Biomedical subjects

L Bouwens

Publications and source records attributed to L Bouwens.

At least 37 records · Page 2Linked to original sources

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

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

Proliferation and differentiation in the human fetal endocrine pancreas.

The morphogenesis and growth of the endocrine pancreas has not been well investigated in man although it represents an important issue in diabetology. We examined human fetal pancreas from 12 to 41 weeks of gestation immunocytochemically to evaluate proliferative activity with the Ki-67 marker, and cytodifferentiation with cytokeratin 19 (ductal cells), synaptophysin (all endocrine cells), and insulin, glucagon, somatostatin and pancreatic polypeptide (islet cell types). Ki-67 labelling was found in all these cell types but was much higher in ductal cells than in islet cells. An intermediate population expressed synaptophysin but lacked islet hormones. With increasing gestational age the Ki-67 labelling index decreased from 17 to 4% in ductal cells, from 9 to 1% in synaptophysin-positive cells, and from 3 to 0.1% in insulin- or glucagon-positive cells. From 12 to 16 weeks, all epithelial cells including the endocrine islet cells expressed cytokeratin 19. Thereafter cytokeratin 19 expression decreased and eventually disappeared from most islet cells, whereas strong expression remained in the ductal cells. We show that differentiated human islet cells have only very limited proliferative capacity, and we demonstrate the existence of transitional differentiation stages between ductal and islet cells.

Biomarkers↗

PubMed1997

Effect of interferon-gamma and glucose on major histocompatibility complex class I and class II expression by pancreatic beta- and non-beta-cells.

Surface major histocompatibility complex (MHC) class I and class II expression by pancreatic islet cells is considered a local initiator or regulator of immune processes that can lead to diabetes. Locally released cytokines, in particular interferon-gamma, are known to stimulate MHC antigen expression by islet cells. The present study quantifies MHC expression in cultured pancreatic beta- and non-beta-cells from both rat and human organs. Interferon-gamma increased MHC class I expression in endocrine beta- and non-beta-cells as well as in pancreatic ductal cells. The cytokine induced a 6-fold increase in the MHC class I messenger ribonucleic acid levels in pancreatic beta-cells; this effect was 2-fold amplified in the presence of elevated glucose levels (20 mmol/L instead of 6 mmol/L). No MHC class II expression was observed in endocrine beta- or non-beta-cells; human, but not rat, ductal cells exhibited MHC class II expression that increased in the presence of interferon-gamma. These data indicate that the increase in beta-cell MHC class I expression described in the pancreata of diabetic patients may result from stimulated transcription after exposure to locally released interferon-gamma and/or to a hyperglycemic state. The association of human islets with ductal cells in which MHC class II expression is stimulated by interferon-gamma makes these cells potential participants in the autoimmune process in diabetes.

Adolescent↗

PubMed1997

Effect of vascular endothelial growth factor on growth and differentiation of pancreatic ductal epithelium.

Endocrine and exocrine pancreatic morphogenesis is known to occur from ductal epithelium, but the factors that regulate this process are unknown. Vascular endothelial growth factor (VEGF)/vascular permeability factor has recently been reported to affect fetal islet ontogenesis. VEGF is an angiogenic factor with a growth-promoting effect that is thought to be restricted to vascular endothelial cells. We demonstrated that VEGF is also a mitogen for adult rat pancreatic duct epithelial cells in primary culture. VEGF supplementation to a serum-free culture medium increased the 5-bromo-2'-deoxyuridine-pulse labeling index of ductal cells more than 2-fold. Immunohistochemical staining and protein blots revealed that pancreatic duct cells express fetal liver kinase-1 high-affinity receptors for VEGF. In pancreatic tissue, immunohistochemistry shows that VEGF peptide is expressed in normal pancreatic islet cells. In duct ligation-induced acute pancreatitis, numerous inflammatory leukocytes containing VEGF were seen to infiltrate between hyperplastic ducts. In the latter model, islet neogenesis has previously been observed. Our data indicate the possibility that VEGF plays a role in the paracrine regulation of ductal growth and differentiation in vivo, eg, in pancreatitis. In vitro, however, VEGF did not induce endocrine differentiation of ductal cells, indicating that it is not the only factor required for the activation of islet neogenesis.

Animals↗

PubMed1996

Sensitivity of human pancreatic islets to peroxynitrite-induced cell dysfunction and death.

Nitric oxide and peroxynitrite (generated by the reaction of nitric oxide with the superoxide anion) may both be mediators of beta-cell damage in early insulin-dependent diabetes mellitus. We observed that acute exposure of primary cultured human pancreatic islets to peroxynitrite results in a significant decrease in glucose oxidation and islet retrieval. DNA strand breaks in single human and rat islet cells are detectable after acute peroxynitrite exposure, followed by a decrease in islet cell survival after 1 h and 24 h. Cell death appeared to occur via a toxic cell death mechanism (necrosis) rather than apoptosis, as suggested by vital staining and ultrastructural evidence of early membrane and organelle degradation, mitochondrial swelling and loss of matrix. This study demonstrates for the first time that cultured human pancreatic islets are susceptible to the noxious effects of peroxynitrite.

Animals↗

PubMed1996

The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.

Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.

1-Methyl-3-isobutylxanthine↗

PubMed1996

The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver.

Previous studies showed that blood large granular lymphocytes (LGL), which possess natural killer (NK) activity, develop within rat liver sinusoids into high-density (HD) and subsequently into low-density (LD) pit cells which show an increasing level and spectrum of tumor cytotoxicity. In this study, we investigated the role of adhesion molecules, such as CD2, CD11a, CD18, and CD54 in the recruitment of pit cells to the liver. Immunostaining for electron microscopy, and two color flow cytometry showed that most pit cells expressed CD2, CD11a, CD18, and CD54. After intravenous injections into rats with anti-CD2, anti-CD11a, and anti-CD18 antibodies, the number of pit cells per square millimeter in frozen sections of liver tissue decreased. Treatment of rats with zymosan increased the number of pit cells fivefold, whereas subsequent treatment with anti-adhesion-molecule antibodies resulted in approximately 60% lower number of pit cells. Anti-CD54, supposed to block CD54 expression on sinusoidal endothelial cells, also decreased the number of pit cells. The number of blood LGL was, however, not affected by these antibodies. These results indicate that blocking of CD2, CD11a, CD18, and CD54 antigens on blood LGL and/or liver endothelium decreased the number of pit cells in the liver. These adhesion molecules therefore play an important role in the recruitment of pit cells in the liver.

Animals↗

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

Islet cell neogenesis in the pancreas.

The beta cell population of the endocrine pancreas may expand by either of two processes, neogenesis or replication. While replication requires the existence of an already differentiated beta cell, neogenesis depends on the presence of active stem cells. Since the replicative activity of highly specialized cells such as beta cells seems to be limited, it is interesting to study the potential of the endocrine pancreas for beta cell neogenesis. In this article we review both the current state of knowledge of beta cell neogenesis under natural conditions and in response to stimulation, and the significance of neogenesis for beta cell growth in health and disease.

Animals↗

PubMed1996

Beta-cell growth in adolescent and adult rats treated with streptozotocin during the neonatal period.

Regeneration of neonatal beta cells after subtotal streptozotocin (STZ)-induced destruction is incomplete but nevertheless leads to rapid remission of hyperglycaemia. To study the proliferative and functional capacity of regenerated beta cells in adolescent and adult rats after early beta-cell damage and to determine the time point after birth which is decisive for regeneration, beta-cell growth and metabolic capacity were analysed in rats treated during the neonatal period with STZ (100 micrograms/g body weight) and studied over 3 to 20 weeks. Using immunocytochemistry combined with morphometry we found that the regenerated beta cells continue to increase in number till week 6 of life, when they reached values of more than 50% of those of controls. After week 6, the regenerated beta cells had enlarged in size but failed to further increase their number, although their proliferative activity, determined by bromodeoxyuridine (BrdU) pulse labelling, was still higher at 6 and 10 weeks than that of normal rats. The inability of regenerated beta cells to further increase their number coincided with a deterioration of their function (week 10, male rats; week 20, female rats). When beta cells were destroyed on day 2 or 5 instead of the day of birth, regeneration of beta cells markedly decreased and the rats were already on the threshold of development of glucose intolerance at 3 weeks of age. We concluded that the partially regenerated beta-cell population in rats treated with STZ on the day of birth ceases to grow 10 to 20 weeks later. This growth arrest may be due to the sustained functional demand to which these beta cells are exposed in order to compensate for their reduced number. Beta-cell capacity for regeneration declines rapidly during the first days of life.

Aging↗

PubMed1996

Islet morphogenesis and stem cell markers in rat pancreas.

During embryonic development, and possibly also later in life, pancreatic islets of Langerhans originate from differentiating epithelial stem cells. These stem cells are situated in the pancreatic ducts but are otherwise poorly characterized. We found by immunohistochemical staining that protodifferentiated pancreatic epithelial cells from rat embryos of Day 13-Day 15 express the cytoskeletal protein keratin 20, similar to mature duct epithelium. During the period of islet morphogenesis, which occurs between Day 17 and birth, large aggregates of K20-positive duct cells were formed, which gradually differentiated into endocrine cells. This islet morphogenic mechanism has not been described thus far and we did not observe it in postnatal rats. During fetal islet formation, transient expression of vimentin was noted in the duct cells but not in endocrine cells. This intermediate filament protein is not observed in duct epithelial cells after birth. The proto-oncogene product bcl-2, a putative epithelial stem cell marker, was detected in duct cells from fetal and postnatal pancreas. We conclude that K20, vimentin, and bcl-2 are markets for the pancreatic (islet) stem cells.

Animals↗

PubMed1995

Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats.

We investigated the growth of islet beta and alpha cells in adult rats which had undergone partial pancreatic duct ligation. Whereas the non-ligated head portion of the pancreas remained unaffected in terms of histology and cell population dynamics, the ligated tail part of the pancreas showed pronounced changes in histology and cell growth. These changes included replacement of exocrine acini by ductal complexes and significant growth of islet cells. Using immunocytochemistry and morphometry, we found that the beta-cell population had nearly doubled within 1 week and that a smaller, but also significant growth of the alpha-cell population had occurred. In addition, small islets and islet-cell clusters were more numerous in the pancreatic tail, indicating islet neogenesis. The bromodeoxyuridine (BrdU) pulse labelling index of beta and alpha cells increased five fold and threefold, respectively, in the tail. However, the observed beta-cell labelling index remained below 1% which was largely insufficient to explain the increased number of beta cells. This indicates that recruitment from a proliferating stem-cell compartment was the main source for the beta-cell hyperplasia. A tenfold-elevated BrdU labelling index (18%) was observed in the duct-cell compartment which was identified by specific immunostaining for cytokeratin 20. Transitional cytodifferentiation forms between duct cells expressing cytokeratin 20 and beta cells expressing insulin, or alpha cells expressing glucagon, were demonstrated by double immunostaining. Pancreatic duct ligation also induced the expression of the beta-cell-specific glucose transporter type 2 (GLUT-2) in duct cells, indicating their metaplastic state. We concluded that in this adult rat model, the proliferation and differentiation of exocrine duct cells represents the major mechanism of endocrine beta-cell neogenesis. Our study thus demonstrates that in normal adult rats islet-cell neogenesis can be reactivated by stimulation of pancreatic duct cells.

Animals↗

PubMed1995

Human and rat beta cells differ in glucose transporter but not in glucokinase gene expression.

Glucose homeostasis is controlled by a glucose sensor in pancreatic beta-cells. Studies on rodent beta-cells have suggested a role for GLUT2 and glucokinase in this control function and in mechanisms leading to diabetes. Little direct evidence exists so far to implicate these two proteins in glucose recognition by human beta-cells. The present in vitro study investigates the role of glucose transport and phosphorylation in beta-cell preparations from nondiabetic human pancreata. Human beta-cells differ from rodent beta-cells in glucose transporter gene expression (predominantly GLUT1 instead of GLUT2), explaining their low Km (3 mmol/liter) and low VMAX (3 mmol/min per liter) for 3-O-methyl glucose transport. The 100-fold lower GLUT2 abundance in human versus rat beta-cells is associated with a 10-fold slower uptake of alloxan, explaining their resistance to this rodent diabetogenic agent. Human and rat beta-cells exhibit comparable glucokinase expression with similar flux-generating influence on total glucose utilization. These data underline the importance of glucokinase but not of GLUT2 in the glucose sensor of human beta-cells.

Animals↗

PubMed1995

Identification of rat pancreatic duct cells by their expression of cytokeratins 7, 19, and 20 in vivo and after isolation and culture.

Cells from the excretory ducts of the pancreas are thought to be capable of differentiating into exocrine and endocrine cells. To study this in rat models, markers must be found to identify the cells under different experimental conditions. We tested antibodies to different cytokeratins (CKs) by immunocytochemical staining on pancreatic tissue sections from normal rats, after partial pancreatectomy, and after isolation and culture of duct fragments. Monoclonal antibodies to human CK7, CK19, and CK20 were found to react specifically on rat pancreas tissue, as shown by Western blotting. CK20 and CK19 were immunocytochemically detected only in cells of the ductal system, from centroacinar cells to main ducts. CK7 was expressed by islets of Langerhans and by duct cells from main, inter-, and intralobular ducts, but not by centroacinar and terminal duct cells. CKs 7, 19, and 20 were also expressed in proliferating duct cells during tissue regeneration and after isolation and different periods of culture. We conclude that CKs 7, 19, and 20 are very useful markers to study the differentiation of rat duct cells under experimental conditions in vivo and in vitro.

Animals↗

PubMed1995

The role of Kupffer cells in the differentiation process of hepatic natural killer cells.

Pit cells, or hepatic natural killer (NK) cells, present in rat liver sinusoids, represent an organ-associated NK cell population, with a higher level of activation and a different morphology when compared with peripheral blood NK cells. These cells are the result of an influx of peripheral blood NK cells in the liver microenvironment, followed by an activation or differentiation process toward the highly activated phenotype. In this work we investigated the role of Kupffer cells in this differentiation process of NK cells in the liver sinusoids. In vivo elimination of Kupffer cells with the macrophage cytotoxic drug dichloromethylene diphosphonate induced a decrease in number of hepatic NK cells that paralleled that of Kupffer cells. This effect was further investigated in vitro. Kupffer cell-conditioned medium appeared to enhance the viability, tumor-cytotoxic activity, and adherence of hepatic NK cells to liver endothelial cells in vitro. We conclude therefore that Kupffer cells, present in the microenvironment of the liver sinusoids, play an essential role in the differentiation process of peripheral blood NK cells to the highly activated hepatic NK cell population.

Animals↗

PubMed1995

The number and distribution of hepatic natural killer cells (pit cells) in normal rat liver: an immunohistochemical study.

Pit cells are a unique population of cells in sinusoids and peripheral blood, which can be considered natural killer (NK) cells with large granular lymphocyte (LGL) morphology. The aim of this study was to investigate the use of the monoclonal antibody (MAb) 3.2.3 as a specific marker of rat pit cells to detect their number and distribution in the liver. The number of 3.2.3-positive cells was comparable to the number of LGL in liver low-density (LD) and high-density (HD) pit cell fractions and in blood lymphocytes (P > .05). Immunoelectron microscopy showed that nearly all LGL in hepatic LD and HD and in blood fractions were 3.2.3 positive. Using MAb 3.2.3 immunoperoxidase staining, the mean number of pit cells in liver frozen sections was determined to be 13.7 +/- 1.1/mm2. The number of pit cells was similar in the different liver lobes (P > .05). Reduced nicotinamide-adenine dinucleotide (NADH) oxidase histochemical staining, to visualize a portal to central vein gradient, combined with immunostaining was used to analyze the lobular distribution of pit cells. We found that 61.3% (17.1/mm2) of pit cells were in the periportal area and 38.7% (10.8/mm2) in the central area. We conclude that MAb 3.2.3 can be used as a specific marker of rat pit cells and therefore can be used to quantify rat pit cell number in various experimental models.

Animals↗

PubMed1994

Tumor-derived transforming growth factor-beta 1 and interleukin-6 are chemotactic for lymphokine-activated killer cells.

Adherent lymphokine-activated killer (A-LAK) cells are purified IL-2 activated natural killer (NK) cells with potent anti-tumor cytotoxic activity. They have been used in the adoptive immunotherapy of metastatic cancers. However, it has been shown that intravenously transferred LAK cells have a poor homing capacity to tumor sites. For the present study, the effects of tumor-derived factors on the in vitro migratory capacity of A-LAK cells was investigated. In a micropore migration assay the conditioned medium from 3LL Lewis lung carcinoma cell cultures was found to exert a strong chemotactic, but not chemokinetic effect on A-LAK cells. This effect was partially inhibited by neutralizing antibodies against the cytokines TGF-beta 1 and IL-6. A combination of the 2 antibodies completely suppressed the chemotactic activity of tumor-cell-conditioned medium. Purified TGF-beta 1 and recombinant IL-6 were chemotactic for A-LAK cells. Biological activities of both cytokines were detectable in the tumor-cell-conditioned medium. The in vivo relevance of these findings, with respect to tissue infiltration of NK cells and LAK cells in inflammation or cancer, remains to be elucidated.

Animals↗