Wayne J. Hawthorne

Loss of ARNT/HIF1β Mediates Altered Gene Expression and Pancreatic-Islet Dysfunction in Human Type 2 Diabetes

beta cell dysfunction is a central component of the pathogenesis of type 2 diabetes. Using oligonucleotide microarrays and real-time PCR of pancreatic islets isolated from humans with type 2 diabetes versus normal glucose-tolerant controls, we identified multiple changes in expression of genes known to be important in beta cell function, including major decreases in expression of HNF4alpha, insulin receptor, IRS2, Akt2, and several glucose-metabolic-pathway genes. There was also a 90% decrease in expression of the transcription factor ARNT. Reducing ARNT levels in Min6 cells with small interfering RNA (siRNA) resulted in markedly impaired glucose-stimulated insulin release and changes in gene expression similar to those in human type 2 islets. Likewise, beta cell-specific ARNT knockout mice exhibited abnormal glucose tolerance, impaired insulin secretion, and changes in islet gene expression that mimicked those in human diabetic islets. Together, these data suggest an important role for decreased ARNT and altered gene expression in the impaired islet function of human type 2 diabetes.

Hypoxia-inducible factor-1α regulates β cell function in mouse and human islets

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1alpha protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1alpha is required for normal beta cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1alpha protein is present at low levels in mouse and human normoxic beta cells and islets. Decreased levels of HIF-1alpha impaired glucose-stimulated ATP generation and beta cell function. C57BL/6 mice with beta cell-specific Hif1a disruption (referred to herein as beta-Hif1a-null mice) exhibited glucose intolerance, beta cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1alpha levels by inhibiting its degradation through iron chelation markedly improved insulin secretion and glucose tolerance in control mice fed a high-fat diet but not in beta-Hif1a-null mice. Increasing HIF-1alpha levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1alpha was bound to the Arnt promoter in a mouse beta cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1alpha in beta cell reserve and regulation of ARNT expression and demonstrate that HIF-1alpha is a potential therapeutic target for the beta cell dysfunction of T2D.

T Cell-Activated Macrophages Are Capable of Both Recognition and Rejection of Pancreatic Islet Xenografts

Macrophages have been proposed as the major effector cell in T cell-mediated xenograft rejection. To determine their role in this response, NOD-SCID mice were transplanted with fetal pig pancreas (FPP) before reconstitution with CD4+ T cells from BALB/c mice. Twelve days after CD4+ T cell reconstitution, purified macrophages (depleted of T cells) were isolated from CD4+ T cell-reconstituted FPP recipient mice and adoptively transferred to their nonreconstituted counterparts. After adoptive macrophage transfer, FPP recipient mice transferred with macrophages from CD4+ T cell-reconstituted mice demonstrated xenograft destruction along with massive macrophage infiltration at day 4 and complete graft destruction at day 8 postmacrophage transfer. By contrast, FPP recipients that received macrophages from nonreconstituted mice showed intact FPP xenografts with few infiltrating macrophages at both days 4 and 8 after macrophage transfer. The graft-infiltrating macrophages showed increased expression of their activation markers. Depletion of endogenous macrophages or any remaining CD4+ T cells did not delay graft rejection in the macrophage-transferred FPP recipients, whereas depletion of transferred macrophages with clodronate liposomes prevented graft rejection. Our results show that macrophages primed by FPP and activated by CD4+ T cells were attracted from the peripheral circulation and were capable of specific targeting and destruction of FPP xenografts. This suggests that in xenograft rejection, there are macrophage-specific recognition and targeting signals that are independent of those received by T cells.

The role of the fibrocyte in intimal hyperplasia

Summary.  Background: Experimental animal studies have shown that the intimal hyperplasia (IH) responsible for occlusion after successful revascularization procedures may be partially caused by a bone marrow‐derived cell that migrates to the site of vascular injury. Concurrent studies have demonstrated an extensive role in wound healing for the circulating fibrocyte. Objectives: We aimed to trace the path of the circulating cell that contributes to IH and determine if it is the fibrocyte. Methods and results: We established an in vitro model whereby purified monocytes from six healthy human volunteers were cultured into fibrocytes. These cells were morphometrically similar to the vascular smooth muscle cell (VSMC) found in IH and expressed alpha‐smooth muscle actin (α‐SMA) as well as CD34, CD45 and Collagen I (Col I), markers indicative of the fibrocyte. In an in vivo ovine carotid artery synthetic patch graft model, carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeled circulating leukocytes were observed throughout the graft as well as in the neointima in 18 sheep. These cells were shown to produce collagen and α‐SMA at 1, 2 and 4 weeks. These cells then underwent immunohistochemical analysis and were found to express a set of markers unique to the fibrocyte (CD34, CD45, Vimentin and α‐SMA) and also to double stain for CD34 and α‐SMA. Conclusions: IH in an ovine carotid artery patch graft model is partially derived from a hematopoietic circulating progenitor cell that acquires mesenchymal features as it matures at the site of injury.

Control of IBMIR in neonatal porcine islet xenotransplantation in baboons.

The instant blood‐mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose‐α1,3‐galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti‐thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal‐deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet‐poor plasma. Recipients of GM NICC developed a variable anti‐pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell‐mediated rejection, although scattered insulin‐positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long‐term graft survival may require more effective immunosuppression or further donor genetic modification.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 4: pre-clinical efficacy and complication data required to justify a clinical trial.

In 2009, the International Xenotransplantation Association (IXA) published a consensus document that provided guidelines and “recommendations” (not regulations) for those contemplating clinical trials of porcine islet transplantation. These guidelines included the IXAs opinion on what constituted “rigorous pre‐clinical studies using the most relevant animal models” and were based on “non‐human primate testing.” We now report our discussion following a careful review of the 2009 guidelines as they relate to pre‐clinical testing. In summary, we do not believe there is a need to greatly modify the conclusions and recommendations of the original consensus document. Pre‐clinical studies should be sufficiently rigorous to provide optimism that a clinical trial is likely to be safe and has a realistic chance of success, but need not be so demanding that success might only be achieved by very prolonged experimentation, as this would not be in the interests of patients whose quality of life might benefit immensely from a successful islet xenotransplant. We believe these guidelines will be of benefit to both investigators planning a clinical trial and to institutions and regulatory authorities considering a proposal for a clinical trial. In addition, we suggest consideration should be given to establishing an IXA Clinical Trial Advisory Committee that would be available to advise (but not regulate) researchers considering initiating a clinical trial of xenotransplantation.

An increased concentration of rifampicin bonded to gelatin-sealed Dacron reduces the incidence of subsequent graft infections following a staphylococcal challenge

The purpose of this study was to determine if 10 mg/ml rifampicin bonded to gelatin-sealed Dacron (Gelsoft) reduced staphylococcal infection. Grafts soaked in rifampicin were interposed in the left carotid artery of 20 merino sheep and then inoculated with 10(8) colony-forming units of MRSA (10 sheep) or a slime producing Staphylococcus epidermidis (10 sheep). Grafts were harvested at 3 weeks, and perigraft abscess, anastomotic disruption and graft occlusion recorded. Swabs were taken to assess bacterial growth of the perigraft tissues, and external and internal graft surface. Grafts segments were incubated in broth medium. Results were compared with previously published results that used graft that were not soaked in rifampicin (control) and grafts soaked in 1.2 mg/ml rifampicin. A total of 4/50 cultures were positive and significantly reduced for S. epidermidis compared with the control group of 30/50 (P < 0.05) and the 1.2 mg/ml group of 13/45 (P < 0.05). For the methicillin resistant staphylococcus aureus (MRSA) group, 6/40 cultures were positive, which was significantly reduced compared with the control group (38/40, P < 0.05) and the 1.2-mg/ml group (19/32, P < 0.05). In conclusion an increased concentration of rifampicin significantly reduced the incidence of prosthetic vascular graft infection following a challenge of MRSA or S. epidermidis.

Adoptive Transfer With In Vitro Expanded Human Regulatory T Cells Protects Against Porcine Islet Xenograft Rejection via Interleukin-10 in Humanized Mice

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ−/− mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.

Multicenter Australian trial of islet transplantation: Improving accessibility and outcomes

Whilst initial rates of insulin independence following islet transplantation are encouraging, long‐term function using the Edmonton Protocol remains a concern. The aim of this single‐arm, multicenter study was to evaluate an immunosuppressive protocol of initial antithymocyte globulin (ATG), tacrolimus and mycophenolate mofetil (MMF) followed by switching to sirolimus and MMF. Islets were cultured for 24 h prior to transplantation. The primary end‐point was an HbA1c of <7% and cessation of severe hypoglycemia. Seventeen recipients were followed for ≥12 months. Nine islet preparations were transported interstate for transplantation. Similar outcomes were achieved at all three centers. Fourteen of the 17 (82%) recipients achieved the primary end‐point. Nine (53%) recipients achieved insulin independence for a median of 26 months (range 7–39 months) and 6 (35%) remain insulin independent. All recipients were C‐peptide positive for at least 3 months. All subjects with unstimulated C‐peptide >0.2 nmol/L had cessation of severe hypoglycemia. Nine of the 17 recipients tolerated switching from tacrolimus to sirolimus with similar graft outcomes. There was a small but significant reduction in renal function in the first 12 months. The combination of islet culture, ATG, tacrolimus and MMF is a viable alternative for islet transplantation.

Management of Primary Symptomatic Lymphocele After Kidney Transplantation: A Systematic Review

Background. Management of lymphoceles after kidney transplantation is highly variable. The aim of this study was to evaluate and compare the different approaches of lymphocele management among kidney transplant recipients. Methods. MEDLINE and EMBASE were systematically searched for case studies published between 1954 and 2010. Inclusion criteria were symptomatic lymphoceles developing in recipients of deceased or living donor kidneys with specified intervention and outcome. Primary outcome was the rate of recurrence. Secondary outcomes were the rate of conversion from laparoscopic to open surgery, hospital stay, and complication rates. Results. Fifty-two retrospective case series with 1113 cases of primary lymphocele were selected for review. No randomized controlled trials or prospective cohort studies were located. Primary treatment modalities included were as follows: aspiration (n=218), sclerotherapy (n=155), drainage (n=219), laparoscopic surgery (n=333), and open surgery (n=188). Of the 218 cases of lymphocele managed with aspiration alone, 141 recurred with a recurrence rate of 59% (95% confidence interval [CI]: 52–67). Among those who received laparoscopic and open surgery, the recurrence rates were 8% (95% CI: 6–12) and 16% (95% CI: 10–24), respectively. The conversion rate from laparoscopic to open surgery was 12% (95% CI: 8–16). Conclusions. Laparoscopic fenestration of a symptomatic lymphocele is associated with the lowest risk of lymphocele recurrence. However, the evidence base to support a recommendation for laparoscopic surgery as first line treatment is weak and highlights the need for a multicenter prospective cohort study to examine the benefits of incorporating initial simple aspiration into the management of lymphocele after kidney transplantation.