Characterization, isolation, and culture of spermatogonial stem cells in / 亚洲男科学杂志(英文版)

Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kit

Do males bond? A study of male-male relationships in Nicobar long-tailed macaques Macaca fascicularis umbrosus

Due to their unique properties, carbon nanotubes (CNTs) are being widely explored for industrial and medicalapplications. This has necessitated a thorough assessment of the effect of CNTs on human and animalphysiology and health. Impact of CNTs on epithelial tight junctions has not been evaluated in the context oftheir toxic effects in many biological systems. In the present study, we examined the effect of acid functionalized single-walled carbon nanotubes (AF-SWCNTs) on the function and expression of two tight junctionproteins (ZO-1 and occludin) in the Madin-Darby canine kidney (MDCK) cell line. Treatment of MDCK cellswith AF-SWCNT resulted in a downregulation of tight junction proteins, decreased trans-epithelial electricalresistance (TER), increased paracellular permeability, and disruption of tight junctions. Taken together, ourdata demonstrate that AF-SWCNT disrupts tight junction barrier by downregulating tight junction proteins inMDCK epithelial cells.

Comparative safety assessments of the biosimilar APZ001 and Erbitux in pre-clinical animal models

Abstract Purpose: To evaluate the toxicity of Erbitux as well as its biosimilar APZ001 antibody (APZ001) in pre-clinical animal models including mice, rabbits and cynomolgus monkeys. Methods: We performed analysis of normal behavior activity, autonomic and non-autonomic nervous functions, nervous-muscle functions, nervous excitability and sensorimotor functions on CD-1 mice. Subsequently, we studied that effects of APZ001 and Erbitux on respiratory system, cardiovascular system and kidney in Cynomolgus monkey models and performed local tolerance experiments on New Zealand rabbits. Results: The comparisons between APZ001 and Erbitux showed no significant differences in mice autonomic nervous system, nervous muscle functions, non-autonomic nervous functions, nervous excitability and sensorimotor functions between treated and untreated group (p>0.05). APZ001 and Erbitux showed negative effect on CD-1 mice in the present of pentobarbital sodium anesthesia (p>0.05). Single administrations of high, medium or low doses of APZ001 did not lead to monkey urine volume alterations (p>0.05). In human tissues, APZ001 and Erbitux showed positive signals in endocardium, lung type II alveolar epithelial cell and surrounding vessels, but showed negative results in kidney and liver tissues. No hemolysis phenomenon and serious side-effects in vessels and muscles were observed in rabbits when administrated with APZ001 and Erbitux respectively. Conclusion: The safety comparisons between APZ001 antibody and Erbitux showed that these two antibodies showed highly similarities in mice, rabbits and cynomolgus monkey animal models in consideration of pharmaceutical effects, indicating APZ001 might be a suitable substitute for Erbitux.

Investigation of natural depression model under the attack-yield behavior of caged cynomolgus monkeys / 上海交通大学学报（医学版）

Objective • To investigate the natural depression model of Macaca fascicularis induced by submissive-aggressive behaviors. Methods • In a simulated wild environment, two Macaca fascicularis groups (G1 and G2) were respectively established. The two groups consisted of 19 and 14 Macaca fascicularis respectively. The effective frequency of each group's submissive-aggressive behaviors was observed and recorded, the matrix of submissive-aggressive behaviors was analyzed, each individual of the group was calculated separately by David's score(DS), according to the level of individual DS, each group was divided into attack group and yield group; the behavior changes of each individual in 7 periods were recorded by the focus observation and the correlation analysis of 11 different behaviors was carried out. Results • After the formation of these groups, there was fierce conflict between the cynomolgus monkeys. 1 122 and 1 409 submissive-aggressive behaviors were recorded in the two groups respectively. In the submissive-aggressive behavior matrix, the differences between high and low DS in anxiety behavior (t1=-4.053, P1=0.005; t2=-3.041, P2=0.012), conflict behavior (t1=8.478, P1=0.018; t2=7.651, P2=0.002), depression behavior (t1=-3.691, P1=0.006; t2=-2.431, P2=0.045) and exercise behavior (t1=9.639, P1=0.007; t2=3.568; P2=0.002) were statistically significant. Conclusion • The natural depression model of caged Macaca fascicularis is a social defeat model induced by yield-attack behavior.

Assessment of effects of 7,8-dihydroxyflavone on the striatum in normal cynomolgus monkeys with 99TCm-TRODAT-1 SPECT/CT imaging / 中华核医学与分子影像杂志

Objective To assess the effects of 7,8-dihydroxyflavone (7,8-DHF) on the striatum (ST) in normal cynomolgus monkeys using 99Tcm-TRODAT-1 imaging.Methods A total of six healthy female cynomolgus monkeys were included in this study.Three of them were fed with normal food (control group),and the other three were given oral administration of 7,8-DHF in addition to normal food (experimental group).The SPECT/CT imaging was performed at different time after 99Tcm-TRODAT-1 injection.The ROI of ST was drawn on images of 3 consecutive transverse slices that could be visualized best.The cerebellum (CB) was taken as the background reference area.The radioactivity uptake ratios of ST/CB at 1,3,4 and 5 h were calculated respectively.Paired-t test was used to analyze the data.Results ST radioactive uptake ratios showed continuing increase on the delay images.ST/CB uptake ratios of the control group at 1,3,4 and 5 h were 1.43±0.04,1.82±0.06,2.04±0.12,2.42±0.23,respectively,and those of the experimental group were 1.35±0.08,2.40±0.09,2.74±0.13 and 3.25±0.15 respectively.There was no significant difference between the two groups at 1 h (t =2.57,P＞0.05),while ST/CB uptake ratios of the experimental group at 3,4 and 5 h were significantly higher (t values:2.77,2.87 and 2.92,all P＜0.05).Conclusion 99Tcm-TRODAT-1 SPECT/CT imaging can be used to assess the DAT activation effect by 7,8-DHF on ST of cynomolgus monkeys.

Safty evaluation, migration and distribution of human bone marrow derived-mesenchymal stem cells in the cns of young macaca fascicularis / 中国比较医学杂志

Objective To evalutate the safty of hBMSCs transpalntation and to observe their migration and distribution in the brain of young macaca fascicularis. To establish a new technology platform and theoretical basis for the treatment of central nervous system diseases in children. Methods Labelled hBMSCs were transplanted into the striatum of young macaca fascicularis. Brain sections were examined to evalutate the inflammatory reaction and immunological rejection of local injection sites by HE observation and immunohistochemical staining. Migration and distribution of transplanted?hBMSCs was observed by real?time fluorescence quantitative PCR of male DNA and fluorescence microscope. Results The results showed that the direct intracerebral injection of hBMSCs did not cause systemic symptoms in animals. There is no inflammatory reaction and immunological rejection was detected, and degeneration and necrosis of neural cells and proliferation of glial cells were absent in the local injection sites. The transplanted hBMSCs survived, and migrated into the brain after 4 weeks transplantation. Its migration and distribution have certain regularity and were overlapping between transplant recipients. In addtion, hBMSCs tended to extend rostrally into the forebrain and showed preference of migrating toward the blood vessels and below the ependyma. Conculsions Intracerebral transplantation of hBMSCs is safe. And hBMSCs can survive and migrate into the brain.

Small hairpin RNA targeting inhibition of NF-κB gene in endometriosis therapy of Macaca fascicularis / 中华妇产科杂志

Objective To observe the therapeutic effect of NF-κB gene short hairpin RNA (shRNA) on endometriosis and identify the function of NF-κB on the maintenance and development of endometriosis in Macaca fascicularis.Methods The Macaca fascicularis model of endometriosis was developed,which divided into experimental group,negative control group and simple model group.The high specificity adenovirus vector mediated shRNA targeting NF-κB gene and negative control shRNA adenovirus with no-load NF-κB gene were synthesised.The experimental group injected the adenovirus which carried the NF-κB shRNA into the endometriosis lesions under laparoscopy surgery,the negative control group with no-load shRNA adenovirus and the simple models group injected with normal saline.Four weeks later after the injection,an observed operation was performed through laparoscopy and some lesions were collected.The CD34 immunohistochemistry of these lesions were done to detect the microvessel density,then the variation of the microvessel density among each group were observed.The expression of the NF-κB and proliferating cell nuclear antigen (PCNA) were detected through western blot.Results First,the Macaca fascicularis model of endometriosis was successful developed,and the experimental group has an evident atrophy in ectopic lesions compared with the previous.The lesions' microvessel density in experimental group decreased evidently compared with the negative control group and simple model group (0.002 0±0.000 3 versus 0.021 9±0.002 6 versus 0.024 5±0.003 3),and the differences was statistically significant (P＜0.01).The expression of PCNA (0.37±0.17 versus 0.57±0.26 versus 0.57±0.28) and NF-κB (0.338 ± 0.174 versus 0.678 ± 0.021 versus 0.645 ±0.098) in experiment group was lower than the negative control group and simple model group,the differences were statistically significant (all P＜0.01).Conclusion Through targeting suppressed the NF-κB gene expression by NF-κB shRNA,we can inhibit the development of endometriosis through reducing the ability of angiogenesis and cell proliferation of ectopic endometrial cells.

Long-term toxicity of fully humanized anti-human tumor necrosis factor-αmonoclonal antibody for injection in cynomolgus monkeys / 中国药理学与毒理学杂志

OBJECTIVE To evaluate the long-term toxicity of fully human anti-human tumor necrosis factor-α monoclonal antibody(anti-hTNF-α FHMA)for injection in cynomolgus monkeys. METHODS Forty cynomolgus monkeys were randomly divided into 5 groups (4 males and 4 females in each group):negative control group,adalimumab 10 mg·kg-1 group,anti-hTNF-αFHMA 2,10 and 50 mg·kg-1 groups. Cynomolgus monkeys in each group were injected sc once a week for 5 consecutive times, followed by 4 weeks of recovery. During the test,general clinical observation,body mass,body temperature,electrocardiogram(ECG),hematology,coagulation function,blood biochemistry,urine, ophthalmology,immune index,and pathological changes in organs and tissues were observed. At the same time,plasma drug concentrations were detected and the toxicokinetics parameters were analyzed. RESULTS No significant toxicological changes related to drugs were observed in general clinical observation,body mass,body temperature,ECG,ophthalmic examination,blood cell counts,coagu?lation function,blood biochemistry,urine analysis,lymphocyte subsets,cytokines,serum immuno?globulin,serum complement. Neutralizing anti-drug antibody(ADA)could be detected in adalimumab group and anti-hTNF-αFHMA groups. Anti-hTNF-αFHMA showed linear dynamic characteristics in cyno?molgus monkeys. At the same dose(10 mg·kg-1),anti-hTNF-αFHMA had similar immunogenicity and kinetics characteristics to adalimumab. CONCLUSION The level of anti-hTNF-α FHMA at which no adverse effect was observed was 50 mg · kg-1,which is equivalent to 75 times clinical dosage of quasi (0.67 mg·kg-1),which suggests that anti-hTNF-αFHMA be safe in clinical use.

Applied Anatomy of the Femoral Veins in Macaca fascicularis / Anatomía Aplicada de las Venas Femorales en Macaca fascicularis

The aim was to understand the anatomical features of the venous valve in Macaca fascicularis and to compare it with that of humans. The bilateral lower limbs (24 limbs from 12 animals) of Macaca fascicularis cadavers were dissected, and the femoral veins (FVs) were equally divided into distal, intermediate, and proximal sections. The external diameter of the FV in each section was measured. The venous valves were observed microscopically and stained with hematoxylin and eosin as well as trichrome. Data describing the human venous valve were collected from the current literature. No great saphenous veins were found among the 24 lower limbs from the Macaca fascicularis cadavers. The external diameters of the FVs in the distal, intermediate, and proximal sections were 3.53 ± 0.37 mm, 3.42 ± 0.55 mm, and 3.37 ± 0.54 mm, respectively. In most cases, there was one venous bivalve located in the FV approximately 0-2.71 mm below the junction of the FV and the deep femoral vein. Endothelium covered the luminal and sinusal surfaces of the leaflets. Abundant collagen fibers were found under the endothelial cells beneath the luminal surface of the leaflets. An elastin fiber network was located under the sinus endothelial surface. Smooth muscle cells in the FV extend to the edge of the valve. The venous valve of Macaca fascicularis is similar to that of humans, both morphologically and histologically. However, there is only one venous bivalve and no great saphenous vein in Macaca fascicularis.

El objetivo fue comprender las características anatómicas de la válvula venosa en Macaca fascicularis y compararla con la de los humanos. Fueron disecados bilateralmente los miembros pélvicos (24 miembros de 12 animales) de cadáveres de Macaca fascicularis; las venas femorales (VF) fueron divididas en secciones distal, media y proximal. Se midió el diámetro externo de las VFs en cada sección. Las válvulas venosas se observaron microscópicamente y se tiñeron con H-E y tricrómico. Los datos para describir la válvula venosa humana se obtuvieron desde la literatura. No se encontraron venas safenas magnas entre los 24 miembros inferiores. Los diámetros externos de las VFs en las secciones distal, media y proximal fueron 3,53±0,37 mm, 3,42 mm±0,55, y 3,37±0,54 mm, respectivamente. En la mayoría de los casos, hubo vena bivalva situada aproximadamente 0-2,71 mm debajo de la unión de la VF y la vena femoral profunda. El endotelio cubrió las superficies luminal y sinusal. Se observaron abundantes fibras de colágeno en las células endoteliales bajo la superficie luminal de las válvulas. Una red de fibras de elastina se encontró bajo la superficie del seno endotelial. Las células musculares lisas en las VFs se extiendían hasta el margen de la válvula. La válvula venosa del Macaca fascicularis es similar a la de los seres humanos, morfológica e histológicamente. Sin embargo, sólo hubo una vena bivalvular, y no se observaron venas safenas en Macaca fascicularis.

MR experiments study on the brain of cynomolgus of diabetic models / 中国医学影像技术

Objective To investigate the value of MRI and~1H-MRS in diagnosis of early stage of diabetic encephalopathy by detecting regional metabolite in cynomolgus diabetes models. Methods Five pathogen-free male adolescent cynomolgus were made type 1 diabetes mellitus models (T1DM) by intravenous injection of streptozotocin (STZ) (100 mg/kg), and the reliability and stability of the modes were assessed with long term follow-up of blood glucose and intravenous glucose tolerance tests. MRI and ~1H-MRS were performed to evaluate the volume, signal intensity and metabolic ratios of NAA/Cr, mI/Cr and Cho/Cr at hippocampus, lateral temporal lobe and occipital lobe 3 years after model establishment. Cortisol in serum was detected with immunoradiometric assay. In addition, 5 normal adult cynomolgus monkeys were selected in the control group and accepted the same examination above. Results ①Intravenous administration of STZ could made stable T1DM monkey model. ②Only mI/Cr ratio increased at hippocampus of diabetic monkeys compared to the control group (P<0.05). ③There was no statistical difference of cortisol in serum between the diabetic group and the control group (P＞0.05). Conclusion ~1H-MRS may detect the metabolic changes of the hippocampus in STZ-induced diabetic adolescent cynomolgus monkeys and may contributes to the early diagnosis of diabetic encephalopathy.

Changes of Circulating Immune Complex in Monkeys Infected by Simian Immunodeficiency Virus / 广州中医药大学学报

[Objective] To observe the dynamic changes of circulating immune complex (CIC) in monkeys infected by simian immunodeficiency virus (SIV). [Methods] Agglutination test of complement-sensitized yeast cell was used to determine the serum CIC level in 30 cases of monkeys, which were infected with SIVmac251 and sampled in different time-points after infection. Sixty-eight cases of normal monkeys were also examined as controls. [Results] After SIV infection, CIC can't be detected in all 30 monkeys until the 4th week, the total positive rate being 30% . In the 8th week, CIC were detected in 46.7% of these monkeys and then declined gradually in the following 12 weeks. Since the 20th week, the CIC in these monkeys maintained lower liter and lower positive rate which was close to that of the normal monkeys (about 10%). [Conclusion] CIC appeared and increased during the primary SIV infection and declined accompanying with the virus clearance from the circulalion. The formation of CIC may not benefit to the control of virus replication and the induction of anti-virus immunity; CIC has a role in the pathogenesis after SIV infection.

Research on activity of IL-2,IL-6,IL-10 experimental allergic encephalomyelitis in monkey / 中华神经科杂志

Objective To investigate the change of the concentration of IL-2?IL-6?IL-10 in peripheral blood and cerebral spinal fluid (CSF) in monkey's experimental allergic encephalomyelitis (EAE),as to explaining it's function in mechanism and prognosis of the disease. Methods First set up animal models of monkey's EAE. Then separate the animals by their onset symptoms according to the level and the different stages of disease. At the same time survey the blood and CSF concentrations of IL-2?IL-6?IL-10, finally process the findings statistically. Results IL-2 in the first or the third week in the acute stage of EAE (5.0?0.8 and 5.3?1.2,respectively) was obviously higher than that (0.7?0.3) before the onset,P=0.045,0.041 respectively,both IL-6 and IL-10 in recuperation or chronic stage were higher significantly than before the onset,P=0.004 3,0.006 5 respectively. The findings were significant statistically. Conclusion IL-2 up-regulates immunologically,which accelerates the disease. IL-6 and IL-10 down-regulate immunologically,which reduce the disease. The findings may play an important role in studying immunological mechanism of EAE or MS in the future.

Macaca irus bone mesenchymal stem cells differentiate into neuron-like cells induced by cryptotanshinone in vitro / 中国病理生理杂志

AIM: To determine the optimal protocol and condition in which macaca irus mesenchymal stem cells (MSCs) are induced to differentiate into neuron-like cells by cryptotanshinone in vitro. METHODS: MSCs from macaca irus bone marrow were generated in vitro and induced with cryptotanshinone. The morphological changes of MSCs were evaluated by microscope. The positive percentages of neurofilament (NF), neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression were measured by immunocytochemistry with ABC staining. RESULTS: The result showed that MSCs were positive for CD29, CD44, CD105, CD166, and negative for CD34, CD71, CD80 and CD86. After induced with cryptotanshinone, MSCs began to display neuronal morphologies, such as contracted multipolar cell body and formed extensive networks. The percentages of positive NSE, NF expression were 68.3%?3.5%, 70.3%?1.5%, respectively. CONCLUSION: Macaca irus MSCs could be induced to differentiate into neuron-like cells in vitro by cryptotanshinone and might be applied in cell transplantation and gene therapy in nervous system disorders.