---start--- the final is closed book, in class exam, not to memorize details but to understand concepts, things you should take home from this class. funding for training in LAM requires postdocs to spend a large amount of time in research, and study of clinical and administrative side of veterinary medicine, and really requires preparation as a primary investigator on research projects intended to benefit animals. Dr Norm Peterson - he is funded as a special career research veterinarian this fully supports his salary, his lab, his research. he is on faculty here, and is going to talk to us about research in LAM and how animals and veterinarians interact in research programs from the perspective of the LAM veterinarian. a few housekeeping things: he has no handout. finals are coming up. you don't need another piece of paper to keep track of. the stuff to be covered today is simplistic; the exam should be such that you could figure out the answers even if you missed class today. Today: diseases - mostly human diseases. do not memorize these diseases. do not remember specific models we discuss. do not memorize that this animal model is used for that disease. if there is anything major to know he will tell you. This is a subject on animal models, and it raises controversy, so a quick brush-up on ethics: everyone has different ideas. some people say we shouldn't use animals at all in research. others say we should use animals for whatever we want and to hell with an IACUC. veterinarians come in all flavors. today's lecture is sort of biased, you will see that, but you have to form your own opinions. Dr P feels we need to use animals in order to make progress. you have plenty of education in animal systems; you know it is difficult to use computers, other models, tissue assays, etc, to reproduce functions of living organisms. the LAM vet helps ensure this is done responsibly. a lot of people are going out into practice and aren't interested in research. why is this lecture important? you are going to be the expert in your community on animal issues. clients might ask you about animal research, will want your opinion. you need a background to form your own opinions. also it's important b/c you will read research journals, use drugs and medications that are important for your family or patients, etc. talking about models - what are they? engineers use'em, architects use'em, meteorologists use'em - they help give us a format, help us figure out what will happen in the thing we're really interested in. by definition, it isn't identical to that which it represents, so it has shortcomings, questions it can't answer, etc. animal rights people sometimes say animal models are inappropriate b/c mouse is so different from human. Dr P thinks that is untrue. we can get some information, just not all the information. an animal model is a naturally occuring or induced disease in an animal that is useful for studying the phenomenon as it exists in humans. so the lipemic hamster which is resistant to atherosclerosis might be a good model for a project attempting to find ways to prevent atherosclerosis. slide: squid a squid at first might not seem like an appropriate animal model, but nervous system studies on the squid gave a lot of useful information regarding the generation of action potentials, and helped us to understand various neurodegenerative/demyelinating diseases. considerations: alternatives: if you want to use primate - could you use a mouse instead? what about a roach? how about a computer? pathogenesis: is it similar in the animal and the human? normal parameters: useful to know for your model spp - this is why mouse is so popular - we know their normals very well. but we don't know what a sick woodchuck looks like, or what the normal white count is, etc. husbandry: special facilities add into the expense. in Illinois, they are using black bears, and had to build special pens. expense: animals are expensive. bigger ones are more expensive. Three Rs: know these!! reduce, refine, replace reduce: the number of animals used. look at the protocol and do statistical analysis - reduce numbers to minimum which show statistical significance. also inbred rodent use helps reduce animal numbers because it reduces the variability. refine: that means we refine it to make it somewhat better. a PI might be doing surgery on an animal and then put it back in cage, alone. refinement might be to also give an analgesic. or if a dog is on a study and needs blood taken every hour, and you are doing repeat blood draws, a good refinement would be to place a catheter so animal has fewer sticks. replace: replace animals in the study - the ultimate goal. we try to do all these things without compromising the study. oone example of replacement: soft agar assay, in vitro assay that replaces some tumor growth assays in mice. tumor cells are plated out on the soft agar to grow for ten days. two plates have anticancer agents in the agar - no growth there. before anyone thought of this assay, it was done in mice - mice were injected with tumor cell and given various drugs. this assay helps figure out what dose might work, etc. you still eventually have to use an animal but this gives you a ballpark idea of what's going on and reduces # ultimately needed. keep in mind - an animal will eat, digest food, etc. in tissue cultures, you change media/feed cells every 2 or 3 days, and oxygenation is via diffusion...only cells that can grow on plastic survive. it's a different situation. animal studies vs alternatives: macro/microenvironmental differences: dog sees cat - response to that will change cellular reactions. a cat in the environment of a computer model or tissue culture won't change anything. microenvironment: cellular level of dog - moment to moment changes occur, etc. ethical concerns: we don't like to use animals cost: animals are more expensive interacting systems: immunologic, metabolic, neurologic, endocrine - not present in in vitro system. approaches to developing animal models: obtain from natural population: gets hard b/c disease occurs with low frequency, but if you have good screening method as at VHUP in medical genetics program where they actively screen for metabolic diseases, can work. mating and colony formation: need to create a colony of affected animals genetic manipulation: if you can't find it in natural population, can do this - transgenic mice, etc. chemical induction: radiation, too. artificially produce disease by outside means. infection: can administer infectious agent to the animal surgical Genetic diseases: mutation - induce via radiation, or mutagenic chemical. disadvantage - uses a lot of animals, hard to predict response. 20-30 yrs ago they would irradiate mice, let them have pups, and see what diseases occured in the pups, and study if interesting. used a lot of animals. today we use specific genetic manipulations naturally occuring: screening methods, case reports of specific diseases. genetic manipulation: transgenics, knockouts, recombination, gene therapy animal models of atherosclerosis (hardening of arteries) rabbits - Watanabe rabbit - has no LDL receptors, can't absorb cholesterol from blood, tends to get clogged arteries NHP - N.C. colony, fed high fat diet, over time they develop atherosclerosis. mice - difficult to induce in mice dogs - have to be hypothyroid to get atherosclerosis pigeons - also will get atherosclerosis monkeys - deposit fat in different areas of vessels depending on spp - cyno different from african green. can study deposits at molecular level to get more information about why it deposits where it does. the monkeys in NC are good to work with b/c they establish hierarchies, so they wanted to measure stress effect on atherosclerosis, so they disrupted the hierarchy to see what happened. pigeon - show racer pigeon gets atherosclerosis whereas its cousin the white something or other does not. so you can do comparative studies in them. Chemically induced animal models: phenylketonuria - inborn errors of impaired phenylalanine oxidation causing mental retardation, microcephaly, congenital heart disease, and low birth weight. in primates they took this chemical that inhibits the metabolism of phenylalanine to tyrosine - and they induced the disease. they did this b/c they figured somewhere out there is a phenylketonuric monkey but they didn't know how to recognize it or make a screening test. so they created the artificial dz to figure out how to make a screening test. diabetes - streptozotocin can be given to mice or primates - acts on islet cells to knock out their function parkinsonism: MPTP attacks dopaminergic neurons which deteriorate in parkinson's patients IBD can be induced by DSS in mice - DSS enema in mice causes increased mononuclear infiltrate, etc. or, in cotton topped tamarin, the disease occurs naturally chemical inductions create rapid onset of disease as opposed to the natural gradual onset. that's the shortfall here. also the other shortfall is that some chemicals have other effects so you have to screen what's happening from the disease and what is another effect of chemical. transgenic animals - produced by transfer of DNA into the oocytes, embryonic stem cells, or tissues. standard procedure is to inject into oocyte. transgenic mouse sickle cell anemia model: they knew the gene for sickle cell anemia, so they introduced it into a transgenic animal. realize that isn't removing the normal copy - so this mouse was still normal, phenotypically even though SNN genotypically. so the PIs took these naturally occuring beta-thalassemic mice (NB). the SNB offspring of the transgenic mice and beta-thalassemic mice were phenotypically sickle cell anemics. homologous recombination of stemm cells: you have a chromosome - you know the gene sequence - you want to get rid of one gene. so you make a construct with a blank spot where that gene is. you add that to your cells and by natural homologous recombination you get a knockout of the gene in question. then you take those embryonic stem cells and put them into the mother mouse. they used this for HOX-/HOX- mice. they noted a number of developmental abnormalities involving the neck. then they went to human literature and found something called DiGeorges syndrome which produced a similar phenotype in people. gene therapy for atherosclerosis: they took watanabe rabbits and stuck a protein which is preferentially taken up by hepatocytes and they added onto it a gene for the LDL receptor. so the protein was absorbed by hepatocytes, gene was integrated into hepatocytes, and liver started to make LDL receptors. the circulating LDL levels significantly decreased in these rabbits. gene therapy in tumor cell biology: they took tumor cells from mice, grew in culture, transfected with IL-4, put them back into the mouse. IL-4 is a Tcell stimulant, so the tumor itself stimulated the local T cells to attack the tumor. the T cells also then destroyed non-transfected tumor. Surgical animal models: used in a variety of ways. to develop a technique or approach develop a disease: such as coronary dz, to produce an infarction in the heart, etc. auxiliary: certain sx techniques that make it easier to do research - like putting in a vascular access port or something. transplantation: tissue or synthetic. with tissues, worry about diseases... SCID mouse - remember this. severe combined immunodeficiency mouse used often. has immune defects - lacks recombinase for immunoglobulins to form properly so that it doesn't have B or T cells. used to study diseases secondary to AIDS. these mice get pneumocystis carinii as do human AIDS patients. also useful for transplantation biology b/c you can grow human tumor cells in these mice. SCID-hu mouse is a SCID mouse which has had various human tissues implanted - this is also used to study AIDS and cancer. SCID-hu mouse: fetal thymus, fetal liver, lymph nodes. to study AIDS they put these human tissues into the SCID mice. so you had a SCID mouse with a human immune system, so you could infect it with the HIV. but T cell levels dropped too rapidly and had some other shortcomings so it wasn't such a great model after all. Infectious disease models: natural infection or experimental infection natural: woodchuck hepatitis virus. with natural infections you may not know when the infection occurred, or if you are in the early or late stage of disease. you don't know the dose of infectious agent and may not know the strain. things you don't know in natural infection but do with experimental infection: time of infection dose of infectious agent strain of infectious agent route of infection Hepatitis B models: chimpanzee - the only one that can be given human HBV but clears infection within 3 wks/1 month woodchuck- WHV peking duck DHV beechey ground squirrel GSHV viruses tend to be spp specific, so we can't study our viruses in them, in general. slide: woodchuck in a cage. normally a wild animal so he experiences stress in the lab. also will hibernate at various times. disease probably progresses differently than in wild or in human. chronic WHV infection produces an increased risk of hepatocellular carcinoma, as we see in people with HBV.so there are some studies going on trying to figure out what happens to cause the carcinoma in woodchucks. animal models of AIDS: chimps again, can be infected with HIV - most successful animal model - but clear it within a month. may get rash and fever but not AIDS. cats - FIV - kind of unique in that they often get the neural form of AIDS and can be used to study those aspects of the infection cynomolgus monkeys get SIV which mimics AIDS in humans - fever, rash acutely, then later immunosuppression. but it's a different virus. cows - BIV (?) so from this lecture, hopefully you gathered enough bits and pieces. you take results from in vitro systems in lab and combine them with in vivo work to put the puzzle together and get better insight into what's going on in the situation in humans. ----end----