Rapid cloning and comparative sequence analysis of full-length cDNA of Rhesus monkey (Macaca mulatta) resistin.

Resistin protein is thought to link insulin resistance in murine models of obesity and type-2 diabetes, but the role of resistin in human studies of inflammatory metabolic disorders have generated conflicting data. Here, we describe the structure of the resistin gene using adipose tissue from non-human primates (NHPs), which have been used extensively to model a host of human diseases. Full-length cDNA from rhesus macaque resistin obtained by rapid amplification of cDNA ends (RACE) is comprised of 526 nucleotides covering an open-reading frame (ORF) that encodes a 108-amino-acid protein that is 92% homologous with the human counterpart but only 60% homologous with the murine counterpart. Using a modified polymerase chain reaction technique, we identified single nucleotide polymorphisms and a 78-bp deletion within resistin cDNA of nine rhesus macaques. Comparisons of the full-length cDNA sequence and an amplified 569-bp genomic DNA sequence revealed an error in published predictions arising from genomic studies about the gene's exon 3 region. Our data show, for the first time, the full-length macaque resistin cDNA sequence (GenBank: JF740676.1). These findings will illuminate future studies into the role of resistin in NHP models of inflammatory metabolic diseases.

Effects of aqueous extracts of raw pu-erh tea and ripened pu-erh tea on proliferation and differentiation of 3T3-L1 preadipocytes.

Pu-erh tea has shown anti-obesity effects but little is known about its effect on proliferation and differentiation of preadipocytes. This study investigated the effects of the aqueous extracts of raw pu-erh tea and ripened pu-erh tea on proliferation and differentiation of murine 3T3-L1 preadiopocytes. We examined dose and time effects of both aqueous extracts on proliferation of 3T3-L1 preadipocytes. The contents of triglycerides in cytoplasm and the mRNA expression of critical transcriptional factors involved in differentiation were determined. Cytotoxicity and apoptosis rate of preadipocytes by pu-erh tea extracts treatment were test for toxic and pro-apoptotic effects. Both aqueous extracts of pu-erh tea inhibited the proliferation of 3T3-L1 preadipocytes at the selected time points. At lower concentration of raw pu-erh tea extracts (less than 300 µg/ml) and ripened pu-erh tea extracts (less than 350 µg/ml), no significant cytotoxic and pro-apoptotic were observed. Ripened pu-erh tea was more effective with lower IC50 than raw pu-erh tea. Both extracts suppressed the differentiation and down-regulated the gene expression of peroxisome proliferator-activated receptor-γ and CCAAT/enhancer binding proteins-α. Therefore, these results indicate that both aqueous extracts of pu-erh tea can inhibit proliferation and differentiation with ripened pu-erh tea more potent. Polyphenol rich in both extracts may play a role in the inhibition of proliferation and differentiation of 3T3-L1 preadipocytes.

Nonhuman Primate Model of Oculocutaneous Albinism with TYR and OCA2 Mutations.

Human visual acuity is anatomically determined by the retinal fovea. The ontogenetic development of the fovea can be seriously hindered by oculocutaneous albinism (OCA), which is characterized by a disorder of melanin synthesis. Although people of all ethnic backgrounds can be affected, no efficient treatments for OCA have been developed thus far, due partly to the lack of effective animal models. Rhesus macaques are genetically homologous to humans and, most importantly, exhibit structures of the macula and fovea that are similar to those of humans; thus, rhesus macaques present special advantages in the modeling and study of human macular and foveal diseases. In this study, we identified rhesus macaque models with clinical characteristics consistent with those of OCA patients according to observations of ocular behavior, fundus examination, and optical coherence tomography. Genomic sequencing revealed a biallelic p.L312I mutation in TYR and a homozygous p.S788L mutation in OCA2, both of which were further confirmed to affect melanin biosynthesis via in vitro assays. These rhesus macaque models of OCA will be useful animal resources for studying foveal development and for preclinical trials of new therapies for OCA.

Dysbiosis of gut microbiome affecting small intestine morphology and immune balance: a rhesus macaque model.

There is a growing appreciation for the specific health benefits conferred by commensal microbiota on their hosts. Clinical microbiota analysis and animal studies in germ-free or antibiotic-treated mice have been crucial for improving our understanding of the role of the microbiome on the host mucosal surface; however, studies on the mechanisms involved in microbiome-host interactions remain limited to small animal models. Here, we demonstrated that rhesus monkeys under short-term broad-spectrum antibiotic treatment could be used as a model to study the gut mucosal host-microbiome niche and immune balance with steady health status. Results showed that the diversity and community structure of the gut commensal bacteria in rhesus monkeys were both disrupted after antibiotic treatment. Furthermore, the 16S rDNA amplicon sequencing results indicated that Escherichia-Shigella were predominant in stool samples 9 d of treatment, and the abundances of bacterial functional genes and predicted KEGG pathways were significantly changed. In addition to inducing aberrant morphology of small intestinal villi, the depletion of gut commensal bacteria led to increased proportions of CD3 + T, CD4 + T, and CD16 + NK cells in peripheral blood mononuclear cells (PBMCs), but decreased numbers of Treg and CD20 + B cells. The transcriptome of PBMCs from antibiotic-treated monkeys showed that the immune balance was affected by modulation of the expression of many functional genes, including IL-13, VCAM1, and LGR4.

Long-term toxicity, pharmacokinetics and immune effects of a recombinant adenovirus vaccine expressing human papillomavirus 16 E6 and E7 proteins (HPV16 E6E7-Ad5 Vac) in primates.

OBJECTIVE: This study aimed to: evaluate long-term toxicity and pharmacokinetic parameters; to identify the target organ of toxicity of a recombinant adenovirus vaccine expressing human papillomavirus 16 E6 and E7 proteins (HPV16 E6E7-Ad5 Vac) in primates; and to determine the specific immune response of this recombinant adenovirus vaccine. METHOD: HPV16 E6E7-Ad5 Vac (dose 4.68 × 109 IU/bottle) was administered to Macaca fascicularis (M. fascicularis) to evaluate its long-term toxicity. The Cynomolgus Monkeys were divided into a negative control group (sodium chloride injection group), a low-dose group (4.68 × 108 IU/macaque), and, a high-dose group (4.68 × 109 IU/macaque). The drugs were administered at intervals of once every three weeks (D1, D21, D42). The macaques were observed until the sixth week of the recovery period (D84) for safety and toxicological indicators and pharmacokinetic indicators. To study the specific immune response in Rhesus Macaque, empty viruses (rAd5-null) and buffer were inoculated as controls, respectively. Two doses of the vaccine were given at 1.0 × 108 IU/ml and 1.0 × 109 IU/ml and theHPV-16 E6-/HPV-16 E7-specific IFN-γ productions were measured. RESULTS: The macaques of both the high-dose group and the low-dose group did not exhibit any systemic toxic response. The administered safe dose of the vaccine was 4.68 × 109 IU per animal. Following vaccination, HPV16 E6/E7-specific antibodies were observed to be generated in both groups, indicating an immune response of the lymphocytes targeting HPV16 E6 and HPV16 E7 epitopes (specific NF-r) was elicited. The peak level of HPV-16 E6-/HPV-16 E7-specific IFN-γ production was observed in the ninth week.

Neonatal rhesus monkeys as an animal model for rotavirus infection.

AIM: To establish a rotavirus (RV)-induced diarrhea model using RV SA11 in neonatal rhesus monkeys for the study of the pathogenic and immune mechanisms of RV infection and evaluation of candidate vaccines. METHODS: Neonatal rhesus monkeys with an average age of 15-20 d and an average weight of 500 g ± 150 g received intragastric administration of varying doses of SA11 RV ( 107 PFUs/mL, 106 PFUs/mL, or 105 PFUs/mL, 10 mL/animal) to determine whether the SA11 strain can effectively infect these animals by observing their clinical symptoms, fecal shedding of virus antigen by ELISA, distribution of RV antigen in the organs by immunofluorescence, variations of viral RNA load in the organs by qRT-PCR, histopathological changes in the small intestine by HE staining, and apoptosis of small intestinal epithelial cells by TUNEL assay. RESULTS: The RV monkey model showed typical clinical diarrhea symptoms in the 108 PFUs SA11 group, where we observed diarrhea 1-4 d post infection (dpi) and viral antigen shed in the feces from 1-7 dpi. RV was found in jejunal epithelial cells. We observed a viral load of approximately 5.85 × 103 copies per 100 mg in the jejunum at 2 dpi, which was increased to 1.09 × 105 copies per 100 mg at 3 dpi. A relatively high viral load was also seen in mesenteric lymph nodes at 2 dpi and 3 dpi. The following histopathological changes were observed in the small intestine following intragastric administration of SA11 RV: vacuolization, edema, and atrophy. Apoptosis in the jejunal villus epithelium was also detectable at 3 dpi. CONCLUSION: Our results indicate that we have successfully established a RV SA11 strain diarrhea model in neonatal rhesus monkeys. Future studies will elucidate the mechanisms underlying the pathogenesis of RV infection, and we will use the model to evaluate the protective effect of candidate vaccines.

Nasal Infection of Enterovirus D68 Leading to Lower Respiratory Tract Pathogenesis in Ferrets (Mustela putorius furo).

Data from EV-D68-infected patients demonstrate that pathological changes in the lower respiratory tract are principally characterized by severe respiratory illness in children and acute flaccid myelitis. However, lack of a suitable animal model for EV-D68 infection has limited the study on the pathogenesis of this critical pathogen, and the development of a vaccine. Ferrets have been widely used to evaluate respiratory virus infections. In the current study, we used EV-D68-infected ferrets as a potential animal to identify impersonal indices, involving clinical features and histopathological changes in the upper and lower respiratory tract (URT and LRT). The research results demonstrate that the EV-D68 virus leads to minimal clinical symptoms in ferrets. According to the viral load detection in the feces, nasal, and respiratory tracts, the infection and shedding of EV-D68 in the ferret model was confirmed, and these results were supported by the EV-D68 VP1 immunofluorescence confocal imaging with α2,6-linked sialic acid (SA) in lung tissues. Furthermore, we detected the inflammatory cytokine/chemokine expression level, which implied high expression levels of interleukin (IL)-1a, IL-8, IL-5, IL-12, IL-13, and IL-17a in the lungs. These data indicate that systemic observation of responses following infection with EV-D68 in ferrets could be used as a model for EV-D68 infection and pathogenesis.

Dopaminergic neuron-like cells derived from bone marrow mesenchymal stem cells by Lmx1α and neurturin overexpression for autologous cytotherapy in hemiparkinsonian rhesus monkeys.

Bone marrow-derived mesenchymal stem cells hold great potential for cytotherapeutics of neurodegenerative disorders, including Parkinson's disease. The neurotrophic factor neurturin can rescue dopaminergic neurons damaged during the disease process. Lmx1α can promote mesencephalic dopaminergic differentiation during embryogenesis. In this study, we tested a cytotherapeutic strategy combining NTN/Lmx1α gene therapy and cell transplantation to ameliorate disease progression in hemiparkinsonian rhesus. Rhesus BMSCs were prepared for autologous grafting by transfection with recombinant adenoviral vectors expressing secreted NTN and Lmx1α,and cultured in the presence of induce factors, particularly the Lmx1α regulatory factor sonic hedgehog, to guide dopaminergic differentiation. These induced rh-BMSCs exhibited gene/protein expression phenotypes resembling nigral dopaminergic neurons. They survived and retained dopaminergic function following stereotaxic injection into the MPTP-lesioned right-side substantia nigra as indicated by SPECT measurement of DAT activity. Injected cells preserved and supplemented the remaining endogenous population of dopamine neurons (TH-positive cell ipsilateral/contralateral ratio was 56.81% ± 7.28% vs. 3.86%±1.22% in vehicle-injected controls; p<0.05). Cell injection also partially restored motor function and reduce apomorphine-evoked rotation (p<0.05). Moreover, function recovery occurred earlier than in previous studies on injected BMSCs. Our findings demonstrate a promising strategy for restoration of PD-associated motor dysfunction by transplantation of autologous BMSCs overexpressing NTN/Lmx1α.

Type 2 diabetes mellitus non-genetic Rhesus monkey model induced by high fat and high sucrose diet.

To build an ideal animal model for studying the mechanism of occurrence, developing and treating of diabetes become a more important issue, facing with the fact that the big threat of diabetes to human health has been worsen. First, we used the normal control diets or the high-fat/high-sucrose diets to feed the adult rhesus monkeys and the macaques induced by the high-fat/high-sucrose diets in the high-fat/high-sucrose group and the type 2 diabetes mellitus (T2DM) group developed the hyperglycemia, hyperinsulinemia at 6 months in accordance with the precious researches that reported that minipigs, rats and mice could develop hyperglycemia, hyperinsulinemia, hyperlipidemia and obesity after being induced with high-fat/high-carbohydrate diets. Second, the rhesus monkeys in T2DM group were injected STZ at a low dosage of 35 mg/kg BW to induce glucose persistent elevation which maintained pretty well after 12 months. Third, we took the assay of glucose tolerance test and insulin resistance index, assessed the changing tendency of serum resistin and analysed the pathological characteristics of the tissues like pancreas and liver by staining in different ways. The results indicate the rhesus monkeys in T2DM group have lots of clinical features of T2DM. The experimental non-genetic T2DM rhesus monkeys model not only contribute to simulating of clinical manifestations and pathological features of human T2DM, but also may be a good kind of model for research on the treatment of T2DM and for new drugs evaluation.

[Effect of simian vacuolating virus 40 on development and differentiation of dendritic cells from Rhesus macaque].

To study the effect of simian vacuolating virus 40 (SV40) on development and differentiation of dendritic cells (DC) from rhesus macaque, the peripheral blood-derived dendritic cells from rhesus monkey were pulsed with inactivated SV40 and infective SV40, respectively at the 5th day post DC cultivation. Expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface at the 7th, 9th day post DC cultivation were analyzed by flow cytometry (FCM). The results showed that expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface in the inactivated SV40-pulsed experimental group were higher than those in the infective SV40-pulsed experimental group (P < 0.05). These cell surface molecules represented characteristic development and differentiation phase of DC. Down-regulation of expressions of these cell surface molecules indicated that infective SV40 might hamper differentiation and maturation of dendritic cells from rhesus monkey.

[The changes of stem cells, immune cells and cytokines in peripheral blood after marrow stem cell mobilization in macaqne].

AIM: To explore the changes of white blood cells(WBC), neutrophils, stem cells, immune cells and cytokines after marrow stem cell mobilization by GM-CSF in macaqne. METHODS: Macaqne were injected with GM-CSF 8 mug/(kg.d) for 5 days. The ratio, number and subpopulation of WBC were observed by Blood Cell Autoanalyzer; the ratios of CD133(+), CD34(+), CD3(+), CD4(+), CD8(+), CD56(+) cells were identified by FCM; and levels of TNF-alpha, IL-1beta, IL-2 were tested by ELISA on 0, 2, 4, 6, 8, 10 day after stem cell mobiliation by GM-CSF. RESULTS: The ratios and number of CD133(+) cells, CD34(+) cells, WBC and neutrophils were significantly increased (P<0.01). The number of above cells was elevated to 6.4, 9.1, 117 and 163.3 times of normal at 6 day after stem cell were mobilized, but the ratios of CD133(+) cells and CD34(+) cell were decreased to the normal level after stem cell mobilization stopped while that of WBC and neutrocytes kept increasing over 8 d . The ratios of CD3(+), CD4(+), CD8(+), CD16(+) were decreased during 1 to 6 d, but came to the normal level at 8 d and increased at 10 d after stem cell were mobilized and the number of those cells in blood increased to 4.1, 4.0, 2.9 and 4.3 times when compared with the normal level. The concentration of TNF-alpha, IL-1beta, IL-2 in peripheral blood was significantly increased and IL-2 level was increased higher and longer than that of TNF-alpha and IL-1beta. CONCLUSION: CD133(+), CD34(+) cells and WBC, neutrophils in peripheral blood are greatly increased after GM-CSF mobilization, but WBC and neutrophils keep increasing longer than that of CD133(+), CD34(+) cells. The results of the number of CD3(+), CD4(+), CD8(+), CD16(+) cells and the levels of TNF-alpha, IL-1beta, IL-2 in peripheral blood indicate that marrow stem cell mobilization by GM-CSF can also stimulate immune cell proliferation and differentiation while increasing cytokine producing.