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Skeletal lesions and anemia associated with ascorbic acid deficiency in juvenile rhesus macaques.

Young rhesus macaques housed in outdoor corn cribs and fed a commercially prepared primate diet became weak, depressed, were reluctant to move, and expressed locomotor abnormalities. Thirteen severely affected animals were hospitalized for evaluation. Physical examination disclosed swellings and instabilities involving the ends of long bones. Radiography confirmed physeal fractures in 11 of 13 animals. Affected bones included the distal femur, proximal humerus, distal tibia/fibula, and distal radius/ulna. Other, less obvious changes were noted on radiographs. Anemia was a consistent finding. Ascorbic acid deficiency was suspected and therapy was initiated that consisted of vitamin supplements, diet change, cage rest, and support bandages. Feed samples were submitted to a laboratory for analysis and were confirmed deficient in vitamin C. Follow-up radiographs showed large calcifying subperiosteal hematomas in epiphyseometaphyseal regions, consistent with a diagnosis of scurvy. Twelve of 13 animals recovered clinically. Subsequent radiographs documented improvement of initially severe angular deformities associated with displaced fractures.

Anemia

Effect of staphylococcal enterotoxin B on cardiorenal functions and survival in X-irradiated rhesus macaques.

Pretreatment of rhesus macaques with nonlethal total-body x-irradiation (400 R) prolonged survival time from an average of 15 hours to 101 hours after intravenous (IV) inoculation of 50 microgram of staphylococcal enterotoxin B (SEB)/kg of body weight. Radiation exposure per se did not produce detectable cardiorenal changes; however, the longer survival after SEB challenge exposure in x-irradiated rhesus macaques was associated with improved cardiorenal functions if compared with that of nonirradiated macaques given the same dose of SEB. Total-body radiation exposure 4 days prior to IV SEB inoculation prevented typical SEB-induced decreases (where measured at 5 hours) in cardiac output, stroke volume, TcH2O, CPAH, Cosm, and urine flow, as well as increases in total peripheral and renal resistance. A theory concerning the significance of radiation-induced leukopenia on modification of SEB-induced cardiorenal functions is postulated.

Animals

Sera from simian immunodeficiency virus-infected rhesus macaques inhibit lymphocyte proliferation.

Rhesus macaque monkeys infected with the simian immunodeficiency virus (SIV) develop a syndrome mimicking acquired immunodeficiency syndrome (AIDS) in humans. We had demonstrated previously that sera from individuals infected with human immunodeficiency virus (HIV) inhibit the proliferation of lymphocytes from healthy noninfected subjects and that this phenomenon was associated with the development of clinical AIDS. Thus, we sought to determine whether sera from SIV-infected monkeys would also inhibit lymphocytes from healthy humans and SIV-negative rhesus monkeys. Sera from SIV-infected monkeys were compared with sera from uninfected animals and cultured with cells from healthy human volunteers or SIV-negative monkeys in the presence or absence of phytohemagglutinin (PHA). Cell proliferation was determined by measuring the incorporation of radiolabeled thymidine into cellular DNA. Sera from SIV-infected monkeys suppressed the proliferation of human and non-human primate lymphocytes. This activity appears to be similar to that described for sera from HIV-1-infected humans. Therefore, rhesus macaques infected with SIV provide a model for the study of serum inhibitory factors previously reported in AIDS patients.

Animals

Lymphocyte subpopulations in spontaneous disease of rhesus macaques.

The immunologic status of rhesus macaques (Macaca mulatta) with naturally occurring disease was evaluated by determining the percentages of B and T lymphocytes and mitogen responsiveness of lymphocytes in peripheral blood and lymph nodes. The B lymphocytes were identified by the presence of cell surface immunoglobulin and receptors for complement. The T lymphocytes were identified by their ability to form spontaneous rosettes with sheep red blood cells. Rhesus macaques with idiopathic or primary amyloidosis had normal lymphocyte characteristics. Peripheral blood lymphocytes from rhesus macaques with atypical tuberculosis had decreased percentages of spontaneous rosette-forming cells and depressed responses to concanavalin A, and those with chronic diarrhea or chronic arthritis were also found to have abnormal peripheral blood lymphocyte characteristics. The percentage of B and T lymphocytes in normal lymph nodes was variable, making simultaneous histologic examination necessary for evaluation of diseased animals.

Amyloidosis

Genetic variation in transferrin alleles of rhesus macaques, Macaca mulatta.

The rhesus macaque displays an extensive polymorphism at the transferrin locus. A principal components analysis describes the variance and covariance of alleles at the transferrin locus in eight widely dispersed sample populations. Using an eigenvectorial representation of the covariance matrix and systematically approximated geographical locations the distribution of populations and transferrin alleles is compared. Alleles with high variance prove to be the determining factor in the placement of populations in a "genetic map" and provide a means for interpreting the low congruence of genetics and geography found.

Alleles

Comparison of cardiorenal functions between women and female rhesus macaques.

Selected cardiorenal functions for conscious and chaired rhesus macaques were compared directly and indirectly with corrections for body weights and surface areas to well-established base-line values for human subjects. Certain functional differences between species and conditions of measurement do not allow all experimental results from the rhesus macaque to be extrapolated to man. However, this comparative study provides evidence that can be useful in attempting to evaluate cardiorenal data from macaques in relationship to human studies.

Animals

Potential significance of the cellular immune response against the macaque strain of simian immunodeficiency virus (SIVMAC) in immunized and infected rhesus macaques.

The cellular immune response of seven rhesus macaques immunized with Tween-ether-treated macaque strain of simian immunodeficiency virus (SIVMAC) and three non-vaccinated control animals was investigated. Immunization elicited antigen-specific proliferating CD4+ cells in five of seven monkeys. Proliferating T cells were found in all animals protected from a first virus challenge. Cytotoxic T lymphocytes (CTLs) were not induced by the immunization. After the second challenge, the four formerly protected animals became infected, despite a strong proliferative CD4+ cell activity in three of them. All animals lost their proliferative activity 2 weeks after infection. After the first challenge four of the six infected animals exhibited a CTL response and after the second challenge, one of four newly infected macaques acquired a CTL response. The five animals with a CTL activity against SIVMAC proteins were protected from severe thrombocytopenia, which appeared in the five CTL-negative animals after infection. Our data show the induction of proliferative T cells by immunization with soluble SIVMAC antigen. This T cell reactivity was found in all animals protected from the first virus challenge, but did not confer protection from the second challenge. Interestingly, the proliferative T cell reactivity disappeared 2 weeks after virus infection. Furthermore a CTL response against viral proteins seems to protect infected animals from severe thrombocytopenia which is an early sign of AIDS in monkeys.

Animals

Estradiol concentrations, fat deposits, and reproductive strategies in male rhesus macaques.

Squirrel monkeys (Saimiri spp.) are thought to undergo a unique pattern of seasonal weight change among the Primates that reflects a fatted male phenomenon. But many male mammals, including rhesus macaques (Macaca mulatta), undergo circannual weight changes concurrent with mating season activity. Four adult male rhesus macaques living in a heterosexual social group in an outdoor enclosure were studied over a 2-year period in order to ascertain potential mechanisms underlying seasonal weight changes. Alterations in weight were significantly correlated with changes in abdominal fat levels and in body mass index. Neither testosterone nor estradiol fluctuations were correlated with weight changes, but estradiol levels were significantly correlated with body mass index. The annual profile of changes in abdominal fat level reflected the annual profile of variation in circulating estradiol levels. The fatted male phenomenon appears to characterize circannual weight fluctuations in rhesus macaques. Seasonal weight changes resulting from fat deposition are suggested to provide a substrate for adipose tissue aromatization that yields elevated estradiol levels and enables males to forego feeding and concentrate their energy budgets on activities directly related to mate acquisition and retention.

Adipose Tissue

Role of TAR RNA splicing in translational regulation of simian immunodeficiency virus from rhesus macaques.

The untranslated leader sequences of rhesus macaque simian immunodeficiency virus mRNAs form a stable secondary structure, TAR. This structure can be modified by RNA splicing. In this study, the role of TAR splicing in virus replication was investigated. The proportion of viral RNAs containing a spliced TAR structure is high early after infection and decreases at later times. Moreover, proviruses containing mutations which prevent TAR splicing are significantly delayed in replication. These mutant viruses require approximately 20 days to achieve half-maximal virus production, in contrast to wild-type viruses, which require approximately 8 days. We attribute this delay to the inefficient translation of unspliced-TAR-containing mRNAs. The molecular basis for this translational effect was examined in in vitro assays. We found that spliced-TAR-containing mRNAs were translated up to 8.5 times more efficiently than were similar mRNAs containing an unspliced TAR leader. Furthermore, these spliced-TAR-containing mRNAs were more efficiently associated with ribosomes. We postulate that the level of TAR splicing provides a balance for the optimal expression of both viral proteins and genomic RNA and therefore ultimately controls the production of infectious virions.

Animals

Localization of SIV in the genital tract of chronically infected female rhesus macaques.

Despite the fact that human immunodeficiency virus (HIV) is transmitted primarily by sexual contact, the biology of the sexual transmission of HIV is poorly understood. Simian immunodeficiency virus (SIV) can be transmitted to female rhesus macaques by placing cell-free virus into the vaginal canal, and SIV can be isolated from the vaginal secretions of infected rhesus macaques. The authors examined the genital tracts from 16 chronically infected female rhesus macaques and localized SIV-infected cells using in situ hybridization and immunohistochemistry. SIV-infected cells were found in the genital tract of 13 of the 16 animals examined, and in most cases the SIV-infected cells were located in the submucosa of the cervix and vagina. However, SIV-infected cells were also found in the vaginal epithelium. SIV-infected cells were more common in sites of inflammation than in normal areas. These findings suggest that SIV gains access to genital tract secretions from the cervix and vaginal epithelium.

Animals

Pre-clinical immunogenicity and safety evaluation of H2 strain Hepatitis A Inactivated Vaccine in rhesus macaques.

BACKGROUND: Hepatitis A is a viral infection of the liver that can cause mild to severe illness. Currently, two types of HAV vaccines are used worldwide, inactivated hepatitis A vaccines, which are used in most countries, and live attenuated vaccines (H2 and L-A-1 strain), which are mainly used in China. The major disadvantage of live attenuated virus to cause secondary infections among contacts and mutation shifts of the live vaccine strain. The H2 strain was selected for the development of an inactivated hepatitis A vaccine to further reduce biosafety risks. Rhesus macaques high genomic homology with humans and the incubation period after hepatitis A vaccination and human natural infections are similar. We use rhesus macaques to assess immunogenicity and safety of the H2 strain Hepatitis A Inactivated Vaccine. METHODS: The vaccine was assessed in rhesus macaques, divided into four groups (n = 10 per group): the control group (adjuvant buffer; aluminum content 0.35 mg/mL; 2 mL per dose), the low-dose group (320EU, 0.5 mL of 640EU/mL with aluminum content 0.35 mg/mL), the medium-dose group (640EU, 1 mL of 640EU/mL with aluminum content 0.35 mg/mL), and the high-dose group (1280EU, 2 mL of 640EU/mL with aluminum content 0.35 mg/mL). Animals were injected intramuscularly at multiple sites in the hind limbs and received four inoculations at 4-week intervals. Test items including Clinical indicators, immunogenicity indicators and Histopathological examination. RESULTS: No abnormalities were observed in any group in terms of general clinical condition throughout the study period except for slight decreases in body temperature after immunization. Hematological parameters, serum biochemistry indices, and histopathological findings showed fluctuated to different degrees of fluctuation after immunization across all groups. Immunogenicity assessments showed that the inactivated hepatitis A vaccine (H2) induced both humoral and cellular immune responses effectively, and the levels of antibodies increased with certain dose- and time-response trends. CONCLUSION: The inactivated hepatitis A vaccine (H2 strain, human diploid cell) was safe and immunogenic in non-human primates. The results provide strong preclinical support for the further clinical development of this vaccine candidate.

Animals

Proteomic Characterization of the Rhesus Macaque Lens Nucleus: Similarity to Human Lens, Age Effects on Protein Solubility, and Trends in Post-Translational Modifications.

PURPOSE: Proteomes of lens nuclei from young (4 years old) and old (15-16 years old) rhesus macaques (Macaca mulatta) were analyzed to determine similarity of the proteomic profile to that of human lenses, age-related differences in protein solubility, and association of various post-translational modifications with age and protein solubility. METHODS: Lens core proteins were separated into water-soluble and water-insoluble fractions using aqueous buffer and centrifugation. The water-insoluble fraction was solubilized using sodium dodecyl sulfate (SDS). Proteins were processed using S-trap columns, and peptide digests were analyzed using high-resolution, label-free data-dependent acquisition (DDA) proteomics. Open modification searches were performed using MSFragger to identify possible post-translational modifications (PTMs). The number of modified peptide tandem mass spectra confidently assigned to samples by age or solubility were compared to find PTMs with statistically significant count differences. RESULTS: The overall proteomic profile of rhesus macaque lenses was very similar to human lenses, consisting of 80.2% crystallins, 1.1% beaded filament proteins, and 18.7% other proteins. The crystallin fraction consisted of 27% alpha crystallins, 67.6% beta/gamma crystallins, and 5.4% taxon-specific psi crystallin. Glycolytic enzymes, beta/gamma crystallins, and a few glutathione-related enzymes were found to have age-related shifts to the water-insoluble fraction. There were significant differences in deamidation, dioxidation, carbamylation, carboxymethylation, and trioxidation based on age and/or solubility of proteins. CONCLUSIONS: These data indicate a high level of conformity between rhesus macaque and human lens proteomes, and a few key differences. We identified several age-related differences in protein solubility and PTM that may contribute to lens pathology.

Animals

Control of nipple and body contact by mothers and infants in rhesus macaques.

While 3-month-old infant rhesus macaques (Macaca mulatta) were awake and active in social interactions away from their mothers, body and nipple contacts with their mothers were nevertheless made from time to time. In each dyad the proportions of contacts made by the mother nearly equalled those broken by her, suggesting a meshed interaction in which each partner accepted most of the other's contact initiations and terminations. Passive prevention of nipple contact by a mother reduced the frequency of nipple contact by her infant in the first 5 s after the infant had made body contact. Passive prevention occurred after fewer than 1 in 6 body contacts initiated by infants, and--even without its occurrence--most infants were less ready to take the nipple after their own initiatives than after maternal initiatives. Once nipple contact had been made, the probability of breaking body contact was reduced. The role of maternal rejection both in the control of nipple contact in the short term and in determining (through its effect on the sucking pattern) whether the mother gives birth in the next birth season or later is discussed. We suggest that, by the age of 3 months, the infants had already learned when and how often nipple contact with their mothers would be acceptable during their awake and active periods, and we suggest that subsequent decreases in the frequency of nipple contact were partly the results of maternal rejections which were accepted by the infants.

Animal Communication

Cellular immunologic studies of malignant lymphoma in rhesus macaques.

Cells from malignant lymphoma in 10 rhesus macaques were examined for lymphocyte surface markers. Three had features of T cells, 5 had features of B cells, and 2 lacked evidence of either B- or T-cell differentiation. Correlation between the histologic classification of cell type and the B- or T-cell nature of the neoplasms was not evident. Evaluation of serum electrophoresis, mitogen responses tests, and previous histologic studies suggest that the development of the neoplastic lymphocyte proliferation occurred following or during an abnormal immunologic response.

Animals

Molecular cloning and expression of the rhesus macaque D1 dopamine receptor gene.

Using homologous probes for the cloning of related genes within the family of guanine nucleotide-binding protein-coupled receptors, we have cloned the gene for the rhesus macaque D1 dopamine receptor. By using the rat D1 receptor coding sequence as a probe under high stringency conditions, the rhesus D1 receptor gene was isolated from a lambda EMBL3 rhesus genomic DNA library. The rhesus D1 dopamine receptor gene is intronless and encodes a 446-amino acid protein that contains two consensus sites for asparagine-linked glycosylation (Asn-5 and Asn-176) and two consensus sites for cAMP-dependent protein kinase phosphorylation (Thr-136 and Thr-268). The primary amino acid sequence of the rhesus D1 dopamine receptor shows an extremely high degree of similarity (99.6%) to the human D1 receptor. Genomic DNA analyses conducted with high and reduced stringency hybridizations indicate that the rhesus macaque D1 receptor is a member of a large multigene family. Like the human D1 receptor mRNA, the rhesus D1 receptor mRNA is approximately 4 kilobases in size and is localized predominantly in the caudate, with lesser amounts in the hippocampus and cortex. The rhesus D1 receptor coding region was inserted into the cytomegalovirus promoter-driven expression vector pcDNA-1, and the recombinant (pcDNA-D1) was cotransfected with the selectable marker pRSVneo, conferring G418 resistance, into D1 receptor-deficient C6 glioma cells. Analyses of the selected transfectant demonstrate the expression of a high affinity, functional D1 dopamine receptor. The D1 receptor radioligand [3H]SCH 23390 bound transfectant membranes with an affinity (Kd), of 0.3 nM; the D2-selective ligand spiperone, the dopamine receptor ligand clozapine, and the serotonin receptor antagonist ketanserin bound with considerably lower affinities (102, 80, and 95 nM, respectively). Both dopamine and the D1-selective agonist SKF 38393 inhibited the binding of [3H]SCH 23390 to transfectant cell membranes; the binding of these agonists was sensitive to GTP. Dopamine potently stimulated the accumulation of cAMP in transfected C6 cells, whereas SKF 38393 was a partial agonist in these cells. Also, the density of recombinant D1 receptors on the transfectant cells was decreased 40% upon treatment with 10 microM dopamine, indicating that occupation of recombinant D1 receptors by agonists alters surface expression of the receptors.

Adenylyl Cyclases

Candid No. 1 Argentine hemorrhagic fever vaccine protects against lethal Junin virus challenge in rhesus macaques.

The protective efficacy of Candid No. 1, a live-attenuated vaccine against Argentine hemorrhagic fever (AHF), was evaluated in non-human primates. Twenty rhesus macaques immunized 3 months previously with graded doses of Candid No. 1 (16-127, 000 PFU), as well as 4 placebo-inoculated controls, were challenged with 4.41 log10 PFU of virulent P3790 strain Junin virus. All controls developed severe clinical disease; 3 of 4 died. In contrast, all vaccinated animals were fully protected; none developed any signs of AHF during a 105-day follow-up period. Viremia and virus shedding were readily detected in all placebo-vaccinated controls, while virus could be recovered only once (by amplification) from throat swabs of 2 Candid No. 1 vaccinees on day 21. Vigorous secondary-type neutralizing and immunofluorescent antibody responses were seen in most vaccinees that had received 3 log10 PFU Candid No. 1 or fewer; all others, including those receiving 127,200 PFU, maintained relatively stable titers during follow-up. Candid No. 1 was highly immunogenic and fully protective against lethal Junin virus challenge in rhesus macaques, even at extremely low (16 PFU) vaccine doses.

Animals

Maternal plasma catecholamines in the rhesus macaque during late gestation: effect of photoperiod and timed melatonin infusion.

This study tested the hypothesis that changes in photoperiod alter plasma catecholamine concentrations in the rhesus monkey during late gestation. Twelve chronically catheterized pregnant rhesus macaques were acclimated to a 12-h photoperiod (lights-on, 0700-1900 h). Under the control L:D cycle, blood samples were collected at 3-h intervals over 24 h for catecholamine analysis. Plasma concentrations (mean +/- SEM, pg/ml) ranged from 678 +/- 90 to 928 +/- 142 for norepinephrine; 230 +/- 22 to 631 +/- 141 for epinephrine; and 282 +/- 70 to 1090 +/- 362 for dopamine. A diurnal rhythm was observed in epinephrine with peak concentrations during lights-on (0900-1800 h; p less than 0.05, compared to lights-off). After the first sampling protocol, the animals were divided equally between two groups: phase shift, in which lights-on was shifted 11 h (2000-0800 h) and constant light, with lights on continuously. After the phase shift, a parallel shift in the plasma epinephrine rhythm was noted, with peak levels observed between 2200 and 0700 h (p less than 0.05). Constant light abolished the rhythm in epinephrine, with an overall reduction in mean basal levels of all three catecholamines. Daily melatonin infusions (0.2 micrograms/kg/h, 1900-0630 h) under constant light failed to restore the epinephrine rhythm or to return basal catecholamine concentrations to control photoperiod levels. These data suggest that photoperiod entrains the rhythm in epinephrine secretion, but the rhythm is ablated under constant conditions. Further, melatonin does not appear to play a role in the regulation of catecholamine secretion in the pregnant rhesus macaque.

Animals

Restraint inhibits luteinizing hormone and testosterone secretion in intact male rhesus macaques: effects of concurrent naloxone administration.

The present study investigates how restraint affects the hypothalamo-hypophysial adreno-cortical axis and the hypothalamo-hypophysial gonadal axis in intact, adult male rhesus macaques. Restraint was chosen because it is not physically painful or harmful to the animal, but rather serves both as a physical and psychological stressor. Blood samples were collected from a remote site at 15-min intervals beginning at 07.00 h from tethered adult male rhesus macaques. Each of 4 animals was subjected to 6 h of chair restraint after a 3-hour control period in the animals' home cage. Samples were collected for an additional 6 h at the end of the restraint period when the animal was returned to its home cage. Brief anesthesia with ketamine (administered through the indwelling catheter) facilitated transfer of the animals to and from the chair. Blood samples were collected from 4 undisturbed males to document LH and testosterone secretion throughout the day. Plasma ACTH and cortisol, measured as indexes of stress, were elevated within 15 min after initiation of restraint and remained elevated for most of the restraint period. Conversely, LH and testosterone began to fall immediately after restraint and remained suppressed for several hours after the animals were removed from restraint and returned to their home cage. Testosterone levels were more consistently inhibited than were LH levels, a reflection of the fact that in some animals, testosterone remained low after the return of pulsatile LH secretion. In studies with naloxone (Nx), the opiate receptor antagonist (5 mg bolus plus 5 mg/h) was given beginning either at the initiation of restraint (n = 2) or 2 h thereafter (n = 2), and continued until the end of the restraint period. With Nx treatment of the restrained animals, both ACTH and cortisol were elevated as in the controls and LH and testosterone secretion were significantly increased within 1-2 h. However, after the Nx treatment was terminated and the animals were returned to their home cages, plasma levels of LH and testosterone were not different from levels in restrained animals and were significantly less than levels in untreated animals. These data show that restraint is a potent stimulus for activation of the HPAC axis and inhibits both LH and testosterone release. The pathway through which restraint inhibits LH release probably includes endogenous opiate suppression of hypothalamic GnRH release since Nx partially blocks the effect of stress.

Adrenocorticotropic Hormone