u4 Flashcards
endemic
Disease is constantly present in a population usu. at low frequency
epidemic
Disease suddenly increases in a population
pandemic
Disease increases within large widespread populations usu. worldwide
MORTALITY
MORTALITY
Leading infectious killers
– acute respiratory infections: pneumonia, influenza
+ HIV AIDS, diarrhoeal diseases, TB, malaria, measles
MORBIDITY
MORBIDITY
Infections = huge burden
(measured as disability adjusted life years; DALYs)
= so sick -> cannot contribute to the community • cannot work
• cannot care for children
major factors contibuting to the emergence of infectious disease
- human demographics and behavior
- technology and industry
- economic development and land use
- international travel and commerce
- microbial adaptation and change
- breakdown of public health measures
aim of active immunisation
Aims
• to produce long lasting immunity to pathogens in individuals and the community
• to eliminate the pathogen (where possible)
symbiosis
= Living together of organisms
Commensalism:
Commensalism: ‘commensal’ = lives in / on another
ie Interaction between normal flora and host One partner benefits
Other partner not affected
Mutualism
Mutualism: ‘mutualist’ / ‘symbiont’ ->Both partners benefit
Often obligatory eg ruminants, GIT flora
Parasitism
Parasitism: ‘parasitic organisms’
Pathogen = parasitic organism causing specific disease
- One partner benefits
- Other harmed
Benefits: of normal flora
Benefits: • Compete with pathogens for attachment sites, nutrients • Produce anti-microbial compounds = toxic to invaders, pathogens • Aid digestion • Supply essential growth requirements • Stimulate immune system Aid resistance to infection
factors governing symbiosis exam
- No. organisms:
Increase numbers = shift to parasitism eg poor hygiene - Virulence of organisms
Increase virulence = shift to parasitism - Host’s defence / Resistance
Healthy = high resistance
Decrease resistance = shift to parasitism
what is virulence
the degree or intensity of pathogenicity.i.e. increased virulence-> likely to cause harm and result in parasitism
drift
Drift = small antigenic changes
→ alteredprotein
= not effectively recognised by immune system
shift
Shift = drastic antigenic changes
→ large scale altered proteins
= not recognised at all by immune system
what is the result of shift and drift
major epidemics (or pandemics)
transmission of influenza virus
• Air-borne:
– aerosols: coughing, talking, sneezing: ‘flu, chicken pox, mumps, measles etc
– dust: hospitals = nosocomial infection
• Contact: Direct usu. skin to skin; utensils, STI’s
• Vehicle: contaminated food / water: cholera, food poisoning
• Vectors: malaria, trypanosomiasis
control of infecious disease from the infectious disease cycle. EXAM
Source: reduce / eliminate
– Eliminate contaminated food or water: food poisoning
– Quarantine carriers and diseased: smallpox
– Destroy animal carriers: mad cow disease, chicken ‘flu HK – Identify reservoir: bats in SARS outbreak
Transmission (KEY ROLE): stop spread one host to next – Change behaviour: HIV protection
– Destroy insect vectors: DDT mosquitoes
– Control animal vectors: cattle: brucellosis, TB
Host susceptibility
– Improve nutrition
– Vaccinate
– Problem with lack of immunocompetence
virulence and mode of transmission EXAM
Virulence =intensity of pathogenicity OR degree of ability to cause disease
Affected by ability to live outside host
Eg Common cold:
virulence low / direct contact low level transmission host still active, moves around, spreads virus
cv. Increase virulence, host ill (bed-ridden), transmission drops
Conversely, vector transmission eg African sleeping sickness: host can’t transmit, reliant on vector
= Virulence not connected with transmission
Pathogenicity
Pathogenicity
= the ability to cause disease
Virulence
Virulence
= degree or intensity of pathogenicity of a microbe»_space; indicated by:
fatality rates
ability to invade host tissues & cause disease
- What are virulence factors?
1.Microbial strategy / Trait
• Product eg toxin
2. Contribute to virulence = virulence mechanism
• Clear connection: virulence infection
3. Plus “Housekeeping” functions
• Derive nutrients, energy for survival in host
virulence factors
- Aid colonisation
- Allow penetration of host tissue
- Prevent/reduce host response
- Cause direct damage eg.toxicity
colonisation
Attachment / Adhesion -Non-specific
-Specific
give e.g of non-specific attachment
NON-SPECIFIC: SLIME/CAPSULE/GLYCOCALYX
1. dental plague
e.g. the attachment of slime layer derive from polysacchride cause the acummulation of the plague
2. glycocalyx
the intestinal epithelium hide under glycocalyx of the E.coli->protection
give e.g. of specifc attachment
influenza virus protein spikes
- the spikes bind to the receptor initiate infection.
HA trimer for attachment and NA tetramer for puncture of the wall
adheren mechanism
- pilli for sexual conjugation
2. fimbrial for adhesions
host cell penetration and growth
Uptake via • Invasins: bacterial surface proteins: promote ingestion • Endocytosis: non-phagocytic cells Exocytosis: actin tail propulsion • Phagocytosis. e.g. influenza virus:endocytic uptake Measles virus: membrane fusion uptake
coagulase
coagulates blood
kinases
digests fibrin clots
hyaluronidase
hydrolyses hyaluronic acid
collagenase
hydrolyzes collagen
IgA proteases
destroy IgA antibodies=survivall
4.Evading the Immune Response
Capsules Virulence protective anti-phagocytic
Capsules Virulence
-e.g. Crytococcus neoformans forming capsule, capsule can prevent phagocytosis therefore escape the immune response
protective
anti-phagocytic
- damaging the host
- Microbial Toxins
i. Exotoxins
ii. Endotoxins - Pathogenicity Islands
describe exotoxins EXAM
source: mostly G+ metabolic product: by-products of growing cell chemistry: protein fever: NO neutralized by antitoxin: YES
e.g. of a disease caused by exotoxin
exfoliative toxin: Scalded skin syndrome caused by the exotoxin of staph.aureus in immuno naive infant
microbial exotoxins classification
3 types based on structure & action:
- A-B
- Membrane-disrupting 3. Superantigens
Microbial Exotoxins: A-B Toxins
Active-binding protein
- B involve in binding, A invove in damage the cell
e.g. of A-B toxin
cholera exotoxin
- the exotoxin cause the protein to be inactive result in the disease Vibrio cholerae.
membrane disrupting
Exotoxins and e.g
Channel-forming (pore- forming) -Phospholipase hydrolysis of membrane: * Destabilises * Destroys integrity e.g.Clostridium perfringens alpha-toxin: phospholipase action causing gas gangrene
superantigen-exotoxin
Rare strains of Staphylococcus aureus produce TSS-toxin, In the tempon, the Mg stimulate the production of an organism causing peelin skin
endotoxin
Source: Gram – Metabolic product: Present in LPS of outer membrane Chemistry: Lipid Fever? Yes Neutralized by antitoxin: No
inflammatory action of endotoxins
- macrophage ingestes a G- bacterium
- the bacterium is degraded in a vacuole, releasing endotoxins that induce the macrophage to produce IL-1
- IL-1 is released by the macrophage into the bloodstream, through which it travels to th ehypothalamus of the brain
- IL-1 induces the hypothalamus to produce PG, which reset the body’s thermostat to a higher temperature, producing fever
what is the inflammatory components of G+ bacteria
- peptidoglycan
- lipoteichoic acid LTA
- teichoic acid TA
mechnism of pathogenicity islands exam
• Contribute to characteristics of pathogenicity – Absent in non-pathogenic strains
• Large segments of DNA
– Contain insertion-like sequences = mobile – Encode major virulence factors
– Associated with tRNA encoding genes
• Pathogens may have >1 PI
• Maybe plasmids
• Acquired by horizontal gene transfer
e.g. of pathogenicity islands
Examples
– alter actin microfilaments to mediate adherence
– modulate host activities
example of pthogenicity island:Protein secretion: Modulates host activities. exam
Yersinia pestis (causes plague): Type III secretion system: Function: • delivers effector proteins • secretes plasmid-encoded outer membrane proteins into phagocytic cells Action: • counteracts natural defence mechanisms • helps Y. pestis multiply and disseminate in the host
endotoxin
Gram –ve: LPS
Gram +ve: Peptidoglycan, LTA,TA
Non-specific host defences
- Physical barriers
– Skin / Mucosal membranes / Flushing mechanisms - Chemical Barriers
– Proteins / pH / Hormones / Others: bile, urea - Biological Barriers
– Normal flora / Inflammation / Phagocytosis
Host factors affecting successful establishment of infection
- Socio-economic status: - Nutrition
- Living conditions: poverty, overcrowding 2. 2. Age:
- Very young
- Aged - Gender
- Occupation
- Race
- Genetic factors
- Defective immune systems
physical barriers
- Skin
* Mucous membranes • Flushing mechanisms
defence of skin EXAM
- dry acidic environment: prevent growth of many bacteria
- dead, keratinized cells: keratin is hard to degrade, and dead cell diacourage colonization
- slouphing of surface cells: remove bacteria that adhere
- toxic lipids, lysozyme: protect hair follicles, sweat glands and sebaceous gland
- normal microbiota
- underlying immune cells (langerhans): combat bacteria that mannage to reach the dermis and tissue below it
defenses of mucosal membrane
- thick layer of mucin
- trap bacteria before reach the cell
- contain antimicrobial compounds such as lysozyme and lactoferrin
- MALT mucosal-associated lymphoid tissue
Muco-ciliary escalator
move and repel foreign particle
what does lysozyme do in mucus
digest peptidoglycan
what does lactoferrin do in mucus
sequester iron, prevent growth of bacteria
chemical barriers
• Antimicrobial factors in body fluids: mucous, tears, saliva – Lysozyme • Proteins – Enzymes – Bacteriocins – Complement – Fibronectin – Cytokines: eg Interferon • Hormones • LowpH • Others – Bile, urea
Lysozyme Breaks Down __ ?
Gram +ve Cell Walls
complement
• Series of 9 serum enzymes, act in cascade
• Always present in serum
• Activated when Ag/Ab reactions occur
• In association with Ab, causes bacterial lysis
• Aids phagocytosis
»»> RESULT: Kills bacterial cells
glycoproteins
= proteins + polysaccharide moieties eg Fibronectin - Mediatesnon-specificclearance - Coatsforeigncellsclotting - Blocksattachmentofforeignorganismsto epithelial cells >>>>> RESULT: limits colonisation
cytokines
• Intercellular signal
• Bindtoreceptorsoncells
• Triggers cellular behaviours:
– Initiate signal transduction pathway
» regulates specific transcription, translation events • Soluble small protein or glycoprotein
• Can induce production of other cytokines
• Produced in response to non-specific stimulus from: – microbes: bacteria, viruses, parasites
– cancer
– inflammation
– action of specific immune cells
interferon
• antiviral proteins
• formed in response to viral infection • excreted by infected cell
• species specific, not virus specific
• interesting as anti-viral drug:
expensive; genetic engineering
»»> RESULT: Protects other cells from viral infection
Anti-viral Action of Interferon 1
- virus RNA enters the cell
- infecting virus replicates
- the infecting virus induces RNA to produce alpha and beta interferon
- interferons released by the virus-infected cell bind to plasma membrane of neighbouring uninfected cells inducing them to produce anti-viral proteins (AVPs)
- new viruses infect neighboring cells
- AVP’s degrade new viral mRNA, inhibiting protein synthesis and replication
Biological Barriers
• Normal flora
• Inflammation – Acute
– Chronic
• Phagocytosis
acute inflammation
– – – Results from tissue injury and infection See redness, swelling, warmth, pain 4 steps: 1. Increasedbloodflowanddilation 2. Riseintemperature 3. Formationoffibrinclot 4. Phagocyticaction >>>>> RESULTS: destruction of invader localisation of infection
4 steps in acute inflammation
- Increasedbloodflowanddilation 2. Riseintemperature
- Formationoffibrinclot
- Phagocyticaction
Acute Inflammation: Capillary penetration
- damaged tissue
- inflammatory signals
- dialation/permeability
- chemotaxis
- attaction of pahacytes to site
- destruction of foreign microbe
• Chronic
– Slow
– Intracellular pathogens eg TB, syphilis
outcome of phagocytosis
- nonencapsulated bacterial engulfed by pathocyte and digested
- bacteria protected by capsule or by M protein cannot be engulfed by phagocytes (e.g. streptococcus phemoniae/pyogenes)
- pathogens engulfed but produced leukocidin and destroy phagocyte (Staph.aureus, strep pyogenes)
- pathogens engulfed but gorwn within pjagocyte (e.g. mycobacterium tuberlosis, salmonella typhi)
natural killer cells
• Non-phagocytic lymphocytes (more on lymphocytes next lecture)
• RecognisecellswithdefectiveMHCclass1protein on cell surface
• Result in production of pore-forming perforin proteins and granzymes = lyse target cells = apoptosis
• Attackanddestroycells: – Malignant
– Containing microbes
– Opsonised with antibody
Revision: How are Bacteria Classified?
1. Phenetic (phenotype): – Gram reaction and morphology – Carbon sources, energy sources – Electron acceptors (eg. aerobic/anaerobic) 2. Phylogenetic (genotype): – Ribosomal RNA sequence – Other DNA, RNA, protein sequence
diverse G- bacteria can cause disease
- proteobacteria
- gamma-proteobacteria: Escherichia, salmonella, vibrio, pseudomonus
- beta-proteobacteria:neisseria
- alpha-proteobacteria: Rickettsia - spirochaetes: e.g. treponema
Escherichia
- gamma-proteobacteria:
- Facultative anaerobic, heterotrophic, G-ve rods
- Found in gut of humans and animals
-Part of family Enterobacteriaceae: contains many other pathogens: Salmonella, Klebsiella, Yersinia
• Widely used in microbiology as a model organism
• Motile by peritrichous flagella e.g. e.coli
the polar flagellum e.g. pseudomonus is non-motile
Transmission of Disease
Transmission of Disease
1.Inoculation =directcontact
eg staphyloccal skin infections, STI’s, leprosy, anthrax, Herpes cold sores & genital herpes, tetanus
2. Inhalation = air-borne, ~10um diameter droplets -> respiratory tract eg TB, chickenpox , influenza, diphtheria, Legionnaires disease, meningococcal disease
Ingestion = food/water-borne -> gastrointestinal tract
eg botulism, food poisoning, typhoid, cholera, viral gastroenteritis, polio
Vector-borne = minor = arthropod or mosquito bite ->skin
eg plague, malaria, sleeping sickness, Ross River fever, Murray Valley encephalitis
Combination = both air-borne & direct contact transmission
eg Ebola & Marburg viruses
= haemorrhagic fevers involving massive haemorrhage throughout the body
Inoculation
Inoculation =directcontact
eg staphyloccal skin infections, STI’s, leprosy, anthrax, Herpes cold sores & genital herpes, tetanus
Inhalation
air-borne, ~10um diameter droplets -> respiratory tract eg TB, chickenpox , influenza, diphtheria, Legionnaires disease, meningococcal disease
ingestion
Ingestion = food/water-borne -> gastrointestinal tract
eg botulism, food poisoning, typhoid, cholera, viral gastroenteritis, polio
Vector-borne = minor = arthropod or mosquito bite ->skin
eg plague, malaria, sleeping sickness, Ross River fever, Murray Valley encephalitis
vector-borne
Vector-borne = minor = arthropod or mosquito bite ->skin
eg plague, malaria, sleeping sickness, Ross River fever, Murray Valley encephalitis
combination
Combination = both air-borne & direct contact transmission
eg Ebola & Marburg viruses
= haemorrhagic fevers involving massive haemorrhage throughout the body
Staphylococcal Infections
Transmission: Inoculation, Direct Contact
bacterial skin infections
When the stratum corneum is disrupted:
– Trauma: abrasion, insect bite, burn…
– Maceration due to excess moisture: blisters… – Inflammation: injury, allergy…
Two common bacterial pathogens:
Staphylococcus aureus
– Present in anterior nares of 20-40% of people
– Can spread person-person
– Usu. colonises nose first then spreads to skin
Streptococcus pyogenes (= Group A Streptococcus) Refer to U3L20
− Rarely persists for long
− Generally acquired from others
− Usu. goes to skin first; later colonises respiratory tract
Staphylococcus aureus exam
Staphylococcus aureus
– Present in anterior nares of 20-40% of people
– Can spread person-person
– Usu. colonises nose first then spreads to skin
Streptococcus pyogenes
Streptococcus pyogenes (= Group A Streptococcus) Refer to U3L20
− Rarely persists for long
− Generally acquired from others
− Usu. goes to skin first; later colonises respiratory tract
Epidermis: Impetigo exam
Caused by S. pyogenes alone or with S. aureus
Does not scar as infection is superficial
Resolves spontaneously or with antibiotic treatment
Dermis: Folliculitis exam
- Staphylococcus aureus is most common cause of infection and inflammation of hair follicle
- Stye = infection of oil gland of Zeis on base of eyelid
Furuncles:
Furuncles:
extension of folliculitis
carbuncles
coalescence of furuncles; associated chills, fever indicate systemic spread of Staphylococcus
Virulence Factors of S. aureus: Cell wall
- Cell wall components:
Capsule / slime: Anti-phagocytic, masks cell, adherence - Protein A:
Anti-phagocytic, binds host Ig, prevents complement activation - Peptidoglycan: Stimulates endotoxin-like activity; attracts polymorphs (abscess formation); inhibits phagocytosis; stimulates complement cascade
- Teichoic acid:
Invasion; mucosal cell attachment
action of S.aureus capsule/slime
Anti-phagocytic, masks cell, adherence
action of S.aureus protein A
Protein A:
Anti-phagocytic, binds host Ig, prevents complement activation
action of S.aureus peptidoglycan
Peptidoglycan: Stimulates endotoxin-like activity; attracts polymorphs (abscess formation); inhibits phagocytosis; stimulates complement cascade
action of S.aureus Teichoic acid
- Teichoic acid:
Invasion; mucosal cell attachment
virulence factor os S.aureus:enzymes
Enzymes:
1. B-lactamase :
Penicillin; inactivates antibiotic
2. Catalase: Lethal H2O2; protects against killing by oxidative burst in phagocytosis
3. Coagulase
Host fibrinogen fibrin coating on Staph. cells clumping; anti-phagocytic
4. DNase: DNA; destroys host nucleic acid
5.Hyaluronidase: Hyaluronic acid; spreading factor
6. lipase: Lipids; affects cell membranes Nucleic acid
7. protease: Proteins
S.aureus coagulase production, covert fibrinogen into fibrin
• Clots plasma:
Coagulase + prothrombinblood > staphylothrombin + fibrinogenblood > fibrin
• Related to virulence:
Surrounds Staph cell with fibrin meshwork: Increased diameter > ineffective phagocytic uptake = protective persistence infection
Enterotoxins of S.aureus
toxins of S.aureus as part of virulence factors
GIT, cause food poisoning
cytotoxic haemolysins (toxins of S.aureus as part of virulence factors)
alpha: erythrocytes, skin
beta: erythrocytes, sphingomyelin
alpha and gamma: erythrocytes
leukocidin (toxins of S.aureus as part of virulence factors)
Cytotoxic; inhibits phagocytosis by granulocytes
Exfoliative toxins A & B (toxins of S.aureus as part of virulence factors)
Surface layers of skin; shedding
Toxic shock syndrome toxin-1 (toxins of S.aureus as part of virulence factors)
Systemic effects, causes fever, shock
virulence factors of S.aureus: toxin
enterotoxins: GIT, cause food poisoning
cytotoxic haemolysins : alpha: erythrocytes, skin
beta: erythrocytes, sphingomyelin
alpha and gamma: erythrocytes
leukocidin: Cytotoxic; inhibits phagocytosis by granulocytes
Exfoliative toxins A & B:Surface layers of skin; shedding
Toxic shock syndrome toxin-1:Systemic effects, causes fever, shock
S. aureus Leukocidin
leuko = white; cide = kill: kills white blood cells eg PMNs, neutrophils
Postulated actions:
• causes polymorphonuclear leukocyte (PMN) lysis
• low concentrations mediate PMN apoptosis: bind directly to mitochondrial membranes
• creates pus: pus contains pus cells = necrotic PMNs
• tissue necrosis results from release of reactive oxygen species from lysed PMNs
• release of granule contents from lysed PMNs
->inflammatory response ->tissue necrosis
»»> RESULT: cell death, pus formation
S. aureus Exfoliative Toxin:
Ritter’s Disease aka Scalded Skin Syndrome
toxin-mediated disease: caused by exfoliatin toxin produced by certain strains of S. aureus
Occurs in young children: spontaneous recovery in 7-10d; scarring absent
Taken from: Emond et al
Secondary complicating bacterial infections sometimes cause more serious disease
STI: Chlamydia Transmission: Intimate Direct Contact exam
• Acquired via intimate contact
• Usu. microbes unable to survive outside human body
• Several important bacterial causes of STI:
– Neisseria gonorrhoea,Treponema pallidum(syphilis), Chlamydia trachomatis
• Several important viral causes of STI: – HIV, genital herpes, papilloma virus, HepB
• Globally 300 million new cases/year
• Many unreported, undiagnosed ->sequelae = sterility, scarring
why chamydial infection increase from yrs to yrs
- Increased recognition, better testing → increased diagnoses → increased notifications
- Underestimate because often asymptomatic
Chlamydial Infection
Males: • Painful urination • Inflammation • Discharge: mucopurulent = v. similar to gonorrhoea Females: • Slight discharge, 80% asymptomatic • Inflammation of cervix • Burning on urination
Who is at risk?
Who is at risk?
• People in contact with asymptomatic, infected persons
• People with multiple partners
• Newborns of infected mothers
Chlamydia trachomatisEXAM
• Small
C. trachomatis Bind to Mucosa
Problems: • scarring ♀ blocks Fallopian tubes ♂ blocks urethra, vas deferens • infection spreads - salpingitis PID Result >>> infertility
TB transmission: aerosols
• Oldest documented infectious disease
• ‘Great White Plague’ / ‘Consumption’ (Satine in ‘Moulin Rouge’)
• Linked to poor sanitation / bad living conditions
• Chronic, slowly progressing infection – usu. of lungs
– also: kidneys, spine, brain
• Incidence:
– 16th-18th C: 25% with TB died – Today
• Globally: Each year:
– 2 billion infected (1/3 world’s pop.) – 2 million die
• Australia: 1300 new cases / year
contributors to increased incidence
• Immigration from endemic TB areas
• The human immunodeficiency virus epidemic – AIDS: infection with atypical strains
• Antibioticresistance
– development of multi-drug resistant TB
• TBtransmissionininstitutionalsettings – hospitals and prisons
• Reducedemphasisoncontrol&prevention
– Deterioration of the infrastructure for TB services: • No mobile clinics in Australia
– No new drugs
– Need for more research
causative organis for TB
Mycobacterium species
• Usu. M. tuberculosis
• M. bovis: cattle assoc’d:
– Vaccination / herd testing / milk pasteurisation
• Atypical strains (eg M. avium):
– Immunosuppression eg AIDS, cancer drugs – RT defect eg COAD
Pathogen:
Mycobacterium tuberculosis
• Aerobe • Fastidous • Slow-growing: 12-24hours/generation • Waxy lipids (mycolic acid) in cell wall – 40% lipid Hydrophobic – Resistant to disinfectants and stains – Acid fast
Identification
dentification Specimen = Sputum/phlegm – Stain: Acid fast rod in smear – Culture: Lowenstein Jensen medium (SLOW) • Confirmatory • Drug resistance – Molecular techniques (QUICK) • PCR
establish of TB infection important NO toxins produced
Enters URT in small aerosol droplets tubercle
phagocytosis: selective uptake
- Preferential uptake via complement and mannose receptors
- Inhibits uptake via Fc receptor
- Inhibits phagosome / lysosome fusion
- Possibly escapes from phagosome into cytoplasm
Tubercles
Tubercles
= Area of dead tissue surrounded by layer of macrophages and T lymphocytes
• Walled off, calcified
• Interior ‘cheese-like’: caseous necrosis – Trap bacteria
Essentials of CMI response to TB EXAM
• T helper cells and macrophages interact via receptors and cytokines
• Th1 cells secrete INTERFERON GAMMA (IFN-y)
• Macrophages secrete TUMOUR NECROSIS FACTOR (TNF)
• IFN-y plus TNF → OPTIMAL MACROPHAGE ACTIVATION
• Macrophage activation →
↑↑ proteolytic enzymes
↑↑ ROI
↑↑ RNI
some tubercle bacilli killed → giant cells & epithelioid cells
• Granulomas form
– consist of activated macrophages, T helper & T cytotoxic cells – may contain the infection
Outcomes of Cell-mediated Immune Response
• Control (latent infection):
– Activated macrophages kill M. tuberculosis
• Tubercles calcify = granuloma
• No symptoms, not contagious
• Reactivation ~20yrs / immunosuppression OR
• Uncontrolled (TB disease):
– Insufficient immune response
• Centre of tubercles liquefy
• Bacteria coughed up in sputum • Spread to blood, organs
• 50% mortality rate
Outcomes of Cell-mediated Immune Response control (latent infection)
• Control (latent infection):
– Activated macrophages kill M. tuberculosis
• Tubercles calcify = granuloma
• No symptoms, not contagious
• Reactivation ~20yrs / immunosuppression
Outcomes of Cell-mediated Immune Response uncontrolled (TB disease )
• Uncontrolled (TB disease): – Insufficient immune response • Centre of tubercles liquefy • Bacteria coughed up in sputum • Spread to blood, organs • 50% mortality rate
latency
• Bacteria survive:
– inside walled-off lesions in lungs
– in lung-associated lymphoid tissue – in other sites
• Reactivation up to 20 years later
TB disease Pulmonary Symptoms
- Chronic, bad cough: lasts > 3 weeks
- Chest pain
- Coughing up blood/sputum * Weakness or fatigue
- No appetite weight loss
- Chills, fever, night sweating
Symptoms
cough and weight loss
Extrapulmonary TB
Active TB in : – Kidneys
– Skin
– Lymph nodes – Bones / joints – Brain
screening for TB
- ChestX-ray
- Tuberculintest(Mantoux):
– Intradermalskininoculationoftuberculo- protein PPD
– Indicatescell-mediatedhypersensitivity
– DetectslatentandactiveTBorsuccessful BCG vaccination
-ve result:
• No skin reaction after 2d
• Never infected with M. tuberculosis
• Sometimes false –ve in tuberculous meningitis
prevention of TB
• Limit transmission • Improve living conditions – Housing – Nutrition – Social deprivation • BCG (Bacille Calmette-Guérin) vaccine
BCG Vaccination
• Attenuated vaccine: Live strain of M. bovis reduced in virulence
• Local reaction at site within 2-6 weeks: Papule ulcerates, heals, scar
• Initiates cell-mediated immune response
• Administration:
– Developing countries / endemic areas: children at birth
– Developed countries: high risk individuals: contacts, healthcare workers, teachers, immigrants from endemic countries
– Not used for:
• Immunosuppressed people • Asymptomatic HIV +ve
– Variable effectiveness:
• Research studies: UK 70% / 20years; India 0 - 90%
»> BCG provides some protection against TB because:
• M.tuberculosisandM.bovisBCGsharesomeantigenicepitopes • MemoryTcellsconferprotection
treatment TB
• Isoniazid,rifampicin,pyrazinamide ethambutol
• Two or more drugs given together – Minimises development of resistance
• Long-term: ~1year – Poor compliance – Resistance
• Spontaneous chromosomal mutations
• 2007 in Aust.: >11% strains showed some
resistance (Lumb et al)
The clinical specimen should be:
The clinical specimen should be: 1. Appropriate and adequately represent the diseased area
- Of sufficient quality /quantity for all tests
- Free of contamination
- Collected into appropriate containers 5. Obtained before antibiotics are administered (if possible) 6. Accompanied by notes from the physician
Transport
Transport Must be quick because pathogens can - Die quickly outside the body - Be overgrown by contaminants Minimise time lag between sampling and testing using: • special transport media • preservatives • dip slides • refrigeration/freezing
Laboratory reception:
Laboratory reception:
Need good documentation on receipt of specimen,
Assignment of ID no. and labeling
Examination:
Examination:
- Macroscopic
- Microscopic
- Using specific lab tests
Additional tests and reporting
Additional tests and reporting Initial report Preliminary report Presumptive diagnosis Final report Confirmatory diagnosis Supplementary report
B. Laboratory Testing Techniques
B. Laboratory Testing Techniques I. Microscopic methods II. Cultural methods III. Serological/immunological methods IV. Molecular methods V. MALDI-TOF VI. Typingsystems:strainswithinspecies VII. Antibiotic sensitivity testing
B. Laboratory Testing Techniques:I. Microscopic methods
- Unstained preparations
- Simple stains: Gram
- Special stains: specific features
- Immunofluorescence
I. Microscopic methods:
General stains
Haemophilus influenzae
present in CSF seen with Gram stain
Fungal cell staining pink with the H&E stain used in histology
I. Microscopic methods:
Special stains
Ziehl-Neelsen’s acid fast stain shows the presence of Mycobacterium in a sputum sample
Negative staining of a CSF sample with India ink shows encapsulated Cryptococcus cells
I. Microscopic methods:
Immunofluorescence
M. tuberculosis - labelled with fluorescent antibod
B. Laboratory Testing Techniques cont.
II. Cultural methods:
These allow:
- Isolation on solid media
- Identification through morphological and biochemical characteristics
- Pure isolate for other testing
(eg. biochemical tests, antibiotic sensitivity tests)
Three Types of Culture Media:
- Enrichment media
• For low numbers or fastidious organisms • Can be liquid or solid
• Enriched with blood, tissue infusions, etc
Three Types of Culture Media:
- Selective media
Separation of pathogen from mixture Selective ingredients - antibiotics, dyes, bile salts etc
Mannitol salt agar; Staphylococci can tolerate high salt media while most other bacteria are inhibited
Three Types of Culture Media:
- Indicator media
• Identify particular colonies among a mixture • Contain sugars or other substrates and dyes • Indicator media are usually selective as well
Chromogenic agar
MacConkey agar – selective and indicator medium
Culture: Biochemical Tests
• Series of tests allow presumptive ID – Sugar usage
– Chemical compound usage
Test for indole
- separate enterics such as E. coli (Ind+) from Enterobacter (Ind-)
Salmonella in Rappaport - Vassiliadis medium
+ve (left) & -ve (right)
Fermentation reactions
- used to separate enterics
Culture: Biochemical Tests
Multitest systems combine tests in a single kit eg. API
Limitations of culturing exam
Limitations of culturing
• Incubation time delays diagnosis and treatment
• Labour-intensive
• Improvements include automation and computer analysis
Laboratory Testing Techniques cont
III. Serological / Immunological methods:
eg precipitatin, haemagglutination, complement fixation, ELISA
• GenerallyusedtodetectvirusesorAbtoviruses • Useful for organisms that are difficult to
culture or dangerous
• Disadvantages:
- retrospective
- +ve result ≠ active infection
Laboratory Testing Techniques cont
IV. Molecular methods (Genus & species ID)
- Probe for genes
- specific virulence factors of pathogen - commercially available
- quick, accurate so long as gene present in high numbers - DNA Amplification
- increase copies of gene before probe - difficult/slow, dangerous pathogens - Microarrays
- simultaneous screening for different pathogens
IV. Molecular methods (Genus & species ID)Advantages and dis
IV. Molecular methods (Genus & species ID) • Advantages - highly automated - high sensitivity & specificity • Disadvantages - contamination->false +ve - inhibition->false –ve - can only detect known microbes - expensive equipment and reagents
V. MALDI TOF
Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight
- MALDI TOF detects abundant housekeeping proteins in the sample, especially ribosomal proteins
- Developed for bacteria; can also be used for fungi/yeasts; not very good for viruses as they don’t have much protein
- Now adopted by many large diagnostic labs worldwide due to speed, sensitivity and low cost
MALDI TOF limitation
Limitations:
• Doesn’t work for viruses
• Only as good as your database
• Can’t handle mixed samples where there are >1
organism present
• Doesn’t work on solid samples (stool; tissue)
VI. Typing systems - strain within species
• Tracesourceofoutbreak • Biochemical,culture,genetic - biotyping – phenotypic markers - serological typing – agglutination - molecular typing – restriction digestion-based eg. Pulsed Field Gel Electrophoresis (PFGE), sequence-based, eg. MultiLocus Sequence Typing (MLST)
VII. Antibiotic sensitivity testing Identified pathogen – which antibiotic?
• Tests ensure
appropriate treatment -> minimise severity of
disease
-> shorten recovery time
• Especially valuable for organisms with unpredictable AB sensitivity
C. Treatment
C. Treatment
• If disease is life threatening – initiate antibiotic therapy without waiting
• Antibiotic sensitivity test: - confirm treatment OR
- recommend more effective therapy
• Importance of fungi to human health
- Importance of fungi to human health
- Poisonous mushrooms (mycetism) • Food spoilage fungi and mycotoxins • Allergenic fungi and hypersensitivity • Commensal and pathogenic fungi
fungi
- Eukaryotic
- No chlorophyll
- Moulds / yeasts
- Usually found in moist dark habitats
- Usually aerobic, though some are anaerobic • Cell walls contain chitin
- Reproduce by spores
Fungal nutrition
Fungal nutrition
• Heterotrophic = other feeding
• Absorbtive nutrition - digest then ingest their
food
• Can live off dead matter = saprotrophic
• Or living matter - harm host = parasites - help host = mutualists
(symbionts)
Fungal classification
- where fungi fit with other organisms
Most fungi fit other place
Microsporidia are considered to be fungi as well despite a different phylogenetic placement
In fact, fungi are closer to animals than to plants…
…which helps explain why fungal diseases are so difficult to treat
- Mycetisms
Amatoxins • LD50 = 0.1 mg/kg • Damage the intestine, kidney and liver, by inhibiting RNA polymerase • Current treatments: 1) remove toxin from system 2) increase excretion of toxin 3) supportive care
- Food spoilage fungi and mycotoxins
Fungi can grow on and damage all types of foods – is this a problem?
Yes – especially if they make mycotoxins…
Mycotoxicoses
Acute • moderate-high levels toxin • specific, observable disease Chronic: • low-moderate levels of toxin • weight loss, infertility, failure to thrive Mycotoxin-associated diseases: • low-very low levels of toxin • general immunosuppression, increased susceptibility to infectious disease
Aflatoxins
Aflatoxins
• First discovered as “Turkey X disease”
• Aflatoxin = Aspergillus flavus toxin
• Most potent natural carcinogen known; associated with high levels of liver cancer in some developing countries
• Mycotoxins could be potential bioterrorist agents
- Allergenic fungi and hypersensitivity
3. Allergenic fungi and hypersensitivity • Main genera causing problems: § Aspergillus § Penicillium § Alternaria § Cladosporium • Grow under damp conditions and make allergenic spores and volatiles
- Commensal and pathogenic fungi – the discipline of medical mycology
- Superficial, cutaneous and subcutaneous mycoses – infect skin and underlying tissue
- Invasive fungal infections - The “Big Three”:
- Candidiasis
- Aspergillosis
- Cryptococcosis
Superficial mycoses
• Limited to the outer surface of hair or skin
• Infection of hair shafts known as piedras
• Some superficial mycoses known as tineas (Latin for grub or worm)
• Generally limited to tropics; Prevented by good personal hygiene; Treated with cleansing agents
Black piedra on hair shaft
Tinea versicolor caused by Malassezia furfur
Cutaneous mycoses: Dermatomycoses, (= ringworm, tinea)
Involve hair, skin and nails and evoke various cellular responses Tinea corporis – ringworm of the body Tinea capitis Ringworm of the scalp hair Tinea pedis – athletes foot
Dermatomycosis (cont) Tinea unguium – ringworm of the nails Tinea cruris – ringworm of the groin, or “jock itch”
Tinea unguium
– ringworm of the nails
Tinea cruris
– ringworm of the groin, or “jock itch”
The fungi that cause dermatomycoses
• “Dermatophytes” :
Trichophyton, Microsporum, Epidermophyton
• Characterised by distinctive spore structures • Easily passed from person-to-person via infected surfaces (eg locker rooms, showers)
Clinical aspects of dermatomycoses
• Blister-like lesions dry to leave scaly ring
• Without treatment may get hair loss, change
of hair colour, local inflammatory reactions
• Fungi shed on flaking skin and transmitted by
direct contact and via intermediates • Can be highly adapted to humans of
transmitted from animals or soil • Treat by changing environment
• Griseofulvin from Streptomyces griseus used for severe cases
• Blister-like lesions dry to leave scaly ring
• Without treatment may get hair loss, change
of hair colour, local inflammatory reactions
• Fungi shed on flaking skin and transmitted by
direct contact and via intermediates • Can be highly adapted to humans of
transmitted from animals or soil • Treat by changing environment
• Griseofulvin from Streptomyces griseus used for severe cases
Subcutaneous mycoses
Subcutaneous mycoses
• Caused by numerous soil fungi - introduced via puncture wounds; not spread from person-to-person
• Disease develops very slowly with fungi spreading along lymphatic channels - produce subcutaneous nodules that can drain to the skin surface
• Generally difficult to treat - some treated with antifungals; may require surgical excision
• Diagnosis is important as some bacterial infections mimic subcutaneous mycoses
Types of subcutaneous mycoses
• Chromoblastomycosis
– Caused by black moulds that
exist worldwide
• Maduromycosis
– Caused by Madurella mycetomatis; destroys subcutaneous tissue
– Often called a eumycotic mycetoma (fungal tumor)
• Sporotrichosis
– Caused by Sporothrix shenkii; can spread throughout body
Invasive Fungal Infections (IFIs)
• Few fungi can cause IFIs and these are mostly rare or geographically confined
• The Big Three of global importance are:
1. Aspergilus
2. Candida
3. Cry[tococcus
Aspergillus - Aspergillosis
- Primarily Aspergillus fumigatus (~85%) and A. flavus (~5%); other Aspergillus sp. ~5%
- Acquired from the environment as Aspergillus makes millions of tiny spores
Aspergillus - Aspergillosis
• Aspergillosis is an opportunistic infection – gets more serious with the level of immunocompromise of the host
– Aspergilloma (fungus ball) – Invasive pulmonary
aspergillosis
– Disseminated aspergillosis
IFIs: Candidiasis
• Candida albicans and other Candida species cause candidiasis
• Single celled yeast that often forms pseudohyphae • Is a commensal so can
be acquired endogenously • Can be passed from
person-to-person
• 4th most common cause
of bloodstream infections in hospitals
Candidiasis takes many forms
• Candidiasis of the vagina = vulvocandidiasis
• Candidiasis of the mouth
= oral candidiasis or thrush
• Candidiasis of skin - fingers (paronychia) and nails (onychomycosis)
Candidiasis cont.
• Chronic mucorcutaneous candidiasis
• Disseminated candidiasis following immuno-
suppression, surgery, severe burns, drug abuse
Cryptococcosis
• Caused by two medically important species of Cryptococcus :
- C. neoformans - C. gattii
• Both species have a large capsule, which is an important virulence factor
• Cause cryptococcal pneumonia, cryptococcal meningitis and meningoencephalitis
Process of cryptococcal infection
- Acquired from the environment (trees, soil, guano)
- The first site of infection is the lungs
- From there it can disseminate to other parts of the body, particularly the brain
Pathogenic Cryptococcus species
1.Cryptococcus neoformans • Opportunistic • Major AIDS pathogen • Worldwide 2.Cryptococcus gattii • Primary pathogen • Restricted distribution • Important in Australia
Diagnosis and treatment of fungal infections
• Diagnosisdonebycultureand microscopy
• Todayimmunologicalandmolecular
techniques are often used to speed up
diagnosis – eg ELISA with fungus-specific
antibodies or PCR with fungus-specific
primers (will discuss these further in the lecture on clinical microbiology)
• Treatmentisdifficultastherearefew effective anti-fungals (amphotericin, azoles, echinocandins)
- Often toxic since fungal cells are quite like our own
Factors contributing to the emergence of infections Change in the ecological niche or global environment
Change in the ecological niche or global environment - Weather patterns (droughts, floods), deforestation
- Hantavirus disease: increase mouse population after
heavy rainfall 1991-92 (El Niño phenomenon); first ever outbreak of Hantavirus pulmonary syndrome
- Cholera: El Niño phenomenon of 1997-98 large areas Africa flooded
Factors contributing to the emergence of infections
! Human behavior
! Human behavior
- AIDS epidemic in the wake of sexual behaviors and IV
drug users
- Population growth: overcrowding, poor sanitation (cholera)
- Population shifts and urbanization: disturb natural habitats & increase human contact remote environments- SARS
- Travel, globalization of world commerce: SARS
- Cultural practices: ritual preparation of bodies for burial (Ebola)
Factors contributing to the emergence of infections
! Technology applied to industry
! Technology applied to industry
- Enterohemorhagic E. coli: “hamburger diseases”
- Viral transmission with medical use of blood and tissues
Factors contributing to the emergence of infections ! Microbial adaptation and evolution
! Microbial adaptation and evolution
- Antibiotic resistance: vancomycin resistant enterococci (VRE)
post-operative hospital patients, UTIs, septicaemia, wound infections - Bird flu
Factors contributing to the emergence of infections
! Relaxation of public health controls
Multi-drug resistant tuberculosis
Factors contributing to the emergence of infections
! Bioterrorism
- Smallpox, Anthrax
Severe Acute Respiratory Syndrome-SARS
! Viral respiratory illness caused by SARS-associated coronavirus (SARS-CoV)
! First reported in Asia in February 2003 ! Outbreak contained by end of 2003
=
Symptoms of SARS
! High fever, headache, discomfort, body aches ! 10-20 percent of patients have diarrhea
! After 2 to 7 days, may develop a dry cough ! Most patients develop pneumonia
Transmission of SARS
! close person-to-person contact
- Primarily healthcare workers and household members
- Infrequent instances of community transmission
! respiratory droplets when infected person coughs or sneezes
- Fecal-oral - Fomites
! Where did SARS come from: jumped from animal host?
- SARS virus not related to other coronavirus subfamilies
SARS Containment Strategies
! Locally
- Home quarantine - Hospital closures - School closures
! Internationally - Travel advisories
- Health Alert Notices
- Screening departing airline passengers
- Information to passengers arriving from affected areas
Bird Flu- definition
! Viral disease affecting respiratory, enteric or nervous system of many kinds of poultry and birds
! Can be a virulent and acute disease with a short course and extremely high mortality
! Recognised in birds over 100 years ago, widespread in 1930s (‘fowl plague’), then sporadic and localised
! Influenza A: causes most influenza in humans
! All Influenza A subtypes are present in birds, particularly
waterfowl (principal reservoir)
! Influenza pandemics often caused by hybrid viruses - combination of avian & human viral genes (antigenic shift)
! Typically avian species need a period of adaptation to human host- limited ability to directly infect humans
T
seasonal H1N1
easily spread rarely fatal
H5N1
spreads slowly often fatal
Bird Flu and Humans
! Guangdong Province, China (1996): H5N1 virus in ducks
! Hong Kong (1997):H5N1 virus chickens and humans
- First example avian influenza transmisson birds to humans - 18 people hospitalised, 6 deaths
! Hong Kong (1999), Avian influenza H9N2 two children, both recover
! Netherlands (2003) H7N7 virus poultry workers and families - More than 80 cases reported, 1 death
Why are we concerned? H5N1
!Human to human transmission observed but still inefficient. What if:
1) recombination with readily transmissible human strain? 2) mutation (probably of H5)?
!H5N1 in pigs, are a ‘breeding ground’ for human and avian virus recombinations
!Virus is in migratory birds
!Worst case scenario: Initial estimates predicted 150 million deaths!!!
Swine Flu! (Pandemic H1N1/09 virus)!
!First reports outbreak in March 2009 (Mexico City)
! novel Influenza A H1N1 strain
! Reassortment of 4 known influenza A strains (1 human, 1
avian, 2 swine)
!Transmission is human to human (not from pigs)
! disease generally mild (compared to H5N1); death rate ~1%
Treatment for swine flu
!Tamiflu, Relenza
! global stockpiles are too small
! production has been increased: but can we make enough?
! drug resistance?
Ebola
Ebola
! RNA virus of the filovirus family, 5 subspecies identified
! Zaire Ebola virus cause of
current epidemic, ~70% mortality rate
! Human to human transmission occurs through direct blood or other body fluids of an infected human or other animal contact with infected bodily fluids
!Incubation period ~11 days, diagnosis by PCR
! Symptoms non-specific- fever, weakness, diarrhoea; internal
external bleeding, death from fluid loss/organ failure ! no licensed prophylaxis or treatment; supportive
How can we deal with emerging and re-emerging infections?! exam
! Enhanced surveillance (e.g. SARS, bird flu)!
!
!
! More effective control measures- new drug and vaccine development (e.g tuberculosis)
!
!Improvements in health standards: sanitation, living conditions (e.g cholera)
! Increased education into the risk and spread of infectious diseases (e.g HIV/AIDS, Ebola)
!
!
Why did it work?! ! the small pox vaccination and eradication
” Effective vaccine (life-long immunity)! !
“ No animal reservoir!
!
“ No subclinical infections, no carriers! !
“ One Variola serotype!
!
“ Major commitments by governments!
Aim of vaccination (immunisation ) exam
- Induce specific immunity to protect against infection/disease!
- Induce protective immunity without immunopathology!
- Eradication of major diseases in which humans are the only natural host!
- Immunisation can be ACTIVE or PASSIVE!
Passive Immunization!
• Confer protective immunity by transfer of specific antibodies!
!
• Rapidly effective but short lived life of antibody!
!
• No specific immunological memory!
!
• Immunity can be acquired either naturally or artificially !
Passive Immunization!
Natural Process!
Natural Process!
– Transplacental transfer!
– IgA in colostrum during breastfeeding!
E.g. Maternal Abs to diptheria, tetanus, streptococci, rubella, mumps and poliovirus!
Passive Immunization!
artificial
artificial
• Injection of recipient with preformed antibodies!
!
• Several conditions warrant the use of passive
immunization! !
– Deficiency in synthesis of Abs (agammaglobulinaemia,
hyper IgM syndrome)! !
– Exposure to a disease that will cause complications (e.g.
child with leukemia exposed to varicella or measles)! !
– Protection from poisonous snakes (anti-venom) and spider bites!
Passive Immunization!
Associated risks!
Associated risks!
• Immunization with Abs of different species (eg. horse) results in anti-isotype Abs (“serum sickness”)!
• May lead to immune complexes depositing in blood vessel walls and tissue-> tissue and organ damage!
Active Immunisation! !
!
• elicit long lasting, protective immunity in individuals !
!
• Eliminate pathogen (where possible)!
and in the community (“herd immunity”)!
• Can be achieved by natural infection or it can be acquired artificially by the use of VACCINES!
Criteria for useful active vaccine:! ! exam
Criteria for useful active vaccine:! !
! Safe- low risk of side effects, disease!
! Effective- stimulate good immunity in individuals at risk and !
!interrupt transmission of disease; eliminate disease if possible! !
!Long-lasting immunity!
!
!Cheap and readily available! !
! Stable!
!
!Easily administered!
Killed Inactivated Vaccines! !
!
! Micro-organisms killed by physical or chemical inactivation !
!(eg formaldehyde)! !
!eliminate infectivity without affecting antigenicity!
!
- Examples:e.g.SalkPoliovaccine(Inactivatedpoliomyelitis(IPV):
poliovirus type 1, type 2 and type 3; monkey kidney cells, inactivated with formaldehyde)!
- cholera, HepA!
Killed Inactivated Vaccines adv and dis exam
advantage:
safe from reversion to virulence!
!”acceptable for immuno-compromised individuals!
!”stable for transport and storage!
disavantage
“ less effective than live vaccines (cell-mediated I.R)!
!”multiple doses required !
!”requires adjuvants to enhance responses!
Live Attenuated Vaccines!
! Micro-organisms lose pathogenicity but retain capacity for
transient growth in host! !
! Extensive culture in “abnormal” media- accumulation of many mutations; or targeted mutations!
!
! Examples: Sabin polio vaccine: passage of virus through monkey kidney cells (e.g. 57 independent mutations in Sabin 1 strain)!
- measles, mumps, rubella, BCG!
Live Attenuated Vacci
Advantage
““authentic” immune response elicited!
!!
!”long-term protective immunity (replicate)! !!
!”usually single-dose, no adjuvants! !!
!”transfer non-immune contacts (consent?)
disvantgae
!” possible reversion virulent form (1 in 4 mill for Sabin) !
!”complications similar natural disease! !!
!”growth media/culture cells produce adverse reactions! !!
!”limited shelf life; refrigeration in tropics!
attenuated VS inactivated vaccines
killed virus vaccine need multipe dose to amplify
live virus vaccine have natural amflification
Attenuated vs Inactivated vaccines: The Polio Story!
Polio: major killer in 1950s!
!Salk vaccine (1954)- inactivated poliovirus!
!”injected, very safe, high levels circulating IgG, little mucosal immunity! ! “ ∴ stops paralysis, not virus spread in GI tract (virus can infect others)!
! Sabin vaccine (1961; OPV) - attenuated poliovirus !
!”oral vaccine; stimulates high level IgA, control virus GI, limit spread!
BUT: !
!”some Sabin vaccine-induced polio cases (8-10 per year in USA)! !”countries don’t use Sabin, Polio effectively controlled!
!”since 2005 in Oz only inactivated Salk vaccine used in children!
Other vaccine types (in use)! !
!Subunit vaccines (purified macromolecules)! !
a) Toxoids! !
a) Toxoids! !
“Exotoxins from organisms chemically inactivated (formaldehyde)!
“Develop Abs to toxins, neutralise effect of pathogen
e.g. DTPa
e.g. DTPa: Diptheria toxoid, tetanus toxoid, pertussis toxoid (+ other components of Bordetella pertussis)!
!
- 3 doses (im) before 6 months, booster at 4 years!
- second booster with adult formulation at 12-17 years
Other vaccine types (in use)! !
!Subunit vaccines (purified macromolecules)!
Recombinant or purified antigens !
Recombinant or purified antigens !
- Cloned genes expressed in bacteria, yeast or mammalian cells !
- Very safe, limited CTL response (processed as exogenous proteins)!
- Generally require adjuvants!
Subunit vaccines: Examples!
1Recombinant proteins!
2.Purified proteins !
Recombinant proteins!
HepB: recombinant hepatitis B surface antigen (HBsAg) protein! !
!Purified proteins !
Influenza vaccines: Fluvax, one of 7 available in Australia! !
- haemagglutinin of each of the 3 recommended strains.!..
Other vaccine types (in use)! !
!Virus-like particles!
!
!Virus-like particles!
- viral protein(s) derived from structural proteins of a virus. !
- resemble virus but lack viral nucleic acid: non-infectious. ! !
- as vaccines effective eliciting T and B cell immune responses. ! !
- Human papillomavirus & Hepatitis B vaccines approved for use!
Todays Vaccine Challenges!
• Millions of people worldwide die from malaria, tuberculosis and AIDS!
• No effective vaccines for these diseases; Why?!
• New vaccine approaches: improve current vaccines and
develop new ones!
• Lower vaccine costs and availability!
• Using knowledge of how immune system works to develop new vaccines!
cellular compartment and virus replication
All virus replication occurs within the host cell
• The cell provides:
– Nucleotide substrates – Energy
– Enzymes
– Other proteins
All virus genomes must produce mRNA which can be translated by host ribosomes
Virus life-cycle
Viruses need to:
- Get their genome into a host cell
- Produce mRNA; leads to viral protein synthesis
- Replicate their own genome
- Assemble new virions and leave the cell
Biosynthesis
Biosynthesis
ß synthesis of viral mRNA
ß synthesis of viral proteins
ß synthesis of viral nucleic acids
1.Attachment to host cells
Attachment to host cells
• Mediated by interactions between specific cellular receptors and proteins on the virion surface
• One virus may use more than one cellular receptor
• A cellular receptor may be shared by multiple viruses
• Co-receptors can be involved e.g. HIV uses CD4 as a primary receptor and CCR5 or CXCR4 as co-receptors
• The receptor can determine cell-type specificity and host range (other factors such as a post-entry block may also play a role)
Attachment of enveloped viruses
- Viralglycoproteinsarepresentintheviralenvelope
- Glycoproteinsinteractwithcellularreceptorstoattach
- Sometimesalsouseco-receptorsegHIV
Attachment of non-enveloped viruses
e.g. adenovirus, no envelope
polio bind to receptor
Entry into cells
Can happen in 2 ways
– Fusion of virus
envelope with cellular membrane
– Receptor mediated endocytosis, generally for non-enveloped viruses
fusin for entry
e.g. herpesvirus entry
Endocytosis
- Endocytosis is a fundamental process by which macromolecules are taken into cells
- After attachment all non-enveloped viruses (and some enveloped viruses) are taken up by endocytosis
- Release of the viral genome is dependent on conformational changes in viral proteins triggered by receptor binding or the low pH of the vesicle
- Enveloped viruses which are endocytosed can utilise fusion to cross the vesicle barrier
Virus life-cycle
1.Attachment and entry to host cell • Receptors • Membrane fusion • Endocytosis 2. Uncoating 3.mRNA synthesis- production of viral proteins 4.Replication of the viral genome 5.Assembly of new virions and release
uncoating
• Uncoating: release of viral genome from the capsid
• Generally poorly understood process
• Uncoating can occur at: HIV uncoating at
– the plasma membrane – within endosomes
– the nuclear membrane
biosynthesis
• mRNA synthesis- production of viral proteins • Replication of the viral genome
Biosynthesis
- GenerationofmRNA
- TranslationofmRNAintoviralproteins
- Replicationofthegenome
- Hostcellribosomesareusedfortranslationof mRNA; therefore the virus must make mRNA which can be read and translated by host ribosomes
Central dogma of molecular biology
DNA->RNA ->Protein
DNA viruses
Most DNA viruses must get their DNA genome into the nucleus as they use cellular DNA dependent RNA polymerase to produce viral mRNAs
Exception: poxviruses-brings their own DNA dependent RNA polymerase as it replicates in the cytoplasm and cellular DNA dependent RNA polymerase is found in the nucleus
Advantage of DNA over RNA is increased replication fidelity (accuracy), however this also means that DNA viruses are slower to evolve and adapt than RNA viruses
ss DNA e.g. parvovirus
ss DNA → ds DNA → mRNA → translation
• dsDNAmustbemadefrom ssDNA before mRNA is made
• ssDNAconvertedtodsDNA by cellular enzymes
• CellularDNA-dependent polymerase need to replicate viral genome
special viral proteins
tools of trade
replication
primary aim of all virus is to produce mRNA and to replicate their genome
if you r a DNA virus then generally you need rely on the host machineryto do this for u
however most RNA viruses are more independent
special viral proteins : RNA polymerases
During our own replication cycle we can copy DNA to DNA using a number of different DNA-dependent DNA polymerases (DdDp).
We can also transcribe DNA-> mRNA (DNA-dependent RNA polymerase) However we cannot: copy RNA->RNA
Therefore all RNA viruses (positive and negative sense) must encode for their own RNA-dependent RNA-polymerases (RdRp)
RNA pol
For negative sense viruses,
such as Influenza virus this RdRp must be carried in with the virus particle
i.e. it is a structural component of the virion
It needs to do this to first copy it’s negative sense genome into mRNA
ds DNA herpesviruses
ds DNA → mRNA → translation
ss(-) RNA
Eg: Orthomyxoviridae (influenza)
• Encodes an RNA-dependent RNA polymerase, which is a virion
component
• ss(-) RNA is transcribed by viral RNA-dependent RNA polymerase into (+) mRNA
• ss (+) RNA generated serves as a template to make new ss(-) virion RNA or to make proteins
dsDNA with reverse transcription
- Includes Hepatitis B virus
- partially double stranded DNA
- Cellular DNA-dependent RNA polymerase generates mRNA
- mRNA codes for proteins
- mRNA also reverse transcribed to DNA (brings in its own reverse transcriptase in the virion)
- Cellular DNA polymerase generates dsDNA
Assembly
Assembly
• Virus assembly occurs spontaneously within the infected cell
• Highly ordered process
• Proteins and nucleic acids brought together
– Self assembly
– Assembly using virus encoded proteins
• Not all viral proteins will necessarily be in the virion
1. Release via cell lysis
1. Release via cell lysis
• Infections with many viruses results in cell death and
destruction
• Structural integrity of the cell is damaged
• Poliovirus can induce cell lysis within approximately 8 hrs of infection
2. Budding from the host membrane
2. Budding from the host membrane
} Viral glycoproteins become embedded in host cell membrane
} Other viral structural proteins and viral genome assemble at plasma
membrane
} Viral particle bud from the cell
} Influenza requires neuraminidase (NA) to release it from host cell
3. The secretory pathway
3. The secretory pathway
• The secretory pathway is used by the cell to release proteins outside the cell
• Consists of a series of steps from the ER and golgi to a vesicle which fuses with the plasma membrane
exit via the secretory pathway e.g. hepatitis C
- Somevirusesusethis pathway for egress
* Allowsviralglycoproteinsto accumulate at intracellular membranes rather than at cell surface
cell to cell transmission e.g. HIV
- Some viruses can spread directly from one cell to another neighbouring cell (eg VZV)
- May cause fusion of infected cell with multiple uninfected cells- syncytia
how to stop a virus infection by targeting the replication cycle
• Target steps which are unique to virus lifecycle • Prevent attachment and entry – Neutralising antibodies – Receptor analogues (acts like cellular receptor) – Fusion inhibitors • Prevent biosynthesis – Inhibit viral polymerases – Viral RNA replication – Reverse transcriptase inhibitors-HIV • Prevent assembly – Protease inhibitors – Prevent capsid assembly • Prevent release – Neuraminidase inhibitors-influenza
Virusesentercellviaspecificreceptorswhichdefinehost range and tissue tropism
• Replicationinvolvesbothvirus-encodedandhost- encoded factors
• Pathwaysforsynthesisofprogenygenomedependson nucleic acid type and configuration
• Virusreplicationcycleoffersbothimmunologicaland antiviral drug targets
T
Viral pathogenesis-
important definitions
Pathogenicity [Gr. “pathos”, pain]: ability to cause disease
Pathogen: organism able to cause disease
Pathogenesis: means by which organism produces disease in the host
A result of:
• injury to discrete populations of cells
• in particular target organs
• producing signs & symptoms of disease in a given host
Virulence: “capacity” to produce disease
Pathogenicity
Pathogenicity [Gr. “pathos”, pain]: ability to cause disease
Pathogen:
Pathogen: organism able to cause disease
Pathogenesis
Pathogenesis: means by which organism produces disease in the host
A result of:
• injury to discrete populations of cells
• in particular target organs
• producing signs & symptoms of disease in a given host
Virulence:
“capacity” to produce disease
Key requirements for a virus to establish a successful infection
• Find a way to enter the host - viral entry
• Find a way to get through the host defenses -
immune responses
• Move through the host - dissemination/spread
To spread infection, viruses must:
• Find a way for new viral progeny to exit infected cells and host - transmission
Viral infection
Viral infection • Routes of virus entry into host • Spread of virus within host • Immune response • Types of infections • Spread to others - shedding, transmission • Fate of host
portal of entry
- skin
* respiratory tract • gastrointes0nal tract • genitourinary tract • conjunc0va
routes of virus entry skin
Skin may be penetrated by viruses as a result of:
-‐ mechanical trauma/injec0on (HPV,HIV, HSV, HBV) -‐ by the bite of an infected mosquito (arboviruses) -‐ by the bite of an infected animal (rabies)
Viruses o?en do not mul0ply locally but are carried away from site of infec0on: by bloodstream (HBV, arboviruses) or migration along nerves (rabies)
route of virus entry respiratory tract
• Major route of invasion:
1) For viruses causing local respiratory infection Eg. RSV, influenza, rhinoviruses
• Inhalation of viruses as
aerosolised droplets
– Coughing, sneezing
2) Other viruses causing asymptomatic initial infection then generalised spread
Eg. Chickenpox (VZV), Measles, mumps
• Host physical defenses
– Mucus lining and ciliated cells – Alveolar macrophages
routed of virus entry GIT
Eating, drinking, common route, faecal-oral
Systemic infection; enteroviruses, hepatitis A, poliovirus Localised infection; rotavirus, norovirus
Hostile environment
Virus survival depends on stability against the following Physical host defenses:
– Stomach - acidic
– Intestine - alkaline
– Digestive enzymes
– Bile detergents
– Mucous lining the epithelium
Routes of virus entry: Genitourinary tract
• Infection via sexual activity
– through tears/abrasions in lining - HIV
– infect epithelium - Papillomaviruses (local lesions) – infect underlying tissues
• Sensory neurons - HSV • Lymphoid tissue -HIV
• Host defenses
– Low pH (vagina)
– Mucous layer, macrophages
Routes of virus entry: Conjunctiva
- Abrasion,poorsanitation
- localisedinfections-conjunctivitis
- SpreadtoothersitesegHSV-1spreadtoneurons • Hostphysicalbarriers;tears,mucous,proteases
Types of viral infection
Types of viral infection
Localised infection
– Replication at primary site of infection – Spread to adjacent cells
Disseminated infection*
– Must breech physical and immunological barriers – Extends beyond primary site of infection
Systemic infection*
– Many organs affected
*Viruses need to move to target organ/tissue or preferred site of replication
Systemic infection*
Systemic infection*
– Many organs affected
*Viruses need to move to target organ/tissue or preferred site of replication
Localised virus infection
• Manyvirusesmultiplyinepithelialcellsatsiteofentry – produce a spreading infection
– then shed directly to exterior
• Respiratory infections – Influenza, rhinoviruses
• Gastrointestinal infections- Rotaviruses
• Skin infections- Papillomaviruses
disseminated virus infection
• Via circulatory system • organ invasion from virus migrating from blood vessels • directly through capillaries via bite or puncture • HIV - T lymphocytes, monocytes • EBV - B lymphocytes • Measles – monocytes • VZV- T lymphocytes
Viral dissemination
Via nervous system
Enter sensory or motor nerve ending • Sensory - dorsal root ganglia • Motor - motor neurons Eg. rabies, poliovirus HSV, VZVViral dissemination Via nervous system Enter sensory or motor nerve ending • Sensory - dorsal root ganglia • Motor - motor neurons Eg. rabies, poliovirus HSV, VZV
innate immunity
First line of defense • Skin • Mucous membranes • Gastric juice • Normal bodyflora Passive barriers, immediately effec2ve Second line of defense • Natural killer cells • (Macrophages) • (Eosinophils) • Cytokines (e.g. interferons) • Fever Ac2va2on required, Effec2ve in less than 24 h
adaptive immunity
Third line of defense • T-‐cells (cell killing, cytokines) • B-‐cells (an.body produc.on) Effec2ve after a few days
different types of infection e.g. influenza virus
- Rapid production of infectious virions
- Rapid resolution and elimination of infection
- Typical course for viruses such as rhinovirus, rotavirus, influenza virus
- Infections usually brief in immunocompetent hosts
- Virions and infected cells are completely cleared by the immune response
different tyoes of infections 2. persistent infection e.g. lymphocytic choriomeningitis virus
for slow virus infection e.g.HIV
• •
Unlike acute infection, persistent infection is not cleared effectively by the immune response
Virus particles or products continue to be produced for long periods Two types of persistent infection (i) slow/chronic and (ii) latent
(i) Slow/chronic
Virions produced throughout although symptoms may only appear late (eg lymphocytic choriomeningitis virus)
Alternatively, long periods of time with low virus production (eg HIV) or infectious genome spread (Measles sub acute sclerosing panencephalitis)
persistent infection LATENT
• After initial acute infection, infectious virus is completely cleared by the immune response
• Viral genome persists without any detectable infectious virus production
• Periodically virus can “re-awaken” from latency by a process called
reactivation
• Reactivation leads to new infectious virus with/without disease
• Herpesviruses characterised by their capacity to establish latent infection
Viral shedding:
Release of infectious virus from host
Transmission via aerosols
Some of the most medically important viruses are spread via aerosol droplets (sneezing and coughing) Examples: - Influenza virus - SARS coronavirus - rhinovirus - adenovirus
Viral shedding
Faecal-Oral Transmission
• Mostviralgastroenteritisoccursinplaceswherehumancongregationtakes place in confined places
Eg: school, daycares, cruise ships, restaurants and summer camps
• Thereisusuallyanexceptionallyhighamountofvirusshedinthewatery diarrhoea and vomiting associated with viral gastroenteritis
• However,becauseofthereasonthatthesevirusesareextremelycontagious in nature, they are also spread through other routes:
- Shellfish – when harvested in contaminated sewage waters
- When people have contact with objects or surfaces which are contaminated - Through infected people who may be preparing foods for others.
- When water used is contaminated with sewage
• Viruses spread via the fecal-oral route include: poliovirusHep A and Hep E, rotavirus and norovirus
Viral shedding
Transmission via blood and body fluids
sViral shedding
Transmission via blood and body fluids
• Blood- West Nile Virus, dengue virus, hepatitis C, HIV
• Blood transmission via biting insects, sexual activity, childbirth, exposure to contaminated blood
• Semen- HIV, CMV, hepatitis B
• Breast Milk- CMV
• Urine- hantaviruses
Transmission of viral disease exam
Successful Transmission depends on… 1) number of viruses shed - more is better
2) stability of virus in environment
– those resistant to desiccation better survival rates, most
viruses are unstable on prolonged drying
3) number of virus particles required to infect a new host
- dependent on virus and route of transmission
Routes of Transmission
Routes of Transmission
• Horizontal transmission • Vertical transmission
• Zoonoses
Transmission between humans
Horizontal
– Respiratory tract, cough, sneeze and saliva, blood
– Faecal-oral
– Sexual contact
Vertical Transmission
mother to child
• Prenatal - transplacental – HIV, rubella, CMV
• Perinatal - infected birth canal – Hepatitis B
– HSV
• Postnatal - milk or direct contact – CMV, Hepatitis B
Zoonoses - Transmission from animals
Biting arthropods (e.g.dengue virus) or vertebrate reservoirs (rabies virus)
What causes the symptoms?
1) Damage to cells due to virus replication:
- cell death by rupture of cell during virus release
- cell commits suicide in response to infection (apoptosis)
- infected cell loses function
- infected cell transformed by virus – tumours
What causes the symptoms?
2) Damage due to the host response to infection:
- Immunopathology - antibodies, and immune cells destroy infected cells - tissue damage
- Inflammation - Symptoms of immune cells infiltrating the area where virus infection has occurred
(swelling, rash etc.)
What does “Epidemiology” mean?
• Epidemiology is the study of health-‐event paSerns in a society
• It is the cornerstone method of public health research
• Viral epidemiology uses informa;on about: -‐ the virus and the host
-‐ behavioural and environmental factors
• Helps to determine the risk of disease in the human popula;on
• Helps to determine op;mal treatment approaches to clinical prac;ce and for preven;ve medicine.
Viral Epidemiology – The Virus
• Disease syndrome
Eg fever, encephali;s, lesions, respiratory
• Virulence
Eg subclinical, mild, severe, fatal
• Gene;c and an;genic varia;on Eg Influenza
• Transmissibility
Eg Highly infec;ous (cold flu), poorly infec;ous (rabies, HIV)
Viral Epidemiology – The Host
Innate immunity
-‐ Ability to ac;vate interferon, NK cells
Acquired immunity
-‐ Ability to produce an;bodies and virus specific T cells
Popula;on
-‐ size and dynamics (density, movement)
Eg Acute viral infec;ons (measles, mumps etc.) requires large dynamic popula;on Eg chronic infec;ons (HCMV, EBV, HIV, HPV) can survive in small isolated pops
Viral Epidemiology – Environmental factors
Climate -‐ seasonality Vector/reservoir host breeding -‐ interac;ons with suscep;ble individuals -‐ standing water Virus survival -‐ humidity and temperature Geography -‐ eg. physical isola;on or open borders
Viral Epidemiology – Behavioural factors
Socio-‐economic -‐ living condi;ons, water supply Personal hygiene -‐ hand washing, food prepara;on Travel habits -‐ increase in rapid air travel and tourism (eg SARS-‐coronavirus) Safe sex prac;ces -‐ condoms, health checks
Prevalence
- ‐ number of cases at a point in ;me (chronic illness)
- ‐ expressed as the propor;on of the popula;on who have the infec;on/disease at a given point in ;me
eg. Prevalence of HIV infec;on in Australia in 2004 was 0.07% of popula;on
Incidence
Incidence
- ‐ frequency of new cases over ;me (acute illness)
eg. approx 1.1 new cases of dengue per 100,000 popula;on were no;fied in Australia 2002
pandemic
Pandemic -‐widespreadepidemic
ie. spread of human disease across a large geographical region (eg across borders, con;nents)
Epidemics and pandemics result from outbreaks of disease
epidemics
Epidemic -‐peak in disease incidence in a given human popula;on, and during a given period, that substan;ally exceeds endemic baseline ie. occurrence in a community or regions of cases of disease clearly in excess of normal expectancy
endemics
Endemic -‐con;nuous occurrence of disease
The NaConal NoCfiable Diseases Surveillance System
• NNDSS established in 1990
• Co-ordinates the national surveillance of an agreed list of
communicable diseases or disease groups in Australia
• Provides a means of detecting/monitoring outbreaks of disease, epidemics/pandemics
• Data sent electronically from states and territories daily or several times a week. Published by the Dept Health and Aging
• 65 notifiable diseases (~ 27 are diseases caused by viruses)
whats outbreak
Disease with greater than expected morbidity or mortality
• Increase of cases above background
– background determined by surveillance data
index case
- Index case (also called patient “zero”): the first patient that indicates the existence of an outbreak
- The index case may indicate the source of the disease, the possible spread, and what reservoir holds the disease in between outbreaks
Epidemiological invesCgaCon
Epidemiological invesCgaCon
• Confirm outbreak
– Real or artifactual increase above background?
• Bettertestingfordiseasemayresultinartificialincrease
• Confirm diagnosis
– Consistent with clinical features and history
– Laboratory confirmation (where possible)
• Environmental investigation
– Samples (eg food, water, animals, surface swabs)
– Interview relevant staff
– Policies and procedures
• Patient data
– Demographics
– Onset and symptoms
– Exposure to known risk factors of disease
Outbreak Control Measures
Outbreak Control Measures
• Prophylaxis
• Vaccina;on (target at risk groups)
• Transmission control (eradicate vectors, quaran;ne, improved hygiene/sexual prac;ces)
communication
• Outbreak communication
– Publish, meetings with stakeholders, industry
etc.
• Outbreak documentation
– Especially if new source of infection is identified
– Lessons learned – improve investigation methods for the future
norovirus
• Genus Norovirus, Family Caliciviridae
• Single-‐stranded (+) RNA, non-‐enveloped viruses that cause acute gastroenteri;s
in humans
• responsible for 5-‐10% of serious gastroenteri;s cases
norovirus clinical features
- Adults and Children
* Usual incuba;on period is
virus outbreak investigation
Norovirus outbreak: wedding cake Epidemiological investigation 1. survey 2. same symptoms and time 3. risk factors for developing illeness p less than 0.001 4. environmental investigation 5. laboratory investigation - stool sample
Inflammation includes…
- A tissue-based reaction
- Pain
- Coagulation
- Destruction of microbes and host cells
- Healing of tissues
But, if blockade …inflammation
infiltration of tissues (granulomas, etc.)
particular forms (pannus, etc.)
distortion of tissues (fibrosis, etc.)
(chronic) DNA oxidised->neoplasias
Summary: body’s defence against micro-organisms
- Barriers:
outer covering of body – skin and mucous membranes (e.g. mouth, gastrointestinal tract), and secretions (mucous, tears) - “INNATE IMMUNITY” (OR “Innate anti-microbial response”):
inflammatory phagocytes (neutrophils and macrophages) plus plasma proteins (e.g. complement system), and Natural Killer (NK) cells - Lymphatic system:
drainage to lymph nodes – more phagocytes encountered - “ACQUIRED IMMUNITY” (or “Adaptive immunity”):
humoral (antibody-mediated) and cellular (leucocyte-mediated) immunity
thrombosis
- injury exposes collagen
- blood patelets attach tocollagen, forming initial haemostatic plug
- release of thromboxane A2and ADP from platelet
- activation of thrombin, formation of fibrin
- Aggregation of platelets, deposition of thrombin and stabilisation of initial plug to form “thrombus”.
what is definition of inflammation?
inflammation is the rxn of VASCULARISED tissues to LOCAL injury or infection, characterised by the MOVEMENT OF FLUID and LEUCOCYTES out of the blood into the extravascular tissue
what are the roles of inflammatory response?
- to localise micro-organisms
2. to eliminate micro-organisms, damaged cells, inanimate foreign particles, or antigens
- Chronic inflammation
- Chronic inflammation
• •
Adelayed,prolongedresponsetoinjuryorinfection, taking two major forms:
characterised by the presence in the affected tissue of many macrophages and lymphocytes (chronic granulomatous inflammation)
• •
characterised by the presence of large numbers of neutrophils (chronic suppurative inflammation)
Note:Continuousdestructionandrepairofnormal tissue are always seen in chronic inflammation.
types of leukocytes involved
- Acute = polynuclear
* Chronic = mononuclear
what is characterised for chronic granulomatous inflammation? e.g. TB
- characterised by the presence in the affected tissue of many macrophages and lymphocytes
giant cells
what is characterised for chronic suppurative inflammation ? eg.chronic abscess
charaterised by the presence of large number of neutrophils. and some macrophages rare
importance of inflammation
- Positive roles in promoting host survival
Direct role in host defence against micro- organisms.
Role in initiating adaptive immune response. Role in initiating tissue healing mechanisms. - Deleterious effects on host survival (complications of inflammation)
Acute inflammation:
• appendicitis (possibility of perforation) • meningitis (intra-cranial pressure)
Chronic inflammation:
• tuberculosis (impairment of lung function) • arthritis (incapacitation)
• initiation of cancer?
what causes CELSUS
- RUBOR (redness) caused by vasodilation
- CALOR(heat) caused by vasodilation
- TUMOR (swelling) caused by increase vascular permeability and increased granulation tissue
- DOLOR (pain) caused by physical and chemical stimulation of nociceptors
+ FUNCTIO LAESA (lost/altered function) caused by pain, reflex muscle inhibition, disruption of tissue structure, fibroplasia and metaplasia
what accompanying the fluid movement (exudate)
- inflammatory stimulus
- increase permeability
- increase transudation
- exudation
major forces at vessel wall
going out is the hydrostatic force P,
going inside the vessel is osmotic P (pi)
microcirculation
• blood vessels smaller than 100 μm (i.e. arterioles, capillaries, and venules).
exudation
Following the initial hyperaemia, fluid (containing plasma proteins) moves from the blood into the affected tissue.
This fluid movement leads to slowing of the blood flow in the affected area. The vessels appear “congested”. (Example follows).
Swelling results from the increased fluid content of the tissue.
Accompanying the fluid movement is emigration of neutrophils from the blood into the affected tissue.
• Normal conditions: – Membranes are impermeable to proteins (dots) – Filtration = reasborption • Inflammation: – Gaps – Proteins escape – Result: inflammatory oedema! 
T
oedema
accumulation of fluid extra-vascularly in tissues
PURULENT: adjective from “PUS” SUPPURATION: formation of PUS
T
How exudation?
In mild acute inflammation:
Through inter-endothelial gaps in post-capillary venules only.
In more severe inflammation:
Resulting from damage to endothelial cells in all micro-vessels.
Contraction of endothelial cells!

Carbon particles leave the vascular lumen and
enter in perivascular spaces
The increase in permeability is limited to post-capillary venules !
T
Why exudation?
Fluid: • dilution of toxins • increased flow into lymphatics Plasma proteins: • antibody • complement system components • fibrin system components Neutrophils: • destruction of micro-organisms
Sources of mediators of vascular permeability changes
- amines from mast cells and platelets
- lipid-derived from leucocyte and parenchyma
- plasma derived from plasma protein precursors
histamine
Source:
• mast cells, platelets
Activities:
• vasodilation (all microvessels, but primary action on arterioles)
• increased vascular permeability (post-capillary venules only)
what does vasodilation apply to
arterioles
vasodilation mediators
histamne, kenins, PG E2, PG I2
what does Increased vascular permeability apply to
post-capillary venules
increase vascular permeability mediators
histamine
kenins, C3a, C5a and Leukoreienes B4 and C4
injury and pain
injury cause: 1.release of PG and other mediators 2. platelet aggregation 3. mstocyte degranulation 4. activation of immuno-competent cells these all lead to the stimulation of nociceptive nerve endings
pain sensitisation
Sensitisation of pain receptors by prostaglandins
The 2 major components of inflammation
• VASCULARcomponent – Oedema
– Mediators (U4L11)
• CELLULARcomponent – Platelets
– Leucocytes
what are the neutrophil events in acute inflammation
- neutrophil margination in small vessels
- emigration from vessel into tissue
- chemotaxin release and chemostatic response
- phagocytosis of micro-organism
- killling of micro-organism
- death of neutrophil
Neutrophil adherence to endothelium
• Stage 1: “activation”
• Stage 2 “rolling”
– mediatedbyproteinscalled selectins,newly expressed on the endothelium.
• Stage 3: “adhesion”
– mediatedby integrin proteins,constitutively expressed on the neutrophil, binding to receptors on the endothelium.
• Stage 4: “emigration”
– also mediated by integrins
Phagocytosis & Degranulation
• Phagocytosis (“to eat and destroy”) – Attach
– Engulf
– Kill
• Degranulation and the oxidative burst destroy the engulfed particle
Chemotactic factors include:
- Complement components
- Arachidonic Acid (AA) metabolites • Soluble bacterial products
- Chemokines
Neutrophil movement is influenced by chemicals
CHEMOKINESIS: increased movement in random directions.
CHEMOTAXIS: increased unidirectional movement, towards the source of the signal.
TISSUE ACCUMULATION: nett increase in neutrophils in an anatomical location.
CHEMOTAXIN, CHEMOTACTIN, CHEMOATTRACTANT: agent that can induce chemotaxis in target cells.
direct acting chemotactic on neutrophil
Leukotrienes
Interleukin-8
Formylated peptides*
indirect acting chemotactic on neutrophil
TNF, IL-1 and LPS
“Indirect” agents act on other cells (e.g. macrophages) to induce release of chemotaxins (e.g. interleukin-8).
Interleukin-8, interleukin-1 and tumour necrosis factor are “cytokines”.
polarised neutrophil contain?
- lamellipodium at the front
- nucleus
3.granules
and uropod is at the back
polarised neutrophil contain?
- lamellipodium at the front
- nucleus
3.granules
and uropod is at the back
lacking ER and golgi and mitochondria, cant synthesis proteins, cant aerobic, rely on anaerobic environment
killing of bacteria and fungi by neutrophil
- phagocytosis of particles (forming a vacuole termed a phagosome)
- fusion of cytosolic granules with phagosome forming phagolysosome
3a. release from granules into phagolysosome of antimicrobial proteins
3b. oxidative burst (formation of ROS) directed into phagolysosome - killing of microorganism
Neutrophils never give up…
Neutrophils never give up…
• As they die, neutrophils release “neutrophil extracellular traps (NETs)”
• Made up of DNA strands with anti-microbial proteins attached
• Trap and kill bacteria
Formation of Neutrophil Extracellular Traps
- nucleus break down
- granule protein adhere to DNA strand
- neutrophil lyses, NETs released, can trap bacterial
Tissue accumulation of leukocytes in inflammation
• Early (minutes – hours):
maximum rate of migration of neutrophils.
• Later (hours – days):
maximum rate of migration of monocytes.
• Monocytes differentiate into macrophages.
Structural and biochemical properties of macrophages
Structure • single-lobed nucleus • no obvious cytoplasmic granules • prominent endoplasmic reticulum and Golgi apparatus • abundant mitochondria Biochemistry • large protein synthesis capacity • energy from aerobic respiration or anaerobic glycolysis (glycogen) Consequences for function • long-lived cell • can differentiate and proliferate
Cytokines is protein
Characteristics
• Produced by a cell involved in an immune or inflammatory reaction
• Modifies the activity of a
different type of cell
involved in that reaction
• Stimulates or inhibits cellular functions
Interleukin-1 (IL-1) and tumour necrosis factor (TNF) in inflammation
- Produced by several types of cell macrophages are major source
- Broadly similar activities Often act synergistically Products of different genes Bind to different receptors
- Over-production can contribute to tissue damage e.g. arthritis
Synergistic systemic effects of IL-1 and TNF in inflammation
injury and infection cause macrophage to release TNF and IL-1 and send to
- stem cells->leucocytes
- endothelial cell: expression of adhesion molecules
- T cell-> proliferation
- hypothalamua-> fever
Innate immune cell recognition of pathogens
• Identification of “non self”
– Evolutionarily conserved microbial components or products (“pathogen-associated molecular patterns”, PAMPs)
• Co-evolution of pattern recognition receptors (PRRs)
– Toll-like receptor system – Several others
Pattern recognition receptors:
an interface between the microbial world and immune system
T
Pattern recognition receptors (PRRs): Roles
- Detect microbial infections
* Trigger anti-microbial host defences • Initiate adaptive immune responses
Toll-like receptors recognise components of pathogens
e.g. TLR4 receptor recognise LPS
Regeneration:
Regeneration:
replacement of injured tissue by parenchymal cells of the same type
Repair:
replacement of injured tissue by fibrous tissue
Healing:
regeneration, repair, or some combination of the two
regeration definition
Regeneration: growth of cells and tissues to restore a lost structure
– Amphibians: amputated limb
– Mammals: liver after partial hepatectomy, cells of the haematopoietic system, epithelia of the skin and gastrointestinal tract
Requires intact tissue scaffold
Possible because cells have a high proliferative capacity and/or stem cells are present
Repair by fibrosis: involves granulation tissue
- Digestion of debris by macrophages: starts within 24 – 48 hours.
- Proliferation of fibroblasts and ingrowth of new microvessels, starting around 48 hours. [Vessels are essential to provide nutrients for fibroblasts to synthesise collagen].
- Fibroblasts migrate into margins of wound.
- Fibrosis (fibrous tissue production by fibroblasts – main component is collagen).
healing definition
Healing: tissue response to a wound (skin), inflammatory processes in internal organs, cell necrosis in organs incapable of regeneration (myocardium)
Combination of regeneration and scar formation (fibrous tissue) in variable proportions
– superficial wound of epidermis: healing by regeneration only
– deep wound with damage in the dermis: collagen scar
Mechanisms of
Tissue Proliferation & Growth
Recruitment of quiescent cells into the cycle into G1 phase - Most important
Shortening of cell-cycle time – less important
Proliferative capacity of cells
Labile cells:
• normally proliferate to replace cells that are continually being lost
• examples: gut epithelium; bone marrow stem cells
Stable cells:
• do not normally proliferate but capable of doing so when required
• examples: fibroblasts; endothelial cells; hepatocytes
Permanent cells:
• rarely proliferate
• examples: CNS neurons; cardiac myocytes
Which cells? Which mediators? for renewal
Which cells? Which mediators?
• Vascular cells
– Endothelial cells – Circulating cells
• Cells of the connective tissue (C.T.) • Elements of the C.T. matrix
FOUR stages of healing:
FOUR stages of healing: 1 – HAEMOSTASIS 2 – INFLAMMATION 3 - PROLIFERATION Granulation tissue (soft callus) Scar – Fibrosis (hard callus) 4 – REMODELLING Contraction Scar maturation / wound strength
Cytokines: signals between cells
the stimulus cu=ause effector cell to release cytokines which act on target cel Examples: • Tumour necrosis factor (TNF) • Transforming growth factor (TGF) • Platelet-derived growth factor (PDGF)
First intention for healing
incision wound become a scar, no granulation tissue
second intension for healing
- open would
- granulation tissue
- scar with collagen and fibroblast
Major differences 1st and 2nd intension
Major differences
• First intention:
– Margins can attach – Margins are sutured – No infection
• Second intention:
– Margins not ready to attach
– Infection
– Margins are devitalised: bruised or necrotic
Healing by first intention: closed aseptic wounds
- Haemostasis: within seconds to minutes • Inflammation: within minutes to hours
- Scab formation
- Migration of fixed cells: within 24 hours • Regeneration: 3 days
- Early scarring: 7-10 days
- Scar maturation: 1 month-2 years (very rare)
Healing by first intention: the principal steps

- platelet and fibrin
- neutrophil and macrophages
- fibroblasts
- connection between newly formed vessels, efficient angiogenesis
FOUR Stages of Healing:
- haemostasis
- inflammation
3.-proliferation
Granulation tissue (soft callus)- fibrin
Scar – Fibrosis (hard callus)
4 – REMODELLING
Contraction
Scar maturation / wound strength
haemostasis
first priority
STOP bleeding
- arteriole: vasoconstriction
- blood clt: platelet and fibrin
Phase 1: Haemostasis
Phase 1: Haemostasis
• Plateletplug
• Chemo-attraction
– PolyMorphoNuclear Neutrophils (PMNs) – Monocytes
– Lymphocytes
“chemoattraction”: attraction (of cells) by chemicals (mediators, such as cytokines, chemokines)
Phase 1: Haemostasis
Platelet Aggregation and Activation :
- PDGF: platelet-derived growth factor
- TGF-B: transforming growth factor
- IGF-1: insulin-like growth factor
- EGF: epithelial growth factor
- vWF: von Willebrand factor
- also release serotonin, which causes vasodilation and increased vascular permeability
inflammation
second priority WARD OFF BACTERIA - worst case scenario - recruitment of cells - scavenging of debris
Phase 2: Inflammation
• Neutrophils : 6-48 h
– Kill bacteria
– Secrete cytokines to cause inflammation
• Monocytes : 48-72 h
– Attracted to wound by cytokines (eg TGF-B )
– Extravasate and turn into macrophages
– Secrete more cytokines to initiate proliferative phase
• Lymphocytes
– Modulate extent of inflammation
proliferation
-Granulation tissue (soft callus) Scar
– Fibrosis (hard callus)
- scab: a natural dressing
- new structures
Phase 3: Proliferation
Begins day 2-3 after wounding Lasts 2-4 weeks • Angiogenesis • Fibroplasia • Epithelialisation
angiogenesis
angio=vessel
genesis=development
Phase 3: Proliferation
Angiogenesis - necessary to support a wound environment that can repair the injury
• Stimulated by
– macrophage-derived growth factors (FGF, TGF-α, TGF-B, TNF) – hypoxia, fibronectin and hyaluronic acid (found in the wound
matrix)
• Supplies oxygen and nutrients for fibroblast proliferation and production of wound matrix
phase 3:proliferation
- Fibroblasts migrate into wound on the fibrin scaffold left behind from haemostatic process
- Macrophage-derivedgrowthfactorsstimulateproliferationof fibroblasts (collagen synthesised at an accelerated rate)
Phase 3: Proliferation
Epithelialisation
• Cells in basal layer at wound edge flatten
• De-differentiation
• Pseudopodformation
• Migrate across wound - integrin receptors in underlying extracellular matrix
• Cells along margin divide to reform mature, multilayered epithelium
• For surgical wounds: complete wound coverage in 24-48 hrs.
Phase 3: Proliferation
Epithelialisation
• Process compromised by: • Bacteria
• Protein exudate from leaky capillaries • Necrotic debris
• Delayed epithelialisation = prolonged & profound inflammatory process
• Facilitated by clean moist wound
phage 3 proliferation. all form granulation tissue cause by the following
• Macrophages – growth factors • Epithelial cells – Migrate to the sides of edges • Endothelial cells – Angiogenesis • Fibroblasts – Extra Cellular Matrix formation
remodelling
- Contraction
- Scar maturation / wound strength
modulation of proteins:fibrin and collagen
transformation of cells:myofibroblasts
phase 4: remodelling wound contraction, second wk of healing
- Fibroblasts transform into myofibroblasts, this is stimulated by TGF-B, PDGF.
- Collagen synthesis and degradation is controlled by metalloproteinases (produced by macrophages, epidermal cells, endothelial cells and fibroblasts)
- Wound gains about 20% strength by 3 weeks. A scar is only about 70% of normal skin.
Phase 4: Remodelling
1. Contracture of the wound – Contraction of fibrin – Myofibroblasts – Fibroblasts and collagen lattice 2. re-epithelialisation and 3. organisation that will lead to would strength
Wound Kinetics
- 1 week –no change in the size of a wound
- 3 weeks almost 20% tensile strength
- A scar has only ~70% of strength of normal skin
- Healing time is logarithmically related to wound size
Factors affecting Healing:
Systemic nutrition vitamin def. age immune status other diseases Local necrosis infection apposition blood supply mobility foreign body
Complications of healing
• Infection
– Surgery axiom: “the resistance of wounds to infection is
proportional to their blood supply”
– Leucocytes deprived of oxygen have an impaired respiratory burst
• Foreignbodies
• Metabolic disorders/ microvascular problems: diabetes
• Keloids (hypertrophic scars)
