Genome analysis of four Old World monkey adenoviruses supports the proposed species classification of primate adenoviruses and reveals signs of possible homologous recombination.

Within the family Adenoviridae, presently Simian mastadenovirus A is the single species approved officially for monkey adenoviruses (AdVs), whilst the establishment of six further species (Simian mastadenovirus B to Simian mastadenovirus G) has been proposed in the last few years. We examined the genetic content and phylogenetic relationships of four Old World monkey (OWM) AdV types [namely simian AdV (SAdV)-8, -11, -16 and -19] for which it had been proposed that they should be classified into different AdV species: SAdV-11 to Human mastadenovirus G, and the other three viruses into three novel species. By full genome sequencing, we identified gene contents characteristic for the genus Mastadenovirus. Among the 36 ORFs, 2 genes of different lengths, predicted to encode the adenoviral cellular attachment protein (the fibre), were found. The E3 regions contained six genes, present in every OWM AdV, but lacked the E3 19K gene, which has seemingly appeared only in the ape (hominid) AdV lineages during evolution. For the first time in SAdVs, two other exons belonging to the gene of the so-called U exon protein were also predicted. Phylogenetic calculations, based on the fibre-1 and the major capsid protein, the hexon, implied that recombination events might have happened between different AdV species. Phylogeny inference, based on the viral DNA-dependent DNA polymerase and the penton base protein, further supported the species classification proposed earlier.

Osteosarcoma in Baboons (Papio spp).

Bone neoplasms in baboons (Papio spp) are rare, with only one confirmed case of osteosarcoma previously described in the literature. Over a 12-y period, 6 baboons at a national primate research center presented with naturally occurring osteosarcoma; 3 lesions affected the appendicular skeleton, and the remaining 3 were in the head (skull and mandible). The 6 cases presented were identified in members of a large outdoor-housed breeding colony. The subjects were not genetically related or exposed to the same research conditions. Diagnoses were made based on the presentation and radiographic findings, with histologic confirmation.

Metabolism and disposition of bupropion in pregnant baboons (Papio cynocephalus).

Recent in vitro data obtained in our laboratory revealed similarities between baboons and humans in the biotransformation of bupropion (BUP) by both hepatic and placental microsomes. These data supported the use of baboons to study BUP biotransformation during pregnancy. The aim of this investigation was to determine the pharmacokinetics of BUP in baboons during pregnancy and postpartum, as well as fetal exposure to the drug after intravenous administration. Pregnant baboons (n = 5) received a single intravenous bolus dose of bupropion hydrochloride (1 mg/kg) at gestational ages 94-108 days (midpregnancy), 142-156 days (late pregnancy), and 6 weeks postpartum. Blood and urine samples were collected for 12 and 24 hours, respectively. The concentrations of BUP, hydroxybupropion (OH-BUP), threohydrobupropion, and erythrohydrobupropion in plasma were determined by liquid chromatography-tandem mass spectrometry. Relative to the postpartum period, the average midpregnancy clearance of BUP trended higher (3.6 ± 0.15 versus 2.7 ± 0.28 l/h per kg) and the average C(max) (294 ± 91 versus 361 ± 64 ng/ml) and the area under the curve (AUC) of BUP values (288 ± 22 versus 382 ± 42 h·ng/ml) trended lower. AUC(OH-BUP) also tended to be lower midpregnancy compared with postpartum (194 ± 76 versus 353 ± 165 h·ng/ml). Whereas the observed trend toward increased clearance of BUP during baboon pregnancy could be associated with a pregnancy-induced increase in its biotransformation, the trend toward increased renal elimination of OH-BUP may overshadow any corresponding change in the hydroxylation activity of CYP2B.

Measuring fecal testosterone in females and fecal estrogens in males: comparison of RIA and LC/MS/MS methods for wild baboons (Papio cynocephalus).

The development of non-invasive methods, particularly fecal determination, has made possible the assessment of hormone concentrations in wild animal populations. However, measuring fecal metabolites needs careful validation for each species and for each sex. We investigated whether radioimmunoassays (RIAs) previously used to measure fecal testosterone (fT) in male baboons and fecal estrogens (fE) in female baboons were well suited to measure these hormones in the opposite sex. We compared fE and fT concentrations determined by RIA to those measured by liquid chromatography combined with triple quadropole mass spectrometry (LC/MS/MS), a highly specific method. Additionally, we conducted a biological validation to assure that the measurements of fecal concentrations reflected physiological levels of the hormone of interest. Several tests produced expected results that led us to conclude that our RIAs can reliably measure fT and fE in both sexes, and that within-sex comparisons of these measures are valid: (i) fTRIA were significantly correlated to fTLC/MS/MS for both sexes; (ii) fTRIA were higher in adult than in immature males; (iii) fTRIA were higher in pregnant than non-pregnant females; (iv) fERIA were correlated with 17ß-estradiol (fE2) and with estrone (fE1) determined by LC/MS/MS in pregnant females; (v) fERIA were significantly correlated with fE2 in non-pregnant females and nearly significantly correlated in males; (vi) fERIA were higher in adult males than in immature males. fERIA were higher in females than in males, as predicted, but unexpectedly, fTRIA were higher in females than in males, suggesting a difference in steroid metabolism in the two sexes; consequently, we conclude that while within-sex comparisons are valid, fTRIA should not be used for intersexual comparisons. Our results should open the field to important additional studies, as to date the roles of testosterone in females and estrogens in males have been little investigated.

Smad7 inhibits autocrine expression of TGF-ß2 in intestinal epithelial cells in baboon necrotizing enterocolitis.

Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-ß 2 (TGF-ß(2)) in the developing intestine. We hypothesized that low epithelial TGF-ß(2) expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-ß(2) in these cells. Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-ß(2), Smad7, Ski, and strawberry notch N (SnoN)/Ski-like oncoprotein (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. NEC was recorded in seven premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-ß(2) than term intestine. TGF-ß(2) expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-ß(2) promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-ß(2). Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. Smad7 inhibits autocrine expression of TGF-ß(2) in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.

Dynamic expression of IL-6 trans-signalling molecules in the lungs of preterm baboons undergoing mechanical ventilation.

BACKGROUND: Interleukin (IL)-6, when complexed with soluble IL-6 receptor (sIL-6R), has emerged as an important modulator of chemokine expression and leukocyte recruitment during inflammation and in this state can be specifically antagonised by soluble gp130 (sgp130). The expression of these modifiers of IL-6 activity during ventilator-induced inflammation remains poorly understood. OBJECTIVES: To ascertain the expression pattern of IL-6, sIL-6R and sgp130 in response to mechanical ventilation in the preterm neonatal lung and define its relationship to associated markers of inflammation. METHODS: Inflammatory cell recruitment and expression of IL-6, sIL-6R, sgp130, IL-8 and monocyte chemotactic protein-1 (MCP-1) were quantified in tracheal aspirate fluid collected over a 14-day period from preterm (125 days) baboons undergoing mechanical ventilation. RESULTS: Over the period of ventilation, the ratio of agonistic IL-6/sIL-6R increased 4.3-fold between days 3 and 10-11 (p < 0.01) while the ratio of antagonistic sgp130/IL-6 decreased 2.6-fold over the same period (p < 0.05). Over the same period, the relative numbers of neutrophils compared to mononuclear cells shifted from an excess of 1.8 on day 1 to 0.6 on day 14 (p < 0.01). Both IL-8 and MCP-1 were elevated between days 1 and 10-11 of ventilation (p < 0.01). CONCLUSIONS: In the ventilated preterm baboon lung, expression of sIL-6R and dynamic modulation of sgp130 expression appear to modulate the activity and inflammatory potential of IL-6.

Tracheal stenosis and adenocarcinoma in an olive baboon (Papio cynocephalus anubis).

An adult female baboon (Papio cynocephalus anubis) presented for progressive difficulty in endotracheal intubation. Over a 7-y period prior to presentation, she was anesthetized and intubated 67 times for imaging by using single-photon emission computed tomography or positron emission tomography. Laryngoscopic examination revealed tracheal stenosis. Because of increased anesthetic risk and lack of alternative use, she was euthanized, and partial necropsy focusing on the larynx, trachea, and associated structures was performed. Gross examination revealed rigidity and functional fusion of the proximal 5 or 6 tracheal rings and narrowing of the lumen. Histology revealed ossification of tracheal rings and fibrosis of overlying tissue. In addition, a transmural umbilicated mass was present midway down the cervical trachea on its dorsolateral aspect. Histology of the tracheal mass identified a relatively well-circumscribed transmural adenocarcinoma. The combination of overall histologic pattern, evidence of anaplasia, and results of immunohistochemical staining was consistent with a diagnosis of adenoid cystic carcinoma. Anterior tracheal stenosis is a reported complication of intubation in humans and animals. Primary tracheal neoplasms are rare in domestic and research animals and, to our knowledge, have not previously been reported to occur in nonhuman primates.

Evolution of a malaria resistance gene in wild primates.

The ecology, behaviour and genetics of our closest living relatives, the nonhuman primates, should help us to understand the evolution of our own lineage. Although a large amount of data has been amassed on primate ecology and behaviour, much less is known about the functional and evolutionary genetic aspects of primate biology, especially in wild primates. As a result, even in well-studied populations in which nongenetic factors that influence adaptively important characteristics have been identified, we have almost no understanding of the underlying genetic basis for such traits. Here, we report on the functional consequences of genetic variation at the malaria-related FY (DARC) gene in a well-studied population of yellow baboons (Papio cynocephalus) living in Amboseli National Park in Kenya. FY codes for a chemokine receptor normally expressed on the erythrocyte surface that is the known entry point for the malarial parasite Plasmodium vivax. We identified variation in the cis-regulatory region of the baboon FY gene that was associated with phenotypic variation in susceptibility to Hepatocystis, a malaria-like pathogen that is common in baboons. Genetic variation in this region also influenced gene expression in vivo in wild individuals, a result we confirmed using in vitro reporter gene assays. The patterns of genetic variation in and around this locus were also suggestive of non-neutral evolution, raising the possibility that the evolution of the FY cis-regulatory region in baboons has exhibited both mechanistic and selective parallels with the homologous region in humans. Together, our results represent the first reported association and functional characterization linking genetic variation and a complex trait in a natural population of nonhuman primates.

Cell death-associated ADAMTS4 and versican degradation in vascular tissue.

High blood flow through baboon polytetrafluorethylene aorto-iliac grafts increases neointimal vascular smooth muscle cell (SMC) death, neointimal atrophy, and cleavage of versican to generate the DPEAAE neoepitope, a marker of ADAMTS-mediated proteolysis. In this study, we have determined the effect of high blood flow on transcript abundance in the neointima for ADAMTS1, -4, -5, -8, -9, -15, and -20. We found that after 24 hr of flow, the mRNA for ADAMTS4 was significantly increased, whereas that for the other family members was unchanged. Because vascular SMC death is markedly increased in the graft after 24 hr of high flow, we next examined the possibility that the ADAMTS4 induction and the cell death are causally related. The addition of Fas ligand to SMC cultures increased both ADAMTS4 mRNA and cell death approximately 5-fold, consistent with the idea that ADAMTS4-dependent cleavage of versican may be partly responsible for cell death and tissue atrophy under these conditions.

Regeneration of a spinal ligament after total lumbar disk arthroplasty in primates.

Total disk arthroplasty (TDA) is a new procedure that replaces the intervertebral disk space with an artificial motion segment and necessitates the resection of the anterior longitudinal ligament (ALL). We assessed whether a collagen-based graft made from porcine small-intestine submucosa (SIS) can be used as a regenerative scaffold to restore the function and structure of the ALL in the lumbar spine. A total of 10 mature male baboons underwent TDA at L5-L6 using one of two treatments: (1) TDA only (n = 5) or (2) TDA combined with SIS (n = 5). Six months postoperatively, mock revision surgery was performed to assess tissue adhesions followed by non-destructive multidirectional flexibility testing of the spinal segment. The vertebral segments were then processed for histology. The tissue adhesion score was 2.8 +/- 0.8 in the TDA only group and 1.8 +/- 1.4 in the TDA-SIS group (p = 0.2). Segmental range of motion and the length of the neutral zone were similar in both groups. Histology showed that the SIS scaffold led to an organized ligamentous structure with a significantly (p = 0.027) higher thickness (2.18 +/- 0.25 mm) compared to the connective tissue structure in the TDA-only group (1.66 +/- 0.33 mm). We concluded that using a SIS bioscaffold after TDA did not lead to increased great vessel adhesion while its use facilitated the formation of highly organized ligamentous tissues. However, the SIS- induced and newly formed ligamentous tissue anterior to the spinal segment did not lead to a measurable limitation of spinal extension.

Ontogeny and phagocytic function of baboon lung dendritic cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells, but the ontogeny and functions of lung DCs are not known during prenatal period. Here, we isolated lung DC population from fetal (125-175 days of gestation age) and adult baboons. The cells were stained with fluorochrome-conjugated-HLA-DP, DQ, DR, CD1a, CD11c, CD14, CD40, CD80, CD86, CD209, CMKLR1, ILT7-specific antibodies, and staining was analyzed by flow cytometry. The phagocytic function was investigated by incubating the cells with fluorescent-labeled Escherichia coli bioparticles and analyzed by flow cytometry and fluorescence microscopy. The fetal baboon lung DCs expressed low levels of HLA-DP, DQ, DR, CD11c and CD86 as compared to adult baboon lung DCs and showed distinct DC morphology. The fetal lung DCs were also less capable of phagocytosing E. coli as compared to the adult lung DCs (P<0.05). In conclusion, the fetal lung DCs are not only phenotypically immature, but also less efficient in phagocytosing E. coli.

Evidence of simian virus 40 exposure in a colony of captive baboons.

Simian virus 40 (SV40) is a polyomavirus for which non-human primates are the permissive host. The baboon (Papio spp.) is an old world monkey that is used in a variety of research investigations; however, natural infection of SV40 among baboons has not been thoroughly examined or reported. Initially, we were interested in determining the prevalence of SV40 infection among a captive colony of baboons based on the presence of antibodies to SV40 large T-antigen (Tag). An overall seroprevalence rate of >50% was found after screening sera from 142 baboons in the colony based on ELISA. Endpoint titer values for serum antibody binding to SV40 Tag reached as high as 1280 for 5 out of 142 baboons. Peptide binding assays revealed that a range of SV40 Tag epitopes are immunogenic in the baboon, and that individual animals differ in their humoral immune responses to SV40 Tag based on epitope recognition. Specificity to SV40 Tag and not some other primate polyomavirus encoded large Tag was further examined by serologic reactivity to peptide epitopes unique to SV40 Tag. Additional serology was performed to assess SV40 Tag reactivity by Western blot and whether antibodies were capable of neutralizing SV40 infectivity in vitro. Although antibodies with high levels of SV40 neutralization were observed in a number of the baboons, there was a lack of correlation between viral neutralization and antibodies to SV40 Tag. Further examination using molecular-based diagnosis and SV40 Tag specific real-time quantitative PCR determined that some of the baboons appeared to be exposed to SV40. DNA sequence analysis of the PCR products confirmed that SV40 Tag specific sequences were detected in baboons.

Sepsis-induced coagulation in the baboon lung is associated with decreased tissue factor pathway inhibitor.

Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis.

An updated review of inhalation studies with cigarette smoke in laboratory animals.

Until recently, the published literature on inhalation studies with laboratory animals and cigarette smoke consisted entirely of negative findings, as far as neoplastic disease is concerned. This paper brings readers up to date, with analyses of recent studies that do indeed appear to report success after so many years of failure. The paper consists of a brief analysis of the literature up until a couple of years ago, giving brief, representative examples of inhalation studies with the five main species of laboratory animals that have been used: rat, mouse, hamster, dog, and nonhuman primate. A brief examination of the various technologies used to expose laboratory animals is given, along with an analysis of the histopathology and related toxicology data (specifically, biomarkers of exposure) that have been reported. The paper concludes by briefly mentioning the most recent studies, where positive results have been reported.

Prostaglandin E2 receptor expression in fetal baboon lung at 0.7 gestation after betamethasone exposure.

The fetal lung produces and metabolizes prostaglandin (PG) E2. In vitro PGE2 induces surfactant production via E prostaglandin (EP)1 and cyclic adenosine monophosphate (cAMP)-coupled EP (EP2 and EP4) receptors. Glucocorticoids alter PG function and increase lung function in preterm neonates. We hypothesized that fetal exposure to maternally administered betamethasone (betaM) enhances fetal lung EP1 and cAMP-coupled EP receptor expression. Pregnant baboons were injected intramuscularly (i.m.) with either betaM (n=7) or saline [control (CTR); n=8] at 0.7 gestation. Fetal lungs were removed at cesarean section 48 h after the first injection. We determined mRNA levels, protein localization and abundance for all four PGE2 receptors by real-time polymerase chain reaction (PCR), immunohistochemistry, and Western blot. EP receptors were widely distributed in bronchiolar epithelium, bronchiolar smooth muscle, and endothelium and media of blood vessels, but not alveoli. Compared with CTR, betaM exposure resulted in a twofold EP2 mRNA decrease (p<0.05) in male fetuses only. EP1, EP3, and EP4 receptor mRNA levels were unaffected. Western blot analysis showed no alteration in EP receptor protein expression. In summary, this is the first demonstration of the four EP receptors in fetal lung. The only change after 48-h betaM exposure was a gender-specific decrease in EP2 receptor mRNA.

Coming of age: steroid hormones of wild immature baboons (Papio cynocephalus).

Large gaps exist in our knowledge about common patterns and variability in the endocrinology of immature nonhuman primates, and even normal hormonal profiles during that life stage are lacking for wild populations. In the present study we present steroid profiles for a wild population of baboons (Papio cynocephalus) from infancy through reproductive maturation, obtained by noninvasive fecal analyses. Fecal concentrations of glucocorticoid (fGC) and testosterone (fT) metabolites for males, and of fGC, estrogen (fE), and progestin (fP) metabolites for females were measured by radioimmunoassay (RIA). In males, infancy was characterized by high and declining levels of fGC and fT, whereas steroid concentrations were low during the juvenile years. During the months immediately prior to testicular enlargement, fT (but not fGC) concentration tended to increase. Males that matured early consistently had higher fT and fGC concentrations than those that matured late, but not significantly so at any age. Individual differences in fT concentrations were stable across ages, and average individual fT and fGC concentrations were positively correlated. For females, high and declining levels of fE characterized infancy, and values increased again after 3.5 years of age, as some females reached menarche by that age. Both fP and fGC were relatively low and constant throughout infancy and the juvenile period. During the months immediately prior to menarche, fGC concentration significantly decreased, while no changes were observed for fE levels. fP exhibited a complicated pattern of decrease that was subsequently followed by a more modest and nonsignificant increase as menarche approached. Early- (EM) and late-maturing (LM) females differed only in fP concentration; the higher fP concentrations in EM females reached significance at 4-4.5 years of age. Maternal rank at offspring conception did not predict concentrations of any hormone for either sex. Our results demonstrate the presence of individual endocrine variability, which could have important consequences for the timing of sexual maturation and subsequently for individual reproductive success. Further evaluation of the factors that affect hormone concentrations during the juvenile and adolescent periods should lead to a better understanding of mechanisms of life-history variability.

De novo generation of CD4 T cells against viruses present in the host during immune reconstitution.

T cells recognizing self-peptides are typically deleted in the thymus by negative selection. It is not known whether T cells against persistent viruses (eg, herpesviruses) are generated by the thymus (de novo) after the onset of the infection. Peptides from such viruses might be considered by the thymus as self-peptides, and T cells specific for these peptides might be deleted (negatively selected). Here we demonstrate in baboons infected with baboon cytomegalovirus and baboon lymphocryptovirus (Epstein-Barr virus-like virus) that after autologous transplantation of yellow fluorescent protein (YFP)-marked hematopoietic cells, YFP+ CD4 T cells against these viruses were generated de novo. Thus the thymus generates CD4 T cells against not only pathogens absent from the host but also pathogens present in the host. This finding provides a strong rationale to improve thymopoiesis in recipients of hematopoietic cell transplants and, perhaps, in other persons lacking de novo-generated CD4 T cells, such as AIDS patients and elderly persons.

Comparative transmission of multiple herpesviruses and simian virus 40 in a baboon breeding colony.

Little is known about the natural history of herpesviruses indigenous in baboons. Here, we describe the development of ELISAs for five herpesviruses. These assays were used to test more than 950 serum samples collected from approximately 210 infant/juvenile and 130 adult baboons in a captive breeding colony over a period of seven years. Results indicated that baboon cytomegalovirus, lymphocryptovirus, and rhadinovirus are transmitted efficiently within the colony and are acquired at an early age. Baboon alpha-herpesvirus HVP2 and polyomavirus simian virus 40 (SV40) were acquired later and by fewer juveniles than were the other three herpesviruses. More than 60% of baboons acquired HVP2 before reaching sexual maturity, indicating that oral infection of infants and juveniles, rather than sexual transmission between adults, is the predominant mode of transmission for this virus. Antibody to simian varicella virus (SVV) was found in about 40% of baboons. SVV was acquired principally by infants and juveniles; few adults seroconverted despite seronegative adults being in constant contact with infants and juveniles undergoing primary infection. Time of seroconversion was not statistically correlated to specific individual herpesviruses, suggesting that each virus is acquired as an independent infection event rather than multiple viruses being acquired at the same time. Several baboons that were delivered by cesarean section and were housed separate from, but in close proximity to, other baboons remained free of many or all viruses for several years, suggesting that, similar to human herpesviruses, baboon herpesviruses and SV40 are transmitted principally by direct contact.