Innate Immunity

Question 1

Briefly describe platelets

Answer 1

No nucleus but have granules which secrete substances which control clotting and the breakdown of the blood clot
The platelets themselves Dom a part of the blood clot
They live for 8-12 days and are r over by the macrophages in the liver and the spleen

Question 2

Briefly describe granulocytes

Answer 2

Neutrophils- live in the blood for a few hours and they migrate into the tissues where that can live for 4-5 days
They are phagocytic
Granules contain lysozyme and myeloperoxidase
Eosinophils- parasite infections
Basophils sentinel cells that alert for damage
Both are involved in allergies

Question 3

Briefly describe monocytes

Answer 3

Can migrate into tissues from the blood and become macrophages
They are phagocytic

Question 4

Briefly describe lymphocytes

Answer 4

T cells are early progenitor cells from the bone marrow but develop in thymus
B cells develop in the bone marrow and exit as naive cells
NK cells develop in the bone marrow

Question 5

Describe the sites of haematopoiesis in development

Answer 5

Yolk sac- 17 days-
Aorta-Gonad mesonephros- 2 months-
Placenta- 7 months
Foetal liver- 7 months
Bone marrow- 5-9 months
Infants- bone marrow in vitally every bone 
Adults- bone marrow in axial skeleton

Question 6

Describe the haematopoiesis development tree

Answer 6

LT-HSC➡️ ST-HSC➡️ Multipotent progenitors (MPP)➡️➡️
➡️➡️common myeloid progenitor➡️ granulocyte-macrophage progenitor➡️ ➡️eosinophil, neutrophil, monocytes, basophils
➡️myeloid-erythroid progenitor➡️ erythrocytes, megokaryocytes
➡️➡️lymphoid➡️ T cell, B cell, NK cell

Question 7

How would you measure haemoatopoiesis at different differentiation stages?

Answer 7

Identify by functional assay until they are committed precursor cells- immunotyping- CD34
Then identify with marrow staining- blasts

Question 8

What are the factors that affect HSC differentiation?

Answer 8

Intrinsic factors- transcription factors

Extrinsic factors- the microenvironment- adhesion molecules, growth factor, receptors and surrounding cells

Question 9

Name three growth factors that drive haemoatopoiesis down a particular route

Answer 9

EPO- MEP➡️ RBCs
TPO- MEP➡️ platelets
G-CSF- GMP➡️ neutrophils

Question 10

Briefly describe monocytes maturation

Answer 10

Monoblast➡️ pro monocytes➡️ monocytes

Question 11

Describe erythropoiesis

Answer 11

Committed BFU-E/CFU-E ➡️Proerythroblast ➡️early ➡️ intermediate ➡️ late➡️normoblast ➡️reticulocytes ➡️erythrocyte (reticulocytes have extruded nucleus but still has RNA so it can make haemoglobin- erythrocytes cannot)

Question 12

Which organ stimulates erythropoiesis?

Answer 12

The kidney has oxygen sensing perivascular interstitial cells that produce erythropoietin that upregulate RBC production

Question 13

Describe platelet production

Answer 13

2000-3000 platelets per megakaryocyte
The cells enlarge due to nuclear division- endomitosis
4n-64n- more nuclei=more platelets
Regulated by TPO

Question 14

What clinical techniques have we gained from our knowledge of haematopoiesis?

Answer 14

Erythrocytes transfusion- 1 month
Platelet transfusion- few days
HSC transplant- should last for whole life
Growth factors- EPO mainly used for end stage renal failure and in myelodysplasia, pre-autologous blood donation
G-CSF- prevention of infection in neutropenic patients
And mobilise stem cells into peripheral blood for stem cell harvest and transplantation

Question 15

Give a brief description of haematological malignancies

Answer 15

Leukaemias- malignancies of haematopoietic cells which arise in the bone marrow and spread to involve blood, lymph nodes/spleen
Lymphomas- malignancies of lymphoid cells which arise in lymph nodes/spleen and spread to involve the bone marrow
Myelomas- malignancies of plasma cells in the marrow
Myeloproliferative disease and myelodysplasia- neoplastic chronic abnormal myeloid proliferation

Question 16

What type of cells do you find in the blood?

Answer 16

Erythrocytes
Platelets
Leucocytes
-Lymphoid- T cell, B cell, NK cells
-Myeloid cells- monocytes, granulocytes- eosinophils, basophils, neutrophils

Question 17

What are PRRs and PAMPs?

Answer 17

Pattern recognition receptors (greatest expression on sentinel cells of the innate immune system) recognise pathogen-associated molecular patterns (common microbial components) eg. LPS, flagellin, lipoteichoic acid, beta-glycans or nucleic acid

Question 18

Describe TLRs

Answer 18

Toll-like receptors are surface expressed PRRs that recognise surface/secreted PAMPs
Eg. Viral coat proteins, LPS, lipoteichoic acid, beta-glycan
Can be endosome expressed- found in phagocytic immune cells recognise nucleic acids

Question 19

Describe CLRs

Answer 19

C-type lectin receptors are PRRs found on macrophages and dendritic cells
They recognise sugars- mannose in bacteria, beta-glucan in fungi or fructose

Question 20

Describe NLRs

Answer 20

NOD- like receptors are PRRs expressed in the cytoplasm of macrophages and dendritic cells
Recognise surface PAMPS

Question 21

Describe RLRs

Answer 21

Rig-like receptors are cytosol expressed PRRs expressed by most cells
Rig1 recognises ssRNA and MDA5 recognises dsRNA from viruses

Question 22

Describe DSRs

Answer 22

DNA sensing Receptors are cytosol expressed PRRs found in most cells
Recognise DNA from viruses and bacteria

Question 23

What are the cellular responses to the activation of PRRs?

Answer 23

Recognition of PAMPs- eg. Lipoproteins causes the heterodimerisation of the intracellular domains of TLR1 and TLR2➡️ release of interferon, interleukins, cytokines and chemokines which triggers a response
Also activates the adaptive response
Acute inflammation causes capillaries to become more permeable to fluid and cells

Question 24

What TLR mediated effector functions inhibit virus replication?

Answer 24

IFN-alpha and IFN-beta stimulates the expression of ribonucleases and protein kinases that degrade mRNA and inhibit protein synthesis

Question 25

How do TLR-mediated effector functions promote an adaptive immune response?

Answer 25

Dendritic cells increase CD80/86 expression Present peptide-MHC complex after antigen uptake Produce costimulatory molecules after activation of PRR➡️ activation and migration to lymph node to trigger the maturation of specific T cells

Question 26

What is special about blisters?

Answer 26

Sterile inflammation Cause by physical damage to cells- cel lysis They release Damage Associated Molecular Patterns (DAMPs) Extracellular components exposes following lysis eg. DNA, histones and ATP DAMPs are ligands for some TLRs, CLRs, NLRs and trigger then production of NF-kappa-beta for inflammation

Question 27

Briefly describe opsonisation

Answer 27

Complement and immunoglobulin receptors cover antigens if bacteria- promote phagocytosis

Question 28

Briefly describe how NK cells recognise and kill virus infected cells

Answer 28

MHC1 on normal cells is recognised by inhibitory receptors that inhibit signals from activating receptors If this signal is edited the killer activating signal prevails NK cells can also recognise and kill antibody opsonised virus-infected cells following Fc receptor ligation via Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) NK cells kill virus-infected cells via the release of lyric granules containing perforin, granzyme and FasL which induced apoptosis

Question 29

What is complement?

Answer 29

The humoral components of the innate immune system Present in high concs in plasma and tissue fluids- produced in the liver and local release by macrophages Involved in opsonisation- coating the pathogen so it can be more readily taken up by phagocytes Can kill pathogen directly, indirectly and induce inflammation

Question 30

Describe complement proteins

Answer 30

Pro-enzymes- become active after cleavage Classical pathway- C(number)a/b proteins Small fragment is the 'a' protein, larger fragment is the 'b' protein A is a mediator of inflammation, b has an attachment site to bind to microbe surface and an enzyme site to active the next proenzyme in the cascade

Question 31

Describe opsonisation by complement proteins

Answer 31

``` Eg. C3b,C4b bind to bacterium Complement receptors (eg. CR1,2,3,4,Ig) on phagocytes trigger phagocytosis of complement coated pathogens ```

Question 32

Describe pathogen lysis by complement proteins

Answer 32

Formation of Membrane Attack Complex by C5b,6,7,8,9n | Punctures membrane of bacteria and viral envelops

Question 33

Describe the three pathways of complement initiation

Answer 33

Classical- CRP binds to phospholipids, or specific Ab bound to the pathogen Alternative- spontaneous conversion of C3 to C3b occurs all the time, being inactivated by host proteins. Pathogens lack protein so C3b persist and bind to it. Continuous self-activation of C3 initiated complement cascade and tags surface of cells Lectin- involves Mannose binding lectin (MBL) to recognise surface sugars on pathogens

Question 34

Describe the outcome from the three possible initiation pathways for complement

Answer 34

All pathways generate a C3 convertase which cleaves C3 leaving C3b bound to the pathogen surface and releasing C3a➡️ ➡️C3a and C3b recruit phagocytic cells to the site of infection and promote inflammation ➡️phagocytes with CR to C3b engulf and destroy the pathogens ➡️completion of the complement cascade leads to the formation of a membrane-attack complex which causes cell lysis

Question 35

What are acute phase proteins?

Answer 35

Free floating soluble PRR MBL (mannose-binding lectin) -sugars and CRP (C-Reactive protein)- phospholipids Levels significantly increase during inflammation They don't bind to your own cells- initiators of the complement cascade

Question 36

What else does C3b do in terms of the amplification of complement?

Answer 36

Activate C5 convertase that cleaves C5 to yield C5a and C5b that mediates inflammation and binds to pathogen surfaces C5b forms a membrane attack complex

Question 37

What are the actions of C3a and C5a?

Answer 37

Activate tissue mast cells Activate endothelial cells Attract innate immune cells from the blood to the infection site

Question 38

Briefly describe the increased susceptibility of individuals with complement deficiencies to infections

Answer 38

Early pathway proteins (MBL, C1q, C2,3,4 and C3 convertases)- encapsulated pathogen infections eg. Strep pneumoniae and Haemophilus influenzae Late pathway proteins (C5,6,7,8,9- formation of MAC)- infections with Neisseria spp Early classical pathway proteins (C1q, C2,3,4 and C3 convertase)- autoimmune disease (SLE-like syndrome)

Question 39

What is the role of the macrophage in acute inflammation?

Answer 39

PRR respond to PAMPs and DAMPs induce transcription of cytokines (IL-1beta, IL-6 and TNFalpha) and chemokines (CXCL(IL-8)

Question 40

What is the role of mast cells in acute inflammation?

Answer 40

C3a, PAMPs and DAMPs stimulate mast cell release of inflammatory mediators Early phase- granules Granules contain vasoactive peptides (histamine), cytokines (TNFalpha) and chemokines (IL8) Late phase- de novo synthesis Arachidonic acid➡️ leukotrines (LTB4) or prostaglandins (PGD2) Increase blood flow and permeability

Question 41

What opsonic receptors do phagocytes have?

Answer 41

``` Complement receptors (CRs) Antibody receptors (FcR) ```

Question 42

What is NADPH?

Answer 42

A large multi-subunit enzyme complex whose assembly in the phagosome membrane is triggered by a phagocytic stimulus Gp91 and p21- granule membrane components P47 and p67- cytosolic components It helps to generate chemicals used in the digestion of pathogens

Question 43

Describe the oxidising agents used by neutrophils to kill microbes

Answer 43

O2➡️ (NADPH Oxidase) O2- Superoxide anions ➡️ (superoxide dismuatse (SOD)) H2O2 ➡️ (Myeloperoxidase (MPO) HOCl (hypochlorous acid) OH- by-products (hydroxyl free radical)

Question 44

Describe the generation of oxides of nitrogen in the killing of bacteria in phogocytes

Answer 44

L-arginine + O2➡️ (inducible nitric oxide synthase (iNOS)) citrilline + NO- (nitric oxide)➡️ NO- + O2-➡️ ONOO- (peroxynitrite) Peroxynitrite- lipid perioxidation, protein oxidation, protein nitrification, inactivation of enzymes

Question 45

What are the contents of neutrophil granules?

Answer 45

Cationic peptides- permeabilisation Phospholipase A2- phospholipid cleavage Lysozyme- peptidoglycan cleavage Lactoferrin- iron binding Peroxidase- generation of hypohalites Neutral proteases- digest many cell wall proteins Acid hydrolases- digest many cell wall constituents

Question 46

What happens to neutrophils after phagocytosis?

Answer 46

Apoptosis ACAMPs- Apoptotic Cell Associated Molecular Patterns Eat me signals to macrophages If not removed they degenerate into secondary necrosis

Question 47

Describe NETosis

Answer 47

Activated neutrophils can release strands of nuclear DNA- an active form of cell death Neutrophil Extracellular Traps (NETs) contain histones coated in antimicrobial proteins, proteases and lysozyme Is ROS dependent CGD and MPO deficient cells fail to undergo NETosis

Question 48

How is the generation of diversity for B and T cell receptors generated?

Answer 48

Somatic gene segment recombination Gamma and kappa light-chain locus contains around 30 and 38 V (variable) segments and 4 and 5 J (joining) segments Heavy chain locus contains ~40 V segments, ~23 D (diversity) segments and 6 J segments Immunoglobulin isotypes by the range of heavy chain C region gene segments Recombination by recombinase activating genes (RAG1 and RAG2) Further diversity introduced at the joining regions by terminal deoxynucleotidyl transferase (tdt) Hyper mutation in B cells will further increase diversity

Question 49

What is allelic exclusion?

Answer 49

Prevents gene rearrangement and expression from both maternal and paternal chromosomes Heavy gene, kappa, gene, gamma gene

Question 50

What is clonal selection theory?

Answer 50

Single progenitor cell gives rise to many varying lymphocytes Removal of potentially self-reactive immature lymphocytes by clonal deletion leaving a pool of mature naive lymphocytes Proliferation and differentiation of activated specific lymphocyte to form a clone of effector cells

Question 51

What is a Major histocompatibility complex?

Answer 51

MHC or human Leukocyte antigen (HLA) MHC1= HLA A, B, C MHC2= HLA DR, DP, DQ

Question 52

How do MHC class 1 form and work?

Answer 52

If a cell is infected with a virus, interferon is released which causes proteosomes to degrade viral proteins to produce larger peptides suitable for antigen presentation. MHCs being synthesised in the ER are stabilised in the absence of peptide binding by calnexin and calreticulin Tapasin holds the MHC near a TAP Peptides enter through TAP and bind to the MHC molecule, if it fits there is a conformation change and MHC disassociates with the chaperone proteins and is transported to the surface of the cell, to present to CD8+ Tc.

Question 53

How can viruses interfere with antigen processing?

Answer 53

HIV can interfere with MHC production EBV has a way of preventing viral protein degradation and therefore peptide-antigen production Adenovirus- competitively inhibit tapasin and cause the retention of MHC1 in ER Herpes simplex virus1 and human cytomegalovirus (HCMV) blocks peptide entry to ER by interfering with TAP Adenovirus and Ebola can stop the transformation of the proteosome

Question 54

What is the significance of HLA-E?

Answer 54

NK cells activation is inhibited by HLA-E activity. If a viral infection leads to decreased MHC synthesis then fewer leader peptides will be available to bind HLA-E so it's expression at the cell membrane will decrease. Inhibition of NK cells will stop and the cell will be killed by the release of cytotoxic products from the NK cell.

Question 55

Describe MHC class 2

Answer 55

Activates CD4 cells by presentation. It is restricted to macrophages, Bc, DC and epithelial cells in thymus. MHC2 presents peptides degraded from endocytic vesicles, going through the endosome pathway. MHC alpha and beta chains assemble in the ER and form complex with invariant chain. Associates with Calnexin (a chaperone prot), ensuring proper folding. CLIP blocks the binding site to prevent peptides binding until reaching endosome to meet Ag. Transported to Golgi, CLIP replaced with Ag, and the complex is transported to membrane to be presented to CD4.

Question 56

What the differences between MHC class 1 and 2?

Answer 56

Class 1 recognised by CD8 cytotoxic T cells which kill target cells Class 2 recognised by CD4 helper T cells which secretes and proliferates Class 1 has one membrane-spanning α chain and one β microglobulin chain, and peptide loading occurs in ER Class 2 has two membrane-spanning chains, α and β, of similar size, and peptide loading occurs in vesicles Class1 present endogenous antigens that originate from the cytoplasm, and uses Chaperones, TAP in ER Class2 present exogenous antigens that originate extracellularly from foreign bodies, and uses Chaperones in ER; invariant chain in ER, Golgi and vesicle.

Question 57

Describe polymorphism in MHCs

Answer 57

Affects peptide antigen binding Expression is co-dominant A diverse range is expressed as each locus have many alleles. Differences are localised to the outer domain and peptide-binding groove. The polymorphic residues that line the groove determine the peptide-binding properties of different MHC molecules HLA-A,B,C for MHC1 HLA-DP,DQ,DR for MHC2 The beta chains are polymorphic, the alpha chains are monomorphic

Question 58

Describe some consequences of polymorphism in MHC

Answer 58

HLA-B53 reduced risk of malaria in Africa HLA-B7 and B14 Ebola virus survivors HLA-B67 and B15 Ebola virus deaths HLA-DR2 associated with leprosy in India Zhka-DR4 associated with rheumatoid arthritis

Question 59

Can lipids be presented to T cells?

Answer 59

Yes Dendritic cells and monocytes express CD1 that is structurally similar to MHC that can bind a range of lipid molecules from mycobacteria They can be recognised by a subset of Y cells that express neither CD4 or 8 This activate macrophages to kill the source of lipid