Non-linear multidimensional flow cytometry analyses delineate NK cell phenotypes in normal and HIV-infected chimpanzees.

Natural killer (NK) cells are primary immune effector cells with both innate and potentially adaptive functions against viral infections, but commonly become exhausted or dysfunctional during chronic diseases such as human immunodeficiency virus (HIV). Chimpanzees are the closest genetic relatives of humans and have been previously used in immunology, behavior and disease models. Due to their similarities to humans, a better understanding of chimpanzee immunology, particularly innate immune cells, can lend insight into the evolution of human immunology, as well as response to disease. However, the phenotype of NK cells has been poorly defined. In order to define NK cell phenotypes, we unbiasedly quantified NK cell markers among mononuclear cells in both naive and HIV-infected chimpanzees by flow cytometry. We identified NKG2D and NKp46 as the most dominant stable NK cells markers using multidimensional data reduction analyses. Other traditional NK cell markers such as CD8α, CD16 and perforin fluctuated during infection, while some such as CD56, NKG2A and NKp30 were generally unaltered by HIV infection, but did not delineate the full NK cell repertoire. Taken together, these data indicate that phenotypic dysregulation may not be pronounced during HIV infection of chimpanzees, but traditional NK cell phenotyping used for both humans and other non-human primate species may need to be revised to accurately identify chimpanzee NK cells.

Seroprevalence of neutralizing antibodies to human adenoviruses type-5 and type-26 and chimpanzee adenovirus type-68 in healthy Chinese adults.

Replication-defective adenoviruses have been utilized as candidate vaccine vectors. However, clinical application of the best-studied human adenovirus type-5 (AdHu5) is limited by the high prevalence of preexisting neutralizing antibodies resulting from natural infection. Therefore, rare adenovirus serotypes, such as human adenovirus type-26 (AdHu26) and chimpanzee adenovirus type-68 (AdC68), have been employed as substitutes for AdHu5. However, few studies have described the epidemiology of pre-existing immunity to these adenoviruses in China. Thus, 1,154 participants from six regions in China were examined to assess the presence of neutralizing antibodies against AdHu5, AdHu26, and AdC68. The seroprevalence rates of neutralizing antibodies were as follows: AdHu5, 73.1% (844/1,154) (95% confidence interval: 70.5-75.6%); AdHu26, 35.3% (407/1,154) (95% confidence interval: 32.6-38.1%); and AdC68, 12.7% (147/1,154) (95% confidence interval: 10.9-14.8%), respectively. The most frequently detected and highest titer antibodies were specific for AdHu5. The results indicate that AdHu26 and AdC68 serve as more suitable vaccine vectors than AdHu5.

Brief communication: Adrenal androgens and aging: Female chimpanzees (Pan troglodytes) compared with women.

Ovarian cycling continues to similar ages in women and chimpanzees yet our nearest living cousins become decrepit during their fertile years and rarely outlive them. Given the importance of estrogen in maintaining physiological systems aside from fertility, similar ovarian aging in humans and chimpanzees combined with somatic aging differences indicates an important role for nonovarian estrogen. Consistent with this framework, researchers have nominated the adrenal androgen dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), which can be peripherally converted to estrogen, as a biomarker of aging in humans and other primates. Faster decline in production of this steroid with age in chimpanzees could help explain somatic aging differences. Here, we report circulating levels of DHEAS in captive female chimpanzees and compare them with published levels in women. Instead of faster, the decline is slower in chimpanzees, but from a much lower peak. Levels reported for other great apes are lower still. These results point away from slowed decline but toward increased DHEAS production as one of the mechanisms underlying the evolution of human longevity.

African great apes are natural hosts of multiple related malaria species, including Plasmodium falciparum.

Plasmodium reichenowi, a chimpanzee parasite, was until very recently the only known close relative of Plasmodium falciparum, the most virulent agent of human malaria. Recently, Plasmodium gaboni, another closely related chimpanzee parasite, was discovered, suggesting that the diversity of Plasmodium circulating in great apes in Africa might have been underestimated. It was also recently shown that P. reichenowi is a geographically widespread and genetically diverse chimpanzee parasite and that the world diversity of P. falciparum is fully included within the much broader genetic diversity of P. reichenowi. The evidence indicates that all extant populations of P. falciparum originated from P. reichenowi, likely by a single transfer from chimpanzees. In this work, we have studied the diversity of Plasmodium species infecting chimpanzees and gorillas in Central Africa (Cameroon and Gabon) from both wild-living and captive animals. The studies in wild apes used noninvasive sampling methods. We confirm the presence of P. reichenowi and P. gaboni in wild chimpanzees. Moreover, our results reveal the existence of an unexpected genetic diversity of Plasmodium lineages circulating in gorillas. We show that gorillas are naturally infected by two related lineages of parasites that have not been described previously, herein referred to as Plasmodium GorA and P. GorB, but also by P. falciparum, a species previously considered as strictly human specific. The continuously increasing contacts between humans and primate populations raise concerns about further reciprocal host transfers of these pathogens.

Blood pressure reference intervals for healthy adult chimpanzees (Pan troglodytes).

BACKGROUND: Cardiovascular disease is the primary cause of morbidity and mortality among captive chimpanzees. But there are no clinical definitions of normotension or hypertension in chimpanzees. METHODS: We analyzed 1 year of blood pressure (BP) data from a population of 261 healthy captive adult chimpanzees using a consistent set of criteria to ascertain health. RESULTS: Systolic BP varied by body weight. Diastolic BP varied by age. Median normotension was 126/63 mmHg, with an upper limit of 147/84 mmHg. We defined categories of pre-hypertension (148/85-153/88 mmHg) and hypertension (≥154/89 mmHg). The prevalence of elevated BP was 15%. The relative risk of mortality was 2.60, compared to normotensive animals. CONCLUSIONS: We used contemporary methods from human laboratory medicine to define reliable reference intervals for chimpanzee BP. Results allow accurate diagnosis of hypertension and pre-hypertension, and demonstrate an effect of elevated BP on mortality.

Androgen and androgen metabolite levels in serum and urine of East African chimpanzees (Pan troglodytes schweinfurthii): comparison of EIA and LC-MS analyses.

The primary male androgen testosterone (T) is often used as an endocrinological marker to investigate androgen-behaviour interactions in males. In chimpanzees and bonobos, studies investigating the relationship between T levels and dominance rank or aggressive behaviour have revealed contradictory results. The immunoassays used in these studies were originally developed for the measurement of steroids in serum. Their application to non-invasively collected samples, however, can lead to methodological problems due to cross-reacting metabolites, which might occur in urine or faeces but not in blood. The overall aim of this study, therefore, is to clarify whether a T enzyme immunoassay (EIA) is an applicable method to monitor testicular function in adult male chimpanzees. To estimate the impact of cross-reacting androgens on the used T EIA, we compared the results of an EIA measurement with a set of androgen metabolite levels measured by LC-MS. In urine from male chimpanzees, cross-reactivities appear to exist mainly with T and its exclusive metabolites, 5α-dihydrotestosterone (5α-DHT) and 5α-androstanediol (androstanediol). Both urinary and serum T levels of male chimpanzees were significantly higher than female T levels when measured with the T EIA, indicating a reliable measurement of testicular androgens and their exclusive metabolites with the used EIA. In urine from female chimpanzees, the comparison between LC-MS and T EIA results indicated a higher impact of cross-reactions with adrenal androgen metabolites. Therefore, the investigation of urinary T levels in female chimpanzees with a T EIA seems to be problematic. Overall our results show that a T EIA can be a reliable method to monitor testicular function in male chimpanzee urine and that LC-MS is a valuable tool for the validation of immunoassays.

Leptin associations with age, weight, and sex among chimpanzees (Pan troglodytes).

BACKGROUND: Leptin is a hormone secreted primarily by adipocytes, a lipostatic signal to the hypothalamus, and is often correlated with adiposity. Associations between leptin, age, and development are unknown in human's closest evolutionary relative, the common chimpanzee (Pan troglodytes). METHODS: Serum leptin was assessed cross sectionally in association with age, weight, and sex in healthy captive chimpanzee males (n = 47) and females (n = 49) to test hypotheses related to predicted differences in leptin levels with body mass, development, and sexual dimorphism. RESULTS: Leptin increased with age and weight among females, but not in males. Leptin was overall higher in females compared to males. CONCLUSIONS: Sex differences in leptin were most evident during adolescence and adulthood, despite similar increases in weight in both sexes indicating that sexual maturation is a key divergence point for differential somatic investment in adiposity and leptin levels between male and female chimpanzees.

Analyses of amino acid sequences in hypervariable region-1 of hepatitis C virus (HCV) in sera from chimpanzees infected three times with the same HCV strain.

BACKGROUND: To investigate whether or not the same strain of hepatitis C virus (HCV) can twice re-infect the same chimpanzee, we analyzed nucleic and amino acid sequences in HCV hypervariable region-1 (HVR1). Two chimpanzees were inoculated, three times each, with the same HCV strain during the 1983-1991. After each inoculation, chimpanzees developed acute hepatitis C, and then recovered. METHODS: Using sera, HVR1 cloning and antibody to HVR1 major clone measurement were performed. RESULTS: Clones from the first inoculum were divisible into major and minor types. Clones from the second and third inocula, as well as all post-inoculation sera, were essentially identical to the major type. Titers of antibody to HVR1 major clone were consistently low in pre- and post-inoculation sera. CONCLUSIONS: Both chimpanzees were re-infected twice with the same strain of HCV. The sequences from the second and third infections were similar to the major sequences in the first inoculum.

Comparison of 25-hydroxyvitamin D concentration in chimpanzee dried blood spots and serum.

BACKGROUND: Dried blood spots (DBS) are used in human medicine to measure total 25-hydroxyvitamin D (25-OHD) in the blood. However, this easy and affordable sampling technique has not been evaluated in primates to measure vitamin D concentrations. OBJECTIVES: We aimed to compare 25-OHD measurements in chimpanzee serum at two different laboratories and determine the precision and accuracy of the DBS method by comparing DBS and serum results. METHODS: Blood samples from 17 captive chimpanzees were collected, and 25-OHD3 and 25-OHD2 were measured in serum at two accredited laboratories using liquid chromatography-tandem mass spectrometry. The same analytes were measured on DBS cards, and results were compared with that of serum. Data were assessed using the Spearman correlation, Deming regression, and Bland-Altman analyses. RESULTS: The correlation coefficient between the two measurements in serum was rs  = .51 (P = .04), and the mean bias was -1.25 ± 14.83. When comparing 25-OHD concentrations measured in DBS and serum at the same laboratory, the rs was 0.7 (P = .002), and the mean bias was 1.42 ± 14.58. Estimated intra-assay and inter-assay coefficients of variation for DBS results were 6% and 12.6%, respectively. CONCLUSIONS: Although substantial analytical variability was found in 25-OHD measurements regardless of the sample type, the identification of both constant and proportional error and wider limits of agreement with the DBS technique makes the interpretation of DBS results challenging, especially for values close to clinical cut-off points. The DBS and serum methods were not interchangeable, and further studies are needed to validate DBS samples for vitamin D measurements in chimpanzees.

Seroprimatology of chimpanzees: blood-group distribution as a "racial" characteristic.

Significant differences in the distribution of human-type and simian-type blood groups have been demonstrated in chimpanzees classified into subspecies or "races" on the basis of morphological traits. The differences in chimpanzees are analogous to racial differences in the distribution of blood groups in man.

Evaluation of four hematology and a chemistry portable benchtop analyzers using non-human primate blood.

BACKGROUND: Near patient testing (NPT) and point-of-care testing (POCT) using portable benchtop analyzers has become necessary in many areas of the medical community, including biocontainment. METHODS: We evaluated the Beckman AcT diff, Abaxis Vetscan HMII (two instruments), Abbott Cell-Dyn 1800, and Abaxis Vetscan VS2 for within-run precision and correlation to central laboratory instruments using non-human primates blood. RESULTS: Compared with the central laboratory instruments, the Beckman AcT diff correlated on 80%; the HMII instruments on 31% and 44%, the CD1800 on 31%, and the VS2 on 71% of assays. For assays with published manufacturers precision guidelines, the AcT diff met all nine, the HMII instruments met one and six of six, and the CD 1800 met one of six. CONCLUSIONS: Laboratories using NPT/POCT must test their individual instruments for precision and correlation, identify assays that are reliable, and exclude or develop supplemental procedures for assays that are not.

In vivo hepatitis B virus-neutralizing activity of an anti-HBsAg humanized antibody in chimpanzees.

Previously, we constructed a humanized antibody (HuS10) that binds to the common a antigenic determinant on the S protein of HBV. In this study, we evaluated its HBV-neutralizing activity in chimpanzees. A study chimpanzee was intravenously administered with a single dose of HuS10, followed by intravenous challenge with the adr subtype of HBV, while a control chimpanzee was only challenged with the virus. The result showed that the control chimpanzee was infected by the virus, and thus serum HBV surface antigen (HBsAg) became positive from the 14(th) to 20(th) week and actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 19(th) and 23(rd) week, respectively. However, in the case of the study chimpanzee, serum HBsAg became positive from the 34(th) to 37(th) week, while actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 37(th) and 40(th) week, respectively, indicating that HuS10 neutralized the virus in vivo and thus delayed the HBV infection. This novel humanized antibody will be useful in the immunoprophylaxis of HBV infection.

Diet and reproductive function in wild female chimpanzees (Pan troglodytes schweinfurthii) at Kibale National Park, Uganda.

Human female reproductive function is highly sensitive to current energetic condition, indicating adaptation to modulate reproductive effort in accordance with changing ecological conditions that might favor or disfavor the production of offspring. Here, we test the hypothesis that reproductive capacity in female chimpanzees is likewise limited by current energetic condition. We used 12 years of data on wild chimpanzees (Pan troglodytes schweinfurthii) in the Kanyawara community of Kibale National Park, Uganda, to examine the relationship of dietary quality, as assessed by fruit components of the diet, to the occurrence of sexually receptive females, concentrations of ovarian steroid hormones, and timing of conception. We found that the frequency of females having sexual swellings was positively related to the consumption of drupe fruits. Estrogen levels of both cycling and noncycling females increased during seasonal peaks in the consumption of drupe fruits. When average fruit consumption remained high across months, females conceived more quickly. These results support the hypothesis that cycling and conception in chimpanzees are contingent upon high energy balance, and they indicate that the availability of fruit is a key variable limiting reproductive performance in chimpanzees. Chimpanzees appear to share with humans a reproductive system that is primed to respond to proximate levels of energy acquisition.

Comparative Serum Challenges Show Divergent Patterns of Gene Expression and Open Chromatin in Human and Chimpanzee.

Humans experience higher rates of age-associated diseases than our closest living evolutionary relatives, chimpanzees. Environmental factors can explain many of these increases in disease risk, but species-specific genetic changes can also play a role. Alleles that confer increased disease susceptibility later in life can persist in a population in the absence of selective pressure if those changes confer positive adaptation early in life. One age-associated disease that disproportionately affects humans compared with chimpanzees is epithelial cancer. Here, we explored genetic differences between humans and chimpanzees in a well-defined experimental assay that mimics gene expression changes that happen during cancer progression: A fibroblast serum challenge. We used this assay with fibroblasts isolated from humans and chimpanzees to explore species-specific differences in gene expression and chromatin state with RNA-Seq and DNase-Seq. Our data reveal that human fibroblasts increase expression of genes associated with wound healing and cancer pathways; in contrast, chimpanzee gene expression changes are not concentrated around particular functional categories. Chromatin accessibility dramatically increases in human fibroblasts, yet decreases in chimpanzee cells during the serum response. Many regions of opening and closing chromatin are in close proximity to genes encoding transcription factors or genes involved in wound healing processes, further supporting the link between changes in activity of regulatory elements and changes in gene expression. Together, these expression and open chromatin data show that humans and chimpanzees have dramatically different responses to the same physiological stressor, and how a core physiological process can evolve quickly over relatively short evolutionary time scales.

Relationship between sunlight exposure, housing condition, and serum vitamin D and related physiologic biomarker levels in captive chimpanzees (Pan troglodytes).

In primates, the primary source of vitamin D is synthesis in the skin through sun exposure. Decreased sun exposure may lead to vitamin D deficiency and consequently other health issues. In laboratory, sanctuary, and zoo settings, chimpanzees (Pan troglodytes) may be housed indoors for prolonged periods of time without regular exposure to unfiltered sunlight. However, little research has examined the relationship between housing conditions and vitamin D serum levels in captive chimpanzees. In this study, we retrospectively compared serum levels of total vitamin D, calcium, ionic calcium, phosphorous, albumin, and alkaline phosphatase in 18 female and 12 male chimpanzees as they cycled between indoor-only and indoor-outdoor enclosures. Total vitamin D was significantly lower and alkaline phosphatase significantly higher when subjects were in the indoor-only enclosures compared with when they had regular access to outdoor enclosures. A vitamin D effect occurred only in young and prime-adult animals. Changes were significant in female but not in male chimpanzees. Calcium, ionic calcium, phosphorus, and albumin did not differ between indoor-only and indoor-outdoor enclosures. However, female chimpanzees exhibited significantly lower calcium and phosphorous levels while in the indoor-only enclosures. These results suggest that adult captive chimpanzees experience vitamin D deficiency when housed without regular access to unfiltered sunlight and that these effects may be more acute for adult female animals.

Age- and Sex-associated Differences in Phenotypic and Functional Characteristics of Peripheral Blood Lymphocytes in Chimpanzees (Pan troglodytes).

Chimpanzees are the closest phylogenetic relatives to humans, sharing more than 98% genetic sequence identity. These genetic similarities prompted the belief that chimpanzees can serve as an ideal model for human disease conditions and vaccine development. However, in light of the recent NIH decision to phase out biomedical research in chimpanzees and retire NIH-supported chimpanzees, data from the present study will continue to provide value for the care of aged and sick chimpanzees located in zoos, sanctuaries, and primate centers. Surprisingly little information has been published regarding the normal chimpanzee immune system, and most extant studies have been based on small numbers of animals. In the current study, we provide a better understanding of the chimpanzee immune system with regard to age and sex. We examined immune parameters of chimpanzees (n = 94; 51 female, 43 male; age, 6 to 47 y) by using flow cytometry, immune function analysis, and cytokine analysis. Because lymphocytes are key mediators of cellular immune responses, particularly to intracellular pathogens such as viruses, we surveyed the phenotypic and functional attributes of T and B lymphocytes in this healthy and age-stratified population of chimpanzees. We noted a significantly higher percentage of CD16+T cells in male compared with female chimpanzees but no significant changes in percentages of CD3+, CD4+, CD8+, or CD4+CD8+ T cells with age or sex. In addition, aging was associated with decreased proliferative responses to mitogens in both sexes. Sex-specific differences also were present in the percentage of NK cells but not in their cytotoxic activity and in circulating cytokine levels in plasma. Going forward, the data presented here regarding immune cell changes associated with aging in healthy chimpanzees will serve to enhance the care of geriatric and ill animals.

Plasmodium reichenowi EBA-140 merozoite ligand binds to glycophorin D on chimpanzee red blood cells, shedding new light on origins of Plasmodium falciparum.

BACKGROUND: All symptoms of malaria are caused by the intraerythrocytic proliferation of Plasmodium merozoites. Merozoites invade erythrocytes using multiple binding ligands that recognise specific surface receptors. It has been suggested that adaptation of Plasmodium parasites to infect specific hosts is driven by changes in genes encoding Plasmodium erythrocyte-binding ligands (EBL) and reticulocyte-binding ligands (RBL). Homologs of both EBL and RBL, including the EBA-140 merozoite ligand, have been identified in P. falciparum and P. reichenowi, which infect humans and chimpanzees, respectively. The P. falciparum EBA-140 was shown to bind human glycophorin C, a minor erythrocyte sialoglycoprotein. Until now, the erythrocyte receptor for the P. reichenowi EBA-140 remained unknown. METHODS: The baculovirus expression vector system was used to obtain the recombinant EBA-140 Region II, and flow cytometry and immunoblotting methods were applied to characterise its specificity. RESULTS: We showed that the chimpanzee glycophorin D is the receptor for the P. reichenowi EBA-140 ligand on chimpanzee red blood cells. CONCLUSIONS: We propose that the development of glycophorin C specificity is spurred by the P. falciparum lineage. We speculate that the P. falciparum EBA-140 evolved to hijack GPC on human erythrocytes during divergence from its ape ancestor.

No Distinction of Orthology/Paralogy between Human and Chimpanzee Rh Blood Group Genes.

On human (Homo sapiens) chromosome 1, there is a tandem duplication encompassing Rh blood group genes (Hosa_RHD and Hosa_RHCE). This duplication occurred in the common ancestor of humans, chimpanzees (Pan troglodytes), and gorillas, after splitting from their common ancestor with orangutans. Although several studies have been conducted on ape Rh blood group genes, the clear genome structures of the gene clusters remain unknown. Here, we determined the genome structure of the gene cluster of chimpanzee Rh genes by sequencing five BAC (Bacterial Artificial Chromosome) clones derived from chimpanzees. We characterized three complete loci (Patr_RHα, Patr_RHß, and Patr_RHγ). In the Patr_RHß locus, a short version of the gene, which lacked the middle part containing exons 4-8, was observed. The Patr_RHα and Patr_RHß genes were located on the locations corresponding to Hosa_RHD and Hosa_RHCE, respectively, and Patr_RHγ was in the immediate vicinity of Patr_RHß. Sequence comparisons revealed high sequence similarity between Patr_RHß and Hosa_RHCE, while the chimpanzee Rh gene closest to Hosa_RHD was not Patr_RHα but rather Patr_RHγ. The results suggest that rearrangements and gene conversions frequently occurred between these genes and that the classic orthology/paralogy dichotomy no longer holds between human and chimpanzee Rh blood group genes.

Normal C-reactive protein values for captive chimpanzees (Pan troglodytes).

C-reactive protein (CRP) is used widely as an indicator of infections and recovery from infections in human medicine. It is an acute-phase serum protein, and its concentration increases in response to infection, trauma, and inflammation. In efficacy and safety studies involving chimpanzees (Pan troglodytes) as an animal model to evaluate therapeutic drugs targeting the human population, CRP may be used as a diagnostic tool in assessing animal health. Establishing normal values in healthy populations of chimpanzees is crucial to interpreting changes in CRP serum levels and how they relate to drug safety and animal health.