Impact of sleep and physical activity habits on real-life glycaemic variability in patients with type 2 diabetes.

The aim of this study was to better characterise whether sleep habits, eating schedule and physical activity in real-life are associated with glycaemic control in patients with type 2 diabetes. A total of 28 patients (aged 60 years [58; 66], 54% female) with type 2 diabetes treated with basal-bolus insulin therapy administered by insulin pumps were analysed. Glycaemic data measured by Flash Glucose Monitor System, physical activity and sleep data measured by accelerometer, and meal schedules were simultaneously collated with insulin pump administration data, for 7 days in real-life. Their respective impact on the time spent in target, in hypoglycaemia, in hyperglycaemia and on glycaemic variability was evaluated. Multiple regressions showed that the total daily dose of meal boluses of insulin was inversely associated with the coefficient of variation (CV; coefficient ß = -0.073; 95% confidence interval: -0.130, -0.015; p = 0.016), as well as sleep duration. The higher the sleep duration, the lower the glycaemic variability (coefficient ß = -0.012; 95% confidence interval: -0.023, -0.002; p = 0.027). The mean 7 days physical activity of the subjects was very low and was not associated with glycaemic control on the 7 days mean values. However, days with at least 1 hr spent in physical activity higher than 1.5 METs were associated with less glycaemic variability that same day. This real-life observation highlights the importance of sufficient sleep duration and regular physical activity to lessen the glycaemic variability of patients with type 2 diabetes.

No more hypoglycaemia on days with physical activity and unrestricted diet when using a closed-loop system for 12 weeks: A post hoc secondary analysis of the multicentre, randomized controlled Diabeloop WP7 trial.

A post hoc analysis of the Diabeloop WP7 multicentre, randomized controlled trial was performed to investigate the efficacy of the Diabeloop Generation-1 (DBLG1) closed-loop system in controlling the hypoglycaemia induced by physical activity (PA) in real-life conditions. Glycaemic outcomes were compared between days with and without PA in 56 patients with type 1 diabetes (T1D) using DBLG1 for 12 weeks. After the patient announces a PA, DBLG1 reduces insulin delivery and, if necessary, calculates the amount of preventive carbohydrates (CHO). Daily time spent in the interstitial glucose range less than 70 mg/dL was not significantly different between days with and without PA (2.0% ± 1.5% vs. 2.2% ± 1.1%), regardless of the intensity or duration of the PA. Preventive CHO intake recommended by the system was significantly higher in days with PA (41.1 ± 35.5 vs. 21.8 ± 28.5 g/day; P < .0001), and insulin delivery was significantly lower (31.5 ± 10.5 vs. 34.0 ± 10.5 U/day; P < .0001). The time spent in hyperglycaemia and the glycaemic variation coefficient increased significantly on days with PA. In real-life conditions, the use of DBLG1 avoids PA-induced hypoglycaemia. Insulin adjustments and preventive CHO recommendation may explain this therapeutic benefit.

Assessment of plasma microvesicles to monitor pancreatic islet graft dysfunction: Beta cell- and leukocyte-derived microvesicles as specific features in a pilot longitudinal study.

Markers of early pancreatic islet graft dysfunction and its causes are lacking. We monitored 19 type 1 diabetes islet-transplanted patients for up to 36 months following last islet injection. Patients were categorized as Partial (PS) or complete (S) Success, or Graft Failure (F), using the ß-score as an indicator of graft function. F was the subset reference of maximum worsened graft outcome. To identify the immune, pancreatic, and liver contribution to the graft dysfunction, the cell origin and concentration of circulating microvesicles (MVs) were assessed, including MVs from insulin-secreting ß-cells typified by polysialic acid of neural cell adhesion molecule (PSA-NCAM), and data were compared with values of the ß-score. Similar ranges of PSA-NCAM+ -MVs were found in healthy volunteers and S patients, indicating minimal cell damage. In PS, a 2-fold elevation in PSA-NCAM+ -MVs preceded each ß-score drop along with a concomitant rise in insulin needs, suggesting ß-cell damage or altered function. Significant elevation of liver asialoglycoprotein receptor (ASGPR)+ -MVs, endothelial CD105+ -MVs, neutrophil CD66b+ -MVs, monocyte CD 14+ -MVs, and T4 lymphocyte CD4+ -MVs occurred before each ß-score drop, CD8+ -MVs increased only in F, and B lymphocyte CD19+ -MVs remained undetectable. In conclusion, PSA-NCAM+ -MVs are noninvasive early markers of transplant dysfunction, while ASGPR+ -MVs signal host tissue remodeling. Leukocyte MVs could identify the cause of graft dysfunction.

Liver adenomatosis in patients with hepatocyte nuclear factor-1 alpha maturity onset diabetes of the young (HNF1A-MODY): Clinical, radiological and pathological characteristics in a French series.

BACKGROUND: Liver adenomatosis (LA) is a rare disease resulting from biallelic inactivation of the hepatocyte nuclear factor-1 alpha (HNF1A) gene, which induces the proliferation of adenoma cells in liver parenchyma. Liver adenomatosis has only been documented in case reports from patients carrying a HNF1A germline mutation. We have evaluated the frequency of LA among a large cohort of patients with HNF1A-maturity onset diabetes of the young (MODY), previously termed "MODY3," and herein describe its clinical, radiological, and pathological characteristics. METHODS: In all, 137 HNF1A-MODY subjects from 74 families were screened by liver ultrasonography in 13 centers, and 15 additional cases of LA were later included in the series. Liver adenomatosis was confirmed by liver computed tomography, magnetic resonance imaging (MRI), and/or histopathology. RESULTS: Among 137 carriers of an HNF1A mutation, 9 patients (6.5%) from seven families were diagnosed with LA. Diabetes mellitus was present in 87.5% of patients with LA. In 25% of patients, LA was diagnosed due to intra-abdominal or intratumoral bleeding. Liver biochemistry was near normal in all patients. Liver imaging showed adenomas of various sizes and numbers. On MRI, most nodules had the radiological characteristics of steatotic adenomas. Histopathological confirmation of LA was available in 13 cases, and these adenomas were mostly steatotic. Surgery was initially performed in 37.5% of patients, and liver disease progression was observed in 30%. No disease progression was observed in 14 pregnancies. CONCLUSIONS: The frequency of LA in a cohort of screened HNF1A-MODY patients and the high incidence of LA progression and/or hemorrhage warrants systematic screening for liver adenomatosis in HNF1A-MODY families.

Human mesenchymal stem cells improve rat islet functionality under cytokine stress with combined upregulation of heme oxygenase-1 and ferritin.

BACKGROUND: Islets of Langerhans transplantation is a promising therapy for type 1 diabetes mellitus, but this technique is compromised by transplantation stresses including inflammation. In other tissues, co-transplantation with mesenchymal stem cells has been shown to reduce damage by improving anti-inflammatory and anti-oxidant defences. Therefore, we probed the protection afforded by bone marrow mesenchymal stem cells to islets under pro-inflammatory cytokine stress. METHODS: In order to evaluate the cytoprotective potential of mesenchymal stem cells on rat islets, co-cultures were exposed to the interleukin-1, tumour necrosis factor α and interferon γ cocktail for 24 h. Islet viability and functionality tests were performed. Reactive oxygen species and malondialdehyde were measured. Expression of stress-inducible genes acting as anti-oxidants and detoxifiers, such as superoxide dismutases 1 and 2, NAD(P)H quinone oxidoreductase 1, heme oxygenase-1 and ferritin H, was compared to non-stressed cells, and the corresponding proteins were measured. Data were analysed by a two-way ANOVA followed by a Holm-Sidak post hoc analysis. RESULTS: Exposure of rat islets to cytokines induces a reduction in islet viability and functionality concomitant with an oxidative status shift with an increase of cytosolic ROS production. Mesenchymal stem cells did not significantly increase rat islet viability under exposure to cytokines but protected islets from the loss of insulin secretion. A drastic reduction of the antioxidant factors heme oxygenase-1 and ferritin H protein levels was observed in islets exposed to the cytokine cocktail with a prevention of this effect by the presence of mesenchymal stem cells. CONCLUSIONS: Our data evidenced that MSCs are able to preserve islet insulin secretion through a modulation of the oxidative imbalance mediated by heme and iron via heme oxygenase-1 and ferritin in a context of cytokine exposure.

New Automatized Method of 3D Multiculture Viability Analysis Based on Confocal Imagery: Application to Islets and Mesenchymal Stem Cells Co-Encapsulation.

Co-encapsulation of pancreatic islets with mesenchymal stem cells in a three-dimensional biomaterial's structure is a promising technique to improve transplantation efficacy and to decrease immunosuppressant therapy. Currently, evaluation of graft quality after co-encapsulation is only based on insulin secretion. Viability measurement in a 3D conformation structure involving two different cell types is complex, mainly performed manually, highly time consuming and examiner dependent. Standardization of encapsulated graft viability analysis before transplantation is a key point for the translation of the method from the bench side to clinical practice. In this study, we developed an automated analysis of islet viability based on confocal pictures processing of cells stained with three probes (Hoechst, propidium iodide, and PKH67). When compared with results obtained manually by different examiners, viability results show a high degree of similarity (under 3% of difference) and a tight correlation (r = 0.894; p < 0.001) between these two techniques. The automated technique offers the advantage of reducing the analysis time by 6 and avoids the examiner's dependent variability factor. Thus, we developed a new efficient tool to standardize the analysis of islet viability in 3D structure involving several cell types, which is a key element for encapsulated graft analysis in clinical practice.

Risk of autoimmune diseases and human papilloma virus (HPV) vaccines: Six years of case-referent surveillance.

BACKGROUND: Safety of HPV vaccines is still in question due to reports of autoimmune diseases (ADs) following HPV immunization. OBJECTIVES: To assess the risk of ADs associated with HPV vaccination of female adolescents/young adults in France. METHODS: Systematic prospective case-referent study conducted to assess the risks associated with real-life use of HPV vaccines. Cases were female 11-25 years old with incident ADs [central demyelination/multiple sclerosis (CD/MS), connective tissue disease (CTD), Guillain-Barré syndrome (GBS), type-1 diabetes (T1D), autoimmune thyroiditis (AT), and idiopathic thrombocytopenic purpura (ITP)]. Cases were consecutively and prospectively identified at specialized centers across France (2008-2014) and individually matched by age and place of residence to referents recruited in general practice. Risk was computed using multivariate conditional logistic regression models adjusted for family history of ADs, living in France (north/south), co-medications and co-vaccinations. RESULTS: With a total of 478 definite cases matched to 1869 referents, all ADs combined were negatively associated to HPV vaccination with an adjusted odds ratio of 0.58 (95% confidence interval: 0.41-0.83). Similar results were obtained for CD/MS, AT, CT, and T1D, the last two not reaching statistical significance. No association was found for ITP and GBS. Sensitivity analyses combining definite and possible cases with secondary time window showed similar results. CONCLUSION: Exposure to HPV vaccines was not associated with an increased risk of ADs within the time period studied. Results were robust to case definitions and time windows of exposure. Continued active surveillance is needed to confirm this finding for individual ADs.

RAGE and CYBA polymorphisms are associated with microalbuminuria and end-stage renal disease onset in a cohort of type 1 diabetes mellitus patients over a 20-year follow-up.

AIMS: We investigated the association of polymorphisms of three genes implicated in oxidative stress: CYBA C242T, RAGE -374T/A and -429T/C, and ALOX12 Arg261Gln, with the delay of microalbuminuria onset in patients with type 1 diabetes mellitus (DT1). METHODS: A total of 162 T1D patients presenting with diabetes for 32.9 ± 9 years were included in the study; 53 had persistent microalbuminuria (>30 mg/l) and 109 did not. Onset of diabetes, microalbuminuria and end-stage renal disease (ESRD) were recorded as bio-clinical data. We determined polymorphism association of microalbuminuria with a Cox regression model. RESULTS: All polymorphisms respected the Hardy-Weinberg equilibrium. The Cox regression model validated four significant variables associated with microalbuminuria: RAGE 374AA (HR 4.19 [1.84-9.58] (p = 0.001)), CYBA TT+TC (HR 2.1 [1.16-3.80], p = 0.015), male sex (HR 1.92 [1.07-3.43], p = 0.028) and diabetes diagnosis at the pediatric stage (HR 1.85 [1.03-3.32], p = 0.039). The same association was found with ESRD (p = 0.028 and p = 0.033 for CYBA TC+TT and RAGE 374AA, respectively). CYBA C242T and RAGE 374T/A were not significantly associated with diabetic retinopathy. CONCLUSIONS: CYBA C242T and RAGE -374T/A correlate with microalbuminuria onset in the French DT1 cohort. The same correlation with ESRD onset supports the argument for the involvement of a genetic predisposition involving kidney-specific oxidative stress for diabetic nephropathy.

Respective effects of oxygen and energy substrate deprivation on beta cell viability.

Deficit in oxygen and energetic substrates delivery is a key factor in islet loss during islet transplantation. Permeability transition pore (PTP) is a mitochondrial channel involved in cell death. We have studied the respective effects of oxygen and energy substrate deprivation on beta cell viability as well as the involvement of oxidative stress and PTP opening. Energy substrate deprivation for 1h followed by incubation in normal conditions led to a cyclosporin A (CsA)-sensitive-PTP-opening in INS-1 cells and human islets. Such a procedure dramatically decreased INS-1 cells viability except when transient removal of energy substrates was performed in anoxia, in the presence of antioxidant N-acetylcysteine (NAC) or when CsA or metformin inhibited PTP opening. Superoxide production increased during removal of energy substrates and increased again when normal energy substrates were restored. NAC, anoxia or metformin prevented the two phases of oxidative stress while CsA prevented the second one only. Hypoxia or anoxia alone did not induce oxidative stress, PTP opening or cell death. In conclusion, energy substrate deprivation leads to an oxidative stress followed by PTP opening, triggering beta cell death. Pharmacological prevention of PTP opening during islet transplantation may be a suitable option to improve islet survival and graft success.

Comparative impact on islet isolation and transplant outcome of the preservation solutions Institut Georges Lopez-1, University of Wisconsin, and Celsior.

BACKGROUND: Institut Georges Lopez-1 (IGL-1) is a preservation solution similar to University of Wisconsin (UW) with reversed Na/K contents. In this study, we assessed the impact of IGL-1, UW, and Celsior (CS) solutions on islet isolation and transplant outcome. METHODS: We retrospectively analyzed 376 islet isolations from pancreases flushed and transported with IGL-1 (n=95), UW (n=204), or CS (n=77). We determined isolation outcome and ß-cell function in vitro. Transplanted patients were divided into three groups depending on preservation solution of pancreas, and islet graft function was assessed by decrease in daily insulin needs, C-peptide/glucose ratios, ß-scores, and transplant estimated function at 1- and 6-month follow-up. RESULTS: IGL-1, UW, and CS groups were similar according to donor age, body mass index, and pancreas weight. There was no difference in islet yields between the three groups. Success rates, transplant rates, ß-cell secretory function, and viability were similar for all three groups. We observed no difference in decreased insulin needs, C-peptide glucose ratios, ß-scores, and transplant estimated function at 1- and 6-month follow-up between IGL-1, UW, and CS groups. CONCLUSIONS: Our study shows that IGL-1 is equivalent to UW or CS solutions for pancreas perfusion and cold storage before islet isolation and transplantation.

Impact of the number of infusions on 2-year results of islet-after-kidney transplantation in the GRAGIL network.

BACKGROUND: Insulin independence after islet transplantation is generally achieved after multiple infusions. However, single infusion would increase the number of recipients. Our aim was to evaluate the results of islet-after-kidney transplantation according to the number of infusions. METHODS: Islets were isolated at the Geneva University, shipped, and transplanted into French patients from the Swiss-French GRAGIL network, on the "Edmonton" immunosuppression protocol between 2004 and 2010. RESULTS: Nineteen patients were transplanted with 33 preparations. Fifteen patients reached 24 months follow-up; eight subjects were single-graft recipients and seven were double-graft recipients. Finally, single-graft recipients received a median of 5312 islet equivalents/kg (5186-6388) vs. 10,564 (10,054-11,375) for double-graft recipients (P=0.0003) with similar islet mass at first infusion. Insulin independence was achieved in five of eight single-graft subjects (62.5%) versus five of seven in double-graft subjects (71.4%), not significant. Median insulin independence duration was 4.7 (3.1-15.2) months after one infusion vs. 19 (9.6-20.8) months after two infusions (not significant). At 24 months posttransplant, comparing single- with double-graft patients, insulin doses were 0.23 (0.11-0.34) U/kg vs. 0.02 (0.0-0.23) U/kg, P=0.11; HbA1c was 6.5% (5.9%-6.8%) vs. 6.2% (5.9%-6.3%), P=0.16; and basal C-peptide was 302 (143-480) pmol/L vs. 599 (393-806) pmol/L, P=0.05. Only 37.5% of single-graft patients had a ß-score ≥4 compared with 100% of double-graft patients (P=0.03). Two recipients experienced postinfusion bleeding, and two patients (13%) showed renal dysfunction in the absence of biopsy-proven rejection. CONCLUSIONS: One infusion achieves good glycemic control and sometimes insulin independence. However, double-graft patients remain insulin-free longer, tend to have lower HbA1c, and show better graft function 24 months after transplant.

Comparison of adiponectin concentration between pancreatic cancer and colorectal cancer.

INTRODUCTION: Adiponectin (ADP) is an adipocytokine secreted by the adipose tissue which can be a useful marker in oncogenesis. Preliminary studies suggest that adiponectin rates differ according to the type of cancer. AIM OF STUDY: Compare ADP plasma levels in pancreatic cancer (PC) and colorectal cancer (CRC) in a prospective monocentric study. PATIENTS AND METHODS: The study included all the incident cases of PC gathered from a university hospital in France from January 2006 till September 2007. A control population of incident cases of colorectal cancer (CRC), matching on age, gender, and tumor staging was set in the same period. In addition to demographic data, the other parameters analyzed were: ADP rate, insulinoresistance (Homa-test), presence of a dysmetabolic syndrome, evolution of weight and data concerning the tumor (staging, tumor markers: ACE, CA19.9). RESULTS: 33 CRC and 53 PC were analyzed. Type 2 diabetes was found in 18.2% of the CRC cases and 39.6% of the PC (p = 0.037). The mean ADP level was significantly higher in PC versus CRC (20.9 microgram/l versus 15.9 microgram/l; p = 0.03). In multivariate analysis , after adjusting for gender, age, bilirubinemia and weigth loss, the variables independently associated with a high level of ADP (> 10 microG/L) were type 2 diabetes (OR = 0.05, p = 0.01), insulinoresistance (OR = 0.42, p = 0.05) and PC (OR = 12.03, p = 0.047). CONCLUSION: ADP concentration is higher in PC patients than in CRC patients. ADP concentration > 10 microgram/l was independently associated with pancreatic cancer. Our data confirm that adiponectin rates differ strongly according to the type of cancer.

Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro.

Multipotential stem cells can be selected from the bone marrow by plastic adhesion, expanded, and cultured. They are able to differentiate not only into multiple cell types, including cartilage, bone, adipose and fibrous tissues, and myelosupportive stroma, but also into mesodermal (endothelium), neuroectodermal, or endodermal (hepatocytes) lineages. Our goal was to characterize the multipotential capacities of human mesenchymal stem cells (hMSCs) and to evaluate their ability to differentiate into insulin-secreting cells in vitro. hMSCs were obtained from healthy donors, selected by plastic adhesion, and phenotyped by fluorescence-activated cell sorter and reverse transcription-polymerase chain reaction analysis before and after infection with adenoviruses coding for mouse IPF1, HLXB9, and FOXA2 transcription factors involved early in the endocrine developmental pathway. We found that native hMSCs have a pluripotent phenotype (OCT4 expression and high telomere length) and constitutively express NKX6-1 at a low level but lack all other transcription factors implicated in beta-cell differentiation. In all hMSCs, we detected mRNA of cytokeratin 18 and 19, epithelial markers present in pancreatic ductal cells, whereas proconvertase 1/3 mRNA expression was detected only in some hMSCs. Ectopic expression of IPF1, HLXB9, and FOXA2 with or without islet coculture or islet-conditioned medium results in insulin gene expression. In conclusion, our results demonstrated that in vitro human bone marrow stem cells are able to differentiate into insulin-expressing cells by a mechanism involving several transcription factors of the beta-cell developmental pathway when cultured in an appropriate microenvironment.

Reduction of macrophage activation after antioxidant enzymes gene transfer to rat insulinoma INS-1 cells.

BACKGROUND: After transplantation, islet damage occurs through oxidative stress and host immune rejection mediated in part by macrophage activation. We investigated the influence of the overexpression of catalase (CAT) and Cu/Zn superoxide dismutase (Cu/Zn SOD) by rat insulinoma INS-1 beta cells exposed to oxidative stress on their viability and murine macrophage activation. METHODS: INS-1 cells were infected with adenoviral vectors containing CAT (AdCAT) or Cu/Zn SOD (AdSOD) genes. After 72 hours, noninfected and infected INS-1 cells were exposed to oxidative stress and their viability was assessed using a colorimetric assay. Murine peritoneal exudate macrophages (mPEM) incubated with the supernatant of infected and stressed INS-1 cells were tested for chemotaxis and cytokine release (TNF-alpha, IL-alpha and IFN-gamma). RESULTS: After infection, AdCAT and AdSOD gene transfer protected INS-1 cells from the toxicity of different oxidative reagents. The exposure of non-infected INS-1 cells to oxidative stress stimulated mPEM chemotaxis. INS-1 cells infection with AdCAT or AdSOD reduced significantly mPEM chemotaxis from 2.41 +/- 0.31 to 1.61 +/- 0.17 and from 2.53 +/- 0.24 to 1.27 +/- 0.14 respectively (n = 5; p < 0.05). Cytokine release by mPEM was stimulated after exposure to stressed noninfected INS-1 cell supernatant. CAT and Cu/Zn SOD overexpression by infected INS-1 cells decreased significantly the release of TNF-alpha from 268.18 +/- 30.18 to 81.40 +/- 23.58 pg/ml and from 446.96 +/- 75.47 to 20.37 +/- 2.38 pg/ml respectively (n = 6; p < 0.001). The overexpression of these enzymes also reduced significantly the release of IL-1beta and IFN-gamma. CONCLUSIONS: CAT or Cu/Zn SOD gene transfer to INS-1 cells preserved them from oxidative damage and reduced the macrophage activation induced by these pancreatic cells. Therefore, protection of pancreatic beta cells against oxidative injury by antioxidant enzymes gene transfer is an effective approach to overcome the deleterious actions of macrophages in pancreatic islet transplantation.