Biomedical subjects

D G Pipeleers

Publications and source records attributed to D G Pipeleers.

At least 19 recordsLinked to original sources

PubMed1998

Implantation of standardized beta-cell grafts in a liver segment of IDDM patients: graft and recipients characteristics in two cases of insulin-independence under maintenance immunosuppression for prior kidney graft.

Islet allografts in insulin-dependent diabetic (IDDM) patients exhibit variable survival lengths and low rates of insulin-independence despite treatment with anti-T-cell antibodies and maintenance immunosuppression. Use of poorly characterized freshly isolated preparations makes it difficult to determine whether failures are caused by variations in donor tissue. This study assesses survival of standardized beta-cell allografts in C-peptide negative IDDM patients on maintenance immunosuppression following kidney transplantation and without receiving anti-T-cell antibodies or additional immunosuppression. Human islets were isolated from pancreatic segments after maximal 20 h cold-preservation. During culture, preparations were selected according to quality control tests and combined with grafts with standardized cell composition (> or = 50% beta cells), viability (> or = 90%), total beta-cell number (1 to 2 x 10(6)/kg body weight) and insulin-producing capacity (2 to 4 nmol x graft(-1) x h(-1)). Grafts were injected in a liver segment through the repermeabilized umbilical vein. After 2 weeks C-peptide positivity, four out of seven recipients became C-peptide negative; two of them were initially GAD65-antibody positive and exhibited a rise in titre during graft destruction. The other three patients remained C-peptide positive for more than 1 year, two of them becoming insulin-independent with near-normal fasting glycaemia and HbA1c; they remained GAD65- and islet cell antibody negative. The three patients with surviving grafts presented a history of anti-thymocyte globulin therapy at kidney transplantation. Long-term surviving grafts increased C-peptide release following intravenous glucagon or oral glucose but not following intravenous glucose. Thus, cultured human beta-cells can survive for more than 1 year in IDDM patients on maintenance anti-rejection therapy for a prior kidney graft and without the need for an increased immunosuppression at the time of implantation. The use of functionally standardized beta-cell grafts helps to identify recipient and graft factors which influence their survival and metabolic effects. Insulin-independence can be achieved by injection of 1.5 million beta-cells per kg body weight in a liver segment. These beta-cell implants respond well to adenylcyclase activators but poorly to glucose.

Adolescent↗

PubMed1998

Extra-insular beta cells associated with ductules are frequent in adult human pancreas.

Routine immunohistochemical analysis of human donor pancreata indicated the frequent occurrence of single insulin-immunoreactive cells. In a quantitative analysis of nine organs consecutively recruited from adult donors, 15 percent of all beta cells were found in units with a diameter less than < 20 microm and without associated glucagon-, somatostatin-, or pancreatic polypeptide cells. These single beta-cell units are located in or along ductules, from which they appear to bud as previously noticed in fetal and neonatal organs. They contain significantly smaller beta cells than endocrine aggregates with a larger diameter. The use of ductal cell markers such as cytokeratin 19, carbonic anhydrase-II and carbohydrate antigen 19.9 identified a close topographical association between ductal cells and budding beta cells; it also indicated that pancreatic lobules are composed of nearly one third ductal cells. The presence of Ki67 proliferation marker-immunoreactive ductal cells (0.05 %) and absence of Ki67-immunoreactive budding beta cells is compatible with the view that beta cell neogenesis depends on ductal cell proliferation and differentiation. The high proportion of budding beta cells in the adult human pancreas suggests the presence of numerous loci with a potential for beta cell neogenesis.

Adolescent↗

PubMed1998

Increased prevalence of abnormal immunoglobulin M, G, and A concentrations at clinical onset of insulin-dependent diabetes mellitus: a registry-based study. The Belgian Diabetes Registry.

In view of the reported association of insulin-dependent diabetes mellitus (IDDM) with viral infections, (non-) islet-specific immune changes, and partial immunodeficiencies, we used immunonephelometry to measure circulating levels of IgM, IgG, and IgA in a registry-based group of IDDM patients under age 40 years at clinical onset (n = 397) and in age-matched nondiabetic siblings (n = 316) and control subjects (n = 322). Overall, IgM and IgA concentrations were higher, and IgG concentrations lower, in patients than in control subjects or siblings (P < 0.001). In siblings, IgM concentrations were higher than in control subjects (P < 0.001). Using age-adjusted reference intervals, abnormal Ig concentrations were noted in 27% of the patients at onset versus only 10% of the siblings and 7% of the control subjects (P < 0.001). For onset between 20 and 40 years (n = 220), 30% of the patients presented either increased IgM levels (21%) or decreased IgG levels (11%). Both independent phenomena occurred regardless of diabetes-associated immune and genetic markers. In patients and siblings, IgM levels were positively correlated with pancreatic lipase activity. In diabetic children (0-9 years; n = 65), a subgroup presented increased IgA levels (15%) in association with the highest HLA DQ-linked genetic risk and elevated IgM levels. The changes in total Ig concentrations at onset were largely reversed under insulin therapy. They may reflect exposure to environmental triggers, such as viral infections, or to (relative) insulinopenia prior to clinical disease onset. Whatever their cause, different serum Ig levels exist in different age groups of recent-onset IDDM patients.

Adolescent↗

PubMed1998

Intercellular differences in interleukin 1beta-induced suppression of insulin synthesis and stimulation of noninsulin protein synthesis by rat pancreatic beta-cells.

The normal pancreatic beta-cell population exhibits intercellular differences in its responsiveness to glucose. This cellular heterogeneity allows glucose to regulate, in a dose-dependent manner, total rates of insulin synthesis and release. It may also predispose to intercellular differences in susceptibility to dysregulating agents. The present study examines whether this is the case for interleukin 1beta (IL-1beta), which is known to suppress glucose-induced insulin synthesis and release. The effects of the cytokine were compared on beta-cell subpopulations with, respectively, high and low sensitivity to glucose. These subpopulations were separated on the basis of differences in the cellular metabolic responsiveness to an intermediate glucose concentration (7.5 mmol/liter) and then cultured for 20 h at 5 or 20 mmol/liter with or without IL-1beta. The suppressive action of IL-1beta (0.1 ng/ml) occurred predominantly in glucose-activated beta cells, reducing their high rates of insulin synthesis and release by more than 80%. Glucose-unresponsive cells became subject to a similar inhibition after their activation during culture at 20 mmol/liter glucose. On the other hand, IL-1beta induced or enhanced the expression of several noninsulin proteins in both subpopulations. The IL-1beta-stimulated expression of inducible nitric oxide synthase (iNOS) and heat shock protein 70 was more marked in the glucose-responsive subpopulation; that of heme oxygenase and Mn superoxide dismutase was comparable in the two subpopulations. Exposure to IL-1beta resulted in 10-fold higher medium nitrite levels in both subpopulations; this effect was prevented by the iNOS blocker, N(G)-methyl-L-arginine, which also prevented the IL-1beta-induced suppression in the glucose-responsive subpopulation. This study demonstrates that the cellular heterogeneity in glucose responsiveness predisposes to intercellular differences in the IL-1-induced suppression of insulin synthesis and release. While the cytokine induces the expression of noninsulin proteins such as iNOS in both glucose responsive and unresponsive cells, the subsequent nitric oxide production appears to predominantly affect glucose-stimulated functions in the glucose-activated cells.

Animals↗

PubMed1998

Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta-cells.

It has recently been reported that human adult beta-cells proliferate during culture on an extracellular matrix prepared from rat 804G cells and in the presence of hepatocyte growth factor (HGF). The present study compares the mitogenic effect of this condition on human beta-cells and on neighboring non-endocrine duct cells. Islet cell-enriched fractions were prepared from adult human organ donors and cultured in suspension or on 804G matrix, with or without HGF. The combination of 804G matrix and HGF increased the number of 5-bromo-2'-deoxyuridine-positive (BrdU+) cells within 48 h reaching a maximum after 4 days. In sections, virtually all BrdU+ cells were negative for insulin or glucagon and for preproinsulin mRNA but expressed the ductal cell markers cytokeratin 19 and 7, carbonic anhydrase-II, and carbohydrate antigen 19-9. After 4 days of culture, the cytokeratin 19+ ductal cells exhibited a BrdU-labeling index of 30% (P < 0.01 vs. 2% without HGF and matrix), whereas <0.1% of insulin-positive and <1% of glucagon-positive cells were labeled. Formation of bilayers with ductal cells covering the endocrine cells may cause erroneous interpretation on double positivity in unsectioned tissue. It is concluded that culture of human islet cell preparations with HGF and 804G matrix stimulates the proliferation of the duct cells but not of the underlying beta-cells.

Adult↗

PubMed1997

Assessment of insulin secretion in vitro from microencapsulated fetal porcine islet-like cell clusters and rat, mouse, and human pancreatic islets.

BACKGROUND: The possibility of transplanting microencapsulated pancreatic islets into patients with insulin-dependent diabetes mellitus, either as allografts or xenografts, has attracted great interest. A critical evaluation of the results obtained reveals that the success has been very limited. The aim of the present study was to compare the in vitro function of microencapsulated islets obtained from adult humans, adult mice, adult rats, and fetal pigs. METHODS: Human pancreatic islets were isolated at beta-Cell Transplant in Brussels, Belgium, and sent to the Department of Medical Cell Biology, Uppsala University in Uppsala, Sweden. Rat and mouse pancreatic islets and fetal porcine islet-like cell clusters (ICC) were prepared in Uppsala. All groups of islets were subsequently sent to the Department of Biotechnology, Norwegian Institute of Biotechnology, University of Trondheim, Trondheim, Norway. After 1 day in tissue culture, the islets were microencapsulated in alginate then cultured and sent back to Uppsala the next day. After either overnight culture (day 1) or 6 days of culture (day 6), the microencapsulated islets were examined for their insulin content and insulin release. Nonencapsulated islets from the same isolations were used as controls. RESULTS: The insulin content of rodent and human islets was not affected by microencapsulation, whereas porcine ICC showed a diminished insulin content. Microencapsulated porcine ICC also had a marked reduction in their insulin secretion in response to stimulation with glucose or glucose + theophylline both on days 1 and 6 in tissue culture. Mouse islets showed a reduced insulin response at both time points. Rat islets exhibited an inhibition of insulin secretion on day 1, but this had been restored by day 6. Human islets had well-preserved insulin secretion after both days 1 and 6. Microencapsulated human islets showed a normal morphology 3-4 weeks after intraperitoneal transplantation to nude mice. CONCLUSIONS: Pancreatic islets isolated from human, rat, and mouse donors show a glucose-stimulated insulin release in vitro after microencapsulation and repeated transports between laboratories. The insulin secretory capacity of microencapsulated human and rat islets was preserved best, whereas mouse islets and particularly fetal porcine ICC were impaired by microencapsulation.

Adult↗

PubMed1997

IA-2-autoantibodies complement GAD65-autoantibodies in new-onset IDDM patients and help predict impending diabetes in their siblings. The Belgian Diabetes Registry.

IA-2 has been identified as an autoantigen that is recognized by immunoglobulins from insulin-dependent diabetic (IDDM) patients. Using a liquid phase radiobinding assay, we performed an IA-2-autoantibody (IA-2-Ab) assay in 474 IDDM patients and 482 non-diabetic control subjects aged 0-3 years. IA-2-Ab were detected in 58% of the patients and 0.8% of control subjects. Their prevalence in patients was lower than that of islet cell autoantibodies (ICA; 73%) or glutamic acid decarboxylase (M(r) 65 kDa)-autoantibodies (GAD65-Ab; 82%) but higher than that of insulin autoantibodies (IAA; 42%). IA-2-Ab were more frequent in patients under age 20 years (70%) than between 20 and 40 years (45%; p < 0.001). In the whole IDDM group, 92% of patients were positive for at least one of the three molecular assays, which is higher than the positivity for the ICA assay (73%). Only 1% was negative in the molecular assays and positive in the ICA assay. IA-2-Ab levels were positively correlated with ICA titres (p < 0.001) and HLA DQ A1*0301-DQ B1*0.02 (p < 0.003) by multivariate analysis. In a group of 481 non-diabetic siblings (age 0-39 years) of IDDM patients only 7 were IA-2-Ab positive (1.5%). All seven were under age 20 years and positive for at least two other autoantibodies and for DQ A1*0301-DQB1*0302. Four of these seven developed IDDM during the 6-70-month follow-up period. The positive predictive value of IA-2-Ab (57%) was higher than that of ICA, GAD65-Ab or IAA alone, or in combination (< or = 20%) but these calculations are restricted by the relatively short observation period and the small number of cases. The only IA-2-Ab-negative case of pre-diabetes was also negative for IAA and GAD65-Ab, while it was strongly positive for ICA. In conclusion, IA-2-Ab show a high diagnostic specificity for IDDM and are predictive markers of impending diabetes in siblings of patients. In combination with other molecular antibody assays they may replace ICA testing in future. Our data also indicate that other autoantibodies than IA-2-Ab, GAD65-Ab and IAA contribute to ICA.

Adolescent↗

PubMed1997

Low density lipoprotein binding and uptake by human and rat islet beta cells.

Abnormalities in lipoprotein metabolism are common in diabetes. It is unknown whether variations in form or concentration of lipoproteins influence the function of pancreatic beta cells. This study investigates whether low density lipoproteins (LDL) exhibit specific interactions with islet beta cells. Radioactively labeled LDL (125I-LDL) and fluorescently labeled LDL (DiI-LDL) were used as tracers. Rat islet cells express high affinity LDL binding sites (K(d) = 9 nM), which are also recognized by very low density lipoproteins and which are down-regulated by LDL. Binding of LDL appears restricted to the beta cells, as it was not detected on islet endocrine non-beta cells. At 37 C, LDL is taken up and lysosomally degraded by islet beta cells but not by islet non-beta cells. Human islet cells were also found to present LDL binding, uptake, and degradation. Compared with rat islet cells, human islet cells exhibit 10-fold less binding sites (2.10(7) vs. 2.10(8) per 10(3) cells) with a 2-fold lower K(d) value (5 nM) and an equal sensitivity to LDL-induced down-regulation. In conclusion, human and rat islet beta cells express LDL receptors that can internalize the lipoprotein. This pathway should be examined for its potential role in (dys)regulating pancreatic beta cell functions.

Ammonium Chloride↗

PubMed1997

Cytokines induce deoxyribonucleic acid strand breaks and apoptosis in human pancreatic islet cells.

We have previously observed that a 6-day exposure of human pancreatic islets to a combination of cytokines (interleukin-1beta 50 U/ml + tumour necrosis factor-alpha 1000 U/ml + interferon-gamma 1000 U/ml) severely impairs beta-cell functions. In the present study, we examined whether this condition affects DNA integrity and viability of human islet cells. Cells were studied after 3, 6, and 9 days of cytokine treatment by both single cell gel electrophoresis (the "comet assay," a sensitive method for detection of DNA strand breaks) and by a cytotoxicity assay using the DNA binding dyes Hoechst 33342 and propidium iodide as indices for the number of viable, necrotic, and apoptotic cells. Cytokine treatment for 6 and 9 days resulted in a 50% increase in comet length (P < 0.01 vs. controls), indicating DNA strand breaks, as well as in a significant increase in the number of apoptotic cells (P < 0.02 vs. controls), but not in the number of necrotic cells. The arginine analogs N(G)-nitro-L-arginine and N(G)-monomethyl-L-arginine prevented nitric oxide formation by the cytokines but did not interfere with cytokine-induced DNA strand breaks and apoptosis. The present data suggest that prolonged (6-9 days) exposure of human pancreatic islets to a mixture of cytokines induces DNA strand breaks and cell death by apoptosis. These deleterious effects of cytokines appear to be independent of nitric oxide generation.

Analysis of Variance↗

PubMed1997

Associations of GAD65- and IA-2- autoantibodies with genetic risk markers in new-onset IDDM patients and their siblings. The Belgian Diabetes Registry.

OBJECTIVE: To investigate the association of GAD (65-kDa) autoantibodies (GAD65-Abs) and IA-2 autoantibodies (IA-2-Abs) with human leukocyte antigen (HLA)-DQ and insulin gene (INS) risk markers in patients with recent-onset IDDM and their siblings. RESEARCH DESIGN AND METHODS: Blood was sampled from 608 recent-onset IDDM patients and 480 siblings, aged 0-39 years and consecutively recruited by the Belgian Diabetes Registry, to determine GAD65- and IA-2-Ab (radiobinding assay), HLA-DQ- (allele-specific oligonucleotyping), and INS-genotypes (restriction fragment length polymorphism analysis; siblings, n = 439). RESULTS: At the onset of IDDM, GAD65-Abs were preferentially associated with two populations at genetic risk but only in the 20- to 39-year age-group: 1) their prevalence was higher in carriers of DQA1*0301-DQB1*0302 (88 vs. 73% in non[DQA1*0301-DQB1*0302], P = 0.001), and 2) an association was found in patients lacking this haplotype but carrying DQA1*0501-DQB1*0201, together with INS I/I (87 vs. 54% vs. non[INS I/I], P = 0.003). Siblings of IDDM patients also presented the association of GAD65-Abs with DQA1*0301-DQB1*0302 (13 vs. 2% non[DQA1*0301-DQB1*0302], P < 0.001), while associations with the second genetic risk group could not yet be assessed. At the onset of IDDM, IA-2-Ab prevalence was higher in carriers of DQA1*0301-DQB1*0302 (69 vs. 39% non[DQA1*0301-DQB1*0302], P < 0.001) but not of DQA1*0501-DQB1*0201 or INS I/I. This association was present in both the 0- to 19- and the 20- to 39-year age-groups. It was also found in siblings of IDDM patients (4 vs. 0% non[DQA1*0301-DQB1*0302], P < 0.001). CONCLUSIONS: Both GAD65- and IA-2-Abs exhibit higher prevalences in presence of HLA-DQ- and/or INS-genetic risk markers. Their respective associations differ with age at clinical onset, suggesting a possible usefulness in the identification of subgroups in this heterogeneous disease.

Adolescent↗

PubMed1997

Epidemiology, clinical aspects, and biology of IDDM patients under age 40 years. Comparison of data from Antwerp with complete ascertainment with data from Belgium with 40% ascertainment. The Belgian Diabetes Registry.

OBJECTIVE: To compare the incidence rate of IDDM in the age-groups 0-14 and 15-39 years in Antwerp, Belgium, and to compare demographic, clinical, and biological data from Antwerp IDDM patients with 92% ascertainment with those from a larger Belgian patient group with 40% ascertainment. RESEARCH DESIGN AND METHODS: Incident cases of IDDM were reported by physicians of the Belgian Diabetes Registry and in Antwerp by several other sources. In Antwerp, completeness of ascertainment was calculated by the capture-recapture method. Demographic and clinical data were collected by questionnaire. Blood was sampled for HLA-DQ genotyping and, in new-inset patients, for autoantibodies. RESULTS: In Antwerp, the age- and sex-standardized IDDM incidence rates were similar in both age-groups (0-14 years: 11.8/100,000; 15-39 years: 8.9/100,000). The incidence rate decreased in girls above age 15 years (6.9/100,000; P = 0.003) but not in boys (11.0/100,000). Both in Antwerp and Belgium, IDDM was diagnosed more frequently in the 15-39 years age-group (60% of all cases) than under age 15 years, with a lower prevalence of acute symptoms, ketonuria, high-risk HLA-DQ genotype, and autoantibodies against insulin, islet cells, and IA-2, but with a higher prevalence of GAD65 autoantibodies. CONCLUSIONS: In Antwerp, the incidence rate of IDDM under age 15 years is intermediately high compared with the rates in other European regions. It is similar in the 15-39 years age-group, but with a marked male predominance. Demographic, clinical, and biological data show the same age-dependent heterogeneity as the data collected nationwide, with 40% ascertainment indicating the representativeness of the latter.

Adolescent↗

PubMed1996

Prolonged exposure of human beta cells to elevated glucose levels results in sustained cellular activation leading to a loss of glucose regulation.

Human beta cells can be maintained in serum-free culture at 6 mmol/liter glucose, with 80% cell recovery and preserved glucose-inducible functions after 1 wk. Between 0 and 10 mmol/liter, glucose dose-dependently increases the number of beta cells in active protein synthesis (15% at 0 mmol/liter glucose, 60% at 5 mmol/liter, and 82% at 10 mmol/liter), while lacking such an effect in islet non-beta cells (> 75% activated irrespective of glucose concentrations). As in rat beta cells, this intercellular difference in glucose sensitivity determines the dose-response curves during acute glucose stimulation of human beta cells. During 2-h incubations, human beta cells synthesize 7 fmol insulin/10(3) cells at 0 mmol/liter glucose, 20 fmol at 5 mmol/liter, and 31 fmol at 10 mmol/liter. Culture at higher (10 or 20 mmol/liter) glucose does not affect beta cell recovery but decreases by 50-85% the net effect of glucose upon insulin synthesis and release. These reduced responses to glucose are not caused by diminished cellular activities but are the consequence of a shift of beta cells to a state of sustained activation. The presence of more activated cells at low glucose eliminates glucose-dependent cell recruitment as a mechanism for adjusting beta cell responses to acute variations in glucose concentration. It leads to elevated basal biosynthetic (3-fold) and secretory (10-fold) activities, and, hence, to a 4-fold reduction in the beta cell insulin content and the amount of insulin released at maximal glucose stimulation. Prolonged exposure of human beta cells to high glucose can thus lead to a loss of their glucose regulation as a consequence of sustained cellular activation, without signs of glucose-induced toxicity or desensitization.

Analysis of Variance↗

PubMed1996

Nitric oxide donors decrease the function and survival of human pancreatic islets.

Nitric oxide (NO) has been proposed as a possible mediator of beta-cell damage in human IDDM. This hypothesis is based on in vitro studies with rodent pancreatic islets. In the present study we examined whether human beta-cells are affected by NO. In view of species differences in beta-cell sensitivity to damaging agents, rat islets were investigated in parallel. Isolated islets were exposed for 90 min to different concentrations of three chemically unrelated NO donors, SIN-1, GSNO or RBS. At the end of this incubation, human insulin release was mostly similar in control and NO-treated islets but, 48 h later, islet retrieval, islet DNA and insulin content, and glucose-induced insulin release were markedly lower in islets exposed to NO donors. Rat islets were already inhibited during the initial 90 min; 48 h later their loss in beta-cell function was similar to that in human islets. Nicotinamide or succinic acid monomethyl ester partially protected against SIN-1 induced islet cell loss, but not against the functional inhibition of human pancreatic islets. Exposure of human or rat islets to RBS was associated with significant DNA strand breakage, as judged by the comet assay (single cell gel electrophoresis) and by ultrastructural signs of cell damage. DNA damage was more severe in rat islet cells exposed to similar amounts of RBS. It is concluded that NO donors can damage human pancreatic islets, an effect paralleled by induction of nuclear DNA strand breaks.

Adolescent↗

PubMed1996

Comparative immunocytochemical study of MHC class II expression in human donor pancreas and isolated islets.

Expression of major histocompatibility complex (MHC) molecules by pancreatic islets may influence the survival of pancreas or islet grafts in allogeneic recipients. This study compares the presence of MHC class II (HLA-DP, DQ, DX and DR)-positive cells in 27 pancreases and in 10 isolated islet preparations from human donors. Cells expressing MHC class II were present in all tissues examined as histiocytes located in interstitial areas in both the endocrine and nonendocrine components and as endothelial cells in the nonendocrine part. Endocrine, acinar and duct cells were MHC class II negative. In pancreases from donors under the age of 7 years the frequency of MHC class II-positive histiocytes was only one third of that in adults, and they rarely contained MHC class II-positive endothelial cells. The MHC class II-positive histiocytes were further phenotyped as macrophages positive for LCA and acid phosphatase, or dendritic cells negative for the latter markers. Dendritic cells were frequent in adult organs but rare in organs from donors under 7 years of age. In freshly isolated islet preparations from adult donors, less than 1% of the cells were MHC class II positive. These were identified as resident macrophages and dendritic cells. No MHC class II positive cells were encountered in the islet capillaries. The putative role of MHC class II-positive donor cells in allograft rejection suggests that these differences in MHC class II expression influence the immunogenicity of pancreatic and islet grafts in an age-dependent manner.

Adult↗

PubMed1996

Beta cells are important for islet innervation: evidence from purified rat islet-cell grafts.

Pancreatic islets receive an extensive and complex innervation that includes sympathetic, noradrenergic nerve fibres also storing neuropeptide Y. Islets transplanted to the kidney capsule become progressively reinnervated, mainly by sympathetic fibres and to a lesser extent by parasympathetic and sensory fibres. The density of nerve fibres in the islet grafts is often higher than in the graft-bearing organ, suggesting that the grafted islets contain factors that promote ingrowth of nerve fibres. To find out if beta cells are of any importance for attracting nerve fibres, purified preparations of rat islet beta and non-beta cells were transplanted to the kidneys of nude mice. Some of the mice were rendered diabetic by alloxan injection before transplantation. Immunocytochemical analysis revealed that the beta-cell grafts became richly reinnervated by noradrenergic (tyrosine hydroxylase-containing) nerve fibres, also storing neuropeptide Y. Non-beta islet-cell grafts were virtually devoid of demonstrable nerve fibres. There was no discernible difference in the reinnervation pattern between diabetic and non-diabetic mice. The findings indicate that factors mediating islet neurotrophism are produced by the beta cells.

Animals↗

PubMed1996

Structure and function of macroencapsulated human and rodent pancreatic islets transplanted into nude mice.

Macroencapsulation of human pancreatic islets inside biomembranes is a promising approach to maintain islet allografts in the diabetic recipient without immunosuppression. In order to test this possibility islets isolated from human pancreata were kept in culture before macroencapsulation in a tissue chamber device. The device consisted of two titanium rings, which supported two flat membranes. These membranes have previously been shown to protect pancreatic islets and fetal lung tissue from allograft rejection and also to promote neovascularization at the membrane surface. In a first series of experiments macroencapsulated human islets were implanted into the epididymal fat pad of athymic, nude mice concomitant to an injection from the same batch of islets under the kidney capsule. Light microscopy of encapsulated and subcapsularly grafted human islets showed that the survival inside the membranes was as good as under the kidney capsule. There was an extensive formation of new blood vessels at the membrane outer surface. In a second series of experiments insulin was extracted from encapsulated human islets implanted either into the epididymal fat pad or subcutaneously. The encapsulated human islets contained as much insulin as the non-encapsulated ones. In these experiments mouse and rat islets were also used. Rodent islets, however, survived less well than the human islets as evidenced by the markedly reduced insulin content values. In a third series of experiments human islets were loaded into the chambers and transplanted into nude mice without the concomitant implantation of non-encapsulated islets under the kidney capsule of the recipienets. Measurements of human C-peptide in serum samples obtained 4 to 8 weeks post-implantation showed considerable concentrations (0.70-185 ng/ml) in all animals. We conclude that isolated human islets survive when implanted into nude mice and continue to release insulin for several weeks. There are, however, species differences suggesting that rodent islets are much more susceptible to the environmental stress inside the membranes than human islets.

Adult↗

PubMed1996

Abnormal circulating pancreatic enzyme activities in more than twenty-five percent of recent-onset insulin-dependent diabetic patients: association of hyperlipasemia with high-titer islet cell antibodies. Belgian Diabetes Registry.

Pancreatic amylase and lipase activities were measured in sera of 307 Caucasian insulin-dependent diabetes mellitus patients (IDDM) at clinical onset, 303 nondiabetic siblings of registered patients, and 207 control subjects under age 40 years. In all subject groups lipasemia and pancreatic (but not salivary) amylasemia increased with age and were significantly correlated. Using age-dependent reference ranges, reduced pancreatic enzyme levels were measured in 18% of patients, 6% of siblings, and only 2% of control subjects (p < 0.001). Increased lipase levels were noted in 10% of patients and in only 3% of siblings and 2% of control subjects (p < 0.001). Using both univariate and multivariate statistical analysis, elevated lipase activities at clinical onset were associated with higher titers of autoantibodies against islet cell cytoplasmic antigens and glucagon, but not against insulin or the 65-kDa isoform of glutamic acid decarboxylase (GAD65-Ab), or with markers of genetic predisposition or metabolic dysregulation. These findings indicate the presence of modest, but statistically significant, variations in circulating pancreatic enzyme levels in 28% of IDDM patients at clinical onset (p < 0.001 vs. 5% in control subjects). Increased lipase levels may express a form or a stage of the disease with exocrine cell damage; their association with higher titers of islet cell and glucagon autoantibodies is not yet explained. Lower lipase and isoamylase levels are thought to result from the reduced acinar cell function in the vicinity of insulin-depleted islets. It must be tested whether pancreatic enzyme activities in serum can also be altered during the preclinical stage and can thus be considered as an additional marker for the disease process in the pancreas.

Adolescent↗

PubMed1996

Human pancreatic beta-cell deoxyribonucleic acid-synthesis in islet grafts decreases with increasing organ donor age but increases in response to glucose stimulation in vitro.

Human pancreatic beta-cell proliferation may be crucial for the success of islet transplantation. The aim of this study was to test the hypothesis that adult human beta-cells proliferate in vitro and in vivo and respond with increased rates of replication to factors known to promote rodent islet-cell proliferation, i.e. glucose, human recombinant GH, and FCS. For this purpose, human islets were prepared from a total of 19 adult heart-beating organ donors and cultured for 48 h with or without the additives described above. 3H-thymidine was added to the medium during the last 60 min of culture. After immunohistochemical staining for insulin and autoradiography, the labeling index (LI; i.e. % of labeled beta-cells over total number of beta-cells) was estimated by light microscopy. Islets also were transplanted under the kidney capsule of normal or alloxan-diabetic nude mice. After 2 weeks, 3H-thymidine was injected and the islet grafts prepared for determination of LI, as described above. Islets cultured at 5.6 mM glucose showed an increased beta-cell proliferation compared with islets cultured at 2.8 mM glucose (P < 0.05). However, culture at 11 mM glucose failed to further increase beta-cell proliferation. Addition of GH (1 microg/ml) to the medium, in the presence of 1% FCS and 5.6 mM glucose, did not influence the rate of beta-cell proliferation. In islets transplanted to hyperglycemic nude mice, beta-cell proliferation was similar to that observed in islets grafted into normoglycemic nude mice. Proliferation, however, decreased with increasing organ donor age. This study shows that pancreatic beta-cells from adult man are able to proliferate both in vitro and in vivo. Moreover, beta-cells from adult human donors respond with increased proliferation to glucose in vitro and show a decreased proliferation in vivo with increasing donor age.

Adolescent↗