Mechanisms of Disease Flashcards
(371 cards)
1
Q
What are the 3 general considerations for cell growth?
A
- Growth of the cell population
- Growth @ cellular level (cell cycle)
- Loss of cells by programmed cell death (apoptosis)
2
Q
Characteristics of the growth of a population of cells + what does it depend on?
A
- Distinguish between increased cell #s (hyperplasia)
- And increased cell size (hypertrophy)
- This depends on the integration of intra + extracellular signals
- Checks on cellular physiology, cell growth + inhibitory factors and cell adhesion
3
Q
Characteristics of growth @ a cellular level (the cell cycle)
A
- Cell growth is the increase in size and sometimes the growth to this only + cell division
- Cell cycle phases (G1, S, G2, M)
- Progression is controlled at 3 key checkpoints - which are restriction points
4
Q
Characteristics of the loss of cells by programmed cell death (apoptosis)
A
- Coordinated program of cell dismantling, ending in phagocytosis
- This is distinct from necrosis
- This happens during normal development:
• Separation of digits, involution, immune + nervous system development
• + in response to DNA damage and viral infection
5
Q
Growth factors, cytokines and interleukins characteristics
A
- These are proteins
- Proteins that stimulate proliferation, which are called mitogens + maintain survival
- This is usually named after originally identified target:
• EGF, FGF, IL2 + IL4, NGF - Stimulate differentiation + inhibit proliferation, like TGFb
- Induce apoptosis:
• TNFa + other members of the TNF family
6
Q
What are the 3 broad classes of growth factors, cytokines and interleukins
A
Paracrine
Autocrine
Endocrine
7
Q
What are the 3 broad classes of growth factors, cytokines and interleukins - paracrine characteristics
A
- Produced locally.
- To stimulate proliferation of a different cell type that has appropriate cell surface receptor
8
Q
What are the 3 broad classes of growth factors, cytokines and interleukins - Autocrine characteristics
A
- Produced by cell that also expresses appropriate cell surface receptor
9
Q
What are the 3 broad classes of growth factors, cytokines and interleukins - Endocrine characteristics
A
- Like conventional hormones
- Released systematically for distant effects.
10
Q
What are proteins that stimulate proliferation called + their role
A
- Mitogens.
They maintain survival
11
Q
what are the named 4 steps of DNA replication
A
- SCR of DNA. Daughter cells get 1xparental, 1xnew strand
- New DNA made, in 5 to 3 prime direction. From deoxynucleotide triphosphate precursors @ a replication fork. By a multienzyme complex, replication machine
- Fidelity determined, by base pairing (GCAT). Presence of proof reading enzyme in DNA polymerase
- Synthesis of a new DNA strand, using RNA primer. Occurs continuously on leading strand + discontinously on the trailing strand.
12
Q
What are the 4 main stages of mitosis?
A
- Prophase
- Metaphase
- Anaphase
- Telophase
13
Q
What happens during prophase?
A
- nucleus becomes less definite
- microtubular spindle apparatus assembles
- centrioles migrate to poles
14
Q
What happens during prometaphase?
A
- Nuclear membrane breaks down
- Kinetochores attach to spindle in nuclear region
15
Q
What happens during metaphase?
A
- chromsomes align in the equatorial plane
16
Q
What happens during anaphase?
A
- chromatids separate and migrate to opposite poles
17
Q
What happens during telophase?
A
- daughter nuclei form
18
Q
What happens during cytokinesis?
A
- division of cytoplasma
- chromosomes decondense
19
Q
Describe the drugs that act on the cell cycle:
S phase active
A
- 5 Fluoro uracil
- This is an analogue of thymidine. Blocks thymidylate synthesis
- Bromo deoxyuridine
- Another analogue that might be incorporated into DNA + detected by antibodies, to identify cells that have passed through the S phase
20
Q
Describe the drugs that act on the cell cycle:
- M phase active [PVC]
A
- Paclitaxel • Taxol, stabilises microtubules • Prevents de polymerisation - Vinca Alkaloids • Similar reaction to colchicine - Colchincine • Stabilises free tubulin • Preventing microtubule polymerisation + arresting the cells in mitosis - used in karyotype analysis
5 fluorouracil, paclitaxel, the vinca alkaloids + tamoxifen are used in cancer treatment
21
Q
What are the cell cycle checkpoints?
A
- There are controls that involve specific protein kinases + phosphatases
- These ensure strict alteration of mitosis + DNA replication
> DNA completely repicatied, DNA not damaged
Chromosomes aligned on splindle
Cells responsive to growth factors - this is the main site of control of cell growth
Restriction point - DNA not damaged, cell size, metabolite/nutrient stress
22
Q
Cyclin dependent kinase activity controls cell cycle progression : describe the regulation of cyclin CDK activity
A
- Cyclical synthesis (gene expression) + destruction (by proteasome)
- Post translational modification, by phosphorylation
○ Depending on the modification site might result in:
§ Activation, inhibition or destruction - Dephosphorylation
- Binding of cyclin dependent kinase inhibitors.
23
Q
Describe the characteristics of the retinoblastoma protein
A
- It is a key substrate of G1 + G1/S cyclin dependent kinases
- Unphosphorylated RB binds E2F.
○ Prevents its stimulation of S-phase protein expression - Released E2F stimulates expression of more cyclin E + S phase proteins
DNA polymerase, thymidine kinase, PCNA etc. DNA replication will start
24
Q
Describe the characteristics of cyclin dependent kinase inhibitors
(There are 2 families) - describe Family 1 of cyclin dependent kinases [CDKN1]
A
- CDK inhibitory protein / kinase inhibitory protein (CIP / KIP )family - aka . CDKN1
- Expression of members of this family are stimulated weakly by TGFb + strongly by DNA damage, involving TP53
- Inhibit all other CDK - cyclin complexes : late G1, G2, M
- Gradually sequestered by G1 CDKs thus allowing activation of later CDKs
25
Describe the characteristics of cyclin dependent kinase inhibitors
(There are 2 families) - describe Family 2 of cyclin dependent kinases [CDKN2]
- Old name INK4. Expression is stimulated by TGFb
- Specifically inhibit G1, CDKs
- e.g. CDK4 the kinase activated by growth factors
26
List the sequence of events that are triggered by growth factors
1. Growth factor signalling activates early gene expression
- Transcription factors: FOS, JUN, MYC.
2. Early gene products stimulate delayed gene expression,
- Cyclin D, CDK2/4, E2F transcription factors
3. E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)
4. G1 cyclin CDK complexes will hyperphosphorylate RB + then G1/S cyclin - CDK complexes will hyperphosphorlate RB which will release E2F
5. E2F will stimulate expression of more Cyclin E and S phase proteins
- DNA polymerase, thymidine kinase, proliferating cell nuclear antigen
- 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
- This will drive the cell through S phase and mitosis
27
Summary of the key steps that precede apoptosis
1. Growth factors binding to receptors. Induces gene expression
2. G1 and G1/S Cyclin-CDK complexes phosphorylate RB in the absence of inhibition by CKIs (expression of these is regulated by TP53 or TGFb)
3. E2F released, stimulating expression of genes required for S-phase
4. Cell replicates DNA (expression of S-phase Cyclin-CDK complexes)
5. If all DNA replicated, G2/M Cyclin-CDK complexes cause cell to enter mitosis. If chromosomes aligned on spindle, exit from mitosis is triggered
6. If process fails, TP53 initiates apoptosis
28
Causes/ mechanisms of cell damage + DNA - Genetic problems
- Abnormal number of chromosmes (aneuploidy)
- Abnormal chromosomes (deletions + translocations )
- Increased frailty (fanconis anaemia)
- Failure of repair (Xeroderma pigmentosa)
- Inborn errors (storage disorders i.e. Tay Sachs disease)
29
Causes/ mechanisms of cell damage + DNA - Traumatic damage
- Blood flow interruption
- Direction cell rupture
Entry of foreign agents
30
Causes/ mechanisms of cell damage + DNA - Inflammation
- Vasculitis
- Atherosclerosis
- Trauma
- Thrombo embolism
31
Causes/ mechanisms of cell damage + DNA - Infection
- Toxic agents
- Competition for nutrients, intracellular replication
- Viruses/mycobacteria provoking immune response
32
Causes/ mechanisms of cell damage + DNA - Physical
Irradiation, heat, cold, barotrauma
33
Causes/ mechanisms of cell damage + DNA - Chemical
- Acids + corrosives
- Specific actions like enzymes
- Interference with metabolism like alcohol
34
What are the 3 basic mechanisms that cell death is caused by
1. Necrosis
2. Apoptosis
3. Autophagic cell death
35
What are the characteristics of necrosis
- Most common cause of cell death. Occurs after stresses like:
• Ischaemia, trauma, chemical injury
36
What are the characteristics of apoptosis
- Programmed cell death
- Designed to get rid of unwanted host cells through activation of coordinated + internally programmed series of events
- Effected by a dedicated set of gene products
37
What are the characteristics of autophagic cell death?
- Degradation of normal proteins that are involved in cellular remodelling that are found during:
Metamorphosis, Ageing, Differentiation + digestion and removal of abnormal proteins, that would have accumulated after toxin exposure, cancer, or disease
38
What induces the death of breast cancer cells?
- Autophagic cell death. These is degradation of the normal proteins that are involved in cellular remodelling.
39
What are the causes of necrosis
- Lack of blood supply to cells or tissues. Like injury, infection, cancer, infarction + inflammation.
40
Describe the characteristics of necrosis
1. Whole groups of cells are affected
2. This leads to injurious agent or event
3. Reversible events will proceed irreversible
4. Energy deprivation will cause changes
- e.g. cell unable to make ATP because of oxygen deprivation
5. Cells will swell because of water influx - ATP needed for ion pumps to work
6. There is haphazard destruction of the organelles, and nuclear material by enzymes from ruptured lysosomes
7. Cellular debris stimulates inflammatory cell response
41
What is the microscopic appearance of necrosis : the nuclear changes
1. Pyknosis
- Chromatin condensation + shrinkage
2. Karyorrhexis
- Fragmentation of the nucleus
3. Karyolysis
- Chromatin dissolution by DNAse. Causes fading in basophilia of the chromatin
42
Describe the microscopic appearance of necrosis : describe cytoplasmic changes
1. Opacification
- Denaturation of the proteins, with aggregation
2. Eosiniphilia
- Exposure of basic amino acids
3. Complete digestion of cells by enzymes
- Causes the cell to liquefy - liquefactive necrosis
43
Describe the microscopic appearance of necrosis : describe biochemical changes
1. Release of enzymes like creatine kinase, or lac dehydrogenase
Release of proteins like myoglobin
44
Describe the clinical investigations that are associated with cell death - Muscular dystrophy
- Damaged muscles will release creatine kinase + lac dehydrogenase
- M3 and M3H isoforms
45
Describe the clinical investigations that are associated with cell death - Heart attack
- Damaged muscle cells release lactate dehydrogenase
H3 + H3M isoforms
46
Describe the clinical investigations that are associated with cell death - bone + liver disease
- Damaged tissues. Release alkaline phosphatase + lactate dehydrogenase isoforms
- Different isoforms are specific to various tissues
47
What are the 5 types of necrosis
Coagulative necrosis
Liquefactive necrosis
Caseous necrosis
Fatty necrosis
Fibrinoid necrosis
48
Coagulative necrosis characteristics
- Hypoxic environments
- Cell outlines remain after cell death + can be observed by light microscopy
- Myocardial infarction, infarct of the spleen
49
Liquefactive necrosis characteristics
- Associated with cellular destruction + pus formation
| - Like pneumonia
50
Caseous necrosis characteristics
- Mix of coagulative necrosis + liquefactive necrosis
| - e.g. Tuberculosis
51
Fatty necrosis characteristics
- Results from actions of lipases on fatty tissues
| - Acute pancreatitis
52
Fibrinoid necrosis characteristics
- Caused by immune mediated vascular damage
- Marked by deposition of fibrin like proteinaceous material in arterial walls
Appears smudgy + acidophilic on light microscopy
53
What are the functions of necrosis
- Removes damaged cells from organism: failure to do so might lead to chronic inflammation
54
What are the functions of apoptosis?
- Selective process for deletion of 1. Superfluous 2. Infected 3. Transformed cells
55
What processes are apoptosis involved in?
- Embryogenesis, metamorphosis, normal tissue turnover, endocrine dependent tissue atrophy, variety of pathological conditions
56
Give 8 examples of apoptosis.
1. Cell death in embryonic hand to make individual fingers
2. Apoptosis that is induced by growth factor deprivation (neuronal death from lack of NGF
3. DNA damage mediated apoptosis - if the DNA is damaged due to radiation or chemo therapeutic agents: there is an accumulation of p53 (which is a tumour suppressor gene product).
- This arrests cell cycle and enables the cell to repair damage
- If repair process fails, p53 will trigger apoptosis
4. Cell death in tumours, causing regression
5. Cell death in viral diseases like viral hepatitis
6. Cell death that is induced by cytotoxic T cells
- Like cellular immune rejection or graft vs. host disease
7. Death of neutrophils during actue inflammatory response
8. Death of immune cells (both T and B lymphocytes) after depletion of cytokines as well as death of autoreactive T cells in the developing thymus.
57
Apoptosis role in frog development
- Apoptosis helps to eliminate the tail during the metamorphosis of a tadpole into a frog
58
Apoptosis role in mouse development
- There is apoptotic cell death during the development of mouse paws
59
What are the factors that influence the balance of life and death at the cellular level?
- growth factors
- cytokines
- Death domain ligands
- DNA damaging agents
- lack of growth factors
- disruption of cell-cell and or cell-matrix contacts
- cell-cell and or cell-matrix contacts
60
What are the 2 types of apoptosis
Intrinsic or extrinsic
61
Describe the intrinsic pathway for apoptosis?
- Withdrawal of growth factors, like IL3
- There are extracellular signals e.g. TNF
- T cell or NK (natural killer) like granzyme.
62
What are caspases
- Cysteine aspartate specific proteases
63
What do caspases do (cysteine aspartate specific proteases)
- Cysteine proteases that play a central role in initiation of apoptosis
- Most proteases are synthesised as inactive precursors that need activation - usually partial digestion by another protease.
64
What happens when there is caspase activation
- Caspase activation leads to characteristic morphological changes of the cell like shrinkage, chromatin condensation, DNA fragmentation + plasma membrane blebbing
65
What are the 7 steps of apoptosis
1. Single, or few cells selected
2. Programmed cell death
3. Irreversible once initiated
4. Events are energy driven
5. Cells will shrink, as the cytoskeleton is disassembled
6. There is orderly packaging of organelles + nuclear fragments in membrane bound vesicles
7. New molecules expressed on vesicle membranes stimulate phagocytosis - no inflammatory response.
66
What microscopic changes do you see in apoptosis
1. Nuclear changes 2. Cytoplasmic changes 3. Biochemical changes
67
What microscopic changes do you see in apoptosis - the nuclear changes
1. Nuclear chromatin condenses on nuclear membrane
| 2. DNA cleavage
68
What microscopic changes do you see in apoptosis - the cytoplasmic changes
1. Cell shrinkage, organelles are packaged into membrane vesicles
2. Cell fragmentation, membrane bound vesicles bud off
3. Phagocytosis of cell fragments by macrophage + adjacent cell
4. No leakage of the cytosolic components
69
What microscopic changes do you see in apoptosis - the biochemical changes
1. Expression of charged sugar molecules on outer + inner surface of membranes (recognised by macrophage + enhances phagocytosis)
2. Expression of phosphatidylserine on extracellular leaflet of apoptotic cell
3. Protein cleavage by proteases, caspases
70
How do we activate the initiator caspases
- By induced proximity, for example:
1. In response to receptor dimerisation upon ligand binding
2. Cytochromc C release from the mitochondria
71
What are the characteristics of Cytochrome C
- Mitochondrial matrix protein
- Released in response to oxidative stress by a permeability transition
- Any inducers of the permeability transition also eventually induce apoptosis
72
How is the release of cytochrome c regulated?
BCL-2 family
- BCL-2 is a member of a multi gene family in mammals
| - The BCL-2 family members form dimers
73
How is the release of cytochrome c regulated?
BCL-2 family (Anti-apoptotic)
- BCL-2
- BCL-XL
- Others
74
How is the release of cytochrome c regulated?
BCL-2 family (Pro apoptotic )
- Bax
- Bad
- Bid
75
P53 and apoptosis - describe the link
- Mutations in p53 gene are the most common mutations in cancer
- Some mutations can destroy ability of p53 to induce apoptosis
76
Give a definition of pathogens
Ø Disease causing microorganisms
Ø Opportunistic pathogens
• Will only infect the immunocompromised
• Local/systemic, like Staph Epidermis IV line Pneumocystis in HIV infection
Ø Virulence
• Capacity to cause disease
• e.g. virulent strains of commensal bacteria
77
Describe "Koch Posulates" - what the microorganism has to do
- 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
78
How to study pathogenesis - use of Virulence factors
- Identify factor / gene in virulent vs. non virulent strains
• Biochemical / molecular genetic studies
Pathogenity islands - can get groups of virulence genes
- Test the hypothesis in animal model, or in vitro
Compare the effects of removing / blocking virulence factor
79
What are the 4 stages of pathogenesis
1. Adherence +/- entry
2. Multiplication
Ø Local / general spread
3. Evasion of host defences
Ø Each stage
4. Tissue damage
Not all microbes need all 4 stages
80
Describe Stage 1 Adherence
```
he bacterial "adhesins" will attached to the surface, e.g. the:
• Skin
• Urogenital
• GI
• Respiratory tracts
```
E coli "P" fimbrae "Pili US"
- Bind P blood group of the uroepithelial cells
Neisseria Gonnorrhoeae fimbrae
- Attach to mucosal cells
- Non fimbriated mutants are less pathogenic
Vibrio cholera fimbriae - Bind intestinal epithelial cell receptors
Strep pyogenes - Lipoteichoic acid binds to fibronectin receptors in pharynx.
81
describe tears as a non immunological host defence
- cleansing action
| - also contains antibacterial substances, e.g. lysosome
82
describe mucus as non immunological host defence
- barrier to contact of organisms + cell surfaces. May block carb ligand-receptor interactions
83
describe defensins as non immunological host defence
- antibacterial peptides produced by certain epithelial cells, e.g. intestinal epithelium
- and of potential importance in the control of colonisation
84
describe intestinal peristalsis as non immunological host defence
- propels microorganisms that do not have a mechanism for colonising the small or large bowel
85
describe ciliated epithelium as non immunological host defence
- component of the mucocilary ladder which encases upper respiratory pathogens in bronchial mucus
- moves them to the posterior pharynx, where they can be swallowed + disposed of
86
describe gastric acid as non immunological host defence
- lethal to micro organisms without protective mechanisms
87
describe microbial flora as non immunological host defence
- on skin + certain mucosal membranes
- able to occupy niches + produce metabolic products that regulate other organisms e.g. antimicrobial substances such as colcins
88
Describe the process of multiplication (stage 2)
Local surface infection only
- Vibrio cholera
- Some strains of N gonorrhoeae
Local Invasion
- Shigella
- Some Staph Aureus
Wider Invasion
- Through blood, lymph, (nerves)
- S typhi, N meningitidis, Staph aureus
May be critical for transmission
- Respiratory
- Faeco oral + sexual content
89
Describe the process of evasion of host defences (stage 3)
- Each step in specific & non specific immunity
• Antigenic variation (e.g. N meningitidis)
• Capsules can stop contact with the phagocyte
Ø S Penumoniae B anthracis
• Inhibit phagolysosome formation
Ø M tuberculosi, Listeria monocytogenes
• Immunosuppress host (some of the toxins will act as superantigens)
90
Describe anti phagocytic actions
```
Ø Toxin release
Ø Opsonisation prevented
Ø Contact with phagocyte prevented
Ø Phagolysosome fusion inhibited
Ø Escape into the cytoplasm
Ø Resistance to killing
```
91
Describe anti phagocytic actions - 1. Toxin release
- The organism will release the toxin
| - The phagocyte is killed by this toxin
92
Describe anti phagocytic actions - Opsonisation prevented
- Organism makes protein. This prevents contact with the phagocyte
• e.g. streptococcus penumoniae
• Haemophilus
Bacillus anthracis
93
Describe anti phagocytic actions - contact with phagocyte prevented
- Organism possesses a capsule
- Which prevents contact with the phagocyte
• Streptococcus pneumoniae
• Haemophilus
Bacilus anthracis
94
Describe anti phagocytic actions - phagolysosome fusion inhibited
- There is fusion of phagosome + lysosome inhibited by organism
• e.g. Mycobacterium Tuberculosis
• Toxoplasma
Chlamydia
95
Describe anti phagocytic actions - escape into the cytoplasm
- The organism escapes from the phagolysosome ==> into the cytoplasm
- & replicates within the phagocyte
• e.g. leishmania, T cruzi
96
Describe anti phagocytic actions - 6. Resistance to killing
- The organism will resist killing by producing antioxidants
• e.g. by catalase in Staphylococci, or by scavenging free radials
By phenolic glycolipid of M. Leprae
97
What can virulence factors do? - Pilus
Ø Aid attachment to human mucosal epithelium. Contains constant + hypervariable regions - analogous to immunoglobulins (Igs), that contribute to antigenic diversity in gonococci
98
What can virulence factors do? - Por proteins
Ø Forms pores through the outer membrane
Ø Antigenic
Ø Specific serotypes associated with virulence
99
What can virulence factors do? - opa proteins
Ø Assist binding to epithelial cells
100
What can virulence factors do? - LOS
Ø Lipo oligo saccharide
| Ø Endotoxin activity
101
What can virulence factors do? - Rmp proteins
Ø Inhibits cidal activity of serum
102
What can virulence factors do? - IgA protease
Ø Core contains enzyme
| Ø Released by cell to destroy IgA1
103
What can virulence factors do? - capsule
Ø Resists phagocytosis
104
Stage 4. Damage to host : describe what occurs
1. Direct damage by microbe or toxins
- Locally
○ Enzymes : Staph aureus
○ Toxins : Clostridium perfringens, V cholera
- Systemically
○ Exotoxins C diptheriae C tetani
2. Caused by hosts immune response
- Immunopathology
105
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 & other causes of sepsis
5. Superantigen mediated, e.g. toxic shock syndrome
6. Immunopathology:
Ø Immune complex disease (Type 3 hypersensitivity)
Ø Molecular mimicry
Ø Cellular immune response (Type 4 hypersensitivity)
106
Acute inflammatory changes: symptoms of infections (Local symptoms)
Ø Inflammation
- Redness, swelling, warmth, pain, loss of function
- Pus - pyogenic infection
107
Acute inflammatory changes: symptoms of infections (systemic symptoms)
Ø Fever, rigors, chills, tachycardia, tachypnoea
108
Acute inflammatory changes: causes of local symptoms
- Mainly secondary to response of the local small blood vessels with:
• Increased blood flow
• Increased permeability to fluid & plasma proteins
• Increased stickiness of vascular endothelium
• Emigration of phagocytes to site of infection
109
Acute inflammatory changes: causes of inflammatory response
- Inflammatory response = triggered by release of products from the bacteria, e.g. toxins & enzymes
- & amplified by release of products from host cells
• Histamine, prostaglandins, leukotreines, kinins
110
Describe acute inflammatory changes
- Results in accumulation of: phagocytes - mainly neutrophils (pus cells) + some monocytes, complement and other factors & exudate at the site of infection
- Pyogenic infection
- Pyogenic organisms include staphylococci + streptococci & meningococci
111
Acute inflammatory changes characteristics
- Host fights the bacterium
- Inflammation = weapon of the host
- Damage to the hosts tissues may be as a result of the weapons of the bacterium, such toxic products
• e.g. leukocidin from staphylococci
- Results in dead & dying neutrophils - pus
- Or damage may be due to inflammation itself
112
Bacterial enzyme profile:
Hyaluronidase
Origin of hyaluronidase
Ø Streptococci
• e.g. streptococcus pyogenes
Action of hyaluronidase
Ø Will break down hyaluronic acid
Result
Ø Disruption of tissue mosaic
Ø Allowing bacteria & inflammatory exudate to travel deeper and further
113
Bacterial enzyme profile:
Alpha-Lecithinase
Source of alpha-lecithinase
Ø Clostridium perfringens
Action of alpha-lecithinase
Ø Splits lecithin
Ø Found on the surface of many cells
Result
Ø Major tissue damage caused
114
Describe the characteristics of bacterial exotoxins
Ø Most exotoxins = proteins that are secreted by the bacterium
The ways that bacterial exotoxins behave
Ø Enzymatic lysis (e.g. alpha-lecithinase)
Ø Pore formation
Ø Inhibition of protein synthesis
Ø Hyperactivation
Ø Effects on nerve-muscle transmission
115
How else can bacterial exotoxins be classified
1. Molecular structure e.g. subunits
2. Site of actions e.g. enterotoxins
Ø Exotoxins are made by many bacteria including gram positive and gram negative species
116
What is an endotoxin
- An integral part of the bacterial 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
117
Give examples of endotoxin mediated disease
```
Ø Neisseria meningitidis
• Meningococcal meningitis
• Meningococcemia
Ø Escherichia coli
Ø Klebsiella
Ø Pseduomonas aeruginosa
```
118
What are the actions of endotoxin?
- Activation of the macrophage/monocyte cells.
- These macrophage/monocyte cells will release:
• IL-1
• IL-6
• IL-8
• Platelet activating factor
• Tumour necrosis factor (TNF-alpha)
- These also stimulate production of prostaglandins & leukotrienes.
- Cytokines act at various sites, including the endothelium, liver, clotting cascade.
- Results in:
Ø Increased vascular permeability
Ø Hypotension; which leads to shock, fever, disseminated intravascular coagulation (DIC), multiple organ failure
119
What is bacteraemia?
Ø Bacteria in the blood
120
What is septicaemia?
Ø Bacteria in the blood; with symptoms
121
Sepsis: systemic inflammatory response syndrome (SIRS) characteristics
```
- Gram positive organisms, e.g.
Ø Staphylococcus Aureus
Ø Streptococcus Pyogenes
Ø Streptococcus Pneumoniae
○ May also cause septicaemia/SIRS
```
122
What is Toxic Shock Syndrome caused by
- Toxins, that are produced by certain strains of:
Ø Staphylococcus aureus: toxic shock syndrome toxin (TSST)
Ø Streptococcus pyogenes: Streptococcal pyrogenic exotoxin (SPE)
- These toxins might act as superantigens
123
Describe the actions of superantigens?
- They are able to act simultaneous with MHC class 2 antigens, on
• Antigen presenting cells and
• Specific Vbeta regions, of T lymphocytes
- This will activate macrophages/monocytes to elicit IL-1, IL-6, TNF-alpha and interferon-y
124
Multiorgan pathology: immunopathology
Humoral immunity
Ø Production of antibodies by B-lymphocytes
Cellular immunity
Ø T-lymphocytes
125
What are the characteristics of immune complex disease
- Type 3 hypersenstivity reaction Stretococcus pyogenes & glomerulonephritis
- Host produces antibodies against streptococcal antigens
- Antibodies bind to antigens to form immune complexes
- Immune complexes are 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.
126
Describe the characteristics of molecular mimicry
- Throat infection with Streptococcus pyogenes
- Antibodies against streptococcal antigens (cell wall) are produced
- These antibodies will cross-react with antigens of the host, (similar molecular structure)
- Sites of cross reactivity:
• Myocardium
• Synovium
• Brain
127
Examples of molecular mimicry?
- Rheumatic heart disease/ rheumatic fever
- Cross reactions demonstrated between
Ø Group A carbohydrate of streptococcus & structural glycoprotein of heart valve
Ø M-protein of streptococcus and cardiac muscle
- Binding of antibodies, to host antigen will activate complement and will lead to inflammatory response
- Cell mediated response, also probaly will occur
- Granulomas form in the tissue - Aschoffs nodules
- Cross reacting antibodies in synovium lead to inflammation of joints - arthritis
- Antibodies may also cross react with neurons in the caudate & subthalamic nuclei, leading to involuntary movement:
Ø Syndenhams Chorea
Ø St. Vitus's dance
128
Cellular immune responses
- Type 4 hypersensitivity reactions.
• T helper cells react to specific antigens. e.g. Mycobacterium Tuberculosis
• T cells will release cytokines including (TNF-alpha) that activate macrophages
• These + other toxic products might cause tissue damage
• In chronic infections, degree of tissue damage may be extensive
129
Cellular immune responses
- Characteristic response is the granuloma containing epitheliod & giant
- In TB, necrosis is characteristic
- Macroscopically, granulomas form "tubercules"
- Necrosis is described as caseous (cheesy)
130
Granulomas characteristics
- May form with other infections e.g. leprosy syphilis (gummas) and schistosomiasis
- Necrosis may be absent in these conditions
- This may reflect different spectrum of cytokine release and cellular activation
131
How does the immune system defend against bacteria?
1. Recognises bacteria. Attaches/ invades
2. Finds bacteria + ensures recruitment of defence measures to that site.
3. Kills bacteria or cells containing bacteria
4. Mops up released toxins that still damage after bacteria are killed - defuse
5. Mechanism to remember the bacteria - if they invade again our defences can be mobilised rapidly - memory
132
Describe the balance between the microbe and the host - properties of the microbe - pathogenic mechanisms)
- adhesins, toxins, capsules
133
Describe the balance between the microbe and the host - properties of the host : defensive mechanisms
- natural barriers, defensive cells, complement, immune response
134
What are the stages of infection?
1. Acquisition
2. Colonisation - adherence
3. Penetration
4. Spread
5. Immune evasion
6. Damage
7. Transmission
8. Resolution
Immune response:
Ø Site of infection, stage, prior exposure
Ø Damage limitation, clearance
Ø Immune protection
135
What are the types of infection?
Local
- Surface infection; wound
Invasive
- Penetrates barriers, spread
Systemic
- Via blood to other sites
- Effects at different site from colonisation: toxins and endotoxins
Immunopathology
Ø Inflammation
Ø Cross reaction antigens (rheumatic heart disease)
Ø Autoimmunity
Ø Granuloma
136
How to design an immune system to defend self against bacteria? When bacteria are on the epithelial surfaces
• If not attaching, or signalling there is no harm done.
137
How to design an immune system to defend self against bacteria? When bacteria are Free living, in the blood, EC
• As plantonic growth, or biofilms
138
How to design an immune system to defend self against bacteria? When bacteria are inside cells
• Cytoplasmic and vacuolar
139
Surface defences: mucosal and epithelial surfaces
Act as watchdog
- Cells + signalling
Shedding
- Removal and threat of rapid rep
Normal flora
- Microbial antagonism & microbiome balance
- There are 3 examples of this:
○ Immunological - innative and adaptive
○ Physical barriers
○ Vascularity
140
Defences of the tissue & blood - - Usually involves tissue damage + controlled by feedback mechanisms
(Non-specific feedback mechanisms)
- Transferrin
- Complement
- Acute phase proteins (released by the liver)
- Phagocytes - monocytes and macrophages
- PMNs - neutrophils
141
Defences of the tissue & blood - - Usually involves tissue damage + controlled by feedback mechanisms
(Adaptive feedback mechanisms)
- Antibodies
- Macrophage activation
T cells
142
Recognition, danger and signalling: orchestration of innate and adaptive immune defences
- There is orchestration of innate and adaptive immune defences
Continuous, active, seamless, regulated
143
What are the characteristics of inflammation
- Response to tissue injury. The functions are to bring serum molecules and cells, to the site of infection
Ø Increases blood supply
Ø Increases capillary permeability
Ø Migration of cells from the blood to the tissue (PMNs, Mps,)
- This is an ordered, regulated proecss.
- The cardinal signs of inflammation are heat, pain, redness and swelling (calor, dolor, rubor, tumour)
- Vasodilation, oedema, complement activation
- Mast cell degrannulation, PMNs recruitment, clotting
144
the virulence factors actions
1. Promote colonisation and adhesion, to establish infections (e.g. adhesins)
2. Promote tissue damage
3. Transmission, e.g. toxins
4. Subvert immune defences
Immune response against these might protect from disease
145
The roles of complement in non-adaptive (innate) immunity
- Induces inflammatory response
- Promotes chemotaxis
- Increases phagocytosis by opsonisation
- Increases vascular permeability
- Mast cell degranulation
- Lysis of cell membranes
• Pro inflammatory and antibacterial
146
Describe the complement pathways + bacterial capsules
- There are many ways that bacteria can interact with complement
- Mannose binding lectin, is an acute phase protein.
• Will bind MBL associated serine protease (MSP)
147
Describe the process of opsonisation and phagocytosis
- Uncoated (by antibody) bacteria, are phagocytosed poorly
| - On coating with antibody, adherence to phagocytes is enhanced. And will increase complement fixing.
148
Actions of macrophages and neutrophils (PMNs)
Phagocytose free bacteria
149
Actions of cytotoxic T cells CD8+
Kills cells expressing antigen
150
Actions of CD4 helper cells
- Activate macrophages
| - Stimulates the B cells
151
Describe human immunodeficiency virus (HIV)
```
Ø Envelope
Ø Viral gp120 & gp41
Ø 2 copies of RNA
Ø Reverse transcriptase
Ø Integrase
```
Protease
152
Give the characteristics of Hepatitis A virus
- Most common viral hepatitis
- Picornaviridae family
- Naked
- Icosahedral
- Single stranded RNA
+ve sense
153
Give the characteristics of Hepatitis B virus
- Hepadnaviridae family
- Enveloped
- 42nm
Ø Icosahedral
Ø Circular DNA partially double stranded
Ø Complete virus & incomplete particles
Tubular filaments & spherical particles composed of envelope proteins - hepatitis B surface antigen
154
Give the characteristics of Hepatitis C virus
- Flaviviridae family
- Enveloped
- Icosahedral
- Single stranded RNA
- NS1 non structural protein 1
- E proteins are major envelope proteins of the virus
155
Give the characteristics of the gastrointestinal infection, Norovirus
- Diarrhoea
- Calciviridae
- 27nm
- Icosahedral
- Non enveloped
- Single stranded RNA
156
Give the characteristics of the gastrointestinal infection, Rotavirus
- Fever + vomiting and diarrhoes
- Abdominal pain
- No antivirals
- Reoviridae, double stranded RNA
- RNA = segmented - 11 segments
- Non enveloped
- 75nm
- Triple layer capsid
- Icosahedral structure
157
Give the characteristics of viral proteins
- Structural proteins: VP1-VP7
| - Non-structural proteins: NSP1-NSP6
158
Describe the process of virus replication in Rotavirus
- Infects the cells of intestinal epithelium
- Outmost layer has 2 important proteins, VP7 and VP4
- These are important in virus attachment and entry
159
What is the replication cycle?
1. Virus binds to receptors
2. Enters cell by endocytosis
Ø Loses its outer layer
Ø Unusual virus replication cycle
3. Within virus structure dsRNA can replicate
4. This is because the virus has a protein VP6 which acts as a channel
5. VP6 allows movement of RNA
6. Inside the virus core, there are VP1, VP2 and VP3
7. These are involved in the process of transcription
Ø Viral proteins are made in infected cell cytoplasm
Ø Core assembly of single and doubled shelled particles in the cytoplasm
Ø There is entry of double layered particle into endoplasmic reticulum
Ø Acquires outer shell
Ø Released from cell
160
Describe the symptoms of measles
- Fever, cough, runny nose, red eyes, sore throat
- 2-3 days later get small white spots (Kopliks spots) - might appear inside the mouth
- 3-5 days after the start of the symptoms, rash appears on face and spreads downwards to the neck, trunk, arms, legs and feed
- When rash appears, fever can spike
After a few days the fever will subside and the rash will fade.
161
Characteristics of measles virus
```
Ø Enveloped
Ø RNA single stranded
Ø Negative sense
Ø Paramyoxviridae
Ø Pleomorphic
Ø 100-300nm
```
162
Describe 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
163
Mumps characteristics
- Fever + headache, muscle aches and 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
Ø Paramyxovirus
Ø Pleomorphic
Ø Enveloped
Ø Helical nucleocapsid
Ø Ss RNA linear genome
164
Rubella virus characteristics
- Acute viral disease. Causes fever + rash
- Mild disease in children and young adults
- Rash + fever for 2-3 days
Spread through coughing and sneezing
165
Describe congenital rubella
- Birth defects, if acquired by a pregnant women
- Deafness, cateracts, heart defects, damage to foetal brain, liver and spleen damage, more significant if infection happens early in pregnancy
Ø Togavirus
Ø Enveloped
Ø ssRNA
Ø icosahedral
166
Explain adenoviruses
- Different serotypes exist. Most commonly cause respiratory illness.
- May cause various other illnesses:
• Gastroenteritis, conjunctivitis, cystitis, rash
- Adenovirae
- No envelope
- Icosahedral
DNA double stranded, linear
167
Papillomavirus characteristics
- No envelope
- Icosahedral
- DNA genome, circular genome
- Double stranded
- Family papovavridae
- Different serotypes
- Some cause infections in the genital tract
- Cervical cancer
168
Parvovirus characteristics
- Non enveloped
- Small, 22nm
- DNA ss
- Nucleocapsid icosohedral
- Virus has a receptor which allows it to attach to erythrocyte progenitor cells
- Parvovirus B19 causes inhibition of erythropoesis
- Shortened life span of red blood cells to less than 120 days
169
Clinical manifestation of Parvovirus
- Most of the infections in children 5-15 years old. Erythema Infectosum. "slapped cheek" rash on face + red rash on trunk and limbs.
- Rash may itch
- Infections = asymptomatic
- Might have low grade fever, malaise a few days before the rash breaks out and resolves in 7-10 days.
Ø Glove + sock syndrome
Ø Arthropathy
Ø Transient aplastic crises
Ø Chronic red cell aplasia
Ø Neutropenia, thrombocytopenia, pancytopenia.
170
Infections that can occur in pregnancy
- Non immune hydrops foetalis
- Miscarriage + intrauterine foetal death.
Mode of transmission
- Respiratory route. Mother => foetus transmission
- Incubation period between 10-21 days
- Viraemia present.
171
Treatment of parvovirus
- No specific drug to treat Parvovirus B19 infection
- In immunocompetent individuals
- Immune system produces antibodies
- T cell response against virus
Viral clearance occurs
172
Influenza characteristics
- Airborne virus producing feverish illness
- Causes immunocompromisation
- Secondary bacterial pneumonia = cause of death through influenza
- Orthomixoviridae (family)
- A, B, C subtypes
Ø Lipid enveloped
Ø This is derived from the host cell membrane
Ø Proteins including
○ Hemagglutinin (HA)
○ Neuraminidase (NA)
○ Ion channel protein (matrix protein 2, M2)
○ These are embedded in the lipid bilayer of the viral envelope
Ø Ribonucleoprotein complex comprises viral RNA segments associated with the viral proteins
Ø The matrix M1 protein is associated, with both ribonucleoprotein and the envelope.
173
Describe the influenza subtypes
| - Influenza Type A
- Most serious => affects mammals and birds
- Genetic crossover between strains can lead to pandemic
- Subtyped according to surface antigens - haemagglutinin (HA) and neuraminidase (NA)
174
Describe the replication cycle
1. Viral attachment - uses haemagluttinin to attach to sialic acid
2. Internalised by endocytosis
3. Inside endosome pH is low
4. Virus envelope fuses with the endosome membrane
5. Triggers uncoating
6. Viral nucelocapsid released into cytoplasm
175
What happens during the replication cycle?
- The viral RNA is single stranded - negative sense
- 8 segments
- Copies of viral RNA are made in nucleus
Ø First from negative sense a positive sense copy is made
- This is then copied into a negative sense to make the viral genome
- Mrna also synthesised
- Mrna translated in cytoplasm
- Early viral proteins = those that are required for replication + transcription are transported back to the nucleus.
- Late in the infection cycle, the M1 and NS2 proteins facilitate the nuclear export of newly synthesised viral RNPs
176
Describe the process of viral assembly
Ø RNA segments assembled within the nucleocapsid
| Ø Assembly + budding of progeny virions occurs at the plasma membrane
177
Why do the surface antigens of Influenza A mutate rapidly?
1. Virus has enzyme involved in virus replication, RNA polymerase
2. This enzyme has low selectivity
3. Enzyme has no proof reading mechanism
Ø Mutants generated
Ø New mutants spread rapidly
Ø 2 patterns of mutation
Antigenic drift + antigenic shift
178
What is antigenic drift
- Continual viral mutation
- Mutations are often minor, with no effect on function
- Changes accumulate to create new "drifted strains"
- The drifted strains produce illness
- Some strains co-exist
179
When does antigenic drift occur in Influenza
- Happens in type A Influenza only.
- Genetic reassortment - there is mixing of genetic material between strains, when they infect the same host
- This creates novel strains
- Lack of immunity
- Rapid spread will lead to pandemic
- Mixed strains from different species = more virulent and harder to control
180
What is a primary infection
```
- 1st encounter with virus. The options are:
Ø Replication @ the site of entry
Ø Remain @ site of entry
Ø Influenza virus
Ø Rhino virus
```
181
Primary infection characteristics
- First encounter with virus
- Options are to replicate @ site of entry and then spread
• Varicella zoster virus
• Infection started in the respiratory tract
• Asymptomatic
• The patient infectious in this phase
• Local replication of the virus
• Spread
• Secondary site of infection is the skin
• Patient infectious
182
Characteristics of secondary infections
- Infections with second organism
- e.g. following antibiotic treatment
- Candida albicans - thrush
- Following an infection that compromises immunity
- Bacterial + fungal infections in HIV infected patients
- Bacterial pneumonia following viral respiratory tract infections
183
Re- infection
- Influenza virus - once infected you are not permently protected
- Can get re-infected with the same virus
- Rhinovirus - common cold
184
Give examples of
Viruses that cause acute disease
- Rabies
- Rhinovirus
- Influenza
- Rotavirus
- Infected - recover + clear virus
- Or die
185
Give examples of
Viruses that cause chronic disease
- Hepatitis B and Hepatitis C
- Infection can in some cases be cleared
- Often infections remain and immune system does NOT clear infection
186
What factors determine pathogenesis of virus
- Nature of virus
- Compare influenza with herpes viruses
- Entry site
- Tissue tropism
- Cell damage caused
- Ability of immune response to clear the virus
187
Can the virus evade the immune response?
- Large dose of virus
- Perinatal HSV
- Infect @ a time when ability to produce defence is NOT optium
• This is what perinatal infections do
188
What else can determine pathogenesis
Ø Vertical transmission
Ø Foetus not able to mount a protective immune response
Ø Mode of entry also different to norm - via placenta
Ø Example, rubella virus - parvovirus
189
How would you do evasion of immune response - Establish latency example HSV
- Do not replicate fully
| - Only make a few viral proteins
190
How would you do evasion of immune response - Cytomegalovirus
- Down regulate MHC class 1
- Hinders antigen presentation
- Evades immune response
191
Describe the process of immune evasion
- Replicate in privileged sites, e.g. leucocytes
- HIV
- Cytomegalovirus
Ø Change viral proteins
Ø Immune response cannot recognise the virus
Ø Takes time to generate humoral and cell mediated immunity
Ø Virus is @ an advantage
192
Describe the characteristics of papillomavirus
- Non enveloped
- Circular ds DNA
- More than 100 HPV types identified
- Classified into low and high risk
- High risk ones include HPV 16, 18, 31, 45
- Linked to development of cervical carcinoma and other malignancies
193
How does HPV cause cancer? Cancer causing mechanism.
- Virus infects epithelial cells
- Epithelial cells have different layers
- Epithelial cells differentiate
- Normal squamous human epithelia grow as stratified layers
- Only the cells that are in the basal layer are actively dividing
- As cells leave the basal layer and move up, they stop dividing
- This is NOT helpful for the virus
- Virus replication process, depends on the cell machinery
- If the cell is NOT replicative virus cannot replicate.
194
Describe how papillomavirus has to override the cell cycle
Ø Viral infects the basal layers
Ø Migrates to the cell nucleus
Ø Genome established as independent episome
Ø Copies of the viral DNA made
195
Important information about 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:
• E2, E6, and E7
196
Describe the genome of virus that is not integrated
- The viral DNA exists as circular plasmid
- Genes E6 + E7 are important
- They are transforming genes
- E6 interferes with a host cell protein - p53
Ø P53 = tumour suppressor protein
Ø P53 = regulates the cell cycle
Ø E7 = also interfere with a host cell protein - retinoblastoma tumour suppressor protein
- The function of Rb is to regulate the cell cycle
- E7 binds with Rb & stops it functioning
- Viral gene E2 is also important
- Products of the E2 gene down regulate expression of the E6 and E7 gene
- Therefore E2 is down regulating expression of cancer causing genes
197
What happens when HPV integrates into host genome
- Virus DNA not circular any more
- Integrated into host DNA
- Virus genome splits at about the middle of the E2 gene
- Linear fragment produced
198
Integrated HPV- cancer
- The E2 gene is split
- It cannot function & regulate transcription any more
- Upstream regulatory protein (URR) is free to act.
- It allows expression of E6 and E7
- These 2 are the transforming genes
199
What is the cancer causing mechanism of integrated HPV
- The virus produces uncontrolled amounts of E6 & E7 the oncoproteins
- Papillomavirus stops the epithelial cells from exiting the cell cycle
200
Regulation of cell division - virus interference
- Telomeres are structures @ the end of chromosomes
- The telomeres can regulate how many times an individual cells can divide
- Telomeric sequences shorten each time the DNA replicates
- Once telomere shrinks to a certain level: 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
201
Characteristics of HPV and telomerase
- Telomerase = enzyme that stabilised telomere length and stops its erosion
- HPV - E6 activates the telomerase gene
- Cancer cells produce telomerase. This keeps the telomere intact.
- Cells do not age and continue to divide.
202
Possible test types for viruses
- Electron microscopy
- Virus isolation (cell culture)
- Antigen detection
- Antibody detection
- Nucleic acid amplification tests (NAATs e.g PCR)
Sequencing for genotype & detection of antiviral resistance
203
Describe the use of electron microscopy to diagnose viral infection
1. Beams of electrons used to produce images
2. Wavelength of electron beam = shorter than light. This results in much higher resolution than light microscopy
3. Electrons scatter when they pass through thin sections of the specimen
4. Transmitted electrons (those that do NOT scatter) are used to produce an image
5. Denser images in specimen scatter MORE electrons & appear darker
204
Use of electron microscopy for visualising viruses
Ø The virus can be visualised with electron microscope
Ø Many different viruses
Ø Can be visualised but now rarely performed
Ø Possibly still useful for faeces and vesicle specimens
Ø Replaced by molecular techniques
205
Describe virus isolation in cell culture
- Viruses require host cells to replicate + may cause a cytopathic effect (CPE) of cells
• This is when a patient sample containing a virus incubated with a cell layer
- Old method now replaced by molecular techniques, but still needed for research or for rare viruses
- Use different cell lines in test tubes or plates
- Slow but occasionally useful in anti-viral sensitivity testing
206
Describe the cytopathic effect
- Different viruses might give different appearances.
- Different cell liens might support growth of different viruses
- Identify virus using antigen detection techniques or neutralisation of growth.
207
Describe the process of antigen detection?
- Viral antigens, usually proteins (either capsid structural proteins), secreted proteins can be detected.
- Infected cells might display viral antigens on their surfaces. Vesicle fluid
208
Describe the methods that can be used for antigen detection
- A variety of different methods can be used
1. Direct immunofluoresence
2. Enzyme immunoassay
3. Immunochromatographic methods
209
Characteristics of immunofluoresence
- Antigen (from infected host cells in sample), bound to slide
- Specific antibody (polyclonal or monoclonal), to that antigen is tagged to a fluorochrome & mixed with sample
- Viewed using microscope equipped to provide ultraviolent illumination
210
ELISA for antigen detection
```
- Enzyme linked immunosorbent assay
• A component of reaction is adhered to solid surface
- 3 formats
• Indirect
• Direct (primarily antigen detection)
• Sandwich
```
211
What are the steps involved in detection of antigen by ELISA
1. Plate is coated with a capture antibody
2. Sample is added + any antigen that is present binds to capture antibody
3. Enzyme-conjugated primary antibody is added. Binds to detecting antibody
Substrate is added, and is converted by the enzyme to detectable form
212
Immunochromatographic methods of looking at viruses
- Diagnosis of dengue
- Flavivirus
- Arthropod vector
- Common infection in returning travellers
213
Diagnosis by antibody detection
- When infected with a virus the humoral immune response will take place resulting in production of immunoglobulins
- IgMs antibodies = specific to the I virus are produced 1st
- IgM = present for variable period. Usually 1 to 3 months.
- As IgM declines, IgG is produced
- Quantity of IgG rises
Diagnosis can be made by:
Ø Detection of IgM
Ø Or by demonstration of seroconversion
• Negative antibody first
• Then presence of antibody
214
Serology characteristics
- When infected with a virus the humoral immune response will take place resulting in production of immunoglobulins
- IgMs antibodies = specific to the I virus are produced 1st
- IgM = present for variable period. Usually 1 to 3 months.
- As IgM declines, IgG is produced
- Quantity of IgG rises
Diagnosis can be made by:
Ø Detection of IgM
Ø Or by demonstration of seroconversion
• Negative antibody first
• Then presence of antibody
215
Describe the characteristics of serum
- Produced from processing blood
• Blood = coagulated with micronised silica particles
• Gel used to trap cellular components
- Routinely serum tubes are centrifuged for 10 minutes @ 1000g
- Supernatant (serum) is removed and stored
Ø 4 degrees celsius short term
Ø 20 degrees celsius long term
- Routinely serum tubes are centrigued for 10 minutes at 1000xg
Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes
216
Modern laboratory detection of antibodies & antigens in blood : serology
- Detection of antibody and or antigens
- Usually by enzyme immunoassays. e.g. ELISA or related technology
• e.g. microparticle immunochemiluminescence
217
Describe the detection of antigen and antibody
- This is useful for some infection such as:
Ø Hepatitis B, C and HIV
- This is because it allows us to establish whether acute or chronic infection
Might have therapeutic implications
218
Describe molecular diagnostic tests
- Nucleic acid amplification (NAAT)
Ø PCR: although there are other examples
Ø Can detect RNA or DNA
Ø Ability to multiplex using fluorescence probes i.e. can look for several targets in one sample
Ø May be qualitative or quantitative
Ø Requires nucleic acid extraction prior to the amplification
219
What are the advantages of using NAATS, Nucleic acid amplification
- Might be automated
- Highly sensitive and specific. Generates huge numbers of amplicons
- Rapid
- Useful for detecting viruses to make a diagnosis
Ø @ first time of infection like measles and influenza
Ø During reactivation e.g. cytomegalovirus
- Useful for monitoring treatment response
Ø Quantitative e.g. HIV, HBV, HCV, CMV viral loads
220
Describe the process of 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
• May be selected by treatment
- Useful for outbreak investigation by showing identical sequences in suspected source + recipient
221
Combinations of methods: HIV
- Antibody + antigen detection for initial diagnosis
• Screening test (EIA)
• Confirmatory test (EIA)
- Viral load (NAAT) @ baseline and to monitor treatment response.
• Quantification of virus in blood
- Resistance testing (sequencing)
• Look for mutations known to confer resistance before and during treatment
222
Screening for specific infections
- Testing for specific infections in @ risk groups
• HIV, HBV, HCV
- Testing because it may have an implication for others
• e.g. antenatal. HIV, HBV, rubella
- In these situations the patients are asymptomatic
- Needs a sensitive screening test
- Might have some false positives, so need a specific confirmatory test
223
What do capsules do?
Ø Block opsonisation + phagocytosis
Ø Serum resistance
Ø Capsules & capsulated bacteria
• Like N. meninigitidis
224
What does growing inside cells require?
Ø Different types of immunity
Ø CD4 & CD8 effector cells
• Which does not touch TB
225
What do toxins & enzymes do?
Ø Those that target innate + adaptive immunity lukocidins; pertussis toxin ciliostatic
226
Give an example of mimicry?
Ø Steprococcus M proteins (tolerance, suppressed response?)
227
What is the action of Staph. Aureus coagulase
- There is fibrin deposition which avoids phagocytosis
228
Give examples of antigenic diversity
Ø Strep pneumonia serotypes
| Ø Menigitis capsules C & B
229
What is the action of HIV
Ø Intergration into the host DNA - persistence
Ø Damage to CD4 T cells
Ø Escape mutants
230
What is the action of VZV + herpes zoster
Ø Latency, intermittent shedding and immunological privileged sites
231
Describe Non adaptive (innate) immunity - what does complement do?
Failure to trigger
Ø LPS, capsules
Negative binding
Ø Coating with non fixing with IgA
Ø Capsule blocks C3b binding
Ø Capsule prevents C3b receptor access
Block & expel MAC
Ø C5a proteases
232
What are the roles of complement?
- Induces inflammatory responses
- Promotes chemotaxis
- Increases phagocytosis by opsonisation
- Increases vascular permeability
- Mast cell degranulation
- Lysis of cell membrane
233
Describe the players in Non-adaptive (innate) immunity phagocytosis
Kill cell - leucocidins
- Staphs
Prevents opsonisation
- Protein A (will bind the Fc portion of IgG)
- Staphs
Blocks contact
- Capsules (meningococcus, Hib)
234
What is the role of intracellular pathogens in Non-adaptive (innate) immunity
Promote own uptake (safe)
Ø C3R, Fc receptors
Prepares the cell for invasion
Ø Shigella
Negative P-L fusion
Ø M. tuberculosis
Escape P-L to cytoplasm
Ø Listeria
Resist oxidative killing
Produces catalases + peroxidases
- The immune response depends on localisation within the cell
Hidden from serum killing, complement, antibodies
235
Describe the characteristics of "lifers on the inside"
1. Directs phagocytosis via CR3 - no ROI
2. Actin rearrangement - positive engulfment
3. Type 3 secretion systems to prepare the cell
4. Resists digestion & ROIs in PLs - SOD, catalase
5. Escape into cytoplasm (e.g. Listeria)
6. Inhibits PL fusion maintains early endosome - blocks acidification e.g. mycobacteria
7. Controls antigen presentation, stops CTLs or Po activation
Ø There is release of antigen, or secreted proteins
236
Give examples of the intracellular pathogens
Ø Mycobacterium tuberculosis
Ø Listeria
Ø Salmonella
Ø Immune response depends, on the localisation within the cell
Ø Hidden from serum killing, complement, antibodies
237
Describe the components of adaptive immunity
```
Concealment of antigen
Ø Hide inside the cells
Ø Priveliged sites
Ø Block MHC antigen presentation - Herpes - ve TAP protein
Ø Surface uptake of host molecules
• e.g. CMV & beta2microglobulin
Immunosuppression
Ø Decrease in: MHC + Receptors
Ø Apoptosis
Ø Th1-TH2 switch in IgA proteases
Antigenic variation
Persistence/latency/reactivation
```
238
Paradigms of immune evasion
```
Ø Streptococcus pneumonia
Ø Neisseria gonnorrhoeae
Ø VZV and herpes simplex
Ø HIV
Ø Influenza virus
Ø Mycobacterium tuberculosis
```
239
Describe Streptococcus pneumonias pathogenic mechanisms
1. Colonisation
2. Bypass defences
3. Survival
4. Damage
Ø Pneumonia, otitis media, meningitis
240
Viral immune evasion
Ø Intracellular pathogens need adaptive cell mediated immunity
1. Latency
Ø VZV, herpes simplex
2. Decreased antigenic presentation
Ø By binding to TAP
Ø Inhibits peptide transfer to MHC
Ø Herpes simplex
3. Decreased MHC expression
Ø Cytomegalocirus (CMV)
4. MHC mimics
Ø CMV
3. Mutation of epitopes
Ø B cells = neutralisation escape
Ø T cells = CD8+ escape mutants of HIV
241
Describe persistence as an immune evasion strategy
Ø Small community
1. Microbes infects susceptibles
2. Microbe remains latent
Microbes reactivates (zoster) and infects next generation of susceptibles
242
What are the characteristics of Herpes Simplex Virus 1
- The nerves are immunologically priviliged site
- Poor protective immunity for reactivation
- There are creeping lesions, which are scars from previous episodes
243
Viral immune evasion
- Intracellular pathogens
- This requires adaptive cell mediated immunity
1. Latency
Ø VZV, herpes simplex
2. Decrease in antigenic presentation Ø By binding to TAP
• Inhibits the peptide transfer to MHC
• Herpes simplex
3. Decrease in MCH expression
Ø Cytomegalovirus (CMV)
4. MHC mimics
Ø CMV
5. Mutation of epitopes
Ø B cells => Neutralisation escape
Ø T cells => CD8+ escape mutants of HIV
244
MHC and NK cells characteristics
- There is a CD8+ CTL cell, which is expressing TCR
- There is antigen peptide
- In the infected cells there is no MHC expression, due to virus inhibition
- The NK cells will then kill the infected cells
- There is "missing self", therefore foreign.
245
Describe the process of viral immune evasion
- There is mutation of the epitopes
- The B cells => neutralisation escape
- The T cells => CD8+ escape mutants of HIV
246
Definitions within antigenic variation - what are phenotype changes
- Colony morphology
- Virulence
- Serotype loose flagella
- Change surface sugars
Strategy for immune evasion and pathogenesis
247
What is antigenic diversity / polymorphisms
- Genetically stable + alternative forms of antigens in a population of microbes
e. g. serotypes of Strep. Pneumoniae
248
What is antigenic variation
Successive expression of alternative forms of an antigen in a specific clone or its progeny
249
What is phase variation?
- ON/OFF of an antigen @ low frequency occurs in 2 situations
1. During course of infection of an individual host
2. During spread of microbe through a community
250
What are the characteristics of Gonorrhoea - an STD
- Urethritis - discharge of pus - from scanty, to copius and purulent
- There is inflammatory & pyogenic infection of the anterior urethra
- Will infect the mucosal surfaces with columnar epithelium
• Urethra, cervic, rectum, pharynx, conjunctiva
251
What are the symptoms of Gonorrhoea
- Dysuria, redness, swelling, pain on urination, destruction of mucosa
- Prostatitis, orchitis, strictures, ovaritis, fistuals, PID, proctitis, sterility
Disseminated infections => arthritis, endocarditis, meningitis.
252
Characteristics of neisseria gonorrhoeae
- Surface components interact with host cells
- Components vary @ high frequency in a population of bacteria
Variation to avoid immune response
253
Phase and antigenic variation in Neisseria affects cell surface components
1. Phase variation
- ON-OFF switch (capsule, Opa's)
2. Antigenic variation
- e.g. pilins (or both phase and antigenic)
254
What are the characteristics of Influenza virus haemaglutinin
- In the receptor site, there are 4 major antigenic variable regions. Lipid bilayer of the envelope
- There is point mutation and immune selection + Antigenic Drift
255
How do bacteria avoid the immune response - innate & adaptive mechanisms
- There is a bacterium that is surrounded by a capsule
Ø Prevention of opsonin binding
Ø C3a & C5a proteases Anti Inflammatory and anti chemoattractant
Ø Inhibits opsonisation
Ø Inhibits complement activation
Ø Ig binding proteins e.g. protein A
Ø sIgA proteases
Ø Inhibition of antigen presentation
Ø Superantigens & innappropriate immune activation
Ø Blockage of cell cycle progression
Ø Induction / inhibition of apoptosis
256
How to viruses avoid the immune response - innate and adaptive mechanisms
Antigenic variation
Ø MHC mimics. This blocks killing by NK cells
Ø Downregulate MHC
Ø Block antigen processing by TAP
Ø Block MHC, presentation of antigen
Ø Induce immune suppression (decrease in CMI , CD4+)
Ø External host proteins e.g. B2 microglobulin and CMV
Ø Host mimicry
Ø Cytokine mimics and binding proteins
Ø Hide inside the cells + survival inside the cells
Ø Blockage of cell cycle progression
Ø Induction/inhibition of apoptosis
Ø Rapid growth + transmission prior to adaptive immunity e.g. colds
Ø Latency reactivation (e.g. VZV, Herpes Simplex)
Ø CTL escape mutants, quasi species swarms
257
Give examples of chemical carcinogens
- Coal tar
- Cigarette smoke
- Aflatoxins
258
Give examples of physical carcinogens
- UV light
Asbestos
259
Give examples of viral carcinogens
- Hepatitis B
| - Epstein Barr
260
Describe the characteristics of tumour promoters
- Exposure to agent => development of cancer
- This shows the initiators and the promotors
- Example of initiators = benz(a)anthracene
- Example of promoter (e.g. phorbol esters)
261
Explain how radiation can lead to DNA damage
- Ionising radiation (X-Rays, nuclear radiation, UV radiation)
• Causes damage, DNA breaks pyrimidine dimers, which causes failed repair, then there are translocations mutations
262
Describe the viruses that cause cancer in human cancer - DNA viruses
DNA viruses
Epstein-Barr virus
Ø Burkitt's lymphoma
Ø Nasopharyngeal carcinoma
Papilloma viruses,
Ø Cervical carcinoma, warts
Hepatitis B
Ø Hepatoma
263
Describe the viruses that cause cancer in human cancer - RNA retroviruses viruses
RNA retroviruses viruses
SLOW acting
Ø Leukaemia
Ø Mammary tumours
ACUTE, transforming
Ø Sarcoma
Ø Carcinoma
Ø Leukaemia
264
Describe the properties that tumorigenic viruses need
Stable association with cells
Must not actually kill the cell
Must evade immune survaillence
265
What is Knudson's Hypothesis for Hereditary Cancers
- "2 hits" are required for the development of the sporadic cancer
- In hereditary cancers, the first event is provided in the germline
- This single hit will lead to loss of heterozygosity (allelic loss) + cancer
266
Describe the mechanism for cytogenic mechanisms for LOH (loss of heterozygosity - allelic loss )
- Homozygotes without effective normal TS gene, develop cancer
- Point mutations may not give rise to easily detectable LOH
- (LOH = loss of heterzygosity)
267
Describe the process of carcinogenesis by mutation and selection
- There is sequential accumulation of mutations due to exposure to carcinogens
- Tumour cells will get selected for ability to grow and invade
- Selection will include resistance to therapy
- Some mutations may be deleterious for the tumour
268
What is hyperplasia
- Increase in tissue or organ size due to increase in cell number
269
Describe pathological hyperplasia
- This is often driven by a tumour elsewhere e.g. pituitary
| - Or autoimmune phenomenon (e.g. Graves disease or Cushings syndrome)
270
What are the examples causes of thyroid hyperplasia
1. Lack of secreting tissue (cretinism)
2. Lack of substrate (iodine deficiency)
3. Lack of enzymes in pathway - dyshormonogenetic goitre
Ø All of these cause hyperplasia, via normal feedback mechanisms
271
Describe the ophtalmopathies that happen in Graves disease
Ø Periorbital oedema
Ø Exopthalmos
Ø Lid retraction
272
What is hypoplasia
- Decrease in organ or tissue size
| - Due to the loss of cells
273
Give examples of physiological hypoplasia
- Endometrial hypoplasia after the menopause
| - Accompanies by atrophy of endometrium + myometrium
274
Give examples of pathological hypoplasia
- Loss of stimulation e.g. panhypopituitarism after pituitary infarct @ parturition leads to atrophy of target organs
- Pressure on organ from adjacent structures
- Insufficient blood supply
- Destruction of cells, e.g. autoimmune gastritis or thyroiditis.
275
What are the characteristics of hypertrophy
Ø Is an enlargement because of an increase in cell size
- Generally affects muscle
- Can by physiological, as in skeletal muscle
- Hypertrophy with exercise
- Myometrial hypertrophy in pregnancy
- Pathological hypertrophy might occur when an increased load is placed on the organ or tissue
• e.g. left ventricular hypertrophy in hypertension
- In pathological states there may be a combination of hypertrophy & hyperplasia.
276
What is atrophy
- The shrinkage of a tissue or organ
Due to the loss of cells & a decrease in cell size
277
Describe physiological atrophy
Thymic atrophy at puberty
278
Describe pathological atrophy
- Disease, e.g. muscle wasting in an immobilised fracture, or following nerve severance
- Response to pressure e.g. renal atrophy in ureteric obstruction
279
What is heterotopia/ectopia
- Well developed tissue or organ at the wrong site
- e.g. gastric or pancreatic heterotopia within a Meckels diverticulum
- A persistent vitellointestinal duct remnant
Ø Meckels diverticula are in 2% of people - usually are clincally silent
Ø 10% contain heterotopic stomach or pancreas
Ø Some might develop peptic ulcer
280
What is hamartoma
Ø Tumour like masses of tissue, appropriate to a size
| Ø But haphazardly arranged.
281
Describe bronchial hamartoma
- Benign coin like lesion that is often picked up on routine chest X-Ray
Below: Bronchial hamartoma composed of islands of cartilage + respiratory epithelium embedded in smooth muscle and fibrous tissue
282
What is metaplasia
- Reversible replacement of one mature tissue type by another
283
What is squamous metaplasia?
- Bronchus of smokers
- N respiratory ciliated pseudostratified columnar epithelium
- Or of the endocervix (N columnar), as it everts at puberty
284
What is intestinal metaplasia?
- In Barrets oesophagus (N squamous epithelium)
| - And helicobacter-infected stomach (columnar mucosa of specialised or non-specialised type)
285
What is osseus metaplasia?
- In the bronchial cartilage or in calcfied atheromatous deposits in aterial walls
286
What is dysplasia?
- Premalignant state (as in cervical dysplasia)
- Characterised by disordered maturation of cells within a tissue
- Dysplastic cells show cytological features of malignancy but cannot metastasise.
287
What is koilocytosis
Ø Warty changes in the epithelium
| Ø Spiky nuclei and perinuclear haloes
288
Metaplasia characteristics
- Occurs due to environmental stimulus
- Protective mechanism that is characterised by a switch of mature cell type (usually epithelial)
- Because of damage by an injurious agent
• Heat in bronchus (ciliated columnar to squamous)
• Exposure to acid vaginal secretions in the everting cervix (columnar to squamous)
• Oesophageal exposure to acid or bile reflux (squamous to columnar)
289
Dysplasia characteristics
- Often, but not always affects areas with metaplasia
- Microscopically shows abnormal tissue maturation
- Cells have mutations - dysplasia is a step towards the development of cancer
- Mutations, like errors in DNA replication are more likely to occur in tissue which is rapidly dividing
- Obviously the case in tissues which are damaged & undergo metaplasia
290
Describe premalignancy vs predisposition to malignancy
- Infection of the cervix with HPV 16 & 18 predisposes to the development of dysplasia. Dysplasia = premaligant condition
- Squamous carcinoma of cervix = premaligant
- Some inherited mutations (germline mutations) might confer an increased risk of developing maligancy
- I.e. a predisposition to malignancy (e.g. family polyposis coli, retinoblastoma)
291
Describe the process of field change - similar exposure to carcinogens
- Similar exposure to carcinogens - risk of multifocal primary malignancy
- Urothelium: carcinogens in urine
- Gut: carcinogens in diet
- Perineum and cervix : exposed to wart virus
292
What are the characteristics of neoplasms
- Autonomous growth - which is not sensitive to feedback mechanisms
- DNA mutation in a cell (usually several mutations) confers survival advantage
- DNA mutations = often caused by carcinogens but might arise spontaneously
- Monoclonal tumour cell population because all are derived from one original mutated cell
• Further mutations give the daughter cells new characteristics however known as "tumour heterogenity"
293
Stem cell characteristics
- Enable tissues to renew themselves
- They proilferate + differentiate into mature tissue types
- Stem cells have cytoprotective mechanisms which prevent harm from drugs
- Not all stem cells are locally derived. Marrow derived stem cells participate in normal wound healing + in angio and fibrogenesis in tumours
294
Stem cells and their link with cancer
- Cancer stem cells = mutated versions of normal stem cells
| -
295
Abnormal growth: uncontrolled cell division involving stem cells and daughter cells, e.g. colon
Stem cell @ crypt base divides
- This process = unregulated and excess cells are produced
- Stem cell can be mutated. Mutation may affect daughter cells, which might continue to divide instead of ceasing cell division after differentiation
296
What is a neoplasm
- "new growth" that is loosely applied.
297
What is cancer
- Malignant tumour not otherwise specified
298
What does the -oma ending mean
- Suffix that is applied to a lump
299
What does -carcinoma ending mean
- Suffix applied to malignant epithelial tumours
300
What does -sarcoma ending mean
- Suffix that is applied to malignant connective tissue tumours
301
What does -melanoma mean
- Malignant.
302
What does -granuloma mean
- Inflammatory collection of eptiheloid macrophages, like in Tuberculosis
303
How do tumours develop?
- Accumulation of mutations which override the normal mechanisms which control cell proliferation
304
What will make a normal cell divide?
- Normal tissue = needs to repopulate if the cells die of old age/ get damaged
- Each tissue contains stem cells which respond to growth factors, ceasing to divide once the requisite number of cells has been produced
- These signals are transmitted by protein molecules which stimulate receptors on a cell surface
- The signal is transmitted via intermediate receptors to the nucleus => where replication + cell division is initiated.
305
What are the sites of action of oncogene products
- Undetected, unrepaired oncogene mutation will begin the neoplastic process
- M RNA will carry mutated transcript for protein synthesis by the ribsome
- The mutated protein is synthesised in or outside the endoplasmic reticulum and then folded + modified in the golgi apparatus
- Mutated gene product is either secreted, incorporated, in the cell surface or forms an intracellular molecule
Ø Protection from apoptotic destruction
Ø Initiation of cell division by nuclear oncoproteins
306
What are the typical features of benign tumours?
Tumours can be classified into benign & malignant neoplasms.
1. Expansile growth
2. Bland cut surface
3. May be encapsulated
4. No lymph node or vascular invasion
307
What are the typical features of malignant tumours?
1. Irregular infiltrating edge
2. Foci of necrosis and haemorrhage
3. Adjacent organs
4. Lymph nodes
5. Satellite nodules
6. Distant sites via blood
308
Describe the differences between benign vs. malignant in gross macroscopical features - Benign features
1. Circumscribed - sometimes encapsulated
2. "pushing" margin
3. Homogenous cut surface, with little haemorrhage or necrosis
4. No obvious spread into adjacent organs or nodes
309
Describe the differences between benign vs. malignant in gross macroscopical features - Malignant features
1. Ill defined margin or obvious penetration of capsule
2. Infiltrative margin
3. Heterogenous cut surface, due to focal necrosis and haemorrhage
4. Infiltration of adjacent organs or lymph nodes
310
The differences in the microscopical features of benign and malignant features - Microscopical benign features
1. Tumour resembles original tissue
2. Low tumour cellularity
3. Cells have low NCR (1:4)
4. Nuclei are generally round or oval, with well dispersed chromatin
5. Scanty, normal, mitoses
6. No dysplasia in adjacent tissue
7. No vascular invasion
311
The differences in the microscopical features of benign and malignant features -
Microscopical malignant features
1. Often little resemblance to tissue of origin
2. High cellularity
3. Cells have High NCR (1-2:1)
4. Cells have atypical nuclei
Ø Pleomorphism
Ø Hyperchromatism
Ø Abnormal nucleoli
5. Numerous, often atypical, mitoses
6. Dysplasia may be present in adjacent tissue
7. Vascular invasion often present
312
Epithelial tumour characteristics - Benign epithelial tumours
Ø Squamous papilloma
Ø Adenoma
Ø Transitional papilloma
313
Epithelial tumour characteristics - Malignant Benign epithelial tumours
Ø Squamous cell carcinoma
Ø Adenocarcinoma
Ø Transitional cell carcinoma
314
Describe the characteristics of squamous epithelium
- Covers the skin, oropharynx, oesophagus, anal canal, vagina, external auditory canal
- Benign & malignant tumours might arise as a result of viral infection (e.g. HPV)
- Squamous cell carcinoma may arise @ an inappropriate site following squamous metaplasia
• e.g. bronchus of smokers
315
Describe the characteristics of the glandular epithelium
Ø Covers entire GI tract (from stomach to rectum)
Ø Lines the ducts & acini of glands
Ø Forms tubular structures such as renal tubules
Ø Glandular tumours may arise @ innappropriate sites following metaplasia, e.g. Barretts metaplasia in gastro oesophageal reflux disease (GORD)
316
Intestinal type adenocarcinoma appearance
Ø Characterised by glandular structures resembling normal intestinal gland formation
Ø This subtype is most commonly encountered in tumours that are related to Helicobacter Pylori gastritis
317
Appearance of renal cell adenocarcinoma (clear cell carcinoma):
Ø Characterised by glandular structures resembling normal intestinal gland formation
Ø This subtype is most commonly encountered in tumours that are related to Helicobacter Pylori gastritis
318
Appearance of renal cell adenocarcinoma (clear cell carcinoma): why do the tumour cells appear clear
- The tumour cells appear clear due to their high glycogen content
319
Urothelium (used to be transitional epithelium) characteristics
- The urothelium covers the urothelial tract, e.g. renal pelvis, ureter, bladder + urethra
- Benign tumours rarely if ever encountered.
320
Give examples of non epithelial tumours
```
Ø Connective tissue tumours
Ø Embryonal tumours
Ø Germ cell tumours
Ø Lymphohaemopoeitic
Ø Glial and neuronal tumours
```
321
Describe the classification by molecular features of tumours
- Soon, a tumours molecular characteristics which define its prognosis + response to treatment
- Can design drugs to target particular receptors.
1. Tamoxifen
- Blocks estrogen receptor
- In ER+ tumours of the breast
2. Herceptin
- Targets EGF-R in Her2Neu + tumours of the breast
3. Glivec
- Blocks tyrosine kinase receptor in CD117+ tumours
• Chronic myeloid leukaemia
• Gastrointestinal stromal tumours
322
Factors that a patient prognosis are influenced by
- Grade, stage, tumour site
- Production of ECM (desmoplastic reaction). This enhances tumour spread + limits access of chemotherapeutic drugs
- Other tumour effect: e.g. hormone secretion, cachexia
- Host response to tumour:
• Immunosuppresion - lymphocytic reaction to melanoma might cause regression @ the primary site; often after the tumour has spread.
323
What are the major functional changes that occur in cancer
1. Increased growth
Ø Loss of growth regulation
Ø Stimulation of environment that promotes growth e.g angiogenesis
2. Failure to undergo programmed cell death (apoptosis) or senescence
3. Loss of differentiation
Ø Including alterations in cell migration and adhesion
4. Failure to repair DNA damage
Ø Including chromosomal instability
324
Describe the characteristics of proto oncogenes
- Tightly regulated physiological function
- Usually in growth stimulation e.g. the growth factor pathway
- Proto-oncogenes may be mutated to become oncogenes & they were precursors of the retroviral oncogenes
- Mutations causing cancer have a dominant phenotype & are rarely inherited (i.e. they are somatic mutations)
325
Describe the characteristics of acutely transforming oncogenic retroviruses
- e.g. avian erythroblastosis virus (v-erbB)
- Avian myelocytomatosis virus (v-myc)
- Cause tumours, by the transduction of the viral oncogene
326
Describe the chromosomal translocations in Burkitt's lymphoma
- There is a translocation between chromosome 7 (MYC) & chromosome 14 (Ig heavy chain)
- Or less commonly, chromosome 8 & chromosome 2 or 22 (Ig light chain)
- In all cases, the result will be the unregulated expression of MYC.
327
Describe chromosomal translocations in chronic myeloid leukaemia's
- Fusion protein between ABL (9) and BCR (22); has enhanced tyrosine kinase activity
328
Describe the cell transformation by DNA from human cancers
Ø Transfect the DNA
Ø Culture it for 2 weeks, to allow transformed cells to grow
Ø Do an analysis of transformed cells, identified mutated RAS genes
329
Describe the oncogenic mutations that occur in KRAS
- There are mutations @ Gly12. Abolished GTPase activity, this prevents KRAS switching off
330
Mutated KRAS has dominant phenotype - describe this process
- Beta galactosidase postive and K-ras oncogene active
Ø Switching of KRAS in tissue showing somatic mosaicism
Ø There is adenoviral vector delivery of activated KRAS and beta galactosidase in patches of mouse colon (presumably cells transduced during development)
331
Describe the characteristics of tumour suppressor genes
1. Usually cell cycle checkpoint regulators (e.g RB1) differentiation (e.g. APC), or DNA repair (e.g. BRCA1)
2. Phenotype of mutations causing cancer is usually recessive @ the cellular level, but dominant at the level of the organism
3. Inheritance of a non-functinoal Tumour suppressor gene is the most common cause of heritable cancers.
Ø Transmission might be determined by screening a blood sample for the mutated allele
4. Heritable cancers develop after loss of functional allele (loss of heterozygosity)
332
Describe regulation of the cell cycle
1. Growth factor signalling
2. Expression of early response genes
3. G1 cyclin-dependent kinase (CDK) activity
Transcription of genes encoding proteins, required for DNA synthesis
333
Describe oncogenes in DNA tumour viruses
- There is binding of TP53 & targeting for degradation
- Inactivation of RB1
- Upregulation of EGF receptor
334
Describe the process of the dysregulation of Beta catenin
Ø Cell cycle arrest, differentiated cells
Ø APC or beta catenin mutation: progenitor like phenotype, site of future polyp formation.
Ø There are proliferating undifferentiated progenitors
Ø Beta catenin- Tcf/Lef target genes induced by Wnts
335
Describe the steps involved in Heritable breast cancer
- Radiation, free radicals causes damage to DNA => DNA double strand breaks .
Ø Can be repaired by homologous recombination
Ø In event of failed repair, translocations mutations can lead to either apoptosis or cancer.
336
Describe the therapy targeting oncogenes - Monoclonal antibodies (mab)
- Usually the target cell surface receptor proteins
- Trastuzumab: Herceptin - HER2/ERBB2
- Cetuximab: (EGFR/HER1/ERBB1)
337
Describe the therapy targeting oncogenes - Small molecular inhibitors (nib)
- Commonly kinase inhibitors
- Imatinib (Gleevec - BCR-ABL)
- Vemurafenib (BRAF)
338
Describe therapy by synthetic lethality
- Formation of DNA nicks during BER
- There is base excision repair (BER)
- Nicks can be converted to breaks by DNA replication
- Lack of BRCA1 causes death of cancer cell
- Repaired by homologous recombination in normal cells
339
Describe the genes controlling proliferation and differentiation
- Growth factors
- Cell cycle regulators
- Transcription factors
- Apoptotic factors
- Angiogenic factors
340
Describe the genes preventing mutations in the above + controlling the stability of the genome
- DNA repair genes
Checkpoint regulators (controlling passage through the cell cycle and chromosome segregation)
341
The characteristics of malignant tumours:
| Ø Growth
- Unlimited growth
- Not self-limited as in benign tumours
- As long as an adequate blood supply can prevent hypoxia
342
The characteristics of malignant tumours:
| Ø Invasiveness
- Migration of tumour cells into the surrounding stroma
| - Where they are free to disseminate via vascular / lymphatic channels to distant organs
343
The characteristics of malignant tumours:
| Ø Metastasis
- Spread of tumour cells from the primary site to form secondary tumours at other sites in the body
344
Explain the steps of classical progression in colorectal cancer
There is loss of SMAD2/SMAD4 in lots of colorectal cancers
345
What is the molecular basis of tumour progression
- Acquision of specific mutations: carcinogens, multiple hits
- Clonal expansion: tumour promoters
- Genomic instability: DNA repair defects, aneuploidy, LOH
- Epigenetic changes - promoter methylation
346
Describe genomic instability in colorectal tumours - Chromosomal Instability (CIN)
Ø Metaphase checkpoint failure leads to aneuploidy and non disjunction (loss of heterozygosity)
Ø LOH @ 18q might involve the loss of DCC or SMAD2/SMAD4 (TGF beta pathway functions)
347
Describe genomic instability in colorectal tumours - Microsatellite Instability (MIN)
Ø Mismatch repair (MMR) gene mutations cause increased mutability (particularly noticeable in repetitive, microsatellite DNA sequences)
Ø Hereditary non-polyposis colorectal cancer (HNPCC); caused by loss of MMR gene function
348
Describe what happens in the changes during tumour progression
- Progression will lead to heterogeneity & seletive pressures will determine the cellular composition of tumours which might vary in properties
• Antigenicity
• Growth rate
• Response to hormones
• Response to cytotoxic drugs
• Capacity for invasion & metastasis
- Some heterogenity in tumour cells might have no obvious benefit to the tumour but will confound attempts to identify changes that are potential targets for treatment
349
Explain the steps that are involved in cancer dissemination
1. Primary tumour formation
2. Localised invasion
3. Intravasation
Ø Interactino with platelets, lymphocytes, and other blood components
4. Transport through the circulation
5. Arrest in microvessels of various organs
6. Extracasation
7. Formation of micrometastasis
8. Colonisation - formation of macrometastasis
Ø Overall process = highly inefficient
Ø Tumours cells injected intravenously can extravasate successfully (>80%)
Ø But 2 fast steps are very inefficient (
350
Describe the process of neovascularisation of tumours
- Tumours will not grow beyond size of about 2mm without their own blood supply
- Angiogenesis = promoted by hypoxia
351
What are the angiogenic factors? And what do they do?
- Some tumour cells might produce factors
- They simulate the directional growth of endothelial cells.
1. Vascular endothelial growth factor (VEGF1)
2. Fibroblast Growth Factor 2 (FGF2)
3. Transforming Growth Factor Beta (TGF-b)
4. Hepatocyte growth factor/scatter factor
- They are mainly stored + bound, inactive to components of the Extracellular matrix (e.g. heparan sulphate)
- And may be released by matrix metalloproteinases
- Angiogenesis inhibitors are important therapeutically e.g. antibodies to VEGF (bevacizumab) thalidomide
352
What are the mechanisms of tumour cell invasion?
1. Increased mechanical pressure caused by rapid cellular proliferation
2. Increased motility of the malignant cells (epithelial to mesenchymal transition)
3. Increased production of degradative enzymes by tumour cells and stromal cells
353
Describe the process of epithelial-mesenchymal transition
Ø There is loss of:
• Epithelial shape + cell polarity
• Cytokeratin intermediate filament expression
• Epithelial adherens junction protein (E-cadherin)
Ø There is acquisition of:
• Fibroblast-like shape + motility
• Invasiveness
• Vimentin intermediate filament expression
• Mesenchymal gene expression (fibronectin, GDGF receptor, avbeta6 integrin)
• Protease secretion (MMP-2 and MMP-9)
354
Cell adhesion molecules and invasion:
| Ø What are the characteristics of E-Cadherins
- Homotypic adhesion molecule (adhesion of cells with the same cadherin)
- Calcium dependent
- Inhibits invasiveness
- Binds beta catenin
355
Cell adhesion molecules and invasion:
| Ø What are the characteristics of integrins
- Heterodimers (alpha and beta subunits)
- Heterotypic adhesion molecule
- Adhesion to extracellular matrix (via collagen, fibronectin, laminin)
- Cell migration
356
Describe the activation of extracellular proteases in tumours
- Tumour cells will activate plasminogen activator, released by stromal cells
- This results in plasmin production
- Plasmin activates matrix metalloproteinases - which permit invasion & release matrix bound angiogenic factors
357
What is the importance of stromal cells in the tumour
- Many tumours stimulate a host inflammatory response
- Stromal cells include:
Ø Macrophages
Ø Mast cells
Ø Fibroblasts
- These cells release factors:
Ø Angiogenic factors
Ø Growth factors
Ø Cytokines
Ø Proteases
○ That have important effects on tumour progression
358
Describe the regulation of proteolytic activity
- The extent of proteolysis depends on the relative amounts of proteinase & inhibitors of proteinases
- Most tissues = have large amounts of a family of inhibitors calls TIMPS (Tissue Inhibitor of Metalloproteinases)
- Some tumours, e.g. pancreatic tumours = decreased TIMPs level
- Synthetic, low molecular weight inhibitors of MMPs, e.g. Marimastat, have entered clinical trials
359
What determines the pattern of tumour spread:
Ø The mechanical hypothesis
- Anatomical considerations
- Blood + lymphatic systems
- Entrapment in capillary beds (20-30 micrometres carcinoma cells, about ~8micrometres capillary)
- Genetic alterations acquired during progression allows tumour cells to metastasize
360
What determines the pattern of tumour spread:
Ø The seed & soil hypothesis
- There are specific adhesions between tumour cells & endothelial cells in the target organ
- Favourable environment in target organ for colonisation
- Genetic alterations acquired during progression allows tumour cells to metastasize
361
Describe the key steps in progression of a tumour
Ø Angiogenesis (overcomes hypoxia)
Ø Acquisition of ability to undergo epithelial to mesenchymal transition (invasive properties allowing intravasation and extravasation)
Ø Colonisation (ability to expand from a micrometastasis in the target organ)
Ø Release of metastatic cells that have acquired ability to colonise
Ø Resistance to drug interventions
• Expression of MDR, or loss of drug receptors
362
How do tumours spread?
- There are easy routes of spread:
1. Directly into adjacent tissues (basal cell carcinoma of skin)
2. Via lymphatics (Ca breast, colon)
3. Blood vessels - (renal cell Ca, small cell Ca lung, prostatic Ca, all sarcomas)
4. Along nerves (eg Ca pancreas, prostrate)
5. Across coelomic cavities (e.g Ca stomach, ovary)
363
Describe the interesting behaviour of tumours
- CNS tumours can spread widely within the CNS compartment, but are usually confined to the Blood brain barrier
- Some tumours have a particular propensity to spread to bones
• e.g. breast, lung, thyroid, prostate
364
What is TNM?
```
T = tumour
Ø General tumour size, e.g. Ca breast
N = nodes
Ø Regional lymph node involvement is N1, distant nodes N2
M = metastases
Ø M0 = none
Ø M1 = present
Ø MX= not known
- It is possible to detect some tumours @ the T0 non invasive
```
365
Describe the Ann Arbor staging system for Hodgkins diease
Stage 1 => one node group
Stage 2 => more than 1 node group, same side of diaphragm
Stage 3 => node groups either side of diaphragm
Stage 4 => non lymphoreticular organs involved
- The spleen counts as lymphoreticular organ
- Presence of B symptoms a poor prognostic feature
366
What are the effects of chemotherapy
- Might modify the stage of a tumour
| - The prognosis is determined by the pre-treatment stage
367
What are the main factors important in predicting tumour prognosis
- Classification: histological subtype
• e.g. small cell anaplastic carcinoma of lung very poor prognosis
- Grade (differentiation)
- Stage (spread)
• General: TNM, clinical stage I-IV
• Specific, e.g: Duke's (colorectal adenocarcinoma), Ann Arbor (Hodgkin's lymphoma)
- Molecular features, e.g. expression of receptor molecules
• Oestrogen receptor, Her-2-Neu
368
Other influences on a patients prognosis - on the following factors
Ø Tumour site
• Local effects, e.g. astrocytoma
Ø Production of extracellular matrix (desmoplastic reaction) which enhances tumour spread & limits access of chemotherapeutic drugs
Ø Other tumour effect
• Hormone secretion, cachexia
Ø Host response to tumour
• Lymphocytic response
○ e.g. regressed melanomas might be the source of metastatic disease
• Immunosuppression
○ e.g. renal transplant & HPV-driven SCC
○ HIV/AIDS and cerebral lymphoma
369
Desmoplastic stroma
- Has hypovascular microenvironment
- Which will promote tumour growth and invasion
- Cancer cells survive better if accompanied by pancreatic stellate cells - these then migrate with them to metastatic sites, and generate similar stroma
- Targetting the stromal microenvironment
• e.g. with anti-MMP (matrix metalloproteins)
• May permit better access for chemotherapeutic drugs
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Describe what cancer cachexia is?
- Cachexia (kaos + hexis = bad conditions) - 20% cancer deaths are associated with cachexia. Characterised by unintentional weight loss.
- Cachexia conditions worst in upper GI cancer
- Unlike starvation which depletes body fat stores and tries to conserve lean body mass, in cachexia there is depletion of host reserves of fat + muscle
- Driven by cytokines, including TNF + interleukins. So will not response to extra nutrition alone.
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Summary of factors important in predicting tumour prognosis
- Classification: histological subtype
- Grade (differentiation)
- Stage (spread)
• General: TNM, clinical stage I-IV
• Specific, e.g. Dukes' (colorectal adenomacarinoma), Ann Arbour (Hodgkins lymphoma)
- Molecular features, e.g. expression of receptor molecules e.g. oestrogen receptor, Her-2-Neu
- Host response to tumour, e.g. desmoplasia
- Tumour effects on host, e.g. cachexia
