COVID-19 is an emerging, rapidly evolving situation. MLD-STZ in wild type mice induced islet leukocyte infiltration, cytokine production, beta-cell apoptosis, and hyperglycemia. Endothelial cells have also been used to coat the islets; transplantation of these coated islets has reduced coagulation, complement activation, and leukocyte infiltration [53, 54]. Protease- and protease inhibitor–specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. A 3-dimensional projection of a whole mount of a healthy mouse pancreas and visualization of the peri-islet capsule by immunofluorescence staining and electron microscopy are shown. Protease- and protease inhibitor–specific microarray analyses (CLIP-CHIP) of laser-capture microdissection (LCM) material revealed upregulation of cathepsin S, W, and C in leukocyte-infiltrated islets compared with noninvaded pancreatic islets. In contrast, the group with IL-33 treatment showed well-preserved islet morphology with reduced/minimal leukocyte infiltration (Fig 1F). CLIP-CHIP microarray … This suggests that the cells responsible for formation of the peri-islet BM are not lost due to the inflammation and can regenerate the BM once the inflammation has subsided. In marked contrast, a marginal-to-modest islet destruction and leukocyte infiltration were found in the pancreatic islets of NOD mice treated with catenarin at 4 mg/kg and over (Figure 2 (b)). 1C and D) but not laminin α1, α3, and α5 chains (Fig. 5A–C). MLD-STZ in wild type mice induced islet leukocyte infiltration, cytokine production, beta-cell apoptosis, and hyperglycemia. Recent studies have reported the existence of laminins, collagen type IV, and perlecan surrounding the pancreatic islets and the absence of staining for these antigens in pancreata of NOD mice, however, without correlation to disease progression in mice or with type 1 diabetes in humans (23,24). 1A), are associated with the peri-islet BM (Fig. provided antibodies and advice on the cathepsins. cells of the pancreatic islets of Langerhans. 5D–H are from one sample (nPOD case 6052), a 12-year-old donor with type 1 diabetes who died 1 year after diagnosis, which still contained some insulin+ islets surrounded by leukocytes, according to the nPOD histological analysis (http://www.jdrfnpod.org/). Sections from the pancreas head, body, and tail were analyzed by immunofluorescence staining. The islet surface has been coated with heparin complex by exploiting biotin-avidin chemistry to inhibit coagulation [52]. The primary and secondary antibodies used are listed in Supplementary Table 1. Upon MLDS induction, Robo4‐deficiency resulted in increased pancreatic vascular permeability, leukocyte infiltration to the islets and islet apoptosis, associated with reduced insulin levels and faster diabetes development. CD4 and CD8 T cells and macrophages were excluded from islets and remained entrapped in a peri-islet location as inactive exiles, no longer expressing normal levels of interferon-gamma, interleukin-4, and iNOS. (H) The number of apoptotic β cell per islet. Analyses of the laminin family of BM proteins together with endothelial cell markers revealed that the peri-islet BM was biochemically distinct to the BMs surrounding blood vessels and contained laminin α2, α4, β1, β2, γ1, γ2, and γ3 chains (Fig. Analysis of Inflammatory Leukocyte Infiltration Into IFN Receptor–Deficient Mice In normal mice, macrophages are the major leukocyte throughout the life of the mouse ( 13 ). Triple-staining for F4/80, as a marker for macrophages, the nuclei marker DAPI and cathepsin C (B), cathepsin S (C), or cathepsin W (D) show colocalization of all three cathepsins, with a subpopulation of macrophages. National Library of Medicine It also suggests the involvement of several proteases or of proteases with broad specificity. In contrast, Jnk1-/- mice were substantially protected from a loss of insulin producing cells and hyperglycemia in the MLD-STZ model despite a marked islet T cell and macrophage infiltrate. Protease- and protease inhibitor–specific microar-ray analyses (CLIP-CHIP) of laser-dissected leukocyte … Severe islet destruction and leukocyte infiltration was found in the pancreata of NOD mice treated with vehicle, 0.4 mg/kg catenarin and acetylsalicylic acid (Figure 2(b)). NCI CPTC Antibody Characterization Program. However, once autoaggressive T cells are activated, several steps are crucial for disease progression, including extravasation of the CD4+, CD8+ T lymphocytes from the postcapillary venules surrounding the islets of Langerhans and their subsequent infiltration of the islets, leading to β-cell destruction and onset of disease symptoms. A major impediment to advances in understanding, preventing, and curing T1D has been the inability to "see" the disease initiate, progress, or … The boxed areas are shown at higher magnifications to the right. On the one hand, the effects of … B: An optical section of a 3-dimensional projection of a whole-mount pancreatic islet shows in a simultaneous view of XY, XZ, and YZ planes, illustrating the peri-islet BM and endothelial BMs. Parallel analyses of the expression of the major ECM receptors revealed localization of Lutheran blood group glycoprotein and β-dystroglycan on endothelial cells and a weak immunosignal for β-dystroglycan on cells that were close to the peri-islet BM (Table 1). 2A and B). The present work addresses this important step in the development of type 1 diabetes in NOD mice and in humans, in particular, the contribution of the peri-islet basement membrane (BM) to this step. Finally, histological examination for insulitis corroborated reduced leukocyte infiltration within the islets of the AIF1-silenced cohort relative to siScramble-treated NOD controls . A: Immunofluorescence staining reveals the BM component laminin (LM) α5 surrounding the insulin and glucagon-containing islet. Penetration of the peri-islet BM is a critical step in disease progression in NOD mice. Samples for electron microscopy were prepared according to standard protocols and analyzed with an EM-410 (Philips). The mean fluorescence intensity per micrometer of degraded peri-islet BMs is significantly lower than that of intact peri-islet BMs. For confocal microscopy of whole mounts, pancreata were fixed in 4% paraformaldehyde and blocked in 1% BSA/0.3% TritonX-100. F: Electron micrography illustrates two BMs: the peri-islet BM (arrow) that envelops the β-cells and the endothelial BM of blood vessels (arrowhead; inset 1). Although most ECM proteins analyzed showed the same localization pattern as observed in NOD pancreata, only laminin α5 was found to mark the peri-islet BM, which was not the case in mouse samples, a result consistent with findings from previous studies (21,22). that islet architecture was lost, with only a few remaining insulin-positive cells and massive leukocyte infiltration. 2004 Spring;1(1):9-17. doi: 10.1900/RDS.2004.1.9. 1A and C). Mean fluorescence values of peri-islet BM staining for laminin were therefore used to define healthy versus invaded pancreatic islets. This was attributed to inhibition of leukocyte infiltration, production of pro-inflammatory cytokines and islet cell apoptosis at day 14 of MLD-STZ. Such islets dominated the pancreata of donors who had been diagnosed with the disease for several years (nPOD cases 6062–6064; Supplementary Table 2) and type 1 diabetic medalists samples that were diagnosed with diabetes for more than 50 years (nPOD cases 6085 and 6086). Lymphocyte Infiltration in Pancreatic Islets Mediates [beta]-Cell Proliferation in NOD Mice Type 1 Diabetes (T1D), is a T- Type 1 Diabetes (T1D), is a T-cell mediated disease characterized by selective destruction of [beta]-cells. Additionally, FTY720 stimulated insulin secretion from isolated islets by approximately twofold under both normoglycemic and hyperglycemic conditions. Islet leukocyte infiltration and cytokine mRNA expression MLD-STZ induced an infiltrate of F4/80+ macrophages, CD4+ T cells, and CD8+ T cells in the islets of WT mice ( Fig. Secondly, thalidomide treatment reduced leukocyte infiltration of the islet graft as evidenced by reduced MPO activity. The IM and BM influence immune cell migration into or within inflamed tissues (14). Type 1 diabetes (T1D) is an autoimmune disease characterized by leukocyte infiltration into the pancreatic islets, and we have previously shown that treatment of adult NOD mice with a vitamin D analog arrests the progression of insulitis, blocks Th1 cell infiltration into the pancreas, and markedly reduces T1D development, suggesting inhibition of chemokine production by islet cells. (G) PCNA + islet cells. Infiltrating leukocytes retained a peri-islet location, even 215 days following suspension of antibody treatment, potentially forming a barrier to the entry of active, autoantigen-reactive T cells. The authors thank Hartmut Halfter (Department of Neurology, University of Münster, Münster, Germany) for use of the LCM; Friedemann Kiefer, Stefan Volkery, and Ruben Böhmer (Max Planck Institute for Molecular Medicine, Münster, Germany) for assistance in the use of the LSM510 microscope; Karin Loser (Department of Dermatology, University of Münster, Münster, Germany) for help with quantitative real-time PCR; Horst Robenek and Karin Schlattmann (Arteriosclerosis Research, University of Münster, Münster, Germany) for help in the use of the electron microscope; and Frank Arfuso (Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany) for critical reading of the manuscript. To identify the immune cell-type(s), and … J.W. These results demonstrate how quantitative light-sheet imaging can capture changes in individual islets to help … Diabetes 2013;62:531–542 - July 01, 2013, http://clinicaltrials.gov/ct2/show/NCT01319331, http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0432/-/DC1, http://creativecommons.org/licenses/by-nc-nd/3.0/. Laminins are composed of an α, β, and γ chain, and 5α, 4β, and 3γ laminin chains exist that combine to form at least 18 different isoforms. 2 ). performed the quantitative real-time PCR experiments. Data shown in Fig. Leukocytes infiltrate the pancreatic islets of nonobese diabetic mice, causing β‐cell destruction and autoimmune Type I diabetes. Cytokine Secretion, Leukocyte Infiltration, and Beta-Cell Damage. Combination treatment was effective against spontaneous disease when administered from 7 days of age but ineffective when initiated late in the prediabetic period (day 40 or 70). A: Glucagon+ cell clusters are already detectable in heavily inflamed islets of nondiabetic mice, which are surrounded by a peri-islet BM shown here by immunofluorescence for laminin α2 (LMα2). Type 1 diabetes is the clinical manifestation of aberrant leukocytic infiltration of the pancreatic islets; it is usually diagnosed only very late in disease progression, after the critical autoimmune phenomena have mostly played out. The islet surface has been coated with heparin complex by exploiting biotin-avidin chemistry to inhibit coagulation [52]. Examples of this peri-islet BM staining are shown in Fig. Quantitative analyses revealed significantly reduced mean fluorescence intensity of the peri-islet BM pan-laminin staining at sites of leukocyte invasion of islets and loss of insulin+ cells and that this correlates with disease progression, with a higher incidence of insulitis when more islets showed penetration of the peri-islet BM. Menu en zoeken; Contact; My University; Student Portal To investigate whether leukocyte infiltration into the human pancreatic islets is also associated with loss of peri-islet ECM components, human type 1 diabetic samples were analyzed (Supplementary Tables 2 and 3). Stereological analyses reveal a correla-tion between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. Enter multiple addresses on separate lines or separate them with commas. 2). Pancreata from 4- and 12-week-old NOD mice were snap frozen, 12–16 μm cryosections were cut and mounted on polyethylene naphthalate membrane-coated slides (PALM MicroLaser Systems, Bernried, Germany), immediately fixed, dehydrated, and hematoxylin stained using the HistoGene Frozen Section Staining Kit (Arcturus). After sacrifice, the islet grafts were retrieved for analysis of infection and leukocyte infiltration. We do not capture any email address. REFERENCES. 2004 Oct;47(10):1657-60. doi: 10.1007/s00125-004-1518-0. A summary of the BM and IM components of the peri-islet capsule and their receptors are listed in Table 1. For investigation of correlations between peri-islet BM integrity and disease progression, frozen pancreata from 5-, 14- (normoglycemic) and 21-week-old (diabetic) NOD mice were sectioned completely, and every 10th section was immunofluorescently stained with pan-laminin antibody to label BMs, CD45, a pan-leukocyte marker, and insulin. We demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte penetration of the peri-islet capsule, which contrasts to leukocyte extravasation from blood vessels and demonstrates fundamental differences in these two processes. D.H. and S.C. were involved in project development and contributed significantly to compilation of the manuscript. Interestingly, in samples from humans with long-term type 1 diabetes, we found insulin− islets surrounded by an intact peri-islet BM and underlying IM. ACKNOWLEDGMENTS. These results suggest that the cells responsible for producing the peri-islet BM are not lost due to the inflammation and can reconstitute the peri-islet BM once the inflammation has subsided. Leukocytes infiltrate the pancreatic islets of nonobese diabetic mice, causing beta-cell destruction and autoimmune Type I diabetes. C: Meca32 staining marks the endothelial layer of blood vessels (insets show images at higher magnification). 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CMV infection of the graft was only observed prior to complete graft failure. The ECM surrounding the islet is destroyed during islet isolation for transplantation experiments, leading to integrin-mediated cell death; however, preservation of an ECM mantel significantly increases the survival rate of transplanted islets (50). Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. We show that the peri-capsular ECM is composed of a peri-islet BM embedded in an IM layer that interconnects with the surrounding blood vessels and exocrine tissue. Curr Pharm Des. At 14weeks, 40% of the small islets, but more than 80% of large islets, showed leukocyte infiltration. Following full immunological activation, there was a progressive enhancement of inflammatory signatures, culminating in the destruction of the islet PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e59701. Macrophages and DCs are among the first cells recruited to pancreatic islets (34,35), and their localization was shown here to correlate with sites of loss of peri-islet BM (Fig. 1D). 2006 Sep;178(1-2):1-8. doi: 10.1016/j.jneuroim.2006.05.030. As in NOD samples, the healthy human pancreatic islet was surrounded by a capsule composed of a peri-islet BM and subjacent IM (Fig. These results showed that IL-33 treatment prevented islet allograft rejection and NOD mice are one of the best-studied animal models of type 1 diabetes (2) due to similarities with the pathogenesis and genetics of human type 1 diabetes. Taken together, these data suggest a correlation between degradation of the peri-islet capsule and disease progression and that penetration of the peri-islet BM is a critical step in disease progression. hibiting diabetes and determined the fate of leukocytes respon- Serial sections were stained for CD4 and CD8 cells and macrophages, by the sible for the insulitis. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Scale bars are 100 μm. Consistent with the results of the PAS staining, MLD-STZ Jnk1−/− mice also showed a macrophage and T cell infiltrate, the composition of which was not different to that seen in WT mice ( Fig. Only IL-10 expression was retained, which could aid immunosuppression. In all tissues, the extracellular matrix (ECM) acts to separate tissue compartments but also provides specific molecular signals that control processes such as cell migration, differentiation, and survival (8). R.K. and C.M.O. Precisely how the cathepsins function in leukocyte penetration of the peri-islet BM is not clear. E.W. Differential localization of laminin (LM) α4 and α5 chains in the pancreatic islet is shown by immunofluorescence staining of thin sections (C) and whole-mount staining (D). Immunofluorescence staining of a whole mount (A) and thin sections (B; boxed areas show higher magnification) of leukocyte-invaded islets for pan-laminin (PLM) as a marker of the peri-islet BM, and the pan-leukocyte marker CD45 illustrates the correlation between the loss of the peri-islet BM and leukocyte infiltration into the pancreatic islet. As for the NOD mouse, triple-immunofluorescence staining in samples from humans with type 1 diabetes revealed that some of the CD45+ cells were also positive for cathepsin S and W staining at sites of peri-islet BM loss (Fig. In opposition to this proposal. Several members of the cathepsin protease family, including cathepsin C, H, S, and W, showed a four- to sixfold increase in mRNA expression in infiltrated islets (Table 2), which was confirmed by quantitative real-time PCR (Supplementary Fig. Surprisingly, in several human diabetic pancreas samples we observed many islets that were insulin−/glucagon+ but also lacked CD45+ leukocytes and were encased in an intact peri-islet BM (Fig. In pancreatic islets showing disrupted panlaminin staining at sites coincident with CD45+ cells and loss of insulin+ cells, the mean fluorescence value per micrometer was significantly reduced (P < 0.0007; Supplementary Fig. We therefore used immunofluorescence microscopy to investigate whether cathepsins C, S, and W colocalized with the islet-invading leukocytes. Islet leukocyte infiltration and cytokine mRNA expressionMLD-STZ induced an infiltrate of F4/80+ macrophages, CD4+ T cells, and CD8+ T cells in the islets of WT mice (Fig. The upregulation of MMP-14 is consistent with its proposed role in T-cell extravasation into the pancreas via CD44 cleavage (41) but can be excluded as a candidate for degradation of the peri-islet capsule. L.S. These isoforms are differentially expressed in different BM types where they perform different functions (10) mediated by direct binding to a variety of different integrin and nonintegrin receptors (11). The peri-islet BM remains detectable (arrows) at sites of leukocyte accumulation around the islet but not where leukocytes have invaded the islet (arrowhead). The reduced neutrophil infiltration by thalidomide provides additional protection to the islet graft.
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