----start---- 9.3.98 biochemdz atchison multiple handouts are given out... note regarding this course schedule: in this course, each lecture is basically unrelated (except for the first two which are a bit related). So, the unifying principle is that each lecture will look at a particular disease, the biology of the dz, the biochem of it, figuring out a cause, and how that allows you to treat the disease rationally. There is an outline for each lecture in the schedule packet (plus the separate one from cristafalo) Thursdays: B101 Otherwise: C, but occasionally A. or 13. the other big handout says "Final Examination" on it. There is a page for cristafolo separate from it, too. This is a book of all the final exam questions. These are the actual questions. On the final exam day, you will come in to wherever you take it (here, B101), armed with the knowledge of the questions...and you will be asked to answer 5 of them. TOday we are scheduled for two hours but it isn't going to take two hours. AUTOIMMUNITY: the body is attacking its own tissues. two types: organ specific- only a single organ is attacked- eg multiple sclerosis afffecting neural tissue; or systemic - many tissues are affected - eg SLE (lupus). there are a variety of immune diseases, some of them are listed in the handout and are on the first slide which is hard to read but is the same information. You see the name of the disease,and the target organ. these are pretty common. Multiple sclerosis - myelin basic protein is attacked Myasthenia gravis - ACH receptor is attacked Rheumatoid arthritis - peripheral joint tissue is attacked juvenile diabetes - beta cells of pancreas are attacked these diseases are a problem in veterinary as well as human medicine. dogs get a lot of hypothyroidism. CANINE HYPOTHYROIDISM: most common k9 endocrine disorder; 80% due to autoimmune thyroiditis. How is it that autoimmunity arises? your immune system shouldn't be doing this! it was thought before that our immune systems were really precise wrt self vs world, and that there had to be a slip up for autoimmunity to occur. but it isn't that simple. 1.reminders about the immune system: 2.how to treat these diseases: 3.experimental approaches: Summary: regarding the regulation of autoreactive B lymphocytes, we have observed that a spontatneous generation of anti idiotypic antibodies may have a particular bias for autoantibody idiotypes. the above statement is included to remind you that the terminology is confusing. Recall the two kinds of lymphocytes - B and T. each recognizes antigen in a different way. B cells have an Ig molecule on their surface which is made of two chains - heavy and light - and each chain has a variable and a constant portion. depending on if you are heavy or light you have a few hundred variable segments, a dozen or so diversity segments, and anwhere from 1 to 7 constant regions. these all rearrange during development to make a heavy or light chain gene....recombination occurs...all this somatic rearrangement produces the huge amt of diversity that B cells recognize. T cells have a TCR (t cell receptor) with an alpha and beta chain. same story, two chains, similar structure, variable and constant regions. main difference is number of segments. So there is this somatic rearrangement process again, giving a lot of diversity. all this diversity allows the immune system to recognize about anything. How do TcR see antigen? in association with MHC. APCs have MHC on the surface, complexed with Ag, and the Tcell sees Ag in that MHC/Ag complex. MHC is very polymorphic between individuals. the haplotype of MHC is specific to an individual. This is MHC restriction- a T cell will only recognize Ag in the context of MHC of its own haplotype. so remember MHC restriction. so how does autoimmunity arise? there are a few models/theories ofhow you get rid of "bad" antiself T cells. -clonal deletion: in neonatal animal, reactive T cells are killed when they go through the thymus, because anything a T cell sees in a neonate is going to be self. some of this does occur. but you sometimes find autoreactive T cells that aren't active even in adult animals. they are specific for a self antigen but not doing anything - how? -clonal suppression - this is how. the body somehow is suppressing these anti-self T cells. we think. maybe. -the anti-idiotype network. this may sound like a TV channel for morons, but really it is a complex immune function - a way the system regulates itself. remember the epitope is what is recognized by the TcR. that TcR is of a particular idiotype. when a TcR recognizes an epitope,that idiotype can make a reaction against itself. another T cell makes an anti-idiotype to suppress the first one. then you can get an anti-anti-idiotype idiotype. this is complex but it does go on. so probably all three of these occur. all are mechanisms to suppress autoreactive cells. there could be an anti-idiotype that cross reacts with a normal host protein...an infection could occur, and one of the infecting epitopes may look similar to a self epitope - many autoimmune episodes occur after an infection. this shoudl be generally review... so regardless of how immune dysfunction arises, you have to treat it. how? you can use steroids. you can use other drugs like cyclosporine, FK506, other immunosuppressive agents. most common drugs: CyA (cyclosporin), FK506, rapamycin, and glucocorticoids. these all suppress the immune system by shutting down the activation of T cells. when T cells are activated they make cytokins - IL 1,2, 3; TNFa, IFNg, other. these drugs shut down the productionn of some of these cytokines. so you do not get an active T cell out of the deal. the drugs do this by inhibiting certain signalling events within T cells that lead to certain transcription factors - this is your worst nightmare isn't it? cell signalling and transcription factors. oy. but that is how they work. CyA and FK506 shut down a particular factor called NFAT, a nuclear factor of activated T cells. this is usually in the cytoplasm, and then it goes into the nucleus when the cell is activated, to activate cytokine genes. the drugs prevent the nuclear translocation of NFAT, so the cytokine genes aren't turned on, so cell isn't activated. how do the drugs sstop the signalling? well, they are large complex molecules. for a long time, people wanted to know how they worked and only recently have we learned how FK506 and CyA regulate immune function. it is now relatively clear. (great - a few years ago, we wouldn't be talking about this...) the drugs are hydrophobic and enter cells readily, where they bind to proteins - CyA binds cyclophilin, and FK506 binds to FKBP. Five years ago when we first noticed this, we noted that cyclophilin and FKBP have the same enzymatic activity, although they are different proteins. the activity of these proteins is that they are proline isomerases that change the conformation of proline - they are PPIases, more specifically. So, signal transduction involves conformational changes, right? so we thought this was the answer,but it isn't. there is more. the drug/protein complexes interact with another protein called calcineurin. Calcineurin is a phosphatase. It turns out that one of the substrates for it is NFAT. that's the transcription factor that needs to translocate to the nucleus, remember. NFAT is in the cytoplasm in a phosphorylated form. if/when it is dephosphorylated, it moves to the nucleus to turn on genes. the drug complexes deactivate calcineurin - which is required for the dephosphorylation of NFAT. so it prevents that whole thing. Glucocorticoids work similarly. they can inhibit activation of T cells in two ways. first,they can activate AP1, a dimer of leucine zipper proteins. they dimerize and bind DNA and activate transcriptoin, remember? well glucocorticoid receptors also have leucine zippers, which disrupt interaction of AP1, and prevent activation of cytokine genes. that's one way to inhibit T cell activation. also, the glucocorticoids inhibit function of NF-KB, which has a subunit in cytoplasm and in nucleus which must bind together. the cytoplasmic subunit is bound by a protein called IKB which holds it in cytoplasm. Glucocorticoids increase synthesis of IKB, which inhibits the KB subunit from going to the nucleus to bind the other subunit - prevents activation of cytokine genes, again. so these drugs disrupt the signalling events which would have activated transcription factors, preventing cytokine production. what's the problem with this? well, these are nonspecific. they shut down ALL activated T cells. so patient is immunocompromised. can't fight any infections. so they are effective but there are serious drawbacks to these treatments. how do you treat these diseases more specifically? depends... there are two model systems used to decipher autoimmune function. One is EAE: experimental autoimmune encephalomyelitis. The other is AA: adjuvant arthritis. these systems have been valuable for coming up with new treatments. EAE: first demonstrated in 1949. if you take CNS tissue and inject it into a mouse, you get EAE. why this was done we don't know, but it was. the mice get paralyzed and then die. so they took brain extract and fractionated it, trying to figure out what particularly caused the disease. turned out to be MBP myelin basic protein - caused EAE. in some strains of mice, the first 9 AAs of MBP alone will cause the dz. this dz is seenin only some MHC haplotypes - so likely a T cell dz. so when you inject the mice, you shoudl find T cells that react to the MBP. they did. they isolated T cells that react against MBP from these mice. then,they took those T cells and gave them to other mice - who got the disease. so that proved that these MBP reactive T cells did cause the disease. so now, they had cells that caused the disease - so, it was possible to characterize them to find out how to use the informatoin to treat the disease. they looked at the cell surfaces and found CD4 and they were all IAu positive (that's a haplotype). so they were able to cause the Dz, and then they injected Ab to IAu and they cured the disease. but they also eliminated the immune system, since all immune cells were of that haplotype, so they effectively killed the entire immune system. they did the same thing with the CD4 molecule - antiCD4 Ab also cured the disease. only 30-50% of the immune system is CD4+ so this is better but not great. so you still want something more specific. what is more specific? the TcR. how about a common receptor on these overreacting T cells? they looked at the cells and found that the TcR on 8 different T cells were almost identical - 7 out of 8 were identical. so they thought they could make a monoclonal antibody against that. they tried this. 13 out of 16 mice were cured. this was impressive. there was only one relapse in those mice too. also, this part of the TcR was rare, so it was a pretty specific treatment. but is this what really happens when you inject MBP? yes. they induced disease, and cured disease, and there was much rejoicing. this was however an experiment. what about more complex diseases? what if two epitopes are being recognized/? they could mimic this using Gpig MBP which has 2 epitopes that cause disease, only one of which is recognized by the part of the TcR they were giving Ab against. Now, only 6 out of 19 animals could be cured this way. so that kind of sucked. only two epitopes still, and cure rate is way down. but maybe in the general population we only have one epitope causing disease? we don't know . people have looked at MS patients...they do have T cells reacting against MBP. they generally use only one TcR that is reactive against the MBP - but there is big variability from person to person. you can't use one Ab to treat everyone. it is difficult to treat disease that way because each individual is different. but maybe there are other ways.... AA model: this model works by taking an extract of mycobacteria TB and injecting into mice - causes arthritis - bad inflammation of joints. that looks like a case of crossreactive anti-TcR reaction. when you fractionate the bacterial extract, find the causative protein,and sequence it, it is similar to collagen(type II). so it likely a cross reaction of anti TB antibody with collagen in joints. so this is also a T cell disease. so they isolated the reacting T cells. they could then induce disease by injecting these T cells. how do you fight this disease? how about using the autoreactive T cells as a vaccien? kill them, crosslink them, inject them- maybe they will cause production of an anti-idiotype response? and they do. you take those T cells and inject them and animal makes anti-idiotypic T cell response. you don't need to analyze the TcR this way. to prove that you can make an anti-idiotypic Tcell response, you can inject [something i didn't hear] and cure the disease. that's more work than injecting dead T cells. so you need a lot of information. you need to isolate T cells and make a vaccine. another approach - you don't even have to isolate t cells to use therapeutically. This is old info - in 1911 it was noted that if animals ate an antigen, their immune response to it was suppressed. Feed it, then inject it, they don't make immune response. only inject it, they do. so going through the gut causes some kind of suppressing reaction. so what if you say, fed MBP to mice. then inject them with it. is that protective? yes. it's pretty amazing. problem with that is it is prophylactic. you aren't going to sit around and eat self-antigens to protect from autoimmunity (is that how cannibalism started?) so can it reverse the disease? yes. inject and induce disease. then feed the protein. the disease is repressed. there was much rejoicing. what about the AA disease? what do you feed that animal? collagen. this also worked. woo hoo. autoimmune uveitis, against a retinal protein called the S antigen, can also be prevented in this way. the feeding regimen is important- you have to follow a dose and schedule. low doses are effective - they induce inhibitory cytokines - IL4, IL10, TGFb...seem to modulate the immune system in a suppressive way. feeding regimen is sort of unclear wrt optimal regimen. in one case a certain schedule cause disease enhancement. so this is tricky. but animal models are very promising; there have been human trials in a number of diseases. a couple of years ago one was completed - MS patients were tested - remember they react against MBP - so they took bovine MBP and fed it to people (30 people; 15 controls, 15 experimental). in this trial, 6/15 people receiving MBP had an episode; 12/15 controls had an episode. so that's not clinically significant. there is another trial of 515 patients going on. also a rheumatoid arthritis study of people getting chicken collagen. and a uveal retinitis study where people are getting bovine S antigen. AND a diabetes trial, feeding patients human insulin. lots of work is being done. mouse work - myasthenia gravis, feeding them ACH receptor, thyroiditis,feeding thyroglobulin. so this is pretty exciting - it might work, it's relatively easy. you still need to know the antigen. you still need to figure out the basic problem. but it's hopeful. Dr. A is optimistic. question: if you are feeding them - do you have to feed prior to disease? answer: well, animal models say no. feeding will suppress the disease. in humans, we do not know. question: doesn't the antigen have to be species specific? answer: you'd think the best thing would be individual antigen- not just spp specific. right now, it is easier to use other animals. getting human myelin is not trivial. you need a lot of brains. although the ability to clone proteins and make them with bacteria is there, you should be able to use human proteins ultimately. but it is very tricky. but in experimental trials they are using the easiest thing. (gee, but what if that doesn't work). ----end----