We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet ...allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.
Type 2 diabetic subjects manifest both disordered insulin action and abnormalities in their pancreatic islet cells. Whether the latter represents a primary defect or is a consequence of the former is ...unknown. To examine the beta-cell mass and function of islets from type 2 diabetic patients directly, we isolated islets from pancreata of type 2 diabetic cadaveric donors (n = 14) and compared them with islets from normal donors (n = 14) matched for age, BMI, and cold ischemia time. The total recovered islet mass from type 2 diabetic pancreata was significantly less than that from nondiabetic control subjects (256,260 islet equivalents 2,588 IEq/g pancreas versus 597,569 islet equivalents 6,037 IEq/g pancreas). Type 2 diabetic islets were also noted to be smaller on average, and histologically, islets from diabetic patients contained a higher proportion of glucagon-producing alpha-cells. In vitro study of islet function from diabetic patients revealed an abnormal glucose-stimulated insulin release response in perifusion assays. In addition, in comparison with normal islets, an equivalent number of type 2 diabetic islets failed to reverse hyperglycemia when transplanted to immunodeficient diabetic mice. These results provide direct evidence for abnormalities in the islets of type 2 diabetic patients that may contribute to the pathogenesis of the disease.
Acute allograft rejection requires the activation of alloreactive CD4 T cells. Despite the capacity of B cells to act as potent APCs capable of activating CD4 T cells in vivo, their role in the ...progression of acute allograft rejection was unclear. To determine the contribution of B cell APC function in alloimmunity, we engineered mice with a targeted deficiency of MHC class II-mediated Ag presentation confined to the B cell compartment. Cardiac allograft survival was markedly prolonged in these mice as compared to control counterparts (median survival time, >70 vs 9.5 days). Mechanistically, deficient B cell-mediated Ag presentation disrupted both alloantibody production and the progression of CD4 T cell activation following heart transplantation. These findings demonstrate that indirect alloantigen presentation by recipients' B cells plays an important role in the efficient progression of acute vascularized allograft rejection.
Islet amyloid is hypothesized to play a role in nonimmunologic transplanted islet graft loss. We performed a quantitative histologic analysis of liver biopsies from intrahepatic islet grafts ...transplanted in streptozotocin-induced diabetic cynomolgus macaques. Seven animals treated with antithymocyte globulin (ATG) and rapamycin or ATG and rituximab experienced islet graft rejection with lymphocytic infiltrates present on islet graft biopsies. Except for one case involving the oldest and largest donor where amyloid was present on initial biopsy 1 month after transplant, none of the six other cases with rejection contained amyloid, including one case biopsied serially to 25 months. In contrast, four out of six animals treated with ATG and rituximab and rapamycin had no evidence of rejection at the time of biopsy (two animals that discontinued rapamycin had mild periislet lymphocytes), and all four cases followed more than 4 months demonstrated amyloid deposition at subsequent time points. Amyloid severity increased with time after transplant (r = 0.68; P < 0.05) and with decreasing islet β-cell area (r = −0.68; P < 0.05). In two islet recipients with no evidence of rejection and still normoglycemic and insulin independent at the first detection of amyloid, β-cell secretory capacity declined over time coincident with increasing amyloid severity and decreasing β-cell area, with both animals eventually becoming hyperglycemic and insulin dependent. Transplanted islet amyloid also developed in autologous islets placed sc. These results indicate that in cynomolgus macaques, transplanted islets may accumulate amyloid over time associated with subsequent decline in β-cell mass and function and support the development of intrahepatic islet amyloid as a potential mechanism for nonimmunologic islet graft loss.
Enzymatic activity of glucokinase was demonstrated, quantitated, and characterized kinetically in rat and mouse pituitary extracts using a highly specific and sensitive spectrometric assay. A ...previously proposed hypothesis that the glucokinase gene might be expressed in the pituitary corticotrophic cells was therefore reexamined using mRNA in situ hybridization and immunohistochemical techniques. No evidence was found that corticotrophs are glucokinase positive, and the identity of glucokinase-expressing cells remains to be determined. The findings do, however, suggest a novel hypothesis that a critical subgroup of anterior pituitary cells might function as glucose sensor cells and that direct fuel regulation of such cells may modify the classical indirect neuroendocrine pathways that are known to control hormone secretion from anterior pituitary cells.
In transplantation, there is a critical need for noninvasive biomarker platforms for monitoring immunologic rejection. We hypothesized that transplanted tissues release donor-specific exosomes into ...recipient circulation and that the quantitation and profiling of donor intra-exosomal cargoes may constitute a biomarker platform for monitoring rejection. Here, we have tested this hypothesis in a human-into-mouse xenogeneic islet transplant model and validated the concept in clinical settings of islet and renal transplantation. In the xenogeneic model, we quantified islet transplant exosomes in recipient blood over long-term follow-up using anti-HLA antibody, which was detectable only in xenoislet recipients of human islets. Transplant islet exosomes were purified using anti-HLA antibody-conjugated beads, and their cargoes contained the islet endocrine hormone markers insulin, glucagon, and somatostatin. Rejection led to a marked decrease in transplant islet exosome signal along with distinct changes in exosomal microRNA and proteomic profiles prior to appearance of hyperglycemia. In the clinical settings of islet and renal transplantation, donor exosomes with respective tissue specificity for islet β cells and renal epithelial cells were reliably characterized in recipient plasma over follow-up periods of up to 5 years. Collectively, these findings demonstrate the biomarker potential of transplant exosome characterization for providing a noninvasive window into the conditional state of transplant tissue.
The physiological performance of an organ depends on an interplay between changes in cellular function and organ size, determined by cell growth, proliferation and death. Nowhere is this more evident ...than in the endocrine pancreas, where disturbances in function or mass result in severe disease. Recently, the insulin signal-transduction pathway has been implicated in both the regulation of hormone secretion from beta cells in mammals as well as the determination of cell and organ size in Drosophila melanogaster. A prominent mediator of the actions of insulin and insulin-like growth factor 1 (IGF-1) is the 3'-phosphoinositide-dependent protein kinase Akt, also known as protein kinase B (PKB). Here we report that overexpression of active Akt1 in the mouse beta cell substantially affects compartment size and function. There was a significant increase in both beta -cell size and total islet mass, accompanied by improved glucose tolerance and complete resistance to experimental diabetes.
B lymphocytes are required for the pathogenesis of autoimmune diabetes in NOD mice. Previous studies established that a lymphopenic transitional (TR) B cell compartment reduces the competitive ...constraint on the entry of newly emerging TR B cells into the splenic follicle (FO), thereby disrupting a peripheral negative selection checkpoint in NOD mice. Thus, development of clinically feasible immunotherapeutic approaches for restoration of appropriate negative selection is essential for the prevention of anti-islet autoimmunity. In this study we hypothesized that in vivo neutralization of the B lymphocyte stimulator (BLyS/BAFF) may enhance the stringency of TR-->FO selection by increasing TR B cell competition for follicular entry in NOD mice. This study demonstrated that in vivo BLyS neutralization therapy leads to the depletion of follicular and marginal zone B lymphocytes. Long-term in vivo BLyS neutralization caused an increased TR:FO B cell ratio in the periphery indicating a relative resistance to follicular entry. Moreover, in vivo BLyS neutralization: 1) restored negative selection at the TR-->FO checkpoint, 2) abrogated serum insulin autoantibodies, 3) reduced the severity of islet inflammation, 4) significantly reduced the incidence of spontaneous diabetes, 5) arrested the terminal stages of islet cell destruction, and 6) disrupted CD4 T cell activation in NOD mice. Overall, this study demonstrates the efficacy of B lymphocyte-directed therapy via in vivo BLyS neutralization for the prevention of autoimmune diabetes.
Immunologic rejection is a major barrier to successful long-term outcomes in clinical transplantation. The importance of B lymphocytes-and their secretory products, alloantibodies-in the pathogenesis ...of allograft rejection is accepted. Furthermore, it is now clear that the dominant regulator of peripheral B-cell homeostasis and tolerance is the B-lymphocyte stimulator (BLyS), also referred to as the B-cell activating factor (BAFF). Recently, a novel class of clinical immunotherapeutic agents specific for BLyS, and its family of cytokines, has emerged for the treatment of B-cell-mediated diseases. In this study, we demonstrate the potential utility of BLyS-directed immunotherapy in preventing allograft rejection using a murine islet transplantation model.
A transient period of mature peripheral B-cell depletion was induced by means of in vivo BLyS neutralization using a murine analog of the monoclonal antibody, Benlysta. Subsequently, fully major histocompatibility complex-mismatched islets were transplanted into naïve diabetic mice followed by a short course of rapamycin.
After BLyS neutralization, indefinite islet allograft survival was achieved. Induction therapy with rapamycin was necessary, but not sufficient, for the achievement of this long-term graft survival. The tolerant state was associated with (1) abrogation of the donor-specific antibody response, (2) transient preponderance of immature/transitional B cells in all lymphoid organs, (3) impaired CD4 T-cell activation during the period of B-cell depletion, and (4) presence of a "regulatory" cytokine milieu.
In vivo BLyS neutralization effectively induces humoral tolerance and promotes long-term islet allograft survival in mice. Therefore, B-lymphocyte-directed immunotherapy targeting the homeostatic regulator, BLyS, may be effective in promoting transplantation tolerance.
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