which decrease mRNA binding of protein and leads to more ferritin being synthesized and less transferrin receptors being made

Fibrinous pericarditis

The figure on the left shows a build-up of collagen and scarring on the heart ∙This affects the motility and function of the heart Fibrinous exudate is typically observed in linings of the body cavities ∙Seen in pleura, pericardium, meninges High vascular permeability results in fibrinogen exiting the vascular network Build-up of collagen and/or some type of scarring occurs

Staining Characteristics

The gram stain separates bacteria into 2 major groups based on differences in cell wall structure ∙Gram negative vs. gram positive *Some bacteria cannot be classified using gram stain:* ∙Mycobacterium (acid-fast) ∙Mycoplasma (no cell wall) ∙Chlamydia (spore-like structure) ∙Treponema (very thin)

Herd Protection

The herd's immunity prevents diarrhea in older children and others who have no received the rotavirus vaccine Herd protection accounts for 80% reduction of rotavirus disease in children >1 year (only 1% vaccinated) in the US

Pathogens

The immune system protects against pathogens Any organism that has the potential to cause disease is called a pathogen Some pathogens cause disease if they enter the body, others can colonize the body w/ no ill effect but cause disease if the body's defenses are weakened or the microbe get in the wrong place (opportunistic pathogens)

Apoptotic Pathways

The intrinsic pathway: ∙Changes are first seen in the mitochondrial membrane ∙Cytochrome c is released and it will complex with another subset of molecules to activate the initiator caspase (pro-caspase 9) The extrinsic pathway: ∙There's receptor ligand interaction with either Fas or TNF-R →Fas removes immune cells after an adaptive immune response is complete ∙Intracellular signaling events take place while death inducing sequencing complex (DISC) brings together death domains associated with intracellular portion of TNF or fas receptors →This allows multiple molecules of pro-caspase 8 or pro-caspase 10 to come in to close proximity to initiate the pro-caspase phase ∙Once the initiator phase takes place, execution caspases are activated →They act on multiple molecules to give us changes to the intercellular and extracellular environment →Proteolytic cleavage can activate and inactivate events →The cytoplasm and subcellular organelles will be rearranged, but their normal function will stay intact

General considerations: Lymphocyte development

The maturation of B and T lymphocytes involve a series of events that occur in the central lymphoid organs ∙T cells = thymus ∙B cells = bone marrow Maturation of lymphocytes depends on three key processes ∙Proliferation of immature cells ∙Expression of antigen receptor genes ∙Selection of lymphocytes that express useful antigen receptors

Major metabolic distinctions b/w prokaryotic and eukaryotic cells

The metabolism of most bacteria is geared to rapid growth and proceeds to 10-100 times faster than that of our cells Bacteria are more versatile in their ability to use various compounds as energy sources, and in their ability to use oxidants other than molecular O2 to metabolize substrates Bacteria are more diverse in their nutritional requirements ∙Thus, they are more diverse w/ respect to the completeness of their biosynthetic pathways The simple prokaryotic cell structure (i.e. lack of nuclear membrane, golgi, ER, mitochondria) makes it possible for bacteria to synthesis macromolecules by far more streamlined means than human cells Some biosynthetic processes are unique to bacteria such as those producing peptidoglycan, lipopolysaccharide (LPS), and teichoic acid

There are two basic strategies for holding cells together

The strength of the extracellular matrix ∙Complex network of proteins and polysaccharide chains that the cells secrete Strength of the cell cytoskeleton inside the cells and on cell-cell adhesions that tie the cytoskeletons of neighboring cells together

Leukocyte responses to pro-inflammatory stimuli

The term leukocyte is used to describe any white blood cell ∙The leukocyte in the figure above will be referred to as a neutrophil Redness at the site of insult/injury is due to erythrocytes being pushed out of the vessel due to the pressure ∙Erythrocytes don't really have any function at the site of injury When activated white blood cells move through the tissues to the site of insult/injury, the process occurs in two waves ∙Neutrophils initially move to the tissue ∙Macrophages move in the second wave Neutrophil activation and response: ∙Neutrophils respond to factors produced by local tissue insult/injury ∙Neutrophils also respond to factors produced by the endothelial cells →The endothelial cell factors change in response to stimuli that promote inflammation ∙The vascular caliber is changed so that the blood is flowing more slowly through the vessel →Anything heavy (such as a large neutrophil) can now drop out and come into contact with endothelium →There are large numbers of neutrophils now coming into contact with the endothelium ∙Now the endothelium will change its surface cell marker expression, which will promote extravasation and transmigration of the neutrophil →Extravasation - a WBC moving from the circulatory system (a vessel) to the surrounding injured tissue ∙The endothelium expresses P and E selectin on its surface, which allows the neutrophil to start making contact with the endothelium. →Initially this interaction is similar to someone giving successive high-fives to many people, they are touching the hands but not holding on →Eventually, this goes from unstable touching to integrin-mediated stable binding of neutrophil to endothelium →The stable binding can be described as now holding hands with someone; it is a more tight interaction ∙Neutrophils start to flatten out and change shape as they form more stable binding to the endothelium ∙Finally, the neutrophil finds a gap between endothelial cells and dumps all of its lytic granules into the extracellular matrix to help it cut its way through to the injured tissue ∙Neutrophils respond to and migrate toward chemotactic factors such as direct cell damage products, DAMPs(danger associated molecular patterns), factors you see very early in inflammation events, and direct bacterial products ∙Chemokines = cytokines that are specifically produced to draw specific subsets of leukocytes to an area of injury

Types of necrosis

There are a variety of sequences that undergo cell death There are various types of necrosis, the problem initiating the necrosis all carry different features - **KNOW THE TYPES AND THEIR MORPHOLOGY Types: ∙Coagulation necrosis ∙Caseous necrosis ∙Fat necrosis ∙Liquefactive necrosis ∙Fibrinoid necrosis ∙Gangrenous necrosis

Fat Necrosis

There are focal areas of fat destruction Not entire areas of fat, it is very localized An example of this is pancreatitis: you have a release of pancreatic enzymes, like lipases which breakdown the fats, then the fat around the pancreas becomes necrotic ∙It is not the pancreas that is necrotic, it is the fat around the pancreas that is necrotic This could also be caused be blunt trauma to fatty tissue, such as to the breast or buttocks, where you would get a pocket of fat necrosis ∙This is the fatty portion of the bowel and the white chalk is the fat necrosis

Macrophage activation

There are two types of macrophages that play a role in acute inflammation ∙Classically activated macrophages are phagocytic and perform antigen presentation ∙Alternatively activated macrophages are involved in wound healing and repair A classically activated macrophage, an M1, is activated by microbial TLR-ligands and interferon gamma ∙An M1 generates reactive oxygen species, lysosomal enzymes, and products needed to destroy microbes ∙When chronic inflammation occurs, IL-23 will be produced which leads to pathologic inflammation An alternatively activated macrophage, an M2, is activated by IL-13, IL-4 ∙The effects of M2 are anti-inflammatory and wound repair

Serous inflammation: A skin blister

There is transudate fluid present in the tissue of the figure above There is not a lot of damage to the tissue, and little arachidonic acid products

Caspases: Mediators of Apoptosis

These are cysteine proteases that cleave after aspartic acid residue (Asp-X) ∙At least 15 members have been identified ∙Highly conserved across species Additional roles: ∙Promote inflammation (caspase-1) via activation of IL-1beta and inflammasomes →Caspase 11 also plays a role in inflammasome activation ∙Respond to PRR (pattern recognition receptors) and PAMP or DAMPs (Pathogen or Danger activated molecular patterns) →These are also both related to inflammation Non-apoptotic functions for caspases 3 and 7: ∙Proliferation, inflammation, and differentiation of many different cell types ∙Keratinocytes (-3), muscle progenitors (-3 and -9), osteoblasts (-2, -3, and -8), neurons (-3), embryonic stem cells (-3) Involvement at two or more levels: ∙"Initiator" caspases - primary signal at receptor ligand involved in decision/commitment to apoptosis ∙Caspase 8, 10 and 9 →→→Caspase 8 and 10 are involved in initiation phase with cell membrane →→→Caspase 9 is involved in the intrinsic pathway ∙"Effector/ executioner" caspases - involved in execution of apoptosis →They can trigger either the extrinsic and intrinsic pathway →Cleavage and proteolytic events occur, activating certain molecules for fragmentation →Caspase 3, 6 and 7 Caspases exist in pro-enzyme or zymogen form ∙In order to be activated, they undergo cleavage by other caspases or autocatalytically ∙Ex: Caspase 8 needs receptor-ligand interaction and intracellular events to occur in order to be activated

Acute phase proteins (APP)

These are proteins that may increase in concentration or decrease during inflammatory processes or chronic disorders ∙If the proteins are up-regulated by 25%+, then they are positive APPs ∙If they are down-regulated by 25%+, they are called negative APPs As little as 2-fold change or as much as 1000-fold change may occur in response during inflammation APPs are synthesized in the liver except γ globulins

Liquefactive Necrosis

This occurs in organs that are soft, without a strong parenchyma Example of this is the CNS, like the brain Complete digestion of dead cells from bacterial or fungal infections Someone who has HIV/AIDS or is immune compromised can have a fungal infection of the brain and have widespread areas of liquefactive necrosis ∙Here is a slide of the kidney, where dead cells are accumulating ∙Bacteria release enzymes as they proliferate and destroy the tissue

Gram positive cell wall

Thick layer: protects from drying and desiccation More cross-linking b/w linear sugar molecules than in gram negative cell walls Susceptible to lysozyme and more sensitive to cell wall targeting antibiotics than gram negatives, b/c the peptidoglycan is outside Teichoic acid and lipoteichoic acid are only part of gram positive cell walls

Caseous Necrosis

This is most commonly caused by tuberculosis Caseous refers to the appearance of it, it looks like cheese The tissue becomes necrotic and soft Image is caseous necrosis in lung tissue

Fibrinoid Necrosis

This is most commonly seen in the blood vessels It is the result of an immune reaction Because in the immune reaction, antigens and antibodies are being deposited The fibrin leaks out of the damaged blood vessels Bright pink amorphous material in the wall, leading to thickening of the wall and narrowing of the lumen of the blood vessel

Processing of endogenous antigen for presentation on Class I MHC

This pathway is used for processing of cytosolic proteins for presentation at the cell surface on Class I MHC ∙These proteins would include viral gene products, proteins from phagocytosed microbes that have either leaked out of or been transported from vesicles into the cytoplasm (more about this later) or mutated/altered host cell genes You should be aware that a portion of all proteins synthesized within a cell and are present in the cytoplasm are targeted for destruction ∙proteins are complexed with ubiquitin ∙Targets these proteins for transport to a proteosome ∙Proteosome: 28 subunit cylindrical protease complex →Degrades protein into peptide →Peptides transported to endoplasmic reticulum by TAP transporter Meanwhile, newly sythesized MHC Class I and b2 microglobulin are transported to the ER where they form a loose association with TAP and other chaperone molecules Tapasin forms a bridge between Class I and TAP transporter and allows transfer of a peptide into the peptide binding cleft Class I MHC: b2 microglobulin complex then becomes properly folded and is exported to the cell surface

Chronic inflammation

This will occur along w/ functions of adaptive and innate immunity Longer duration ∙Occurs after typical length of immune response Associated with: ∙Presence of lymphocytes and macrophages ∙The proliferation of blood vessels ∙Fibrosis and tissue necrosis

β1 Integrins

Three integrin families were originally described of which of the three β chains were used to form the heterodimers ∙More recently 5 additional β chains have been identified β1 containing integrins are also called VLA molecules (Very late activation) b/c they are expressed on T cells after 2-4 weeks of repetitive stimulation in vitro ∙other VLA are constitutively expressed on leukocytes and rapidly induced in others β1 integrins also called CD49a-g (depending on which α-chain is attached) VLA4 (α4β1) is one of the principle surface proteins that mediate homing of lymphocytes to endothelium at peripheral sites of inflammation

Spread/Transmission of S. aureus

Through direct contact w/ carrier or aerosols associated w/ respiratory infections Facilitated by people more prone to be colonized or carriers (i.e. hospital workers, diabetics, IV drugs users)

Review: Structure of immunoglobulin proteins and genes

To achieve the very large number of antibody specificities, genes encoding immunoglobulin molecules are organized differently from other genes In cells other than B cells, the immunoglobulin genes are in a fragmented form that cannot be expressed Instead of containing a single complete gene, immunoglobulin heavy and light chain loci consist of families of gene segments. This configuration of immunoglobulin genes is said to be in the GERMLINE configuration, because they are inherited in this configuration through the egg and sperm In order to be expressed, they must rearrange

Bacterial Genome

Total genetic material found in bacteria consist of: ∙Chromosome ∙Plasmids ∙Bacteriophage (prophage) ∙Transposons ∙Integrons

Staphylococcal toxin diseases

Toxin causes disease Effects of toxin are remote from site of infection Diseases: ∙Staphylococcal Scalded Skin Syndrome (SSSS) ∙Toxin Shock Syndrome (TSS) ∙Staphylococcal food poisoning

Transduction

Transfer of genetic information from one bacterium to another by a bacteriophage DNA incorporated into chromosome ∙Called a prophage Process called lysogenic conversion ∙Bacteria are called lysogenic bacteria ∙Ex: only Corynebactium diphtheria that have been lysogenized w/ beta prophage produce diphtheria toxin

Blood Vessel Structure

Tunica Adventitia: outermost layer, composed mostly of collagen fibers Tunica Media: middle layer, composed of smooth muscle and elastic fibers ∙Thicker in arteries than in veins Tunica Intima: endothelium that lines the lumen of all vessels

Basic structure of immunoglobulin

Two heavy chains ∙Composed of 3-4 globular constant (C) domains/regions ∙One variable (V) domain ∙Can be one of 5 classes: μ, δ, ε, α or γ Two light chains ∙Composed of one V and one C domain (region) ∙Can be either κ or λ

Recruitment of leukocytes to the site of infection

Two major mechanisms ∙Binding to adhesion molecules on epithelia ∙Chemoattractants produced in response to infection Recruitment is a multi-step process involving adherence of the circulating leukocytes to the luminal surface of the epithelium and migration through the vessel wall ∙Selectin-mediated rolling of leukocytes ∙Endothelium up-regulates expression of selectins (P-selectin and E-selectin) in response to microbial infection ∙Process involves cytokines (most notably IL-1 and TNF) ∙Chemokine-mediated increase in the the affinity of integrins (expressed on leukocytes) ∙Leads to stable binding of leukocytes to endothelium Transmigration

Clinical disease associated w/ Streptococcus pneumoniae

Typical lobar pneumonia (leading cause) Meningitis (most common cause in children and young adults) Otitis media Sinusitis Bacteremia

Acinetobacter baumannii

Ubiquitous Gram (-) Oxidase (-) Can survive on moist surfaces & dry surfaces, like human skin (unusual for gram negatives) Part of normal oropharyngeal flora ∙Can proliferate to large #'s during hospitalization Setting for infection = patients receiving broad spectrum antibiotics, recovering from surgery, or on respiratory ventilation Clinical disease ∙Pneumonia: always nosocomial, usually in ICU ∙Wound infections in hospitalized solders is a problem ∙UTI, septicemia Often resistant to antibiotics ∙Susceptibility testing required

Enterobacteriaceae

Ubiquitous organisms - can be found EVERYWHERE ∙Environment ∙Normal intestinal flora in humans & animals Clinical disease ∙Sepsis (1/3), UTI's (70%), GI's, pneumonias, meningitis, device associated infections ∙Community & hospital-acquired by primary and opportunistic pathogens All ferment glucose and are facultative organisms All are oxidase negative (no cytochrome C oxidase) Motility and ability to ferment lactose is species-specific Some produce prominent capsules (klebsiella pneumonia forms a copious capsule, as an example) Many possess common and sex pili - allows these organisms to exchange genetic material E. coli belongs to the Enterobacteriaceae family

Screening for lactose fermentation

Use selective & differential media →Growth of gram (+) organisms are suppressed →Distinguish b/w a lactose fermenter & non-lactose fermenter ∙MacConkey agar ∙EMB (eosin-methylene blue) agar ∙TSI slants

3rd Generation Spectrum of Cephalosporins

Usually less Gm (+) activity than 1&2 gen., however, used in Gm + meningitis (e.g. pneumonococci) due to good lipid solubility to penetrate CNS Ceftriaxone doc for resistant gonorrhea (N. gonorrhoeae), single IM dose (t1/2= 8 hrs.) for uncomplicated cases and meningitis Cefixime can be used orally in place of ceftriaxone Increased Gm (-) potency, covers most Enterobacteriaceae & anaerobes (Cefotaxime) Some activity against P. aeruginosa, but only ceftazidime and Cefoperazone

Metastatic infections of S. aureus

Usually requires implantation and previous damage to tissues Abscesses in bones (osteomyelitis), joints, lungs and kidneys Elaboration of serine proteases, hyaluronidases, lipases, DNases, facilitates spread

Overview: Stages of T cell differentiation

VERY IMPORTANT

Thrombin

VERY VERY IMPORTANT Begins at very low levels in the clotting cascade ∙Important for levels to be delicately controlled Thrombin is a serine protease ∙Inactive form: pro-thrombin ∙Active form: thrombin Thrombin plays a critical role in blood coagulation activating clotting factors XI, VIII, V, XIII, and platelets Main job: converts fibrinogen to fibrin monomers (fibrin clot)

Immunoglobulins

Variable, antigen-specific glycoproteins made by B cells Major component of the plasma in blood and lymph When secreted, called antibodies Immunoglobulin may also be bound to surface of B cells ∙Serves as B cell receptor for antigen Secreted when B cells are: ∙Stimulated by relevant antigen ∙Get "help" from T cells ∙Differentiate into plasma cells

α2-macroglobulin

Very large APP It consists of a large glycoprotein with four identical subunits It comprises 8-10% of total plasma proteins Levels are shown to increase in any nephrotic syndrome ∙This is due to the large size ∙α2 band in profiles increases significantly It is synthesized only in hepatocytes, monocytes, and also astrocytes but to a lesser degree Panprotease inhibitor to buffer the effect of acute inflammatory processes Necessary to serve as a transporter for zinc ∙Albumin is the other transporter →Even low specificity is good enough for albumin to transport and exchange most of the zinc in blood ∙α2-macroglobulin transports about 10% ∙It binds cytokines and growth factors appearing to target them to particular cell types

Aminoglycoside dosing based on creatinine clearance

Very straight-forward w/ every 24 hour regimen, but can only do this for so long Need to adjust dosage and reduce frequency if administering for long time

Biofilm Formation

Viable bacteria are protected from immune cells and antibiotics in the biofilm Biofilms can form on foreign bodies (i.e. catheters)

Measurement of Microbial Growth

Viable plate count Cell density ∙As the bacteria grow, the turbidity of the broth culture increases ∙Bacterial growth becomes visible as a turbid solution when there are about 10^7 cells/mL If a culture is required, pure cultures are needed for biochemical testing (i.e. antibiotic susceptibility testing) ∙Samples purity varies depending on where sample comes from ∙i.e., CSF is isolated fluid, but getting culture from lung or stomach may contain multiple organisms

Example of a Regulated Bacterial Operon

Virulence genes can be regulated and expressed in this manner We are not responsible for the mechanism of the lac operon, this is just an example ∙The gist is that it is very tightly controlled ∙RNA polymerase only functions here in presence of lactose and in absence of glucose

Bacteriophage (Phage)

Viruses that infect bacteria ∙Specific, used to subtype bacteria (phage typing) Structure ∙Protein capsid that houses genome ∙May have base & tail ∙No membrane envelope Genome ∙DNA or RNA ∙Single or double stranded ∙Linear or circular

Aminoglycosides: Pharmacokinetics

Water soluble, polar aminosugars, poor PO, significant dosage remains in GI/excretion Little if any CNS penetration, high concs in renal cortex, endolymph/perilymph(ear) T1\2- 2-3 hr, low plasma protein binding 10% ∙however, residual bactericidal effect that lasts longer than MIC in plasma (post-antibiotic effect) →allows for large once/day dosing, concentration dependent Excretion unchanged in urine ~= GFR; *must adjust dosage in renal dysfunction proportional to creatinine clearance*

Globulin bands

We always use serum for this test Serum inactivates clotting factors in comparison to plasma which can lead to incorrect diagnoses if plasma is used for these banding patterns

Phagocytosis and Destruction

When C3 is attached to surface of microbe, it is recognized by phagocytic cells for destruction ∙This process is called opsonization Particular Fc receptor domains can be expressed on cells that need to be phagocytized Attachment of C3 and Fc receptors to microbes promotes attachment to the phagocytic cell and eventual phagocytosis The destruction of the phagocytized material happens via different mechanisms: ∙Generation of reactive oxygen species ∙Change the pH of the phagocytic vacuole ∙Transfer of unstable bonds to the microorganism and breakage of its covalent bonds Internalization of microorganism is shown in the second figure Once the organism is internalized, the phagosome will fuse with the lysosome Inside the lysosome, halides, hydroxyl radicals, and inducible nitric oxide synthase contribute to kill the phagocytized organism NADPH oxidase system generates oxygen radicals that create a hazardous environment that will kill the phagocytized microorganism The very low pH of 2-3 is also generated to kill the microorganism

Dynamics of Infection

When an infectious disease is first introduced to a population, it has the greatest opportunity to spread b/c all hosts are susceptible

Receptor-mediated adhesion

When an organism has a specific factor on it that allow it to bind specific receptors/cells Adhesion ∙Macromolecule(s) on the surface of bacteria (i.e. pili, MSCRAMMs) Receptor ∙Specific carbohydrate or peptide on the surface of the host cell that is bound by the adhesin

Fluoroquinolones - 2nd Generation: Pharmacokinetics

Wide distribution, but low CNS levels; t1/2's range 3-8hrs Good PO, but interference by antacid formulations with Al, Mg, Zn, Fe, Ca may result in 50% decrease. This is also true of most fluoroquinolones Mixed excretion: urine/GI, unchanged and metabolized; all reach effective conc in urine/GI and accumulate in renal failure except moxifloxacin (biliary excretion) Ciprofloxacin inhibits CYP-450 drug metabolizing enzymes, may increase t1/2 of other drugs

Activation of Platelets

When platelets come in contact with a damaged vascular surface they recognize the collagen fibers in the vascular wall are a surface for the platelets to bind to Platelets immediately change their own characteristics drastically ∙Begin to swell ∙Assume irregular forms with numerous irradiating pseudopods ∙Contractile proteins contract forcefully and cause release of granules that contain multiple active factors (degranulation)

Fluoroquinolones - 2nd Generation: Clinical Uses

Wide use: skin / soft tissue, bone / joint, UTI / GI (Travelers diarrhea), respiratory, otitis media; advantage of PO in serious gram negative infections, e.g., Pseudomonas; use for prophylaxis in neutropenic patients (e.g. Ofloxacin) Not indicated for CNS/meningitis due to low penetration of CSF/CNS

Why do changes in APP occur?

Why increase positive APP? ∙Positive APPs are important in traumatic or inflammatory events ∙Increase the body's defenses ∙They may act as preformed mediators Why decrease negative APP? ∙It may be necessary to divert available amino acids for the synthesis of positive APP ∙Albumin is a well-known negative APP →Even less than 50-fold down regulation will cause changes and allow for available amino acids to synthesize positive APPs

Pseudomembranous Colitis

Yellowish plaques composed of fibrin w/ cellular debris that overlay the ulcerations in the colonic mucosa Can be caused by Clostridium difficile

Bacteria can quickly modulate gene expression in response to environmental signals

i.e. carbon source, O2, iron concentration, temperature, pH, antibiotics will lead to activation of response regulator, which will bind upstream of gene and cause activation/repression of gene

The alternative pathway: Formation of the alternative C3 convertase

iC3 binds to the inactive serum factor B Makes serum factor B susceptible to cleavage by Factor D ∙Makes smaller Ba and larger Bb Factor Bb has protease activity and cleaves more C3 into C3a and C3b ∙iC3Bb is a soluble C3 convertase C3b gets deposited on pathogen surface ∙Recruits more factor B ∙Facilitates the cleavage of Factor B by Factor D ∙Resulting complex of C3bBb is the alternative C3 convertase

Interpreting the slant

if lac or suc is fermented, acid is produced; slant & butt will be yellow if gases are produced, there will be bubbles in the butt if glu, but not lac or suc, is fermented, the O2-deficient butt will be yellow; the slant will be red if none of the sugars are fermented, both the butt & slant will be red if H2S is produced, the agar will turn black

Genetics of MRSA resistance

mecA encodes for PBP2a, on a pathogenicity island w/ other exotoxin genes ∙staphylococcal cassette chromosome (SCCmec) ∙10 different SCCmec types (I to X)???? Hospital acquired strains ∙Carry large SCCmec types I to III (34 to 67 kb) ∙Extensively drug resistant Community acquired strains ∙Type IV SCC mecA (shorter piece of DNA ~ 24kb) ∙Resistance to meth & other β-lactams and not other classes of antibiotics

Transthyretin (prealbumin)

migrates slightly ahead of albumin during electrophoresis is a negative APP It predominantly transports vitamin A (retinol) and thyroxine It inhibits IL-1 production by monocytes and endothelial cells ∙Possibly acts in the resolution phase of acute inflammatory events

Streptococcus pyogenes (GAS)

β-hemoyltic (GABHS) Bacitracin sensitive M protein ∙100 antigenic types ∙PCR now used to assign types to GAS strains ∙Antibodies are protective against re-infection w/ the same type but not different types ∙Possesses epitopes immunologically similar to human epitopes → ARF, PSGN?

Antibiotic resistance factors

β-lactamase ∙Hydrolyzes classical penicillins ∙Plasmid encoded ∙~90% of strains Pencillin-binding protein 2a (PBP2a) ∙Transpeptidase w/ an alteration in binding site that results in reduced affinity for penicillin and cephalosporin-class antibiotics ∙PBP2a encoded on different cassettes on staph chromosome (SCCmec) ∙Referred to as MRSA strains (HA- or CA-)

Globulins

γ globulin is the predominant immunoglobulin There are various sizes of globulins ∙Smaller ones include albumin and hemoglobin ∙Fibrinogen is one of the largest globulins Image = peak changes in disease states ∙A is a graph of a normal pattern, the expected inflammatory response →The γ globulin band is comprised primarily of immunoglobulins (IgM, IgA, and IgG) ∙Graph B: An immediate response (acute response) shows an increase in α2 globulin band with a decrease in albumin ∙Graph C: Delayed immune response shows an increase in β and γ globulins with a decrease in albumin →The delayed immune response can be caused by: →→→Possible Immune response of multiple weak duration OR →→→Primary immune response that fails to resolve →The body is producing antibodies against the foreign challenge ∙Graph D has gamma globulin deficiency (Hypogammaglobulinemia) →Detected in a small child could indicate an immune deficiency because there are zero immunoglobulins present →There is no skewed pattern besides the absent gamma globulin peak →→→Absence of any type of immune/inflammatory response →→→The patient would present and not necessarily complain to the doctor about any specific illness ∙Graph E: Hepatic Cirrhosis profiles look as if the gamma band bleeds into the beta bands →Get immunoglobulin response to peptides →Indication that we should test for specific autoantibodies ∙Graph F: Paraprotein AKA Monoclonal Gammopathy →There is a single clonal population i.e. a single isotope of immunoglobulin has risen, which indicates a transformation event →It is probably a leukemia or lymphoma ∙Graph G: Nephrotic Syndrome →Will lose smaller components like albumin →Large fibrinogens apparent in nephrotic syndrome cause lower, smaller peaks with a strong β2 peak and almost no γ peak →→→Fibrinogen is being retained →This is a very severe profile that is definitely not acute ∙Graph H: Protein-Losing Enteropathy →Loss of albumin and gamma globulin →Can be associated with nephrotic syndrome →Likely an acute, injurious event →Not as severe as nephrotic syndrome

Classes of adhesion molecules

∙Cadherins ∙Selections ∙Membranes of the immunoglobulin superfamily ∙Integrins

Macrolides/Miscellaneous Antimicrobials

∙Clindamycin ∙Metronidazole ∙Vancomycin ∙Quinupristin / Dalfopristin ∙Linezolid ∙Chloramphenicol ∙Fosfomycin

Stages of Infectious Disease

∙Encounter ∙Entry ∙Colonization ∙Multiplication ∙Invasion/Dissemination

Pathogenesis of Infection

∙Entering a host ∙Establishing a unique niche ∙Evading, subverting, or circumventing initial host defense mechanisms ∙Multiplying and persisting ∙Causing overt disease (optional step) ∙Exiting the host to continue transmission cycle

Majority of bacteria divided into two cell wall types

∙Gram positive ∙Gram negative

Ideal Infectious Agent

∙Highly contagious ∙Low infectious dose ∙Environmentally stable ∙Having different modes of transmission ∙Rapidly and prolifically shed ∙Constantly evolving ∙Evoking limited immunity ∙Moderately virulent ∙Allowing most of those infected to fully recover, thereby maintain a large susceptible host pool (i.e. norovirus)

There are 5 classes (isotypes) of immunoglobulin

∙IgM ∙IgA ∙IgD ∙IgG ∙IgE

Infection Control

∙Immunization and chemoprophylaxis →Out of the three leading infectious killers worldwide, we only have a vaccine against TB, though not commonly administered in USA ∙Short term home curfew ∙Restriction on assembly of groups (school, entertainment site, etc.) ∙Closure of mass public transportation ∙Chlorination of water ∙Appropriate sewage disposal/organized solid waste disposal ∙Food safety ∙Screening blood and blood products ∙Vector control ∙Public health education and counseling ∙Surveillance

Two types of immune responses

∙Innate (or natural) ∙Acquired (specific or adaptive)

Mobile Genetic Elements

∙Insertion sequences ∙Transposons ∙Pathogenicity Islands ∙Integrons ∙Plasmids ∙Bacteriophage (Phage)

Evasion of antibody

∙Intracellular residence ∙Antigenic and phase variation ∙Bind antibody inappropriately ∙IgA protease ∙Molecular mimicry

Exotoxin Routes of Entry into the Body

∙Mucosal infections ∙Wound infections ∙Intestinal infections ∙Food Poisoning (Intoxication)

Clinical indications for Penicillins

∙Otitis media (ear infection) ∙Sinusitis (sinus infection) ∙Pharyngitis (throat infection) ∙Lower respiratory infections ∙Urinary tract infections ∙Systemic infections

Microscopic Distinctions in Bacteria

∙Size ∙Staining ∙Shape and arrangement

Special features used to identify bacteria

∙Spore formation ∙Enzyme production ∙Antibiotic resistance

Tests for Primary hemostasis

∙platelet count ∙platelet aggregation assay ∙von Willebrand Factor function studies ∙Bleeding time: test of platelet vessel wall interactions