Rafael Valdés-González

No Evidence of Porcine Endogenous Retrovirus in Patients With Type 1 Diabetes After Long-Term Porcine Islet Xenotransplantation

Xenotransplantation is a promising alternative for donor shortage to ameliorate physiologic and metabolic disorders. The major concern for xenotransplant is the risk of zoonosis mainly by the porcine endogenous retrovirus (PERV), presentation in the piglet genome. Twenty‐three patients with type 1 diabetes were transplanted with porcine islets using collagen‐generating devices which were implanted subcutaneously in the anterior wall of the abdomen. Clinical characteristics and metabolic tests were recorded in each visit. They were tested for PERV using PCR and RT‐PCR from blood pretransplantation and every 3 months during a 4.6‐ to 8‐year follow‐up after their first xenotransplant. Tests by PCR of every DNA sample (780 samples) revealed that there was no PERV infection in the DNA of white cells. No evidence of PERV activation was found in this group of patients with type 1 diabetes during clinical long‐term follow‐up. J. Med. Virol. 82:331–334, 2010.

Long-term follow-up of patients with type 1 diabetes transplanted with neonatal pig islets

Pancreas transplantation is an option to achieve better metabolic control and decrease chronic complications in patients with diabetes. Xenotransplantation becomes an important alternative. In this study, we show the clinical outcome of patients with type 1 diabetes transplanted with neonatal pig islets without immunosuppression. In a longitudinal study of 23 patients with type 1 diabetes, who received porcine islets between 2000 and 2004, we registered demographic and clinical characteristics every 3 months and chronic complications evaluation yearly. Porcine C‐peptide was measured in urine samples under basal conditions and after stimulation with l‐arginine. More than 50% were female, median current age was 20·8 years, median diabetes duration at transplantation 5·5 years, median current diabetes duration 11 years and median time post‐transplantation 5·7 years. Their media of glycosylated haemoglobin reduced significantly after the first transplantation. Insulin doses remain with a reduction greater than 33% in more than 50% of the patients. Before transplantation, 14 of the 21 patients presented mild chronic complications and currently only two patients presented these complications. Porcine C‐peptide was present in all urine samples under basal conditions and increased post‐stimulation with l‐arginine. These patients achieved an excellent metabolic control after the first transplantation. This could explain, as well as the remaining function of transplanted cells, the low frequency of chronic complications compared to patients with similar diabetes duration and age.

Three-yr follow-up of a type 1 diabetes mellitus patient with an islet xenotransplant

Abstract:  In order to alleviate the shortage of human donors, the use of porcine islets of Langerhans for xenotransplantation in diabetic patients has been proposed as a solution. To overcome rejection, we have developed a procedure for protecting the islets by combining them with Sertoli cells and placing them in a novel subcutaneous device, that generates an autologous collagen covering. A type 1 diabetic woman was closely monitored for 10 months, and then transplanted in two devices with two months of difference and a third time after 22 months. Here we present a three‐yr follow‐up. The close monitoring induced a rapid decrease in exogenous insulin requirements, which stabilized between 19 and 28 IU/d for nine months. After transplantation, the requirements reduced further to below 6 IU/d and for some weeks she was insulin free. Glycosylated hemoglobin levels decreased concomitantly. Porcine insulin could be detected in the serum after a glucose challenge and insulin positive cells inside a removed device after two yr. No complications have arisen and no porcine endogenous retrovirus infection has been detected through PCR and RT‐PCR. This case demonstrates the feasibility of using the xenotransplantation of porcine cells to alleviate metabolic complications and insulin requirements in type 1 diabetic patients.

Method for evaluating quality of cultured neonatal pig Sertoli cells.

Abstract:  Background:  Sertoli cells (SC) in the testis secrete factors that nourish and immunoprotect developing spermatozoa, which have made them the focus of studies that aim to generate localized tolerance, particularly for transplantation and perhaps autoimmunity. Several methods have been described to isolate these cells, which include a two‐step enzymatic digestion with limited assessment of the culture. Here we describe a one‐step method, and a series of tests for determining purity, viability, and function of the cultured cells.

Improved method for isolation of porcine neonatal pancreatic cell clusters.

Abstract:  Based on the described methods for the isolation of neonatal pancreatic cell clusters (NPCCs), we have developed modifications in order to improve their quality, functionality, and process reproducibility in the isolation technique, for potential use in research. In addition, we indicate techniques for describing yield, functionality, viability and purity of the NPCCs.

Unexpected Immunoresponse to Gal and APA Antigens in Diabetic Type 1 Patients Receiving Neonatal Pig Islets After 6 Years

Cotransplantation of porcine islets and Sertoli cells into preimplanted subcutaneous devices improve metabolic control in type 1 diabetic patients, and survive grafted for more than 4 years. We report here, further assessment of the endocrine and porcine nature of the surviving cells and the immune responses elicited toward Gal α(1,3)-Gal β(1,4)-GlcNAc (Gal) antigen in patients who received a second and third transplants. No immunosuppressive drugs were administered. We were able to immunostain insulin- and glucagon-positive cells in all biopsies of patients and Sertoli cell markers in 60.9% of biopsies. Additionally, all biopsies tested, amplified the porcine COII gene. Patients demonstrated an increase in antipig antibodies in response to the first transplant with a decreasing response toward the second and third transplants. In all transplants, the IgG levels promptly returned to basal values after 3–4 months. The long-term survival of porcine cells and the reduced humoral immune response to multiple transplants indicate a form of tolerance. We have not been able to find CD25-positive cells, indicating that it is probably an immune accommodation of the graft.

Correlation between insulin requirements and anti‐galactose antibodies in patients with type 1 diabetes transplanted with neonatal pig islets

Pig xenografts represent an alternative source of organs for transplantation. Immunosuppression can prevent rejection, but involves high risk and cost. New anti‐rejection techniques have been developed; however, results have not been successful. Few studies have reported xenoantibody levels in xenotransplanted patients with diabetes and no patients have reported a clinical correlation. We analysed anti‐galactose (Gal) and other anti‐pig antibody (APA) levels in xenotransplanted patients with type 1 diabetes and the relation to the clinical outcome. Twenty‐three patients with type 1 diabetes were transplanted with porcine islets inside a device without immunosuppression. Demographic characteristics, insulin dose and xenoantibody levels at different periods were recorded. Anti‐Gal and anti‐pig antibodies were measured through indirect enzyme‐linked immunosorbent assay (ELISA) and haemolytic anti‐pig antibody assay. More than 50% were female; the mean current age, current diabetes duration, diabetes duration at xenotransplantation and time post‐transplantation were: 20·8, 11, 5·5 and 5·7 years, respectively. Insulin doses remained with a mean reduction greater than 33% in more than 50% of the patients. The lowest anti‐Gal antibody levels were related to the highest insulin dose reductions. This relationship could be explained by the device, Sertoli cells and accommodation process.