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H Kolb

Publications and source records attributed to H Kolb.

At least 109 records · Page 6Linked to original sources
PubMed1997

MHC class II-dependent abnormal reactivity toward bacterial superantigens in immune cells of NOD mice.

Superantigens have been implicated in the pathogenesis of type I diabetes and other immune-mediated diseases. We therefore tested the hypothesis of an abnormal reactivity of the immune system toward bacterial superantigens during the prediabetic phase. For this purpose, splenocytes from NOD (H-2g7) mice were exposed to two well-characterized superantigens: Staphylococcal aureus enterotoxin-B (SEB) and toxic shock syndrome toxin-1 (TSST-1). Cells from BALB/c (H-2d) and C57BL/6 (H-2b) mice as well as those from NON (H-2non) and NOR (H-2g7) mice were used as controls. After 72 h of co-culture with the superantigens or the mitogen concanavalin A (Con A), proliferative response and mitochondrial activity were determined. In the culture supernatants, the cytokines gamma-interferon (IFN-gamma) and interleukin 10 (IL-10) were measured. Striking similarities between NOD cells and major histocompatiblity complex (MHC)-identical NOR cells could be observed with regard to a low proliferative and mitochondrial response to SEB, accompanied by a normal response to TSST-1 and Con A, respectively. In addition, only NOD and NOR spleen cells were low producers of the T-helper 1 (Th1) cytokine IFN-gamma in response to SEB. Conversely, abnormally high IFN-gamma levels were induced by TSST-1 in NOD and NOR spleen cells. The cytokine response to Con A was also biased toward IFN-gamma in both NOD and NOR. Since IFN-gamma and IL-10 are crucial disease-promoting or -protecting mediators in prediabetic NOD mice, superantigens may affect pathogenesis by acting on the Th1/Th2 cytokine balance. The low responder status toward SEB in NOD spleen cells may be of pathogenetic relevance in view of recent findings that the insulin B-chain also interacts with the SEB binding site on MHC class II molecules. In conclusion, we show here that immune cells from mice with a diabetes-associated MHC type respond differently to common environmental superantigens than do immune cells from control strains.

Animals↗
PubMed1997

Potential mechanisms by which certain foods promote or inhibit the development of spontaneous diabetes in BB rats: dose, timing, early effect on islet area, and switch in infiltrate from Th1 to Th2 cells.

Certain diets can have major effects on the development of IDDM in DP-BB rats, but data are scant on the timing, dose, and mechanisms involved. We therefore determined the dose response, timing, and duration of exposure required to induce diabetes, and characterized the effects of nutritionally adequate diets with widely different diabetogenicity on the pancreatic islet area and cytokines. DP-BB rats were fed a diabetogenic, cereal-based, NIH-07 (NIH) diet or a protective, casein or hydrolyzed casein (HC)-based, semipurified diet. Rats were fed from weaning to 50 or 100 days with the HC diet and then switched to the NIH diet, or fed the NIH diet from weaning to 50 days and switched to the HC diet. Pancreas histology and diabetes outcome were determined. Semiquantitative morphometric analyses of hematoxylin and eosin-stained sections of pancreas from 41-day-old rats were also carried out. Diet-induced effects on pancreatic cytokine levels were measured at 70 days using reverse transcriptase-polymerase chain reaction analysis of gamma-interferon (IFN-gamma), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-beta). Long-term daily exposure, particularly around the beginning of puberty to late adolescence (50-100 days), was important for development of diabetes. DP-BB rats could be rescued from diabetes development by feeding them a low-diabetogen HC diet as late as 50 days. Diabetes frequency was highest in rats fed 70% and 100% NIH diets. By age 41 days, before classic insulitis, the islet area in HC-fed DP-BB rats was 65% greater than in NIH-fed rats. By 70 days, when mononuclear cells were visible in the islets of most NIH-fed, but not HC-fed rats, the more pronounced inflammatory process in NIH-fed rats was associated with a Th1 cytokine pattern (high IFN-gamma and low IL-10 and TGF-beta), whereas the pancreases of HC-fed rats showed fewer infiltrating cells, low levels of IFN-gamma, and high levels of TGF-beta, typical of a Th2 cytokine pattern. Thus dietary modification can occur as late as puberty. Further, long-term exposure to sufficient amounts of food diabetogens between 50 and 100 days was required for maximum diabetes induction. The islet area was modified by diet before signs of classic insulitis. Pancreatic inflammation in NIH-fed animals is a Th1-dependent phenomenon. The HC diet inhibited insulitis and was associated with a Th2 cytokine pattern in the pancreas, protecting diabetes-prone rats from developing diabetes.

Age Factors↗
PubMed1997

Insulin therapy of prediabetes suppresses TH1 associated gene expression in BB rat pancreas.

Subcutaneous insulin treatment of young diabetes prone BB rats has been shown previously to suppress the development of autoimmune diabetes. In this study the hypothesis was tested that exogenous insulin may deviate the autoimmune process by acting on the Th1/Th2 cytokine balance in the pancreas. BB rats were implanted with pellets which continuously released insulin, at 50 d of age. Three weeks later cytokine mRNA expression in the pancreas and insulitis score were determined. While in control BB rats high levels of IFNgamma mRNA were detectable by RT-PCR, insulin treatment almost completely suppressed IFNgamma mRNA levels without concomitant upregulation of counterregulatory IL-10 and TGFbeta gene expression. Insulin also suppressed gene expression of inducible nitric oxide synthase. Mean insulitis scores were decreased after insulin treatment. We conclude that the protective effects of insulin treatment may not be due to the induction of protective Th2 immune reactivity but to general downregulation of immune activation in the pancreas, and hence also of Th1 autoimmunity.

Animals↗
PubMed1996

Primary nonfunction of islet grafts in autoimmune diabetic nonobese diabetic mice is prevented by treatment with interleukin-4 and interleukin-10.

In isologous islet transplantation in spontaneously diabetic nonobese (NOD) mice, destruction of the islet graft is caused by recurrence of T helper (Th)1-driven insulitis[fnc,1. We established a model of transplantation in which female NOD recipients were rendered diabetic by a single injection of cyclophosphamide (250 mg/kg). Under these conditions, 500 freshly isolated islets from young NOD mice transplanted under the kidney capsule did not lead to normoglycemia within 3 day after transplantation, but underwent immediate impairment of function. This primary nonfunction was seen in > 80% of the recipients. Treatment of the recipients with the Th2-associated cytokine interleukin (IL)-4 alone did not prevent primary nonfunction, whereas treatment of the recipients with a combination of IL-4 and IL-10 restored immediate function of the grafts. Cytokine treatment did not prevent later rejection of grafts. Histological analysis of the grafts revealed less severely infiltrated islets, with well preserved islet architecture, in only normoglycemic animals treated with IL-4 or with IL-4 and IL-10. Staining for lymphocytes, macrophages, and tumor necrosis factor (TNF)-alpha did not show differences between the groups, but IFN-gamma was markedly less expressed in IL-4- and IL-10-treated grafts. Concomitantly, analysis of animals treated for 8 days after injection of cyclophosphamide, with IL-4 and IL-10, revealed a reduction of IL-12 mRNA in the pancreas. We conclude from these data that primary nonfunction of islet grafts is prevented by treatment of the recipients with a combination of IL-4 and IL-10, via downregulation of Th1 cytokines.

Animals↗
PubMed1996

Heat shock protein hsp70 overexpression confers resistance against nitric oxide.

Heat stress is known to render rat islet cells resistant against the toxic effects of nitric oxide, reactive oxygen intermediates and the islet cell toxin streptozotocin. We report here for the first time that protection against nitric oxide is mediated by the major heat shock protein, hsp70, even in the absence of heat stress. The human hsp70 gene was stably transfected into the rat insulinoma cell line RINm5F. Constitutive expression of hsp70 caused protection from NO-induced cell lysis which was of the same extent as seen after heat stressing cells. Our results identify hsp70 as a defence molecule against nitric oxide.

Animals↗
PubMed1996

Hyperpolarizing, small-field, amacrine cells in cone pathways of cat retina.

Intracellular recording and horseradish peroxidase (HRP) staining of amacrine cells in the isolated arterially perfused cat retina have revealed examples of small-field cells that hyperpolarize to light. Two were examined in detailed electron microscopic reconstructions to determine patterns of synaptic relationships within the inner plexiform layer (IPL). The cells were morphologically similar to A8 and A13 types as described in Golgi-impregnated material (Kolb et al. [1981] Vision Res. 21:1081-1114). Both types received ribbon synaptic input from rod and cone bipolar cells. The latter input was numerically predominant, occurred in both a and b sublaminae of the IPL, and arose from at least three cone bipolar types. Reciprocal synapses were evident between A13 cells and cone bipolar cells. Amacrine input occurred throughout the dendritic tree of both A8 and A13 types, and numerically exceeded bipolar cell input for A13. Gap junctions between stained, and similar-appearing unstained dendritic profiles were observed for both amacrine types. In addition, A8 engaged in gap junctions with cone bipolar profiles in sublamina b which also provided ribbon input. Synaptic output for both amacrine types occurred primarily upon amacrine and ganglion cells in sublamina a. Both cells were presynaptic upon single OFF-center beta ganglion cells running through the middle of their dendritic trees. Mixtures of rod and cone signals were found in the centrally evoked hyperpolarizations of each type. Center mechanism space constants of such types ranged from 100 to 400 microns, with antagonistic surround in 1 of 5 cases. Dopamine (250 microM) reduced receptive field space constants by one-third in one case. The synaptic organization and potential circuitry implications of these cone system-dominated amacrine types are compared and contrasted to the better-known AII and A17 types previously described for the rod system.

Animals↗
PubMed1996

Interleukin-12 gene-expression of macrophages is regulated by nitric oxide.

Interleukin-12 is a heterodimeric cytokine, mainly produced by macrophages. In our present study we demonstrate that interleukin-12 expression is regulated by nitric oxide. Incubation of the macrophage cell line IC 21 with interferon-gamma gave rise to both interleukin-12 p40 mRNA and nitric oxide production. The concurrent addition of the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine inhibited nitrite production and in parallel completely suppressed interleukin-12 p40 mRNA formation. This indicated that endogenous nitric oxide synthase activity was required for IL-12 p40 gene expression. Exposure of the cells towards the nitric oxide generating compounds nitroprusside or S-nitroso-N-acetyl-penicillamine induced interleukin-12 p40 mRNA. Maximal mRNA levels were induced with nitric oxide donors at 1 microM concentration. We conclude that nitric oxide may exert an autoregulatory and paracrine control of interleukin-12 gene expression.

Animals↗
PubMed1996

Late pulmonary impairment following allogeneic bone marrow transplantation.

The pulmonary function of 88 consecutive leukemic patients who had undergone allogeneic bone marrow transplantation (BMT) was studied beforehand, at 3 months, at 6 months, and annually thereafter until 5 years after grafting. The parameters for function which are indicative for obstructive and restrictive lung disease deteriorated in all patient groups during the first 3 to 6 months after BMT but partially recovered within one year. Long-term decline in lung function was similar in all patient groups, and neither the onset nor the magnitude of pulmonary dysfunction was related to the occurrence of pulmonary impairment within 6 months after grafting. Multivariate analysis was then employed to assess predictors for long-term pulmonary disease. Despite the obvious effect of chronic graft versus host disease on the course of lung function, it was in itself not a significant predictor of long-term pulmonary outcome. Rather, the conditioning regimen turned out to be indicative; compared with busulfan, fractionated total body irradiation was demonstrated to be clearly superior with a lower incidence of both restrictive and obstructive long-term lung impairment. Our data indicate a previously unknown long-term side effect of busulfan conditioning.

Adult↗
PubMed1996

ON-OFF amacrine cells in cat retina.

We studied the morphology, photic responses, and synaptic connections of ON-OFF amacrine cells in the cat retina by penetrating them with intracellular electrodes, staining them with horseradish peroxidase, and examining them with the electron microscope. In a sample of seven cells, we found two different morphological types: the A19, which ramifies narrowly in stratum 2 (sublamina a) of the inner plexiform layer, and the A22, which ramifies mostly in stratum 4 (sublamina b) but extends some dendrites to sublamina a. Both of these cell types have axon-like processes that extend > 800 microns from the conventional dendritic arbor. ON-OFF amacrine cells in our sample had receptive fields (1.7 +/- 0.3 mm diameter) that were broader than their dendritic arbors (425 +/- 35 microns diameter) and that extended over the region of axon-like processes. In addition, we found many features in common with ON-OFF amacrine cells in poikilotherm vertebrates: a broad receptive field without surround antagonism, two sizes of spike-like events, narrow dynamic range (1 log unit intensity), and excitatory postsynaptic potentials at light on and light off. Two A19 amacrine cells were examined in the electron microscope: most synaptic inputs (93 and 76%, respectively) to either cell were from amacrine cells, with minor inputs from cone bipolar cells. Synaptic outputs were to bipolar, amacrine, and ganglion cells, including the OFF-alpha cell.

Animals↗
PubMed1996

HLA-associated inverse correlation between T cell and antibody responsiveness to islet autoantigen in recent-onset insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is a T cell-dependent immune-mediated disease. Recently, a novel islet cell antigen (ICA69) recognized by autoantibodies was described. We tested T cell responsiveness to ICA69 in peripheral blood of patients with recent onset IDDM (n = 46), patients with long-standing IDDM (n = 44), non-diabetic age-matched, islet cell autoantibody- and glutamic acid decarboxylase (GAD)65 antibody-negative first-degree relatives of IDDM patients (n = 15) and rheumatoid arthritis patients (n = 22). T cell responsiveness was significantly higher in recent onset IDDM patients, compared to IDDM patients post-disease onset, non-diabetic first degree relatives and rheumatoid arthritis patients (p < 0.001). In responding IDDM patients a significant inverse correlation between T cell and autoantibody responsiveness to ICA69 was observed (p < 0.0005). Immunogenetic evaluation revealed an association of HLA-DR3 with T cell responsiveness to ICA69 (p < 0.02) and absence of ICA69-reactive autoantibodies (p < 0.04). The increased T cell reactivity to ICA69 in the absence of antibody reactivity at onset of IDMM is associated with an HLA class II immune response gene, and therefore suggestive of a genetically controlled selective activation of T helper subsets to a specific autoantigen in humans.

Antibody Formation↗
PubMed1996

Differential expression of ICAM-1 and LFA-1 versus L-selectin and VCAM-1 in autoimmune insulitis of NOD mice and association with both Th1- and Th2-type infiltrates.

The infiltration of pancreatic islets by mononuclear cells is the hallmark of the development of insulin dependent diabetes mellitus (IDDM) in the NOD mouse, an animal model for human IDDM. The aim, of this study was to correlate adhesion molecule expression with the degree of islet infiltration and to compare Th1- and Th2-driven islet inflammation. Cryostat sections of NOD mouse pancreata before and after diabetes development were analysed by semiquantitative immunohistochemistry. NOD mouse islets did not show the expression of ICAM-1, LFA-1, L-selectin and VCAM-1 prior to infiltration by mononuclear cells. Furthermore, islets with early stage insulitis (grade 1, periinsular location of small infiltrates) still were devoid of adhesion molecule expression. ICAM-1 and LFA-1 were first demonstrable in islets with strong periinsular infiltrates (insulitis grade 2) while L-selectin and VCAM-1 were only seen in islets with mild or strong intraislet infiltration (grade 3-4). Adhesion molecules were demonstrable in areas of macrophage and T-lymphocyte infiltrates but not in adjacent endocrine islet tissue. Islets of all infiltration stages contained Th2 lymphocytes (positive for IL-4). Substantial numbers of Th1 cells (positive for IFN-gamma, TNF-alpha, IL-2 and/or IL-2 receptor) were observed only after acceleration of diabetes development by a single injection of cyclophosphamide (250 mg/kg i.p.). Interestingly, the adhesion molecule expression pattern in islets with "Th1' versus "Th2 insulitis' was not different. In conclusion, the expression of adhesion molecules in islets during the development of autoimmune diabetes does not precede mononuclear infiltration but probably occurs in response to the activation of initial small infiltrates. ICAM-1 and LFA-1 expression is seen prior to L-selectin and VCAM-1. However, adhesion molecule expression during Th1 versus Th2 cell infiltration is very similar, suggesting similar adhesion molecule requirements of the two Th subsets.

Animals↗
PubMed1996

Interleukin-12 gene expression is associated with rapid development of diabetes mellitus in non-obese diabetic mice.

A single dose of cyclophosphamide (250 mg/kg) is known to synchronize and accelerate development of diabetes in non-obese diabetic mice. We have reported previously that cyclophosphamide treatment of 10-week-old female non-obese diabetic mice induces a shift from T-helper type 2 to T-helper type 1 activity in islet lesions. We now show that this shift in regulatory T-cell function is preceded by the expression of interleukin-12 in the islets as well as in the spleen. In the spleen macrophages were identified as the interleukin-12 expressing cell type. At the same time there was little induction of tumour necrosis factor alpha gene expression by macrophages. Since interleukin-12 is well known to drive T-helper cell type 1 responses we assume that interleukin-12 released by macrophages mediates the accelerating effect of cyclophosphamide on islet inflammation in non-obese diabetic mice. mRNA expression of the p40 chain of interleukin-12 in response to cyclophosphamide was not seen in macrophages of Balb/c mice and thus represents an immune abnormality of non-obese diabetic mice favouring T-helper type 1 reactions.

Animals↗
PubMed1996

Cytokine gene expression in the BB rat pancreas: natural course and impact of bacterial vaccines.

In diabetes prone BB rat pancreas the Th1/ Th2 cytokine balance and the expression of inducible nitric oxide synthase (iNOS) was determined by mRNA analysis before and after the onset of insulitis. Specific mRNA was amplified by reverse transcriptase polymerase chain reaction, quantitated with radiolabelled probes by phosphoimaging and calibrated with the amount of co-amplified beta-actin mRNA. At 50 days of age, prior to recognizable insulitis, there was already significantly enhanced expression of both, Th1 and Th2 cytokines, and of iNOS mRNA, when compared to Wistar rat pancreas (p < 0.001). This supports the concept of an inconspicuous early phase of islet infiltration by single immunocytes, called single cell insulitis. At 70 days of age mononuclear infiltration of islets had begun and was associated with upregulation of interferon gamma (IFN gamma) and iNOS, but downregulation of interleukin-10 and transforming growth factor beta mRNA (p < 0.001). These findings correlate the onset of insulitis with a shift of the Th1/Th2 cytokine balance towards Th1 cell reactivity. Indeed there was a close correlation of the Th1/Th2 cytokine ratio but not of absolute IFN gamma mRNA levels with the insulitis score. Vaccination at day 50 with tetanus toxoid did not affect cytokine gene expression while diphtheria toxoid and even more strongly BCG administration induced a shift towards Th2 reactivity (p < 0.001) while iNOS mRNA was decreased (p < 0.01). Oral dosing with immunostimulatory components of Escherichia coli also changed the quality of inflammation. Oral lipopolysaccharide (LPS) from E. coli and OM-89, an endotoxin free extract containing immunostimulatory glycolipopeptides and heat shock protein (hsp) 65, both downregulated IFN gamma mRNA while only OM-89 in addition suppressed iNOS mRNA and enhanced Th2 cytokine gene expression (p < 0.001). We conclude that the onset of insulitis is associated with a shift towards Th1 cytokine and iNOS gene expression. Diphtheria toxoid and BCG vaccination stimulates Th2 reactivity but does not downregulate Th1. The latter can be achieved through oral administration of LPS or a glycopeptide fraction (OM-89) from E. coli.

Administration, Oral↗
PubMed1996

Metabolic activation of islet cells improves resistance against oxygen radicals or streptozocin, but not nitric oxide.

In the present study we investigated the effect of metabolic activation on the susceptibility of isolated rat pancreatic islet cells to the alkylating beta-cell toxin streptozocin (SZ), reactive oxygen intermediates (ROI), and nitric oxide (NO). The latter two represent physiologically occurring mediators involved in the autoimmune destruction of islet cells. ROI were generated by the enzyme xanthine oxidase, and NO was released from sodium nitroprusside. During 18 h of culture at a physiological glucose concentration (5 mmol/L), 75% of the islet cells were lysed by SZ, 81% by ROI, and 74% by NO, as determined by the trypan blue exclusion assay. Increasing concentrations of glucose or the nonnutrient stimulators theophylline and glibenclamide dose-dependently reduced SZ- and ROI-mediated islet cell lysis. In the presence of 29 mmol/L glucose, 5.5 mmol/L theophylline, or 10 micrograms/mL glibenclamide, SZ-induced lysis was reduced to 15%, 22%, or 15%, and ROI-induced lysis was reduced to 20%, 34%, or 15%, respectively. In contrast, stimulation by glucose, theophylline, or glibenclamide did not improve resistance against NO. The protection against SZ and ROI was associated with preserved mitochondrial activity, as determined by the ability of the islet cells to convert the tetrazolium salt 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide into its formazan. Elevation of the glucose concentration from 5.5 to 29 mmol/L increased the residual mitochondrial activity from 45% to 80% in SZ-exposed islet cells and from 21% to 78% in ROI-exposed cells. Conversely, the lack of protection against NO correlated with no preservation of mitochondrial activity in the presence of high concentrations of glucose, theophylline, or glibenclamide. In conclusion, our results show that metabolic stress does not render islet cells more susceptible to inflammatory insults in vitro. Rather, an increased mitochondrial energy supply improves the resistance against SZ and ROI, whereas the toxicity of NO was independent of islet cell activity.

Animals↗