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Gastro BSc > Module 3.1: Basic Immunology > Flashcards

Module 3.1: Basic Immunology Flashcards

1
Q

Define T-Cells

A

Lymphocytes with key importance to the immune system.

They are at the core of ADAPTIVE immunity, the system that shapes the body’s immune response to specific pathogens

Mature T-cells are derived from the THYMUS gland and participate in a variety of cell-mediated immune reactions

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2
Q

Subsets of T-cells

A

CD4+ —> T-helper cells

  • They lead the attack against infections
  • Four fundamental subsets
  • – Th0
  • – Th1
  • – Th2
  • – Th17

CD8+ –> cytotoxic T-lymphocytes (CTLs)

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3
Q

Subsets of T-helper cells

A

Th0 - undifferentiated

Th1

  • responsible for cell-mediated mechanisms
  • eliminate INTRACELLULAR pathogens

Th2

  • role in regulating Ab production
  • eliminate EXTRACELLULAR pathogens

Th17
- key regulator of inflammation

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4
Q

Describe the polarisation of Th cells

A

Th0 can differentiate into Th1, Th2 or Th17 depending on the cytokine environment that it is in

Signature cytokines will be produced as a prime response of the different T-helper cells

In IL-12/STAT4 –> Th1 –> secrete IL-2/IFNg/TNF (transcription factor Tbet)

In IL-4/STAT6 –> Th2 –> secrete IL-4/IL-5/IL-10 (transcription factor GATA-3)

In TGFb/IL6/+-DC/IL-23/IL-1b –> Th17 –> secrete IL-17/IL-21/IL-22 (transcription factor RORg/RORa)

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5
Q

Describe the role of T-cells in immune responses

A

ANTIGEN recognition on the cell surface by MHCI or MHCII

The antigen specific TCR on the T-cell surface interacts with the appropriate peptide-MHC complex

T-Cells play a critical role in the regulation of immune responses and are responsible for mediating many of the effector mechanisms of the immune system

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6
Q

Describe the steps required to take before assessing T-cell function

A

Isolation - Measure - analyse and interpret

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7
Q

Describe how T-cells can be isolated

A

For in vitro assays

  • Human or animal blood
  • Animal spleen

Sorted by

  • magnetic bead separation
  • flow cytometry sorting
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8
Q

Describe the principles of Ficoll Paque Isolation of peripheral blood mononuclear cells in T-cell extraction

A

Completed via density gradient centrifugation using Paque/Ficoll-Paque Plus

  • Layer blood on Ficoll-Paque and centrifuge
  • Due to the density gradient between whole blood and Ficoll-Paque, you end up with different layers based on the density of each component

Allows for purer isolation and enables freezing to be used at a different time

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9
Q

Describe the principles of Conjugated Magnetic Beads in T-cell extraction

Give two examples in commercial use

A

EXAMPLES: Dynabeads, MACS system

DYNABEADS

  • add beads to sample
  • they bind to desired target
  • respond to magnetic field allowing bound material to be rapidly separated
  • – These beads are quite big –> don’t want them in culture

Soooo

MACS microbeads

  • smaller, biodegradable beads (won’t be in culture)
  • easier separation
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10
Q

Positive selection vs negative selection

A

Positive: target cells isolated directly

  • best purity
  • best recovery
  • fast

Negative: all the unwanted cells are collected as the fraction
- may be considered for avoiding activation of cells

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11
Q

Describe cell sorting via Flow Cytometry

A

Flow sorting = fluorescence microscopy whereby single cells in liquid suspension can be identified and physically separated from each other according to unique charateristics

e.g. for CD4+ cells: stain the cells and sort them

Start with a mixed population
Stain cells with colour conjugated Ab againt CD4+
Incubate
Wash and sort cells

Data = DOT plot or HISTOGRAM

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12
Q

Describe methods of measuring T-cell function

A

3H-thymidine incorporation assays

CFDA-SE (CFSE) labeling

Limiting dilution analysis

Measurment of cytokine production

  • immunoassays or bioassays
  • ELISPOT
  • intracellular cytokine staining
  • chromium release
  • ELISPOT for CD8+ T cells activity
  • Tetramer analysis for direct visualisation of CD8+ T cells
  • RNA extraction and real time PCR
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13
Q

Describe the principles of 3H-thymidine incorporation assays in T-cell function measurments

A

measures the strength of response of T-helper cells by measuring the proliferation of T-cells

Occurs following stimulation of cells using different stimuli

Hence T cells are counted
- Positive control: incubated in the presence of a known mitogen e.g. ConA - used to ensure that cells are not responding due to a specific defect they have in proliferation if they are blind to specific antigen hence the need for a positive control

  • Negative control: incubated in the abcence of any stimuli
  • incubated in the presence of specific antigen being tested

When cells proliferate, they will incorporate the radioactive thymidine into the DNA of the dividing cells

Incubated foe 3-7 days and harvested

Radioactivity is measured

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14
Q

Describe the principles of CFDA-SE in T-cell function measurments

A

CFDA is not radioactive

passively diffuses into cells

its acetate groups are cleaved by intracellular esterases

highly fluorescent amine-reactive carboxyfluorescent succinimidyl ester interacts with intracellular amines to form fluroescent conjugates

The label is inherited by daughter cells

HOWEVER, cell division causes halving of the fluorescnece - limited to 8-10 divisions

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15
Q

Describe the principles of ELISA in T-cell function measurments

A

Enzyme linked immunosorbent assays

specific Abs are coated on a plate

specimens are incubated on the plate to measure cytokines directly

another layer of Ab conjugated to an enzyme is added

Followed by the addition of a substrate to the enzyme

ELISA uses a standard curve –> cytokine conc can be extrapolated

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16
Q

Describe the principles of ELISPOT in T-cell function measurments

A

similar to ELISA

looks at the response of SINGLE CELLS that are cultured with other cells

Data will be quantified differently through DETECTION OF SPOTS on nitrocellulose lined microtitre wells

Allows for qualitative and quantitative analysis

Detects cytokine release at single cell level

each spot is the footprint of a single cell that reacted to the antigen

Can be quantified by eye or automated readers

Amount of cytokine released is proportional to spot size and intensity

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17
Q

ELISA vs ELISPOT

A

ELISPOT 200x more sensitive

however the scoring of the spots involve manual enumeration in ELISPOTT

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18
Q

Describe the principles of MSD (meso scale discovery) in T-cell function measurments

A

Multiplexing platorm of more than one cytokine –> uses electrochemiluminescence (ECL) detection

ultra-low detection limit and minimal background

Up to 5 logs of linear dynamic range

minimal sample - as low as 10-25 microlitres

easy handling and protocol

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19
Q

Describe the principles of intracellular staining in T-cell function measurments

A

Flow cytometry used

T cells are stimulated in vivo

the transport cytokines through the golgi is blocked in order to prevent the secretion of cytokines

T cells are then fixed and permeabilised to allow cytokine specific Abs to enter the cell

Directly conjugated anti cytokine Abs are then used for staining

Large no of T cells can be analysed in a short time

More than one cytokine can be assessed at a time

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20
Q

Describe the use of Chromium release assays and ELISPOT assays in CD8+ cell analysis

A

Chromium release assays:

  • radiolabel the target cells with sodium chromate
  • incubate with test effector cells for 4-16h
  • the amount of Chromium released in the supernatant is then quantified to provide a measure of target cell lysis

ELISPOT

  • where the antigen is a peptide known to be an epitope recognised by CD8+ CTL
  • look at CD8+ cells producing IFNg and/or TNFa
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21
Q

Describe the use of limiting dilution anaysis in T cell analysis

A

not favourable

LDA are used for quantitative estimates of the number/frequency of T cells that are specific for a particular antigen

Positive results indicate the presence of antigen specific precursor cells in PBMCs population at the start

Long incubation period - 2-3 weeks and risk to over/underestimate cell numbers

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22
Q

Summarise the characteristics of H. pylori

A

G-ve
spiral
microaerophilic
multiflagellated GI pathogen

colonises gastric mucosa

Colonises 50% of the world population, only 15% symptomatic

90% prevalence in developing countries, 30% in developed

Causes:

  • gastritis
  • gastric and duodenal ulcers
  • MALT lymphoma
  • atrophy
  • non-ulcer dyspepsia
  • adenocarcinoma
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23
Q

Summarise the virulence factors of H. pylori

A

Motility

Urease
- enzyme that hydrolises urea into ammonia and CO2 - provides protective cloud allowing it to move from acidic enviornments to epithelial cells

Adhesins
- Helicobacter outer membrane proteins (HOP) –> allows it to stick to epithelial surfaces

Pathogenicity islands (PAI)

  • include the cytotoxin-associated gene A (CagA)
  • immunodominant protein into target cells
  • 90% CagA+ from ulcer patients - contributes to virulence and damage
Vacuolating cytotoxins (VacA)
- cytoplasmic toxic vacuoles inside target cells

Lipopolysaccarides

  • site of antigenic variation
  • Formed by lipid A, core oligosaccharide and O-chain (most external part seen by host)
  • O side chain expresses LEWIS ANTIGENS - extended chains with fucosylated carbohydrate structure
  • phase variation is the occurence of reversible high frequency on and off switching of cell surface receptors
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24
Q

Define collectins

A

components of innate immune system (PAMP receptors)

innate immunity is an antigen-non-specific defence mechanism used by a host after exposure to antigen

Initial response by the body to eliminate microbes and prevent infection

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25
Q

Desribe the importance of collectins in the gut

A

the gut has many challenges
- food contains microoganisms that could thrive in rich growth medium of digested nutrients

Abundant potential sites of infections in the long tube

oral mucosa, intestinal mucosa comprised of only a single layer of epithelial cells

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26
Q

Describe the type of collectin family found in mammals

A

Mammalian C-type collagenous carbohydrate binding proteins

C - as they require calcium for their action

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27
Q

Which chromosome is the family of proteins implicated in innate immunity encoded on?

A

10

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28
Q

List the proteins implicated in innate immunity on Chr10

A 
surfactant protein A (SP-A)
SP-D
Mannan binding lectin (MBL)
Collectin Liver 1 (CL-L1)
Collectin Placenta 1 (CL-P1)
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29
Q

Describe the 4 domains of collectins

A
  • Carboxy Terminal C-Type Lectin Domain
  • Alpha-helical coiled coil
  • Collagen-like triple helix
  • N-Terminal collagenous region
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30
Q

Describe the role of SP-D in immunity

A

Recognises and binds SELECTIVELY to the surface of viruses, bacteria and fungi and directly to the LPS on G-ve bacteria

Results in aggregation of microorganisms with ENHANCED PHAGOCYTOSIS by neutrophils and macrophages

First discovered in alveolar space, secreted by alveolar type II cells and non-ciliated bronchial epithelial cells

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31
Q

Describe the role of SP-D in H. pylori infections

A

SP-D is expressed and upregulated in the presence of H. pylori infection

binds and agglutinates and inhibits motility in a calcium dependent, lectin specific manner (Murray et al 2002)

Not all H. pylori agglutinate in the presence of SP-D - escape variants present (Khamri et al 2005)

Higher proportion of SP-D binding organisms in the mucus - suggesting that SP-D binding organisms are trapped in the mucus where they can be cleared through physical elimination (Khamri et al 2005)

H. pylori evade SP-D by varying the LPS antigenic structure

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32
Q

Describe the role of dendritic cells in innate and adaptive immune response

A

link between the two

Uptake of CFDA-SE labelled H.pylori by transferrin-Alexa fluor labelled DCs

After 15m of incubation in the absence and after 15m incubation of SP-D and calcium

Uptake by DC was significantly enhanced in the presence of SP-D

Concluded that:

In the absence of SP-D, mice are more suceptible to low dose of H. pylori

Neutrophil responses are diminished in the absence of SP-D due to the absence of chemotactic function of SP-D

In the absence of SP-D, T cell responses are diminished probably due to impaired Ag uptake by DCs

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33
Q

Summarise the differences between innate and adaptive immune responses

A 
INNATE:
immediate
low specificity
same every time
hard wired
reflex
ADAPTIVE:
delayed
high specificity (recognises specific aa sequences)
increases with repeat exposure
sophisticated control (e.g. T-reg)
"voluntary complex movement"
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34
Q

What are the two divisions of the innate immune system?

A

Sensing and Responding

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35
Q

Describe innate immune sensing

A

Senses

  • self vs non-self
  • danger signals - you can present foreign antigens without a danger signal hence not responding e.g. chronic HepB infection

mainly related to detecting PAMP receptors
- LPS, flagellin, RNA

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36
Q

Describe the different types of PAMP receptors and what they recognise

A

Collectins (mannose binding lectin, SP-A, SP-D)
- bacterial/fungal carbohydrates

C-type lectins - recognise carbohydrates
- fungal cell wall (b-glucan)

Pentraxins
- phosphocholines

TLRs

  • microbial genomes
  • bacterial/fungal cell walls
  • flagellin

NOD-like receptors (intracellular)

  • Viral RNA
  • bacterial cell wall

RIG-1 receptors (intracellular)
- viral RNA

AIM2-like receptors (intracellular)
- cytosolic DNA

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37
Q

Define Dendritic cells’ role in immunity

A

immature DCs are not localised and sample antigenic proteins in tissues

interaction with foreign Ag leads to MATURATION –> localisation to LNs

present the antigen to CD4+ –> proliferation

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38
Q

What is the danger model of DCs

A

Matzinger 2002

DCs want to find a non-self Ag or a danger signal

The immune system is more concerned with damage than with foreignness –> called into action by alarm signals from injured tissues rather than the recognition of non-self

HYPOTHESIS OF DAMPs - Danger associated molecular pattern receptors

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39
Q

How does the danger model work?

A

DCs stimulate CD4+ and co-stimulate it with CD40 only in the presence of a danger response –> appropriate Th1 response

If it doesnt encounter a danger response –> regulatory response instead –> Tr1 for immunoregulation activated

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40
Q

Describe the structure of TLRs

A

have an extracellular region with contains Leucine rich repeats (LRRs) + cytoplasmic tail with Toll/interleukin-1 receptor (TIR) domain

Different TLRs recognise different surface and intracellular components of microorganisms

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41
Q

Summarise the types of TLRs

A

TLR2 + TLRX

  • lipoproteins
  • peptidoglycan
  • zymosan
  • present in Gm4ve, yeast, fungal infections

TLR3
- double stranded RNA

TLR4

  • LPS
  • present in G-ve bacteria

TLR5
- flagellin

TLR7
- single stranded (viral) DNA

TLR9

  • hypomethylated CpG DNA
  • hypomethylation indicates bacterial DNA (not host)
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42
Q

Describe the distribution of TLRs

A

most expressed on cell surface

Some only useful inside endosomes - related to bacterial/viral invasions thar are engulfed in phagocytic cells
- 3,7,9 implicated

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43
Q

Describe the roles of TLR5

A

involved in FLAGELLIN DETECTION

non-flagellated e.coli is not detected by TLR5

If induction of listeria flagellin to e.coli – TLR5 expression increases

Salmonella –> normally has flagella and expresses TLR5
– KO flagella causes dampening of TLR5 expression

TLR5 muttion - Hayashi et al 2001

  • causes non-response
  • associated with recurrent UTIs
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44
Q

Describe the roles of TLR2

A

Detects a range of bacterial components

Arg677Trp mutation predisposes pt towards lepromatous (rather than tuberculoid) leprosy

tuberculoid leprosy: immune response can contain infection

lepromatous leprosy: inadequate T-cell response due to inadequate innate response

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45
Q

Describe the roles of TLR9

A

TLR9 KO - decreases response to Helicobacter + decreased inflammation

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46
Q

Describe the roles of TLR4

A

Does not induce signal by itself and REQUIRES CO-FACTORS

  • Bacterial LPS interacts with liposaccharide binding protein (LBP)

LBP interacts with CD14 on cell membrane

this causes interaction with TLR4 with adaptor molecule MD2

SO: multiple host proteins are needed to respond to LPS - not single ligand

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47
Q

Describe the roles of TLR3

A

involved in recognition of double stranded RNA

HCV blocks TLR3 signalling via NS3/4 protease production thereby preventing body from inducing IFN responses via IFR3 and leading to PERSISTENT VIRAL INFECTION

2003 - FIND THE REFERENCE?????

Imran et al 2012?

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48
Q

Describe the interactions between TLRs and the TIR domain

A

triggers the activation of the innate immune system + development of acquired immunity

Cytoplasmic tails of TLR show similarities to IL-1 receptor

TLR signalling pathways originate from the TIR domain (cytosolic component) as a result of its recruitment of TIR-domain-containing adaptors

Crucial proline residue in all TLR TIR domains, except TLR3

All TLRs likely have a MyD88 pathway (Except TLR3)

MyD88 - common adaptor to TLRs - myeloid differentiation primary-response protein 88

MyD88 KO mice have no response to LPS - ESSENTIAL TO INFLAMMATORY SIGNAL (to activate NFkB)

MyD88s –> splice variant of MyD88 - downregulates the inflammatory response

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49
Q

Describe the role of TIRAP, TRIF, TRAM

A

MyD88 independent pathway

  • TIRAP -

TIR domain-containing adaptor protein

Common mutation: S180L

Wild-type TIRAP –> strong NFkB signal

  • TRIF -

TIR domain containing adaptor protein inducing intrferon (TLR-3)

TRIF binds to IRF-3 to produce IFN

  • TRAM -

TRIF related adaptor molecule

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50
Q

What regulates the TLR-signalling pathways?

A

TLR-inducible molecules NEGATIVELY regulate TLR signalling

IRAK-M
SOCS1
MyD88s
SIGIRR
ST2
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51
Q

What genes are regulated by NFkB?

A

Activation of NFkB by binding of TLRs (except TLR3) leads to the activation of:

Inflammatory cytokines - TNF, IL-1, IL-6, IL-12, IFNb

Chemokines - IL8

Adhesion molecules - ICAM-1, VCAM-1

Immune effector mlecules - FasL, iNOS

Pro-survival molecules - Bcl-XL, A1, cIAP1,2

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52
Q

Summarise the role of TLRs in immunity

A

TLRs have an extracellular region containing LRRs and a cytoplasmic tail which has a Toll/IL1 receptor (TIR) domain

Different TLRs recognise different surface and intracellular components of microorganisms

The interaction between TLR and a microbial component triggers the activation of the innate immune response as well as the development of acquired immunity

TLR-signalling pathways originate from the TIR domain

as a result of its recruitment of TIR-domain-containing adaptors such as MyD88, TIRAP, TRIF and TRAM

Signalling through each TLR requires MyD88 for the production of inflammatory cytokines

HOWEVER a MyD88-independent pathway exists for signalling through TLR3 or 4 and leads to the production of IFN1

The TLR ignalling pathways are negatively regulated by TLR-inducible molecules such as IRAK-M, SOCS1, MyD88s, SIGIRR, ST2

53
Q

Examples of cytoplasmic PAMPr

A

NOD2

RIG1

54
Q

Describe the function of NOD2

A

Bacteria cell walls contain peptidoglycan which can be broken down to MDP

MDP interacts with NOD2 through the LRR region which can lead to two pathways

1-
binding results in oligomerisation of NOD2 and recruitment of RIP2 through homotypic CARD-CARD interactions

RIP2 activates IKK complex through IKKy/NEMO, resulting in phosphorylation, ubiquitination and degradation of IkB and release of associated NFkB members

2-
NOD2 activation can also result in the activation of MAPkinase pathways resulting in induction of AP-1 transcription factors

Peptidoglycan can also activate the TLR pathway (TLR2/6)

Signals via MyD88 to stimulate MAP kinases: p38, JNK, ERK causing crptidins and defensins

55
Q

Describe the function of RIG-I

A

RIG-1 signalling is in the same pathways as TLR - NS3/4 pathways also interferes with this system

HCV proteins NS3/4A eliminates antiviral signalling by cleavage of MAVS and TRIF

Production of dsDNA by HCV during infection should result in synthesis of IFNb which in turn leads to production of antiviral proteins known as ISGs

PAMP receptors RIG1 and TLR3 signal through kinases IKKe and TBK1 to activate IRF3 and cause the phosphorylation and degradation of IkB which allows for the nuclear translocation of NFkB.

HCV overcomes this response and establishes chronic infection through cleavage of MAVS and TRIF

56
Q

Give examples to the innate immune effectors

A

PHYSICAL BARRIERS

  • skin
  • oral and GI mucosa - acid and pepsin
  • mucus membranes - mucociliary clearance

CHEMICAL BARRIERS

  • complement system
  • defensins
  • proteases, DNAses, RNAse

CELLULAR BARRIERS

  • macrophages
  • NK cells
57
Q

Describe the complement system in immunity

A

• Key role in innate and antibody-mediated immunity
• Made up of a complex series of proteins and glycoproteins
- Majority are found in solution in the blood
- Some are membrane bound
- These compounds are mainly produced by the liver, some by monocytes and macrophages

  • Triggered by enzyme cascade system in blood
  • Once triggered, produces a very rapid and highly amplified response
  • Activation occurs through three distinct pathways:
  1. Alternative (main)
    - Continuous tick-over production
    - Full activation upon fixation to foreign substances (bacteria)
  2. Classical pathway
    - Activated by antigen-antibody complexes
  3. Lectin pathway
    - Antibody-independent activation of Classical pathway

Classical and Alternative pathways converge at C3, the third component of complement, leading to the final common pathway

The common pathway leads to the formation of the Membrane Attack Complex (MAC), which causes cell lysis and death

58
Q

What are the roles of the complement pathway?

A
  • Opsonisation of microorganism for phagocytosis
  • Direct killing via MAC
  • Promotion of inflammation
  • Chemotaxis of neutrophils and leukocytes
  • Processing of immune complexes (dysregulation of this can lead to conditions such as SLE)
  • Augments induction of specific antibody
59
Q

Describe the recruitment of monocytes and macrophages

A

Inflammation leads to chemokine release, which recruits monocytes via concentration gradient

recruitment also involves using adhesion molecules that attache these and allow transmigration into the cell

Monocytes are localised and enter tissue to become macrophages

Different signals result in different types of macrophages (M1 + M2)

M1

  • Pro-inflammatory
  • Phagocytose bacteria/viruses/dead cells

M2

  • Involved in the resolution of inflammation
  • Deactivated, alternatively activated, activated (more complex)
  • Produce anti-inflammatory cytokines + TGFb + efferocytosis (apoptotic/necrotic cells are removed by phagocytic cells)
60
Q

Describe how microbes are killed by monocytes and macrophages

A

The microorganism is enclosed in a phagolysosome within the phagocyte

Many hostile factors within the lysosome

  • Low pH
  • Hydrolyases, proteases, defensins
  • ROS (most important  chronic granulomatous disease = enzyme producing ROS Is defective)

Some microorganisms can escape/survive

  • TB can survive acidity with the waxy coat
  • TB also inactivates IFN-y signalling, which is required for ROS production

When the microorganisms are broken down, their antigens are loaded onto MHC II, to be presented to Th molecules

61
Q

Describe how monocytes and macrophages achieve antigen uptake

A

Phagocytosis: Involves the ingestion of particulate material including whole pathogenic microorganisms. The plasma membrane expands around the particulate material to form large vesicles called phagosomes (10-20times larger than endosome) [endocytosis is macromolecule ingestion]

Phagocytosis begins with engagement of the bacteria with cell surface receptors

These trigger changes in the cytoskeleton thereby triggering phagocytosis

End result: pathogen broken down in the endosomal compartment so that it is expressed on cell surface receptors MHC Class I and II so that it can stimulate T-cell responses (link between innate and adaptive immunity)

62
Q

Describe the role of defensins in immunity

A
  • Short (25aa) peptides, containing multiple disulphide bonds
  • Very toxic to bacterial cell walls

“punch holes in bacterial cell walls”

• Produced by Paneth cells in the crypts

63
Q

Describe the applications of Molecular Diagnostic Methods in Pathology

A

• Infectious disease
o Virus, Bacteria, Parasites, Fungi
• HLA typing (transplantation)
• Cancer (genomics)
• Genetic diseases (e.g. cystic fibrosis)
• Chromosomal abnormalities (Trisomy 21)
• Immune deficiencies diagnosis
o (e.g Severe combined immunodeficiency – “bubble baby disease”)

64
Q

What is the Key to Success of Molecular Methods

A
  • For a new diagnostic technology to become a mainstream tool it has to be faster, cheaper and better than existing alternatives
  • Concept coined as the iron triangle (Daniel Goldin – NASA)
65
Q

Advantages to Molecular Diagnostic Methods

A

• Fast results
o Results in 2h some POC systems in 15-20 min

• More specific and sensitive than traditional tests
o Viral culture / Minimal Residual Disease Monitoring

• Can screen multiple targets / pathogen at the same time
o (Multiplex e.g. Respiratory viruses)

• Tests can be developed quickly in response to a need
o H1N1 swine flu outbreak

• Cost effective
o Can be integrated to full automation minimising costs

Automation has superceeded previous techniques so that a lot of molecular diagnostics is carried out in massive automated machines (even in a single cartridge). Future direction: moving this to point of care/bedside, away from a lab for the quickest results.

66
Q

What are the variants in molecular testing

A

• Qualitative - Detection presence or absence of DNA or RNA
• Quantitative - Measurement levels of DNA/RNA in a sample
• Genomic analysis – Mutation screening / HLA-typing transplantation/pathogen analysis
• Detection of gross chromosomal abnormalities - (Trisomy 21)
o Array-based comparative genomic hybridization (CGH)
o Chromosomal translocation – i.e. no need to amplify just looking for a mass abnormality

67
Q

Describe the technology used in quantitative and the qualitative technology used in molecular testing

A
  1. Extraction
    a. Isolation and purification of DNA and/or RNA
  2. Amplification
    a. DNA – PCR
    b. RNA – RT-PCR (reverse transcription to convert to cDNA + PCR)
  3. Detection
    a. Real-time PCR (amplification by fluorescent dyes light emission)
    b. Detection by gel PCR product separation (Multiplex)
    c. Microarrays/Next Generation sequencing (fluorescent dye)
    d. Luminex (laser light emission/detection – Multiplex)
    e. Electrospray Ionization Mass Spectrometry
    f. Electrochemical – pH change – nanotechnology
68
Q

Describe the process of EXTRACTION in molecular testing

A

High quality is a key element to a reliable molecular test

Steps involve mainly lysing the cells and solubilise the DNA/RNA and removing contaminating molecules [silica resin is used to separate DNA/RNA from other molecules, and centrifugation to remove the purified DNA (by changing pH)]

Available isolation kits from a number of companies (eg:Roche, Qiagen, Ambion) – so it can be done manually or automated

  • Current automated platforms use liquid handling with packed silica resin or magnetic bead technology
  • – Reduces staff time: can take as little as 5-10mins to 2 hours
  • – Introduces traceability
  • – Minimises potential contamination
69
Q

Describe the process of AMPLIFICATION in molecular testing

A

In order to amplify a specific genomic region, you need to design forward and reverse primers by starting and finishing thee primers where differences in bases lie between different types of viruses

E.g. if designing a sequence for Neisseria menigitidis, you will pick primers where sequences differ from Neisseria gonorrhoea

If using real-time PCR you need to use a method of detection hence the use of a fluorescent DNA probe that can be detected by the machine as amplification carries on (instead of forward and reverse primers)

Amplification process:
PCR detects by making replicates of the target region to the point that it is visible to the naked eye or instrumentation

70
Q

Describe the basic PCR protocol

A

A tube that contains reagents to produce copies of DNA:

Buffer –> create optimal conditions for activity of Taq DNA polymerase
Mg++ –> acts as a cofactor and is a catalyzer in PCR. That means, higher concentrations of MgCl2 increases higher productivity of Taq polymerase.

Nucleotides (A, C, T, G)

Taq DNA polymerase enzyme

Primers located at two defined positions

A thermocycler (35-40 cycles)

Heats the tube >95oC for extracted DNA to open the double strand to two single strands (Denature)

Cools tube to a temperature optimum for “complementary” Primer DNA single strand to bind to one of each open extracted DNA strands (Anneal)

Raises the temperature marginally to optimum for the Polymerase enzyme to make copies of DNA starting from the each primer (Extension)

Repeats the same process 35 to 40 times

• Key points:
o Detection of the unique region is defined by designed short
o single strands of “complementary” DNA Primers
o Primers are designed to produce products of a specific size
o (nucleotide base pairs (bp) (typically: 80bp -500bp -2000bp)
o PCR only works with DNA. RNA has to be converted to DNA by the Reverse Transcriptase Enzyme before PCR

71
Q

Describe the process of DETECTION in molecular testing:

Agarose Gel Electrophoresis

A

Agarose Gel Electrophoresis – End Point Detection
• Primers binding on specific locations on each DNA strand and define the size of PCR product
• DNA has Negative Charge
• Small DNA PCR products move faster in a separation matrix than larger products
• Dyes such as Ethydium bromide or SybrGreen bind to double strand DNA and fluoresce when exposed to UV light
• Example: detection of chromosomal translocation (T: 11, 14) in a patient with suspected Mantle Cell Lymphoma

Real Time PCR
• Useful when time-constrained
• Detection of amplification as it is occurring by PCR machine (in real life report not made until the process is finished)
• Machine: TaqMan Probe
o Addition of primers with labelled flourescent dyes on the 5’ end and a quencher at 3’ end
o At the 5’ end  fluorescence emits a wavelength to quencher at 3’ end  quencher absorbs this and emits a second wavelength  detector records second wavelength emitted by quencher
• Fluorescence increases in the reaction vessel and accumulates at the end of each PCR cycle.
• Fluorescence increases exponentially due to logarithmic increase in PCR product
• CT= Cycle Threshold - PCR cycle where you observe the first detectable level of fluorescence above background (baseline) – Lower CT equates to higher the DNA concentration input
• Quantitation requires a reference standard to read level against

72
Q

Describe the process of DETECTION in molecular testing:

Real Time PCR

A

Real Time PCR
• Useful when time-constrained
• Detection of amplification as it is occurring by PCR machine (in real life report not made until the process is finished)
• Machine: TaqMan Probe
o Addition of primers with labelled flourescent dyes on the 5’ end and a quencher at 3’ end
o At the 5’ end  fluorescence emits a wavelength to quencher at 3’ end  quencher absorbs this and emits a second wavelength  detector records second wavelength emitted by quencher
• Fluorescence increases in the reaction vessel and accumulates at the end of each PCR cycle.
• Fluorescence increases exponentially due to logarithmic increase in PCR product
• CT= Cycle Threshold - PCR cycle where you observe the first detectable level of fluorescence above background (baseline) – Lower CT equates to higher the DNA concentration input
• Quantitation requires a reference standard to read level against

73
Q

Describe the process of DETECTION in molecular testing:

Digital PCR

A
  • Digital PCR is a real-time PCR amplification of single template molecules
  • Nucleic acid molecules are partitioned to hundreds to thousand reaction wells  dilution
  • Some wells receive 0,1,2, 3 or more molecules.
  • Following PCR, positive and negative wells are counted and Poisson distribution is used to identify how likely it is that a positive well contains one or more templates given the fluorescence data.
  • The key to accurate Poisson analysis lies in optimizing the ratio of the number of positive
  • events to the total number of independent events
  • Unlike real time PCR - No need to rely on references or standards to quantify
  • Enables linear detection of small-fold changes
74
Q

Describe the process of Sanger sequence analysis

A
  • Termination sequencing reaction template DNA (double stranded)
  • Primer targeting one strand (specific to your target)
  • DNA polymerase (heat stable, e.g., Taq), Mg2+, Suitable buffer
    • a small amount ofdeoxytriphosphates  ddATP / ddCTP/ ddGTP/ddTTP [stops further nucleotide addition each fluoresce differently]

End Result: generation of a range of primer lengths that can be separated so that each base can be read out to determine the sequence of the template

Sequencing Products separated on Size by Acrylamide Gel Seperation

• Capillary sequencer modification  one sequencing reaction containing dideoxy nucleotides ddATP, ddTTP, ddCTP ddGTP
• Each terminator with a different label  previously sequences read by hand but now substituted by fluorescent dyes that can be translated by software (see right)
• This is useful to determine defects in single nucleotide sequences
o E.g. complement defect in a patient  this can be sequenced and compared to normal and you can see SNP difference in the 80th position
• Useful for determining sequence for 1-6 genes

75
Q

Describe the process of next generation sequence analysis

A

• Next generation sequencing principle à take whole DNA and chop it into different pieces using a microchip that separates each reaction
• Each position on the microchip will complete seperate individual reactions (can do around 30000 reactions for one particular position), amplifying through primers
• Hence you are looking at multiple sequences, so must use bioinformatics to assemble the information together
o Examples: identify a new species that causes disease, identify something in the DNA that is causing a specific disease
• Process takes weeks and months, however if you have a target NGS this allows for a quicker processing (used in cancer e.g. chronic leukaemia panel)
• Useful for determining whole genome sequencing i.e. several thousands of genes

76
Q

Describe the procedure of DNA/CGH Microarrays

A
  • Array comparative genomic hybridisation (CGH)
  • Analyse whether sections of DNA are either missing or present in extra copies
  • The main benefit of array CGH is that it is able to detect small genetic changes and can provide accurate information on the size and the possible effects of the genetic changes found.

• Process:
o Exploits the ability of a DNA molecule (strand) to bind/ hybridise to another DNA
o molecule
o DNA from the patient is “digested” to small fragments
o These fragments are labelled with a coloured fluorescent dye
o Reference DNA, from a person / pool of people without genetic abnormalities is labelled with a different coloured fluorescent dye.
o Reference and patient samples are mixed together and applied to the chip and hybridisation takes place
o The chip is then scanned in a microarray machine scanner which measures the amount of red and green fluorescence on each probe.
o The scanner calculates the ratio of the red to green fluorescent dyes
o Correct amount of DNA (YELLOW) -Too much DNA (RED = duplication) - Too little DNA (GREEN = deletion)

77
Q

Describe the procedure of Flourescent in situ Hybridisation (FISH)

A
  • Is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity
  • Uses fluorescent probes to targets a specific nucleic acid sequence within a histology sample / chromosomes
  • Useful in detecting translocation in cancer  i.e. if you paint chromosomes in different colours, you can see where big translocations have taken place as two different colours will be present on a single chromosome (cancer)
78
Q

Describe the applications of Molecular Diagnostics in Pathology

A
  • Diagnosis
  • Therapy selection
  • Therapy monitoring
  • Surveillance
  • Infection control
  • Predisposition
79
Q

Application of PCR product size separation

A
  • Malignant lymphoma - clonal rearrangement analysis B and T cell Lymphoma
  • Thalassemia detection
  • HLA tissue typing for transplantation (these days using luminex)
80
Q

Application of real time PCR

A

• Virology viral detection and viral loads
o HIV, HCV, HBV, CMV, VZV, HPV, EBV, FluA, FluB, etc
• Microbiology
o Screening tests Gonorrhoea/Chlamydia (GC/CT)
o Mycobacterium Tuberculosis (antibiotic resistance)
o Invasive fungal disease post bone marrow transplantation
o Genito-Urinary Medicine - Sexual Health associated pathogens – rapid diagnostics
• Oncology (diagnosis, staging, prognosis, monitoring) (+/- ) DIGITAL PCR
o Colorectal cancer-KRAS & NRAS -Activating mutation-Resistance to anti-EGFR treatment
o Non-small cell lung cancer –EGFR- Activating mutation- Response to anti-EGFR TKI treatment
o Non-small cell lung cancer –ALK- Translocations -Response to crizotinib treatment
o Leukaemia BCR-ABL Fusion Gene level detection

81
Q

Application of PCR + Sequencing or NGS

A

• HIV treatment resistance mutation analysis
o Allows practitioner to change the regime in HIV therapy if patient is not responding  sequence baseline virus typing to identify mutations that can be targeted by specific regimes

• HCV genotype identification
o Some drugs work better under certain mutations

  • Identify by sequencing bacteria / fungi from aseptic fluids
  • HLA–> High resolution tissue typing to enable bone marrow transplantation (need very good match if you do BM transplant so you need sequence a few target regions)
  • Risk factors / predisposition (e.g. Coeliac Disease HLA-DQ2.5, DQ8 or DQ2.2 – used particularly in paediatrric patients)
  • Mutation screening in cancer cells and gene defects in primary immunodeficiency (e.g complement genes)
82
Q

Application of FISH

A
  • Chromosomal translocation in various solid tumors and lymphomas
  • Breast cancer and herceptin treatment - her-2/neu testing
  • Glioma diagnosis and prognostication - chromosome 1p19q deletion
83
Q

Other benefits of molecular diagnostics

A
  • The ability to scale up rapidly a molecular method makes it ideal for screening programs
  • Used for personalised treatment
84
Q

Summarise innate vs adaptive immunity

A

INNATE

  • primitive
  • cellular contents: monocytes, macrophages DCs, NK, mast cells
  • humoral contents: complement, cytokines, acute phase reactants, antimicrobial peptides
  • receptors: PRR receptors e.g. TLRs, phagocytic receptors
  • rapid
  • limited diversity
  • low specificity
  • some memory
  • no self-discrimination

ADAPTOVE

  • advanced
  • cellular: T, B cells, APCs, NKT cells
  • humoral: immunoglobulins
  • receptors: TCR, BCR
  • slow
  • diverse
  • specific
  • long lived memory
  • self-discriminatory
85
Q

Describe the structure of the TCR

A

95% composed of a and b chains
(5% y and d chains)

diversity arises from genetic recomination of the DNA encoded segments in individual T cells by recombination using RAG1 and RAG2 recombinases

86
Q

Describe T cell development

A

T cells derive from haematopoetic stem cells from BM

migrate and colonise thymus

undergo beta selection, positive selection and negative selection

  • DN development and Beta selection

first, they lack both CD4 and CD8 = DN

progress through development characterised by CD44 and CD25 expression

During DN2/DN3 RAG dependent recombination, B-chain production occurs

Signal for proliferation, survival and upregulation of CD4 and CD8 = DP

Cells that don’t suceed die by apoptosis

  • Positive selection

DP move deeper into cortex of thymus

interact with cTEC presenting self-antigen

2% receive survive signal

98% die by apoptosis

ones that recognise MHCII downregulate CD8 –> become SP CD4 cells

ones that recognise MHCI downregulate CD4 –> become SP CD4 cells

migrate into medulla of thymus

  • negative selection

process of autoregulation

DCs in medulla cross present antigen to CD4+ thymocytes on MHCII

Ones that react with too high an affinity are eliminated by apoptosis or commited to become Tregs

Cells that survive exit as naive t cells

87
Q

B cell central tolerance

A

heavy chain rearrangement by RAG1 and 2

Formation of a pre-BCR with heavy chain pairing with a surrogate light chain

Antigen independent triggering of pre-BCR promotes RAG downregulation and proliferation

Differentiation to small pre-B-cell with downregulation of SLC and reexpansion of RAG - light chain rearrangement

Autoreactive BCR eliminated by apoptosis OR receptor editing of light chain

88
Q

Compare B cell and T cell central tolerance

A

SITE:
Thymus vs BM

RAG-mediated recombination

  • T: sequential RAG mediated recombination of B chain locus then a chain locus
  • B: sequential RAG mediated recombination of heavy chain and then light chain

Formation of pre-receptor

  • T: pre-TCR formed by b chain plus pre-Ta
  • B: pre-BCR formed by heavy chain plus surrogate light chain

Positive selection

  • T: MHC recognition and binding
  • B: tonic BCR signalling

Negative selection

  • T: death by neglect, no editing
  • B: receptor editing alter antigen specificity by changing light chain structure OR death by neglect

Regulatory cell formation:

  • T: Treg produced during -ve selection
  • B: none
89
Q

Methods of peripheral tolerance

A
  • Ignorance
  • Anergy - lack of co-stimulation
  • Anergy – engagement of inhibitory receptors
  • APC control of T cell activation (Innate:adaptive interface)
  • Regulatory T cells
90
Q

process of ignorance

A
  • Naïve T cells circulate blood to secondary lymphoid organs to lymph then back to blood
  • Limited access to many TRA
  • Immunologically privileged sites e.g testes, brain, placenta, where T-cells can’t cross to
91
Q

Anergy in the absence of appropriate co-stimulation

A

Two Signal Model
o In the interaction between an APC and naïve T cell, two signals are required in order to activate the T cell:

Signal 1: TCR receptor ligation with MHC-peptide complex

Signal 2: Appropriate co-stimulation via CD80/CD86 interacting with CD28

If both signals are present T cell activation and clonal expansion will occur

In the absence of signal 2 T cell anergy (functional inactivation) occurs

NB Kupffer cells and LSEC – induce tolerance to gut derived and dietary antigen

92
Q

Anergy due to enagement of inhibitory molecules

A

‘Signal 2’ can also be actively blocked by an inhibitory receptor called CTLA4

Competitive inhibition of CD80/86 for binding to CD28

Sends inhibitory signal which induces T cell activation

CTLA4 is itself induced by T cell activation – provides a natural negative feedback loop (brake/checkpoint)

Multiple other inhibitory receptors exists, which limit T cell activation or effector functions.

93
Q

Anergy in absence of appropriate co-stimulation & engagement of inhibitory receptors

A

APC, such as DCs, will alter their immunogenicity depending on the local milieu

In the presence of innate immune stimulation in a pro-inflammatory environment (presence of pathogens, necrotic cells – TLR stimulation) DCs will upregulate co-stimulatory factors and down regulate co-inhibitory receptors.

94
Q

How to Treg limit T cell activation

A

o anti-inflammatory cytokine production
o Expression of co-inhibitory molecules eg CTLA4
o Direct cytotoxicity against T effector cells

95
Q

Peripheral Tolerance Summary

A
  • Multiple peripheral mechanisms exist to limit T cell activation and prevent autoimmune mediated tissue destruction
  • T cell anergy (functional deactivation) can be induced during antigen presentation in the absence of appropriate co-stimulation or presence of inhibitory molecules
  • Important role for innate:adaptive interaction, with APCs sensing inflammatory milieu and altering threshold of T cell activation
  • Negative feedback mechanisms through immune checkpoints (eg CTLA4) limit excess T cell activation
  • Regulatory T cells (both central and peripheral) have important roles in maintaining tolerance through their control of T cell activation
96
Q

Consequences of breakdown in tolerance

A
  • Impaired thymic negative selection…
    • APECED – Autoimmune polyendocrinopathy, candidiasis and ectodermal dystrophy OR autoimmune polyglandular syndrome 1 (APS1
    • AIRE (Autoimmune Regulator) gene mutation
    • mTEC unable to generate tissue restricted antigens to developing T cells
    • Peripheral escape of autoreactive T cells
  • Impaired regulatory T cell function
    • IPEX Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome
    • Rare X linked recessive condition
    • Mutation in FoxP3 gene which is essential (master transcription factor) for Treg development
    • Patients develop multiple autoimmune complications including enteropathy, type 1 diabetes, dermatitis and autoimmune anaemias
  • Impaired peripheral negative regulation
    • Spontaneous autosomal dominant CTLA4 mutations described – Schubert et al Nat Medicine 2014
    • Multiple autoimmune phenomena including enteropathy, thyroiditis, arthritis
    • Immune checkpoint blockade for cancer therapy associated with Immune mediated tissue injury…
97
Q

Induction and Function of Cytokines

A

A stimulus (e.g. viral RNA) induces receptor (e.g. TLR) on a cell triggering a response  intracellular signalling pathways are activated and cytokine gene is activated, transcribed, translated and cytokines synthesised + released

Receptors for stimulus can be membrane bound at the cell surface, membrane bound in intracellular vesicles or soluble in cytoplasm

Cytokine then binds to target cell by a specific receptor (e.g. Type 1 interferon) and intracellular pathways are activated (e.g. JAK/STAT pathway)  target genes synthesised (e.g. PKR) and tagrget genes will provide effects (e.g. intracellular inhibition of virus replication)

Cytokine effects:
o Autocrine: self stimulation
o Paracrine: stimulation of local cells
o Endocrine: distant cells

98
Q

Nomenclature of cytokines

A

• Interferons
o Type I (IFN,a,b,o,t)
o Type II (IFNy)
o Type III (IFN-lambda1,2,3)

  • Interleukins (IL-1, IL-2… IL-36)  related in structure
  • Tumour necrosis factors (TNFa, TNFb, FASL [FAS ligand], TRAIL [TND-related apoptosis-inducing ligand])
99
Q

Classificaiton of cytokines

A
  • Initial and innate cytokines  act straight away
  • Adaptive cytokines  second layer of defence
  • Chemokines  attract cells of immune system when infection occurs
  • Haematopoietic growth factors  stimulate cells of blood system (crosses barrier of hormones and cytokines)
100
Q

Initial and Innate Cytokines

A

IFNa/b

TNFa

101
Q

Roles of IFNa/b

A
  • There is one IFNb and multiple IFNa genes
  • Made by most cells in response to viral infection
  • Stimulates specific expression of interferon specific genes (ISG) (100s of ISGs)

• Other activities
o Activates NK cell cytotoxic activity
o Enhances MHC I antigen presentation
o Facilitation of T-cell IFNy responses

102
Q

Roles of TNFa

A
  • Upregulation of MHC I
  • Immunoregulatory, antiviral pathways activated
  • Stimulates cell proliferation
  • Anti-apoptotic factors
  • Stimulates liver cells  produce C-reactive proteins
103
Q

What are adaptive cytokines

A

• Produced mainly or exclusively by T cells

IL2, IFNy

IL2
•	T-cells express IL2R
•	Autocrine growth factor
•	T-cell proliferation
•	Induces IFNy expression

IFNy
• Enhanced antigen processing and MHC presentation
• Switching of immunoglobulin classes
• Stimulates nitric oxide synthase (iNOS)  required for T-cell killing of infected cells

104
Q

What are chemokines

A
  • Chemotactic cytokines of 8-12kd in size (smaller than other cytokines)
  • Four families: (named according to number of intramolecular cysteine bonds by letter C)

C: one disulphide bridge
CC: two disulphide bridges
CXC: 2 disulphide bridges separated by any amino acid
CX3C: 2 disulphide bridges separated by any three amino acids

• Chemokines are a homing beacon for cell migration – concentration increases with the direction of chemotaxis

105
Q

What is CCL3

A

macrophage inflammatory protein

  • Induces the synthesis and release of other pro-inflammatory cytokines IL1, IL6 and TNFa from fibroblasts and macrophages
  • Migration of protective NK cells into CMV infected liver
  • Tissue inflammation – influenza, HSV, Coxsackie infections
106
Q

Summarise when each cytokine is secreted

A

in all cases IFN-a/b are stimulated as initial and innate cytokine and IFN-y are simulated in adaptive response. These 3 are central to many virus infections.

107
Q

Describe the role of TLRs in infection

A
  • Currently 13 which recognise different PAMPs (microbial components e.g. dsRNA)
  • Role in viral recognition on endosomal membrane
  • Forms a major part in innate immune responses

This may result in interferon production

108
Q

Explain how HCV is able to block TLR/RIG-I activation

A
  • NS5A blocks Myd88 when TLR7/8 is activated, reducing its activity
  • NS3/4A cleaves both TRIF and MAVS
  • This therefore shuts off IFNa/b production and is one of the ways it can persist
  • Virus has multiple layers of defences to host responses  outcome of infection will depend on which way the balance of these two will tip
109
Q

Describe IFN function

A
  • IFNa binding its receptor causes activation of Janus kinase pathways or JAK/STAT pathway
  • JAK is present in the intracellular part of the IFN receptor, and binding causes its binding to the STAT molecules in the cytoplasm (phosphorylation)
  • STAT activation causes dimerisation, which migrates the nucleus –> stimulation of ISG expression  expression of IFN effectors (antiviral, antiproliferative, immunoregulatory responses)
  • Different IFNs stimulate different JAK molecules (Tyk2, part of the Jak family) AND different STAT molecules –> different effects
Cytokine   	Jak           	 STAT
IFNy         	Jak1/2      	1
IFNa/b	        Tyk2, Jak1	1, 2, 3
IL2	                Jak1, Jak3	3, 5
IL6	                Tyk2, Jak1/2	1, 3
IL12	                Tyk2, Jak2	3, 4
110
Q

Summarise the different mechanisms of IFN action

A
  • 2’,5’-oligoadenylates (2-5A)
  • Protein kinase R (PKR)
  • Adenosine Deaminase (ADAR)
  • Mx Proteins (MxA)  binding proteins
  • Others (e..g inos)
111
Q

Describe how the 2-5A pathway works in IFN action

A
  • IFN1 stimulated
  • Enzymes stimulated by dsRNA binding
  • Binding synthesises 2-5 adenosine from ATP
  • 2-5 adenosine binds and activates RNAaseL (ribonuclease L) –> dsRNA degradation –> this will reduce viral replication (if this does not occur, the cell will apoptose)
112
Q

Describe how the PKR pathway works in IFN action

A
  • Activated when it binds dsRNA
  • PKR is normally inactive in uninfected cells
  • Two PKR molecules bind to the segment of dsRNA, and phosphorylate each other (autophosphorylation)  activation
  • Activated PKR causes inactivation of eIF-2a  inhibition of translation

• Other viral inhibitors:
o Adenovirus: VA RNA
o Influenza: P58IPK

113
Q

Describe how the ADAR pathway works in IFN action

A

Catalyses the deamination of adenosine to inosine in viral dsRNA –> changes protein coding capacity of the RNA (site specific amino acid substitutions)

114
Q

Describe how the Mx protein pathway works in IFN action

A
  • MxA and MxB GTPases are induced by type 1 IFNs are involved in endocytosis and vesicular transport
  • Mx protein monomers oligomerise, trapping viral components and inhibiting transcription, transport and assembly
  • So although the viral particles are created, they cannot be exported out of the cell
115
Q

Describe the role of SOCS in immunity

A

Suppressors of Cytokine Signalling (SOCS)

• Induced by cytokines and negatively regulate their activity, which viruses can exploit to reduce antiviral responses (HIV1, HCV, HBV, HSV1, RSV)

116
Q

Describe the process of SOCS degradation of JAK/STAT proteins

A

• SOCS protein contains:
o Kinase inhibitory region (KIR)
o SH2 region which binds phosphotyrosine
o SOCS Box which binds ubiquitination complex

  • SH2 region binds the phosphotyrosine on JAK + the SOCS Box binds a ubiquitination complex –> JAK degraded by proteasome
  • Also by binding to JAK, reduces its binding to STAT –> competitive inhibition
  • So: both competitive inhibition of binding + degradation of JAK –> decreased JAK/STAT response
117
Q

Describe how SOCS can be used as an antiviral therapy

A
  • HCV core protein induces SOCS3  increases virus replication
  • It can lead to glucose intolerance, as SOCS mediates ubiquitination of the insulin receptor
  • Genotype 1 patients have highest SOCS3 level and are most resistant to IFNa therapy
  • Suppression of SOCS, using SOCS siRNAs which mimic the peptide of phosphorylated JAKs activation loop are being trialled
118
Q

What are NK cells?

A
  • Mononuclear lymphocytes
  • Innate immune system
  • Immediate immune response to pathogens
  • No need for selection or clonal expansion
  • Activation depends on a fine balance between signals from activating and inhibitory surface receptors
  • Presentation of self peptides on MHC class I provides POTENT inhibition of NK cells via KIR (Killer cell Immunoglobulin-like receptors)
  • Note that almost all cells in the body present MHC class I
  • Loss of inhibition via KIR plus positive stimulation via activating receptors results in activated NK cells
  • = the MISSING SELF hypothesis
  • Virally infected or transformed malignant cells express abnormal peptides on MHC class I resulting in loss of NK inhibition via KIR

• NK cells activated against cells identified as ‘non-self’ i.e. virally infected, transformed cells or cells from allogeneic transplants

119
Q

NK cell functional outputs

A

cytotoxicity

Cytokine release

120
Q

cytotoxicity by NK cells

A

o release of lytic granules forming perforations in the target cell wall
 Lytic granules contain perforin and granzyme
o measured experimentally directly (chromium release), indirectly (CD107a expression on NK cell surface, target cell death)
o Antibody mediated cytotoxicity
 Antibodies produced by plasma cells (activated B cells) coat infected cells expressing antibody specific antigen
 CD16 receptors on NK cells bind the Fc portion of the antibody and induce NK cell mediated cytotoxicity and lysis of the target cell

121
Q

Cytokine release by NK cells

A

o IFN gamma supporting monocyte/macrophage responses
o TNF alpha – proinflammatory, triggers apoptosis/cell death in target cells
o Chemokines – recruit other immune cells

122
Q

NK cells in Liver Immunology

A 
-	NK cells make up 50% of the normal lymphocyte infiltrate 
o	Liver is inherently tolerogenic to prevent abnormal activation
	Inhibitory Cytokines:
•	IL18
•	IL10
•	TGFβ1
	Inhibitory ligands:
•	PD-L1
•	B7H6
123
Q

Differences between hepatic and peripheral NK cells

A

o Stegmann et al
o CXCR6 defines liver NK cells
o Relative anti-inflammatory function in liver NK cells compared to peripheral NK cells

o Reduced CD107a post activation on CXCR6+ Liver NK cells compared to CXCR6- NK cells (peripheral NK cells)
o Reduced Granzyme B and Perforin secretion by CXCR6+ Liver NK cells – key molecules within lytic granules released by NK cells when killing target cells
o Increased TRAIL expression in liver NK cells – alternative method used by liver NK cells to induce target cell death by inducing apoptosis
 Controlled apoptosis  less inflammatory response

124
Q

Cirrhosis mediated immune dysfunction

A
  • Patients with cirrhosis are susceptible to infection
  • Patients with cirrhosis are susceptible to hepatocellular carcinoma
  • Some changes might be aetiology dependent:
    o Chronic alcohol intake in mice impaired NK cell function
    o Reduced NK cell cytotoxicity in chronic HCV infection despite increase activating receptor NKG2D expression
125
Q

Why are NK cells important in Cancer?

A
  • NK cells make up 50% of the normal lymphocyte infiltrate – cytotoxic tumour immunosurveillance
  • NK cells within the tumour are hypofunctional and low in number – correlates with poorer HCC survival
  • Reduced number of NK cells in advanced HCC tumour tissue
126
Q

The ‘Missing Self’ Hypothesis

A
  • Tumours express abnormal peptides on MHC-1 and downregulate MHC-1 expression
  • Allows tumours to escape T cell immunosurveillance
  • NK cells are potently activated when they interact with cells lacking the inhibitory MHC-1 expression

• BUT- Tumour infiltrating NK cells are not activated despite this – Why?

The Tumour Microenvironment
- Immunosurveillance
o Cytotoxic immune cells
- Immunoediting
o interplay between malignant and non-malignant cells resulting in modification of non-malignant cells
o Dysfunctional NK cells within HCC tumour – high levels of surface CD107a but low levels of perforin

127
Q

NK cells in hepatocellular carcinoma – how are they modulated?

A

Kumar and Khakoo, 2018
- NK cells can produce stress ligands (MICA), which should activate them, but they also secrete
- Activated NK cells which are further away from the tumour
- By the time they get to the cell, they are exhausted
- The expansion of MDSCs (myeloid derived stem cells) they have an inhibitory effect on NK cells
o Express TGFb-1  directly inhibits NKG2d and inhibits cytotoxic granular release
o Interaxts with NKP40  inhibitory effect on interaction with MDSCs
- Formation of nest with increased expression of CD48/2B4 which interacts with tumour associated monocytes, resulting in a potent activation, resulting in exhaustion before being able to exert effects.

128
Q

Can we use what we know about NK cells for immunotherapy?

A
  • Pre-activating and expanding NK cells ex-vivo
  • Manipulate the balance of activating and inhibitory signals – blocking inhibitory receptors and over expressing activating receptors
  • Allogenic NK transplants – recognising ‘non-self’cells and being activated by the over expression of stress ligands on tumour cells
  • Up-regulating TRAIL expression
  • Specific antibodies against tumour antigens – CD16 mediated NK killing
  • Adapting NK cell lines (eg NK-92) to express chimeric antigen receptors (CARs) that target tumour cells for a more specific response

Gastro BSc (13 decks)

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