Block E - lecture 2

Question 1

what does specific immunosupression lead to ?

Answer 1

Specific in this case means specifically targeted at cells of the immune system and responsible for organ rejection.

Question 2

ALG ?

Answer 2

ALG – Anti – lymphocyte Globulin

Less damaging side effects using this. There is still some, but they are less damaging as the therapy is directly towards immune cells

Question 3

source of ALG ?

Answer 3

Comes from large Animals such as sheep or cattle, IgG Fraction is purified from blood from the animals after they’ve been immunised with T lymphocyte antigens.

Question 4

antigen used ?

Answer 4

Antigen used is Human Lymphoid Cells recently human foetal thymus (extremely potent). It is infused and not given orally , therefore it enters the blood quickly.

Question 5

short term target ?

Answer 5

small long-lived Peripheral Lymphocytes circulating between Blood and lymph node (Ag – recognising cells). These are the specific antigen recognising cells.

Question 6

Long term target ?

Answer 6

Thymus-dependent lymphocytes from the “Cuffs” of lymphoid Follicles, they are depleted as they participate in recirculating pool.

Question 7

mechanism - leading to removal of those cells ?

Answer 7

ALG-Bind to surface of Ag-recognising T-cells (exact molecule for binding is unknown)- the removal of these cells is by Cytotoxicity by serum complement activation.

Question 8

cellular consequences ?

Answer 8

Specific destruction of T-cells is the first thing that occurs , this is the desired effect, consequently specific impairment of DTH (type 4 mediated by CD8 cells) and cellular immunity.

Antibody response is intact as B cells are not targeted , the T cells are the target

Question 9

side effects ?

Answer 9

Side-effects essentially due to foreign protein injection i.e. acquire anti-Abs to the antibodies injected. Also serum sickness Ag-Ab precipitates and can be caught up in different areas of the body such as Kidney and leads to kidney damage. Also lymphoma (histiocytic) at injection site.

Question 10

limitation of use ?

Answer 10

Because of above reasons, eventually the ALG becomes ineffective due to the immune response, a useful first line treatment. So will prevent the organ being rejected but it is not a long term strategy

Question 11

macrolides derivitation ?

Answer 11

more recent , fungal derivitives which are less damaging

Question 12

why are kidneys sometimes rejected ?

Answer 12

The right is a kidney that has been rejected 17 days after transplant, the immune system destroys the kidney. On the left is a healthy kidney, on the right it is red due to good blood supply and not shrivelled.

Question 13

most common macrolide ?

Answer 13

main one is cyclosporin A, which was the first discovered compound, it is an 11 membered ring compound with amino acids. After discovery of cyclosporin A

Question 14

after discovery of CsA , what were developed?

Answer 14

discovery of cyclosporin A this led to the production of other macrolides such as FK-506 and rapamycin. These are termed as agonists, as they actively induce a state of immunosuppression.

Question 15

antagonists such as L-585 and 506BD ?

Answer 15

Antagonists such as L-585,816 and 506BD antagonise the effects of the agonists mentioned before, they do not contain intrinsic activity to produce an immunosuppressive state.

Question 16

with the use of CsA , what is the kidney survival ?

Answer 16

leads to a 1-year Kidney survival due to immunosuppressive effects.

Question 17

pre CsA kidney from cadaver survival ?

Answer 17

50%

Question 18

before CsA - living donor ?

Answer 18

75%

Question 19

after CsA cadaver ?

Answer 19

85%

Question 20

after CsA living donor ?

Answer 20

95%

Question 21

dose of CsA?

Answer 21

20mg/kg

Question 22

what does CsA lead to potent supression of ?

Answer 22

potent suppression of

alloantigen-specific T-cell numbers - T cells against antigen leading to rejection decrease.

Decrease in T-cell proliferation.

Decrease in cytotoxic T-cell numbers.

Suppression of IL-2 and IFN-g which are important T cell cytokines.

Question 23

FK506 compared to CsA ?

Answer 23

Almost identical to CsA

Mainly as CsA except:

University of Pittsburg Hospital — Clinical Study 1989 looking at liver and liver / intestinal transplants. Following the use of FK506 the success rate was 100 %.

typical dose of FK 506 is 150 µg/kg , 100 x times more potent compared to CsA.

FK506 contains side-effects of nephrotoxicity but far less than CsA. Due to the lower dose required.

Question 24

rapamycin clinical profile ?

Answer 24

effective even after T-cell activation unlike Csa and FK506, especially highly histoincompatible organs (heart).

Greater potency & graft survival time than FK506.

no major renal toxicity but: in experimental animals

GI ulceration

vasculitis

testicular atrophy in male animals

Question 25

how are T cells activated ?

Answer 25

The T cell can be activated by Ca2+ ionophores, antigen or mitogens. Once activated there are a set of pathways that are calcium dependent and calcium independent. The normal stimulus for the T cell is antigen, and this will activate the T cell down the T cell receptor route, including the Calcium

Question 26

calcium dependent pathways ?

Answer 26

The calcium dependent pathways, at different levels are activated by calcium ionophores are drugs that punch a hole in the membrane of any cells allowing calcium entry which increases intracellular levels of calcium, antigen and mitogens.

Question 27

calcium independent pathways ?

Answer 27

The calcium independent pathways are activated by anti CD28 antibodies

Question 28

what does CsA and FK506 do for T cell activation

Answer 28

Cyclosporin and FK506 prevent the activation of T cells by inhibiting the calcium dependent pathway, they have no effect on the CD28 pathway. They target pathways after Ca2+ release

A) affect Ca2+ pathways

B) inhibit Ca2+-ionophores i.e, direct Ca2+

C) do not affect generation of Ca2+

Question 29

rapamycin on T cell activation ?

Answer 29

Rapamycin inhibits the proliferation of T cells at a much later stage, it prevents the proliferation even after the production of IL-2, once they have been activated and committed to divide.

Question 30

how does the search for a receptor start ?

Answer 30

Involves the production of radioactive versions or labels of CsA and FK506 and carrying out binding studies. The first experiment carried out, revealed that there was no binding in the plasma membrane portion of a cell. However, it was found out that CsA and FK506 bound to a protein present in the cytoplasm of these cells.

Question 31

binding protein for CsA ?

Answer 31

cyclophilin is a 18kD protein that binds to CsA

Question 32

binding protein for FK506 ?

Answer 32

FKBP is a 12kD protein which binds to FK506 and rapamycin.

Question 33

what type of binding proteins are Cycophillin and FKBP ?

Answer 33

Cyclophilin and FKBP proteins were peptidyl-prolyl isomerases (PPIs), normally called rotamases. Their job is to rotate proteins as they are folding. They take a protein and help form the tertiary structure of a protein, by speeding up the reaction of the protein folding into its final 3D shape.

Question 34

summary of protein binding experiments ?

Answer 34

A) CsA/ FK506/ Rap all inhibit Rotamase activity – CsA inhibits cyclophilin and Rap/FK506 inhibits FBP.

B) antagonists also inhibit Rotamase activity

C) antagonists are not immunosuppressive

Rotamase activity not involved in suppression.

Question 35

role of calcium in the pathway of activating T cells ?

Answer 35

The role of calcium is to activate calcineurin, the phosphatase which removes the phosphate from NFATc , the nuclear factor for activating T cells, allowing it to translocate into the nucleus.

Question 36

what does CsA and FK506 do when bound to their binding proteins ?

Answer 36

CsA and FK506 if bound to their respective binding proteins will inhibit calcineurin and prevents the phosphate being removed from NFATc , therefore it cannot translocate into the nucleus.

This inhibits the post calcium signalling pathway

Question 37

when rapamycin us bound to FKBP what does it act on ?

Answer 37

Rapamycin binds to FKBP and acts on mTOR (mammalian target of rapamycin), this is to be expected as it inhibits at a later stage than CsA and FK506.

Question 38

intracellular target of Rap/FKBP complex ?

Answer 38

Rapamycin outside of the cell binds to its cytoplasmic receptor FKBP by travelling through the membrane, then this complex inhibits the activity of mTOR. MTOR effects a variety of proteins which regulate cell cycle progression. MTOR allows regulation of the cell cycle between the G1 and S phase. When mTOR is inhibited, this affects several downstream proteins such as ribosomal protein

Question 39

downstream proteins affected?

Answer 39

proteins such as ribosomal protein, which isn’t activated if mTOR is inhibited, the S6 kinase is involved in protein synthesis and essential in cell division.

cyclin proteins which are involved in cell cycle progression.

cyclin E, downstream of this there are cyclin dependent kinases (cdk’s) = which can be inhibited.

Also inhibited is a protein involved in protein translation, eIF-4F, (elongation initiation factor 4F).

Question 40

can CsA,FK506 and rapamycin effect the targets directly ?

Answer 40

CsA , FK506 or Rapamycin cannot bind directly to targets. Only the drug/ binding protein complexes suppress the downstream enzymes.

Question 41

CsA and FK506 binding to calcineurin ?

Answer 41

The main target is inhibition of calcineurin, a phosphate as it is critical for T cell activation. If the calcineurin is inhibited, then you don’t get the transcription factors produced which are needed for IL-2 and T cell proliferation.

These binding proteins are called target proteins and present inside the cells, as they present the molecules forward and slightly change their conformation, so they can fit in a particular site.

The immunosuppressive complexes both bind to particular subunits of the calcineurin phosphatase. The calcineurin has 2 subunits, CnA at the bottom and CnB , binding to the complexes occur at the CnB subunit which is a pocket between the 2 subunits. The normal function of the calcineurin B subunit, is to bind calcium shown in blue in the above diagram at the top.

Therefore, the FK506 and CsA complexes do not affect the ability of calcium to bind to calcineurin. At the Calcineurin A site this is where the phosphatase catalytic site is, shown in a pink star. It is this catalytic site that is hindered by CsA and FK506 complexes and this is what inhibits the phosphatase action of removing the phosphate from NFATc , they are allosteric inhibitors.