Human multipotent adult progenitor cells enhance islet function and revascularisation when co-transplanted as a composite pellet in a mouse model of diabetes

AIMS/HYPOTHESIS: Hypoxia in the initial days after islet transplantation leads to considerable loss of islet mass and contributes to disappointing outcomes in the clinical setting. The aim of the present study was to investigate whether co-transplantation of human non-endothelial bone marrow-derived multipotent adult progenitor cells (MAPCs), which are non-immunogenic and can secrete angiogenic growth factors during the initial days after implantation, could improve islet engraftment and survival.METHODS: Islets (150) were co-transplanted, with or without human MAPCs (2.5 × 105) as separate or composite pellets, under the kidney capsule of syngeneic alloxan-induced diabetic C57BL/6 mice. Blood glucose levels were frequently monitored and IPGTTs were carried out. Grafts and serum were harvested at 2 and 5 weeks after transplantation to assess outcome.RESULTS: Human MAPCs produced high amounts of angiogenic growth factors, including vascular endothelial growth factor, in vitro and in vivo, as demonstrated by the induction of neo-angiogenesis in the chorioallantoic membrane assay. Islet-human MAPC co-transplantation as a composite pellet significantly improved the outcome of islet transplantation as measured by the initial glycaemic control, diabetes reversal rate, glucose tolerance and serum C-peptide concentration compared with the outcome following transplantation of islets alone. Histologically, a higher blood vessel area and density in addition to a higher vessel/islet ratio were detected in recipients of islet-human MAPC composites.CONCLUSIONS/INTERPRETATION: The present data suggest that co-transplantation of mouse pancreatic islets with human MAPCs, which secrete high amounts of angiogenic growth factors, enhance islet graft revascularisation and subsequently improve islet graft function

Type 1 diabetes: entering the proteomic era

During the last decade, a major breakthrough in the field of proteomics has been achieved. This review describes available techniques for proteomic analyses, both gel and non-gel based, particularly concentrating on relative quantification techniques. The principle of the different techniques is discussed, highlighting the advantages and drawbacks of recently available visualization methods in gel-based assays. In addition, recent developments for quantitative analysis in non-gel-based approaches are summarized. This review focuses on applications in Type 1 diabetes. These mainly include proteomic studies on pancreatic islets in animal models and in the human situation. Also discussed are mass spectrometry-based studies on T-cells, and studies on the development of diagnostic markers for diabetic nephropathology by capillary electrophoresis coupled to mass spectrometry.status: publishe

Proteomics as a tool to discover biomarkers for the prediction of diabetic complications

BACKGROUND: The incidence of diabetes is increasing rapidly, owing to unhealthy lifestyles. The human suffering and healthcare burden caused by diabetes are mainly a consequence of its microvascular and macrovascular complications. Late diagnosis and lack of strict metabolic control lead to increased morbidity and mortality. A problem is the insidious course of the complications, diagnosed in advanced stages, when end organ damage is already present. OBJECTIVE: Discovery of early markers of microangiopathy and macroangiopathy may help to identify patients at risk of organ damage. METHODS: This review focuses on recent developments in proteomics, performed on body fluids and different tissues implicated in microvascular and macrovascular complications. RESULTS: The results provide important information for the early detection of diabetic complications as well as for better understanding of their pathophysiology.status: publishe

UVB-induced 1,25(OH)2D3 production and vitamin D activity in intestinal CaCo-2 cells and in THP-1 macrophages pretreated with a sterol Delta7-reductase inhibitor

Epidermal keratinocytes are able to produce 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and induce vitamin D activity upon UVB irradiation. To find out whether this property is keratinocyte specific, we investigated this characteristic in two other cell types, namely intestinal CaCo-2 cells and the macrophage-like differentiated THP-1 cells. THP-1 macrophages and preconfluent CaCo-2 cells contain the vitamin D receptor (VDR), possess 25-hydroxylase (CYP2R1 and CYP27A1) and 1alpha-hydroxylase (CYP27B1) activity, and survive the low UVB doses essential for vitamin D3 photoproduction. Upon irradiation, 24-hydroxylase (CYP24) mRNA is induced in both cell types pretreated with the sterol Delta7-reductase inhibitor BM15766 whereby the 7-dehydrocholesterol (7-DHC) content was increased. Transfection studies in CaCo-2 cells with a vitamin D response element-containing construct revealed the involvement of the VDR in this UVB-dependent CYP24 induction. The CYP24 inducing activity in BM15766-pretreated UVB-irradiated CaCo-2 cells and THP-1 macrophages was identified as 1,25(OH)2D3 by combined high-performance liquid chromatography radioimmunoassay. Addition of vitamin D binding protein to the CaCo-2 cells attenuated UVB-induced CYP24 induction suggesting the possibility of a paracrine or autocrine role for the photoproduced 1,25(OH)2D3. In conclusion, preconfluent CaCo-2 cells and THP-1 macrophages are able to induce vitamin D activity upon UVB irradiation and hence combine all parts of the vitamin D photoendocrine system, a characteristic which is therefore not keratinocyte specific.status: publishe

Neuropeptides of the islets of Langerhans: a peptidomics study

Neuropeptides from the endocrine pancreas (the islets of Langerhans) play an important role in the regulation of blood glucose levels. Therefore, our aim is to identify the "peptidome" (the in vivo peptide profile at a certain time) of the pancreatic islets, which is beneficial for medical progress related to the treatment of diabetes. So far, there are few neuropeptides isolated and sequenced from the endocrine pancreas and mainly in situ hybridisation and immunocytochemical techniques have been used to demonstrate the occurrence of peptides in the pancreas. These techniques do not allow for unequivocal identification of peptides. In contrary, mass spectrometry identifies peptides unambiguously. We have analysed the peptidome of the islets using peptidomics, i.e. a combination of liquid chromatography, mass spectrometry and bioinformatics. We are able to identify the peptidome of islets extracts. We not only confirm the presence of peptides with a well-known effect on blood glucose levels, but also identify new peptides, which are unknown to affect blood glucose levels.status: publishe

Novel insights in the immune function of the vitamin D system: Synergism with interferon-beta

The 1,25(OH)(2)D(3) analog, TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)), has an interesting dissociation profile between its potent immunomodulatory and its calcemic effects in vivo. The strong immunomodulatory potency of TX527 is reflected by its ability to attenuate experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). At present most MS patients are being treated with systemic IFN-beta administration. The aim of this study was to investigate whether combining IFN-beta with TX527 could empower its EAE-protective effects. We evaluated also combinations with the standard immunosuppressant cyclosporin A (CsA). EAE was induced in SJL mice by PLP immunization, treatment was started 3 days before disease induction. The TX527+IFN-beta combination resulted in significant disease protection which was superior to the effect of both treatment separately. No disease amelioration, even aggravation, was obtained with the IFN-beta+CsA combination. By adding TX527 to the IFN-beta+CsA combination near complete protection from EAE was achieved (100% protection from paralysis, mean maximal score of 1.8+/-1.5, both p<0.05 versus controls and all individual treatments). From these data we conclude that adding TX527 to an IFN-beta and/or CsA treatment results in clear additional immunomodulatory effects in EAE prevention and is therefore a potentially interesting candidate to be considered in clinical intervention trials in MS.status: publishe

High glucose induces dysfunction in insulin secretory cells by different pathways: a proteomic approach.

Chronic hyperglycemia is a hallmark of type 2 diabetes and can contribute to progressive beta cell dysfunction and death. The aim of the present study was to identify pathways mediating high glucose-induced beta cell demise by a proteomic approach. INS-1E cells were exposed to 25 mM glucose for a sustained period of 24 h. Protein profiling of INS-1E cells was done by two-dimensional difference gel electrophoresis, covering the pH ranges 4-7 and 6-9 (n = 4). Differentially expressed proteins (P < 0.05) were identified by MALDI-TOF/TOF and proteomic results were confirmed by functional assays. High glucose levels impaired glucose-stimulated insulin secretion and decreased insulin content. 2D-DIGE analysis revealed 100 differentially expressed proteins that were involved in different pathways. Chaperone proteins were down-regulated, protein biosynthesis and ubiquitin-related proteasomal degradation were attenuated and perturbations in intracellular trafficking and vesicle transport and secretion could be observed. Moreover, several pathways were confirmed by functional assays and a direct role for eEF2 in insulin biosynthesis was demonstrated. The present findings provide new insights in glucotoxicity and identify key target proteins for the prevention and treatment of beta cell dysfunction in type 2 diabetes.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Blockade of CTLA-4 enhances allergic sensitization and eosinophilic airway inflammation in genetically predisposed mice

CTLA-4 (CD152) expression is restricted to subsets of activated T lymphocytes and shares homology with CD28. CTLA-4 and CD28 molecules both bind to B7 molecules on antigen-presenting cells. Whereas CD28-B7 interaction enhances T cell activation, cytokine production and survival, CTLA-4 signaling down-regulates T cell responses. Here, we studied the involvement of CTLA-4 triggering in the pathogenesis of allergen-induced airway inflammation in mice. Anti-CTLA-4 mAb were injected during i.p. sensitization with ovalbumin (OVA). This treatment favored OVA-specific IgE production and augmented blood eosinophilia in BALB/c mice. In BALB/c mice, enhanced Th2 sensitization after anti-CTLA-4 mAb injections resulted in more severe airway inflammation, and increased airway hyperresponsiveness to metacholine, bronchial eosinophilia and IL-4 and IL-5 levels in broncho-alveolar lavage (BAL) fluid following repeated allergen inhalations. Importantly, aggravation of airway inflammation and enhancement of Th2 responses were accompanied by a significant reduction of pulmonary TGF-beta levels at protein level in BAL fluid as well as on mRNA level in inflamed lung tissue. In contrast to BALB/c mice, blockade of CTLA-4 did not alter IgE production nor the phenotype of airway inflammation or TGF-beta production in C57BL/6 mice. Our data suggest that CTLA-4 triggering represents an important regulatory mechanism for Th2 sensitization in genetically predisposed mice by modulating TGF-beta productio

Interferon regulatory factor-1 is a key transcription factor in murine beta cells under immune attack.

AIMS/HYPOTHESIS: IFN-gamma, together with other inflammatory cytokines such as IL-1beta and TNF-alpha, contributes to beta cell death in type 1 diabetes. We analysed the role of the transcription factor interferon regulatory factor (IRF)-1, a downstream target of IFN-gamma/signal transducer and activator of transcription (STAT)-1, in immune-mediated beta cell destruction. METHODS: Islets from mice lacking Irf-1 (Irf-1 (-/-)) and control C57BL/6 mice were transplanted in overtly diabetic NOD mice. Viability and functionality of islets were evaluated in vitro. Chemokine expression by Irf-1 (-/-) islets and INS-1E cells transfected with Irf-1 short interfering RNA (siRNA) was measured by real-time PCR as well as in functional assays in vitro. RESULTS: IRF-1 deletion in islets was associated with higher prevalence of primary non-function (63% vs 25%, p <or= 0.05) and shorter functioning graft survival (6.0 +/- 2.6 vs 10.4 +/- 4.8 days, p <or= 0.05) in contrast to similar skin graft survival. Although Irf-1 (-/-) islets were resistant to cytokine-induced cell death, insulin secretion by them was lower than that of control C57BL/6 islets under medium and cytokine conditions. IL-1 receptor antagonist partly restored the cytokine-induced secretory defect in vitro and completely prevented primary non-function in vivo. Cytokine-exposed Irf-1 (-/-) islets and INS-1E cells transfected with Irf-1 siRNA showed increased expression of Mcp-1 (also known as Ccl2), Ip-10 (also known as Cxcl10), Mip-3alpha (also known as Ccl20) and Inos (also known as Nos2) mRNA and elevated production of monocyte chemoattractant protein-1 (MCP-1) and nitrite compared with controls. In vivo, Irf-1 (-/-) islets displayed a higher potential to attract immune cells, reflected by more aggressive immune infiltration in the grafted islets. CONCLUSIONS/INTERPRETATION: These data indicate a key regulatory role for IRF-1 in insulin and chemokine secretion by pancreatic islets under inflammatory attack.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe