Gregory William Warr - US grants

Abstract 9722996 Warr The bony fish have immune systems which are similar to those of higher vertebrates but seem to be less complex. The channel catfish is a well developed experimental fish model, and also a commercially important aquacultured species. This laboratory has previously isolated and mapped the gene that encodes the heavy chain of the major antibody (IgM) of the channel catfish, and the proposed studies are focused on the mechanisms by which this gene is expressed. In this grant they will investigate the nature of the enhancer that drives transcription of this gene, and the transcription factors that are essential for the expression of the gene. In preliminary experiments they have identified the catfish transcription factor that bind to the "octamer: motif which is characteristic of immunoglobulin genes. This transcription factor and the octomer motif to which it binds appear to have undergone co-evolution. The catfish oct transcription factor has a strong functional preference for a motif that has a different sequence from that found in mammals. They propose to determine the basis for this preference by looking at the binding affinity and other protein factors that bind to DNA. The role of the transcription factor or other possible transcription factors will also be determined. These studies will not only provide information on the regulation of the catfish immunoglobulin heavy chain gene, but will provide information on regulation of immunoglobulin genes in general, and will also indicate how immunoglobulin gene expression has evolved in vertebrates. %%% Studies of the immune system in fish, show similarities and differences from the immune system of mammals. Understanding the relation of the immunoglobulin genes of the catfish, will provide information on the general regulation of immunoglobulin genes, and also will enable us to understand how immunoglublin heavy chain gene expression has evolved in vertebrates. ***

9420481 Warr This three-year award will support U.S.-Sweden-Estonia cooperative research in immunology. The investigators are Gregory Warr of the Medical University of South Carolina, Lars Pilstrom of Uppsala University in Sweden, and Sirje Timmusk of the Estonian Marine Institute. The objective of their research is the study of antibody gene expression in transgenic rainbow trout. They propose single cell studies of the following: (1) channel catfish immunoglobulin enhancer in tissue-specific gene expression in rainbow trout; (2) impact of transgenic expression of the endogenous immunoglobulin genes of rainbow trout; and (3) functional chimeric antibodies in transgenic rainbow trout. The U.S. investigator brings to this collaboration expertise with in vitro studies of gene expression and catfish immunoglobulin genes and enhancers. This is complemented by the Swedish and Estonian investigators' experience in growing transgenic trout and expertise with in vivo transgenic studies of immunoglobulin expression. The transgenic fish are grown and maintained at the Estonian Marine Institute. The project will determine whether the catfish heavy chain gene can be expressed and if so can it assist with the formation of a functional antibody in rainbow trout. The project will advance our understanding of the immune system and comparative immunology. It also has potentially important commercial applications if transgenic fish with desired traits can be raised. ***

9807531 Warr Transcriptional control is now recognized to be important for all aspects of immunoglobulin (Ig) gene expression: that is, enhancer function is important not just for driving functional expression of the gene, but it is also essential for the processes of V (D) J recombination, heavy chain class switching, and somatic hypermutation of the Ig genes to occur. The channel catfish provides a well-developed and unique model to study the function of Ig genes at the phylogenetic level of teleost fish: the immunobiology of this species has been extensively studied and well documented, and the Ig genes (for mu, delta, and L chains) have all been cloned. The overall objective of the project is to understand the function of the IgH enhancer of the channel catfish, which has been shown in prior studies to occupy an unusual position in the locus, and to possess an unusually diffuse structure. The following questions will be addressed in this project. 1) Which motifs in the catfish enhancer are important in driving transcription? 2) What is the role played by Oct1 in driving expression from the promoter-associated octamer found in all VH genes (including those of the catfish)? Is a co-activator such as Bob 1 involved in the function of Oct1 and Oct2 in the catfish? 3) What is the nature of the factors (other than octamer transcription factors) that may drive transcription from the catfish enhancer? The approaches to be employed include site-directed mutagenesis to delete individual or multiple motifs (such as octamer, muE3, muE5) from the enhancer to determine their contribution to function, and the cloning of transcription factors (such as Oct1) and the co-activator Bob-1 (a co-activator of Oct transcription factors) from the catfish, in order to define their roles in driving transcription of the Ig genes at this level of evolution. Antibodies have two polypeptide chains, a heavy chain and a light chain. The expression of heavy chain genes occurs largely at the level of transcript ion, the process by which information in DNA is copied into mRNA. Transcription is regulated by sequences in the DNA, called enhancers, which interact with protein transcription factors. This project is investigating the enhancers and transcription factors that regulate the expression of antibody heavy chains in catfish.

The duck's immune system functions poorly, and has been described as inept. This species is susceptible to infectious disease, which has an impact on human health through the farming of ducks and because of the important role of ducks in the ecology of the influenza A virus. The long-term objective of these proposed studies is to understand the immunogenetic basis of the duck's antibody response. Initial studies have shown that 2 of the genes encoding antibody heavy chain constant (C) regions in the duck (the alpha and upsilon genes) are in head-to-head orientation. The specific aims of the proposed work seek to answer the following questions. 1) Is the order of C region genes in the duck IgH locus (mu-alpha-upsilon) or (mu-upsilon- alpha)? Did the recombination events that produced the reorganization of the duck IgH locus result in the loss of the membrane exon associated with the alpha gene? 3) Is there a homolog of the delta gene present in the duck IgH locus? 4) In what manner does the head-to-head orientation of the upsilon and alpha genes affect the process of class switching to IgY or IgA in duck B cells? These questions will be approached through cloning and mapping of the duck IgH locus (by use of recombinant genomic phage and bacterial artificial chromosome libraries) and sequencing of critical regions of the locus. The nature of class-switching mechanisms will be investigated by the isolation and analysis of the circular DNA products of class switch recombination. The results of these proposed studies should provide insight into the manner in which specific aspects of C gene organization, such as head-to-head orientation, affect the functional expression of these genes, and add to our knowledge of the functional significance of different gene organizations found in the vertebrate IgH locus.

A grant has been awarded to Dr. Eric R. Lacy at the Medical University of South Carolina to fund equipment for studying the genetic responses of marine organisms to environmental stress. Increased pressure on the coastal environment has focused attention on how marine organisms respond to this stress. It has long been known that animals defend themselves against environmental insults through hundreds if not thousands of molecular and cellular responses. Until recently scientists had to laboriously measure each response of individual animals to various environmental challenges to try to understand which physiological systems (e.g., immune, respiratory, reproductive) were protected and which had failed. With the advent of molecular genetics, scientists have new tools to look at genes and determine which ones are turned off and turned on when the animals are environmentally stressed.

However, there are tens of thousands of genes and examining them manually, a few at a time, would take years to get the answers needed. The gene arrayer and reader obtained from this grant will be used by the Marine Genome Project in Charleston, SC, to simultaneously examine thousands of genes from shrimp, oysters, dolphins, stingrays, corals, and algae. The experimental goals of this group are to use "functional genomics" (changes in gene expression correlated with changes in marine environmental stress) to: 1) find early genetic markers of stress in marine organisms, 2) use the genetic information to diagnose and predict the particular stress or infection the animal may have, 3) identify new genes that might protect these marine animals from infection and stress, and 4) detect interactions among genes. Expressed genes are isolated from target tissues in the animals before, during and after stress. Then a comparison is made for each animal to see which genes are turned on and which are turned off under each of these conditions.

The results of these studies will show which genes are important in an animal's defense mechanisms. These findings have broad implications for environmental and human health because the two are intimately linked. For example, early genetic changes in an organism may predict changes occurring in the environment that cannot be monitored in any other way. This information also should help selective marine animal breeding programs for aquatic food suppliers. Furthermore, the information from this study should assist scientists in better understanding the mechanisms of the current world-wide decline of coral reefs. The equipment purchased under this award will reside in the newly constructed Hollings Marine Lab, an inter-institutional lab that houses all partners of the Marine Genome Project and the College of Charleston. Students at all levels (high school, undergraduate and graduate) will be trained to use this equipment through internships.

APPLICANT'S DESCRIPTION: Although transcriptional enhancers are essential for the expression of the immunoglobulin heavy chain (IgH) locus, they are also needed for the additional functions of V(D)J joining, V-region hypermutation and immunoglobulin (Ig) class switch recombination. This research is directed towards understanding the function of the enhancer that drives expression of the IgH locus in the channel catfish, a teleost fish and therefore lacking Ig class switching. Preliminary results suggest that the catfish IgH enhancer functions through a novel interaction of transcription factors bound to multiple interacting octamer and muE5 sites. The role of these sites in the function of the enhancer will be dissected by site-directed mutagenesis, and the function of the mutated enhancer will be assessed in transient expression assays upon transfection into catfish B-cells. Catfish muE5-binding factor(s) will be isolated and characterized. The interactions between Oct transcription factors, muE5binding factors and the DNA motifs that they recognize will be measured directly using the BiaCore 2000 instrument that utilizes surface plasmon resonance detection. Such studies will define whether or not cooperative binding reactions occur between the Oct and muE5-binding transcription factors. Gene expression driven by the interaction of octamer-binding (Oct) transcription factors with those binding to muE5 sites (typically bHLH or zinc-finger transcription factors) has not been reported in mammalian systems. Thus, these studies should shed light on novel' transcription factor interactions in Ig enhancers, and will also provide insight into how enhancer function has influenced the evolution of the IgH locus.

The central question of Comparative Immunology is how immune mechanisms have evolved. How do they change and adapt in response to the diverse microbial challenges present in the many and different environments that are occupied by animals? The tremendous diversity of animal life poses, in itself, a great challenge to such studies. Although it is clearly impossible (indeed counterproductive) to investigate the immune system of all animals, are we seriously restricting our knowledge by studying relatively few species? Are we missing important aspects of the diverse immune systems that function so effectively in animals? This workshop will discuss the current status and potential future developments of evolutionary immunobiology while coming to grips with the following two issues. First, are there model species that, while offering ease of experimental manipulation and/or potential economic importance, occupy key phylogenetic positions? Second, can the chosen model species be used to establish paradigms (the equivalent of the Rosetta Stone) through which evolutionary and immunological processes in other species can be interpreted? In addition, the final session of the proposed Workshop will deal with the application of concepts from evolutionary immunobiology to understanding and managing problems of infectious diseases that affect and alter ecosystems. Biology is going through a revolution resulting from the application of genome-wide approaches to understanding organisms. Results from those projects which have been, or are about to be, accomplished (including human, mouse, Drosophila, C. elegans, pufferfish, zebrafish) have demonstrated the extraordinary power of genomics to contribute to the solution of problems in genetics, and molecular cell biology. Genomic approaches yield detailed information about a species, answering questions and generating new hypotheses at a remarkable rate. Yet the advances to be gained from genomic approaches can go far beyond simple analyses of the genes present in any given species, and an understanding of the control of expression of those genes. For example, we now have opportunities to study evolutionary processes through comparisons between complete genomes. In addition, as patterns of organization become apparent at the level of the whole genome, we will be able to extrapolate efficiently and effectively from the findings in favored key species, to make novel and testable predictions about other species that have been studied less intensively. This workshop has, as a major theme, the actual and potential impact of genomics on the field of evolutionary immunobiology.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of the proposed studies is to understand the genetic mechanisms controlling the expression of duck immunoglobulin genes. The duck has an immune response that can be characterized as ineffectual, which has significance for human health given the role of ducks (and other waterfowl) as carriers (or reservoirs) of diseases infectious for man. E.g., ducks constitute the only year-round reservoir of avian influenza A viruses, which are a source of genetic variation implicated in the emergence of novel influenza strains pathogenic to man. Understanding the molecular basis of the expression of duck antibodies will permit an understanding of their ineffectual antibody response and also contribute significantly to our understanding of the evolution of transcriptional control, an important aspect of comparative genomics. The duck has an unusual Ig heavy chain (IgH) locus, which has undergone significant rearrangement in its germline organization. The 3 constant (C) region genes are arranged mu-alpha-upsilon, with the alpha gene in reverse transcriptional orientation. This leads to unique problems of transcriptional control associated particularly with the control of class switch recombination. These are addressed in the Specific Aims of this proposal, and the hypotheses to be tested can be phrased as 3 questions: 1) Does the alpha gene have its own I-exon or does it rely on the bi-directional I-upsilon promoter to initiate class switching to IgA? 2) Have the signal transduction pathways that control Ig class-switching, by transcription from I-exon promoters, been conserved in vertebrate evolution? 3) The mammalian IgH locus contains transcriptional enhancers in both the JH to mu intron and 3' of the C-gene cluster. Where, in the duck IgH locus are the enhancers, what transcription factor-binding motifs do they contain, and how do they function? The approach involves identifying transcriptional control elements through functional analysis (i.e. their ability to drive transcription of a reporter gene), and the responsiveness of I-exon promoters to cytokine-induced class-switch signals. The specific motifs involved will be identified by site-directed mutagenesis, and the transcription factors binding these sites will be identified through EMSA/antibody supershift approaches. The results of these proposed studies will enhance our understanding of the molecular basis of the unusual antibody response of the duck, and provide insight into the evolution of transcriptional control in the vertebrate immune system. [unreadable] [unreadable]

This research is focused on understanding the immune response of a crustacean, the marine shrimp, Litopenaeus vannamei to virus infections. Crustacea are essential components of the marine environment and marine shrimp are of commercial significance, especially since these animals are widely aquacultured. These animals are highly susceptible to numerous viruses, including the Taura Syndrome Virus and White Spot Syndrome Virus, and these have devastating impacts on aquaculture and possible unknown consequences for the health of the marine environment. This research addresses the hypothesis that 2 shrimp molecules (STAT and IkK, which are families of immune function proteins) are components of the molecular machinery by which shrimp recognize and respond to viral infection. There are two Specific Aims. 1) To reduce, by RNA interference methods, the expression of STAT and IkK, and establish the effect of this reduced expression on shrimp resistance to viral infection. 2) To identify the genes regulated by STAT and IkK that are important in viral immunity. "Functional genomics" approaches will be employed, using DNA microarrays to measure the levels of expression of approximately 3,000 shrimp genes simultaneously. Bioinformatic analyses of the resulting data will identify genes participating in the host response to viral infection, and the role of STAT and IkK.

This research will increase our understanding of marine invertebrate immunity, and assist in the management of marine ecosystems and aquaculture resources. The broader impacts of this work include: 1) the dissemination of information on marine organisms and the marine environment to professionals and the public, through coordination with the South Carolina Department of Natural Resources and 2) the education and training of graduate students who will work on the project. In addition, through participation in summer research exposure programs, undergraduate students will have the opportunity to work on this project and to gain experience in research.

meeting /conference /symposium

The International Society for Developmental and Comparative Immunology (ISDCI) have been meeting every 3 years for the last 27 years through an International Congress in order to bring together the community of researchers working in developmental, comparative and evolutionary immunobiology for exchange of information and new ideas and to develop new collaborative projects. The previous Congress was held at St. Andrews in Scotland in 2003 and brought together delegates from 28 countries. This Congress provides 1) a forum in which this community of scientists can assess, collectively, the state of the field and the direction in which it is moving 2) unique opportunities for the exchange of information in both formal and informal settings, and 3) the chance for young researchers to present their ideas to established workers in their field, and to engage in scientific debate. The Congress has traditionally been a venue where many new research collaborations, both national and international, have been incubated. The Society is committed to providing, in its Congress, a research forum in which all scientists working in developmental, comparative and evolutionary immunobiology can actively participate. Thus, while invited plenary speakers and chairs of sessions will mostly be drawn from the pool of senior researchers, the Congress will be structured to allow many short oral presentations, which provide valuable experience for younger scientists, especially postdoctoral researchers and graduate students, to present their work. The poster sessions will be structured to ensure that everyone who wishes to present a poster will be able to do so. In order to maximize the value of the poster sessions, the Congress will set aside two blocks of time that are scheduled only for poster viewing. In addition to the oral and poster presentations, the Congress will also schedule Workshops. The Workshops will deal with more focused topics and will permit researchers who have very specific interests in a particular area to exchange information and ideas in an informal atmosphere. As part of its major commitment to the future of the discipline, ISDCI is particularly interested in providing opportunities for young scientists, women and under-represented minorities to participate in their meetings. ISDCI traditionally provides substantial numbers of travel bursaries to young scientists to facilitate their participation, and makes every effort to contribute to the participation costs of every applicant who is either a graduate student or a postdoctoral fellow.

This project is directed towards the derivation of longterm in vitro cultured cell lines from the Pacific whiteleg shrimp, Litopenaeus vannamei, a marine crustacean. This is high-risk research because no established cell lines have yet been derived for any marine invertebrate. This proposed task is of high potential value because the availability of even a single cell line would take research on this organism to a different level than is currently possible, where investigators are restricted to experiments either on whole organisms or on short-term primary cell cultures. A novel and previously undescribed general innate anti-viral immune response, induced by double-stranded RNA (dsRNA), has been described in L. vannamei. The occurrence and biological significance of this immunity has been clearly established in vivo, but without a cell line it will not be possible to investigate and understand the molecular mechanisms underpinning this novel phenomenon. The ability to understand this innate anti-viral reaction is important for two reasons 1) it has the potential to fill a major gap in our understanding of the evolution of anti-viral immunity 2) many viral diseases of shrimp are highly virulent, causing significant economic loss to the aquaculture industry and potentially causing significant damage to the coastal marine ecosystem. Given the importance of crustaceans to the food-web in the ocean, the potential environmental threat that emerging viruses have on wild populations should not be underestimated. The development of long-term shrimp hemopoietic cell lines will be explored by a variety of complementary approaches. The PIs have developed a set of tissue culture media formulations that permit the survival of shrimp cells for over 12 months, but which do not support continuous cell division. Development of these media will be continued so that they better support growth and cell division. A major aspect of this work will be the addition to the medium of purified recombinant L. vannamei homologue of astakine, a cytokine recently isolated from a freshwater crayfish and which has been reported to promote crayfish hemopoietic cell proliferation in tissue culture. In parallel with improved media formulations, the chances of obtaining long-term cell cultures will be enhanced by transforming shrimp hemopoietic cell precursors using plasmids constitutively expressing oncogene products, with the initial studies focusing on SV40 T antigen. Broader Impacts: The broadest impact of the project will be on the field of research in marine crustaceans. The availability of a cell line (or cell lines) will enable researchers in the field to extend their investigations to the cellular and molecular level, which to this point has been very difficult because of the necessity to use whole animal models or shortterm primary cell cultures. The PIs on this application consider both graduate student education and the exposure of undergraduate students to research important components of their research program. Additionally, the PIs will participate in NSF-funded programs for undergraduate students including the NSF-REU program and the MIMES program run by the South Carolina Department of Natural Resources. The MIMES program provides an introduction to research for minority and underrepresented students, and funds summer research internships to undergraduate students from these groups.