MICN201 Infection + Immunity Lecture key notes PDF

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Lecture notes from the infection and immunity vertical module...

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Intro to Defensive Pathways of Immunity Macrophages detect infection or tissue damage via PRRs pattern recognition receptors, which recognise: - Pathogen associated molecular patterns PAMPs - common microbial molecular patterns e.g. LPS lipopolysaccharide - Danger associated molecular patterns DAMPs - markers of tissue damage e.g. DNA, ATP, uric acid --> activation of macrophages - phagocytosis and killing, release of inflammatory cytokines: ● Phagolysosome for killing and antigen presentation: - low pH acid environment, reactive oxygen and nitrogen intermediates, enzymes B cell antibodies bind to epitopes on unprocessed antigens TCR recognises peptide epitopes bound to MHC - CD4 T cells recognise MHC class II (14-20 amino acids) - help other cells - CD8 T cells recognise MHC class I (8-9) - kill other cells Selection of T cells occurs in the thymus

Microbial virulence Host factors that influence susceptibility and severity of disease - Age, comorbidities, lifestyle, genetics, immunisation, medical interventions, medications, trauma, SES Microbe-mediated pathogenesis - directly damages host cells Host-mediated pathogenesis - prolonged/delayed/inappropriate inflammatory/immune response - Both dependent on: microbial virulence factors + host response - toxins/inflammation cause cell death → further inflammation/damage Host defences: 3 layers of protection Intrinsic (non-immune)

Innate immunity

Adaptive immunity

Physical barriers, secretions, physical removal, pH, microbiome

Phagocytosis, inflammation, complement, cytokines / chemokines, pattern recognition systems

Activated APC, T cells (CD4/8), B cells (plasma, antibodies), cytokines, complement

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Medical interventions, treatments and procedures can disrupt these barriers

PAMPs -> PRRs -> proinflammatory (innate immunity) Gram (+) : lipoteichoic acid -> TLR2 Gram (-) : lipopolysaccharide LPS; endotoxin -> TLR 4 Virulence factors are gene products made by microbes to colonise host: ➢ Biofilms & capsules - alginate/polysaccharide, immune evasion (phagocytes, complement, antibodies), antimicrobial resistance ➢ Motility - flagella (PAMP-> TLR5) - chemotaxis ➢ Adhesins - LPS, pili, fimbriae, capsule ➢ Invasins - enzymes acting locally to break down barriers -> spreading factors ➢ Affecting host response - leukocidins, proteases, pyocyanin

➢ Toxins - act at site distant to bacteria Virulence factors are encoded on MGE mobile genetic elements & readily transmissible - Antimicrobial resistance! - Difficult infections to treat: multidrug resistant, multiple drug resistance mechanisms, biofilms, comorbidities - combination of drugs

Pyogenic infections of skin + soft tissue Pus = bacteria + immune cells (PMNs) + fibrin-rich inflammatory exudate Abscess = localised collection of pus Gram (+) cocci are most likely to be: Staphylococcus or Streptococcus Staph. aureus & Strep. pyogenes Commonalities: - Facultative anaerobic, Gram (+) cocci - Beta haemolysis on blood agar - Microbiome, opportunistic pathogens - Produce biofilms, toxins (superantigens - non-specifically activate T cells) - Cause similar infections - Similar cell wall (PAMPs lipoteichoic acid and peptidoglycan -> proinflammatory) Differences: Staphylococcus aureus (clusters)

Streptococcus pyogenes (pairs/chains)

Often localised infections (abscesses), catalase production and penicillin resistant - use flucloxacillin

Often spreading infections, no catalase production and not penicillin resistant

Infections - Most common cause of wound, skin and nosocomial (HAI) infections, osteomyelitis/septic arthritis, - food poisoning, pneumonia, endocarditis, abscesses, sepsis

Infections - pharyngitis, pneumonia, endocarditis, skin infections, scarlet fever, puerperal fever, sepsis Post infections - RF, acute glomerulonephritis

Microbiome: 30% nasal carriage - skin -> apocrine, oral cavity, URT, GIT, UGT

5-15% pharyngeal carriage (skin, URT) Colonise skin -> entry via break in skin

Selected Virulence factors Toxins - panton valentine leukocidin PVL -> kills PMNs, pore-forming (causes lysis) Exoenzymes - coagulase - Free secreted -> prothrombin to thrombin (abscess formation) - Bound cell wall -> fibrinogen to fibrin (immune evasion/clumping)

M protein -ve charge = repels phagocytes Inhibits C3b deposition, protein A like properties = prevents opsonisation Binds fibronectin = adhesin - rheumatic fever - cross reactive antibody mediated inflammation - glomerulonephritis - immune complexes - treatment of pharyngitis stops RF

Major virulence traits of Staph aureus and Strep pyogenes = secreted enzymes, toxins, molecules which inhibit the immune system (M protein in Strep pyogenes, protein A in Staph aureus, both have capsule), surface adhesion molecules which allow the bacteria to adhere to each other and human cells. M protein also in rheumatic fever - antibodies produced against M protein cross-react with self tissues (heart, joints) Antimicrobial therapy

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Superficial skin infections respond to topical antimicrobials; oral/IV may be required if the infection fails to respond or progresses. Surgery may be needed in addition to antimicrobials to treat more serious infections (drain abscesses or debride necrotic tissue) Infections caused by methicillin (flucloxacillin) resistant Staph aureus MRSA (developed resistance to beta-lactam antibiotics - penicillins and cephalosporins) require vancomycin -

Carbuncles/furuncles (boils) and scalded skin syndrome are caused by Staph aureus Erysipelas (form of cellulitis which also involves the lymphatics system) - Strep. Pyogenes Scarlet fever occurs in pharyngitis (strep throat) when the infecting Strep pyogenes produces erythrogenic toxin Staph aureus producing an epidermolytic toxin is associated with scalded skin syndrome and severe forms of impetigo Both may be found in necrotising fasciitis, cellulitis, impetigo and wound infections; if cellulitis and necrotising fasciitis untreated - sepsis may occur

Immunology of HIV Key antigens expressed on HIV - Inside = p10 (protease), p24 (nucleocapsid), p32 (integrase), p64 (reverse transcriptase) - Outside = envelope glycoproteins gp41 (allows complete fusion of particle to membrane) and gp120 (binds to CD4 on T cells causing conformational shift, complex binds CCR5 coreceptor) Gp120 binds CD4 with high affinity - conformational change exposes coreceptor CCR5 and gp120 binds -- exposes gp41 -> membrane fusion and entry of nucleocapsid - CCR5 inhibitor (maraviroc) prevent binding of gp120 to coreceptor - Peptide analogue of fusion domain of gp41 inhibits fusion (enfuvirtide) Uncoating Reverse transcriptase (RNA genome -> cDNA) - Reverse transcriptase inhibitors RTI: - Nucleoside analogues (NRTI) - compete with natural substrates for HIV RT and incorporated into DNA -> chain termination as no 3’OH (zidovudine) - Non-nucleoside RT inhibitors (NNRTI) - bind to hydrophobic pocket near catalytic site of HIV-1 RT (efavirenz, nevirapine) Formation of pre-integration complex (PIC) Nuclear import Integration of HIV cDNA into host genome (provirus) - integrase - Integrase inhibitors (raltegravir) Transcription - only from integrated provirus, only in activated cells, transcription factors bind promoters in viral LTR (NFkB, NFAT), by host RNApol II Translation - gag and pol translated as polyproteins, env-gp160 (cleaved by host protease) - Protease inhibitors - prevent gag and pol from being cleaved for virus assembly by binding to active site as substrate analogues (nelfinavir) Viral assembly and release Maturation - protease activated with budding, cleaves gag and gag-pol into respective proteins ● ●

High rate of mutation (RT and host RNA pol II both lack proof reading ability) High rates of viral turnover - generation of many variants - accumulation of many mutations which can lead to resistance

Diagnosis of HIV Detection of antibody response to HIV or Detection of HIV - antigen (p24), RNA genome 1. ELISA - detects antibodies against p24 nucleocapsid. (antibody made early in infection) - one well coated with p24 antigen, another well with patient serum + wells washed x 3 to remove any unbound antigen - enzyme labeled with anti-p24 IgG antibody added to wells, wells washed x 3 to remove unbound antibody - substrate added which interacts with enzyme to make coloured product if p24 is present -> Colour change = HIV positive test Problem with ELISA = false positives (low sensitivity) - Initially test with combined antigen/antibody EIA (4th generation) - Confirmation with second EIA that differentiates HIV-1 and HIV-2 2. Western blot - separates proteins of HIV using sodium dodecyl sulphate then separate them using electrophoresis based on size and charge using SDS-PAGE technique. - final result will have bands in the same location as HIV proteins 3. PCR - using HIV specific probes and fluorescent nucleotides to detect level of virus in patient’s serum. HIV RNA is converted to cDNA, PCR amplification produces a large number of DNA sequences and these are then bound to oligonucleotides which are visualised - Measurement of HIV RNA levels by quantitative RT-PCR: - Acute infection, blood bank screening, confirmation in antenatal testing, infants, monitoring of treatment Seroconversion - production of antibodies in the serum after infection or immunisation. 3 reservoirs of HIV infection - Latently infected T cells - Macrophages - HIV resides in their phagocytic vesicles - Follicular DCs - different from normal APDCs as they express Fc and complement receptors that bind opsonized HIV (so major reservoir of HIV particles). Treatment strategies - HAART (highly active anti-retroviral therapy) - combines protease inhibitors with reverse transcriptase inhibitors. Combination therapy has resulted in a reduction in HIV-related deaths. - important to provide the antiviral therapy immediately after infection or after seeing clinical signs (early = fever, sore throat, headache, sores on mouth or genitals, nausea/vomiting, weight loss, dry cough) Management of patient with HIV: 1. Assessment - determine the initial CD4 count and viral loads, signs and symptoms of HIV related disease, which of the 3 stages of HIV infection they are at (asymptomatic, symptomatic, AIDS) 2. Monitoring - of CD4 and viral load every 3-4months 3. Prevention a) to individual - Hepatitis ABC screening, vaccination, stop smoking b) to others - condom se, STI screening, pregnancy, safe injection practice 4. Support - help patient adjust to illness and encouraging them to disclose to others at risk 5. Treatment - antiretroviral therapy - start when CD4 falls 95% adherence = lowest virologic failure

Bone and joint infections Osteomyelitis - inflammatory process of bone secondary to bacterial infection

Septic arthritis - invasion of joint by infectious agent which produces arthritis

Reactive arthritis - autoimmune inflammatory joint disease (unknown cause - TypeII hyper?)

Acute, chronic or subacute Staph aureus most common. Hematogenous spread most common in children (monomicrobial); exogenous in adults (polymicrobial)

Bacteria most damaging - Staph aureus most common Increasing association with joint prostheses

After genitourinary or GI infection. Triad of symptoms (not always) urethritis/cervicitis, conjunctivitis/uveitis, no symmetrical arthritis of large joints

Clinical diagnosis confirmed by culture & Gram stain of aspirates, bone biopsy; imaging (Xrays, CT, MRI), bone scans (scintigraphy)

Diagnosis - arthrocentesis (gram stain, culture - blood culture useful), imaging/ultrasound

Strong association with HLA-B27 (antigen on MHC-I) so affects antigen presentation to T cells

Treatment: 4-6w, initially IV (children oral) Empiric first -> change once sensitivities known Surgical debridement

Treatment: Rapid prevents joint destruction. As osteomyelitis eliminate meningococcal nasal carriage if treated with penicillin/treat contacts - Penicillin resistant Strep pneumoniae requires IV vancomycin - Haemophilus influenzae -> IV ceftriaxone for min 7 days - Listeria monocytogenes -> IV ampicillin or amoxicillin 2-3 weeks ➢ Neonates: combine primary antimicrobial with gentamicin (synergy) -> 2-3 weeks ➢ Adjunctive therapy: support CVS -> BP, blood volume, platelets (coagulation), respiratory, kidneys (sepsis) ➢ Steroids: dexamathasone -> maybe beneficial (don’t affect survival but decrease complications) ➢ Vaccination: recommended for risk groups

Immunity in meningitis Cycle of meningococcal infection 1. Nasopharyngeal mucosa - attach to nasopharyngeal epithelium and pass through the mucosa 2. Blood - their capsule protects them from phagocytosis and complement-mediated bacteriolysis. Their LPS endotoxin causes production of cytokines (TNFa, IL1,6,8) and complement 3. CSF - they enter CSF from blood through BBB and multiply in subarachnoid space

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Meningococcal meningitis is an ‘endotoxic disease’ where there is an increase in LPS inside blood vessels which causes extracellular growth of Neisseria meningitidis; the increased LPS in the blood activates macrophages through TLR4. Macrophages produce cytokines called monokines: - TNFa - causes margination of PMNs such as neutrophils and by chemotaxis - IL-1 - causes endothelial cells to become more adhesive to the PMNs and also stimulates procoagulant activity in the endothelium; causes fever

Host defense mechanisms in subarachnoid space are inadequate to control infection - no/minimal complement in CSF even with inflammation - IgG concentrations are low in normal CSF - increased in meningitis but still low compared with serum Capsules of S. pneumoniae, N. meningitidis, and H. influenzae mask complement and antibody, inhibiting opsonisation and phagocytosis - Anti-capsular antibody required for efficient opsonization and phagocytosis (IgG) and complement deposition (IgM and IgG) Spleen is main organ for clearing poorly and non-opsonised bacteria from the blood - liver is important for clearance of opsonised bacteria - Slow passage of blood through spleen -> prolonged contact time with lots of macrophages to detect and remove bacteria Marginal Zone B cells - Produce IgM to polysaccharide capsule - polysaccharide is a thymus-independent antigen: B cell activation without T cell help - Conjugate vaccines: polysaccharide conjugated to a carrier protein Examples of vaccinations for: - N. meningitidis - capsular and conjugate polysaccharide vaccines for ⅘ of the most common serotypes, no conjugate vaccine for serotype B Bexsero = a Neisseria meningitidis group B vaccine - 3 recombinant proteins, outer membrane vesicle containing PorA (+ others) - S. pneumoniae - pneumococcal capsular polysaccharide vaccine (antibodies short lived, no response in children herd immunity)

Immunodiagnostics Measuring antigen and antibody - Agglutination - Immunofluorescence - directly detecting antigen or indirectly through detecting antibody - Neutralisation - Enzyme immunoassay - detecting antibody: antigen-coated well -> serum added -> enzyme conjugated antibodyspecific antibody added -> enzyme substrate added - detecting antigen: antibody-coated well -> serum added -> enzyme conjugated antigenspecific antibody added -> enzyme substrate added

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Western blot - used to detect antibody only

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Interferon gamma release assays IGRAs - APC processes antigen and presents it to antigen specific T cell - activated and produces IFN-y

Did the infection occur recently? Measure early (acute) and 2-3 weeks later (convalescent) Seroconversion - negative -> positive = 4 fold increase in titre IgG avidity (accumulated strength of multiple affinities) - Early in infection = low avidity; distant infection = high avidity - Measured using chaotropic agent (urea)

Infective endocarditis = infection of heart valves and mural endocardium. Formation of vegetations -> thrombotic material and bacteria (native/prosthetic valves/pacemakers) Bacteria enter bloodstream and are transported around the body - in health this is common but transient; persistent entrance into the blood leads to DIC, inflammatory cascades and infection of internal organs - heart = infective endocarditis, which increases the risk of stroke and MI. - Bacteraemia = viable bacteria in blood secondary to infection/injury at mucosal site, transient - removed by appropriate, localised response; disease in susceptible host - Septicaemia = virulent bacteria/bacterial products in blood, pathological - unregulated, generalised, intravascular response -> pro-inflammatory - DIC, embolism, thrombosis, ischaemia, necrosis SEPSIS stages 1. Systemic inflammatory response syndrome SIRS: 2 out of: hypo/hyperthermia (38), tachycardia (HR >90bpm), tachypnoea (RR >20), increased WBC/immature PMNs 2. Sepsis: 2/+ of SIRS criteria + proven infection (+ve blood culture/gram stain/PCR) 3. Severe sepsis: sepsis + cardiac dysfunction (hypotension + hypoperfusion -> organ dysfunction) 4. Septic shock: severe sepsis + organ dysfunction despite fluid resuscitation 5. Multi-organ dysfunction syndrome MODS: - organ failure due to hypoxia - direct correlation between number of organ systems failed and mortality Bacteria-platelet-endothelium interaction in IE = - Bacteria adhere to platelet receptors via surface molecules or bridge and activate them leading to aggregation; also activate coagulation cascade - Platelets are attracted to and activated by areas of damage via fibrin and platelet deposition. Presence of bacteria increases vegetative growths via fibrin & platelet deposition. 4 things can happen after vegetations form 1 - spread into heart (heart valves mitral/aortic most common site) 2 - bacteria break off and released into bloodstream causing sepsis 3 - macro or micro-emboli released causing occlusion of capillaries such as in lung/brain 4 - deposition of immune complexes can occur (especially in kidneys -> glomerulonephritis)

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Bacteria rich in virulence factors adhere to and damage the endothelium directly. Platelet interaction and toxin production causes vegetations to form, inflammation and endothelial cell death follow (acute)

Types of infective endocarditis ● Acute short incubation - rapid course (days-weeks), destruction previously normal heart valves, virulent pathogens, difficult to treat with antimicrobials -> surgery, significant mortality -> even if treated appropriately - common symptoms = febrile, chills, weight loss, cardiac murmur/failure ● Subacute long incubation - prolonged course (weeks-months), previously damaged valves, lower virulence organisms, responds to antimicrobials - common symptoms = low grade fever, anaemia, weight loss Risk factors - injection drug use, haemodialysis and invasive procedures, most significant = underlying cardiac disease or presence of a prosthetic valve Most common microbes = Staph aureus, coagulase negative staphylococci, streptococci and enterococci; in IDUs it is usually G- rods Virulent bacteria have different mechanisms - Bind directly to endothelium by adhesion molecules MSCRAMMS (to cell surface) and SERAMS (excreted then rebind) - Bacterial toxin release -> inflammation, cell death Device associated/Prosthetic valve endocarditis -> biofilm infection caused by coagulase negative staphylococci which originates in the skin and mucous membrane (part of normal microflora). Platelets/fibrin are deposited in the sewing cuff Diagnosis using: - Modified Duke criteria: clinical, microbiological, echocardiogram - Major = 2-3 positive cultures with organisms typical for IE (gram + cocci) - Minor = PCR, serology - ECG/CXR/Echocardiogram important - pathology suggestive of damage to heart Treatment: - Initial treatment empiric based on likely cause - IE is a biofilm infection - difficult to treat -> prolonged (4-6 weeks), high dose, bactericidal IV therapy - Staphs = flucloxacillin +/- gentamicin MRSA/S.epidermis ...