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Leonard C Harrison

Publications and source records attributed to Leonard C Harrison.

At least 37 records · Page 2Linked to original sources
PubMed2004

Cytokines activate caspase-3 in insulinoma cells of diabetes-prone NOD mice directly and via upregulation of Fas.

In type 1 diabetes, autoimmune inflammation of pancreatic islets of Langerhans ('insulitis') results in destruction of insulin-producing beta cells. Cytokines released from islet-infiltrating mononuclear cells are known to be cytotoxic both directly and by upregulating Fas for FasL-induced apoptosis. To investigate the role of caspase-3, a major effector of apoptosis in beta-cell death, we asked whether cytokine- and/or FasL-induced apoptosis was associated with increased activity of caspase-3 in NIT-1 insulinoma cells and islets of autoimmune diabetes-prone NOD mice. Measurement of caspase-3 activity using a fluorogenic cleavage assay was validated in NOD mouse thymocytes undergoing dexamethasone (Dex)-induced apoptosis. For cytokine-induced apoptosis, NIT-1 cells or islets were exposed to IL-1 beta and IFN-gamma for 24 h. Caspase-3-like activity was increased 2.1+/-0.7 and 2.4+/-0.9-fold in lysates of cytokine-treated NIT-1 cells and NOD mouse islets, respectively. However, NIT-1 cells exhibited 2.1% (4.7 pg active caspase-3/microg protein) and islets 0.8% (1.9 pg active caspase-3/microg protein) of the active caspase-3 content observed in Dex-treated thymocytes (225.1 pg active caspase-3/microg protein). After 24 h cytokine-exposure, the percentage of Fas-positive NIT-1 cells increased from 1.4+/-1.1 to 29.7+/-11.6%. Addition of FasL for a further 3 h increased caspase-3-like activity an additional 1.8-fold in cytokine-treated NIT-1 cells. In summary, exposure of NOD mouse insulinoma cells or islets to IL-1 beta and IFN-gamma for 24 h induced caspase-3-like activity that, in the case of insulinoma cells at least, can be further enhanced by interaction of cytokine-induced Fas receptor with FasL. Compared to thymocytes, insulinoma cells and islets from NOD mice were characterised by low basal and cytokine-induced caspase-3 activity.

Animals↗
PubMed2004

Harp (harmonin-interacting, ankyrin repeat-containing protein), a novel protein that interacts with harmonin in epithelial tissues.

Mutations in the triple PDZ domain-containing protein harmonin have been identified as the cause of Usher deafness syndrome type 1C. Independently, we identified harmonin in a screen for genes expressed in pancreatic beta cells. Using a yeast two-hybrid assay, we show that the first PDZ domain of harmonin interacts with a novel protein, designated harp for harmonin-interacting, ankyrin repeat-containing protein. This interaction was confirmed in an over-expression system and in mammalian cells, and shown to be mediated by the three C-terminal amino acids of harp. Harp is expressed in many of the same epithelia as harmonin and co-localization of native harp and harmonin was demonstrated by confocal microscopy in pancreatic duct epithelium and in a pancreatic beta-cell line. Harp, predicted molecular mass 48 kDa, has a domain structure which includes three ankyrin repeats and a sterile alpha motif. Human harp maps to chromosome 16, and its mouse homologue to chromosome 7. Sequences with similarity to harp include the sans gene, mutations of which are responsible for deafness in the Jackson shaker 2 (js) mutant mouse and in human Usher syndrome type 1G. The functional domain structures of harp and harmonin, their interaction under native conditions and their co-localization suggest they constitute a scaffolding complex to facilitate signal transduction in epithelia.

Amino Acid Sequence↗
PubMed2004

CD4+ T cell proliferation in response to GAD and proinsulin in healthy, pre-diabetic, and diabetic donors.

The ability to measure proliferation of autoantigen-specific T cells is critical for the evaluation of cellular immune function. Using a novel, sensitive, CFSE-based assay, we were able to directly quantitate autoantigen-specific CD4(+) T cell proliferation. However, peripheral blood cells from healthy, pre-diabetic and diabetic donors exhibited overlap in responses to glutamic acid decarboxylase (GAD65) and proinsulin (PI). This indicates that autoantigen-induced CD4(+) T cell proliferation in a functionally complex cell population may not discriminate disease in the general population. Clear discrimination was found between diabetic and healthy sibs, suggesting the need to standardize the genetic and environmental background. In addition, the ability of the CFSE assay to allow analysis of the phenotype and function of autoantigen-responsive T cells may improve discrimination.

Blood Donors↗
PubMed2004

Pancreatic beta-cell function and immune responses to insulin after administration of intranasal insulin to humans at risk for type 1 diabetes.

OBJECTIVE: Mucosal administration of insulin retards development of autoimmune diabetes in the nonobese diabetic mouse model. We conducted a double-blind crossover study in humans at risk for type 1 diabetes to determine if intranasal insulin was safe, in particular did not accelerate beta-cell destruction, and could induce immune effects consistent with mucosal tolerance. RESEARCH DESIGN AND METHODS: A total of 38 individuals, median age 10.8 years, with antibodies to one or more pancreatic islet antigens (insulin, GAD65, or tyrosine phosphatase-like insulinoma antigen 2) were randomized to treatment with intranasal insulin (1.6 mg) or a carrier solution, daily for 10 days and then 2 days a week for 6 months, before crossover. The primary outcome was beta-cell function measured as first-phase insulin response (FPIR) to intravenous glucose at 0, 6, and 12 months and then yearly; the secondary outcome was immunity to islet antigens, measured monthly for 12 months. RESULTS: No local or systemic adverse effects were observed. Diabetes developed in 12 participants with negligible beta-cell function at entry after a median of 1.1 year. Of the remaining 26, the majority had antibodies to two or three islet antigens and FPIR greater than the first percentile at entry, as well as beta-cell function that generally remained stable over a median follow-up of 3.0 years. Intranasal insulin was associated with an increase in antibody and a decrease in T-cell responses to insulin. CONCLUSIONS: Results from this pilot study suggest that intranasal insulin does not accelerate loss of beta-cell function in individuals at risk for type 1 diabetes and induces immune changes consistent with mucosal tolerance to insulin. These findings justify a formal trial to determine if intranasal insulin is immunotherapeutic and retards progression to clinical diabetes.

Administration, Intranasal↗
PubMed2003

Human T-cells recognise N-terminally Fmoc-modified peptide.

We aimed to generate T-cell clones specific for human pre-proinsulin. An HLA DQ8, CD4+ T-cell clone that recognised a 10mer (C65-A9) peptide from pre-proinsulin was isolated. Further analysis revealed that the clone responded neither to recombinant proinsulin nor to re-synthesised C65-A9 peptide. Analysis of the original peptide revealed minor contamination (<0.5%) with an N-terminal Fmoc adduct. This peptide was synthesised and shown to stimulate the clone. Thus, Fmoc-modified peptides, which are common contaminants in synthetic peptides, can stimulate human CD4+ T-cells. This finding has important implications for the use of synthetic peptides in screening and epitope mapping studies and their use as vaccines in humans.

Autoantibodies↗
PubMed2003

A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.

The ability to measure proliferation of rare antigen-specific T cells among many bystanders is critical for the evaluation of cellular immune function in health and disease. T-cell proliferation in response to antigen has been measured almost exclusively by 3H-thymidine incorporation. This method does not directly identify the phenotype of the proliferating cells and is frequently not sufficiently sensitive to detect rare autoantigen-specific T cells. To overcome these problems, we developed a novel assay for antigen-specific human T-cell proliferation. Peripheral blood mononuclear cells (PBMC) were labelled with the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) and cells that proliferated in response to antigen, with resultant reduction in CFSE intensity, were measured directly by flow cytometry. This assay was more sensitive than 3H-thymidine incorporation and detected the proliferation of rare antigen-specific CD4(+) T cells at 10-fold lower antigen concentrations. It also allowed the phenotype of the proliferating cells to be directly determined. Using the CFSE assay we were able to measure directly the proliferation of human CD4(+) T cells from healthy donors in response to the type 1 diabetes autoantigens glutamic acid decarboxylase (GAD) and proinsulin (PI).

Autoantigens↗
PubMed2003

Proinsulin-a pathogenic autoantigen in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterised by immunity to pancreatic beta-cell autoantigens, associated with beta-cell destruction leading to insulin deficiency and hyperglycaemia. The rigorous definition of an autoimmune disease requires evidence that an autoantigen elicits pathological immune responses. Using criteria for the pathogenicity of an autoantigen, we examine the evidence for proinsulin as an autoantigen in T1D. We conclude that proinsulin satisfies these criteria. As a corollary, proinsulin is a potential immunotherapeutic tool for the prevention of T1D.

Amino Acid Sequence↗
PubMed2003

Antigen-induced regulatory T cells in autoimmunity.

The ultimate goal of any treatment for autoimmune diseases is antigen- and/or site-specific suppression of pathology. Autoaggressive lymphocytes need to be eliminated or controlled to prevent tissue damage and halt the progression of clinical disease. Strong evidence is emerging that the induction of regulatory T (T(Reg)) cells by autoantigens can suppress disease, even if the primary, initiating autoantigens are unknown and if inflammation is progressive. An advantage of these autoreactive T(Reg) cells is their ability to act as bystander suppressors and dampen inflammation in a site-specific manner in response to cognate antigen expressed locally by affected tissues. In this review, we consider the nature and function of such antigen-specific T(Reg) cells, and strategies for their therapeutic induction are discussed.

Animals↗
PubMed2003

Transfer of hematopoietic stem cells encoding autoantigen prevents autoimmune diabetes.

Bone marrow or hematopoietic stem cell transplantation is a potential treatment for autoimmune disease. The clinical application of this approach is, however, limited by the risks associated with allogeneic transplantation. In contrast, syngeneic transplantation would be safe and have wide clinical application. Because T cell tolerance can be induced by presenting antigen on resting antigen-presenting cells (APCs), we reasoned that hematopoietic stem cells engineered to express autoantigen in resting APCs could be used to prevent autoimmune disease. Proinsulin is a major autoantigen associated with pancreatic beta cell destruction in humans with type 1 diabetes (T1D) and in autoimmune NOD mice. Here, we demonstrate that syngeneic transplantation of hematopoietic stem cells encoding proinsulin transgenically targeted to APCs totally prevents the development of spontaneous autoimmune diabetes in NOD mice. This antigen-specific immunotherapeutic strategy could be applied to prevent T1D and other autoimmune diseases in humans.

Animals↗
PubMed2003

Disabling an integral CTL epitope allows suppression of autoimmune diabetes by intranasal proinsulin peptide.

Insulin is a major target of the autoimmune response associated with destruction of pancreatic beta cells in type 1 diabetes. A peptide that spans the junction of the insulin B chain and the connecting (C) peptide in proinsulin has been reported to stimulate T cells from humans at risk for type 1 diabetes and autoimmune diabetes-prone NOD mice. Here we show that proinsulin B24-C36 peptide binds to I-A(g7), the MHC class II molecule of the NOD mouse, and, after intranasal administration, induces regulatory CD4(+) T cells that, in the absence of CD8(+) T cells, block the adoptive transfer of diabetes. Curiously, however, intranasal B24-C36 did not inhibit development of spontaneous diabetes in treated mice. We then determined that B24-C36, and its core sequence B25-C34, bind to K(d), the NOD mouse MHC class I molecule, and elicit CD8(+) CTLs. When the CD8(+) T lymphocyte epitope was truncated at the C34 valine anchor residue for binding to K(d), the residual CD4(+) T cell epitope, B24-C32/33, significantly inhibited diabetes development after a single intranasal dose. This study identifies a novel CTL epitope in proinsulin and demonstrates that the therapeutic potential of a "tolerogenic" autoantigen peptide can be compromised by the presence of an integral CTL epitope.

Administration, Intranasal↗
PubMed2003

Late-onset autoimmune diabetes in relatives of people with type 1 diabetes.

The Melbourne Prediabetes Family Study, a prospective study of first-degree relatives of people with type 1 diabetes (T1D), provided an opportunity to examine late-onset autoimmune diabetes within the context of a family history of T1D. We compared genetic, immunologic, and clinical features in relatives of people with T1D, who developed early- versus late-onset diabetes.

Adult↗
PubMed2003

(Pro)insulin-specific regulatory T cells.

Regulatory anti-diabetogenic T cells (T(reg)) can be induced by the mucosal administration of insulin or proinsulin peptides, in the non-obese diabetic (NOD) mouse model of autoimmune type 1 diabetes. Naso-respirtory insulin (which avoids insulin degradation) induces CD8+ alpha(alpha) TCR gamma(delta) T(reg) whereas peptides that bind to the NOD MHC class II molecule, I-Ag7, insulin B9-23 and proinsulin B24-C36, induce CD4+ T(regs) Following naso-respiratory delivery of insulin to NOD mice increased numbers of CD8+ gamma(delta) T cells expressing interleukin (IL)10 are detected in the pancreatic lymph nodes. Neonatal (3 day) thymectomy (NTX) dramatically accelerates diabetes development in NOD mice, associated with lymphopaenia and a block in the maturation of mucosal intrepithelial lymphocytes (IEL), especially extrathymic-derived CD8+ alpha(alpha) TCR gamma(delta) IEL. Regulatory anti-diabetogenic T cells cannot be elicited by naso-respiratory insulin in NTX-NOD mice. Reconstitution of NTX-NOD mice with CD8+ alpha(alpha) TCR gamma(delta) T cells prevents diabetes. CD8+ gamma(delta) T(reg) are conceivably physiological and insulin-specific, induced by exposure to insulin in maternal milk. These findings infer an immunoregulatory role for extrathymic-derived IEL, developing under the influence of the thymus and conditioned by early exposure to the exogenous environment.

Adoptive Transfer↗
PubMed2002

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice.

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonobese diabetic (NOD) mice, recombinant congenic nonobese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [(3)H]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-generative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.

Animals↗
PubMed2002

Bone morphogenetic proteins promote development of fetal pancreas epithelial colonies containing insulin-positive cells.

Extracellular signals that guide pancreas cell development are not well characterized. In an in vitro culture system of dissociated pancreas cells from the E15.5 mouse fetus we show that, in the presence of the extracellular matrix protein laminin-1, bone morphogenetic proteins (BMPs-4, -5 and -6) promote the development of cystic epithelial colonies. Transforming growth factor beta1 (TGF-beta1) and activin A antagonise this effect of BMP-6 and inhibit colony formation. Histological analysis revealed that the colonies are composed of E-cadherin-positive epithelial cells, which in localised areas are insulin positive. The colonies also contain occasional glucagon-positive cells, but no somatostatin- or alpha-amylase-positive cells. These findings indicate that members of the TGF-beta superfamily regulate pancreas epithelial cell development and can promote the formation of islet-like structures in vitro.

Activins↗
PubMed2002

Understanding autoimmune diabetes: insights from mouse models.

Type 1 or insulin-dependent diabetes is an autoimmune disease that causes the selective destruction of insulin-secreting beta cells in the pancreatic islets. Although this is a polygenic disease, with at least 20 genes implicated, the dominant susceptibility locus maps to the major histocompatibility complex (MHC), both in humans and in rodent models. However, in spite of progress on several fronts, the molecular pathology of autoimmune diabetes remains incompletely defined. Major areas of research include environmental trigger factors, the identification and role of beta-cell antigens in inducing and maintaining the autoimmune response, and the nature of the pathogenic and protective lymphocytes involved. In this review, we will focus on these areas to highlight recent advances in understanding the pathogenesis of autoimmune diabetes, drawing extensively on insights gained by studying the non-obese diabetic (NOD) mouse.

Alleles↗
PubMed2002

High avidity antibodies to fetal pig pancreas endocrine cells transfer rejection but are not normally generated to fetal pig pancreas xenografts.

Previous studies on the contribution of T cell-dependent antibody (Ab) to non-vascular xenograft rejection have yielded conflicting results, being confounded by the presence of recipient T cells and the use of different tissues and immunizing regimens to generate Ab. In the present study, the effect of adoptive transfer of Ab on fetal pig pancreas (FPP) and pig PK15 cell xenografts was examined in T cell-deficient severe combined immune deficiency (SCID) mice. T cell-dependent Abs raised by hyperimmunization with different cell types and by FPP transplantation were compared. Ab raised by hyperimmunization with pig thymocytes exhibited strong binding to pig thymocytes and PK15 cells but did not transfer FPP rejection. IgG1 and IgM, but not IgG3, Abs bound strongly to FPP exocrine and connective tissue, whereas binding to endocrine cells in vitro and in vivo was weak or absent. This pattern of Ab binding was similar to that observed after transplanting FPP into BALB/c mice. Furthermore, serum recovered from BALB/c mice 20 days after FPP transplantation bound strongly to non-endocrine but not endocrine cells and did not transfer FPP rejection. In contrast, serum from mice hyperimmunized with PK15 cells bound strongly to PK15 cells and transferred rejection of intraperitoneal PK15 cells. Furthermore, this serum contained IgG1 and IgM Abs that bound strongly, and IgG3 Abs that bound weakly, to endocrine cells in FPP, and also transferred rejection of FPP in SCID mice. These results indicate that endocrine cells express low concentrations of xenoreactive Ab epitopes and that high Ab concentrations and/or high avidity Abs are required for sufficient endocrine cell binding to cause damage and rejection in the immunodeficient mouse model. Such Abs are not elicited by transplanting FPP into immunocompetent mice. Nevertheless, a contribution of Ab to rejection in immunocompetent mice cannot be excluded.

3T3 Cells↗