George Happ - Mammalian Research 

Grants
MHC research
Canine research
Infectious agents
Insect research

MHC Diversity at high latitudes

To mount a defense, you must identify an attack.
For defense from infection, the body must detect foreign antigens. Detection requires processing of the antigens derived from bacteria or other pathogens and then their presentation to T cells on a platform of proteins encoded by genes of the Major Histocompatibility Complex . We focus on the DR and DQ MHC loci of the MHC.

Success of presentation depends on the right fit of foreign peptide to the MHC platform. The surface of that platform is defined by the sequence of amino acids in the MHC-DR and -DQ proteins. We study the immunogenetics of the MHC (DRB and DQB loci) with emphasis on three themes in high latitude species. The drastic hammer of extreme seasonality (especially winters) has provided a suite of natural experiments, awaiting our analyses of the results. .

• Theme 1: Evolutionary Immunology- From his experience with population of wildlife (deer, wild sheep), Valerium Geist (Wildl. Soc. Bull. 13:351-359, 1985) suggested that immunocompetence is somehow compromised in wild mammals at high latititudes and that perhaps the loss of immune competence is related to the passage of ancestral mammals through the Beringean filter in the Pleistocene. We propse that such apparent "weakness" of the immune system might be related to diversity of MHC genes. MHCs highly polymorphic loci in globally-distributed species like cows, dogs, or mice. Over a hundred alleles have been reported at the DRB locus in humans. When temperate species are compared with related arctic species, it does appear that MHC diversity decreases as latitude inccreases, consistent with Geist's hypothesis.
  • In contrast to temperate species, moose (relatives of deer) express only a few DRB alleles while muskoxen (relatives of goats) are completely monomorphic - only one DRB protein product. Lower MHC diversity might explain compromised immune defense.
  • More species need systematic investigation. Our lab now studies moose, muskoxen, caribou, bears, foxes, and wolves. Preliminary data suggest high diversity exists at DRB loci of caribou, bears and wolves, in contrast to that in muskoxen and moose. Geist's hypothesis may account for low MHC diversity in some species, but the social structure and parasite/food spectra may favor higher diversity in other species.
  • With increasing information, the pattern becomes more complex. DQB loci are more diverse than DRB loci in moose and muskoxen. Most intriguing is the fact that both of these 21st century species share a common DQB allelic lineage which apparently has been strongly conserved through tens of millions of years in two families of artiodactyls.
  • MHC genotype may also affect reproductive biology. In mice and humans, MHC loci have been linked to pheromone blends or to development in utero. There would be important implications for mate choice and population health if MHC proteins play such reproductive roles in large mammals.
• Theme 2: Autoimmunity- In some instances, self-antigens are confused with foreign antigens. When MHC proteins present self-antigens as though they were foreign antigens, the consequence can be self-destruction. The resulting autoimmune diseases are akin to damage by friendly fire. Our aim is to evaluate the role of specific MHC alleles (especially alleles at the DLA-DRB and DLA-DQB loci) in autoimmune thyroiditis of dogs, both Alaskan huskies and Doberman Pinschers.
• Theme 3: Ecological immunology- Our investigations are especially timely in the 21st century as fragile high latitude ecosystems are threatened by global climate change. Thus we are drawn to evaluation of the impact of anthropogenic atmospheric contaminants on evolutionary immunology. Parasite spectra may be reduced at high latitudes where there are fewer intermediate hosts. A change of climate can profoundly affect the balance of parasites and hosts.

On the basis of data on genotype frequency in arctic animals, we will attempt to gauge the physiological and ecological ssignificance of specific MHC alleles in natural populations and to evaluate the resulting hypotheses in future laboratory experiments. We hope to exploit these Alaskan species as models for general questions about the diverse functions of MHC polymorphisms in vertebrates.

Is MHC diversity low at high latitudes?
(presented at the International Meeting on the Comparative Evolution of the Mammalian MHC, Manchester, UK September, 2000).

Geist’s Hypothesis: “Having expanded into the southern refugium only post-glacially, moose encountered the white-tailed deer… “Because evolution in cold climates may entail a loss of parasites and pathogens with a subsequent loss of immunity, a northern species may be considered at risk if it moves south, particularly if it meets members of its own lineage…” [emphasis added] Valerius Geist, Wildl. Soc. Bull. 13:351-359, 1985

Our research question is the immunogenetic correlate of Geist’s hypothesis: Does a decline in MHC diversity underly the apparent "loss of immunity" .

Rationale: The Major Histocompatibility Loci (MHC) encode membrane proteins that present foreign antigens to T lymphocytes. MHC loci (e.g. DRB and DQB) are among the most polymorphic genes known in mammals ( > 200 DRB alleles in humans). Low MHC polymorphism suggests high disease susceptibility. Some endangered populations (e.g. Serengeti lions and South Amerinds) have low diversity, but very little data unequivocally link parasite spectra in the field to MHC diversity. Rationale: If the immunocompetence appears to be compromised, the first thing to investigate is MHC diversity.

Methods: Exon 2 of DRB and/or DQB loci was amplified via the PCR with dog and cattle primers. We used dye-terminator Sequence-Based Typing (SBT) for all species and Single Stranded Conformational Polymorphism (SSCP) for caribou. Homozygotes were identified by nucleotide sequence. When the SBT revealed heterozygotes, we cloned and then sequenced.

Summary of preliminary results

Comparisons of allelic diversity

• Moose and musk oxen show low polymorphism at the DRB locus, and somewhat greater diversity at the DQB locus. Low MHC diversity in moose and musk oxen suggests lowered immunocompetence, consistent with Geist’s hypothesis.
• Wolves, bears,and caribou show typical MHC polymorphism at the DRB locus (and at DQB for wolves). The higher MHC diversity in wolves, bears, and caribou does not support Geist’s hypothesis.
• Data on individual herds of caribou [not shown] suggest that each herd has narrow DRB diversity.
• Herd-specific antibody data suggest differences in pathogen/parasite spectra among caribou herds.
• Reduced MHC diversities at high latitudes probably were driven by brutal weather incidents (akin to those affecting Peary musk oxen and caribou in 1973 and 1995) and produced genetic bottlenecks.
• In moose and muskoxen, an explanation must be found for the relatively high diversity at the DQB locus. Did DQB escape the bottleneck? Is this DQB diversity driven by positive selection?

Conclusions and Prospects

• Geist’s hypothesis emphasizes the fact that high latitude produces unique and attractive biomedical models -- a suite of natural experiments in evolutionary immunology. Some species and loci have very low polymorphism. These simple MHC systems offer opportunities to ask questions about the specific roles of DQB and DRB loci, both in wild populations and in a captive experimental population.
• The data also suggest explanations for differential herd health, may lead to possible new management strategies and may offer ways to monitor success of management practices.

Canine Research

This research is a collaboration with Christine Yuncker Happ.

Our canine program has concentrated on physiology and genetics of dogs and wolves.  We study the immunogenetic predisposition to disease and with the impact of seasonal and exercise stress on the immune system.  Our canine model for exercise stress is the racing Alaskan husky.

Our genetic research questions include:

• Do particular genotypes (especially haplotypes of class II histocompatibility genes)  predispose dogs to autoimmune thyroiditis?    Our experimental materials are Alaskan Huskies and Doberman Pinschers.
• What is the extent of  polymorphism and how does this suggest the evolutionary history of class II histocompatibility genes in dogs and wolves?
• What are the sequences and structure of sundry immune system genes of canines?

• Is plasma glutamine an indicator of  physiological stress and does dietary glutamine alleviate physiological stress?
• What is the impact of exercise stress on phenotypes of canine lymphocytes?
• Does exercise stress perturb plasma leptin levels?

Glutamine and exercise stress

• Speed dogs can run marathon distances in 90 minutes, almost twice the speed of human marathoners.  After a "marathon-distance run" in the first day of the Fairbanks Open North American or the Anchorage Fur Rendezvous, the dogs can repeat their performance on the second race day and once more on the third and final day.
• Distance dogs cover over 1000 miles in as little as 10 days on the Iditarod (Anchorage to Nome) or the Yukon Quest, between Fairbanks and Whitehorse.

     Bloody diarrhea and susceptibility to infection are common symptoms of exercise stress in sled dogs.  It has been suggested that glutamine supplementation could be an effective treatment for perforated intestines or for immunosuppressed animal and human patients.  However, the glutamine supplied in the diet appears to be  retained in the wall of the gut and never reach the circulation.  Nonetheless, the additional glutamine could directly nourish the enterocytes and the mucosal lymphocytes and thus alleviate stress and intestinal disease.

    Dietary glutamine is now under investigation as a treatment for the "leaky gut syndrome" in people.  In a case report, oral glutamine appeared to improve survival in parvovirus-infected puppies.  In sled dogs, dietary glutamine could prevent exercise-induced bloody diarrhea.

With the support of a grant from the Ralston Purina Company, we are employing sled dogs in training as an experimental model for a clinical trial of the potential importance of glutamine supplementation for improving gut integrity in stressed dogs.  If glutamine is absorbed into the circulation, plasma glutamine levels might rise, and thus immunosuppression might also be addressed.
 

Genetics of autoimmune thyroiditis

In humans and in mice, particular combinations of alleles for the Class II histocompatibility genes predispose toward autoimmune disease.  Our efforts will focus on the canine Class II histocompatibility alleles, beginning with those at the DLA-DRB locus.  The DLA genes (responsible for rejection  of organ or bone marow transplants) are among the best studied canine loci.  On the basis of the published sequence data for nine DLA-DRB alleles, we have developed a protocol, using reverse transcription and a nested PCR (polymerase chain reaction), that amplifies 334 bases of the polymorphic second exon of the DBR locus.  From the amplification product, we can identify each of the 9 alleles, in heterozygotes or homozygotes, either by restriction enzyme digestion or by direct cycle sequencing.

  With the help of grants from the Morris Animal Foundation, teh Doberman Pinscher Foundation, and the Canine Research Foundation of the American Kennel Club, Leslie Fox, Lorna Kennedy, and George Happ are attempting to correlate DLA-DRA, DRB, DQA, and DQB genotypes with thyroid phenotypes in Doberman Pinschers and other breeds.