Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic α cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated ...with a null mutation of the glucagon receptor (Gcgr-/-). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr-/-mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to α cell, and to a lesser extent, δ cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. Gcgr-/-mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and α and δ cell numbers. Furthermore, the lean phenotype of Gcgr-/-mice suggests glucagon action may be involved in the regulation of whole body composition.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Summary
CD4+CD25+ regulatory T cells (Tregs) are involved in the maintenance of peripheral tolerance and ensure a balanced immune response competent of fighting pathogens and at the same time ...recognizing commensals as harmless. This feature is lost in Crohn's disease (CD). The forkhead/winged helix transcription factor FoxP3 is a master gene for Treg function and defects in the FoxP3 gene lead to a clinical picture similar to inflammatory bowel disease (IBD). Murine colitis can be cured by adoptive transfer of Tregs and ex vivo‐generated gut‐specific Tregs represent an attractive option for therapy in CD. Thus, defective Tregs could contribute to the development of CD. We cultured biopsies of colonic mucosa in the presence of high concentrations of interleukin (IL)‐2 and IL‐4 to overcome the anergic nature of naturally occurring CD4+CD25+ Tregs in the mucosa. We investigated the expression of FoxP3 and regulatory potential of gut‐derived CD4+CD25+ T cells cultured from patients with CD and healthy individuals. The FoxP3 expression was analysed by reverse transcriptase polymerase chain reaction (RT‐PCR), and the suppressive effect of FoxP3+CD4+CD25+ T cells on proliferation and cytokine production of autologous CD4+ T cells was assessed by flow cytometry. Cultured gut‐derived T cells with CD4+CD25+ phenotype expressed FoxP3 and were able as the freshly isolated Tregs from peripheral blood to suppress proliferation and cytokine production of autologous CD4+ T cells. Thus, we demonstrate that FoxP3+CD4+CD25+ T cells with regulatory properties can be propagated in vitro from inflamed mucosa of CD patients, which may be of interest in adoptive immunotherapy.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Background Interleukin (IL)‐20 is a recently discovered cytokine displaying increased levels in psoriatic lesions. Interestingly, IL‐20 levels decrease with antipsoriatic treatment, ...correlating with clinical improvement. However, the role of IL‐20 in the aetiology of psoriasis is unknown.
Objectives In this study, we investigate the effects both of blocking IL‐20 signalling in psoriatic plaques and of adding IL‐20 to nonlesional psoriasis skin.
Methods We employed the human skin xenograft transplantation model in which psoriatic plaques and nonlesional keratome skin biopsies obtained from donors with moderate to severe plaque psoriasis were transplanted on to immuno‐deficient mice. The transplanted mice were treated with anti‐IL‐20 antibodies or recombinant human IL‐20.
Results We demonstrate that blocking IL‐20 signalling with anti‐IL‐20 antibodies induces psoriasis resolution and inhibits psoriasis induction. We also demonstrate that continuous IL‐20 infusion, together with injection of additional nonactivated leucocytes, promotes induction of psoriasis in nonlesional skin from patients with psoriasis.
Conclusions The results suggest that IL‐20 plays a critical role in the induction and maintenance of psoriasis, and IL‐20 is suggested as a new possible specific target in psoriasis treatment.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic ...degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when beta-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when beta-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1 beta converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin-1 and uPA were weakly induced, if at all, in hydrocortisone-treated mice. Furthermore, mRNA for membrane-type matrix metalloproteinase decreased after hydrocortisone treatment and paralleled the almost complete inhibition of activation of latent gelatinase A. Concomitantly, the gland filled with an overabundance of milk. Our data support the hypothesis that there are at least two distinct phases of involution: an initial phase, characterized by induction of the apoptosis-associated genes SGP-2 and ICE and apoptosis of fully differentiated mammary epithelial cells without visible degradation of the extracellular matrix, and a second phase, characterized by extracellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptosis of cells that are losing differentiated functions.
Background: Degradation of extracellular matrix proteins, such as fibrin, is pivotal to tumor invasion. Inhibition of the interaction between urokinase plasminogen activator (u‐PA) and its receptor ...(u‐PAR), and hence pro‐u‐PA activation, is an attractive approach to anti‐invasive cancer therapy. A number of inhibitors exist for the human system, but because of species specificity none of these are efficient in mice. We have recently generated an inhibitory monoclonal antibody (mAb) against mouse u‐PAR (mR1) by immunization of u‐PAR‐deficient mice. Objectives: To evaluate the effect of mR1 in vivo in a physiological setting sensitive to deregulated fibrinolysis, we have administered mR1 systemically and quantitated the effect on liver fibrin accumulation. Methods: Wild‐type and tissue‐type plasminogen activator (t‐PA) deficient mice were administered with mR1, or control antibody, during 6 weeks. Thereafter, the livers were retrieved and the amount of liver fibrin measured by unbiased morphometrical analysis of immunofluorescence signal. Results: Systemic administration of mR1 caused significantly increased fibrin signal in anti‐u‐PAR treated t‐PA‐deficient mice compared to mock‐treated, which mimics the phenotype of u‐PAR;t‐PA double‐deficient mice. Fibrin and fibronectin accumulated within the sinusoidal space and was infiltrated by inflammatory cells. Analysis of small and rare hepatic fibrin plaques observed in t‐PA‐deficient mice showed infiltrating macrophages that, contrary to surrounding Kuppfer cells, expressed u‐PAR. Conclusion: We show that u‐PAR‐expressing macrophages are involved in cell‐mediated fibrinolysis of liver fibrin deposits, and that the antimouse–u‐PAR mAb is effective in vivo and thus suited for studies of the effect of targeting the u‐PA/u‐PAR interaction in mouse cancer models.
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FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Growth hormone and prolactin are important growth factors for pancreatic β-cells. The effects exerted by these hormones on proliferation and on insulin synthesis and secretion in β-cells are largely ...mediated through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Suppressors of cytokine signaling (SOCS) proteins are specific inhibitors of the JAK/STAT pathway acting through a negative-feedback loop. To investigate in vivo effects of SOCS-3 in growth hormone (GH)/prolactin signaling in β-cells we generated transgenic mice with β-cell-specific overexpression of SOCS-3. The relative β-cell proliferation and volume in the mice were measured by morphometry. β-Cell volume of transgenic female mice was reduced by over 30% compared with β-cell volume in wild-type female mice. Stimulation of transgenic islets in vitro with GH showed a reduced tyrosine phosphorylation of STAT-5 when compared with wild-type islets. Transduction of primary islet cultures with adenoviruses expressing various SOCS proteins followed by stimulation with GH or glucagon-like peptide-1 (GLP-1) revealed that SOCS-3 inhibited GH- but not GLP-1-mediated islet cell proliferation, indicating that the decreased β-cell volume observed in female transgenic mice could be caused by an inhibition of GH-induced β-cell proliferation by SOCS-3. In spite of the reduced β-cell volume the transgenic female mice exhibited enhanced glucose tolerance compared with wild-type littermates following an oral glucose-tolerance test. Together these data suggest that SOCS-3 modulates cytokine signaling in pancreatic β-cells and therefore potentially could be a candidate target for development of new treatment strategies for diabetes.
Recombinant human gamma 2 chain of laminin-5 was expressed in Escherichia coli, and used to generate specific polyclonal antibodies which were used to study the distribution of the protein in human ...cancers. A total of 72 biopsies of human cancers were stained, including 23 cases of colon adenocarcinomas, 16 ductal breast carcinomas, 9 malignant melanomas, 14 squamous cell carcinomas of the skin and cervix, and 10 sarcomas. As a control for the specificity of the antibodies, we performed in situ hybridization on adjacent sections of a number of the cases, and in all of these cases the localization of the gamma 2 chain protein and mRNA was identical. We found gamma 2 chain immunoreactivity in cancer cells in all cases of colon adenocarcinomas and squamous cell carcinomas but not in any of the sarcomas, supporting the view that the laminin-5 protein is specific for cells of epithelial origin. Notably, in all of the cases of colon adenocarcinomas, the positive staining was invariably associated with budding cancer cells located at the tip of invading malignant epithelium, whereas the cancer cells deeper in the tumors were most often negative. The staining was cytoplasmic in all cases and only in one case did we see additional extracellular immunoreactivity, indicating that this laminin isoform in cancer tissue is not laid down in the extracellular matrix but probably exerts its function at the cell surface or in its immediate vicinity. Using in situ hybridization to analyze the coexpression of laminin-5 and components of the plasminogen activation system, we found that the histological distribution of laminin-5-positive budding cancer cells at the invasion front in colon adenocarcinomas was identical to that of the receptor for urokinase-type plasminogen activator. These findings suggest that laminin-5 is a marker of invading cancer cells in at least some human malignancies, and that it therefore might represent a valuable marker for the invasive potential of these cancers. The colocalization of laminin-5 and urokinase-type plasminogen activator receptor in a subset of cancer cells in colon cancer also suggests that a controlled up-regulation of a number of gene products is a characteristic of budding colon cancer cells, and that these gene products serve functions crucial for the invasive phenotype of these cancer cells.
Combination therapies are increasingly common in the clinical management of type 2 diabetes. We investigated to what extent combined treatment with the human glucagon-like peptide-1 (GLP-1) analogue ...liraglutide and the dual PPARα/γ agonist ragaglitazar would improve glycaemic control in overtly diabetic Zucker diabetic fatty (ZDF) rats. Ninety overtly diabetic male ZDF rats were stratified into groups with matched haemoglobin A1c (HbA₁c) (9.0 ± 0.1%). Liraglutide (15 and 50 μg/kg subcutaneously twice daily), ragaglitazar (1 and 3 mg/kg perorally once daily) and their vehicles were studied as monotherapy and in combination in a 3 x 3 factorial design. After 4-week treatment, synergistic effects on HbA₁c, non-fasting morning blood glucose (BG) and/or 24-h BG profiles were observed with three of the four combinations. The relationship between plasma insulin and BG in combination-treated animals approached that of historical lean ZDF rats representing normal glucose homeostasis, suggesting that insulin secretion and insulin sensitivity were markedly improved. Increased insulin immunostaining in islets further supports the improved beta-cell function and/or insulin sensitivity in combination-treated animals. The synergistic effect on glycaemic control was found without a similar synergistic increase in beta-cell mass in the combination groups. Our data demonstrate that combination treatment with a human GLP-1 analogue and a dual PPARα/γ agonist through distinct mechanism of actions synergistically improves glycaemic control in the ZDF rat.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Growth hormone secretagogues (GHS) are small, synthetic compounds which have the potential of releasing growth hormone (GH) from the pituitary. The mechanism of action of GHS has not been fully ...elucidated. A specific GHS receptor (GHS-R) is expressed in the pituitary gland and in several areas of the brain including the hypothalamus. We have characterized the GHS-R-mRNA-expressing neurons with respect to co-expression of selected neurotransmitters in the hypothalamus. This was done by dual chromogenic and autoradiographic in situ hybridization with riboprobes for GHS-R mRNA and neuropeptide Y (NPY), pro-opiomelanocortin (POMC), somatostatin (SRIH) or GH-releasing hormone (GHRH) mRNA. In the arcuate nucleus, GHS-R mRNA was expressed in 94 +/- 1% of the neurons expressing NPY, 8 +/- 2% of those expressing POMC and 30 +/- 6% expressing SRIH mRNA. 20-25% of the GHRH- mRNA-expressing neurons contained GHS-R mRNA, whereas the vast majority of the arcuate GHS-R-mRNA-containing cells did not contain GHRH mRNA. The finding of a significant co-expression of GHS-R and NPY mRNA in the arcuate nucleus is in accordance with the previous demonstration by Dickson et al. that c-Fos is induced in NPY neurons following GHS administration. These results indicate that GHS have other effects on neuroendocrine regulation than GH release via GHRH neurons. Stimulation of the arcuate NPY neurons via GHS-R may explain the increased appetite and the cortisol release seen after administration of some GHS compounds.
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