Bashoo Naziruddin

Improvement in Outcomes of Clinical Islet Transplantation: 1999–2010

OBJECTIVE To describe trends of primary efficacy and safety outcomes of islet transplantation in type 1 diabetes recipients with severe hypoglycemia from the Collaborative Islet Transplant Registry (CITR) from 1999 to 2010. RESEARCH DESIGN AND METHODS A total of 677 islet transplant-alone or islet-after-kidney recipients with type 1 diabetes in the CITR were analyzed for five primary efficacy outcomes and overall safety to identify any differences by early (1999–2002), mid (2003–2006), or recent (2007–2010) transplant era based on annual follow-up to 5 years. RESULTS Insulin independence at 3 years after transplant improved from 27% in the early era (1999–2002, n = 214) to 37% in the mid (2003–2006, n = 255) and to 44% in the most recent era (2007–2010, n = 208; P = 0.006 for years-by-era; P = 0.01 for era alone). C-peptide ≥0.3 ng/mL, indicative of islet graft function, was retained longer in the most recent era (P < 0.001). Reduction of HbA1c and resolution of severe hypoglycemia exhibited enduring long-term effects. Fasting blood glucose stabilization also showed improvements in the most recent era. There were also modest reductions in the occurrence of adverse events. The islet reinfusion rate was lower: 48% by 1 year in 2007–2010 vs. 60–65% in 1999–2006 (P < 0.01). Recipients that ever achieved insulin-independence experienced longer duration of islet graft function (P < 0.001). CONCLUSIONS The CITR shows improvement in primary efficacy and safety outcomes of islet transplantation in recipients who received transplants in 2007–2010 compared with those in 1999–2006, with fewer islet infusions and adverse events per recipient.

Pig cells that lack the gene for alpha1-3 galactosyltransferase express low levels of the gal antigen.

Background. The major antigen recognized on pig tissue by primate antibodies is a terminal gal&agr;1-3gal carbohydrate structure (gal antigen) present on glycolipids and glycoproteins. The production of animals from somatic cells allows for the inactivation of specific genes. It is anticipated that the complete inactivation of the gene encoding &agr;1-3 galactosyltransferase, the enzyme that synthesizes the gal&agr;1-3gal linkage, will result in loss of that antigen from pig organs and tissue and will provide a survival benefit in pig-to-primate xenotransplants. Methods. Positive-negative selection was used to produce fetal-pig fibroblasts that were a heterozygous knockout (+/−) of the &agr;1-3 galactosyltransferase gene. Nuclear transfer of these cells generated pig embryos and live born pigs with the appropriate genotype. Using a novel selection method with cells from (+/−) embryos, we produced homozygous (−/−) fetal-pig fibroblast cells. Results. Southern blot analysis of the &agr;1-3 galactosyltransferase gene showed that we had produced (+/−) pig embryos, (+/−) live born pigs, and (−/−) pig-fetal fibroblast cells. Fluorescence-activated cell sorter (FACS) analysis with some, but not all, mouse anti-gal monoclonal antibodies and sensitized human serum showed that (−/−) cells still synthesized the gal antigen at 1 to 2% of the level of control heterozygous cells. Conclusions. Fetal-pig fibroblasts homozygous for the knockout of the &agr;1-3 galactosyltransferase gene appear to express low but detectable levels of the gal antigen.

Shipment of Human Islets for Transplantation

The use of regional human islet cell processing centers (ICPC) supporting distant clinical islet transplantation programs (CITP) has proven successful in recent clinical trials. Standardization of islet shipping protocols is needed to preserve cell product identity, quantity, quality and sterility, and to meet criteria for transplantation. We evaluated the use of gas‐permeable bags for human islet preparation shipment from a single ICPC to two remote CITPs. Product release tests (counts, purity, viability, sterility and potency) were performed at both centers using identical protocols to determine adequacy for transplantation. Thirty‐five islet preparations were shipped either immediately after isolation (n = 20) or following culture (n = 15). Islet recovery rate after shipment was higher in cultured preparations, when compared to those not cultured (91.2 ± 4.9% vs. 72.9 ± 4.7%, respectively; p < 0.05), though the overall recovery rate based on isolation and pre‐transplant counts was comparable (72.9 ± 4.7% vs. 70.4 ± 3.5%, respectively; p = N.S.). All preparations met product release criteria for transplantation. Additional experiments showed that gas‐permeable bags led to improved recovery and potency, when compared to 50‐mL conical tubes or to non‐gas‐permeable bags for shipment. Collectively, our data demonstrate that the use of gas‐permeable bags is efficient for clinical‐grade and should be preferred also for the shipment of research‐grade islet preparations.

Stimulation of Human and Rat Islet β-Cell Proliferation with Retention of Function by the Homeodomain Transcription Factor Nkx6.1

ABSTRACT The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet β-cell development, but its effects on adult β-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3H] thymidine and 5-bromo-2′-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-βGAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are β cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [3H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating β-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of β-cell mass in treatment of both major forms of diabetes.

Recognition of porcine major histocompatibility complex class I antigens by human CD8+ cytolytic T cell clones.

To evaluate the nature of the human cellular immune response to porcine xenoantigens, cytolytic T lymphocyte (CTL) cell lines were generated against porcine aortic endothelial cells (PAEC). After four stimulations, the phenotypes of the T cell lines were primarily CD8+ (79.7+/-19.6%). Natural killer cells were not detected. Functional analysis of the T cell lines showed specific cytotoxicity against syngeneic porcine targets with no lysis of unrelated porcine cells, human cells, or K562, a natural killer target. The major histocompatibility complex (MHC) specificity of this response was confirmed when T cell lines established against PAEC from partially inbred SLAdd miniature swine lysed only PAEC and phytohemagglutinin-stimulated lymphocytes from SLAdd origin but not SLAgg targets. Both CD8+ (7/12) and CD4+ (5/12) T cell clones were generated from the bulk cell lines. All of the CD8+ T cell clones specifically lysed stimulator PAEC and swine leukocyte antigen (SLA)-matched, phytohemagglutinin-stimulated lymphocyte targets but not unrelated porcine targets. CD4+ T cell clones, as expected, showed no lysis of any porcine target cells. The lysis of porcine targets by the human CD8+ cytotoxic T lymphocyte clones was inhibited by monoclonal antibodies against SLA class I antigens and human CD8, which indicates that human CD8+ T cells recognize porcine MHC class I molecules. These results, which show that human T cells differentiate between porcine MHC alleles, have relevance in the clinical application of xenografts.

Improving efficacy of clinical islet transplantation with iodixanol-based islet purification, thymoglobulin induction, and blockage of IL-1β and TNF-α.

Poor efficacy is one of the issues for clinical islet transplantation. Recently, we demonstrated that pancreatic ductal preservation significantly improved the success rate of islet isolation; however, two transplants were necessary to achieve insulin independence. In this study, we introduced iodixanol-based purification, thymoglobulin induction, and double blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression to improve the efficacy of clinical islet transplantation. Nine clinical-grade human pancreata were procured. Pancreatic ductal preservation was performed using ET-Kyoto solution in all cases. When the isolated islets met the clinical criteria, they were transplanted. We utilized two methods of immunosuppression and anti-inflammation. The first protocol prescribed daclizumab for induction, then sirolimus and tacrolimus to maintain immunosuppression. The second protocol used thymoglobulin for induction and tacrolimus and mycophenolate mofetil to maintain immunosuppression. Eternacept and anakinra were administered as anti-inflammatory drugs. The total amount of insulin required, HbA1c, and the SUITO index were determined to analyze and compare the results of transplantation. All isolated islet preparations (9/9) met the criteria for clinical transplantation, and they were transplanted into six type 1 diabetic patients. All patients achieved insulin independence with normal HbA1c levels; however, the first protocol required two islet infusions (N = 3) and the second protocol only required a single infusion (N = 3). The average SUITO index, at 1 month after a single-donor islet transplantation, was significantly higher in the second protocol (49.6 ± 8.3 vs. 19.3 ± 6.3, p < 0.05). Pancreatic ductal preservation, iodixanol-based purification combined with thymoglobulin induction, and blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression dramatically improved the efficacy of clinical islet transplantations. This protocol enabled us to perform successful single-donor islet transplantations. Further large-scale studies are necessary to confirm these results and clarify the mechanism of each component.

Seven consecutive successful clinical islet isolations with pancreatic ductal injection.

Inconsistent islet isolation is one of the issues of clinical islet transplantation. In the current study, we applied ductal injection to improve the consistency of islet isolation. Seven islet isolations were performed with the ductal injection of ET-Kyoto solution (DI group) and eight islet isolations were performed without the ductal injection (standard group) using brain-dead donor pancreata. Isolated islets were evaluated based on the Edmonton protocol for transplantation. The DI group had significantly higher islet yields (588,566 ± 64,319 vs. 354,836 ± 89,649 IE, p < 0.01) and viability (97.3 ± 1.2% vs. 92.6 ± 1.2%, p < 0.02) compared with the standard group. All seven isolated islet preparations in the DI group (100%), versus only three out of eight isolated islet preparations (38%) in the standard group met transplantation criteria. The islets from the DI group were transplanted into three type 1 diabetic patients and all three patients became insulin independent. Ductal injection significantly improved quantity and quality of isolated islets and resulted in high success rate of clinical islet transplantation. This simple modification will reduce the risk of failure of clinical islet isolation.

Islet product characteristics and factors related to successful human islet transplantation from the collaborative islet transplant registry (CITR) 1999-2010

The Collaborative Islet Transplant Registry (CITR) collects data on clinical islet isolations and transplants. This retrospective report analyzed 1017 islet isolation procedures performed for 537 recipients of allogeneic clinical islet transplantation in 1999–2010. This study describes changes in donor and islet isolation variables by era and factors associated with quantity and quality of final islet products. Donor body weight and BMI increased significantly over the period (p < 0.001). Islet yield measures have improved with time including islet equivalent (IEQ)/particle ratio and IEQs infused. The average dose of islets infused significantly increased in the era of 2007–2010 when compared to 1999–2002 (445.4 ± 156.8 vs. 421.3 ± 155.4 ×103 IEQ; p < 0.05). Islet purity and total number of β cells significantly improved over the study period (p < 0.01 and <0.05, respectively). Otherwise, the quality of clinical islets has remained consistently very high through this period, and differs substantially from nonclinical islets. In multivariate analysis of all recipient, donor and islet factors, and medical management factors, the only islet product characteristic that correlated with clinical outcomes was total IEQs infused. This analysis shows improvements in both quantity and some quality criteria of clinical islets produced over 1999–2010, and these parallel improvements in clinical outcomes over the same period.

Evidence for Instant Blood-Mediated Inflammatory Reaction in Clinical Autologous Islet Transplantation

A nonspecific inflammatory and thrombotic reaction termed instant blood‐mediated inflammatory reaction (IBMIR) has been reported when allogenic or xenogenic islets come into contact with blood. This reaction is known to cause significant loss of transplanted islets. We hypothesized that IBMIR occurs in patients undergoing total pancreatectomy followed by autologous islet transplantation (TP‐AIT) and tested this hypothesis in 24 patients and in an in vitro model. Blood samples drawn during the peritransplant period showed a significant and rapid increase of thrombin–anti‐thrombin III complex (TAT) and C‐peptide during islet infusion, which persisted for up to 3 h, along with a decreased platelet count. A concomitant increase in levels of inflammatory proteins IL‐6, IL‐8 and interferon‐inducible protein‐10 was observed. An in vitro model composed of pure islets plus autologous blood also demonstrated significantly increased levels of TAT (p < 0.05), C‐peptide (p < 0.05), tumor necrosis factor‐alpha (p < 0.05) and MCP‐1 (p < 0.05), as well as strong tissue factor expression in islets. Islet viability decreased significantly but was rescued by the presence of low‐molecular‐weight dextran sulfate. In conclusion, AIT‐induced elevation of TAT and destruction of islets suggests that IBMIR might occur during AIT. Modulating this process may help improve islet engraftment and the insulin independence rate in TP‐AIT patients.

Activation of human airway epithelial cells by non-HLA antibodies developed after lung transplantation: a potential etiological factor for bronchiolitis obliterans syndrome.

Background. The main cause of morbidity and mortality after lung transplantation (LT) is bronchiolitis obliterans syndrome (BOS). Anti-HLA antibodies development after LT has been shown to play an important role in BOS pathogenesis. However, the nature of non-HLA antibodies developed after LT and their role in BOS pathogenesis have not been determined. Methods. Sera from 16 BOS+ patients and 11 BOS− patients were collected at 12, 24, 36, and 48 months after LT. Anti-HLA class I and class II antibodies were absorbed with pooled human platelets and pooled human lymphoblastoid cell lines, respectively. Then, the presence of non-HLA antibodies against several cell lines from different origin was determined by flow cytometric analysis. Antibody-positive samples were tested for induction of proliferation and growth factor production in two selected airway epithelial cell (AEC) lines. Results. Five of 16 BOS+ patients (31.2%) and 0 of 11 BOS- patients (0%) developed anti-AEC antibodies after LT (P =0.05). No reactivity against endothelial cells, lymphocytes, monocytes, or granulocytes was detected. Further analysis of two selected sera demonstrated the development of reactivity against a 60-kDa antigen expressed by 60% of AEC lines and only 12% of cell lines from other tissues. Antibody binding to this antigen induced intracellular Ca++ influx, tyrosine phosphorylation, proliferation, and up-regulation of transforming growth factor-&bgr; and heparin-binding epidermal growth factor mRNA transcription in AECs. Conclusions. These results indicate that anti-AEC antibodies may play a role in the immunopathogenesis of BOS in the absence of anti-HLA antibodies.