Immunology Flashcards
innate immune system
sentinel innate immune cells present in all externally exposed tissue detect DAMPs/PAMPs via TLRs to allow broadly-specific and consistent responses
DAMPs
damage associated molecular patterns
PAMPs
pathogen associated molecular patterns
(eg flagellin & lipopeptides)
Toll-like receptors
present in sentinel immune cells on surface and in vesicles/endosomes and bind PAMPs/DAMPs
Apoptosis (Immune)
triggered by NK/CTL (external) or DNA/mitochondrial (internal) signals
= cell shrinkage & chromatin condensation -> nuclear fragmentation & blebbing -> apoptotic body formation
Necrosis (Immune)
cell lysis caused by external (trauma, temp, ischaemia, NO, ROS, NK) or internal (viral) damage
= damage -> organelle/cell swelling, cell membrane ruptures, cell contents release (DAMPs)
innate immune response
sentinel cells produce inflammatory mediators in blood and surrounding tissue
- causes vasculature leakage, enabling immune cells to locate site of damage via inflammatory gradient
- complement proteins and antibodies bind microbes to neutralise and enable detection by neutrophils which phagocytose, and undergo apoptosis
- NK cells detect viruses/mutated cell surface structures, and release perforin and granzyme B to form pores causing apoptosis of infected cell
innate return to homeostasis
monocytes differentiate into macrophages which phagocytose complement/antibody-microbe complexes
- differentiate to produce IL-10, VEGG, TGFb to cease inflammatory actions and promote tissue repair
complement/antibodies
binds microbes to neutralise them and allows detection by neutrophils/macrophages
anti-inflammatory mediators
IL-10, VEGF, TGFbeta
adaptive immune response
defends against specific repeated threats
adaptive immune response activation
dendritic cells ingest proteins from pathogens and dying cells and process them into peptides, which are displayed on MHC complexes
DCs migrate to lymph node and present peptide-MHC complex to T helper and effector cells. T helper cells activate B cells.
B cells
exhibit specific antibodies, once antigen detected under go clonal expansion into
- antibody producing plasma B cells (amplification of response)
- memory B cells
antibodies
specialised proteins that recognise specific antigens
- contain a Fv region for specific recognition and Fc region for complement binding/Fc receptors on macrophages/NK cells
IgM
transient (allows detection of duration of infection)
governs initial recognition
IgG
longer-lasting specific antibodies, common drug target
IgA
antibody specific to GI and respiratory illnesses
IgE
allergic reactions
IgD/basophils
govern a different arm of allergic reaction that IgE
neutralisation
antibodies directly bind an antigen to block interactions
- IgM, IgG, IgA
complement fixation
antibodies bind complement to induce membrane attack complex, which forms pores in bacteria to cause death
- IgG, IgM
opsonisation
coating of pathogen with antibody and complement to increase recognition for phagocytosis
- IgG, IgM, + complement
antibody-dependent cellular toxicity
Recognition of IgG by NK cells causing perforin and granzyme B release to form pores and cause apoptosis
T cells
recognise peptide-MHC complexes to destroy pathogens (CD8/CTL) or help other immune cells (CD4)
activation of T cells
TCR binding to peptide-MHC complex
co-stimulation from CD80/86-CD28 and CD40-CD40L
co-stimulation from cytokines
activation of CD4 T cells
TCR binding to peptide-MHCII complex
co-stimulation from CD80/86-CD28 and CD40-CD40L
co-stimulation from cytokines (IFNa/b, IL-12)
action of CD4 T cells
differentiate into effector T helper (Th) cells which migrate to site of inflammation/MHC presentation through circulation
IFNy
cytokine produces by Th
promotes inflammatory response by activating macrophages and enhancing phagocytosis
IL4/13
cytokines produced by Th
activate eosinophils to kill parasites, and act of macrophages to promote tissue repair and fibrosis
IL-17/22
cytokines produced by Th cells
- promotes inflammatory response & recruits neutrophils to phagocytose bacteria & stimulates epithelial cell barrier responses
IL-10/TGFb
cytokines produced by Th cells
maintains and restores homeostasis by regulating other T cells, preventing self-reactive T cell action (autoimmunity)
Cytokines produced by Th Cells
IFNy, IL-4, IL-13, IL-17, IL-22, IL-10, TGFb
CD8 T cells/CTLs
TCR binding to peptide-MHCI complex
co-stimulation from CD80/86-CD28 and CD40-CD40L
co-stimulation from cytokines (IL-2)
CD8 T cell mechanism
detect infected cells, and release cytotoxic IFNy/TNFa/granzymeB/perforin to induce apoptosis
Immune disorders
hyper-stimulation of immune response by external (allergens/irritants) or internal (autoimmune) cues
immune co-morbidites
very high, as immune disorders result from an accumulation of genetic mutations/dysfunctional microflora
Th stimulation of B cell
TCR-MHCII
CD40L/CD40
IL-4, IL-6, IL-21
Types of Th cell that can induce inflammation and tissue damage
Th1, Th7, Th17
blood samples
used to isolate cells, proteins, and drugs circulating in blood. Centrifuge…
+ anticoagulant = erythrocytes, buffy coat, plasma
- anticoagulant = blood clot, serum
serum
contains water, hydrophilic drugs, nutrients, metabolic waste, and proteins (including cytokines and antibodies)
plasma
contains water, proteins (antibodies, cytokines), nutrients & metabolic waste, and electrolytes
buffy coat
contains neutrophils, eosinophils, basophils, monocytes, lymphocytes, platelets, B Cells
swabs
used to isolate live organisms, nucleic acids, proteins, and drugs in secretions
- live organisms (bacteria/viruses) can be cultured
- nucleic acids from dead cells, bacteria, and viruses can be detected by assay
tissue biopsy
used to isolate whole disease tissue or live cells, proteins, or drugs from tissue
- tissue sections
- tissue homogenate
tissue sectioning
tissue (from tissue biopsy) is obtained
- sliced by microtome
- fixed by freezing/paraffin fixation (cross-linking)
- contains fixed cells, nucleic acids, proteins to determine architecture
allows repeated analysis in cases of incorrect diagnosis
!! must reverse cross-linking before analysis!!
tissue homogenate
tissue is homogenised by enzymatic reactions/sepharose beads/sonification
- contains live cells/proteins/chemicals + water
!! must apply protease inhibitor if looking at proteins to prevent protein degradation !!
antibody testing
quick and accurate biochemical testing used to measure presence or concentration of a specific molecue
analyte
specific molecule recognised by antibody
polyclonal antibody generation
- animals are immunised by injection of analyte into tissue over ~ 45 days
- serum from blood is obtained
- use of analyte-coated sepharose beads to obtain antibodies
- obtain polyclonal antibodies (variety)
monoclonal antibody generation
- immunisation of animal over ~45 days by injection of analyte
- collect buffy layer from blood
- isolate antibody secreting plasma cells
- breed plasma cells with myelomas = replicative immortality
- screening and culturing to obtain specific antibodies
- expansion of hybridomas
advantages of monoclonal antibodies
- production limited to mouse/rat/rabbit
- continuous unlimited supply not dependent on animals
- highly selective
- reproducible
disadvantages of monoclonal antibodies
- takes months/years to validate
- hybridoma can be lost/contaminated
- limited use in assays due to specificity
- expensive as hell
advantages of polyclonal antibodies
- rapid production in any species
- quick and easy to purify
- more sensitive; recognises antigens across species
- inexpensive
disadvantages of polyclonal antibodies
- limited to life span of an animal
- batch variation
- greater false positives
- less reliable
ELISA
enzyme-linked immunosorbent assay, used to detect soluble targets in serum/plasma/secretions
- uses horseradish peroxide to convert H2O2 + OPC to a brown product
- performed in a 96 well format
- control with everything except analyte
- use standard curve to analyse absorbance-concentration relationship
indirect ELISA
Analyte fixed on well surface
- primary binds analyte
- secondary binds primary
- secondary has conjugated enzyme that converts a substrate to a coloured product
good for isolating different polyclonal antibodies
sandwich ELISA
antibody fixed in wells
- analyte added & binds
- washing of excess
- addition of secondary antibody
- washing of excess
- add substrates for enzymatic detection
competitive ELISA
known concentration of competitive analyte fixed on plate
- addition of analyte of interest (unknown concentration)
- antibody binds either
- washing of excess
- quantification
- the less signal, the more competitive the analyte of interest, as the detector antibody was bound and washed away with it
rapid antigen test
- A diluted nasopharyngeal sample is dropped into the sample well (S)
- Sample moves up test via capillary flow (wet-?dry)
- Gold-labelled antibodies specific for virus antigen mix with the sample in the conjugate pad
- SARS-CoV-2 antigens bound to gold antibodies are collected by antigen-binding immobilised antibodies in the testing (T) well
- Residual gold-labelled antibodies (that do not bind SARS-CoV-2 antibody) are collected by Fc binding immobilised antibodies in the control (C) well
Western blot
used to detect soluble/cell-surface targets in homogenised tissue/lysed cells
- proteins precipitated with antibody coated sepharose beads
- separation by size and charge via SDS-PAGE
- transferred onto PVDF or nitrocellulose
- immunohistochemistry
- quantification using chemiluminescent system
Immunohistochemistry
used to detect location and abundance of soluble/cell surface target molecules in tissue sections
- cross sectional/longitudal sections of 4-20um
- fixation (cross linked), and frozen or embedded in paraffin wax
- reversal of cross linking
- antibody application (primary+secondary)
- detection via enzymatic or fluoresence
paraffin - polycloncal
frozen - monoclonal
flow cytometry
used to detect cell-surface molecules in live cells from blood/tissue homogenate
- application of fluorescent antibodies
- cell-antibody passes through nozzle in single cells stream and passes through laser beam
FSC (forward scatter): cell size
SSC (size scatter): identified fluorescence/granularity
- # of positive cells/intensity of fluorescence detected
cons of flow cytometry
- limited use in diagnosis (expensive)
- must use small cell count for diagnosis or analysis is complicated
- only measures within/on cell content
downstream adaptor molecules of TLR
- MYD88, TRIF, TRAF
- MAPK
- IFR (interferon regulatory factors)
- NF-kb
drugs that target TLR
- synthetic liposaccharides (CpG, dsDNA, ssRNA)
- antibodes
- antagonists
- small molecule inhibitors of MYD88, TRIF, TRAF
All kinda shit due to broad effect with broad toxicity, most drugs withdrawn
imiquimod
targets endosomal TLR7
only used in skin conditions, topical action = reduced side effects
Nk-kb mechanism
TLR activation initiates IKK complex
- (p) IkB inhibitory subunit of NF-kb complex
- IkB dissociates and is degraded by ubiquitination
- NF-kB enters nucleus
- transcription:
phospholipase A2 & COX2 (prostaglandin synthesis)
chemokines & adhesion molecules (immune recruitment)
cytokines (activate sentinel cells)
Nk-Kb drug targeting
SHIT: damaging effects on immunity, repair and homeostasis = no approved drugs
- inhibits IKK complex, stabilise NF-kB complex, NF-kB translocation into nucleus, gene transcription
glucocorticoid receptor agonists
SHIT: increased infection risk, healing impairments, complications with longterm use/withdrawal
- mimic cortisol action
- reduce NF-kB signalling
Prostaglandin
a type of eicosanoid (lipid mediator of inflammation)
prostaglandin synthesis
- Phospholipase (A2) converts membrane lipids into arachidonic acid
- COX converts into endoperoxidases, which form prostaglandins
NSAIDS
pretty shit: target COX1 & COX2, leads to reduced prostaglandin synthesis
side effects: ulcers, bleeding, kidney issues
COX2 inhibitors (Coxibs)
selectively target COX2, which is only present in inflamed tissue = more specific actions = reduced side effects
PRETTY OK
TNF receptor ligands
TNFa/b, LT, CD40, FasL, BAFF, April, Ox40, GITR, nerve growth factor
TNF action
- downstream signalling via TRAF/TRADD
- activates MAPK, NF-kB
- overactivation = apoptosis
targeting TNF
Target TNF present in blood via chimeric, humanised, truncated receptors.
= rejection and macrophage induced inflammation
= anaphylaxis, infection, bone marrow suppression, heart failure (SHIT)
(unless u conjugate receptor w/PEG)
IL-1b inhibitors
Recombinant IL-1 receptors, receptor-Ig fusion proteins, and antibodies against IL-1b
Reduce T cell activation and inflammation = Risk of infection, bone marrow suppression and heart failure
sIL-6R
antibodies against the soluble IL-6 receptor
Reduce T cell activation and inflammation = Risk of infection, bone marrow suppression and heart failure
IFNa/R1 inhibitors
block cytokine receptor/signalling pathway
= block DC activation & autoimmune responses
= limits comms between adaptive and innate
risk of latent virus activation, and respiratory infection
IL-12/23 inhibitors
target the p40 subunit of IL-12/23 cytokines
= Block T cell activation and inflammation & Risk of latent virus activation and respiratory infection
IL-12/23 inhibitors
target the p40 subunit of IL-12/23 cytokines by modified human IgG
= Block T cell activation and inflammation & Risk of latent virus activation and respiratory infection
Risk of latent virus activation and respiratory infection
best side effects possible for targeting immunity
(respiratory infection always requires a large response so any dampening will affect this)
jakinibs
mimic SOCS
1st gen: non-selective (SHIT)
2nd gen: more selective, block specific cytokine signalling, thus side effects related to cytokine blocked
(topical reduce side effects)
Treg cells
intervenes to stop inappropriate reactions to self-antigen by
- blocking DC binding to immune cells by binding CD80/86/MHCII via CTLA4/TCR
- produces immunosuppressant cytokines IL-10/TGF-b1 (act on effector T cells)
CTLA4-Fc drug
mimics Treg activity by blocking site of co-stimulation on DCs
- risk of anaphylaxis and infection
- maintains basal T cell activation (allows return to homeostasis)
- mainly acts in lymph nodes & blood
- most effective at start of flare up
- lasts 4-8 weeks
Co-stimulation downstream effects
- calcineurin activation
- NFAT activation
- IL-2 transcription
- IL2 acts on JAK-STAT receptor
- mTOR activation
- nucleotide synthesis for clonal expansion
inhibiting nucleotide synthesis
small molecule inhibitors of purine/pyrimidine synthesis
FUCKING AWFUL SIDE EFFECTS: Off-target effects on gastrointestinal function, bone marrow (site of immune stem cells), liver enzymes, fetal development, general immune suppression due to suppression of both B/T cells
inhibiting calcineurin
binding to cyclophilin/FK-binding protein 12
- inhibits IL-2 production; reduces T cell proliferation
- more specific to T cells
STILL SHIT: Off-target effects on kidneys, bone marrow, immune and healing responses (due to lack of T cells) & increased risk of skin cancer
inhibiting mTOR
blocking binding of FK-binding protein 12 to mTORC1 complex
- inhibit IL-2 induced T cell proliferation
SHIT GENERAL SIDE EFFECTS: Off-target effects on gastrointestinal functions, endocrine system, kidneys, bone marrow, immune and healing responses
IL-2R inhibition
binding to receptor with chimeric/humanised antibodies
- inhibit IL-2 induced proliferation
- maintains basal T cell signalling
PRETTY ALRIGHT: Off-target effects include respiratory and skin infections (due to immune suppression, skin and respiratory infection require big immune response)
B cell receptor (BCR/CD20)
binds to antigen to induce proliferation and inflammation
Other B Cell receptors
CD19: recruits signalling molecules triggered by the BCR
- CD21: binds complement, enhancing BCR signalling
- CD22: binds sugars modulating cell adhesion and BCR signalling
BAFF, APRIL & IL-6
soluble factors that regulate B cell proliferation, activation and inflammation
targeting BCR/CD20
by antibodies
= inducing cytotoxic killing of all bound B cells = cytokine storm = massive toxic inflammation and complete B cell depletion
= 15% chance of death by acute/pulmonary toxicity
AWFUL AWFUL DRUG DONT USE IT
Targeting CD19/21/22
via antibody
- moderate B cell depletion
- lacking success
= NOT WORTH YOUR TIME
Targeting IL-6
fusion with human(ised) antibodies with an albumin-fused nanobody
= confined to blood = good t1/2 and reduced toxicity
ITS OK?: Adverse effects include liver damage, gut perforation, bacterial or fungal infection
targeting BAFF/APRIL
via receptor fusion, human antibodies and small molecule inhibitors for B cell -specific targeting
prevents B cell proliferation
=
SHIT: Side effects include gastrointestinal upset, kidney damage, lung and urinary infections, inflammatory reactions (related to essential action of B cells)
cell adhesion/chemotaxis
Rolling adhesion/tight binding
T/B cells enter blood supply and navigate towards site of inflammation by chemotactic gradient (binds chemokines on cell surface receptors)
- Use cell adhesion molecules (selectins and integrins) to move (P/E selectins of endothelial cells bind L-selectins of T/B cells)
- These increase in number and selectivity closer to the site of inflammation
Diapedesis (enters cell) and Migration to infection
targeting chemokines
FUCKING HORRENDOUS IDEA WHAT ARE YOU THINKING?!?!?
chemokines provide directions to ALL immune cells, interconnected effects = redundancy in targeting
L-selectins
present on circulating immune cells, and bind P/E-selectins on endothelium & interact with sulphated polysaccharides
- facilitate rolling adhesion
targeting selectins
EGG! NO! so many cells use this method to move through blood, massively broad side effects on normal and immune cells
