1
Q
What are the considerations for cell growth?
A
• Growth of a population of cells
- Distinguish between increase in cell numbers (hyperplasia) and increase in cell size (hypertrophy)
- Depends on integration of intra- and extracellular signals (checks on cellular physiology, growth and inhibitory factors, cell adhesion etc.)
¥ Growth at the cellular level (the cell cycle)
Ð Cell growth = increase in size (sometimes growth refers to this only) and cell division
Ð Cell cycle phases (G1, S, G2, and M)
Ð Progression controlled at three key checkpoints (restriction points)
¥ Loss of cells by programmed cell death (apoptosis)
Ð A coordinated program of cell dismantling ending in phagocytosis. Distinct from necrosis
Ð Occurs during normal development (e.g. separation of the digits, involution, immune and nervous system development)
Ð And in response to DNA damage and viral infection
2
Q
What are growth factors, cytokines and interleukins?
A
¥ Proteins that:
Ð stimulate proliferation (called mitogens) and maintain survival
¥ usually named after originally identified target e.g. EGF, FGF, Interleukins (IL2 & IL4), NGF
¥ but see also PDGF (platelet-derived GF) and IGF1 (Insulin-like GF – the main effector of pituitary growth hormone)
Ð stimulate differentiation and inhibit proliferation e.g. TGF
Ð induce apoptosis e.g. TNFα and other members of the TNF family
3
Q
What are the three broad classes of growth factors, cytokines and interleukins?
A
Ð Paracrine: produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor
Ð Autocrine: produced by a cell that also expresses the appropriate cell surface receptor
Ð Endocrine: like conventional hormones, released systemically for distant effects
4
Q
What happens in DNA replication?
A
- DNA is replicated semiconservatively (daughter cells inherit one parental and one new strand)
- New DNA is synthesized in the 5’ to 3’ direction from deoxynucleotide triphosphate precursors at a replication fork by a multienzyme complex (a replication machine)
- Fidelity is determined by base pairing (A=T, G≡C) and presence of a proof reading enzyme in DNA polymerase
- Synthesis of the new DNA strand uses an RNA primer and occurs continuously on the leading strand and discontinuously on the trailing strand (giving rise to Okazaki fragments, which are ligated together after removal of the RNA primer)
5
Q
What are the main stages of mitosis?
A
¥ Prophase (1) Ð Nucleus becomes less definite Ð Microtubular spindle apparatus assembles Ð Centrioles (yellow) migrate to poles ¥ Prometaphase Ð Nuclear membrane breaks down Ð Kinetochores attach to spindle in nuclear region ¥ Metaphase (2) Ð Chromosomes (blue) align in equatorial plane ¥ Anaphase (3) Ð Chromatids separate and migrate to opposite poles ¥ Telophase (4) Ð Daughter nuclei form ¥ Cytokinesis Ð Division of cytoplasm Ð Chromosomes decondense
6
Q
What drugs act on S-phase of the cell cycle?
A
• S-Phase active
- 5-Fluorouracil (an analogue of thymidine blocks thymidylate synthesis).
- Bromodeoxyuridine (another analogue that may be incorporated into DNA and detected by antibodies to identify cells that have passed through the S-phase).
7
Q
What drugs act on M-phase of the cell cycle?
A
Ð Colchicine (stabilizes free tubulin, preventing microtubule polymerization and arresting cells in mitosis – used in karyotype analysis)
Ð Vinca alkaloids (similar action to colchicine)
Ð Paclitaxel (Taxol, stabilizes microtubules, preventing de-polymerization)
5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen are used in treatment of cancer
8
Q
What are cell cycle check points?
A
Controls (involving specific protein kinases and phosphatases) ensure the strict alternation of mitosis and DNA replication
9
Q
What is the regulation of cyclin-CDK activity?
A
¥ Cyclical synthesis (gene expression) and destruction (by proteasome).
¥ Post translational modification by phosphorylation – depending on modification site may result in activation, inhibition or destruction
¥ Dephosphorylation
¥ Binding of cyclin-dependent kinase inhibitors
10
Q
What is the retinoblastoma protein?
A
A key substrate of G1 and G1/S cyclin-dependent kinases
- Unphosphorylated RB binds E2F preventing its stimulation of S-phase protein expression
- Cyclin D-CDK4 and cyclin E-CDK2
- Released E2F stimulates expression of more cyclin E and S-phase proteins eg DNA polymerase, thymidine kinase, PCNA etc. DNA replication starts.
11
Q
What are the two families of cyclin-dependent kinase inhibitors (CKIs)?
A
- CDK Inhibitory Protein/Kinase Inhibitory Protein (CIP/KIP) family (now called CDKN1)
¥ Expression of members of this family stimulated weakly by TGF and strongly by DNA damage (involving TP53)
¥ Inhibit all other CDK-cyclin complexes (late G1, G2 and M)
¥ Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs - Inhibitor of Kinase 4 family (INK4) (now called CDKN2)
¥ Expression stimulated by TGF
¥ Specifically inhibit G1 CDKs (e.g. CDK4 the kinase activated by growth factors)
12
Q
What is the sequence of events triggered by growth factors?
A
¥ Growth factor signalling activates early gene expression (transcription factors – FOS, JUN, MYC)
¥ Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors)
¥ E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)
¥ G1 cyclin-CDK complexes hypophosphorylate RB and then G1/S cyclin-CDK complexes hyperphosphorylate RB releasing E2F
¥ E2F stimulates expression of more Cyclin E and S-phase proteins (e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)
S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms. These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis.
13
Q
What are the options after DNA damage is detected at checkpoints?
A
- stop the cycle (CDK inhibitors)
- attempt DNA repair (nucleotide or base excision enzymes)
- programmed cell death if repair impossible (BCL2 family, caspases)
14
Q
What are the causes and mechanisms of cell damage/cell death?
A
- genetic
- inflammation
- physical
- traumatic damage
- infection
- chemical
15
Q
What are the genetic causes of cell damage/death?
A
- Abnormal number chromosomes (aneuploidy)
- Abnormal chromosomes (deletions/translocations)
- Increased fragility (Fanconi’s anaemia)
- Failure of repair (Xeroderma pigmentosa)
- Inborn errors (storage disorders ie Tay Sachs disease)
16
Q
What are the inflammation causes of cell damage/death?
A
- trauma
- thromboembolism
- atherosclerosis
- vasculitis
17
Q
What are the physical causes of cell damage/death?
A
- irradiation
- heat
- cold
- barotrauma
18
Q
What are the traumatic damage causes of cell damage/death?
A
- Interruption of blood supply
- Direct rupture of cells
- Entry of foreign agents
19
Q
What are the infection causes of cell damage/death?
A
- Toxic agents
- Competition for nutrients
- Intracellular replication – viruses/mycobacteria provoking an immune response
20
Q
What are the chemical causes of cell damage/death?
A
- Acids/corrosives
- Specific actions eg enzymes
- Interference with metabolism eg alcohol
21
Q
What is necrosis?
A
most common cause of cell death. Occurs after stresses such as ischemia, trauma, chemical injury
- Whole groups of cells are affected
- Result of an injurious agent or event
- Reversible events proceed irreversible
- Energy deprivation causes changes. (e.g. cells unable to produce ATP because of oxygen deprivation)
- Cells swell due to influx of water (ATP is required for ion pumps to work).
- Haphazard destruction of organelles and nuclear material by enzymes from ruptured lysosomes.
- Cellular debris stimulates an inflammatory cell response
22
Q
What is apoptosis?
A
programmed cell death. Designed to eliminate unwanted host cells through activation of a co-ordinated, internally programmed series of events effected by a dedicated set of gene products
23
Q
What is autophagic cell death?
A
Autophagy is responsible for the degradation of normal proteins involved in cellular remodeling found during metamorphosis, aging and differentiation as well as for the digestion and removal of abnormal proteins that would otherwise accumulate following toxin exposure, cancer, or disease. An example is the death of breast cancer cells induced by Tamoxifen.
24
Q
What are causes of necrosis?
A
- Usually caused by lack of blood supply to cells or tissues eg
- Injury
- Infection
- Cancer
- Infarction
- Inflammation
25
What are nuclear changes in necrosis?
1. Pyknosis: Chromatin condensation/shrinkage.
2. Karyorrhexis: Fragmentation of nucleus.
3. Karyolysis: Dissolution of the chromatin by DNase causing a fading in
the basophillia of the chromatin.
26
What are cytoplasmic changes in necrosis?
1. Opacification: denaturation of proteins with aggregation.
2. Eosinophillia: exposure of basic amino groups.
3. Complete digestion of cells by enzymes causing cell to liquify (liquefactive necrosis).
27
What biochemical changes occur in necrosis?
1. Release of enzymes such as creatine kinase or lactate dehydrogenase
2. Release of proteins such as myoglobin
28
What clinical investigations are associated with cell death?
1. Muscular dystrophy. Damaged muscles release creatine kinase and lactate dehydrogenase (M3 and M3H isoforms).
2. Heart attack. Damaged muscle cells release lactate dehydrogenase (H3 and H3M isoforms).
3. Bone and liver disease. Damaged tissues release alkaline phosphatase and lactate dehydrogenase isoforms (different isoforms specific to various tissues).
4. Haemolytic anaemias. Damaged red cells release LDH1/2.
29
What are the types of necrosis?
* Coagulative necrosis - typically seen in hypoxic environments. Cell outlines remain after cell death and can be observed by light microscopy (e.g. myocardial infarction, infarct of the spleen)
* Liquefactive necrosis - is associated with cellular destruction and pus formation (e.g. pneumonia)
* Caseous necrosis - is a mix of coagulative necrosis and liquefactive necrosis (e.g. tuberculosis)
* Fatty necrosis - results from the action of lipases on fatty tissues (e.g. acute pancreatitis)
* Fibrinoid necrosis - caused by immune-mediated vascular damage. It is marked by deposition of fibrin-like proteinaceous material in arterial walls, which appears smudgy and acidophilic on light microscopy.
30
What are the functions of necrosis?
- Removes damaged cells from an organism
| - Failure to do so may lead to chronic inflammation
31
What are the functions of apoptosis?
```
¥ Selective process for the deletion of superfluous, infected or transformed cells.
¥ Involved in:-
¥ Embryogenesis
¥ Metamorphosis
¥ Normal tissue turnover
¥ Endocrine-dependent tissue atrophy
¥ A variety of pathological conditions
```
32
What are examples of apoptosis?
1. Cell death in embryonic hand to form individual fingers
2. Apoptosis induced by growth factor deprivation (neuronal death from lack of NGF)
3. DNA damage-mediated apoptosis. If DNA is damaged due to radiation or chemo therapeutic agents, p53 (tumour suppressor gene product) accumulates. This arrests that cell cycle enabling the cell to repair the damage. If the repair process fails, p53 triggers apoptosis.
4. Cell death in tumours causing regression
5. Cell death in ciral disease (eg viral hepatitis)
6. Cell death induced by cytotoxic T cells (ie cellular immune rejection or graft vs host disease)
7. Death of neutrophils during an acute inflammatory response
8. Death of immune cells (both T and B lymphocytes) after depletion of cytokines as well of death of autoreactive T cells in the developing thymus.
Apoptosis helps eliminate the tail during the metamorphosis of a tadpole into a frog.
33
What factors influence the balance of life and death at the cellular level?
- cell-cell and/or cell-metric contacts
- growth factors
- cytokines
- disruption of cell-cell and/or cell-matrix contacts
- lack of growth factors
- death domain ligands
- DNA damaging agents
34
What are the types of apoptosis?
- DNA damage - p53-dependent pathway
- interruption of the cell cycle
- inhibition of the protein synthesis
- viral infection
- change in redox state
- withdrawal of growth factors (e.g. IL-3)
- extracellular signals (eg TNF)
- T cell or NK (natural killer)
35
What are caspases?
Cysteine Aspartate-specific proteases
* Caspases are cysteine proteases that play a central role in the initiation of apoptosis
* Most proteases are synthesised as inactive precursors requiring activation (usually partial digestion by another protease)
36
What does caspase activation lead to?
Caspase activation leads to characteristic morphological changes of the cell such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing.
37
What happens in apoptosis?
1. Single or few cells selected.
2. Programmed cell death.
3. Irreversible once initiated.
4. Events are energy driven.
5. Cells shrink as the cytoskeleton is disassembled.
6. Orderly packaging of organelles and nuclear fragments in membrane bound vesicles.
7. New molecules expressed on vesicle membranes stimulate phagocytosis, no inflammatory response
38
What nuclear changes occur in apoptosis?
1. Nuclear chromatin condenses on nuclear membrane
| 2. DNA cleavage
39
What cytoplasmic changes occur in apoptosis?
1. Shrinkage of cell. Organelles packaged into membrane of vesicles.
2. Cell fragmentation, membrane bound vesicles bud off.
3. Phagocytosis of cell fragments by macrophage and adjacent cell.
No leakage of cytosolic components
40
What biochemical changes occur in apoptosis?
1. Expression of charged sugar molecules on outer and inner surface of membranes (recognised by macrophage and enhances phagocytosis)
2. Expression of phosphatidylserine on extracellular leaflet of apoptotic cell
3. Protein cleavage by proteases, caspases
41
How do we activate the initiator caspases?
By induced proximity.
For example:
- In response to receptor dimerization upon ligand binding or
- Cytochrome C release from the mitochondria
42
What is cytochrome C?
* Mitochondrial matrix protein
* Known for many years to be released in response to oxidative stress by a ‘permeability transition’
* Any inducers of the permeability transition also eventually induce apoptosis
43
How is the release of cytochrome C regulated?
- Bcl-2 is a member a multi-gene family in mammals
- anti-apoptotic bcl-2, bcl-XL, others
- pro-apoptotic Bax, Bid, Bad, others
The bcl-2 family members form dimers
44
What is the link between p53 and apoptosis?
Mutations in the p53 gene are the most common mutations in cancer. Some mutations destroy the ability of p53 to induce apoptosis
45
What is asymptomatic carriage?
carrying an organism that can be passed on to others, but without experiencing any symptoms.
46
What is microbial antagonism?
* Balanced ecological niche – good health
* Bacterial overgrowth eg caused by antibiotics
The removal of the normal flora of gut through antibiotics which can cause Pseudomembraneous colitis by the overgrowth as C.difficile. Damages and disrupts the defence mechanism.
47
What are the general concepts of bacterial pathogenicity?
- host susceptibility and defences
- bacterial virulence
- host-mediated pathogenesis
- intracellular growth
- asymptomatic infections and carriage
48
What is host susceptibility and defences?
- Innate and adaptive immunity; immune-statu – young, old, HIV positive genetics
- Barriers; antimicrobial peptides; turnover; iron binding proteins
- Signalling, recruitment and inflammation
49
What is bacterial virulence?
- Capacity to cause disease – virulent strains eg of E.coli
- Genes: chromosomal, plasmids, transposons, bacteriophages, genetic exchange, evolution
- Capacity to cause disease has to be encoded in its genome – virulence factors
50
What are Koch's postulates?
Discovered that microorganisms are responsible for causing disease.
A microorganism has to:
• Be present in every case of the infection
• Be cultured from cases in vitro
• Reproduce disease in an animal
• Be isolated from the infected animal
• Not possible for non-culturable organisms eg leprosy, syphilis
• Need molecular tests eg PCR – eg HepC
• What about food poisoning – B.cereus and toxins – no infection with organism, but disease
51
What types of infection are there?
* Local: surface infection eg from a small wound on the skin – V.cholera, N.gonorrhoeae
* Invasive: penetrate barriers spread, wound – shigella, staph aureus
* Systemic: via blood, lymph to other sites – S.typhi, N.meningitidis
Effects at different site from colonisation – toxins, endotoxins
52
What are the stages of infection?
* Acquisition
* Colonisation – adherence
* Penetration
* Multiplication and spread
* Immune evasion
* Damage
* Transmission – shedding
* Resolution
Not all microbes need all stages
Disease not always required for transmission – asymptomatic shedding.
53
What are virulence factors?
- adherence factors
- invasion factors
- capsules
- endotoxins
- exotoxins
- siderophores
54
What are adherence factors?
colonise mucosal sites by using pills (fimbrae) to adhere to cells
55
What are invasion factors?
surface components and secreted effector proteins
56
What are capsules?
polysaccharides - protect from opsonisation and phagocytosis
57
What are endotoxins?
lipopolysaccharide on gram negatives - cause fever, changes in blood pressure, inflammation, lethal shock
58
What are exotoxins?
protein toxins and enzymes produced and/or secreted e.g. cytotoxins, neurotoxins, and enterotoxins
59
What are siderophores?
iron-binding factors to compete with the host for iron haemoglobin, transferring and lactoferrin
60
How do bacteria attach to host surfaces?
- Complex molecular interactions
- projecting fibrils on surface
- other membrane surface molecules expressed by bacteria
61
What are bacterial toxins?
‘cause damage to cells, tissues or the whole host organisms, thereby contributing to disease’
Exotoxin – released proteins found in Gram positive and gram negative
Endotoxins – powerful immunostimulants, portion of LPS found in gram negative cell walls, (also lipotheicoic acids of gram positive)
Toxins – often described by resultant action on different cells
- Cytotoxins
- Enterotoxins
- Neurotoxins
- Leukocidins
- Ciliostatic toxins
62
What is the functions of toxins in infection?
* Promote adhesion, survival or spread of bacterial – hyaluronidase, collagenases
* Damage or destroy cells cell membranes – phospholipases; pores
* Interfere with cell metabolism – cholera; diphtheria
* Affects nerves – neurotoxins – botulism and tetanus
Survival, growth and transmission?
63
How can toxins be classified by site of action?
* TYPE I – at cell membrane, not transported in
* TYPE II – on cell membrane, membrane damage
* TYPE III – intracellular effect after translocation
* Extracellular – cellular matrix or connective tissue
Exotoxins – make pores in molecules, stop signalling, bind to receptors being internalised and undermined metabolic potential of the cell, direct entry of toxin via a receptor to be released inside cell and then has an effect on a target inside the cell, or can be injected into the cell via injectosomes and the toxin then has a pharmacological action. A range of pathogens have a range of toxins that can do one or more of these mechanisms.
64
How do type I toxins act?
stimulates signalling proteins, guanyl cyclase (GC) changes intracellular cGMP
eg E.coli ST enterotoxin – Travellers diarhhoea
Short polypeptides of about 19 aa – it is heat stable because it cant be destroyed through cooking etc. Binds to and inhibiting a GC protein which normally responds to other signals, but modifying cGMP causes disruption of chloride channels, causing water efflux and acute diarrhoea.
65
What are lysins?
Lysins are toxins that cause membrane damage by forming pores etc. Pneumonia, food poisoning, infants, etc – there is a whole range of pathogens with virulence factors that work like this.
66
How can the same pathogen cause different types of diseases?
because they can bind to different types of cell
67
How can neurotoxins work?
Clostridium tetani and tetanus
Blocks synaptic transmission of things such as glycine, causing muscular spasm.
The tetanus toxin targets synaptobrevin which is part of the presynaptic vesicles. If you release the toxin it blocks the release of inhibitory neurones of the neurotransmitters, causing over excitation giving spastic paralysis.
Clostridium botulinum and botulism
Also used in botox. The toxin produced is also a zinc dependent endopeptidase, the binding component takes it to the neuromuscular junction synapse, blocking the release of acetyl choline. You get flaccid paralysis because of where it has gone to, even though it is a similar metabolic action as previous example – but the binding activity affects the results.
68
About Whooping cough...
Bordetella pertussis
* Invasive adenylate cyclase – injects its own AC to disrupt cAMP levels inside cells.
* Lethal toxin (dermonecrotic toxin) – superantigen – tracheal cytotoxin
* Pertussis toxin, PTx – changes cAMP but also acts as an adhesive molecule – multifunctional
Allows bacteria to adhere to the cilia allowing it to grow and bring more pathogens.
69
What is the complement system?
Originally described as a heat-sensitive component of serum that could augment the ability of antibiotics to inactivate antigen.
A group of plasma proteins; normally inactive
- More than 30 plasma and cell surface proteins
- 9 central components of the complement cascade
- complement component 1 (C1) to C9
Triggers activation of complement pathways
- part of both innate immunity and humoural immunity
Complement activation – purpose
- generation of products that mediate effector functions of complement
70
What are the rules of the complement system?
Complement activation involves a cascade of enzymatic cleavage of complement proteins (proteolytic cascade).
Amplification (each activated protease can generate multiple activated proteases in the following step)
Products of complement proteolysis attach covalently to microbial surfaces (or Abs bound to microbes/other Ags)
Stabilisation (complement proteins are inactive/transiently active when in fluid form; stable activation when deposited on microbes)
Regulatory proteins inhibit complement activation on healthy host cells; absent from microbes.
‘Specificity’ (complement activation on microbial surfaces whilst minimising complement-mediated damage to host cells)
71
What are the roles of the complement system?
To eliminate microbes
1. opsonisation of pathogens (facilitates phagocytosis)
2. inflammation (recruitment and activation of leucocytes)
3. Lysis of microbes (Membrane Attack Complex, MAC)
* To eliminate apoptotic cells/debris
* To promote clearance of Ag:Ab (immune) complexes
* To promote B cell activation
Complement is involved in disease pathogens.
72
What is the classical pathway of complement?
Classical pathway:
- Discovered first
- C1 interacts with antibodies (IgM, IgG) bound to microbes
- Effector mechanism of humoural (adaptive) immunity
- Initiated by binding of C1 to antigen-bound IgG or IgM
- C1 – multimeric protein complex of C1q, C1r or C1s
- C1q binds to antibodies (IgM, IgG1, IgG3)
- C1r and C1s are proteases
- C1 does not bind to soluble (free) antibody molecues
- C1 binds only to antibodies that are bound to Ag
73
What is the alternative pathway of complement?
Alternative pathway:
- Discovered later (but phylogenetically older)
- Direct recognition of microbial structures (innate immunity)
• Spontaneous cleavage of C3 in plasma (C3 tickover)
• C3 contains a reactive thioester bond (hidden)
• C3 cleavage induces conformational change in C3b, which exposes the thioester bond
• Exposed thioester bond reacts with amino or hydroxyl groups on surface of microbes to form ester bonds covalent attachment of C3b to surface of microbes/cells
In the absence of covalent attachment C3b remains in fluid phase, rapidly inactivated by hydrolysis further complement activation is stopped
74
What is the lectin pathway of complement?
Lectin pathway:
- MBL (mannose binding lectin) recognise terminal mannose residues on microbes (innate immunity)
- Ficolin recognises residues on microbes (innate)
75
What are the similar consequences for the different complement pathways?
Similar consequences:
- Generation of proteolytic enzymatic complexes
- C3 convertase: cleaves C3 in C3a and C3b
- C5 convertase: cleaves C5 in C5a and C5b
Proteolytic products of complement proteins identifies by lower case letter suffixes, ‘a’ for the smaller product; ‘b’ for the larger product.
C3 cleavage is crucial for all complement functions.
76
What does C3 converts do in the alternative pathway?
* Covalently tethered C3b binds Factor B
* Bound Factor B is cleaved by Factor D (plasma protease)
* Generates Bb (large) and Ba (small) fragments
* Bb remains attached to C3b
* C3bBb complex is the alternative pathway C3 convertase
*
C3 convertase has the main function to cleave more C3 molecules amplification of complement activation
• Newly generated C3b deposits on microbial surface
• C3a is a soluble fragment; mediates biological activities
77
What are the late steps of the alternative pathway?
* C5 convertase cleaves C5 in C5b (large) and C5a (small)
* C5a is a soluble fragment; has several biological activities
* C5b remains bound to C5 convertase
* C5v recruits C6 and C7
* C7 is hydrophobic inserts into lipid membranes
* C7 recruits C8 (three chains; one inserts into membrane)
* C5b-C8 complex recruits C9 C9 polymerases pores
* C5b-C9 is called MAC (Membrane Attack Complex)
* Lysis of microbe by MAC; entry of H2O via C9 pores
78
What is the C1 complement component?
- C1q hexamer: 6 globular heads connected via collagen-like arms to central stalk (‘brunch of tulips’)
- C1r (x2) and C1s (x2) form a tetramer
79
What happens with C3 convertase in the classical pathway?
- C3 convertase cleaves more C3 molecules
- Newly generated C3b deposits on microbial surface
- C3a is a soluble fragment; mediates biological activities
- Some C3b binds to C3 convertase complex C4b2a3b
- C4b2a3b is the classical pathway C5 convertase
- C5 convertase cleaves C5 and initiates late steps of complement activation
80
What is the complement activation in the lectin pathway?
* Initiated in absence of antibodies
* Triggered by microbial carbohydrate recognition by PRRs
* PRRs involved: MBL (mannose/mannan binding lectin), ficolins, similar structure to C1q
* MBL binds to mannose residues on microorganisms
* Ficolin binds to N-acetylglucosamine
* binding of MBL to mannose residues on microorganisms
* binding of ficolin to N-acetylglucosamine residues on microbes
* activates of MBL-Associated Serine Proteases (MASP1 and MASP2)
* MASPs cleave C4 and then C2
* Then proceeds similarly to classical pathway
81
How does the alternative pathway amplify complement activation?
C3b generated by classical/lectin pathway can bind factor B and generate C3bBb (alternative pathway C3 convertase) amplification
82
How is complement activation tightly regulated?
- Prevent complement activation on healthy cells
| - Limit duration of complement activation on microbes or Ag:Ab complexes
83
How are mechanisms of complement regulated?
- Inhibit formation of C3 convertase
- Break down/inactivate C3/C5 convertase
- Inhibit MAC formation
84
What are fluid phase regulators?
- Present in plasma, body fluids
- Factor H, properdin (activator) alternative pathway
- C1 inhibitor (C1ING), C4 binging protein (C4BP): classical/lectin pathway
85
What are membrane bound regulators?
- CD46 (MCP, Membrane Cofactor Protein), CD55 (DAF, complement Decay Accelerating Factor), CD59 (protectin), complement receptor 1 (CR1)
86
What is C1 INH?
Complement regulators – C1 INH
Inhibits formation of C3 convertase:
- C1INH dissociates C1r and C1s from C1q
- C1NH: blocks proteolytic activity of C1r and C1s
87
What are examples of complement regulators?
DAF, MCP, CR1 – present on surface of healthy cells
Factor H and C4BP – present in plasma
DAF, CR1:
- Displace C2a from C4b2a C3 convertase
- Displace Bb from alternative pathway C3 convertase
C4BP:
- Binds C4b and displaces C2a from C4b2a C3 convertase
Factor H:
- Binds C3b and displaces Bb from alternative pathway C3 convertase
MCP:
- Binds C3b, C4b co-factor for Factor I degradation of C3b and C4b
88
What is C3b degradation?
* Covalently bound C3b is degraded by plasma Factor I
| * MCP, factor H, C4BP and Cr1 are co-factors for Factor I
89
What is CD59 (protection)?
- Present on surface of healthy cells
- Binds to C5-C8
- Inhibits recruitment and polymerisation of C9
- Inhibits MAC formation
90
What can happen with deficiencies in complement proteins?
- C2, C4, C1q deficiency SLE-like syndrome
- C3 deficiency frequent serious infections with pyogenic bacteria
- Properdin, factor D deficiency infections pyogenic bacteria
- C5-C9 (MAC) deficiency disseminated infections with Neisseria (N. meningitides, N. gonorrhoeae)
91
What can happen with deficiencies in complement regulatory proteins?
C1 INH deficiency hereditary angioedema:
- Increased cleavage C4, C2
- Oedema in skin/mucosa abdominal pain, vomiting, diarrhoea, airway obstruction
DAF, CD59 deficiency paroxysmal nocturnal haemoglobulinuria:
- Recurrent intravascular haemolysis (RBC lysis)
Factor I deficiency:
- Uncontrolled cleavage of C3 C3 depletion
- Increased infections with pyogenic bacteria
92
What are the types and consequences of host damage as a result of bacterial infection?
1. Acute inflammatory changes
2. Damage by bacterial enzymes
3. Exotoxins
4. Endotoxin and other causes of sepsis
5. Superantigen mediated eg toxic shock syndrome
6. Immunopathology; immune complex disease (type III hypersensitivity), molecular mimicry, cellular immune response (type IV hypersensitivity)
93
What are symptoms of infection?
Symptoms of infection
Local symptoms (inflammation)
- Redness, swelling, warmth, pain, loss of function, Pus – pyogenic infection
Systemic symptoms
- Fever, rigors, chills, tachycardia, tachypnoea
Local symptoms mainly secondary to response of the local small blood vessels with
- Increased blood flow
- Increased permeability to fluid and plasma proteins
- Increased stickiness of vascular endothelium
- Emigration of phagocytes to site of infection
94
What is the inflammatory response triggered by?
Inflammatory response is triggered by release of products from the bacteria (eg toxins, enzymes) and amplififed by release of products from host cells (eg histamine, prostaglandins, leukotrienes, kinins).
Results is accumulation of phagocytes, mainly neutrophils (pus cells) and some monocytes, complement and other factors, and exudate at the site of infection.
95
What is pyogenic infection?
Pyogenic organisms include staphylococci and streptococci and meningococci.
• Host fights the bacterium
• Inflammation is the weapon of the host
• Damage to the hosts tissues may be as a result of the weapons of the bacterium such as toxic products
• Eg leucocidin from staphylococci
• Results in dead and dying neutrophils – Pus
…or damage may be due to inflammation itself.
96
What are the types of bacterial enzymes?
- protease
- lipase
- amylase
- nuclease
97
In what ways might bacterial endotoxins act in?
- Enzymatic lysis eg alpha-lecithinase
- Pore formation
- Inhibition of protein synthesis
- Hyperactivation
- Effects on nerve-muscle transmission
98
How might bacterial endotoxins be classified?
They may also be classified by:
- Molecular structure eg subunits
- Site of action eg enterotoxins
99
What are endotoxins?
* An integral part of the bacteria cell
* Found only in gram-negative bacteria
* Usually only released when the bacterial cell is damaged
* Evoke a variety of effects at many different sites
100
What are the actions of endotoxins?
Activation of macrophage/monocyte cells
Macrophage/monocytes release IL-1, IL-6, IL-8, platelet activating factor and tumor necrosis factor (TNF-alpha). These also stimulate production of prostaglandins and leukotrienes.
Cytokines act at various sites including endothelium, liver, clotting cascade.
Results in increased vascular permeability, hypotension leading to shock, fever, disseminated intravascular coagulation (DIC), multiple organ failure
- activates complement via alternative pathway
- can directly activate clotting cascade
- cause polyclonal expansion of B-cells and secretion of immunoglobulins
- increase phagocytic activate of macrophages
Results in increased vascular permeability, hypotension leading to shock, fever, disseminated intravascular coagulation (DIC), multiple organ failure
Results in increased vascular permeability, hypotension leading to shock, fever, disseminated intravascular coagulation (DIC), multiple organ failure
101
What is toxic shock syndrome?
Toxins produced by certain strains of:
- staphylococcus aureus – TSST (toxic shock syndrome toxin)
- streptococcus pyogenes – SPE (streptococcal pyrogenic exotoxin)
These toxins may act as superantigens.
102
What are super antigens?
Able to act simultaneously with MHC class II antigens on Antigen Presenting Cells AND specific Vbeta regions of T-lymphocytes
Activates macrophage/monocytes to elicit IL-1, IL-6, TNF-alpha and interferon-gamma
103
What is the different between humeral and cellular immunity?
Humoral immunity
- production of antibodies by B-lymphocytes
Cellular immunity
- T-lymphocytes
104
What is immune complex disease?
* Type III hypersensitivity reaction streptococcus pyogenes and glomerulonephritis
* Host produces antibodies against streptococcal antigens
* Antibodies bind to antigens to form immune complexes
* Immune complexes deposited in glomerular capillary walls in diffuse, irregular (lumpy-bumpy) distribution
* Activation of complement, influx of inflammatory cells and release of tissue-damaging enzymes etc
105
What is molecular mimicry?
• Throat infection with streptococcus pyogenes
• Antibodies against streptococcal antigens (cell wall) are produced
• These antibodies cross-react with antigens of the host (similar molecular structure)
• Site of cross reactivity include;
- Myocardium
- Synovium
- Brain
106
What are the cellular immune responses?
* Type IV hypersensitivity reactions
* T helper cells react to specific antigens eg Mycobacterium tuberculosis
* T cells release cytokines including (TNF-alpha) that activate macrophages
* These and other toxic products may cause tissue damage
* In chronic infection, degree of tissue damage may be extensive
* Characteristic response is the granuloma containing epitheloid and giant cells
* In TB, necrosis is characteristic
* Macroscopically, granulomas form ‘tubercles’
* Necrosis is described as caseous (cheesy)
107
About granulomas...
* Granulomas may form with other infections eg leprosy, syphilis (gummas) and schistosomiasis
* Necrosis may be absent in these conditions
* This may reflect different spectrum of cytokine release and cellular activation
108
How to design an immune system to defend yourself against bacteria?
1. Ability to recognise bacteria – attached or invaded
2. Ability to find the bacteria and ensure recruitment of defence mechanisms to that site
3. Mechanism for killing bacteria or cells containing bacteria
4. Mop up released toxins that still damage after bacteria are killed – defuse
5. Mechanisms to remember the bacteria so if they invade again our defences can be mobilised rapidly – memory
109
What are the stages of infection?
* Acquisition
* Colonisation – adherence
* Penetration
* Spread
* Immune evasion
* Damage
* Transmission
* Resolution
110
What are the types of infection?
* Local – surface infection; wound
* Invasive – penetrate barriers, spread
* Systemic – via blood to other sites
Effects at different site from colonisation – toxins, endotoxins
111
Where can bacteria be?
Epithelial surfaces – if not attaching or signalling = no harm
Free living in blood, extracellular fluid or tissue as planktonic growth or biofilms
Inside cells – cytoplasmic and vacuolar
System must constantly patrol areas of body – mostly mucosal surfaces
Concentrate resources in this vicinity – mostly what happens.
112
What are surface defences?
Surface defences – mucosal and epithelial surfaces
- Act as a watchdog - cells and signalling
- Shedding – removal or threat and rapid repair
- Normal flora – microbial antagonism and microbiome balance
113
What are natural barriers to infection?
- Skin
- Lungs
- Gut
- GU tract
- Eyes
114
What are the defences tissue and blood?
Usually involves tissue damage and controlled by feedback mechanisms
- Non-specific: transferring, complement, acute phase proteins (released by liver), phagocytes – monocytes and macrophages, PMNs – neutrophils
- Adaptive – antibodies, macrophage activation, T cells
115
What do PAMPs and PRRs do?
links inname immunity recognition to adaptive immunity via signalling
116
What is the inflammatory response to tissue injury?
Response to tissue injury – functions to brig serum molecules and cells to the site of infection.
1. Increased blood supply
2. Increased capillary permeability
3. Migration of cells from blood to tissue (PMNs, Mps)
Ordered process – regulated
Cardinal signs of inflammation: heat, pain, redness, and swelling – calor, dolor, rubor, tumor
[vasodilation, oedema, complement activation, mast cell degranulation, PMNs recruitment, clotting]
immune response against these may protect from disease.
117
What are virulence factors?
- promote colonisation and adhesion establish infection - e.g. adhesions
promote tissue damage - transmission? e.g. toxins
- subvert immune defences
118
What are the roles of complement?
Roles of complement:
- Induces inflammatory response
- Promotes chemotaxis
- Increases phagocytosis by opsonisation
- Increases vascular permeability
- Mast cell degranulation
- Lysis of cell membranes
Pro-inflammatory and antibacterial.
119
What is serum resistance?
Bacteria
- Capsules
- Lipopolysaccharides (LPS)
Host
- Complement
- Opsonisation
- Phagocytosis
decrease complement binding/activation
decrease opsonisation
decrease phagocytosis
Ineffective non-specific response…
Requires specific antibodies – opsonisation – phagocytosis and killing. Vaccines promote thi
120
About opsonisation and phagocytosis...
uncoated (by antibody) bacteria are phagocytosed poorly. On coating with antibody, adherence to phagocytes is enhances and increases complement fixing.
121
What are the antibacterial roles of antibodies?
- neutralisation of microbes and toxins
- opsonisation and phagocytosis of microbes
- antibody-dependent cellular cytotoxicity
some are too big to be phagocytosed so we can make antibodies against them. then they are recognised by specialised receptors on eosinophils which will degranulate and kill the organism.
- lysis of microbes
- complement activation
- phagocytosis of microbes opsonised with complement fragments (e.g. C3b)
- inflammation
122
What cells are involved in immune response to bacteria?
- various cytokines
- IFN-gamma
TNF
123
What are the various types of immune response?
Extracellular – staph aureus, strep pyogenes, e.coli
Complement – antibodies – phagocytosis
Cytoplasmic – shigella, listeria
CD8 TCTL – NKs – ADCC
Vacuoles – TB, salmonella, Yersinia
CD4 Th1 – Mp killing
124
What are the criteria for virus classification?
* Type and organisation of genome
* DNA/RNA
* Single stranded/double stranded
* Genome relatedness
* Viral replication strategy
* For example does it go through reverse transcription?
* Structure and size of the virion
* Does it have an envelope?
* Viral structure – most are icosahedral
125
What are the different human hepatitis viruses?
* A – enteric trans – vaccine
* B – non enteric – persistent – vaccine
* C – non enteric – persistent – phase I trial of a vaccine in April 2011
* D – non enteric – persistent
* E – enteric trans
126
About hepatitis A virus...
* Most common viral hepatitis
* Picornaviridae family
* Naked
* Icosahedral
* Single stranded RNA
* Positive sense
127
What are the clinical and epidemiological features of viral hepatitis A?
- Incubation (days) – 15-45, mean 25
- Onset - acute
-
Age preference – children, young adukts
- Transmission
- Foecal-oral - +++
- Percutaneous - unusual
- Perinatal - -
- Sexual- ±
128
About the hepatitis B virus...
* Hepadnaviridae family
* Enveloped
* 42nm
* icosahedral
* circular DNA partially double stranded
* complete virus and incomplete particles
* tubular filaments and spherical particles composed of envelope proteins – hep B surface antigen
129
What are the clinical and epidemiological features of hep B?
- incubation (days) – 30-150, mean 75
- onset – insidious or acute
- age preference – young adults, babies, toddlers
- transmission
- faecal-oral - -
- percutaneous - +++
- perinatal - +++
- sexual - ++
130
About hepatitis C...
- Blood transfusion before 1992
- Haemophilia – controls in place
- Intravenous drug abuse
- Body piercing and tattoos
- Needle stick injury
- Haemodialysis – control measures now in place
- Vertical transmission
- Sexual transmission
- Solid organ transplantation – donors now tested
- Nonsterilised injections – countries where needles are used
131
About the clinical and epidemiological features of hep C...
- Incubation (days) – 15-120 – mean 50
- Onset – insidious
- Age preference – any age, more common in adults
- Transmission
- Fecal-oral - -
- Percutaneous - +++
- Perinatal - ±
- Sexual - ±
- Clinical
- Fulminant 0.1%
- Chronicity >75%
- Cancer +
- Mortality – 0.2%
- Therapy – interferon 50% effective
132
About norovirus...
* Diarrhoea
* Common cause of infectious gastroenteritis in the UK
* Caliciviridase
* 27nm
* icosahedral
* non-enveloped
* single stranded RNA
* old name Norwalk virus
133
About rotavirus...
* infects people worldwide
* hospitalisation and deaths annually
* most common in infants and young children, but adults and other children can also become infected
* reoviridae
* double stranded RNA
* RNA is segmented – 11 segments
* Non-enveloped
* 75nm
* triple layer capsid
* icosahedral structure
134
What are the symptoms of GI infections?
- once exposed takes approximately 2 days for symptoms to appear
- fever
- vomiting
- diarrhoea – may last days
- abdominal pain
- no antivirals
- vaccines available – live oral vaccine
135
What is virus replication of rotavirus?
* infects cells of intestinal epithelium
* outmost layer has two important proteins VP7 and VP4
* these are important in virus attachment and entry
136
What is the replication cycle of viruses?
1. virus binds to receptors
2. enters cell by endocytosis
3. loses its outer layer
- unusual virus replication cycle
- within virus structure dsRNA can replicate
- this is because the virus has a protein VP6 that acts as a channel
- BP6 allows movement of RNA
Inside the virus core there are VP1, VP2 and VP3. These are involved in transcription.
* Viral proteins are made in infected cell cytoplasm
* Core assembly of single and double shelled particles in cytoplasm
- Entry of double layered particle into endoplasmic reticulum
- Acquires outer shell
- Released from cell
137
What are the symptoms of measles?
¥ Fever, cough, runny nose, red eyes, sore throat
¥ 2 or 3 days later small white spots (Koplik’s spots) may appear inside the mouth.
¥ 3-5 days after the start of symptoms, rash appears on face and spreads downward to the neck, trunk, arms, legs, and feet.
¥ When the rash appears, a person’s fever may spike
¥ After a few days, the fever subsides and the rash fades.
138
About the measles virus...
- Enveloped
- RNA single stranded
- Negative sense
- Paramyxoviridae
- Pleomorphic
- 100-300nm
139
What is the transmission of measles?
¥ Highly contagious
¥ 90% of non immune close contacts will become infected.
¥ Virus present in the mucus in the nose and throat of the infected person.
¥ Transmitted by sneezing or coughing, droplets spray into the air.
¥ Virus can live on infected surfaces for up to 2 hours
¥ Measles is a disease of humans – no animal reservoirs
140
About mumps...
* Most common symptoms:
* Fever, headache
* Muscle aches, Tiredness
* Loss of appetite
* Swollen & tender salivary glands under the ears on one or both sides (parotitis)
* Symptoms typically appear 16-18 days after infection.
* Transmission droplets – similar to measles
141
About the mumps virus...
- Paramyxovirus
- Pleomorphic
- Enveloped
- Helical nucleocapsid
- Ss RNA linear genome
142
About the rubella virus...
```
¥ An acute viral disease causes fever and rash
¥ mild disease in children and young adults
¥ Rash and fever for 2-3 days
¥ Spread through coughing and sneezing
¥ Togavirus family
¥ Enveloped
¥ ssRNA
¥ icosahedral
```
143
About congenital rubella?
* Birth defects if acquired by a pregnant woman:
* deafness
* Cataracts
* heart defects
* Damage to foetal brain
* liver and spleen damage
* More significant if infection happens early in pregnancy
144
About adenoviruses...
- Different serotypes exist
- Most commonly cause respiratory illness
- May also cause various other illnesses, such as gastroenteritis, conjunctivitis, cystitis, rash
- Adenoviridae
- No envelope
- Icosahedral
- DNA double stranded, linear
145
About papillomavirus...
* No envelope
* Icosahedral
* DNA genome
* Circular genome
* Double stranded
* Family papovaviridae
* Different serotypes
* Some cause infections in the genital tract
* Cervical cancer
146
About parvovirus...
- Non-enveloped
- Small 22mm
- DNA ss
- Nucleocapsid icosahedral
147
About the virology of parvovirus...
* The virus has a receptor which allows it to attach to erythrocyte progenitor cells
* Parvovirus B19 causes inhibition of erythropoiesis
* Shortened life span of RBCs
148
What is the clinical manifestation of parvovirus?
Most of the infections occur in children aged between 5 and 15 years. Infections are often asymptomatic.
Clinical manifestation can occur mainly in children – Erythema infectosum.
Ill child may have a ‘slapped cheek’ rash on the face and red rash on the trunk and limbs. Occasionally, the rash may itch.
* Can cause other problems
* Gloves and socks syndrome
* Arthropathy
* Transient aplastic crisis
* Chronic red cell aplasia
* Neutropenia, thrombocytopenia and pancytopenia
149
What are the diagnostic techniques of parvovirus?
* Serological
* IgM antibodies recent infection
* IgG past infection
* Viral DNA detection
150
What is the mode of transmission of parvovirus?
- Respiratory route
- Mother to the foetus transmission
- Incubation period between 10-21 days
- Viraemia present
151
What is the treatment of parvovirus?
* No specific drug to treat Parvovirus B19 infection
* In immunocompetent individuals
* Immune system produces antibodies
* And T cell response against virus
* Viral clearance occurs
152
About the influenza subtypes...
* Type A – most serious
* Affects mammals and birds
* Genetic cross-over between strains can lead to pandemic
* Subtyped according to its surface antigens – haemagglutinin (HA) and neuraminidase (NA)
* Type B – affects humans
* Causes minor outbreaks but not pandemic
* Type C – mild, only affects humans
* Endemic, symptoms similar to the common cold
153
What is the replication cycle of viruses (influenza)?
Viral attachment – uses haemagglutinin to attach to sialic acid. Internalised by endocytosis.
Inside endosome the pH is low. The virus envelope fuses with the endosome membrane. This triggers uncoating.
Viral nucleocapsid is released into the cytoplasm.
The viral RNA is single stranded – negative sense. There are 8 segments. Copies of viral RNA is made in nucleus – first from negative sense a positive copy is made. This is then copied into a negative sense to make the viral genome. mRNA is also synthesized.
mRNA is translated in the cytoplasm. Early viral proteins, that is, those required for replication and transcription, are transported back to the nucleus. Late in the infection cycle, the M1 and NS2 proteins facilitate the nuclear export of newly synthesized viral RNPs.
154
About viral assembly...
• RNA segments assembled within nucleocapsid.
| The assembly and budding of progeny virions occurs at the plasma membrane
155
How does the influenza virus mutate?
• The surface antigens of Influenza A mutate rapidly because:
- Virus has an enzyme involved in virus replication – RNA polymerase
- This enzyme has low selectivity
- Enzyme has no proof reading mechanism
• Mutants generated
• New mutant spread rapidly
• Two patterns of mutation: antigenic drift and antigenic shift
156
What is antigenic drift?
* Continual viral mutation
* Mutations are often minor with no effect on function
* Hanges accumulate to create new ‘drifted strains’
* Drifted strains produce illness
* Some strains co-exist
* Occurs ~ 2-8 years
157
What is antigenic shift?
* Type A only
* Genetic reassortment – mixing of genetic material between strains, when they infect the same host
* Creates novel strains
* Lack of immunity
* Rapid spread – pandemic
* Mixed strains form different species are more virulent and harder to control
* Spiral Flu Pandemic 1918 – killed between 40 and 100 million people, mostly young adults
158
What is the current strategy and its limitations?
The global influenza network (GIN) (part of WHO) estimate the strain of virus before the following season and recommend vaccines
Limitations:
- Errors in the estimate
- Manufacturing difficulties
- Time constraints
- Third world don’t have resources
- Antigenic drift continues (vaccine mismatch)
159
What are the early symptoms of Ebola infection?
Symptoms can appear from 2 to 21 days after exposure – only spreads after symptoms begin:
- Fever
- Headache
- Diarhhoea
- Vomiting
- Stomach pain
- Muscle pain
- Unexplained bleeding or bruising
160
About Ebola virus?
* RNA virus
* Enveloped
* Natural reservoir not yet identified
* Possibly that first patient infected through contact with infected animal – bats or primates
* Then person to person transmission
* Direct contact with blood or body fluids
* Contaminated objects – needles, syringes
* Enters through mucous membranes
* Breaks in the skin
* Or parenterally
* Infects many cell types
* Migrates from initial site of infection to lymph nodes then liver, spleen, adrenal gland
* Tissue necrosis
* Inflammation
* Organ failure
161
What is the relationship between lifestyle and infection?
LIFESTYLE
- Papillomavirus
- Genital warts
- Cervical cancer
- Number of sexual partners
EFFECT OF AGE
- Varicella zoster virus
- Shingles
EFFECT OF AGE AND BEHAVIOUR
- Epstein barr virus
- Glandular fever
- Teenagers
- Kissing
Other predisposing factors
CONCLUSION: Multiple factors determine whether we get an infection, and if so what is the severity of the disease likely to be.
162
What are the options of occurrence in primary infection?
• First encounter virus
Options:
- Replication at the site of entry
- Remain at the site of entry
- Influenza virus
- Rhino virus
Eg Replicate at site of entry then spread
o Varicella zoster virus
o Infection started in the respiratory tract
o Asymptomatic
o The patient infectious in this phase
o Local replication of the virus
o Spread
o Secondary site of infection is the skin
o Patient infectious
163
How does replication within the host occur?
1. Penetration of virus into skin. Local replication and entry of virus into cutaneous neurons.
2. Centripetal migration in the axon of uncoated nucleucapsids.
3. Synthesis of infectious virions.
4. Centrifugal migration of infectious virions to epidermis.
164
About secondary infections?
* Infection with a second organisms
* Following treatment
* Candida albicans – thrush
* Following an infection that compromises immunity
* Bacterial and fungal infections in HIV infected patients
* Bacterial pneumonia following viral respiratory tract infections
165
About re-infection?
* Influenza virus – once infected you are not permanently protected
* Can get re-infected with same virus
* Rhinovirus – common cold
166
What are routes of viral entry?
- Respiratory tract eg. Influenza virus, Rhinovirus, Varicella zoster virus
- Fecal-oral route; norovirus, rotavirus
- Blood; HIV, Hep B and C
- Body fluids; Epstein Barr virus
- Cuts in skin; papilloma virus, molluscum contagiousum
- Sexual transmission; papilloma virus, herpes simplex virus, HIV
- Animal bites; Rabies
- Insect bite transmission; haemorrhagic fever, lassa fever, arenaviridae
167
What are examples viruses that cause acute disease?
* Rabies
* Rhinovirus
* Influenza
* Rotavirus
* Infected – recover and clear virus
* Or die
168
What are viruses that cause chronic disease?
Hepatitis B and hepatitis C
Infection can in some cases be cleared
Often infection remains and immune system does not clear infection
169
What determines pathogenesis?
- Nature of the virus
- Compare influenza virus with herpes viruses
- Site of entry
- Tissue tropism
- Cell damage caused
- Ability of immune response to clear the virus
170
About immune evasion...
- Replicate in priviledged sites eg leucocytes
- HIV
- Cytomegalovirus
- Change viral proteins
- Immune response cannot recognise the virus
- Takes time to generate humoral and cell mediated immunity
- Virus at an advantage
- Examples?
- Release decoy particles
- Divert immune response
- Hepatitis B virus
171
About pathogenesis...
* Viruses that replicate in cells that do not divide may be at a disadvantage
* Transform cells
* Cancer causing viruses
* Papilloma virus
* Hepatitis B
* Hepatitis C
172
About papillomavirus...
* Non-enveloped
* Circular ds DNA
* More than 100 HPV types identified
* Classified into low risk and high risk
* High risk ones include HPV 16, 18, 31, 45
* Linked to development of cervical carcinoma and other malignancies
173
What is the cancer causing mechanism of viruses?
1. Epithelial cells differentiate
2. Normal squamous human epithelia grow as stratified layers
3. Only cells I the basal layer are actively dividing
4. As cells leave basal layer and move up they stop dividing
5. This is not helpful for the virus!
6. Virus replication process depends on the cell machinery
7. If cell is not replicative virus cant replicate
174
How does HPV cause cancer?
- Virus infects epithelial cells
| - Epithelial cells have different layers
175
How does papillomavirus have to override the cell cycle?
- Virus infects basal layers
- Migrates to cell nucleus
- Genome established as independent episome
- Copies of viral DNA made
176
What is some important information on the viral genome?
* Encodes 8 open reading frames
* Early genes – E1,2,4,5,6,7
* Late genes – L1,2 – code for structural proteins
* Important ones to remember are the E2, E6 and E7
177
About the genome of the virus that is not integrated...
•
Viral DNA exists as a circular plasmid
• Genes R6 and E7 are important
• They are transforming genes
• E6 interferes with a host cell protein – p53
• P53 is a tumour suppressor protein
• P53 regulates cell cycle
• E7 also interferes with a host cell protein – retinoblastoma tumour suppressor protein
• Function of Rb is to regulate cell cycle
• E7 binds with Rb and stops it functioning
• Viral gene E2 is also important
• Preducts of the E2 gene down-regulate expression of the E6 and E7 genes
• Therefore E2 is down regulating expression of cancer causing genes
178
What happens when HPV integrates into host genome?
- Virus DNA not circular anymore
- Integrated into host DNA
- The virus genome splits at about the middle of the E2 gene
- Linear fragment produced
179
About integrates HPV and cancer...
* E2 gene is split
* It cannot function and regulate transcription any more
* The upstream regulatory region (URR) is free to act
* It allows expression of E6 and E7
* These two are the transforming genes
Cancer causing mechanism
- Virus produces uncontrolled amounts of E6 and E7 the oncoproteins
- Papillomavirus stops the epithelial cells from existing the cell cycle
180
What is the virus interference regulation of cell division?
- Telomeres are structures at the end of chromosomes
- Telomeres regulate how many times an individual cell can divide
- Telomeric sequences shorten each time the DNA replicates
- Once the telomere shrinks to a certain level, the cell can no longer divide.
- Healthy human cells are mortal because they can divide only a finite number of times, growing older each time they divide
181
About HPV and telomerase?
- Telomerase is an enzyme that stabilises telomere length and stops its erosion
- HPV – E6 activated the telomerase gene
- Cancer cells produce telomerase, which keeps the telomere intact
- Cells do not act and continue to divide
182
About zika virus?
- In 1947 new virus was discovered
- The virus remained relatively unknown until recently
- The virus belongs to the Flaviviridae family
- Enveloped
- RNA – single stranded – positive sense
- Small – 50nm
- Transmission via mosquitos
- Initially no indication that it can cause human disease – but later serological surveys found that a high number of individuals in some countries had antibodies to the virus
- number of infants were born with microcephaly high in areas where Zika virus was first reported
- the full spectrum of foetal illness is yet to be determined
- studies looking at timing of infection suggests first trimester of pregnancy may be te greatest risk period
- early foetal loss has also been reported
- disease mechanism under investigation
183
What are the possible virus test types?
* Electron microscopy
* Virus isolation (cell culture)
* Antigen detection
* Antibody detection
* Nucleic acid amplification tests (NAATs eg PCR)
* Sequencing for genotype and detection of antiviral resistance
184
How can viruses be visualised?
they need electron microscopy, x20,000
185
About electron microscopy...
* Beams of electrons are used to produce images
* Wavelength of electron beam is much shorter than light, resulting in much higher resolution than light microscopy
* Electrons scatter when they pass through thin sections of a specimen
* Transmitted electrons (those that do not scatter) are used to produce an image
* Denser regions in specimen scatter more electrons and appear darker
* Viruses can be visualised with electron microscope
* Many different viruses
* Can be visualised but now rarely performed
* Possibly still useful for faeces and vesicle specimens
* Replaced by molecular techniques
186
About virus isolation in cell culture...
* Viruses require host cells to replicate and may cause a cytopathic effect (CPE) of cells when a patient sample containing a virus incudbated with a cell layer
* Old method, now replaced by molecular techniques
* But still needed for research or for rare viruses
* Led to discovery of hMPV and Nipha virus in last 20 years
* Use different cell lines in test tubes or plates
* Slow, but occasionally useful in anti-viral sensitivity testing
Virus alters appearance of cells in cell lines if virus is present.
187
What is the cytopathic effect?
- Different viruses may give different appearances
- Different cell lines may support growth of different viruses
- Identify virus using antigen detection techniques or neutralisation of growth
Depending on what we want to isolate we may choose different cell lines. It is hard to get hold of some lines eg human embryonic lung cells are hard to get hold of ethically, and also very expensive.
188
About antigen detection...
Viral antigens, usually proteins – either capsid structural proteins, secreted proteins can be detected. Infected cells may display viral antigens on their surfaces. Vesicle fluid.
- Nasopharyngeal aspirates (NPA) eg RSV, influenza
- Blood (serum or plasma) eg Hep B, dengue
- Vesicle fluid eg herpes simplex, varicella zoster
- Faeces eg rotavirus, adenovirus
These techniques are being replaced by nucleic acid detection methods due to improved test performance.
```
• A variety of different methods can be used
• Commonest methods are
- Direct immunofluorescence
- Enzyme immunoassay
- Immunochromatography methods
```
189
About immunofluorescence...
* Antigen (from infected host cells in sample) bound to slide
* Specific antibody (polyclonal or monoclonal) to that antigen is tagged to a fluorochrome and mixed with sample
* Viewed using a microscope equipped to provide ultraviolet illumination
190
About ELISA for antigen detection...
```
• Enzyme-linked immunosorbent assay
- A component of reaction is adhered to a solid surface
• Three formats:
- Indirect
- Direct (primarily antigen detection)
- Sandwich
```
191
How is detection of antigens by ELISA done?
1. Plate is coated with a capture antibody
2. Sample is added and any antigen present binds to capture antibody
3. Enzyme-conjugated primary antibody is added, binds to detecting antibody
4. Substrate is added, and is converted by the enzyme to detectable form
We use capture antibody that is specific to the antigen we are trying to find in the sample. Any antigen that is present in the sample will bind to the capture antibody. There is specific binding between the antigen and the antibody. Then we add the enzyme conjugated primary antibody which will bid to detecting Ab. Then we add the enzyme substrate. The substrate will be converted by the enzyme and we will see a colour change – eg colourless to yellow. We can then put it into the spectrophotometer and get the optical density reading from the plate reader.
192
What are immunochromatographic methods?
Eg diagnosis of dengue
- Flavivirus
- Arthropod vector
- Common infection in returning travellers
One of the antigen detection kits might use as point of care
193
What is diagnosis by antibody detection?
• When infected with a virus the humoral immune response takes place resuling in production of immunoglobulins
• IgM antibodies specific to the i virus are produced first
• IgM present for a variable period – usually 1 to 3 months
• As IgM declines IgG is produced
• Quantity of IgG rises
• Diagnosis can be made by
- Detection of IgM
- Or by demonstration of seroconservation
o Negative antibody first
o Then presence of antibody
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About serology for diagnosis?
• Indirect detection of the pathogen
• Diagnostic mode of choice for organisms which are refractory to culture
• Serology can be used to:
- Detect an antibody response in symptomatic patients
- Determine if vaccination has been successful
- Directly look for antigen produced by pathogens
• Serological tests are not limited to blood and serum
- Can also be performed on other bodily fluids such as semen and saliva
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About the use of serum in virus detection...
¥ Produced from processing blood
Ð Blood is coagulated with micronized silica particles
Ð Gel used to trap cellular components
¥ Routinely serum tubes are centrifuged for 10 min at 1000xg
¥ Supernatant (serum) is removed and stored
Ð 4ºC short term
Ð -20ºC long term
Ð Routinely serum tubes are centrifuged for 10 min at 1000xg
¥ Serum contains proteins, antigens, antibodies, drugs (some)
and electrolytes
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What can detection of antibody and antigen be used for?
¥ This is useful for some infections such as
Ð Hepatitis B
Ð HIV
Ð Hepatitis C
¥ This is because it allows us to establish whether acute or chronic infection
¥ This may have therapeutic implications
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What are molecular diagnostic tests?
¥ Nucleic acid amplification (NAAT)
Ð e.g. PCR although there are other examples
Ð Can detect RNA or DNA
Ð Ability to multiplex using fluoresecence probes i.e. can look for several targets in one sample
Ð May be qualitative or quantitative
Ð Requires nucleic acid extraction prior to the amplification
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What are the advantages of using NAATS?
Ð May be automated
Ð Highly sensitive and specific, generates huge numbers of amplicons
Ð Rapid
Ð Useful for detecting viruses to make a diagnosis
¥ At first time of infection e.g. measles, influenza
¥ During reactivation e.g. cytomegalovirus
Ð Useful for monitoring treatment response
¥ Quantitative e.g. HIV, HBV, HCV, CMV viral loads
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What is organism sequencing?
¥ DNA or RNA viruses
¥ Used to predict response to anti-virals e.g. for HIV in Rx naïve patients, or if clinical suggestion of resistance in drug experienced patients
¥ Consensus sequence based on clinical observation of resistance or in vitro evidence
¥ Minority species sequencing
Ð May be selected by treatment
¥ Useful for outbreak investigation by showing identical sequences in suspected source and recipient
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About combinations of methods of detection e.g. in HIV...
¥ Antibody and antigen detection for initial diagnosis
Ð Screening test (EIA)
Ð Confirmatory test (EIA)
Ð Viral load (NAAT) at baseline and to monitor treatment response
Ð Quantification of virus in blood
¥ Resistance testing (sequencing)
Ð Look for mutations known to confer resistance before and during treatment
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About screening for virus detection...
¥ Testing for specific infections in at risk groups
Ð e.g. HIV, HBV and HCV
¥ Testing because it may have an implication for others e.g. antenatal
Ð HIV, HBV, rubella
¥ In these situations the patients are asymptomatic
¥ Needs a sensitive screening test
¥ May have some false positives, so need
¥ A specific confirmatory test
Semester 4 Revision
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