Biomedical subjects

K Buschard

Publications and source records attributed to K Buschard.

At least 55 records · Page 3Linked to original sources

PubMed1997

Total number and size distribution of motor neurons in the spinal cord of normal and EMC-virus infected mice--a stereological study.

The encephalomyocarditis virus of the diabetogenic M-strain (EMC-M) is known to cause diabetes in mice. The EMC-M virus has also been shown to cause paresis in some of the infected animals. The clinical features include an acute ascending predominantly motor paralysis, developing within days. This resembles acute idiopathic polyneuritis. The alpha motor neurons would be a possible target for the virus, so two parameters, the total number and the size distribution of motor neurons, were therefore selected for further investigation in 6 mice with neurological involvement and compared with 6 control mice. The optical fractionator method was applied for estimating the total number of motor neurons and the 3D size distribution was estimated using the rotator method in a vertical design. No difference was found in the total number of motor neurons and the size distributions were similar in the 2 groups. This design can be used as a model for the estimation of the total number of motor neurons and their size distribution in other experimental animal models.

Animals↗

PubMed1997

Adrenaline stimulates glucagon secretion in pancreatic A-cells by increasing the Ca2+ current and the number of granules close to the L-type Ca2+ channels.

We have monitored electrical activity, voltage-gated Ca2+ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na+- and Ca2+-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca2+ influx through omega-conotoxin-GVIA-sensitive (N-type) Ca2+ channels. Stimulation of the A-cells with adrenaline (via beta-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca2+ influx through L-type Ca2+ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca2+ channels.

Animals↗

PubMed1997

Glucagon-like peptide I and glucose-dependent insulinotropic polypeptide stimulate Ca2+-induced secretion in rat alpha-cells by a protein kinase A-mediated mechanism.

High-resolution capacitance measurements were used to explore the effects of the gut hormones GLP-I(7-36) amide [glucagon-like peptide I(7-36) amide] and GIP (glucose-dependent insulinotropic polypeptide) on Ca2+-dependent exocytosis in glucagon-secreting rat pancreatic alpha-cells. Both peptides produced a greater than threefold potentiation of secretion evoked by voltage-clamp depolarizations, an effect that was associated with an approximately 35% increase of the Ca2+ current. The stimulatory actions of GLP-I(7-36) amide and GIP were mimicked by forskolin and antagonized by the protein kinase A (PKA)-inhibitor Rp-8-Br-cAMPS. The islet hormone somatostatin inhibited the stimulatory action of GLP-I(7-36) amide and GIP via a cyclic AMP-independent mechanism, whereas insulin had no effect on exocytosis. These data suggest that the alpha-cells are equipped with receptors for GLP-I and GIP and that these peptides, in addition to their well-established insulinotropic capacity, also stimulate glucagon secretion. We propose that the reported inhibitory action of GLP-I on glucagon secretion is accounted for by a paracrine mechanism (e.g., mediated by stimulated release of somatostatin that in turn suppresses exocytosis in the alpha-cell).

Animals↗

PubMed1997

Mononuclear cytotoxicity and proliferation towards glucose stimulated rodent pancreatic islet cells.

Diabetes is due to an autoimmune cellular immunologic destruction of the pancreatic beta cells. By the use of a chromium release assay and a proliferation assay we have investigated the possible role of beta cell activity for this destruction. Results show that in vitro glucose stimulated pancreatic islet cells are subjects to a slight but significantly higher cellular immunologic destruction by mononuclear spleen cells than unstimulated islet cells. The functional dependency of the islet cell destruction must be a product of both a mononuclear cell dysfunction and a specific islet cell pattern. This is due to the fact that all combinations of mononuclear cells and islet cells from diabetes prone BB rats and non-diabetes prone WF rats tested against each other, results in functional dependent cytotoxicity, except for the assay in which both effector cells and target cells are of WF rat origin. Additional observations indicate, that the diabetes prone BB rat mononuclear cells need previous in vivo activation as only cells from diabetic individuals, and not normoglycemic ones, display the reaction in question. Functional dependent cytotoxicity is validated in an other IDDM animal model--the NOD mouse. NOD mononuclear cells towards the murine MIN-6 beta cell line results in increased cellular cytotoxicity when the latter is glucose stimulated. Also the proliferative response of BB rat mononuclear cells to whole islets tend to show function dependency.

Animals↗

PubMed1996

Presence of sulphatide (3'-sulphogalactosylceramide) in pericytes in the choroid layer of the eye: sharing of this glycolipid autoantigen with islets of Langerhans.

The aim of the study was to investigate the distribution in the eye of sulphatide, an acid glycolipid which has previously been demonstrated in islets of Langerhans, nervous tissue and in kidney glomeruli of diabetic patients, and against which antibodies have been found in patients with newly diagnosed insulin-dependent diabetes mellitus. A specific monoclonal antibody, Sulph I, was used for detection of sulphatide by thin-layer chromatography, and light and electron microscope immunohistochemistry. A distinct, patchy staining was found in the choroid layer and the ciliary processes. The antigen was confirmed to be sulphatide and its concentration in human eyes was 30 nmol sulphatide/g wet tissue. By electron microscopy, anti-sulphatide choroid labelling was demonstrated in pericytes and in smooth muscle cells surrounding vessels. No Sulph I-negative pericytes were seen. Double labelling with Sulph I and anti-smooth muscle actin revealed that only pericytes in the eye contained sulphatide and not those in heart, lung, liver, adrenal, spleen, lymph node, thymus, or pancreatic tissue. Thus, sharing of the autoantigen sulphatide has been demonstrated between islets of Langerhans and pericytes in the choroid layer of the eye.

Aged↗

PubMed1996

GAD65 autoantibodies in women with gestational or insulin dependent diabetes mellitus diagnosed during pregnancy.

We have studied the presence of GAD65 autoantibodies in women with insulin-dependent diabetes mellitus (IDDM) (n = 28) or gestational diabetes (GDM) (n = 139) diagnosed during pregnancy and investigated the temporal relationship between these autoantibodies and the subsequent recurrence or development of IDDM. Among the GDM patients, 4.3% (6 of 139) developed true IDDM during a median follow-up period of 6.3 years (range 4.0-11.0). Of these, 2.2% (3 of 139) were positive for GAD65 autoantibodies at diagnosis of GDM compared to 0% (0 of 27) of healthy pregnant women. All 3 GAD65 autoantibody positive GDM patients subsequently developed IDDM after a median of 14 months (range 4-34). GAD65 autoantibodies were present in 50% (14 of 28) of sera from women with IDDM diagnosed during pregnancy. The non-insulin-requiring remission period was significantly shorter in GAD65 autoantibody positive patients (median 0.5 years [range 0-6.0 years]) than in GAD65 antibody negative patients (median 2.6 years; range 0-9.7 years; p < 0.05). The results suggest that screening for GAD65 autoantibodies in women with GDM or IDDM diagnosed during pregnancy may be useful for predicting the clinical course of the disease.

Adolescent↗

PubMed1996

Diabetic animal models.

Insulin-dependent (type 1) diabetes is a frequent disease with an incidence of up to about 1%. It requires daily treatment and serious late complications are observed. Good animal models exist for studying diabetes. These can be categorized as animals with spontaneously developing diabetes (BB rats, NOD mice) and as animals with induced diabetes (e.g. by virus). Immunodeficient nude mice have also been widely used. None of the models is perfect, but each has contributed to our present knowledge of the disease. Studies on the pathogenesis of type 1 diabetes are given as an example. Recently, experience with prophylactic treatment of animals in order to prevent diabetes has been applied to humans with promising results.

Animals↗

PubMed1996

Sulphatide and its precursor galactosylceramide influence the production of cytokines in human mononuclear cells.

Sulphatide is expressed in the central and peripheral neural system, in islets of Langerhans, and in tissues affected by late diabetic complications. Autoantibodies to sulphatide are present in patients with insulin-dependent diabetes and the Guillain-Barré syndrome. Cytokines influence these disease processes, and we therefore studied whether sulphatide and its precursor galactosylceramide (gal-cer) influence the in vitro production of cytokines by blood mononuclear cells (MNC) originating from 15 healthy persons. Using lipopolysaccharide (LPS)-stimulated cells, sulphatide increased the IL-2 production (163 +/- 17% of controls without sulphatide, p = 0.02), and gal-cer increased the IL-1 alpha production (145 +/- 13%, p = 0.006), whereas neither gal-cer nor sulphatide had an effect on the production of IL-6, IL-10 or TNF alpha. When stimulating cells with phytohaemagglutinin (PHA), sulphatide decreased the production of IL-6 (88 +/- 5%, p = 0.009), IL-10 (66 +/- 3%, p = 0.000003), and TNF alpha (75 +/- 9% p = 0.02). Gal-cer, however, increased the production of IL-6 (188 +/- 13% p = 0.000006), and decreased the production of TNF beta (80 +/- 6%, p = 0.007). Neither gal-cer nor sulphatide had an effect on the production of IL-2 or IFN gamma from PHA-stimulated cells. Northern blot analysis using an IL-6 probe similarly showed an increased amount of IL-6 mRNA after gal-cer incubation (range 469%-150%, n = 3) of PHA-stimulated control. Thus, sulphatide and gal-cer influence the production of several cytokines thought to be involved in immunoinflammatory disease processes.

Cytokines↗

PubMed1996

Fluorescence-activated cell sorted rat islet cells and studies of the insulin secretory process.

Studies of individual cell types in the islets of Langerhans are complicated by the cells' functional coupling by gap junctions and paracrine interaction. Access to purified alpha and beta cells is therefore desirable. We present a simplified and optimized method for fluorescence-activated cell sorting of endocrine pancreatic rat islets. For dispersion of the islets, dispase was superior to trypsin, as the number of vital single cells was higher (1.1 +/- 0.1 x 10(3) vs 0.6 +/- 0.1 x 10(3)/islet, P < 0.05). The purity of the sorted cells was 96.7 +/- 1.2% for the non-beta cells and 97.8 +/- 0.6% for the beta cells (numbers in percentages of endocrine cells). In culture, isolated beta cells, non-beta cells and mixtures of beta and non-beta cells formed aggregates, but not at low temperature (4 degrees C) and not in medium with low serum content (2%). Finally, in pure beta cell aggregates, glucose stimulated changes in cytoplasmic free Ca2+ concentration although both glucose- and arginine-induced insulin secretion was much reduced. We conclude that alpha cells are necessary for insulin secretion but not for glucose sensing.

Animals↗

PubMed1996

Differential responsiveness to interferon-alpha in beta-cells and non-beta cells.

Interferon-alpha (IFN-alpha) is important in the innate immune defense, particularly in viral infections. IFN-alpha induces 2',5'A synthetase, the products of which, 2',5'-oligoadenine nucleotides, activate mRNA degrading enzymes. IFN-alpha is the first detectable cytokine in the insulitis lesion seen in recent-onset IDDM, and insulin promoter directed expression of IFN-alpha in transgenic mice leads to development of IDDM. Here, we demonstrate that IFN-alpha induces 2',5'A synthetase activity only in insulin-producing betaTC3 cells and in isolated single rat beta-cells but not in alphaTC3 cells or in isolated rat non-beta-cells. The increased responsiveness of beta-cells but not non-beta-cells to IFN-alpha with the ensuing activation of the mRNA-degrading 2',5'A synthetase system suggests why only the beta-cells are destroyed in the diabetogenic process.

2',5'-Oligoadenylate Synthetase↗

PubMed1996

Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes.

The establishment of gene delivery systems that result in efficient transfection of the pancreatic beta-cells may generate an important tool for the study of IDDM and may also represent one critical step toward a clinical application of gene transfer for the prevention or early treatment of the disease. Using the reporter gene vectors pCAT and pCMV beta-gal, we have investigated the efficiency of transfection mediated by calcium phosphate precipitation, the monocationic liposome Lipofectin, the polycationic liposome Lipofectamine, and adenovirus-polylysine (AdpL) DNA complexes in human, mouse, rat, and fetal porcine islet cells. In all species studied, calcium phosphate-mediated transfection resulted in lower chloramphenicol acetyl transferase (CAT) activities than the other methods. Intact human, mouse, and rat islets were poorly transfected by Lipofectin, Lipofectamine, and AdpL. When dispersed by trypsin treatment, however, human, mouse, rat, and fetal pig islect cells were efficiently transfected by Lipofectamine. Moreover, transfection of dispersed human and mouse islet cells using AdpL, also resulted in high CAT activities. The percentage of cells staining positively for beta-galactosidase after transfection with Lipofectamine was 49% for mouse, 56% for rat, and 57% for dispersed human islet cells. Transfection of human islet cells using AdpL, however, yielded 70% beta-gal-positive cells. Fluorescence-activated cell sorting-purified rat islet alpha- and beta-cells were transfected with similar efficiency using Lipofectamine. CAT expression in human islet cells transfected with either Lipofectamine or AdpL reached a peak value after 5-7 days, followed by a gradual decline. It is concluded that transfection with AdpL or Lipofectamine are both efficient means to achieve transient expression of gene constructs in human and mouse islet cells, while for rat and fetal porcine islet cells, Lipofectamine is the most efficient of the agents investigated in this study.

Analysis of Variance↗

PubMed1995

Tissue culture supernatants from human islets of Langerhans activate the oxidative burst response of human monocytes in vitro.

Macrophages play a major role in the pathogenesis of insulin-dependent diabetes mellitus in animals. These cells are the first to invade the pancreas and macrophage-eradicating treatments reduce the incidence of the disease. In humans, however, their role is less clear. In this study we investigated the hypothesis that the pancreatic environment per se could activate macrophages. Tissue culture supernatants from human islets of Langerhans were tested for chemotactic activity and oxidative burst response in monocytes isolated from healthy adults. Preincubation with the supernatants enhanced the oxidative burst response evoked by fMLP (up to 379%) and opsonized zymosan (up to 173%). The activity decreased by dilution and was no longer detectable at 1:16. No increased activity was seen in supernatants from a number of other human endocrine and non-endocrine primary cells, suggesting a factor specific for islet tissue. The increased oxidative burst response could partially be eliminated by heat- and proteinase K treatment, suggesting that the activity could be of polypeptide nature. The factor could not be absorbed by polyvalent rabbit antibodies directed towards a variety of cytokines not by a mixture of high-titer anti-cytokine antibodies. It is possible that islet factors could also promote such monocyte activation in vivo in monocytes attracted to the islets of Langerhans by other means. This could contribute to the development of insulin-dependent diabetes in humans.

Adolescent↗

PubMed1995

Age-related changes in T gamma delta cells of NOD mice.

T gamma delta cells have been reported to recognize both mycobacterial and human heat-shock proteins (HSP), and a possible role of 65 kDa HSP has been suggested also in the pathogenesis of insulin-dependent diabetes mellitus. The aim of this study was to investigate age-related changes of T gamma delta cells during diabetes development in non-obese diabetic (NOD) mice. Using FACS analysis relative numbers of T gamma delta + cells from thymus, blood and spleen were determined in a 3-week-old non-diabetic, at onset of diabetes, and 1-week diabetic NOD mice and corresponding BALB/cJ controls. In comparison to BALB/cJ mice, higher values (2.4 +/- 0.2% vs. 1.1 +/- 0.1%) were found in the thymus of 3-week-old NOD mice (P < 0.01) as well as spleens of 22-week-old littermates (1.1 +/- 0.1% vs. 0.6 +/- 0.1%, P < 0.01). In addition, a higher proportion of T gamma delta cells was observed in blood samples of all age groups of NOD as compared to BALB/cJ mice, with values 3.5 +/- 0.7% (P < 0.05) in 3-week-old to 4.4 +/- 0.9% and 3.7 +/- 0.3% (P < 0.01) in 16- and 22-week-old NOD littermates. Differences in TCR gamma delta expression did not influence the whole CD3+ subset of mononuclear cells. Thus, our results show relatively higher numbers of T gamma delta cells in NOD mice and their increase in the periphery at onset of diabetes and later may suggest that T gamma delta cells participate in beta-cell destruction.

Aging↗

PubMed1995

Interleukin-1 beta induces the expression of hsp70, heme oxygenase and Mn-SOD in FACS-purified rat islet beta-cells, but not in alpha-cells.

The cytokine IL-1 beta has previously been demonstrated to induce the expression of the stress genes iNOS, hsp70, heme oxygenase and Mn-SOD in rat pancreatic islets in vitro. The aim of this study was to determine whether the IL-1 beta-induced effects are specific for the insulin producing beta-cell, or whether other islet cells, such as the glucagon-producing alpha-cell, respond to IL-1 beta addition. Purified rat alpha- and beta-cell suspensions were obtained by fluorescence-activated cell sorting and incubated with or without IL-1 beta (25 U/ml) for 24 h. The alpha- and beta-cell contents of hsp70, heme oxygenase and Mn-SOD and medium nitrite levels were determined. It was found that IL-1 beta exposure induced the production of nitric oxide in beta-cells, but not in alpha-cells. Moreover, the expression of hsp70, heme oxygenase and Mn-SOD was also induced in beta-cells, but not in alpha-cells. There were no detectable levels of hsp70 in alpha-cells. It is concluded that the stress gene response following IL-1 beta exposure is markedly different in alpha- and beta-cells. This finding may be of importance for the understanding of the autoimmune destruction of beta-cells in insulin-dependent diabetes mellitus.

Animals↗

PubMed1995

Neonatal treatment of BB rats with sulphatide delays development of diabetes but does not change incidence.

Sulphatide is a newly described autoantigen in insulin-dependent diabetes mellitus; it is present in the islets of Langerhans, and anti-sulphatide antibodies are found in diabetic patients and in spontaneously diabetic BB rats. The aim of the study was to treat neonatal BB rats with sulphatide in order to induce tolerance and thereby possibly influence later diabetes development. One hundred and twelve newborn BB rats, divided into three groups, were treated once daily during the first 6 days of life with intrathymic injections of sulphatide, galactosyl-ceramide (which is similar to sulphatide but without sulphate) or phosphate buffer alone. Although the results showed no difference in diabetes incidence among the three groups, there was a delayed onset of diabetes in the sulphatide-treated group, which developed diabetes on average at 77 +/- 1 days of age, compared to 70 +/- 2 days (p < 0.02) for the galactosyl-ceramide-treated group and 70 +/- 1 days (p < 0.01) for the buffer-treated group. The degree of insulitis and the size of islets of Langerhans were studied histologically for the diabetic animals in all three groups; there were no significant differences although the sulphatide-treated group tended to have a more normal histology. The blood glucose levels for the diabetic BB rats were similar in all three groups. Thus, neonatal treatment with the diabetic autoantigen sulphatide at the chosen dosage does not influence the incidence of diabetes, but delays the onset of disease.

Animals↗

PubMed1995

Cytotoxicity towards neonatal versus adult BB rat pancreatic islet cells.

Cell-mediated autoimmunity is believed to influence the development of diabetes in BB rats. It has been suggested that the autoimmune destruction is due to lack of tolerance induction in early neonatal life caused by delayed maturation of the pancreatic beta cells. The present experiment has been initiated to investigate if there is any difference in in vitro cytotoxicity, and therefore antigenicity, to islet cells from neonatal, young and adult diabetes prone BB rats, and to establish the possible developmental difference in these rats compared to non diabetes prone Wistar Furth rats. Islets from rats of different ages were isolated, dispersed and 51C-labeled. In vitro cytotoxicity mediated by mononuclear spleen cells from newly diabetic BB rats was measured by counting gamma-ray emission from the culture supernatant after 16 h coincubation. We found that full adult-like islet cell maturation in BB rats--as evidenced by sensitivity to cytotoxicity--is not seen before the age of 8 to 21 days after birth. In contrast adult-like cytotoxicity to neonatal islets cells from Wistar Furth rats is seen already at the age of 8 days. Thus delayed islet cell maturation is a fact observed in BB rats.

Aging↗

PubMed1994

Sulphatide in islets of Langerhans and in organs affected in diabetic late complications: a study in human and animal tissue.

Sulphatide has been found in rat islets of Langerhans and anti-sulphatide antibodies have been demonstrated in patients with insulin-dependent diabetes mellitus. Using a specific monoclonal antibody, Sulph I, directed against sulphatide, we investigated the in situ distribution of this glycolipid immunohistochemically; furthermore, the sulphatide concentration was determined in several organs and cells by thin-layer chromatography. The islets of Langerhans in all species examined, mouse, rat, pig, and monkey were intensively stained but exocrine tissue remained unlabelled. The sulphatide concentration in human islets was 150 +/- 46 pmol/100 islets. The only glycolipid-antigen detected was sulphatide. Regarding other tissues, sulphatide was found to be located in distal tubules in the kidney, peripheral nerves, distinct scattered spot-like structures in the choreoid layer of the eye, the ovum, and peripheral granulocytes. Sulph I injection in mice showed homing to kidney tubules, Lung, heart, liver, adrenal, spleen, lymph node and thymus were not stained by Sulph I. Thus, the distribution of sulphatide shows an association with organs known to be affected in diabetes, either initially or in late complications.

Adult↗

PubMed1994

Effects of 1,25-dihydroxyvitamin D3 and the analogues MC903 and KH1060 on interleukin-1 beta-induced inhibition of rat pancreatic islet beta-cell function in vitro.

The cytokine interleukin-1 beta (IL-1 beta) has been proposed to be involved in pancreatic beta-cell destruction during the development of autoimmune insulin-dependent diabetes mellitus. It has been demonstrated that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) inhibits T-lymphocyte and monocyte functions in vitro, probably through an effect on cytokine actions, and that in vivo treatment with vitamin D can prevent pancreatic insulitis in diabetes-prone NOD mice. In this study isolated rat pancreatic islets were exposed to human IL-1 beta (25 U/ml) in the absence or presence of 1,25-(OH)2D3 or the analogues MC903 and KH1060 for 48-72 h in tissue culture, whereupon medium insulin accumulation, islet DNA and insulin contents, glucose-stimulated insulin secretion and glucose oxidation rates were assessed. All three vitamin D derivatives counteracted the suppressive effect of IL-1 beta on medium insulin accumulation, 1,25-(OH)2D3 being active at concentrations down to 0.1 nM, i.e., 1-2 orders of magnitude more efficacious than the analogues. However, only KH1060 opposed the suppressive effect of IL-1 beta on islet glucose-stimulated insulin secretion and glucose oxidation rate despite the fact that KH1060 itself reduced the islet DNA and insulin content by approximately 10% and 30%, respectively. The protective effect observed against IL-1 beta-induced beta-cell dysfunction might be related to a beneficial action of vitamin D3 on the mitochondrial calcium metabolism of the beta-cells.

Animals↗