bromochloroacetic-acid and Diabetes-Mellitus--Type-1

This is a short review of porcine neonatal pancreatic cell clusters (NPCCs) which might eventually be useful for beta cell replacement therapy in people with diabetes. The current success with islet allograft transplantation is reviewed and is problematic because only partial success has been obtained and the shortage of human islet tissue means that only a small fraction of people with diabetes would be able to benefit. For these reasons there is considerable interest in xenotransplantation, with pigs being a particularly attractive source. The relative merits of early fetal, late fetal, neonatal and adult porcine tissue are discussed. Neonatal tissue has several attractive features, with their hardiness and potential for growth being especially noteworthy. NPCCs are harvested after digested and dispersed clumps of cells are kept in culture for 7 days. The NPCCs consist mainly of duct cells, protodifferentiated cells and mature endocrine cells. The protodifferentiated cells are either double or triple stained for insulin, cytokeratin 7, glucagon, pancreatic polypeptide, or somatostatin. When transplanted into diabetic nude mice it usually takes weeks before glucose levels are normalized, and during that time differentiation and growth of the graft can be observed. Potential strategies for controlling xenograft rejection are mentioned, with these being immunosuppression, induction of tolerance, immunobarrier devices, and gene transfer approaches.

Topics: Animals; Animals, Newborn; Diabetes Mellitus, Type 1; Fetal Tissue Transplantation; Glucagon; Humans; Insulin; Islets of Langerhans; Islets of Langerhans Transplantation; Keratins; Mice; Mice, Nude; Pancreatic Polypeptide; Somatostatin; Swine; Transplantation, Heterologous

Noninvasive glucose measurements are possible by analysis of transmitted near-infrared light over the 4000- to 5000-cm(-1) spectral range. Such measurements are highly sensitive to the exact position of the fiber-optic interface on the surface of the skin sample. A critical question is the degree of heterogeneity of the major chemical components of the skin matrix in relation to the size of the fiber-optic probed used to collect noninvasive spectra. Microscopic spectral mapping is used to map the chemical distribution for a set of excised sections of rat skin.. A Fourier transform near-infrared microspectrometer was used to collect transmission spectra from 16 tissue samples harvested from a set of four healthy Harlan-Sprague male rats. A reference point in the center of the tissue sample was probed regularly to track dehydration, changes in tissue composition, and changes in instrument performance. Amounts of the major skin constituents were determined by fitting microspectra to a set of six pure component absorbance spectra corresponding to water, type I collagen protein, keratin protein, fat, an offset term, and a slope term.. Microspectroscopy provides spectra with root mean square noise levels on 100% lines between 418 and 1475 microabsorbance units, which is sufficient for measuring the main chemical components of skin. The estimated spatial resolution of the microscope is 220 microm. The amounts of each tissue matrix component were determined for each 480 x 360-microm(2) location of a 4.8 x 3.6-mm(2) rectangular block of skin tissue. These spectra were used to generate two-dimensional distribution maps for each of the principal skin components.. Distribution of the chemical components of rat skin is significant relative to the dimensions of noninvasive glucose sensing. Chemical distribution maps reveal that variations in the chemical composition of the skin samples are on the same length scale as the fiber-optic probe used to collect noninvasive near-infrared spectra. Analysis of variance between tissue slices collected for one animal and analysis of variations between animals indicate that animal-to-animal variation for all four chemical components is significantly higher than variations between samples for a given animal. These findings justify the collection and interpretation of near-infrared microspectroscopic maps of human skin to establish chemical heterogeneity and its impact on noninvasive glucose sensing for the management of diabetes.

Topics: Animals; Blood Glucose; Blood Glucose Self-Monitoring; Body Water; Collagen Type I; Diabetes Mellitus, Type 1; Humans; Keratins; Male; Microspectrophotometry; Monitoring, Ambulatory; Rats; Skin; Spectroscopy, Near-Infrared

Tubular complexes (TC) in the pancreas contain duct-like structures with low cuboidal or flattened cells surrounding a large lumen and are thought to be a response to pancreatic injury. TC have been studied in animal models of chemical or surgically induced pancreatic damage but their occurrence has not been reported in rodent models of spontaneous autoimmune type I diabetes. We hypothesized that TC would be increased during the active phase of islet destruction in autoimmune diabetes and could contain islet progenitor cells. We analyzed TC in pancreas of Wistar Furth (WF), control (BBc) and diabetes-prone BioBreeding (BBdp) rats using immunohistochemistry and morphometry. TC were observed in all rat strains during active pancreas remodeling ( approximately 13 days). They increased between 60 and 93 days only in BBdp rats coincident with the increase in diabetes cases. Most TC were infiltrated with CD3(+) T-cells. Duct-like cells in the TC had low expression of the exocrine marker amylase, increased expression of epithelial cell markers, keratin and vimentin, and remarkably high cell proliferation and cell death. TC islets contained cells stained positive for insulin, glucagon, somatostatin, pancreatic polypeptide, as well as PDX-1, chromogranin, and hepatocyte-derived growth factor receptor, c-met. Transitional cells that were keratin(+)/insulin(+) and keratin(+)/amylase(+) cells were present in TC. The stem cell marker, nestin was upregulated in the TC region. Duct-like cells in TC of BBdp rats expressed markers of committed endocrine precursors: PDX-1, neurogenin 3 and protein gene product 9.5. This study demonstrates that TC are upregulated during beta-cell destruction and contain potential endocrine progenitors.

Topics: Amylases; Animals; Apoptosis; Biomarkers; Cell Differentiation; Cell Proliferation; Diabetes Mellitus, Type 1; Disease Models, Animal; In Situ Nick-End Labeling; Islets of Langerhans; Keratins; Pancreas, Exocrine; Rats; Rats, Inbred BB; Rats, Inbred WF; Regeneration; Stem Cells; Vimentin

Atrial natriuretic peptide (ANP) has been recently described as an endogenous inhibitor of the synthesis and angiogenic action of vascular endothelial growth factor (VEGF). Given VEGF's key role in promoting neovascularization in proliferative diabetic retinopathy (PDR), this study was designed to evaluate the possibility that ANP could be involved in the neovascular and fibrotic complications of PDR.. We determined ANP by radioimmunoassay in plasma and vitreous humor samples collected from diabetic patients with and without PDR and from non-diabetic subjects. ANP was also immunohistochemically localized in the epiretinal membranes of patients with PDR.. Vitreous ANP concentrations were significantly higher in patients with active PDR compared to patients with quiescent PDR, diabetes without PDR or controls <0.05. Significant differences were also observed between vitreous ANP levels in diabetic patients without PDR and control subjects. There was no significant correlation between serum and vitreous ANP levels in any of the patient groups. ANP was detected in the fibrovascular epiretinal tissue of patients with PDR.. Diabetic patients with active neovascularization have significantly higher levels of ANP in the vitreous humor than those without active PDR. Diabetic patients without PDR were also found to have significantly higher vitreous ANP levels than non-diabetic patients. Since plasma and vitreous ANP concentrations were found to be unrelated, we suggest intraocular ANP synthesis and/or an increase in the release of ANP into the vitreous, as opposed to diffusion from the blood, as the main factors contributing to the high vitreous ANP levels observed in diabetic patients. In the fibrovascular epiretinal tissue of these patients, ANP was found to be localized in vascular, glial, fibroblast-like and retinal pigment epithelium cells. Our findings suggest a role for ANP in PDR.

Topics: Adult; Aged; Atrial Natriuretic Factor; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Epiretinal Membrane; Female; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Radioimmunoassay; Retinal Neovascularization; Vitrectomy; Vitreous Body

Porcine neonatal islet-like cell clusters (NICCs) may be an attractive source of insulin-producing tissue for xenotransplantation in type I diabetic patients. We examined the functional and immunohistochemical outcome of the islet grafts in vitro during long-term culture and in vivo after transplantation to athymic nude mice. On average we obtained 29,000 NICCs from each pancreas. In a perifusion system, NICCs responded poorly to a glucose challenge alone, but 10 mmol/L arginine elicited a fourfold increase in insulin secretion and 16.7 mmol/L glucose + 10 mmol/L arginine caused a sevenfold increase in insulin section indicating some sensitivity towards glucose. Hormone content as well as the number of hormone-containing cells increased for the first 14 days of culture. When NICCs were stained for hormones, proliferation (Ki67), and duct cells (CK7), some insulin- and glucagon-positive cells co-stained for proliferation. However no co-staining was observed between insulin- and glucagon-positive cells or between hormone-and CK-positive cells. Following transplantation of 2000 NICCs under the renal capsule of diabetic nude mice, BG levels were normalized within an average of 13 weeks. Oral and IP glucose tolerance tests revealed a normal or even faster clearance of a glucose load compared with normal controls. Immunohistochemical examination of the grafts revealed primarily insulin-positive cells. In summary, in vitro, NICCs responded to a challenge including glucose and arginine. There was a potential for expansion of the beta-cell mass of NICCs in vitro as well as in vivo where NICCs eventually may normalize blood glucose of diabetic mice.

Topics: Animals; Animals, Newborn; Arginine; Cell Culture Techniques; Cell Differentiation; Cell Division; Cells, Cultured; Diabetes Mellitus, Type 1; Glucagon; Glucose; Glucose Tolerance Test; Graft Survival; Immunohistochemistry; Insulin; Insulin Secretion; Islets of Langerhans; Islets of Langerhans Transplantation; Keratin-7; Keratins; Ki-67 Antigen; Male; Mice; Mice, Nude; Somatostatin; Sus scrofa; Transplantation, Heterologous

The recent discovery of heretofore unknown, multipotential stem cells within the embryonic and adult islets of Langerhans suggests new therapeutic options for the treatment of diabetes mellitus type 1. These cells are characterised by the expression of the neural stem cell marker nestin and by their ability to differentiate ex vivo into pancreatic endocrine, exocrine and hepatic phenotypes with the expression of insulin, glucagon, amylase, cytokeratin-19 as well as liver specific proteins such as alpha-fetoprotein. The insulinotropic hormone glucagon-like peptide-1 enhances the differentiation of these nestin positive islet derived progenitor (NIP) cells into insulin secreting cells by activation of IDX-1, the key transcription factor for the differentiation into a beta-cell.

Topics: Adult; alpha-Fetoproteins; Amylases; Cell Differentiation; Cells, Cultured; Diabetes Mellitus, Type 1; Embryo, Mammalian; Fluorescence; Glucagon; Humans; Immunohistochemistry; Insulin; Insulin Secretion; Intermediate Filament Proteins; Islets of Langerhans; Keratins; Multipotent Stem Cells; Nerve Tissue Proteins; Nestin; Pancreas; Phenotype; Stem Cells; Transcription Factors

Low-angle synchrotron X-ray diffraction has revealed clear and consistent changes in the molecular structure of alpha-keratin of hair in insulin-dependent diabetes (IDDM) both for human IDDM subjects and for baboons with streptozocin induced diabetes. These changes in both meridional and equatorial intensity distributions are fully explained by a newly developed hexagonally packed model for keratin which locates the modification produced in hair in IDDM in the labile structure of the matrix at established intermediate filament linkage sites. The nature of the extracellular bonding suggests that the change is endogenous, occurring via the blood during the aggregation of the IFs in the follicle. The reproducibility of these changes indicate that hair may represent an easily accessible tissue for the study of how hyperglycaemia can modify extracellular matrix materials which lead to diabetic complications.

Topics: Adult; Animals; Child; Child, Preschool; Diabetes Mellitus, Type 1; Hair; Humans; Keratins; Papio; X-Ray Diffraction

In the present study we have evaluated the expression of different beta-cell markers, islet molecules and auto-antigens relevant in diabetes autoimmunity by a human insulinoma cell line (CM) in order to define its similarities with native beta cells and to discover whether it could be considered as a model for studies on immunological aspects of Type 1 diabetes. First, the positivity of the CM cell line for known markers of neuroendocrine derivation was determined by means of immunocytochemical analysis using different anti-islet monoclonal antibodies including A2B5 and 3G5 reacting with islet gangliosides, and HISL19 binding to an islet glycoprotein. Secondly, the expression and characteristics of glutamic acid decarboxylase (GAD) and of GM2-1 ganglioside, both known to be islet autoantigens in diabetes autoimmunity and expressed by human native beta cells, were investigated in the CM cell line. The pattern of ganglioside expression in comparison to that of native beta cells was also evaluated. Thirdly, the binding of diabetic sera to CM cells reacting with islet cytoplasmic antigens (ICA) was studied by immunohistochemistry. The results of this study showed that beta cell markers identified by anti-islet monoclonal antibodies A2B5, 3G5 and HISL-19 are expressed by CM cells; similarly, islet molecules such as GAD and GM2-1 ganglioside are present and possess similar characteristics to those found in native beta cells; the pattern of expression of other gangliosides by CM cells is also identical to human pancreatic islets; beta cell autoantigen(s) reacting with antibodies present in islet cell antibodies (ICA) positive diabetic sera identified by ICA binding are also detectable in this insulinoma cell line. We conclude that CM cells show close similarities to native beta cells with respect to the expression of neuro-endocrine markers, relevant beta cell autoantigens in Type 1 diabetes (GAD, GM2-1, ICA antigen), and other gangliosides. Therefore, this insulinoma cell line may be considered as an ideal model for studies aimed at investigating autoimmune phenomena occurring in Type 1 diabetes.

Topics: Autoantigens; Biomarkers; Chromatography, High Pressure Liquid; Diabetes Mellitus, Type 1; Gangliosides; Humans; Immunoblotting; Immunohistochemistry; Insulinoma; Islets of Langerhans; Keratins; Models, Immunological; Pancreatic Neoplasms; Tumor Cells, Cultured

Quantitative analysis was performed using computerized morphometry on the relationships between residual beta cells, pancreatic exocrine glands, and islet cell antibodies (ICA) in 14 pancreata of insulin-dependent diabetic (IDDM) patients. Both pancreatic exocrine glands and beta cells were markedly reduced in weight in IDDM patients compared with non-insulin-dependent diabetic (NIDDM) patients or nondiabetic controls. beta cells were preserved in six cases and were completely abolished in eight cases. In nine IDDM pancreata weighed at autopsy, the weights of pancreatic exocrine glands in six patients with either no or virtually no residual beta cells (32.3 +/- 1.6 g) were greater than those in three patients with residual beta cells (23.1 +/- 2.5 g, P < .05). Infiltration of lymphocytes positive for leukocyte common antigen (LCA) around the islet was observed in only one case with ICA and residual beta cells. Infiltration of LCA-positive lymphocytes around pancreatic acinar cells was observed in 50% (six of 12) of patients examined. The weight of pancreatic exocrine glands in patients with LCA-positive lymphocyte infiltration (26.2 +/- 2.3 g) was lower than that in patients without this condition (33.2 +/- 2.4 g, P < .05). Pancreatic cytokeratin autoantibodies (PKA) were detected in four of 10 patients examined. In addition, all four ICA-positive patients had residual beta cells, while only one of seven ICA-negative patients had residual beta cells (P = .03).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Autoantibodies; Diabetes Mellitus, Type 1; Female; Humans; Islets of Langerhans; Keratins; Lymphocytes; Male; Middle Aged; Organ Size; Pancreas

Autoantibodies reacting with human pancreatic exocrine cells were investigated by immunofluorescent techniques in 107 patients with Type 1 (insulin-dependent) diabetes mellitus, 20 first-degree relatives of the Type 1 diabetic patients, 347 patients with Type 2 (non-insulin-dependent) diabetes, 34 with alcoholic pancreatitis, 26 with rheumatoid arthritis and 107 normal control subjects. Both immunoblotting analysis and double-immunostaining methods were used to characterize the antigens targeted by the pancreatic exocrine cell autoantibodies. Sera positive for human pancreatic exocrine cell cytoplasm, producing a "fine fibrillar" pattern, were found in 21% (23/107) of the Type 1 diabetic patients. The autoantibodies were present in 39% (15/38) of Type 1 diabetic patients diagnosed within 3 months, and the prevalence decreased with duration of diabetes. The antibodies were of the IgM class in 87% (13/15) of recent-onset Type 1 diabetes cases, but IgG-autoantibodies became more prevalent with increasing duration of diabetes. Three out of 347 (0.9%) Type 2 diabetic patients and 4 of 20 (20%) first-degree relatives of Type 1 diabetic patients had autoantibodies targeted against pancreatic exocrine cells. None of the patients with alcoholic pancreatitis or rheumatoid arthritis and none of the control subjects had these antibodies. Immunoblotting analysis and double-immunostaining demonstrated that the autoantibodies reacted with 40 kilodalton cytokeratin in pancreatic exocrine cell cytoplasm. The antibody was absorbed by the Triton X-100-insoluble fraction of pancreatic extract. These results indicate the presence of distinct autoantibodies to pancreatic exocrine cells in Type 1 diabetes. This suggests the provocative concept that the cytoskeletal system of pancreatic exocrine cells is involved in the pathogenetic process of Type 1 diabetes.

Topics: Adolescent; Adult; Alcoholism; Arthritis, Rheumatoid; Autoantibodies; Chronic Disease; Diabetes Mellitus, Type 1; Family; Female; Fluorescent Antibody Technique; Humans; Islets of Langerhans; Keratins; Male; Pancreas; Pancreatitis; Reference Values