Mechanism of Pathogenicity STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Pathogens & Disease Pathogens are defined as microbes capable of causing host damage. When host damage reaches a certain threshold, it can manifest itself as a disease. The evolution of an infectious disease in an individual involves complex interactions between the pathogen and the host. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

PATHOGENICITY & VIRULENCE Pathogenicity – the ability to cause disease by overcoming the defenses of the host Virulence – the degree or extent of pathogenicity Virulence factors – the various traits or features that allow or enhance the microorganism’s ability to cause disease. These take may forms and include: adhesion organelles, toxin production, evasion of the host’s immune response, resistance to antibiotics, ability to invade host tissues STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

MECHANISMS OF PATHOGENICITY Portal of Entry Adherence Penetration/invasion of host defense Damage to host cell STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala PORTALS OF ENTRY To cause disease, most pathogenic bacteria must gain access to the host including skin and mucus membranes cuts, surgical procedures, catheters, etc may allow bacteria entrance into the host Normal skin flora, including Staphylococcus aureus and Staphylococcus epidermidis, can enter through these barriers and establish an infection STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala PORTALS OF ENTRY Many pathogens have preferred portals of entry that are necessary for disease production If they gain entrance via another portal, disease may not occur Salmonella typhi produces disease when swallowed but not if rubbed on the skin Streptococci that are inhaled can cause pneumonia but, if swallowed, generally do not produce disease Bacillus anthracis can initiate disease from more than one portal of entry (skin inoculation, GI, respiratory) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala ADHERENCE. Means attachment A necessary step in pathogenicity Attachment between pathogen and host is accomplished by means of adhesins or ligands. Most adhesins of microbes are glycoproteins or lipoproteins STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala ADHERENCE The term pili (pilus) is also used to bind the host cells Gram positive organisms use other structures for adhesins (lipoproteins, etc). Streptococcus pyogenes uses lipoteichoic acid to bind to epithelial cells Once attached to target cells, many bacteria can then invade the cell STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

ADHESINS ARE VERY DIVERSE. S. mutans plays a key role in tooth decay attaches to the surface of teeth by its glycocalyx E. coli have adhesins on fimbriae that adhere only to specific kinds of cells STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala INVASION Not all bacteria are invasive. Invasive organisms attach and enter host cells by a number of mechanisms: Production of surface proteins called invasins Production of enzymes: collagenase which breaks down collagen in connective tissue hyaluronidase which breaks down hyaluronic acid that holds cells together (particularly connective tissue cells) Coagulase which converts fibrinogen to fibrin producing a clot (may be protective against phagocytes) Kinases which can break down clots decreasing the isolation of bacteria in clots (spreading effect) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

HOW BACTERIA DAMAGE HOST CELLS. Direct damage The production of Toxins Types of toxins: Exotoxins and Endotoxins. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Bacterial Toxins Many different types of toxins Exotoxins Endotoxins Toxins are are not required for growth Genes for toxins are usually on plasmids STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala EXO and ENDOTOXINS. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala EXOTOXINS. Produced inside some bacteria as part of their growth and metabolism and released into the surrounding medium Are proteins, and many are enzymes Most bacteria that produce exotoxins are gram-positive The genes for most exotoxins are carried on bacterial plasmids or phages. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Neurotoxin. Target the nervous system, and can interfere with normal nerve impulse transmission, e.g. C. tetani, C. botulinum. ENTEROTOXINS. Affect cells lining the gastrointestinal tract. E.g. V. cholerae, C. difficile. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala ACTION OF AN EXOTOXIN. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala NOTABLE EXOTOXINS. Diphtheria toxin. Erythrogenic toxins. Botulinum toxin. Tetanus toxin Vibrio Enterotoxin. Staphylococcal Enterotoxin. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Bacterial Endotoxins Endotoxins Toxin is not internalized Toxin is located on outside of microorganisms (Part of the outer portion of the cell wall of bacteria) LPS of gram – bacteria Lipoteichoic acid or gram + bacteria Only toxic at high levels Liposaccharide STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Exert their effects when the gram negative bacteria dies and their cell wall undergo lysis, thus liberating the endotoxin(e.g use of antibiotics) All endotoxins produce the same signs and symptoms Endotoxins can also induce miscarriage. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Mechanism of Action of Endotoxins Endotoxins bind to Receptors on Macrophages Neutrophils Lymphocytes Proteins of complement Complement is a group of proteins which circulate at constant levels in the blood When activated complement is a powerful tool against invading pathogens Increased inflammation STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Bacterial Endotoxins Endotoxins Host cell receptors (TLR) bind to components of pathogen Pathogen associated molecular patterns PAMPS LPS – gram - cell walls Flagella Lipoteichoic acid – gram + cell walls Signal transduction pathways begin to make a cellular response Production of cytokines STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Bacterial Exoenzymes Enzymes secreted by bacterial cells into the extra cellular matrix of host Membrane Damaging Toxins Enzyme destruction of host cell membranes Lyse red blood cells Membrane pore forming complex Enzymes which act in the extra cellular matrix Spreading factors Breaks down connective tissue Attacks blood clots Enzymes which subvert drug therapy in patients Penicillinase STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Some Common Exoenzymes α toxin Pore forming toxin Common in Staphylococcus aureus Hemolysins Destroy red blood cells Streptolysins – group of hemolysins excreted by Streptococcus Streptokinase Attacks fibrin clots From Streptococcus pyogenes Hyaluronidase Breaks down hyaluronic acids in connective tissue Similar function for Collagenase Elastases DNase DNA is viscous Thins pus (DNA & debris) released from WBC STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Structure of Antibodies STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Antibodies A.K.A Immunoglobulins & gamma globulins Structure variable hypervariable constant STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Figure 21.13a

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Antibody Structure Antibodies Are Made Up Of: 2 Light Chains (identical) ~25 KDa 2 Heavy Chains (identical) ~50 KDa Each Light Chain Bound To Heavy Chain By Disulfide (H-L) Heavy Chain Bound to Heavy Chain (H-H) First 100 a/a Of Amino Terminal Vary of Both H and L Chain Are Variable Referred To As VL , VH, CH And CL CDR (Complementarity Determining Regions) Are What Bind Ag Remaining Regions Are Very Similar Within Same Class STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Antibodies Constant (C) region defines antibody class determines chemical & cellular interactions determines how class functions to eliminate antigens STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Antibody Classes Antibody Classes: IgM, IgG, IgA, IgD, IgE (Ig = immunoglobulin) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Antibody Classes IgG: the most abundant circulating Ig. The dominant circulating Ig of the primary & the secondary response. Crosses the placenta. Complement binding (Monomer). IgA: the Ig of secretions. Helps prevent antigen penetration of membranes (Dimer). IgD: the Ig of B-cell activation. Found on B-cell surface (Monomer). STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Antibody Classes IgM: occurs as a monomer & a pentamer Occurs on the B-cell surface (Monomer). The Ig of early primary plasma cell response, circulating antibody; a potent agglutinator. Complement binding (Pentamer). STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Antibody Classes IgE: the Ig associated with allergies. Stem binds to mast cells & basophils. Receptor binding results in histamine release & inflammation. Found mostly in mucosa of respiratory & GI tract (Monomer). STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Antigen-Antibody interactions Characterized as: Non-covalent interaction (similar to “lock and key” fit of enzyme-substrate) Do not lead to irreversible alteration of Ag or Ab This exact and specific interaction has led to many immunological assays that are used to: detect Ag or Ab diagnose disease measure magnitude of humoral IR identify molecules of biological and medical interest STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Introduction Ag – Ab reactions are one of the most specific noncovalent biochemical reactions known The forces that hold the reactants together are: - van der Waals forces - Electrostatic forces - Hydrophobic forces They can be represented by the simple formula: Ag + Ab ↔ AgAb The reaction is driven to the right but it is reversible STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Strength of Reaction The strength of the reaction (how far it is driven to the right) is referred to as affinity Antibody affinity - A quantitative measure of binding strength - Combined strength of the noncovalent interactions between a binding site on an Ab & monovalent Ag - Affinity varies broadly among immunoglobulins STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Strength of Reaction Antibody avidity - Avidity is often used to describe the collective affinity of multiple binding sites on an antibody molecule - True strength of the Ab -Ag interaction within biological systems - The interaction at one site will increase the possibility of reaction at a second site - High avidity can compensate for low affinity (secreted pentameric IgM has a higher avidity than IgG ) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STAGES OF Ag - Ab REACTIONS Primary reactions Vs secondary reactions: Small Ag - Ab complexes Vs large complexes (The Lattice hypothesis) Development of macroscopic manifestations reactions (e.g. immunoprecipitation) Ag – Ab reactions involving IgM are confined to the blood stream, while those of lower molecular weight (IgG and IgE) can leave the vasculature and enter tissues Time required is hours to days for precipitin formation leading to irreversible immunoprecipitates STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

ZONES OF PRECIPITIN FORMATION Precipitin Curve STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Precipitation Precipitation can take place in capillary tubes, test tubes, and in gel STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Precipitation in gel - Double diffusion - Single (radial) diffusion - Combination of diffusion in gel and electrophoresis STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

SINGLE VS. DOUBLE DIFFUSION Single diffusion Supporting medium (gel) contains one reactant at a uniform concentration Only the unknowns move through the medium Double diffusion Gel is inert (contains no reactants) Both Ag and Ab travel through the medium STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala The region of equivalence STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

RADIAL IMMUNODIFFUSION Ab uniformly distributed in gel; Ag diffuses outward from a well (single diffusion) Ag- Ab complexes form as concentric rings around the well at zone of equivalence At a set time, ring diameters are measured [Ag] is directly proportional to the ring d2 Unknown value is determined by comparing to a 3-standard curve STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

RADIAL IMMUNODIFFUSION Samples Standards Precipitin Rings A B C a b c Standard Curve STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

RADIAL IMMUNODIFFUSION Fahey method (kinetic) Read at 18 hours Plot [std] vs. ring diameter on semi-log paper Mancini method (endpoint) Read at 48 or 72 hours Plot [std] vs. ring diameter squared on graph paper Results reliable only if the ring size is within the range of the standards; if greater than highest std, dilute and repeat test Used to measure IgM, IgG, C4,C3,transferrin, CRP, others STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

OUCHTERLONY DOUBLE DIFFUSION Ag & Ab placed in wells cut into an agarose gel (both reactants diffuse) Precipitin line (or arc) indicates Ab has specificity for Ag Position of precipitin between wells depends on MW and concentration of reactants 3 possible patterns of reaction: identity, non-identity, partial identity STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

OUCHTERLONY DOUBLE DIFFUSION Ouchterlony Plates Precipitin Patterns STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Agglutination Titer Zeta potential Types of Agglutination - Direct agglutination or hemagglutination - Indirect (passive) agglutination or hemagglutination - Agglutination or hemagglutination inhibition The Coombs test - Direct - Indirect STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Agglutination Reactions STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Agglutination Qualitative slide agglutination - identification of bacteria with antisera directed against O, H, K antigens STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Agglutination Latex agglutination Coagglutination STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Agglutination Tube agglutination tests: - Gruber-Widal: typhoid fever (S. typhi) - Weil-Felix: typhus (Rickettsia) - Wright: brucellosis Identify and titrate antibodies in the patient’s serum. Titre: is defined as the reciprocal of the highest dilution of serum showing agglutination. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala 1:200 1:400 1:100 STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Titer

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Agglutination inhibition STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Hemagglutination Inhibition Test To Detect Antibodies (Rubella) - Serum (Ab)+ HA +RBCs= No Hemagglutination = Positive Test - Serum (No Ab)+ HA + RBCs =Hemagglutination =Negative Test To Detect Antigen (HBsAg) - Serum (HBsAg) +Anti HBsAG + HBsAg coated RBCs = No Hemagglutination = Positive Test - Serum (No HBsAg)+ Anti HBsAG + HBsAg coated RBCs = Hemagglutination =Negative Test STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Use of Labels in Ag – Ab Reactions Immunoassays - Radioimmunoassay (RIA) - Enzyme Immunoassys (EIA) Immunofluorescence (IF) - Direct IF - Indirect IF Flow cytometry and Cell Sorting (FACS) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Immunologic Tests 4) Radioimmunoassay (RIA)– a very sensitive test; used for measuring hormones, serum proteins, drugs, etc. at low concentrations (≤ 0.001ug/ml) measures “competitive binding” of radiolabelled Ag + unlabelled (test) Ag to high affinity Ab STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala ELISA STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala ELISA tests Depend on enzyme conjugated to 2 Ab reacting with a specific substrate to produce a color reaction. Variations of ELISA’s: Allows for qualitative or quantitative testing. Each one can be used for qualitative detection of Ag or Ab Also, a standard curve based on known concentrations of Ag/Ab can be prepared and an unknown concentration can be determined Indirect ELISA Sandwich ELISA Competitive ELISA STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Direct and indirect Immunofluorescence STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Immunoprecipitation Provides a quick and sensitive test for finding proteins/Ag’s especially in low concentrations Binds Ab to synthetic bead support  centrifuged Or 2° Ab with bead or magnetic bead and collect by magnetism STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Distribution of selected markers on some leukemia cell types → Immunophenotyping using “flow cytometry & mAb” STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Sensitivity of various immunoassays STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Hypersensitivities: Types Immediate hypersensitivity (Type I): symptoms within seconds of exposure to an allergen (requires sensitization = previous exposure) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala Figure 21.21

Hypersensitivities: Type I Anaphylaxis (IgE mediated; mast / basophils) Local: histamine induced vasodilation & increased permeability. Watery eyes, runny nose, itching & redness. Respiratory  allergy induced asthma Systemic: anaphylactic shock: associated with allergens that have systemic distribution. Widespread vasodilation, airway swelling Atopy: the tendency to display Type I symptoms to certain environmental antigens without prior sensitization STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Hypersensitivities: Types II & III Subacute hypersensitivity (IgG & IgM mediated) Cytotoxic reactions (Type II): antibodies bind to cellular antigens promoting complement fixation / inflammation / phagocytosis (transfusion reaction) Immune complex h. (Type III): widely distributed antigen reacts with antibody. Antigen-antibody complexes cannot be cleared; persistent inflammation / tissue damage (farmer’s lung; associated with autoimmune disorders) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Hypersensitivities: Type IV Delayed hypersensitivity (cell mediated) takes one to three days to react. Involves TC, TH1 & macrophages. Allergic contact dermatitis (poison ivy, heavy metals, TB tine tests). Agents act as haptens & elicit response after binding to tissue STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala