David M. Harlan

Diabetes and cancer: a consensus report.

Epidemiologic evidence suggests that cancer incidence is associated with diabetes as well as certain diabetes risk factors and diabetes treatments. This consensus statement of experts assembled jointly by the American Diabetes Association and the American Cancer Society reviews the state of science concerning 1) the association between diabetes and cancer incidence or prognosis, 2) risk factors common to both diabetes and cancer, 3) possible biologic links between diabetes and cancer risk, and 4) whether diabetes treatments influence risk of cancer or cancer prognosis. In addition, key unanswered questions for future research are posed.

Assessment of Human Pancreatic Islet Architecture and Composition by Laser Scanning Confocal Microscopy

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets α and δ cells resided at the periphery of the β-cell core. However, human islets were markedly different in that α, β, and δ cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of α, β, and δ cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.

results From A Human Renal Allograft Tolerance Trial Evaluating The Humanized Cd52-specific Monoclonal Antibody Alemtuzumab (campath-1h)

Background. Profound T‐cell depletion before allotransplantation with gradual posttransplant T‐cell repopulation induces a state of donor‐specific immune hyporesponsiveness or tolerance in some animal models. Alemtuzumab (Campath‐1H, Millennium Pharmaceuticals, Cambridge, MA) is a humanized CD52‐specific monoclonal antibody that produces profound T‐cell depletion in humans and reduces the need for maintenance immunosuppression after renal transplantation. We therefore performed a study to determine if pretransplant T‐cell depletion with alemtuzumab would induce tolerance in human renal allografts and to evaluate the nature of the alloimmune response in the setting of T‐cell depletion. Methods. Seven nonsensitized recipients of livingdonor kidneys were treated perioperatively with alemtuzumab and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically by peripheral flow cytometry, protocol biopsies evaluated immunohistochemically, and real‐time polymerase chain reaction‐based transcriptional analysis. Results. Lymphocyte depletion was profound in the periphery and secondary lymphoid tissues. All patients developed reversible rejection episodes within the first month that were characterized by predominantly monocytic (not lymphocytic) infiltrates with only rare T cells in the peripheral blood or allograft. These episodes were responsive to treatment with steroids or sirolimus or both. After therapy, patients remained rejection‐free on reduced immunosuppression, generally monotherapy sirolimus, despite the recovery of lymphocytes to normal levels. Conclusions. T‐cell depletion alone does not induce tolerance in humans. These data underscore a prominent role for early responding monocytes in human allograft rejection.

Association of cytokine polymorphic inheritance and in vitro cytokine production in anti-CD3/CD28-stimulated peripheral blood lymphocytes.

Background. Genetic variations in cytokine genes are thought to regulate cytokine protein production. However, studies using T cell mitogens have not always demonstrated a significant relationship between cytokine polymorphisms and in vitro protein production. Furthermore, the functional consequence of a polymorphism at position −330 in the IL-2 gene has not been described. We associated in vitro protein production with cytokine gene polymorphic genotypes after costimulation of cultured peripheral blood lymphocytes. Methods. PBL were isolated from forty healthy volunteers. Cytokine protein production was assessed by enzyme-linked immunosorbent assay. Polymorphisms in interleukin- (IL) 2, IL-6, IL-10, tumor necrosis factor (TNF-&agr;), tumor growth factor (TGF-&bgr;), and interferon (IFN-&ggr;) were determined by polymerase chain reaction (PCR). Results. Statistical difference between protein production and cytokine polymorphic variants in the IL-10, IFN-&ggr;, and TNF-&agr; genes was not evident after 48-hour stimulation with concanavalin-A. In contrast, after anti-CD3/CD28 stimulation significant differences (P <0.05) were found among high and low producers for IL-2, IL-6, and among high, intermediate, and low producers for IFN-&ggr;, and IL-10. Augmented levels of IL-2 in individuals that were homozygous for the polymorphic IL-2 allele were due to an early and sustained enhancement of IL-2 production. No association was found among TNF-&agr; and TGF-&bgr; genotypes and protein production. Conclusion. Polymorphisms in IL-2, IL-6, IL-10, and IFN-&ggr; genes are associated with their protein production after anti-CD3/CD28 stimulation. The profound effect of the IL-2 gene polymorphism in homozygous individuals may serve as a marker for those that could mount the most vigorous allo- or autoimmune responses, or perhaps become tolerant more easily.

Ethnicity greatly influences cytokine gene polymorphism distribution.

Polymorphisms in the regulatory regions of cytokine genes are associated with high and low cytokine production and may modulate the magnitude of alloimmune responses following transplantation. Ethnicity influences allograft half‐life and the incidence of acute and chronic rejection. We have questioned whether ethnic‐based differences in renal allograft survival could be due in part to inheritance of cytokine polymorphisms. To address that question, we studied the inheritance patterns for polymorphisms in several cytokine genes (IL‐2, IL‐6, IL‐10, TNF‐α, TGF‐β, and IFN‐γ) within an ethnically diverse study population comprised of 216 Whites, 58 Blacks, 25 Hispanics, and 31 Asians. Polymorphisms were determined by allele‐specific polymerase chain reaction and restriction fragment length analysis. We found striking differences in the distribution of cytokine polymorphisms among ethnic populations. Specifically, significant differences existed between Blacks and both Whites and Asians in the distribution of the polymorphic alleles for IL‐2. Blacks, Hispanics and Asians demonstrated marked differences in the inheritance of IL‐6 alleles and IL‐10 genotypes that result in high expression when compared with Whites. Those of Asian descent exhibited an increase in IFN‐γ genotypes that result in low expression as compared to Whites. In contrast, we did not find significant ethnic‐based differences in the inheritance of polymorphic alleles for TNF‐α. Our results show that the inheritance of certain cytokine gene polymorphisms is strongly associated with ethnicity. These differences may contribute to the apparent influence of ethnicity on allograft outcome.

Cytokine polymorphic analyses indicate ethnic differences in the allelic distribution of interleukin-2 and interleukin-6

BACKGROUND Polymorphisms in the regulatory regions of cytokine genes affect protein production and are associated with allograft outcome. Ethnic origin has been identified as a significant prognostic factor for several immune-mediated diseases and for outcome after allotransplantation. A clear relationship between cytokine polymorphisms and ethnicity has not been shown. METHODS One hundred sixty subjects including 102 whites and 43 African-Americans were studied. Using polymerase chain reaction-based assays and, in some cases, restriction enzyme digestion, we determined genetic polymorphisms for the cytokines interleukin (IL) -2, IL-6, IL-10, tumor necrosis factor-alpha, transforming growth factor-beta, and interferon-gamma (IFN-gamma). Genetic polymorphism frequencies were then compared to ethnicity using chi-square analysis and Fishers exact two-tailed tests. RESULTS For both the IL-2 and IL-6 genes, we found that whites and African-Americans differed significantly (P <0.05) in their allelic distribution and genotype frequency. A trend toward ethnic distribution was noted among the alleles and genotypes for the IL-10 and IFN-gamma genes. We found no correlation between ethnicity and either allelic distribution or genotype frequency for the tumor necrosis factor-alpha or transforming growth factor-beta genes. When comparisons were made between patients with or without a history of kidney failure, the allelic or genotypic distributions for the IL-6 and IFN-gamma genes were found to significantly differ. CONCLUSIONS Our work demonstrates a correlation between ethnicity and polymorphisms in several cytokine genes. In addition, we found that patients requiring renal transplantation differ from the general population with regard to certain cytokine gene polymorphisms. These findings may have relevance in making prognostic determinations or tailoring immunomodulatory regimens after renal transplantation.

Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion

T cells target peptide combos One of the enduring mysteries of autoimmunity is the identity of the specific proteins targeted by autoimmune T cells. Delong et al. used mass spectrometry to elucidate the peptide targets of autoimmune T cells isolated from a mouse model of type 1 diabetes. T cells targeted hybrid peptides formed by the covalent linking of a peptide derived from pro-insulin to other peptides derived from proteins found in pancreatic beta cells. T cells isolated from the pancreatic islets of two individuals with type 1 diabetes also recognized such hybrid peptides, suggesting that they may play an important role in driving disease. Science, this issue p. 711 Autoimmune T cells recognize covalently linked peptides derived from two distinct proteins. T cell–mediated destruction of insulin-producing β cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in β cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in β cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in β cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.

Induction Therapy With Monoclonal Antibodies Specific For Cd80 And Cd86 Delays The Onset Of Acute Renal Allograft Rejection In Non-human Primates1

CD80 and CD86 (also known as B7–1 and B7–2, respectively) are both ligands for the T cell costimulatory receptors CD28 and CD152. Both CD80 and CD86 mediate T cell costimulation, and as such, have been studied for their role in promoting allograft rejection. In this study we demonstrate that administering monoclonal antibodies specific for these B7 ligands can delay the onset of acute renal allograft rejection in rhesus monkeys. The most durable effect results from simultaneous administration of both anti-B7 antibodies. The mechanism of action does not involve global depletion of T or B cells. Despite in vitro and in vivo evidence demonstrating the effectiveness of the anti-B7 antibodies in suppressing T cell responsiveness to alloantigen, their use does not result in durable tolerance. Prolonged therapy with murine anti-B7 antibodies is limited by the development of neutralizing antibodies, but that problem was avoided when humanized anti-B7 reagents are used. Most animals develop rejection and an alloantibody response although still on antibody therapy and before the development of a neutralizing antibody response. Anti-B7 antibody therapy may have use as an adjunctive agent for clinical allotransplantation, but using the dosing regimens we used, is not a tolerizing therapy in this non-human primate model.

Current advances and travails in islet transplantation.

The successful demonstration that insulin-producing β-cells can be isolated (in the form of cell clusters called islets containing β and other endocrine and nonendocrine cells) from a recently deceased donors pancreas, then transplanted into subjects with type 1 diabetes, and thereby restore, at least temporarily, insulin-independent normoglycemia has firmly established the important “proof of concept.” Even so, worldwide efforts to advance the therapy for widespread applicability have served to focus attention on the hurdles yet to clear. This review will briefly describe the present state of the art and succinctly define the research problems being attacked along with some recent advances that demonstrate significant progress. Since Paul Lacys early rodent experiments in the 1960s established that pancreatic islets could be isolated from one animal and transplanted into a diabetic recipient to restore normoglycemia (1), investigators have pursued efforts to develop the therapy for clinical use. After years of development in various animal models and efforts to improve human islet isolation techniques (see [2–4] for reviews with a historical perspective), the first patient achieving short-term insulin independence was reported by the group at Washington University in St. Louis. That advance was based on new islet isolation technology utilizing islets pooled from several donors, intensive insulin treatment in the peritransplant period, and induction immunosuppression with antithymocyte globulin (ATG) to avoid glucocorticoid therapy (5). The development of new immunosuppressive drugs that allowed patients to remain off glucocorticoid therapy while awaiting subsequent islet infusions (because most recipients require islets from two or more donors) enabled the group in Edmonton to optimize the clinical islet transplantation procedure (6). The approach allowed the group to conclude that about 12,000 islet equivalents per recipient body weight (in kilograms) was required to restore insulin-independent normoglycemia (6) and sparked intense international interest and effort. Current …

Kidney transplantation with rabbit antithymocyte globulin induction and sirolimus monotherapy

Renal allograft recipients generally need to take several immunosuppressive agents for life. Calcineurin inhibitors and glucocorticosteroids are the mainstays of most regimens but have undesirable chronic effects. We postulated that aggressive T-cell depletion combined with the newer immunosuppressant sirolimus would permit transplantation without multidrug treatment. We therefore tested T-cell depletion with rabbit antithymocyte globulin followed by sirolimus monotherapy in 12 patients in an open-label study. This approach was tolerated well, and all patients achieved excellent renal function, and most did not need chronic steroid treatment or calcineurin inhibitors. Rejection was typically correlated with low concentrations of sirolimus, indicating continued dependence on maintenance immunosuppression.