Preliminary report of preclinical trial of multi-genome engineering pig-to-macaque heart, liver and kidney transplantation / 器官移植

Objective To investigate the application prospect of the most extensive genome engineering pig internationally in preclinical xenotransplantation. Methods Porcine endogenous retrovirus (PERV) knockout combined with 3 major heterologous antigen gene knockouts and 9 humanized genes for inhibition of complement activation, regulation of coagulation disorders, anti-inflammatory and anti-phagocytosis were transferred into a pig (PERV-KO/3-KO/9-TG) as a donor, and the heart, liver and kidney were obtained and transplanted to 3 Rhesus macaque recipients respectively to establish a preclinical research model of pig-to-Rhesus macaque xenotransplantation. The functional status of xenografts after blood flow reconstruction was observed and the survival of recipients was summarized. The hemodynamics of xenografts were monitored. The change of hematological indexes of each recipient was compared. The histopathological manifestation of xenografts was observed. Results After the blood flow was reconstructed, all xenografts showed ruddy color, soft texture and good perfusion. The transplant heart, liver and kidney showed full arterial and venous blood flow and good perfusion at 1 d after operation. The postoperative survival time of heart, liver, and kidney transplant recipients was 7, 26, and 1 d, respectively. The levels of creatine kinase, creatine kinase isoenzyme, and lactate dehydrogenase increased in heart transplant recipient at 1 d after operation, and gradually recovered to near normal levels at 6 d after operation. All indexes increased sharply at 7 d after operation. The level of aspartate aminotransferase increased in liver transplant recipients at 2 d after operation, and the alanine aminotransferase basically returned to normal at 10 d after operation, but the total bilirubin continued to increase. Both aspartate aminotransferase and alanine aminotransferase increased at 12 d after operation, and reached a peak at 15 d after operation. The kidney transplant recipient developed mild proteinuria at 1 d after operation, and died of sudden severe arrhythmia. Histopathology showed that the tissue structure of cardiac and renal xenografts was close to normal, and liver xenografts presented with patchy necrosis, the liver tissue structure was disordered, accompanied by inflammatory damage, interstitial hemorrhage and thrombotic microangiopathy. Conclusions PERV-KO/3-KO/9-TG pig shows advantages in overcoming hyperacute rejection, mitigating humoral rejection and coagulation dysregulation. However, whether it can be used as potential donor for clinical xenotransplantation needs further evaluation.

Induction of Rhesus Keratinocytes into Functional Ameloblasts by Mouse Embryonic Dental Mesenchyme

Fast progresses in stem cell-based tooth tissue engineering have been achieved in recent years in several animal models including the mouse, rat, dog, and pig. Moreover, various postnatal mesenchymal stem cells of dental origin have been isolated and shown capable of differentiating into odontoblasts and generating dentin. Meanwhile, human keratinocyte stem/progenitor cells, gingival epithelial cells, and even iPSC-derived epithelium have been demonstrated to be able to differentiate into functional ameloblasts. Translational medicine studies in the nonhuman primate are irreplaceable steps towards clinical application of stem cell-based tissue engineering therapy. In the present study, we first examined the epithelial stem cell markers in the rhesus skin using immunostaining. Keratinocyte stem cells were then isolated from rhesus epidermis, cultured in vitro, and characterized by epithelial stem cell markers. Epithelial sheets of these cultured keratinocytes, which were recombined with E13.5 mouse dental mesenchyme that possesses odontogenic potential in the presence of exogenous FGF8, were induced to differentiate into enamel-secreting ameloblasts. Our results demonstrate that in the presence of appropriate odontogenic signals, rhesus keratinocytes can be induced to gain odontogenic competence and are capable of participating in odontogenesis, indicating that rhesus keratinocytes are an ideal epithelial cell source for further translational medicine study of tooth tissue engineering in nonhuman primates.

Anti-angiogenic mechanism of cordycepin on rhesus macaque choroid - retinal endothelial cell line cultured in high glucose condition / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the angiogenesis effect and protective mechanism of cordycepin on rhesus macaque choroid- retinal endothelial ( RF/ 6A) cell line cultured in high glucose condition. METHODS: Cultured RF/ 6A cells were divided into normal control group, high glucose group and high glucose (HG) + different concentration cordycepin groups (HG+ 10μ g/ mL group, HG+ 50μ g/ mL group, HG+ 100μ g/mL group). The cell proliferation was assessed using cholecystokinin octapeptide dye after treated for 48h. The cell migration was investigated by a Transwell assay. The tube formation was measured on Matrigel. Furthermore, the impact of cordycepin on high glucose - induced activation of VEGF and VEGF receptor 2 (VEGFR-2) was tested by Western blot analysis. RESULTS: Compared with normal control group, cell viability markedly increased in high glucose group ( P CONCLUSION: Cordycepin can suppress the proliferation, migration and tubu formation of RF/ 6A in high glucose condition, might via inhibiting expression of VEGF and VEGFR-2.

Morphology and hemodynamic analysis of vertebral arteries in rhesus macaques by 320-CT and color Doppler ultrasound / 中国实验动物学报

Objective To obtain imaging information of the cervical arteries and vertebral structure of rhesus ma -caques and provide useful reference data for medical research works using macaques as animal models .Methods Six a-dult macaques (3 males and 3 females) in age of 3-5 years, body weight from 3.5 to 5 kg, were used to examine the neck by 320 row spiral CT imaging under general anesthesia , and three-dimensional reconstruction was performed to observe the vertebral artery morphology , and to measure the diameter of vertebral artery and the size of transverse foramen of the ma -caques .Color Doppler ultrasound was combined with 320-CT imaging to assess the blood flow velocity in carotid arteries . After CT scan to make sure that the vertebral artery and cervical vertebra of macaques had no deformity , the size of trans-verse foramen and diameter of vertebral artery were measured in the cross-sectional images .Color Doppler ultrasound was used to analyze the peak systolic velocity ( PSV) , diastolic peak flow velocity ( EDV) and the diameter ( D) of common ca-rotid artery (CCA), internal carotid artery (ICA), external carotid artery (ECA), and vertebral artery (VA) of the ma-caques .Results The 320-CT scan showed that in the monkeys , the vertebral artery enters the cervical spine through the intervertebral foramina at C6, and leaves the cervical spine at C 2 level, and the cervical spine has Luschka joint , which is similar to the anatomic structure in humans .The diameter of left and right vertebral arteries was 1.89 ±0.44 mm and 1.72 ±0.39 mm, respectively , with no significant difference between them in the same segment ( P>0.05 ) , and the size of transverse foramen also had no significant difference in the same segment ( P>0.05 ) .Color Doppler ultrasound showed that the left and right side systolic peak velocity (PSV) and diastolic peak velocity (EDV) of CCA and ICA, the left and right side systolic peak velocity (PSV) of ECA and VA had statistically significant difference (P<0.05), and the left ar-teries were predominant in the macaques .Conclusions In this study we obtained some imaging information of vertebral artery morphology and artery blood flow of macaques and these information may provide some useful basic data for further studies using rhesus macaques as animal models .

Pathogenesis of influenza A (H5N1) virus-induced viral pneumonia in the rhesus macaque / 中国人兽共患病学报

At present ,the mechanism of highly pathogenic avian influenza H5N1 virus causing human infection or death is still not fully clear .In order to better understand the pathogenesis of the disease ,the rhesus macaques were infected with H5N1 virus (AF148678/ACGoose/Guangdong/11961H5N1) .We analyzed the clinical symptoms ,characteristics of the virus invades body ,pathological changes ,and immune response to discuss the pathogenesis of viral pneumonia induced by H 5N1 virus infection from the early time to the recovery time .The rhesus macaques were infected with H5N1 virus through nasal .Clinical signs were assessed daily ,and major organs and blood were collected for detection of blood routine analysis ,viruses were isola-ted and titrated from organs ,and pathologic and immunohistochemical were also conducted .As a result ,the rhesus macaques in-fected with H5N1 virus experienced fever ,dyspnea ,and anorexia .The respiratory tract was the major target of the virus and the virus could not replicate in organs outside the respiratory tract .Positive staining cells by immunohistochemistry were bronchial epithelial cells and alveolar macrophages .Rhesus macaques experienced temporary severe pneumonia after 1-3 days ,mainly be-cause of neutrophils infiltration ;gradual recovery 6 days later ,mainly with macrophage infiltration ;lung tissue presented recov-ery state after 14 days ,mainly with T lymphocytes infiltration .Finally ,we concluded that the predilection of the H 5N1 virus to infect the lower airway suggests that it may be a limiting factor in human-to-human transmissibility of the H5N1 virus .The pathogenesis may include virus invasion ,replication and immune injury .

Shigella flexneri infection in a newly acquired rhesus macaque (Macaca mulatta) / 한국실험동물학회지

A 3.4 year-old rhesus macaque weighing 4.5 kg, was suffering from anorexia, acute mucous and bloody diarrhea. On physical examination, the monkey showed a loss of activity, hunched posture, abdominal pain, dehydration, mild gingivitis and unclean anus with discharge. Whole blood was collected for the examination of electrolytes, hematology and serum chemistry; fresh stool was also collected for bacterial culture. Blood profiles showed leukocytosis (14.5 K/microL) and neutrophilia (11.0 K/microL) on complete blood cell count and imbalanced electrolytes associated with diarrhea. As a result of bacterial culture, Shigella flexneri was identified through Mac/SS, IMVIC test, TCBS and VITEK II. Based on these results, this monkey was diagnosed as having acute enteritis caused by Shigella flexneri. Treatment was performed with enrofloxacin prior to the isolation of Shigella flexneri to prevent the transmission of disease. Fortunately, mucus and bloody diarrhea did not persist and general conditions fully recovered. Our results show that the use of enrofloxacin is effective in controlling Shigella flexneri infection in newly acquired rhesus monkeys.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Pathological changes on SARS-CoV infected Microtus brandtii, Lewis rat and Rhesus macaque / 基础医学与临床

Objective The pathological changes on SARS CoV infected Microtus brandtii,Lewis rat and Rhesus macaque and the possibility as SARS animal model were studied.Methods The amimals were infected with SARS-CoV through nasal cavity and sacrificed for collecting pathologic tissues after inoculation.The pathological changes,immuno-response and the replication of SARS-CoV in vivo were tested with microscopy,ELISA and virus isolation.Results Similar pathological changes were observed in SARS CoV infected Rhesus macaques,Lewis rats and Microtus brandtii and the pathological changes were similar with those of human.Conclusion All of the tree species of animals infected with SARS-CoV showned similar pathological changes with human SARS case.Among them,SARS-CoV infected Rhesus macaques is the best one in terms of pathological changes.Therefor the infected Rhesus macaque may be one of the most ideal animal model to study the pathogenesis and evaluation of anti-SARS-CoV vaccine.