revision so afr

what are the different treatments for IBD

• anti-TNF therapy

• T cell homing/trafficking therapy

• janus kinase inhibitors

what is elevated in the serum of IBD patients

TNF

what did immune cells isolated from gut biopsies of IBD patients spontaneously produce

increased amounts of TNF which correlated with the degree of mucosal inflammation in the tissue

what is TNF also produced by

immune cells in the inflamed gut mucosa of IBD e.g. macrophages, T cells, dendritic cells, and non-immune such as fibroblasts and fat cells and is only present in panted cells during IBD

what are the effects of increased TNF alpha in IBD patients

• activates macrophages to produce pro-inflammatory cytokines

• increases apoptosis of gut epithelial cells

• regulates T cell apoptosis

• causes panted ce;; death via necrosis

what would the desired effects of blocking TNF be

• to induce T cell and inflammatory cell apoptosis

• reduce inflammatory cytokine production

• reduce paneth cell necrosis

• reduce epithelial cell apoptosis

• elevate regulatory macrophages

• reduce MMP-induced tissue destruction

what are the 2 forms of TNF

• membrane bound (mTNF)

• soluble form (sTNF)

what is mTNF cleaved by

TNF alpha converting enzyme (TACE) into the sTNF

how do both TNF forms signal

through 2 distinct TNF receptors TNFR1 and TNFR2

where is TNFR1 expressed

on lymphocytes and endothelial cells

where is TNFR2 expressed

expressed ubiquitously

what are the differences between mTNF signalling and sTNF signalling

• mTNF signals through both receptors

• sTNF signals with greater affinity through the TNFR1

what does TNFR1 activation result in

• results in an intracellular signalling cascade with pleiotropic effects, mainly apoptosis (through a caspase-8 dependent signalling pathway via FADD)

• or cytokine secretion e.g. IL-8, IL-1, and IL-6 (via NF kappa B pathway)

what does the TNFR2 activation pathway not contain and what can this result in

a death domain and can result in cell proliferation, migration, and cytokine production e.g. IL-1 and IL-6

what anti-TNF therapies are used in IBD

• infliximab

• gloimumab (UC only)

• adalimumab

what are drugs like infliximab an example of and what do they do

• monoclonal antibodies

• bind to both the soluble and transmembrane bioactive forms of human TNF alpha

• this interaction prevents the binding of TNF alpha to its receptors, thereby inhibiting the biological activity of TNF alpha

what is the effectiveness of TNF therapy like for UC and CD

relatively similar for both

in IBD what do activated T cells do

migrate or home to the gut tissues where they accumulate in large numbers, secrete inflammatory cytokines (IL-6, IL023, TNF alpha) and result in an un-resolving chronic inflammation

what does T cell homing from the blood to the site of inflammation in the gut require

requires the interaction of 2 molecules; one surface of T cells and one on the surface of endothelial cells of the vessels

what are the different types of T cells

• Treg

• T helper: Th1, Th2, Th17

what do the different types of T cells all express

• common molecule necessary for emigration from blood vessels into gut tissues

• defects in the function of these cells occurs in IBD

what is alpha 4 beta 7

a cell surface integral which is a gut specific adhesion molecule not found in other sites organs of the body

what does the intern alpha 4 beta 7 interact with

mucosal vascular adressin adhesion molecule (MAdCAM1) that is expressed on the endothelium

how are T cells retained within the gut tissue

through binding of alpha 4 beta 7 intern to E cadherin on the basal membrane of epithelial cells

what is a potential therapeutic target for treatment of IBD

blocking T cell homing and retention to the gut

what is vedolizumab

anti alpha 4 beta 7 antibody blocking homing of T cells to the inflamed gut

what does the sphingosine 1-phosphate receptor control

aggression of immune cells from lymph nodes through a concentration gradient of S1P

what do S1PR1 agonists do

render lymphocytes sensing the S1P gradient and exiting lymph therefore causing a reduction in lymphocytes

ozanimod

• currently being appraised by NICE

• for UC immune cell trafficking

• acts as a S1PR antagonist preventing the aggression of immune cells from lymph nodes to inflammatory tissues

what is ustekinumab

monoclonal antibody targeting the p40 subunit of IL-12 and IL-23

what are IL-12 and IL-23 involved in

activating a cascade of inflammatory mediators responsible for the pathogenesis of IBD

what does inhibition of IL-12 and IL-23 do

suppresses the Th1 and Th17 cell lineage of cytokines and chemokine in IBD

what are tofacitinib and filgotinib examples of

janus kinase inhibitors

what type of JAK inhibitor is tofacitinib

JAK 1 and 3 inhibitor

what type of JAK inhibitor is filgotinib

JAK1

what di JAK inhibitors do

inhibit the JAK-STAT signalling pathway

what do IBD patients have alterations in

• alterations in their mucosal layer and microbial dysbiosis

• there’s a decrease in microbial diversity

• in CD- a decrease in firmucites, in UC- reduced bactericides and clostridium genera but increase in enterococcus

what are some emerging therapies in altered epithelial barrier and dysbiosis

• fecal microbial transfer and probiotic transfer

• phosphatidycholine- essential protective component of colonic mucus

what are some drugs used for IBD

• corticosteroids

• azathioprine

• 5-aminosalicylates

• ciclosporin

• methotrexate

what can the glucocorticoid receptor complex inactivate

pro-inflammatory transcription factors such as NF kappa B and activator protein 1 (AP1) preventing them activating inflammatory mediators such as IL-6 and leukotrienes

what are glucocorticoids

potent inhibitors of T cell activation and pro inflammatory cytokines and are a highly effective treatment for active IBD

mechanism of action of glucocorticoids

• passively transport themselves into target cells and binding the intracellular glucocorticoid receptor, also known as the classic GR or GR-alpha which is held in the cytoplasm due to being bound to the heat-shock complex

• glucocorticoid ligand becomes activated when it binds to GR alpha

• formation of a homodimer of 2 activated GRs which is then transported into the nucleus of the target cell

• this then inhibits the promoter regions of genes such as NF kappa B and AP1

what is the heat-shock protein complex compromised of

• chaperone molecules hsp90 and hsp70

• immunophilin FKBP59

what does azathioprine do

inhibits de novo purine synthesis and acts as an anti proliferative agent by interfering with protein, DNA, and RNA synthesis and promoting apoptosis

what is the metabolism pathway of azathioprine

competing pathways result in inactivation by TPMT or XO, or incorporation of cytotoxic nucleotides into DNA

what is azathioprine

an immunosuppressive antimetabolite

what is azathioprine metabolised to

6-mercaptopurine (6-MP)

what does activation of 6-MP occur via

occurs via hypoxanthine-guanine phosphoribzosyltransferase (HGPRT) and a series of multi-enzymatic processes involving kinases via thiopurine S-methyltransferase (TPMT) to form 6-thioguanine nucleotides (6-TGNs) as major metabolites

what is the cytotoxicity of azathioprine due to

in part due to the incorporation of 6-TGN into DNA

what is a different inactivation pathway for azathioprine

• oxidation

• catalysed by xanthine oxidase to form 6-thiouric acid

what do azathioprine and its metabolites play a role in

control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation

how is specific blockade of Rac1 activation achieved

achieved by azathioprine generated 6-thio-GTP that binds to Rac1 instead of GTP

what genes do activation of Rac1 target and what does this leads to

• genes such as MEK, NFkappaB, and Bcl-xL is suppressed by azathioprine

• leading to a mitochondrial pathway of apoptosis

what does azathioprine thus convert

converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity

how are 5-aminosalicylates (5-ASAs) administered

orally and rectally via a suppository or enema

what do 5-ASAs have effects on

prostaglandin synthesis/COX pathway

example of 5-ASAs

• sulphasalazine

• sulphapyridine

what is interesting about the structure of mesalazine

• similar to aspirin

• molecular target therefore similar to NSAIDs

what does 5-ASA affect

• affects the COX-independent pathway

• has effects on reactive oxygen metabolites (ROM)

• can scavenge free radicals

how does 5-ASA affect the COX-independent pathway

• reduces neutrophil chemotaxis to sites of inflammation

• inhibit the survival of immune cells through NF-kappaB signalling

• inhibits TNF-mediated effects on epithelial proliferation

how does 5-ASA have an effect on reactive oxygen metabolites

• increased number of neutrophils and activated monocytes in IBD gut tissue is a source of ROM

• ROM produced during acute and chronic stage inflammation causing DNA and collateral tissue damage and oxidative products

when do 5-ASA scavenge free radicals

during superoxide anion generation in neutrophils

what does cyclosporin modulate

pro-inflammatory cytokine production and T cell survival

what are ciclosporins normally used for

patients with UC but not in CD

what can ciclosporins induce

remission in UC

what is ciclosporin

lipophilic cyclic peptide of 11 amino acids

what sort of effects do ciclosporins have

immunosuppressive effects on cell-mediated and humeral immune responses

what is the mechanism of action of ciclosporin

• binds to cyclophilin

• ciclosporin is a specific competitive inhibitor of calcineurin calcium and calmodulin-dependent phosphatase

• calcineurin normally acts in opposition to the many protein kinases involved in signal transduction

what happens when an antigen binds the T cell receptor

intracellular Ca2+ increases

what enzyme is activated by increased Ca2+ in T cells

calcineurin

what does calcineurin activate in T cells

NF-AT transcription factors, leading to IL-2 gene expression

what key cytokine production is reduced by ciclosporin

IL-2 cytokine synthesis

what is the final effect of ciclosporin on T cells

decreased T-cell proliferation and reduced IL-2 receptor expression

what does ciclosporin inhibit

translocation of NF-AT transcription factors and reduced transcription of cytokine genes for IL-2, TNF alpha, IL-3, IL-4, CD30L, GM-CSF and IFN-gamma

how are folates taken up

actively taken up into cells and then converted to polyglutamates

what are folates essential for

synthesis of purine nucleotides and thymidylate

what is the dihydrofolate reductase enzyme involved in

thymidylate synthesis and is important for transforming an inactive form of folic acid into the active form, which is necessary to make some of the building blocks needed for DNA

what enzyme does methotrexate inhibit

dihydrofolate reductase

what is methotrexate

an antimetabolite and folate antagonist

what is the normal role of DHFR

converts dihydrofolate to tetrahydrofolate

why is tetrahydrofolate important

acts as a cofactor for synthesis of purines and thymidylate needed for DNA

what happens when methotrexate inhibits DHFR

decreases tetrahydrofolate therefore decreasing DNA synthesis

which DNA component synthesis is directly affected by methotrexate

conversion of dUMP —> dTMP

what is the final cellular effect of methotrexate

inhibition of rapidly dividing cells

mechanism of action of methotrexate

• inhibits DHFR

• decreases tetrahydrofolate

• decreases thymidylate and purine synthesis

• decreases DNA replication

what protects the gut epithelium from bacteria

the mucus layer

what are commensal-epithelial interactions

normal interactions between beneficial gut microbes and epithelial cells that maintain gut health

where are intestinal stem cells located in the gut epithelium

• at the base of the intestinal crypts

• Lgr5+ stem cells

what is the order of epithelial cell renewal in the intestine

• stem cells

• proliferation

• differentiation

• migration

• shedding

why do intestinal epithelial cells shed

to maintain a healthy and functional gut lining

what triggers intestinal inflammation

luminal microbes/bacteria breaching the epithelial barrier

what happens when bacteria cross the gut barrier

they trigger an immune response

what major 2 immune responses occur during gut inflammation

1. immune cell recruitment

2. secretion of inflammatory cytokines

what is the role of cytokines in gut inflammation

they signal and amplify immune responses