Search

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose–basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation. In conjunction with the creation of a prevascularized graft bed in a Lewis rat model, posttransplant oxygen inhalation significantly improves the efficiency of subcutaneous islet transplantation.

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose–basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.

Allogeneic islet transplantation is a validated therapy in type 1 diabetes; however, there is decline of transplanted islet graft function over time and the mechanisms underlying this decline are unclear. We evaluated the distinct association between primary graft function (PGF) and 5-year islet transplantation outcomes.

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (?OCRglc) can predict in vivoefficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of ?OCRglcobtained at both institutions were compared. Islet specificity was determined by comparing ?OCRglcin islet and nonislet tissue. The ?OCRglcranged from 0.01 to 0.19 nmolmin100 islets (n 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (?OCRglcwas 0.12 nmolmin100 islets vs. 0.007 nmolmin100 nonislet clusters). The ?OCRglcwas statistically independent of percent beta cells, demonstrating that ?OCRglcis governed more by islet quality than by islet composition. The ?OCRglc, but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ?OCRglcas a measurement of islet quality for human islet transplantation.

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (∆OCRglc) can predict in vivoefficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of ∆OCRglcobtained at both institutions were compared. Islet specificity was determined by comparing ∆OCRglcin islet and nonislet tissue. The ∆OCRglcranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (∆OCRglcwas 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The ∆OCRglcwas statistically independent of percent beta cells, demonstrating that ∆OCRglcis governed more by islet quality than by islet composition. The ∆OCRglc, but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ∆OCRglcas a measurement of islet quality for human islet transplantation.

The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum-free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS-p38IH and islet viability was not significantly different in the three groups. β Cell numbers and function were the highest in islets cryopreserved with ICS-p38IH. Glucose-stimulated human C-peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscidmice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (015), whereas it occurred in 411 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.

A simple radioimmunoassay for the measurement of 17α-hydroxyprogesterone in 0·1–0·25 ml serum is described. An antibody prepared against 17α-hydroxyprogesterone 3(O-carboxymethyl)-oxime-BSA as immunogen was used. A correlation of the 17α-hydroxyprogesterone concentration found in the serum of normal subjects and of patients with that obtained by a more complex immunological purification method was good (r = 0·924, p < 0·001). Accuracy, precision, and specificity were acceptable with the simple method. Normal values for infants, children, and adults have been established, and the individual concentrations of 17α-hydroxyprogesterone in the serum of patients with congenital adrenal hyperplasia were well separated from normal values.