Andrew A. Lackner, D.V.M., Ph.D. - US grants

DESCRIPTION: (Adapted from applicant's abstract) The broad objective of this project is to develop a SIV macaque model of pediatric AIDS that can be used to elucidate the neuropathogenesis of human immunodeficiency virus (HIV) infection. The investigator has three specific aims the first of which is to try to determine the temporal distribution and cellular targets of SIV in the central nervous system (CNS) of fetal and neonatal macaques inoculated with SIVmac at different stages of development. CNS tissue from 12 fetuses, inoculated at different stages of gestation and 10 inoculated on the same day of gestation and serially sacrificed, will be provided for study by collaborators at the California Regional Primate Research Center. The distribution of virus and identity of infected cells will be determined by immunohistochemistry and in situ hybridization. The second goal of the investigator is to attempt to develop an in vitro reaggregate brain culture system to examine mechanisms of SIV neuropathogenesis. This section has three subparts, the first of which is to try to assess the neuropathologic potential of different SIV isolates, molecular clones and recombinant viruses. The second is to evaluate the role of possible viral cofactors such as cytomegalovirus (CMV) in SIV neuropathogenesis. The third is to evaluate possible lentiviral-related toxins such as SIV-gp130, cytokines and quinolinic acid.These tasks will be accomplished by in vitro inoculation of reaggregated brain cultures with various isolates of SIV or potential toxins. The cultures will be evaluated by histopathology, immunohistochemistry for cellular and viral proteins, in situ hybridization for viral nucleic acid and biochemical analysis for potential toxins. The third specific aim is to attempt to test potential SIV-associated neurotoxins for their effect in vivo. This goal is designed to test the in vivo relevance of the findings in the second specific aim. This will be accomplished by chronic intrathecal administration of putative neurotoxins to normal uninfected infant rhesus macaques. The animals will then be monitored using the same neurodevelopmental test used in SIV-infected macaques. This will allow a comparison between the CNS effects of SIV and putative neurotoxins.

DESCRIPTION: (Adapted from applicant's abstract) The broad objective of this project is to develop a SIV macaque model of pediatric AIDS that can be used to elucidate the neuropathogenesis of human immunodeficiency virus (HIV) infection. The investigator has three specific aims the first of which is to try to determine the temporal distribution and cellular targets of SIV in the central nervous system (CNS) of fetal and neonatal macaques inoculated with SIVmac at different stages of development. CNS tissue from 12 fetuses, inoculated at different stages of gestation and 10 inoculated on the same day of gestation and serially sacrificed, will be provided for study by collaborators at the California Regional Primate Research Center. The distribution of virus and identity of infected cells will be determined by immunohistochemistry and in situ hybridization. The second goal of the investigator is to attempt to develop an in vitro reaggregate brain culture system to examine mechanisms of SIV neuropathogenesis. This section has three subparts, the first of which is to try to assess the neuropathologic potential of different SIV isolates, molecular clones and recombinant viruses. The second is to evaluate the role of possible viral cofactors such as cytomegalovirus (CMV) in SIV neuropathogenesis. The third is to evaluate possible lentiviral-related toxins such as SIV-gp130, cytokines and quinolinic acid.These tasks will be accomplished by in vitro inoculation of reaggregated brain cultures with various isolates of SIV or potential toxins. The cultures will be evaluated by histopathology, immunohistochemistry for cellular and viral proteins, in situ hybridization for viral nucleic acid and biochemical analysis for potential toxins. The third specific aim is to attempt to test potential SIV-associated neurotoxins for their effect in vivo. This goal is designed to test the in vivo relevance of the findings in the second specific aim. This will be accomplished by chronic intrathecal administration of putative neurotoxins to normal uninfected infant rhesus macaques. The animals will then be monitored using the same neurodevelopmental test used in SIV-infected macaques. This will allow a comparison between the CNS effects of SIV and putative neurotoxins.

HIV-1 infection is characterized by the gradual loss of the cell-mediated immune response and the ultimate development of opportunistic infections and neoplasms. In addition, patients infected with HIV-1 manifest a number of debilitating inflammatory conditions of the CNS, lung, gastrointestinal tract, skeletal muscle, skin, heart, and joints. Excluding inflammation secondary to opportunistic infections, these inflammatory conditions are believed to represent primary lentiviral-induced lesions. However, the viral and host parameters and pathogenetic mechanisms responsible for the development of these inflammatory conditions are not known. Multiple lines of evidence indicate that inflammatory reactions are modulated by leukocyte interactions with adhesion molecules on endothelial cell membranes. The work outlined in this proposal focuses on the hypothesis that AIDS-related inflammatory disease occurs as a direct result of changes in the adhesive interactions between endothelium and circulating leukocytes. Specifically, to test this hypothesis and to explore the mechanisms involved in the evolution of AIDS-related inflammatory disease, we plan to use the SIVmac model to: 1) examine the viral determinants associated with inflammatory disease in AIDS, 2) determine the relative expression of endothelial-derived leukocyte adhesion molecules in tissues from SIVmac-infected monkeys using immunohistochemical techniques, 3) examine leukocyte-endothelial adhesion in AIDS by determining the immunophenotypes of leukocytes and adhesion molecules involved in AIDS-related inflammatory disease using multiple in vitro and in vivo techniques, 4) investigate the mechanisms responsible for any changes in endothelial adhesion expression in tissues from SIVmac-infected animals by examining these tissues for cytokine elaboration and by examining normal rhesus endothelial cells for adhesion molecule expression after coculturing with SIVmac-infected cells, supernatants, or whole virus, and 5) investigate the roles of other factors in leukocyte extravasation in AIDS by examining vessels for evidence of reductions in flow caliber and for the presence of specific chemotactic factors.

cell cell interaction; disease /disorder model; human immunodeficiency virus; tissue /cell culture

virus; nervous system; AIDS; immunology; mother /infant health care; sexually transmitted diseases; communicable diseases; lymphatic system; Primates; Mammalia;

Infection with human immunodeficiency virus (HIV) commonly results in neurologic disease termed the AIDS dementia complex (ADC). Neuronal loss and injury have been found in the HIV-infected brain, but the underlying mechanisms are poorly understood. The simian immunodeficiency virus (SIV) infected macaque is an excellent model for HIV infection, but neuronal loss has not been demonstrated. To determine whether neuronal damage occurs in the SIV-infected macaque we quantified the neuronal marker n-acetylaspartate (NAA) using proton magnetic resonance spectroscopy in brain extracts of control and SIV-infected macaques and correlated these findings with histologic analyses. We found reduced NAA in the SIV-infected animals compared to controls (2.94+1.37 versus 6.21+1.73 mols/g wet weight; p=0.004). A significant decrease in NAA was also found in SIV-infected animals sacrificed in the acute stages of infection. We conclude that SIV infection of rhesus macaques results in neuronal damage that is demonstrable soon after infection.

Competitive PCR was used to evaluate the expression of cytokine, granzyme B, and chemokine genes in lymph nodes of macaques recently infected with the simian immunodeficiency virus (SIV) pathogenic molecular clone SIVmac239 (n = 16), the nonpathogenic vaccine strain SIVmac239 D nef (n = 8), and the nonpathogenic molecular clone SIVmacA11 (n = 8). For both SIVmac239 and its nef-deleted derivative, strong expression was observed as early as 7 days postinfection for interleukin 1b(IL-1b, IL-6, tumor necrosis factor alpha, gamma interferon and IL-13. The levels of gene induction were equally intense for both viruses despite a lower viral load for SIVmac239 Dnef compared with that for SIVmac239. However, the nature of the cytokine network activation varied with the viral inocula. Primary infection with SIVmac129 was characterized by a higher level of IL-4, IL-10, MIL-1a, MIP-1b, MCP-1, and RANTES gene expression and a lower level of IL-12 and granzyme B gene expression compared with infection with SIVmac239Dnef. Thus, infection with nef-deleted SIV was associated with a preferential TH1 versus TH2 pattern of cytokine production. Infection with SIVmacA11 was characterized by a delayed immune response for all markers tested. The unique patterns of cytokine and chemokine gene expression in lymph nodes correlated nicely with the pathogenic potential of the SIV strains used as well as with differences in their ability to serve as protective vaccines.

HIV infection of children usually results in more rapid and severe disease than is seen in adults To explore the pathogenesis of pediatric AIDS we examined 20 rhesus macaques inoculated intravenously with SIV within 24 hours of birth Viral inocula included the pathogenic molecular clone SIVmac239 (n = 13), the macrophage-competent derivative SIVmac239/316 (n = 2), the minimally pathogenic SIVmac239 nef (n = 2) and uncloned SIVmac251 (n = 3) Infected neonates were sacrificed at intervals over the first 50 days (n = 16) or allowed to progress to terminal disease (n =4) Neonates inoculated with these viruses had higher viral loads than older animals inoculated with the same dose and stock of virus although differences did not reach statistical significance The highest viral loads were present in animals infected with SIVmac239 followed in order by SIVmac251, SIVmac239/316 and SIVmac239 nef The latter, were the only group of animals that showed normal weight gai n By in situ hybridization (ISH) virus was detectable in lymphoid tissues of all animals as early as 3 days postinoculation (dpi) with maximal numbers of infected cells by 14 dpi coincident with peak viremia The majority of infected cells were present in the T-cell zones of the spleen and lymph nodes and in the thymic medulla Rare, SIV-infected cells undergoing mitosis were also observed Combined in situ hybridization/immunohistochemistry experiments revealed that most of the infected cells were T-cells with little evidence of monocyte/macrophage infection Neonates infected with SIVmac239 nef had far fewer infected cells and they were primarily within germinal centers of the spleen and lymph nodes Histologically, spleen and lymph nodes of animals infected with SIVmac239 and SIVmac251 for < 50 days were characterized by an absence of follicular hyperplasia This is in contrast to older animals inoculated with these same viruses and age-matched neonates inoculated with eithe r SIVmac239/316 or SIVmac239 nef which had marked follicular hyperplasia and dysplasia In summary, while viral loads were not significantly different between neonates and older animals inoculated with the same stock and dose of virus, numerous other differences were observed that must be the result of age-specific host determinants

The demonstration, on neurons, of chemokine receptors known to function as coreceptors for HIV and SIV raises the possibility that the neuropathogenesis of HIV/SIV infection is mediated via these receptors. We hypothesize that neuronal damage is determined by the interaction of chemokine receptors on neurons and astrocytes with viral envelope protein or chemokines produced by infected macrophage/microglia in the CNS. These interactions can either result in aberrant signaling through chemokine receptors or interfere with the binding of "trophic factors", either of which may be damaging to neurons. A corollary of this hypothesis that will be tested in vivo is that neurons expressing the highest density of appropriate chemokine receptors will be the most susceptible. To test this hypothesis we will: 1. Examine the regional and cellular distribution of chemokine receptor expression in brains of SIV-infected rhesus macaques at different stages of disease compared to uninfected controls. This will be achieved by using immunohistochemistry, double and triple label immunofluorescence with confocal microscopy and quantitative image analysis to examine expression of chemokine receptors in relation to markers of neuronal injury. This will be done in conjunction with in situ hybridization to localize viral infection and PCR to quantitate tissue viral load. 2. Characterize chemokine receptor expression in immediately ex vivo and in vitro populations of rhesus macaque neurons and astrocytes. 3. Evaluate the effects of chemokine receptor stimulation on neurons and glial cells. This will be achieved by exposing macaque neurons or mixed cultures of astrocytes and neurons to 1) chemokines that function through CCR3, CCR5, or CXCR4, or 2) conformationally authentic, noninfectious SIV virions prepared from closely related molecularly cloned viruses that utilize CCR5 in a CD4 dependent (SIVmac239) or CD4 independent (SIVmac239/316) fashion. We will then assess cultures for cellular injury and induction of apoptosis.

HIV infection of children usually results in more rapid and severe disease than is seen in adults To explore the pathogenesis of pediatric AIDS we examined 20 rhesus macaques inoculated intravenously with SIV within 24 hours of birth Viral inocula included the pathogenic molecular clone SIVmac239 (n = 13), the macrophage-competent derivative SIVmac239/316 (n = 2), the minimally pathogenic SIVmac239 nef (n = 2) and uncloned SIVmac251 (n = 3) Infected neonates were sacrificed at intervals over the first 50 days (n = 16) or allowed to progress to terminal disease (n =4) Neonates inoculated with these viruses had higher viral loads than older animals inoculated with the same dose and stock of virus although differences did not reach statistical significance The highest viral loads were present in animals infected with SIVmac239 followed in order by SIVmac251, SIVmac239/316 and SIVmac239 nef The latter, were the only group of animals that showed normal weight gai n By in situ hybridization (ISH) virus was detectable in lymphoid tissues of all animals as early as 3 days postinoculation (dpi) with maximal numbers of infected cells by 14 dpi coincident with peak viremia The majority of infected cells were present in the T-cell zones of the spleen and lymph nodes and in the thymic medulla Rare, SIV-infected cells undergoing mitosis were also observed Combined in situ hybridization/immunohistochemistry experiments revealed that most of the infected cells were T-cells with little evidence of monocyte/macrophage infection Neonates infected with SIVmac239 nef had far fewer infected cells and they were primarily within germinal centers of the spleen and lymph nodes Histologically, spleen and lymph nodes of animals infected with SIVmac239 and SIVmac251 for < 50 days were characterized by an absence of follicular hyperplasia This is in contrast to older animals inoculated with these same viruses and age-matched neonates inoculated with eithe r SIVmac239/316 or SIVmac239 nef which had marked follicular hyperplasia and dysplasia In summary, while viral loads were not significantly different between neonates and older animals inoculated with the same stock and dose of virus, numerous other differences were observed that must be the result of age-specific host determinants

Human immunodeficiency virus (HIV)-infected patients commonly have unintentional weight loss and low serum micronutrient levels that have been correlated with disease progression. However, the relationship among these nutritional parameters, direct effects of the virus on the intestinal immune system and intestinal dysfunction and the role of opportunistic infections in progressive weight loss are not clear. These issues are extremely difficult to address in persons infected with HIV because a large percentage of HIV/AIDS patients are already taking a variety of nutritional supplements in an uncontrolled manner and dietary intake varies greatly. Furthermore, the rapid evolution of antiretroviral therapy complicates obtaining answers to these questions. To obviate these problems and also to control for additional variable such as time since inoculation and differences in viral inocula, we propose to use the simian immunodeficiency virus (SIV)-infected macaque model of AIDS. The organization of the program project involves three projects and four cores. One project will define the natural history of weight loss and micronutrient deficiency in SIV-infected macaques and conduct two interventions to try and alleviate wasting. The next project will examine direct effects of SIV infection on the intestinal immune system and intestinal function. Another project will determine the relationship between key gastrointestinal opportunists (C. parvum, E. bieneusi, and M. avium) and weight loss. Thus this program project represents a multi-disciplinary approach by a group of interactive, experienced investigators to examine the interplay between 1) nutrition, 2) direct effects of SIV infection, and 3) opportunistic infections of the gastrointestinal tract on the pathogenesis of wasting.

Description (adapted from applicant's abstract): Neurologic abnormalities in HIV-1-infected children are considered to be more sever than in adults with a different spectrum of manifestations. The reasons for these differences are obscure. A likely explanation is the immaturity of the host, specifically the developmental state of the CNS and immune system. In support of this general thesis, we have recently observed significant differences in the pathogenesis of SIV in neonatal vs. adult macaques. In the CNS, the primary differences were in the frequency of infection of monocyte/macrophage/microglia. We hypothesize that these differences are related to the "maturity" of neonatal monocyte/macrophages and their ability to support SIV replication and/or the selective recruitment and retention of mononuclear cells in the CNS. Unifying these apparently disparate possible explanations is the expression of chemokine receptors, particularly CCR5. Furthermore, CCR5 and other chemokine receptors have been demonstrated on neurons suggesting that CCR5 and related molecules may play important roles at multiple steps in the neuropathogenesis of AIDS. To examine our hypothesis and the role of chemokine receptors in the neuropathogenesis of pediatric AIDS we will use the SIV/macaque model to 1). Examine the role of viral determinants in neuroinvasion and neurovirulence in neonatal macaques inoculated at birth. 2). Examine the role of host factors responsible for neuroinvasion and neurovirulence of HIV infection in neonatal macaques by a). Examining the sequential expression of leukocyte and endothelium derived adhesion molecules, and chemokines in the brain and correlate their expression with the onset of cellular infiltrates and SIV infection of the CNS; b). Analyze the regional and cellular distribution of chemokine receptor expression in brains of neonatal SIV-infected rhesus macaques at different stages of disease compared to uninfected controls and c). Characterize chemokine receptor expression in immediately ex vivo populations of brain macrophages from SIV-infected and uninfected macaques and examine their ability to support SIV infection.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The breeding corrals at the TNPRC currently house approximately 4,000 primates ($22 million) in enclosures ranging in size from 1/8 acre to one acre. Significant growth both in numbers of primates and corrals has occurred in the last ten years without any significant improvements to the drainage system. This is a concern both from the standpoint of regulatory issues as well as the health of the colony. Increased demands for NHPs both for research and as part of a national biodefense initiative further justify improvements to their facilities. Additionally, threats from vandals and animal rights activists have increased dramatically in the past ten years. Only very minor security enhancements have been implemented during this time. The current threat level along with the significant value of the colony demands that improvements be made to the physical security of the facility. This application seeks funds to address both the drainage and security concerns of the facility. The proposed improvements to the drainage will require the current system of ditches to be modified to the proper elevations to ensure efficient flow, damaged and improperly sized culverts will be removed and replaced. The storm water collected in the ditches will be directed to a constructed wetland that will be enhanced in this project with new plant life. This "wetland" will serve to retain and treat the storm water from the corrals. The proposed design would ensure that the outflow from the "wetland" has been sufficiently treated to allow its discharge without any regulatory requirements. The security enhancement proposed in this application includes a new perimeter fence, a soil cement perimeter roadway, lighting and surveillance cameras. These improvements will allow for enhanced intrusion detection via manned patrols on the perimeter road and remote monitoring of the cameras. The upgraded perimeter fence will slow unauthorized entrance to the property. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This proposal is a request by the Tulane National Primate Research Center to construct a clinic facility that will support the breeding programs, which have a current census of close to 4,000 nonhuman primates. The rapidly expanding specific pathogen free (SPF) breeding program requires that state of the art facilities be available to support the housing, diagnosis and treatment of animals originating from this and other colonies. The majority of animals used in research programs at TNPRC are derived from the breeding colony. Consideration has been given, during the design phase, to assure that the facility will accommodate the projected growth of the breeding program well into the future. The proposed facility will allow the clinical care and housing of breeding colony animals to occur adjacent to outdoor housing facilities, alleviating the current need to transport animals to housing areas on the main campus. The convenience of a clinical facility constructed adjacent to the breeding colony area will allow more immediately available health care for these animals. Other benefits of the facility would include, provisions for state of the art caging equipment and security, provisions for separation of animals based on viral status and origin in support of the SPF program, provisions for separation of research and breeding colony animals, and for other specialized clinical support such as radiography. In addition to the benefits to the TNPRC breeding program, the proposed facility will support the Washington National Primate Research Center (WaNPRC) breeding colonies, which are housed entirely at the TNPRC. These colonies support the research program of WaNPRC. The proposed construction is a component of a recently developed long-range strategic plan for facilities at the TNPRC. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The research program at the Tulane National Primate Research Center (TNPRC) has been heavily invested in infectious disease research for close to three decades. The AIDS research program is the largest research program at the Center. For core investigators, 73% of current research funding is related to AIDS ($9.8/13.4 million) and 60% of the entire research program, which includes affiliate and core investigators is related to AIDS. The NPRC program provides resources and opportunities for research using nonhuman primates to core faculty and to faculty affiliated with outside institutions. Recent changes in leadership at the TNPRC have resulted in a 70% increase in grant funding over the past 18 months. Most of this funding has been awarded within the large AIDS research program of the Center. The rapid increase in AIDS funding has resulted in a shortage of appropriate BSL2 nonhuman primate housing space to support the AIDS research program. The availability of BSL2 animal housing facilities directly impacts the Center's ability to carry out AIDS research. This proposal is a request by the Tulane National Primate Research Center to acquire funds to purchase equipment (manufactured modular BSL-2 palmate housing equipment) to help meet the current need for BSL2 animal housing for the AIDS research program. The proposed modular housing will provide space for approximately 100 animals assigned to AIDS research projects. The manufactured modular housing equipment will provide much needed additional BSL-2 space particularly during renovation of existing facilities.

AIDS; Primates; animal colony;

ABSTRACT NOT PROVIDED

ABSTRACT NOT PROVIDED

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The Tulane National Primate Research Center (TNPRC) has experienced substantial growth in the last two years which has included a three-fold increase in the research grant portfolio as well as numerous renovation and construction projects (including an $18.6M Regional Biocontainment Laboratory) funded primarily by NIH. These assets (and the status affixed to the Regional Biocontainment Laboratory) necessitate physical security upgrades to the main campus of the TNPRC. The recommendations for these upgrades come from three sources: Physical Security Design Guidelines for NIH-owned Facilities (Draft - January 16, 2004), a physical security threat and assessment report (March 2004) performed as a requirement of the Regional Biocontainment Laboratory award, and from master planning efforts for the Center performed with the assistance of CUH2A of Princeton, NJ and Atlanta, GA. The last few years have seen increasingly more destructive attacks on animal research facilities and biotechnology companies. These incidents have included arsons, bombings (incendiary and fragmentation type) and facility intrusions. Additionally, post-September 11th concerns and the national status of the NPRCs and RBLs have raised legitimate concerns about these sites as potential terrorist targets. This application seeks funds to enhance the Center through a number of security enhancements outlined in the application. These improvements will greatly enhance the security of the facility and allow the Center to meet or exceed security guidelines employed at NIH. [unreadable] [unreadable]

Animal Housing; Animal Husbandry; Animal Model; Animal Models and Related Studies; Animal Welfare; Animals; Animals, Laboratory; Arts; Assay; Bacillus anthracis, protective antigen; Bioassay; Biologic Assays; Biological; Biological Assay; Biopsy Sample; Biopsy Specimen; Blood; Breeding; CD7; CD7 gene; Caring; Clinical; Clinical Management; Development; Equipment; Expertise, Technical; Funding; Future; GP40; Genes; Georgia; Georgia (Republic); Georgia SSR; Georgia, USA; Georgia, Western Asia; Georgian S.S.R.; Georgian SSR; History; Housing; Housing, Animal; Human Resources; Husbandries, Animal; Immune response; Immune system; Immunomodulation; Individual; Infection; Infectious Diseases / Laboratory; Infectious Diseases Research; Institution; Investigators; Joint Ventures; LEU9; Laboratories; Laboratory Animals; Ligands; Louisiana; Macaca mulatta; Mammals, Primates; Manpower; Mission; Modeling; Mucosal Immune Responses; NCRR; NIAID; National Center for Research Resources; National Institute of Allergy and Infectious Disease; PA antigen, Bacillus anthracis; Pathology; Phase; Poxviridae; Poxviruses; Primates; Procedures; Programs (PT); Programs [Publication Type]; Protocol; Protocols documentation; Recording of previous events; Research; Research Personnel; Research Resources; Researchers; Resources; Reticuloendothelial System, Blood; Rhesus; Rhesus Macaque; Rhesus Monkey; Role; Services; TP40; TP41; Technical Expertise; USA Georgia; Universities; Ventures, Joint; Virulence; Western Asia Georgia; Work; animal care; anthrax protective antigen; anthrax protective factor; anthrax toxin PA; anthrax toxins translocating protein, Bacillus anthracis; biodefense; body system, allergic/immunologic; concept; design; designing; experience; host response; immune modulation; immunologic reactivity control; immunoregulation; immunoresponse; model organism; new vaccines; next generation vaccines; non-human primate; nonhuman primate; novel vaccines; organ system, allergic/immunologic; pag protein, Bacillus anthracis; pagA protein, Bacillus anthracis; personnel; pox virus; programs; social role; vaccine development

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The educational mission of the TNPRC is to provide training for undergraduate, veterinary and graduate students, post-doctoral fellows, veterinarians, and visiting scientists. The TNPRC educational effort is further broadened by participation in a T35 training grant in conjunction with the Louisiana State University School of Veterinary Medicine (LSUSVM), a T32 training grant also held together with the LSUSVM, a Summer Fellowship Program, a Pathology Training Curriculum, and a Veterinary Preceptorship. The summer fellowships entail one-on-one participation in a research project with an end-of-summer seminar session by the students. An R25 training grant provides funding for residency training of veterinarians in clinical medicine of nonhuman primates. This program is run in collaboration with the LSUSVM. There is also a Visiting Scientist Program. The basic objective of this program is to provide an understanding of the mission and functions of a National Primate Research Center. The Pathology Training Curriculum is directed toward furthering professional development of staff veterinary pathologists, staff veterinarians, and veterinarians involved in research. Lastly, three Center-wide colloquia address diverse educational interests of TNPRC scientists: 1) a seminar on infectious diseases, with invited speakers, 2) a biweekly research lab meeting, and 3) quarterly pathology and medicine Grand Rounds. This year there were 23 invited speakers. The center hosted 15 graduate students and 18 post-doctoral fellows, 3 laboratory animal medicine residents, 4 T32 trainees, as well as 5 students in the Veterinary Preceptorship Program. The Summer Fellowship Program was subscribed by 9 students (2 veterinary and 7 undergraduate students). There were 3 participants in the Pathology Training Curriculum. We are pleased with the response of students and investigators to our educational efforts, and look forward to maintaining this trend in the future.

The objective of the nonhuman primate core (Core H), located at the Tulane National Primate Research Center (TNPRC), is to provide leadership, collaborative expertise, and highly integrated clinical management and laboratory support to CFAR investigators for research utilizing nonhuman primate models of AIDS. The Core will enhance and facilitate the ability of CFAR investigators to perform studies in nonhuman primates and promote scientific collaborations between the TNPRC and CFAR colleagues in Philadelphia by providing services and expertise to CFAR investigators, andlhrough a Nonhuman Primate"Pilot Grant program (separate from but complementary to the pilot grant program of the CFAR Developmental Core). The core consists of clinical and laboratory components. The clinical component will acquire, house and care for the nonhuman primates, and assist investigators with experimental design. The core will also be responsible for the daily clinical care of animals and animal procedures such as immunization, blood draws, fluid collection, bronchoalveolar lavage, biopsies, etc. The laboratory component of the core will perform routine hematology, clinical chemistry, ova and parasite examination of feces, microbiology, and pathologic examination of all necropsies and biopsies in support of the animal studies. The core will also provide flow cytometry and immunology services, SIV and SHIV viral stocks and isolation, and specialized pathology services including in situ hybridization, immunohistochemistry, confocal microscopy and image analysis. The core also includes animals and animal support for developmental pilot studies. From our experience over the last five years, we expect that the combination of nonhuman primate resources and specialized research expertise with nonhuman primate models of AIDS will enhance the research mission of the Penn CFAR and result in new and stronger collaborations as well as attracting new investigators to use nonhuman primate models of AIDS. In addition to its service related mission and the developmental pilot grant program, the Core stimulates translation of bench-based findings into animal experimentation, a key step prior to studies in humans, by participation via videoconference in CFAR seminars, joint symposia, and extensive interactions that serve to advise CFAR investigators on how in vitro studies can be extended into this critical in vivo model.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A key feature of SIV and HIV infection is the rapid and near complete depletion of mucosal CD4+ T lymphocytes, however, this depletion also occurs in nonpathogenic infections of natural hosts, suggesting that it is a common feature of primate lentiviral infections. Here we evaluate pathological correlates produced by two variants of highly pathogenic SIVmac239, [unreadable]nef and [unreadable]GY, in rhesus macaques. Compared to SIVmac239, [unreadable]nef has a deleted nef gene, critical for virulence in vivo;[unreadable]GY has Gly-Tyr deletion from a conserved trafficking motif, Yxx[unreadable], in the envelope cytoplasmic tail. Acute peak viremia of [unreadable]GY was 1 week later, but comparable to, SIVmac239 (1.1 x 10^7 vs. 1.3 x 10^7) and higher than [unreadable]nef (3.2 x 10^5). Acute infection with [unreadable]GY and [unreadable]nef spared gut CD4+ T lymphocytes, compared to pathogenic SIV, despite high plasma viral loads. Compared to SIVmac239, the gut immune effector sites were infected by [unreadable]GY, however, the infection was less diffuse and rapidly shifted to immune inductive sites. Confocal microscopy identified [unreadable]GY- infected cells as CD3+ T lymphocytes;[unreadable]GY was not observed in macrophages or in the brain, indicating a less diverse target cell population and tissue distribution than for SIVmac239. Over time, the [unreadable]GY-infected animals with the highest viral loads exhibited a slow decline in gut and blood CD4+ T lymphocytes, and in these animals, sequencing identified two recurring mutations in the envelope cytoplasmic tail, either S727P (a previously identified compensatory mutation) or the generation of new Yxx[unreadable] motifs. In all envelope clones the Gly-Tyr deletion remained intact despite a high mutation rate. Interestingly, the S727P mutation also developed in the [unreadable]nef-infected animal with the highest viral load. Our results show [unreadable]GY and [unreadable]nef have a reduced ability to deplete mucosal CD4+ T lymphocytes, suggesting a common defect in entering and/or replicating at this site.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The purpose of the Pilot Research Program is to attract new investigators who show promise of developing a strong career in nonhuman primate biomedical research or who wish to add a nonhuman primate component to their existing research programs. The program is also open to investigators with established research programs using nonhuman primates who wish to develop substantially new research directions. The research must have potential for leading to a strong research grant application to outside funding agencies. Pilot research funds will not provide interim support for established projects or for any projects that qualify for support from other sources. Four pilot projects are ongoing. Peripheral Neuropathy in SIV-infected CD8-depleted rhesus macaques: Four animals have been obtained, inoculated with SIVmac251 and depleted of CD8+ T cells. The in life phase of the study has been completed. Blood and tissue samples are now being analyzed. Genetic Determinants of Pathogenesis and Neuropathogenesis for an Attenuated Variant of SIVmac239: Four rhesus macaques were infected with a mutated SIVmac239 SIVmac239[unreadable]GY S/P) on 7/5/10. Two animals were euthanized at 28 days post inoculation as per the study design. We continue to monitor the two remaining animals for signs of SIV disease progression. Data obtained to date indicates that SIVmac239[unreadable]GY S/P is a more pathogenic virus than SIVmac239[unreadable]GY and that the mutations made in SIVmac239[unreadable]GY S/P are compensatory in nature. Astrocytes are Key Regulators of SIV-related inflammation: This quarter, we have noted that, at the mRNA level, key integrins are down regulated on stellated astrocytes, notably integrin alpha 6. Concomitant with this, there is increased VCAM-1. Experiments continue to confirm this at the protein level. There are also altered expression of the intermediate filaments, including GFAP, nestin and vimentin. Cytokine secretion has also been assessed. We have one paper in press, partially funded by this project: MCP-3/CCL7 production by astrocytes: implications for SIV neuroinvasion and AIDS encephalitis In vivo gene silencing in macaques: We have now received clearance from all the regulatory bodies involved (IACUC, IBC and TRAC). Four cynomolgus macaques have been assigned to the project and are currently in quarantine. We expect to begin Mtb infections in April 2011. Meanwhile we are optimizing the silencing of SOCS3 in macaque cells (bone-marrow derived macrophages) in order to settle on two best pairs of oligonucleotides that will give us a chance to perform in-vivo silencing. Three additional pilot projects were recently awarded: CNS white matter tracts as a novel avenue for gene therapy for Krabbe disease Development of innate immunity in neonatal macaques Replication of sylvatic dengue virus in a natural primate host

DESCRIPTION (provided by applicant): This proposal is to construct a new central surgical facility for the TNPRC by adding on to a recently constructed animal research facility (Building 21) that houses NHP assigned to infectious disease research studies and animals from the TNPRC AIDS SPF breeding colony. The new surgical facility will replace existing square footage in a nearly 50-year-old facility (Building 2) that contains laboratory space, administrative offices, NHP housing, and the central surgical facility. The addition of the surgery facility to Building 21 is the final step necessary to move all animal related activities out of Building 2. As an additional benefit, square footage from the current central surgical facility will be made available for renovation and conversion to laboratory space for NIH funded investigators. The renovation of animal to laboratory space in Building 2 will be performed using funds other than those requested in this proposal. This plan significantly leverages the impact of funds awarded for this proposal by widening the scope of the improvement beyond the construction of the new surgical facility.

The development of HIV vaccines and other prevention strategies relies on the use of nonhuman primates in preclinical studies to advance the development of effective AIDS vaccine candidates and to advance development of effective topical microbicides and other prevention modalities or immune-based therapies. The NIAID Simian Vaccine Evaluation Unit (SVEU) contracts shall provide nonhuman primate resources that primarily support preclinical evaluation of AIDS vaccines. The SVEUs conduct studies in support of vaccines being developed by a wide range of investigators. These studies complement NIAID-supported basic vaccine research and vaccine evaluation studies funded through R01 (investigator-initiated research) grants, HIVRAD (HIV Vaccine Research and Design Program) grants, IPCAVD (Integrated Preclinical-Clinical AIDS Vaccine Development) grants, and NHP Consortium awards. The SVEUs also perform studies of candidate vaccines offered by companies or other researchers.

The development of HIV vaccines and other prevention strategies relies on the use of nonhuman primates in preclinical studies to advance the development of effective AIDS vaccine candidates and to advance development of effective topical microbicides and other prevention modalities or immune-based therapies. The NIAID Simian Vaccine Evaluation Unit (SVEU) contracts shall provide nonhuman primate resources that primarily support preclinical evaluation of AIDS vaccines. The SVEUs conduct studies in support of vaccines being developed by a wide range of investigators. These studies complement NIAID-supported basic vaccine research and vaccine evaluation studies funded through R01 (investigator-initiated research) grants, HIVRAD (HIV Vaccine Research and Design Program) grants, IPCAVD (Integrated Preclinical-Clinical AIDS Vaccine Development) grants, and NHP Consortium awards. The SVEUs also perform studies of candidate vaccines offered by companies or other researchers.

DESCRIPTION (provided by applicant): This R24 application is to promote nonhuman primate (NHP) health and the quality of NHP research resources for translational work by using metagenomics to improve diagnosis of infectious diseases with an emphasis on gastrointestinal (GI) disorders. Despite significant advances in the diagnosis of infectious diseases, unrecognized or adventitious agents in NHPs have the potential to confound experimental work. At the Tulane National Primate Research Center (TNPRC) over the past 3 years, we averaged 467 cases of GI disease/year presenting as animals with diarrhea. The average cost alone for case management of these animals was over $530,000/year. We have developed technologies that can readily, in a single test, detect multiple novel viruses, other potential pathogens, and the structure of the microbiome in fecal samples and intestinal tissues. Leveraging the resources of the laboratory of co-PI Dr. Skip Virgin at Washington University School of Medicine and the resources of the TNPRC under co-PI Dr. Andrew Lackner provides a unique opportunity to expand existing NHP models to include microbiome studies and to bring new approaches to the diagnosis of infections in NHPs. NHPs are used extensively in biomedical research programs. The overall objective of this proposal is to integrate clinical and pathology data with advanced next- generation sequencing and computational methods to facilitate the early recognition and diagnosis of established and novel infectious diseases of NHPs in the context of the microbiome. With these data we will create a central annotated repository of sequence and pathology information that will foster rapid dissemination of information on pathogens, and potential pathogens, across the NHP research community. Our goal is to provide this resource to both commercial and academic concerns to optimize the value of NHPs in translational research (see letters of support for documentation of the need for such a program). To this end we propose to establish a Primate Infectious Disease Resource (PIDR) that takes advantage of unique resources and specialized expertise present at WUSM and the TNPRC through the following aims. Specific aim 1: To establish a Primate Infectious Disease Resource to support NHP national biomedical research objectives. Specific aim 2: To define enteric metagenomic profiles from National Primate Research Center (NPRC) specific pathogen free (SPF) NHP breeding colonies and correlate these profiles with differences in gastrointestinal pathology and disease. Specific aim 3: To define the importance of selected new viruses or other pathogens identified via metagenomics and develop standard diagnostic tools for agents of proven relevance to NHP health.