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A comparison of three methods of decellularization of pig corneas to reduce immunogenicity

International Journal of Ophthalmology(English edition) 2014年第4期7卷 587-593页

作者： Whayoung Lee Yuko Miyagawa Cassandra Long David K.C.Cooper hidetaka hara Department of Surgery Thomas E. Starzl Transplantation Institute University of Pittsburgh

·AIM: To investigate whether decellularization using different techniques can reduce immunogenicity of the cornea, and to explore the decellularized cornea as a scaffold for cultured corneal endothelial cells(CECs).Transplantation of decellularized porcine corneas increases graft transparency and survival for longer periods compared with fresh grafts.·METHODS: Six-month-old wild-type pig corneas were cut into 100-200 μm thickness, and then decellularized by three different methods: 1) 0.1% sodium dodecyl sulfate(SDS); 2) hypoxic nitrogen(N2); and 3) hypertonic NaCl. Thickness and transparency were assessed visually. Fresh and decellularized corneas were stained with hematoxylin/eosin(H&E), and for the presence of galactose-α1,3-galactose(Gal) and N-glycolylneuraminic acid(NeuGc, a nonGal antigen). Also, a human IgM/IgG binding assay was performed. Cultured porcine CECs were seeded on the surface of the decellularized cornea and examined after H&E staining.· RESULTS: All three methods of decellularization reduced the number of keratocytes in the stromal tissue by >80% while the collagen structure remained preserved. No remaining nuclei stained positive for Gal or NeuGc, and expression of these oligosaccharides on collagen was also greatly decreased compared to expression on fresh corneas. Human IgM/IgG binding to decellularized corneal tissue was considerably reduced compared to fresh corneal tissue. The cultured CECs formed a confluent monolayer on the surface of decellularized tissue.· CONCLUSION: Though incomplete, the significant reduction in the cellular component of the decellularized cornea should be associated with a significantly reduced in vivo immune response compared to fresh corneas.

关键词： cornea decellularization immuneresponse pig xenotransplantation

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Induction of diabetes in cynomolgus monkey with one shot of analytical grade streptozotocin

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Animal Models and Experimental Medicine 2020年第1期3卷 79-86页

作者： Zhengzhao Liu Ying Lu Wenbao Hu hidetaka hara Yifan Dai Zhiming Cai Lisha Mou Shenzhen Xenotransplantation Medical Engineering Research and Development Center Institute of Translational MedicineShenzhen Second People's HospitalThe First Affiliated Hospital of Shenzhen UniversityHealth Science CenterShenzhenChina Movement System Injury and Repair Research Center Xiangya Hospital of Central South UniversityChangshaChina Jiangsu Key Laboratory of Xenotransplantation Nanjing Medical UniversityNanjingChina Xenotransplantation Program/Department of Surgery The University of Alabama at BirminghamBirminghamALUSA

Backgrounds:Streptozotocin(STZ)-induced diabetic monkey is a wide used preclinical animal model for the investigation of diabetes such as islet transplantation and development of diabetic *** are serious side effects of this method,including nausea,emesis,weight loss,liver damage,renal failure,and metabolic *** order to reduce the side effects,diabetic monkeys were induced using clinicalgrade ***,clinical-grade STZ is not available in ***,we establised a method by using 100 mg/kg analytical-grade STZ to induce complete diabetes in cynomolgus monkey without generating adverse effects to liver and ***:Three cynomolgus monkeys were used in this study.100 mg/kg STZ dissolved in normal saline and infused through vein line in 5 minutes after indwelling catheter in the carotid artery and jugular *** the STZ administration,blood glucose levels were examined every 1 or 2 hours in the first 48 ***,blood glucose levels were examined twice per day during the first week after the STZ *** and C-peptide levels were measured by *** chemistry of hepatic and renal function tests were *** and glucagon expression in the islet of diabetic monkey and normal monkey were examined by immunohistochemistry ***:The stimulated C-peptide level(Intravenous glucose tolerance test)which is less than 0.5 ng/mL,the triphasic blood glucose response and the destroyed β cell suggested the complete induction of diabetes *** apparent adverse effects were observed including no signs of vomiting and toxicity after STZ ***:In summary,we established a safe and reproducible STZ-induced diabetic cynomolgus monkey model for islet transplantation which will be used to develop novel approaches for the treatment of diabetes.

关键词： analytical grade diabetic model nonhuman primate STZ

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The potential of genetically-engineered pigs in providing an alternative source of organs and cells for transplantation

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The Journal of Biomedical Research 2013年第4期27卷 249-253页

作者： David K.C.Cooper hidetaka hara Mohamed Ezzelarab Rita Bottino Massimo Trucco Carol Phelps David Ayares Yifan Dai Thomas E.Starzl Transplantation Institute University of Pittsburgh Medical Center Division of Immunogenetics Children's Hospital of Pittsburgh University of Pittsburgh Revivicor Inc Jiangsu Key Laboratory of Xenotransplantation Nanjing Medical University

There is a critical shortage of organs, cells, and corneas from deceased human donors worldwide. There are also shortages of human blood for transfusion. A potential solution to all of these problems is the transplantation of organs, cells, and corneas from a readily available animal species, such as the pig, and the transfusion of red blood cells from pigs into humans. However, to achieve these ends, major immunologic and other barriers have to be overcome. Considerable progress has been made in this respect by the genetic modification of pigs to protect their tissues from the primate immune response and to correct several molecular incompatibilities that exist between pig and primate. These have included knockout of genes responsible for the expression of major antigenic targets for primate natural anti-pig antibodies, insertion of human complement- and coagulation-regulatory transgenes, and knockdown of swine leukocyte antigens that stimulate the primate＇s adaptive immune response. As a result of these manipulations, the administration of novel immunosuppressive agents, and other innovations, pig hearts have now functioned in baboons for 6-8 months, pig islets have maintained normoglycemia in diabetic monkeys for 〉 1 year, and pig corneas have maintained transparency for several months. Clinical trials of pig islet trans- plantation are already in progress. Future developments will involve further genetic manipulations of the organ- source pig, with most of the genes that are likely to be beneficial already identified.

关键词： pig blood transfusion pig genetic-engineered pig islets pig organs xenotransplantation

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Papain Activity in Dextran Solution for Keratin Hydrolysis

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Advances in Enzyme Research 2014年第1期2卷 49-53页

作者： Nanami hara Shintaro Morisada Keisuke Ohto hidetaka Kawakita Department of Applied Chemistry Saga University Saga Japan

High concentration of polymer solution in cosmetics has the essential role in keeping water on peel of skin. This study includes the papain activity in the high concentrated dextran solution for keratin hydrolysis. Papain is the industrial and a representative hydrolase. The concentration of dextran was ranged to 100 g/L. In the increasing concentration of dextran, the activity of papain decreased due to the low diffusivity of papain and substrate. Even in concentrated dextran solution, keratin powder was possible to be hydrolyzed at the high efficiency.

关键词： Dextran Papain Keratin Hydrolysis

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