Nicholas Cohen - US grants

The developing frog (e.g., clawed frog, Xenopus laevis) offers unique opportunities to attack fundamental immunological problems that are not restricted by the phylogenetic position of the experimental animal in which they are studied. Research is proposed with amphibians that addresses three such areas of developmental interest. To approach the first - the embryonic origins of T and B lymphocytes - chimeras will be constructed between embryos of different chromosomal complements and the derivatives of each component will be analyzed by flow cytometry. Sites of lymphopoiesis in larvae will also be examined by immunofluorescence. Finally, if a thymus-controlled alternative pathway of lymphopoiesis can be confirmed, its regulation will be examined as will its role in contributing stem cells to larvally thymectomized frogs that differentiate along a pre-T cell pathway. To approach the second area - thymic education of MHC restricted T-cells, chimeras will be created during embryonic life such that all lymphocytes will be of one MHC haplotype and the thymus of another. MHC restriction of T cells from such chimeras that never passed through their own thymus will be evaluated in cell cooperation studies. The third area addresses the issue of the ontogeny of self and nonself tolerance during metamorphosis. Adoptive transfer and in vitro techniques will be applied to analysis of the mechanisms of tolerance and of a disease that may reflect the failure to achieve such self tolerance.

This action is for travel support for American scientists to participate in a symposium on the interplay between the nervous, endocrine and immune systems to be held in conjunction with the annual meeting of the American Society of Zoologists in Vancouver, British Columbia. State-of-the-art lectures will be held on (1) behavioral regulation of immunity and (2) mechanisms and consequences of central nervous system and immune system interactions. The emphasis of these presentations is on the use of diverse biological systems. The goal of this meeting is to encourage greater research participation by zoologists in this new and expanding field. The hope is that conveying the conceptual and empirical advancements in psychoneuroendocrinology will lead to the development of new experimental approaches which, in turn, will be used to unravel the complex mechanisms, pathways and feedback loops that serve to communicate interactions between these three systems. Elucidating the basic mechanisms underlying stress and developing coping strategies will be one outcome of a better understanding of this intimate relationship between the neural, endocrine and immune systems.

DESCRIPTION (Adapted from the Investigator's Abstract): Heat shock proteins (hsps) are highly conserved molecules found in almost all cell types from prokaryotes to eukaryotes where they perform essential functions under normal as well as stressful physiological conditions. In mammals, hsps are involved in multiple facets of immunity including inflammation, autoimmunity, antigen presentation, and tumor immunity. The role of these proteins in immune function is also of clinical interest (e.g., in vaccine development) owing to the recent discovery that some hsps (e.g., gp96) purified from murine cancer cells can elicit a specific protective immunity in mice and rats. The proposed research will test the hypothesis that hsps are ancestral agents of immune surveillance. To this end, a new model of tumor immunity will be studied in the amphibian Xenopus that makes use of stable and well characterized lymphoid cell lines, each of which has been derived from a different spontaneously occurring Xenopus thymic tumor. Two cell lines, 15/0 and 15/40, which differ in expression of MHC antigens, have been derived from tumors in cloned LG-15 frogs. The model is the only one in which immune responses directed against syngeneic lymphoid tumors can be studied in an ectothermic vertebrate. Moreover, since Xenopus tadpoles do not express MHC class I antigens until metamorphosis, the importance of class I peptides in presenting hsps to the immune system can be determined. Given the remarkable similarity between the immune system of frogs and mammals, despite the millions of years that separate their origins, the finding that Xenopus hsps can elicit potent anti-tumor immune responses would certainly support the fundamental immunobiological importance and clinical potential of these molecules. Moreover, the proposed experiments address fundamental questions about hsp immunogenicity that are not phylogenetically restricted. Experiments are planned to: (1) define, in isogeneic adults, the immunogenicity and specificity of anti-tumor immunity elicited by immunization with intact irradiated tumor cells that do, and do not, express MHC class I antigens; (2) define, in isogeneic adults, the immunogenicity, adjuvanticity, and specificity of anti-tumor immunity elicited by immunization with gp96 derived from the MHC class I-negative 15/0 lymphoid tumor cell line; (3) define the effector system(s) that are being stimulated by tumor cell immunogenization and by gp96; and (4) define the immunogenicity of class 1-negative tumor cells and tumor-derived gp96 in immunocompetent naturally class 1-deficient tadpoles.

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Collins et al.

We propose an IRCEB in host-pathogen biology with a strong problem-solving focus designed to enhance collaboration and encourage pursuit of answers beyond the boundaries of traditional scientific disciplines. Host-pathogen interactions alternately fascinate and frustrate biologists. The fascination stems from the opportunities these systems offer as models for understanding at a basic level the complex mechanisms underlying organismal relationships. The frustration stems from not understanding these interactions sufficiently well to anticipate or react to epidemics or epizootics. Ecologists increasingly acknowledge a role for pathogens in population dynamics and in maintaining diverse communities and ecosystems. We have a poor understanding, however, of how pathogens alter host population dynamics, how pathogens infect hosts in a population, and how host and pathogen populations coevolve.
In many populations, hosts and pathogens coexist and each shows regular increases and decreases in population size. At times, however, a pathogen nearly or completely decimates its host population. We propose testing the basic mechanisms underlying each of these patterns using amphibians as a model system. Most amphibian populations regularly fluctuate in numbers of individuals, but beginning about 1989 herpetologists became alarmed by reports that populations and even species were persistently declining, some to extinction. Extreme population fluctuations and declines are centered on a broad region of the Cordilleras of western North America from southern Saskatchewan south to Costa Rica and northern Panama, and the higher elevations of Australia from southeastern to north Queensland. Four main causes seem to be acting alone, sequentially, or synergistically: habitat destruction, exotic species, disease, and anthropogeneic environmental change due to toxic chemicals, UB radiation, or global climate change. Many regions with declines are conservation areas protected from exotic species and habitat destruction suggesting subtle, complex causes like environmental change or pathogens are at work.
We have assembled an international research team to answer the question: Why are pathogens causing some amphibian populations to decline, even to extinction? The evidence suggests a pathogenic chytrid fungus causes populations to plummet, while another pathogen, an iridovirus causes amphibian populations to fluctuate. The proposed research will address the questions: How do pathogens influence host population dynamics? Are these newly-introduced amphibian pathogens, or has the virulence of historically benign amphibian associates changed? Have recent environmental changes altered amphibian-pathogen interactions? Except for some cases involving humans as hosts (e.g., cholera, malaria), few host-pathogen interactions have been dissected from molecular biology to population dynamics. Our team will do just that to advance basic host-pathogen biology with the goal of applying our findings to one of the most significant global biodiversity problems facing us today: Why are amphibian populations declining?
Metaphorically, the declining amphibian problem is like a prism in reverse: instead of dispersing colors, it focuses research disciplines - ecology, evolution, organismal biology, genetics, pathology, and immunology - so that they no longer appear individually. Understanding the basic mechanisms of host-pathogen interactions is central to our proposal and essential for answering the question of why amphibians are declining. The point of this metaphor is that while a spectrum can be "divided" into its colors, the variation is continuous - "divisions" are artificial and at the origin they disappear. Amphibians are integral parts of ecosystems in ways that place them at this metaphorical core. Therefore, we need a diversity of disciplines and a diversity of investigators willing to collaborate on integrative research projects. The declining amphibian case in an exciting, but unfortunate, opportunity to explore the boundaries of host-pathogen biology, and the time is now to understand why amphibians are declining. The point of our IRCEB research program is to meld diverse disciplinary concepts, methods, and traditions in ways that advance our understanding of pathogens as a factor in amphibian declines, a key example of the general loss of biodiversity.

This project addresses the hypothesis that gp96, a heat shock protein that has been implicated in antigen presentation in mammals, is part of an ancestral pathway that is antecedent to, and independent of, the antigen presentation pathway that uses MHC molecules. We will test this hypothesis by exploring the role of gp96 in immune processes in the frog, Xenopus. Moreover, since the Amphibia occupy a pivotal position in the evolution of vertebrates, it is anticipated that if our hypothesis is correct, gp96 should be involved in immune processes in frogs as well as mammals. Since premetamorphic immunocompetent Xenopus tadpoles do not express cell surface MHC class I molecules, the frog model allows us to explore the role of gp96 in immune processes in the absence (larvae) and presence (adults) of MHC class I-dependent presentation pathways. To begin to determine whether gp96 is involved in immune processes in Xenopus, four specific aims will be addressed. Aim 1 deals with the hypothesis that gp96 purified from normal Xenopus tissues can bind peptides. This hypothesis will be tested by examining the ability of Xenopus gp96 loaded with peptides from vesicular stomatitis virus and ovalbumin to prime murine CTL clones. In addition to determining whether Xenopus gp96 is normally associated with native peptides, we will clone the Xenopus gp96 homologue and determine the degree to which its primary structure, particularly its putative peptide-binding domain, has been conserved. In addition, cloning the gp96 homologue of Xenopus will provide useful tools to determine whether this gp96 gene is linked to genes of the MHC (like hsp70) or to genes encoding other members of the hsp90 family; and determine whether its promoter contains an IFN-gamma-responsive element. Aim 2 focuses on testing the hypothesis that if Xenopus gp96 is involved in antigen presentation and/or immunomodulation, it may be detectable as a cell surface molecule. Recent data reveal cell surface gp96 on a subset of adult Xenopus B-cells, on larval lymphocytes, and on lymphocytes from teleosts and hagfish. Experiments are proposed to further characterize the pattern of this cell surface gp96 on lymphocytes from MHC class I+ adults and MHC class I- larvae. Aims 3 and 4 develop the novel hypothesis that in vivo, the immunogenicity of gp96 may be revealed by its capacity to evoke accelerated rejection of minor H-antigen disparate skin allografts in adults, and tolerance of the same grafts in larvae. Experiments to evaluate the specificity and peptide complex-dependency of these putative alloimmune reactivities are presented as are experiments to reveal the nature of effector cells involved.

Frogs and salamanders are key contributors to many ecosystem functions. It is a concern, therefore, that amphibian species are declining worldwide, some to extinction. Among the suspected causes of amphibian declines is the emergence of infectious diseases. The proposed research will answer a series of questions designed to test how extinction, disease, and environmental change are linked. Do novel, highly virulent pathogens increase the chance of extinction? Does the health of the host change the risk of infection? Are there environmental conditions that increase the likelihood animals become ill? Why do emerging diseases drive some populations or species to extinction but not others? An international team of molecular biologists, immunologists, pathologists, population ecologists, and epidemiologists will use experiments and observations in the field and laboratory to answer these and related questions.

The number of species on Earth is diminishing, and these losses affect the capacity of ecosystems to deliver the goods and services required to sustain life. Infectious diseases are likely responsible for species losses in some ecosystems, but relatively little is known about how pathogens increase the risk of extinction. From time to time the decline of a key species, for example the American eagle, signals a degrading environment that also threatens many other organisms. It is important to understand how infectious diseases and amphibian declines are linked if we are to ensure a high quality environment for our generation and those that follow.