Trisomy of chromosome 18 in the baboon (Papio hamadryas anubis).

Trisomy 18 is usually a lethal chromosomal abnormality and is the second most common autosomal trisomy in humans, with an incidence of 1:8000 live births. It is commonly associated with abnormalities of the lower and upper extremities, having the frequency of 95% and 65%, respectively. A newborn female olive baboon (Papio hamadryas anubis) was diagnosed with intrauterine growth retardation and severe arthrogryposis-like congenital joint deformities. Cytogenetic analysis including G-banding and fluorescence in situ hybridization (FISH) revealed that the congenital abnormalities were associated with chromosomal mosaicism for trisomy 18. Genetic analysis with microsatellites from chromosome 18 confirmed the maternal origin of the extra chromosome 18. This is the first report of trisomy 18 in the baboon, which may be a promising animal model of human disease.

CD8+ cells from HIV-2-infected baboons control HIV replication.

OBJECTIVE: To analyze the CD8+ cell antiviral immune response in HIV-2-infected baboons. DESIGN: Baboons were infected with clinical isolates of HIV-2, CD8+ cells were isolated from phytohemagglutinin (PHA)-stimulated baboon peripheral blood mononuclear cells (PBMC). These cells were cultured with PHA-stimulated CD4+ cells acutely infected with HIV-2 at several CD8+:CD4+ cell ratios. Control of HIV-2 replication was determined by comparing peak levels of HIV-2 replication in fluids from CD8+:CD4+ cell cocultures with those in fluids from infected CD4+ cells cultured alone. RESULTS: CD8+ cells from HIV-2-infected baboons inhibited HIV-2 replication in acutely infected autologous CD4+ cells to a significantly greater extent than did CD8+ cells from uninfected baboons (P = 0.0001). At the beginning of the acute phase of HIV-2 infection, CD8+ cells showed either a transient reduction or loss in the antiviral activity. In some cases the CD8+ cell response enhanced HIV-2 replication. Subsequently, the strength of the CD8+ cell antiviral activity increased concomitant with a decrease in the HIV-2 load in the PBMC. Suppression of HIV replication could be demonstrated with filtered fluid from CD8+ cells. Other studies indicated that infected CD4+ cells are lost during coculture of CD8+ cells with infected CD4+ cells. CONCLUSIONS: CD8+ cells of HIV-2-infected baboons develop substantial anti-HIV-2 activity following HIV-2 infection, which may account in part for the low frequency of pathogenesis in HIV-2-infected baboons. Studies to elucidate the mechanism of this CD8+ cell antiviral activity suggest that it is mediated in part by a soluble antiviral factor, but primarily in association with the loss of infected CD4+ cells.

High-efficiency gene transfer into rhesus macaque primary T lymphocytes by combining 32 degrees C centrifugation and CH-296-coated plates: effect of gene transfer protocol on T cell homing receptor expression.

Although steady progress has been made in transducing human T lymphocytes by Moloney murine leukemia virus (Mo-MuLV)-based vectors, few studies have been done to define ex vivo gene transfer protocols to transduce rhesus macaque primary T lymphocytes. Given the fact that simian immunodeficiency virus (SIV) infection in rhesus macaque is a well-characterized model for human immunodeficiency virus (HIV), it is of great interest to develop an efficient protocol to transduce rhesus macaque primary T cells. In this study, we have used MuLV-10A1-pseudotyped retrovirus expressing enhanced green fluorescent protein (EGFP) to evaluate a number of ex vivo gene transfer protocols in rhesus macaque primary T lymphocytes. Our objectives in designing these protocols were (1) to test whether higher efficiency gene transfer could be obtained by combining two previously defined protocols, centrifugation at 32 degrees C and the CH-296-coated plate; and (2) to study the effect of an ex vivo gene transfer protocol on the expression of lymphocyte homing receptors L-selectin and alpha 4 beta 7 and alpha 4 beta 1 integrins. From seven independent experiments we demonstrate by flow cytometry analyses of EGFP expression that whereas centrifugation at 32 degrees C or the fibronectin fragment CH-296-coated plate protocol alone yielded 10-14% transduction efficiency, combining these two protocols resulted in 28.1-51.2% transduction efficiency. EGFP in transduced cells was expressed highly throughout the 14 days of posttransduction expansion. Our results also demonstrate that whereas the transduction procedure per se did not significantly alter the expression of lymphocyte homing receptors, anti-CD3 and anti-CD28 antibody stimulation profoundly reduced the expression of L-selectin. The selective reduction of L-selectin may result in significant in vivo consequences if transduced cells are infused.

Screening donors for xenotransplantation. The potential for xenozoonoses.

Xenotransplantation is a potential solution to the current donor shortage for solid organ transplantation. The transmission of infectious agents from donor organs or bone marrow to the recipient is a well-recognized phenomenon following allotransplantation. Thus the prospect of xenotransplantation raises the issue of xenozoonoses--i.e., the transmission of animal infections to the human host. Anticipating an increasing number of baboon to human transplants, 31 adult male baboons (Papio cynocephalus) from a single colony in the United States were screened for the presence of antibody to microbial agents (principally viral) that may pose a significant risk of infection. Antibody to simian cytomegalovirus, simian agent 8 and Epstein-Barr virus, was found in 97% of animals tested. Antibody to simian retroviruses and Toxoplasma gondii was found in 30% and 32% respectively. Discordant results were found when paired samples were examined by two primate laboratories. This was particularly noted when methodologies were based on cross-reaction with human viral antigens. These results highlight the need to develop specific antibody tests against the species used for xenotransplantation.

Lack of susceptibility of baboons to infection with hepatitis B virus.

Historically, hepatitis B virus (HBV) has been considered species specific and unable to infect baboons. Based on this premise, two patients with HBV endstage liver disease underwent baboon liver xenotransplantation. To study whether baboons are susceptible to HBV infection, four baboons (two receiving immunosuppressive therapy) were inoculated with HBV. Animals were followed for 6 months: clinical examinations and biochemical studies were normal, hepatitis B surface antigen and hepatitis B core antigen staining of biopsies was negative, and HBV serology remained negative. HBV polymerase chain reaction was transiently positive in one animal, which most likely reflects the initial inoculation. This pilot study corroborates historical evidence and beliefs that baboons are resistant to HBV.

PCR Identification and Differentiation of Baboon Cytomegalovirus From Other Human and Nonhuman Primate Cytomegalovirus.

Background: Differential identification of baboon alpha, beta, and gamma herpesviruses is an essential technology in order to monitor xenozoonotic transmission of baboon viruses to foreign species organ and/or cell recipients. We present polymerase chain reaction techniques that will differentiate known baboon cytomegaloviruses (CMV) from their closely related counterparts found in humans. Methods and Results: Polymerase chain reaction techniques for identification of the known beta herpesviruses commonly present in the baboon are reported. The techniques described also permit the unequivocal differentiation of these virus types from closely related human as well as other nonhuman primate viruses in the family herpesviridae. Methods are based upon sequence analysis of specifically selected genes of baboon CMV. Primer pairs from sequence analyses were selected based upon nonhomologous sequences in gene homologues of human CMV. Unique, baboon species specific amplimers were identified both with ethidium bromide staining and with radiolabeled probe analyses. Conclusions: With the described techniques, it is possible to monitor organ/cell recipients of xenograft transplantation for the establishment of baboon CMV in the foreign human host. Monitoring can be performed throughout the life of the recipient in effort to rule out host susceptibility to baboon CMV, as well as to rule out potential host:donor recombinant viruses formed between the closely related members of herpesvirus family.

Human immunodeficiency virus-2 infection in baboons is an animal model for human immunodeficiency virus pathogenesis in humans.

OBJECTIVE: To assess disease progression in baboons (Papio cynocephalus) that were infected with two human immunodeficiency virus-2 (HIV-2) isolates. METHODS: Eight baboons were inoculated intravenously with either HIV-2UC2 or HIV-2UC14 and were followed for a 2- to 7-year period of observation. RESULTS: Six of 8 baboons showed lymphadenopathy and other signs of HIV-related disease, 3 of 8 baboons had an acute phase CD4+ T-cell decline, and 2 of 5 baboons infected with the HIV-2UC2 isolate progressed to an acquired immunodeficiency syndrome-like disease. Human immunodeficiency virus-2-specific pathology in lymphatic tissues included follicular lysis, vascular proliferation, and lymphoid depletion. Both neutralizing antibodies and a CD8+ T-cell antiviral response were associated with resistance to disease. CONCLUSIONS: Disease progression and the development of acquired immunodeficiency syndrome in HIV-2-infected baboons have similarities to human HIV infections.

Experimental pneumonia virus of mice infection of guineapigs spontaneously infected with Bordetella bronchiseptica.

Guineapigs that were intranasally inoculated with pneumonia virus of mice (PVM) seroconverted to PVM by 11 days post-infection. During the course of study (2-60 days post-infection) no gross or histologic lesions were identified within the lungs that could be attributed to PVM infection. Mild rhinitis and tracheitis were found in most animals and acute purulent bronchopneumonia in two animals, which may have resulted from spontaneous subclinical Bordetella bronchiseptica infection. Viral and bacterial respiratory diseases of the guineapig are briefly reviewed.

Eosinophilic bronchitis-like lesion as the cause of death in a Macaca mulatta: a first case report.

BACKGROUND: Eosinophilic bronchitis is a recently described, relatively benign condition in humans that is characterized by a corticosteroid-responsive chronic cough and sputum eosinophilia without the abnormalities of airway function seen in asthma. The exact cause of this condition is currently unknown, however has been associated with various occupational exposures in humans. It has also been reported to progress to irreversible airway obstruction. This disease has been reported in dogs and horses, but not in non-human primates. METHODS: Gross examination of an otherwise healthy 13-year-old, colony-born Macaca mulatta, which died of severe non-responsive respiratory distress revealed that the lungs were markedly inflated and moist. RESULTS: Hematoxylin and eosin-stained sections from the lungs contained widespread accumulation of eosinophils, sloughed epithelial cells, and mucus centered around bronchioles and adjacent airways. There was no evidence of mast cell infiltration of peribronchiolar smooth muscle, goblet cell hyperplasia, or basement membrane thickening. CONCLUSIONS: This ruled out recurrent episodes as would be expected in asthma, favoring the diagnosis of an eosinophilic bronchitis-like lesion. We report a first case of eosinophilic bronchitis-like features in a M. mulatta.

KIT-positive gastrointestinal stromal tumor in a 22-year-old male chimpanzee (Pan troglodites).

Gastrointestinal stromal tumors (GIST), KIT-positive and KIT signaling driven or platelet-derived growth factor receptor alpha (PDGFRA) signaling driven mesenchymal tumors, are poorly known in nonhuman primates. Availability of KIT- and PDGFRA-inhibitor drug imatinib mesylate has greatly raised the interest for these tumors. At necropsy of a 22-year-old male chimpanzee, a round, firm 2-cm intramural tumor was incidentally found in the midbody of the stomach and diagnosed as a GIST. Histologically, the mass was composed of spindle to polygonal epithelioid cells arranged in short to intermediate-length, interlacing streams, bundles, and nodular whorls often separated by hyalinized eosinophilic matrix. The mitotic rate was a maximum 1/50 high-power field. Immunohistochemically, the tumor cells were diffusely positive for KIT and CD34, focally positive for alpha-smooth muscle actin, and negative for muscle specific actin, desmin, S-100 protein, synaptophysin, and glial fibrillary acidic protein. Because the majority of human GISTs have gain-of-function KIT or PDGFRA mutations, genomic sequences of KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18 from this chimpanzee GIST were polymerase chain reaction amplified and sequenced. However, no mutation was identified in the analyzed "mutational hot spots." This study is the first extensive histomorphologic, immunohistochemical, and molecular genetic analysis of a chimpanzee GIST. More cases of nonhuman primate GISTs should be analyzed to discover the clinicopathologic spectrum of GISTs in these species.

Analysis of hepatitis C virus-inoculated chimpanzees reveals unexpected clinical profiles.

The clinical course of hepatitis C virus (HCV) infections in a chimpanzee cohort was examined to better characterize the outcome of this valuable animal model. Results of a cross-sectional study revealed that a low percentage (39%) of HCV-inoculated chimpanzees were viremic based on reverse transcription (RT-PCR) analysis. A correlation was observed between viremia and the presence of anti-HCV antibodies. The pattern of antibodies was dissimilar among viremic chimpanzees and chimpanzees that cleared the virus. Viremic chimpanzees had a higher prevalence of antibody reactivity to NS3, NS4, and NS5. Since an unexpectedly low percentage of chimpanzees were persistently infected with HCV, a longitudinal analysis of the virological profile of a small panel of HCV-infected chimpanzees was performed to determine the kinetics of viral clearance and loss of antibody. This study also revealed that a low percentage (33%) of HCV-inoculated chimpanzees were persistently viremic. Analysis of serial bleeds from six HCV-infected animals revealed four different clinical profiles. Viral clearance with either gradual or rapid loss of anti-HCV antibody was observed in four animals within 5 months postinoculation. A chronic-carrier profile characterized by persistent HCV RNA and anti-HCV antibody was observed in two animals. One of these chimpanzees was RT-PCR positive, antibody negative for 5 years and thus represented a silent carrier. If extrapolated to the human population, these data would imply that a significant percentage of unrecognized HCV infections may occur and that silent carriers may represent potentially infectious blood donors.

DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection.

Hepatitis C virus (HCV) poses a worldwide health problem in that the majority of individuals exposed to HCV become chronically infected and are predisposed for developing significant liver disease. DNA microarray technology provides an opportunity to survey transcription modulation in the context of an infectious disease and is a particularly attractive approach in characterizing HCV-host interactions, since the mechanisms underlying viral persistence and disease progression are not understood and are difficult to study. Here, we describe the changes in liver gene expression during the course of an acute-resolving HCV infection in a chimpanzee. Clearance of viremia in this animal occurred between weeks 6 and 8, while clearance of residual infected hepatocytes did not occur until 14 weeks postinfection. The most notable changes in gene expression occurred in numerous interferon response genes (including all three classical interferon antiviral pathways) that increased dramatically, some as early as day 2 postinfection. The data suggest a biphasic mechanism of viral clearance dependent on both the innate and adaptive immune responses and provide insight into the response of the liver to a hepatotropic viral infection.

Genetic analysis of hematological traits in chimpanzees (Pan troglodytes).

Hematological traits are commonly assessed markers of health status that have been used in a large number of anthropological studies, especially those focusing on high-altitude adaptation. Despite the wealth of literature on environment-associated variation in these traits, relatively few studies have dealt with the underlying genetic components of hematological measures. The purpose of this study is to estimate heritabilities for eight hematological traits using data obtained from a large pedigreed chimpanzee colony. Seven of the eight hematological traits exhibited significant heritabilities, ranging from h2 = 0.308 for mean cell volume to h2 = 0.834 for red blood cell count. The use of multiple measures per individual proved to be essential for the accurate estimation of heritabilities. We conclude that the underlying genetic variation in hematological traits should be considered when these measures are used in study protocols.

Viral persistence, antibody to E1 and E2, and hypervariable region 1 sequence stability in hepatitis C virus-inoculated chimpanzees.

The relationship of viral persistence, the immune response to hepatitis C virus (HCV) envelope proteins, and envelope sequence variability was examined in chimpanzees. Antibody reactivity to the HCV envelope proteins E1 or E2 was detected by enzyme-linked immunosorbent assay (ELISA) in more than 90% of a human serum panel. Although the ELISAs appeared to be sensitive indicators of HCV infection in human serum panels, the results of a cross-sectional study revealed that a low percentage of HCV-inoculated chimpanzees had detectable antibody to E1 (22%) and E2 (15%). Viral clearance, which was recognized in 28 (61%) of the chimpanzees, was not associated with an antibody response to E1 or E2. On the contrary, antibody to E2 was observed only in viremic chimpanzees. A longitudinal study of animals that cleared the viral infection or became chronically infected confirmed the low level of antibody to E1, E2, and the HVR-1. In 10 chronically infected animals, the sequence variation in the E2 hypervariable region (HVR-1) was minimal and did not coincide with antibody to E2 or to the HVR-1. In addition, low nucleotide and amino acid sequence variation was observed in the E1 and E2 regions from two chronically infected chimpanzees. These results suggest that mechanisms in addition to the emergence of HVR-1 antibody escape variants are involved in maintaining viral persistence. The significance of antibodies to E1 and E2 in the chimpanzee animal model is discussed.

Heritabilities of clinical chemical traits in chimpanzees.

Clinical chemical measures are commonly used biomarkers of health status in nonhuman primates and may also serve as important covariates or outcome variables in experimental protocols. There is a considerable range of normal variation in most clinical chemical traits and the determinants of this variation have been relatively unexplored in nonhuman primates used as animal models in biomedical research. This study assesses the evidence for genetic determinants of normal variation in nine clinical chemical traits (blood urea nitrogen, creatinine, potassium, sodium, CO2, glucose, albumin, globulin, and total cholesterol concentrations) in an important animal model, the chimpanzee. We found significant moderate heritabilities for potassium, sodium, albumin, globulin, and total cholesterol. The results provide information useful for addressing issues in both genetic management and experimental research.

Development of a primary tamarin hepatocyte culture system for GB virus-B: a surrogate model for hepatitis C virus.

GB virus-B (GBV-B) causes an acute hepatitis in tamarins characterized by increased alanine transaminase levels that quickly return to normal as the virus is cleared. Phylogenetically, GBV-B is the closest relative to hepatitis C virus (HCV), and thus GBV-B infection of tamarins represents a powerful surrogate model system for the study of HCV. In this study, the course of infection of GBV-B in tamarins was followed using a real-time 5' exonuclease (TaqMan) reverse transcription-PCR assay to determine the level of GBV-B in the serum. Peak viremia levels exceeded 10(9) genome equivalents/ml, followed by viral clearance within 14 to 16 weeks. Rechallenge of animals that had cleared infection resulted in viremia that was limited to 1 week, suggestive of a strong protective immune response. A robust tissue culture system for GBV-B was developed using primary cultures of tamarin hepatocytes. Hepatocytes obtained from a GBV-B-infected animal maintained high levels of cell-associated viral RNA and virion secretion for 42 days of culture. In vitro infection of normal hepatocytes resulted in rapid amplification of cell-associated viral RNA and secretion of up to 10(7) genome equivalents/ml of culture supernatant. In addition, infection could be monitored by immunofluorescence staining for GBV-B nonstructural NS3 protein. This model system overcomes many of the current obstacles to HCV research, including low levels of viral replication, lack of a small primate animal model, and lack of a reproducible tissue culture system.

Mycobacterium tuberculosis-secreted protein antigens: immunogenicity in baboons.

The effective control of tuberculosis (TB) requires the development of improved vaccines. It is now well established that Mycobacterium tuberculosis-secreted antigens represent promising candidates to be included in subunit vaccine preparations. It also is accepted that studies in nonhuman primate models will be required to further develop these vaccine preparations. As a necessary step in this direction, we have assessed the immunogenicity of M. tuberculosis-secreted antigens in baboons. Animals received a total of three intramuscular injections consisting of M. tuberculosis culture filtrate proteins resuspended in an adjuvant formulation (MPL-SE) and were tested for development of specific antibody and cellular responses. All animals produced antibody and cellular proliferative responses in the absence of detectable delayed-type hypersensitivity reactions. Production of gamma-interferon following stimulation of peripheral blood lymphocytes with culture filtrate proteins was either absent or present at low levels. Results from this study show that, although M. tuberculosis-secreted protein antigens are relatively safe and immunogenic in baboons, alternative immunization approaches must be identified for the induction of T-helper type 1 responses.

Clinical parameters of the normal baboons (Papio species) and chimpanzees (Pan troglodytes).

Improved equipment and advanced progressive techniques by scientists using baboons and chimpanzees in biomedical research have resulted in improved clinical laboratory data. The use of state-of-the-art clinical laboratory instruments, methodologies with improved accuracy, and an increased variety of individual tests routinely requested and performed has necessitated the comparison of current data with prior data produced in our laboratory and with those values reported in the literature. In addition to an expanded hematologic profile, including red blood cell distribution width and mean platelet volume, and a more comprehensive chemical profile of 28 individual tests, additional data collected included values for coagulation profiles, arterial blood gases, serum protein electrophoresis, and urine osmolalities. Samples for evaluation were obtained from clinically normal sedated adult baboons (Papio species) and chimpanzees (Pan troglodytes) and processed conventionally according to Good Laboratory Practice Act standards. Arithmetic means were calculated, values of 3 standard deviations or greater were eliminated, and means were recalculated to include 2 standard deviations. All data correlated well with prior in-house values, and no remarkable differences from established data were detected, thus indicating the reliability of past and present data. Data compared favorably with normal clinical values established for humans.