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 (HIF-1α) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1α protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1α is required for normal β cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1α protein is present at low levels in mouse and human normoxic β cells and islets. Decreased levels of HIF-1α impaired glucosestimulated ATP generation and β cell function. C57BL/6 mice with β cell-specific Hif1a disruption (referred to herein as β-Hif1a-null mice) exhibited glucose intolerance, β cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1α 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 β-Hif1a-null mice. Increasing HIF-1α levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1α was bound to the Arnt promoter in a mouse β cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1α in β cell reserve and regulation of ARNT expression and demonstrate that HIF-1α is a potential therapeutic target for the β cell dysfunction of T2D. IntroductionThe transcription factor HIF-1α is important for a range of functions, including cellular responses to hypoxia and other stressors, angiogenesis, and fetal development (1-6). It has strong antiapoptotic effects (7-11) and is implicated in the pathogenesis of cardiovascular diseases and some cancers (12)(13)(14)(15)(16)(17)(18)(19)(20).HIF-1α is a member of the bHLH-PAS family (reviewed in refs. 2, 18, 21) and functions as an obligate dimer with other family members, including aryl hydrocarbon receptor (AhR) nuclear translocator (ARNT). We previously reported that ARNT was decreased in islets isolated from patients with type 2 diabetes (T2D) and that decreasing ARNT in Min6 cells or disrupting it in mouse β cells caused changes in gene expression and glucose-stimulated insulin secretion (GSIS) similar to those seen in islets isolated from humans with T2D (22). Recently, we reported a loss of ARNT expression in the livers of people with T2D, affecting dysregulation of gluconeogenesis (23). Though the specific ARNT partner which is important for its actions in β cells (or liver) is not known, candidates include AhR, HIF-1α, HIF-2α, HIF-3α, and circadian rhythm molecules, e.g., BMAL.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.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-a1,3-galactose (aGal) 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 aGal-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.

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.

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 (a-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 a-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 a-SMA) and also to double stain for CD34 and a-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.

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.

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.

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.