Langerhans In Mice Research Articles

Effect of Ajwa Date (Phoenix dactylifera) Extract on the Histopathology of Pancreatic Islets in Mice with Diabetes Mellitus

Introduction: It is necessary to develop alternative antidiabetic therapies that are safer and more affordable to overcome the high prevalence of diabetes mellitus in Indonesia. Ajwa dates (Phoenix dactylifera) have a high flavonoid content; hence, this study aimed to investigate their effect on streptozotocin-induced diabetes mellitus mice by examining the number of beta cells and the islets of Langerhans. Methods: Twenty-five mice were divided into five groups: a negative control group (K1), a positive control group (K2), and three treatment groups (P1, P2, and P3). The K2, P1, P2, and P3 groups were induced by 100 mg/kg bw of streptozotocin. Additionally, the P1, P2, and P3 groups received oral treatment using ajwa date methanol extract at different doses of 3, 5, and 7 g/kg bw, respectively. The treatment was administered daily for four weeks. The initial analysis included the homogeneity test and the Shapiro-Wilk test. As the data were non-normally distributed, the analysis proceeded with the Kruskal-Wallis test (p<0.05). Results: The comparative analysis revealed significant differences in the number of beta cells among the groups, with a notable decrease observed in the K2 group and an increase in each treatment group. The measurement of the islets of Langerhans exhibited significant differences among the groups, with p=0.001. Conclusion: The administration of ajwa date methanol extract can affect the number of beta cells and the islets of Langerhans in mice with diabetes mellitus. Highlights: This original study examined the antioxidant compounds derived from naturally sourced ajwa date (Phoenix dactylifera) extract. Ajwa date extract has the potential to protect against histological damage, specifically to beta cells and pancreatic islets, in mice induced by streptozotocin.

Identification of MicroRNAs Associated with Prediabetic Status in Obese Women.

MicroRNAs (miRNAs) recently emerged as means of communication between insulin-sensitive tissues to mediate diabetes development and progression, and as such they present a valuable proxy for epigenetic alterations associated with type 2 diabetes. In order to identify miRNA markers for the precursor of diabetes called prediabetes, we applied a translational approach encompassing analysis of human plasma samples, mouse tissues and an in vitro validation system. MiR-652-3p, miR-877-5p, miR-93-5p, miR-130a-3p, miR-152-3p and let-7i-5p were increased in plasma of women with impaired fasting glucose levels (IFG) compared to those with normal fasting glucose and normal glucose tolerance (NGT). Among these, let-7i-5p and miR-93-5p correlated with fasting blood glucose levels. Human data were then compared to miRNome data obtained from islets of Langerhans and adipose tissue of 10-week-old female New Zealand Obese mice, which differ in their degree of hyperglycemia and liver fat content. Similar to human plasma, let-7i-5p was increased in adipose tissue and islets of Langerhans of diabetes-prone mice. As predicted by the in silico analysis, overexpression of let-7i-5p in the rat β-cell line INS-1 832/12 resulted in downregulation of insulin signaling pathway components (Insr, Rictor, Prkcb, Clock, Sos1 and Kcnma1). Taken together, our integrated approach highlighted let-7i-5p as a potential regulator of whole-body insulin sensitivity and a novel marker of prediabetes in women.

High-casein diet restores the redox balance in the liver and pancreatic health of mice when exposed to radiation during call mode from mobile phones

Introduction: The current lives of human beings cannot be imagined without cellular phones and electronic gadgets. Still, the probabilityof being exposed to their harmful radiation is unabated which demands an immediate intervention to shield humankind from beingaffected by using such devices. In our current investigation, we aimed to check the role of a high-casein diet as a protective measureagainst the biological effects of electromagnetic radiation (EMR) emerging from mobile phones on hepatic and pancreatic systemsMethods: Swiss albino mice (n=24) were fed on a standard laboratory diet (ND; n=12) and an isocaloric high-casein diet (HCD; n=12).Six mice from each dietary group were exposed to mobile phone radiation for 3 hours/day within a 3 months span. Histopathologicalalterations were studied in hepatic and pancreatic tissues by using Periodic acid-Schiff (PAS)-hematoxylin and routine H&E stainingmethods. Biochemical evaluation of total serum protein, lipid profile and glucose, total glutathione, oxidized glutathione, and antioxidantenzymatic activities of hepatic tissues were performed. Results: Hyperglycemia was noticeable along with a reduced number and areaof islets of Langerhans in radiation-exposed mice when compared to other groups. This was supported by the observation of depletedglycogen reserves in their liver. Additionally, distorted hepatic morphology with nuclear degeneration was suggestive of apoptoticprogression. The total glutathione pool was reduced on radiation exposure, hint at redox imbalance. HCD was competent in preservingthe normal pancreas and liver functioning in radiation-exposed mice.

Pancreatic Pathological Changes in Murine Toxoplasmosis and Possible Association with Diabetes Mellitus.

Previous studies have reported involvement of Toxoplasma gondii (T. gondii) infections in the pathogenesis of some autoimmune diseases, such as polymyositis, rheumatoid arthritis, autoimmune thyroiditis, and Crohn's disease. However, data on the association between T. gondii infections and Type 1 diabetes mellitus (T1DM) are still controversial. Therefore, in the present study, we aimed to investigate the pancreatic pathological changes in mouse models with acute and chronic toxoplasmosis and their association with T1DM. Three groups (10 mice each) of male Swiss Albino mice were used. One group of mice was left uninfected, whereas the second and third groups were infected with the acute virulent T. gondii RH strain and the chronic less virulent Me49 T. gondii strain, respectively. T. gondii-induced pancreatic pathological changes were evaluated by histopathological examination of pancreatic tissues. Moreover, the expression of insulin, levels of caspase-3, and the pancreatic infiltration of CD8+ T cells were evaluated using immunohistochemical staining. Pancreatic tissues of T. gondii-infected animals showed significant pathological alterations and variable degrees of insulitis. Mice with acute toxoplasmosis exhibited marked enlargement and reduced numbers of islets of Langerhans. However, mice with chronic toxoplasmosis showed considerable reduction in size and number of islets of Langerhans. Moreover, insulin staining revealed significant reduction in β cell numbers, whereas caspase-3 staining showed induced apoptosis in islets of Langerhans of acute toxoplasmosis and chronic toxoplasmosis mice compared to uninfected mice. We detected infiltration of CD8+ T cells only in islets of Langerhans of mice with chronic toxoplasmosis. Acute and chronic toxoplasmosis mice displayed marked pancreatic pathological changes with reduced numbers of islets of Langerhans and insulin-producing-β cells. Since damage of β cells of islets of Langerhans is associated with the development of T1DM, our findings may support a link between T. gondii infections and the development of T1DM.

Quantitative 3D OPT and LSFM datasets of pancreata from mice with streptozotocin-induced diabetes

Mouse models for streptozotocin (STZ) induced diabetes probably represent the most widely used systems for preclinical diabetes research, owing to the compound’s toxic effect on pancreatic β-cells. However, a comprehensive view of pancreatic β-cell mass distribution subject to STZ administration is lacking. Previous assessments have largely relied on the extrapolation of stereological sections, which provide limited 3D-spatial and quantitative information. This data descriptor presents multiple ex vivo tomographic optical image datasets of the full β-cell mass distribution in mice subject to single high and multiple low doses of STZ administration, and in glycaemia recovered mice. The data further include information about structural features, such as individual islet β-cell volumes, spatial coordinates, and shape as well as signal intensities for both insulin and GLUT2. Together, they provide the most comprehensive anatomical record of the effects of STZ administration on the islet of Langerhans in mice. As such, this data descriptor may serve as reference material to facilitate the planning, use and (re)interpretation of this widely used disease model.

Presynaptic-like mechanisms and the control of insulin secretion in pancreatic β-cells.

Exocytotic release of hormones from endocrine cells must encompass mechanisms that direct the hormone into the blood stream. Increasing evidence indicates an intimate link between pancreatic β-cells and the capillary bed of islets of Langerhans in both mouse and human. Integrins are exclusively activated at the region where β-cells contact extracellular matrix proteins that surround the islet capillaries; furthermore, insulin granule exocytosis is targeted to this same region, therefore delivering hormone directly into the blood stream.In this review we discuss evidence suggesting that the capillary interface of β-cells forms a specialised domain that is analogous to the presynaptic active zone of neurones. Pancreatic β-cells possess many of the same proteins as found in the neuronal active zone, including several key presynaptic scaffold proteins. These scaffold proteins are enriched at the capillary interface of β-cells and some have also been shown to control insulin secretion. We present a model that suggests this active zone-like domain in β-cells may anchor key components of the stimulus secretion cascade, to not only target granule exocytosis to this region but also function as a significant regulator of insulin secretion.

Pleiotrophin Expression and Actions in Pancreatic β-Cells.

Pleiotrophin (PTN) is a heparin-binding cytokine that is widely expressed during early development and increases in maternal circulation during pregnancy.Aged PTN-deficient mice exhibit insulin resistance, suggesting a role in metabolic control. The objectives of this study were to determine if PTN is expressed in mouse pancreatic β-cells in young vs. adult animals, and its effects on DNA synthesis, β-cell gene expression and glucose-stimulated insulin secretion (GSIS). The Ptn gene was expressed in isolated fractions of young mouse β-cells, especially within immature β-cells with low glucose transporter 2 expression. Expression was retained in the adult pancreas but did not significantly change during pregnancy. PTN and its receptor, phosphotyrosine phosphatase-β/ζ, were also expressed in the proliferative INS1E β-cell line. Fluorescence immunohistochemistry showed that PTN peptide was present in islets of Langerhans in adult mice, associated predominantly with β-cells. The percentage of β-cells staining for PTN did not alter during mouse pregnancy, but intense staining was seen during β-cell regeneration in young mice following depletion of β-cells with streptozotocin. Incubation of INS1E cells with PTN resulted in an increased DNA synthesis as measured by Ki67 localization and increased expression of Pdx1 and insulin. However, both DNA synthesis and GSIS were not altered by PTN in isolated adult mouse islets. The findings show that Ptn is expressed in mouse β-cells in young and adult life and could potentially contribute to adaptive increases in β-cell mass during early life or pregnancy.

Keratin 7 Is a Constituent of the Keratin Network in Mouse Pancreatic Islets and Is Upregulated in Experimental Diabetes.

Keratin (K) 7 is an intermediate filament protein expressed in ducts and glands of simple epithelial organs and in urothelial tissues. In the pancreas, K7 is expressed in exocrine ducts, and apico-laterally in acinar cells. Here, we report K7 expression with K8 and K18 in the endocrine islets of Langerhans in mice. K7 filament formation in islet and MIN6 β-cells is dependent on the presence and levels of K18. K18-knockout (K18‒/‒) mice have undetectable islet K7 and K8 proteins, while K7 and K18 are downregulated in K8‒/‒ islets. K7, akin to F-actin, is concentrated at the apical vertex of β-cells in wild-type mice and along the lateral membrane, in addition to forming a fine cytoplasmic network. In K8‒/‒ β-cells, apical K7 remains, but lateral keratin bundles are displaced and cytoplasmic filaments are scarce. Islet K7, rather than K8, is increased in K18 over-expressing mice and the K18-R90C mutation disrupts K7 filaments in mouse β-cells and in MIN6 cells. Notably, islet K7 filament networks significantly increase and expand in the perinuclear regions when examined in the streptozotocin diabetes model. Hence, K7 represents a significant component of the murine islet keratin network and becomes markedly upregulated during experimental diabetes.

Natural flavonoid α-glucosidase inhibitors from Retama raetam: Enzyme inhibition and molecular docking reveal important interactions with the enzyme active site

Retama raetam (Forsk.) Webb & Berthel plant has been traditionally used for the treatment of diabetes mellitus and hypertension. Interest in the medicinal chemistry of the plant in the past resulted in the isolation of a number of compounds with anti-hyperglycemic activity. The current work is a further extension of our recent work in which we isolated and characterized seven new flavonoids from Retama raetam with preliminary biological activity screening. It addresses the α-glucosidase inhibitory activity and molecular docking studies of the flavonoids. Retamasin D, G, H, and erysubin A and B noncompetitively inhibited the enzyme whereas retamasin C and F exhibited competitive inhibition. Moreover, retamasin C, F, G, and erysubin A and B carry dual activity in addition to α-glucosidase inhibition. Our previous studies have shown that they also caused significant stimulation of insulin from the blood-perfused pancreatic islets of Langerhans of mice. The C6 and C8 substituent groups greatly influenced the inhibition potency of the compounds. The most potent inhibitor was retamasin H with the γ-lactone ring substituent at C6 position of the main flavonoid moiety. Notable active chemical groups in the target compounds include γ-lactone, dihydropyran and dihydrofuran rings with hydroxyl and geminal methyl groups. Molecular modeling studies revealed that the compounds fit well in the α-glucosidase active site by interacting with important active site residues. These findings will incorporate new chemical, structural and functional diversity to the search and drug development of α-glucosidase inhibitors as anti-diabetic drugs.

Alpha Mangostin and Xanthone Activity on Fasting Blood Glucose, Insulin and Langerhans Islet of Langerhans in Alloxan Induced Diabetic Mice

Objective: This research elaborated role of alpha mangostin and xanthone on fasting blood glucose, insulin and langerhans islet in alloxan induced diabetic mice. Methods: Fasting blood glucose, insulin and langerhans islet test were conducted using male Mus musculus mice, divided into 10 groups randomly, which were normal, control (alloxan induced only), glibenclamide, various doses of α-mangostin and xanthone (5, 10, 20 mg/kgbw). Mice were treated for 21 days. Overnight-fasted mice (12 h) were sacrificed by cervical decapitation on day 21st, following the ethical norms granted by the ethics committee. Fasting blood glucose and insulin plasma were checked. Pancreatic tissues were excised from sacrificed animals, and then fixed in 10 % (v/v) neutral buffered formalin. Histologic observations for Langerhans area were performed after staining using Gomori staining method. Results: The effects of alpha mangostin and xanthone on fasting blood glucose different significantly to control, and were not significantly different from glibenclamide and metformin. Increasing alpha mangostin/xanthone dose from 5 mg/kgbw to 20 mg/bw also did not cause significant differences, although the best results were obtained at a dose of 20 mg/kgbw. Insulin plasma analize showed that there were no significant difference between alpha mangostin/xanthone to normal group, except xanthone 10 mg/kgbw. Langerhans area showed significant difference between alpha mangostin/xanthone to control group. But there’s still had significant difference if we compare to glibenclamid/metformin group. Conclusion: Alpha mangostin and xanthone are two substances that showed antidiabetic effect on fasting blood glucose level, insulin plasma and Langerhans islet.

The anti-aging protein Klotho is induced by GABA therapy and exerts protective and stimulatory effects on pancreatic beta cells

Systemic gamma-aminobutyric acid (GABA) therapy prevents or ameliorates type 1 diabetes (T1D), by suppressing autoimmune responses and stimulating pancreatic beta cells. In beta cells, it increases insulin secretion, prevents apoptosis, and induces regeneration. It is unclear how GABA mediates these effects. We hypothesized that Klotho is involved. It is a multi-functional protein expressed in the kidneys, brain, pancreatic beta cells, other tissues, and is cell-bound or soluble. Klotho knockout mice display accelerated aging, and in humans Klotho circulating levels decline with age, renal disease and diabetes. Here, we report that GABA markedly increased circulating levels of Klotho in streptozotocin (STZ)-induced diabetes. GABA also increased Klotho in the islet of Langerhans of normal mice, as well as the islets and kidneys of STZ-treated mice. In vitro, GABA stimulated production and secretion of Klotho by human islet cells. Knockdown (KD) of Klotho with siRNA in INS-1E insulinoma cells abrogated the protective effects of GABA against STZ toxicity. Following KD, soluble Klotho reversed the effects of Klotho deficiency. In human islet cells soluble Klotho protected against cell death, and stimulated proliferation and insulin secretion. NF-κB activation triggers beta-cell apoptosis, and both GABA and Klotho suppress this pathway. We found Klotho KD augmented NF-κB p65 expression, and abrogated the ability of GABA to block NF-κB activation. This is the first report that GABAergic stimulation increases Klotho expression. Klotho protected and stimulated beta cells and lack of Klotho (KD) was reversed by soluble Klotho. These findings have important implications for the treatment of T1D.

Cbfβ2 deficiency preserves Langerhans cell precursors by lack of selective TGFβ receptor signaling.

The mouse Langerhans cell (LC) network is established through the differentiation of embryonic LC precursors. BMP7 and TGFβ1 initiate cellular signaling that is essential for inducing LC differentiation and preserving LCs in a quiescent state, respectively. Here we show that loss of Cbfβ2, one of two RNA splice variants of the Cbfb gene, results in long-term persistence of embryonic LC precursors after their developmental arrest at the transition into the EpCAM+ stage. This phenotype is caused by selective loss of BMP7-mediated signaling essential for LC differentiation, whereas TGFβR signaling is intact, maintaining cells in a quiescent state. Transgenic Cbfβ2 expression at the neonatal stage, but not at the adult stage, restored differentiation from Cbfβ2-deficient LC precursors. Loss of developmental potential in skin-residential precursor cells was accompanied by diminished BMP7-BMPR1A signaling. Collectively, our results reveal an essential requirement for the Cbfβ2 variant in LC differentiation and provide novel insight into how the establishment and homeostasis of the LC network is regulated.

A novel toolbox to investigate tissue spatial organization applied to the study of the islets of Langerhans

Thanks to the development of new 3D Imaging techniques, volumetric data of thick samples, especially tissues, are commonly available. Several algorithms were proposed to analyze cells or nuclei in tissues, however these tools are limited to two dimensions. Within any given tissue, cells are not likely to be organized randomly and as such have specific patterns of cell-cell interaction forming complex communication networks. In this paper, we propose a new set of tools as an approach to segment and analyze tissues in 3D with single cell resolution. This new tool box can identify and compute the geographical location of single cells and analyze the potential physical interactions between different cell types and in 3D. As a proof-of-principle, we applied our methodology to investigation of the cyto-architecture of the islets of Langerhans in mice and monkeys. The results obtained here are a significant improvement in current methodologies and provides new insight into the organization of alpha cells and their cellular interactions within the islet’s cellular framework.

Identification of 5-hydroxytryptamine-producing cells by detection of fluorescence in paraffin-embedded tissue sections.

5-Hydroxytryptamine (5-HT) produced by enterochromaffin (EC) cells is an important enteric mucosal signaling ligand and has been implicated in several gastrointestinal diseases, including inflammatory bowel disease and functional disorders such as irritable bowel syndrome. The present study reports a new, simple and rapid visualization method of 5-HT-producing EC cells utilizing detection of fluorescence in paraffin-embedded tissue sections after formalin fixation. In human samples, there was a high incidence of fluorescence+ cells in the 5-HT+ cells in the pyloric, small intestinal and colonic glands, while co-localization was lacking between fluorescence+ and gastrin+ cells in the pyloric and small intestinal glands. Fluorescence+ EC cells were detected in the colon of mice and rats. Fluorescence+ cells were also observed in 5-HT+ β cells in the pancreatic islets of Langerhans in pregnant mice, while non-pregnant mouse pancreatic islet cells showed no 5-HT immunoreactivity or fluorescence. These results suggest that fluorescence+ cells are identical to 5-HT+ cells, and the source of fluorescence may be 5-HT itself or molecules related to its synthesis or degradation. This fluorescence signal detection method may be applicable for monitoring of inflammatory status of inflammatory bowel diseases in both the experimental and clinical settings.

Effects of methyl mercury on the activity and gene expression of mouse Langerhans islets and glucose metabolism

Mercury (Hg) is a well-known heavy metal and causes various toxic effects. It is abundantly present in fish in the form of methyl mercury (MeHg). Also, various other forms of mercury can enter human body either from environment like inhalation or through dental amalgams. The present study was designed to assess MeHg induced toxicity in mouse plasma and pancreatic islets with respect to insulin secretion, oxidative balance, glucose tolerance, gene expression, caspases 3 and 9 activities. MeHg was dissolved in tap water and administered at doses 2.5, 5 and 10 mg/kg/day, for 4 weeks. In mice, MeHg significantly caused increase in plasma insulin as well as C-peptides. Glucose intolerance, insulin resistance and hyperglycemia are main consequences of our study that correlate with the gene expression changes of glucose homeostasis as well. MeHg caused increase lipid peroxidation in a dose-dependent manner in plasma as well as pancreatic islets. In addition, total thiol molecules and ferrous reducing antioxidant power in MeHg treated group was decreased in plasma as well as pancreatic islets. Caspases 3 and 9 activities of pancreatic islets were upregulated in MeHg exposed animals. Reactive oxygen species were extremely high in pancreatic islets of MeHg treated groups. MeHg disrupted gluconeogenesis/glycogenolysis pathways and insulin secretory functions of islets by targeting GDH, GLUT2 and GCK genes of pancreatic islets. In conclusion, the current study revealed that insulin pathways, oxidative balance and glucose metabolism encoded genetic makeup are susceptible to MeHg toxicity and the subsequent oxidative stress and alternations in gene expression could lead toward functional abnormalities in other organs.

Two different pathogenic mechanisms, dying-back axonal neuropathy and pancreatic senescence, are present in the YG8R mouse model of Friedreich's ataxia.

ABSTRACTFrataxin (FXN) deficiency causes Friedreich’s ataxia (FRDA), a multisystem disorder with neurological and non-neurological symptoms. FRDA pathophysiology combines developmental and degenerative processes of dorsal root ganglia (DRG), sensory nerves, dorsal columns and other central nervous structures. A dying-back mechanism has been proposed to explain the peripheral neuropathy and neuropathology. In addition, affected individuals have non-neuronal symptoms such as diabetes mellitus or glucose intolerance. To go further in the understanding of the pathogenic mechanisms of neuropathy and diabetes associated with the disease, we have investigated the humanized mouse YG8R model of FRDA. By biochemical and histopathological studies, we observed abnormal changes involving muscle spindles, dorsal root axons and DRG neurons, but normal findings in the posterior columns and brain, which agree with the existence of a dying-back process similar to that described in individuals with FRDA. In YG8R mice, we observed a large number of degenerated axons surrounded by a sheath exhibiting enlarged adaxonal compartments or by a thin disrupted myelin sheath. Thus, both axonal damage and defects in Schwann cells might underlie the nerve pathology. In the pancreas, we found a high proportion of senescent islets of Langerhans in YG8R mice, which decreases the β-cell number and islet mass to pathological levels, being unable to maintain normoglycemia. As a whole, these results confirm that the lack of FXN induces different pathogenic mechanisms in the nervous system and pancreas in the mouse model of FRDA: dying back of the sensory nerves, and pancreatic senescence.

High-fat diet feeding significantly attenuates anagliptin-induced regeneration of islets of Langerhans in streptozotocin-induced diabetic mice.

BackgroundDPP-4 inhibitors reportedly exert effects on both alpha and beta cells, and promote the proliferation and survival of beta cells. We investigated the effects of anagliptin on structurally-impaired islets of Langerhans in streptozotocin (STZ)-treated mice, fed either a normal or a high-fat diet. Pdx-1 expression in the pancreas and serum insulin/glucagon concentrations were also examined.FindingsAnagliptin treatment significantly up-regulated pancreatic Pdx-1 expression, with elevated serum glucagon-like peptide-1 concentrations, regardless of whether the diet was normal or high-fat. However, interestingly, the beta cell regeneration, structural normalization of islets of Langerhans including alpha cell: beta cell area ratios, and serum insulin elevation, all observed with anagliptin administration in the animals fed a normal diet, were markedly suppressed in the high-fat fed group.ConclusionsHigh-fat diet feeding clearly weakened the regenerative effects of anagliptin on the islets of Langerhans in STZ-treated mice. Our findings suggest the importance of normalizing lipid metabolism for full manifestation of DPP-4 inhibitor effects on the islets of Langerhans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13098-015-0047-y) contains supplementary material, which is available to authorized users.

Physiological and clinically attainable concentrations of 1,25-dihydroxyvitamin D3 suppress proliferation and extracellular matrix protein expression in mouse pancreatic stellate cells.

Vitamin D is an antiproliferative and differentiation-promoting secosteroid hormone with pleiotropic homeostatic functions in bone and extraskeletal tissues. Signaling of vitamin D is mediated via its ubiquitously expressed nuclear receptor, the vitamin D receptor (VDR). Pancreatic stellate cells have recently been identified as targets of vitamin D action. Our aim was to elucidate the effectiveness of the most potent endogenous vitamin D metabolite, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the proliferation and extracellular matrix (ECM) protein expression in pancreatic stellate cells (PSCs) using concentrations of the compound from the physiological and clinically attainable range in humans. Culture-activated mouse PSCs were exposed to 1,25(OH)2D3 concentrations ranging from 0.1nM to 10nM for 7 days and subjected to colorimetric crystal violet assay for cell growth assessment and to Western blot and immunohistochemical analyses of VDR, fibronectin and collagen I using protein-specific antibodies. Immunohistochemical localization of VDR was performed on mouse pancreatic tissue and on a set of human specimens obtained at pancreatic surgery. A low basal level of VDR was detected in PSCs that was strongly induced in the presence of ligand. Cell growth was suppressed dose-dependently by 1,25(OH)2D3, the mean percentages of inhibition ranging from 24% at the physiological 0.1nM concentration to around 60% at 10nM. Significant 48% and 40% reductions in fibronectin expression were seen at 0.5nM and 1nM 1,25(OH)2D3. A minor decrease in collagen I expression was detected at 5nM. VDR was predominantly localized in the islets of Langerhans in mouse and human tissues. In the latter VDR was expressed also in the exocrine tissue showing individual variation in its cellular distribution. Mouse PSCs express VDR protein and are sensitive 1,25(OH)2D3 target cells with low levels of 1,25(OH)2D3 exerting antiproliferative and antifibrotic effects on activated PSCs invitro.

3D Ultrastructural Investigation of Entire Pancreatic Islets of Langerhans by Serial Block Face Scanning Electron Microscopy

Serial block-face scanning electron microscopy (SBF-SEM) provides three-dimensional ultrastructure of biological tissues at the nanometer scale (Denk and Horstmann, 2004). Previous studies have applied this technique to visualize the architecture of pancreatic islets of Langerhans in wild-type mice. Initial results indicate that an average beta cell has almost twice the cellular volume of an average alpha cell and four times the mitochondrial volume, whereas the nuclear volumes in both cell types are approximately equal. Comparisons of insulin-secreting beta granules and glucagon-secreting alpha granules show that the beta granules have more pronounced halos and diameters twice that of the alpha granules. In addition, three-dimensional rendering of islet blood vessels reveals that all secretory cells in an islet are in contact with the pericapillary space with an average contact area of 9% +/− 5% of the plasma membrane's surface. We are currently applying SBF-SEM to determine quantitatively how islet morphology differs between genotypes in wild type, non-obese diabetic (NOD) and IA-2/IA-2 beta double-knockout mice. Such quantitative analysis of ultrastructural differences between controls and diabetes-related animal models could help further understanding of disease. The research was supported by the intramural programs of NIBIB and NIDCR.Denk, W., & Horstmann, H. (2004). Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS biology, 2(11), e329.

CCL17 production by mouse langerhans cells stimulated with Staphylococcus aureus cell wall components.

Patients with atopic dermatitis (AD) show increased numbers of Th2 cells in their acute skin lesions and superficial skin colonization by Staphylococcus aureus. The purpose of this study was to clarify the effect of S. aureus cell wall components on Th2 chemokine production by murine Langerhans cells (LCs). Murine LCs were stimulated with peptidoglycan (PEG) and/or muramyldipeptide (MDP) for 24 or 48 h, and Th1 and Th2 chemokine production was investigated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). PEG-stimulation of LCs induced production of the Th2 chemokine CCL17 and this was enhanced in the presence of interleukin (IL)-4. A low-molecular weight PEG fragment, MDP, did not induce CCL17 production by LCs. However, when LCs were stimulated with PEG in combination with MDP, PEG-induced CCL17 production was synergistically enhanced by MDP. Furthermore, PEG- and MDP-induced CCL17 production was enhanced to a greater extent in the presence of IL-4. These results suggest that S. aureus colonization in AD patients may enhance the Th2-prone immune response through upregulation of CCL17 production by LCs, which would occur as a result of simultaneous stimulation with PEG and MDP from S. aureus in a Th2 environment.