Correlative microscopy reveals abnormalities in type 1 diabetes

Microscopische technieken zijn van grote waarde voor onderzoek naar de regulatie van leven en ziekte op het niveau van cellen en moleculen. Ons lab maakt geroutineerd gebruik van een manier om op grote schaal elektronenmicroscopie (EM) data op te nemen, nanotomie voor nano-anatomie. Een database met donor materiaal van type 1 diabetes (T1D) patiënten werd aangemaakt om het ziekteverloop op hoge resolutie te kunnen bestuderen. Echter, deze grote hoeveelheid data is alleen in grijswaardes en daardoor moeilijk te analyseren. Met gecorreleerde licht microscopie (LM) en EM (CLEM) worden moleculen specifiek zichtbaar gemaakt met behulp van fluorescentie wat vervolgens in een hoge resolutie context geplaatst met EM. Het doel van dit proefschrift was om nieuwe CLEM probes en targeting technieken te ontwikkelen en te implementeren om de analyse van onder andere de nanotomie data te verbeteren. Na het labelen van reeds ingebed materiaal met quantum dots, gecombineerd met een nieuw ontwikkelde ‘kleurenEM’ toepassing werden in nanotomie datasets van zowel een T1D rat model als in het humane donormateriaal afwijkende cellen gevonden in de eilandjes van Langerhans met zowel exocriene als endocriene karakteristieken. Eerste resultaten van functionele vervolgproeven suggereren dat schade aan de exocriene pancreas mogelijk een pro-inflammatoire reactie door insuline producerende bètacellen kan veroorzaken in een vroeg stadium van T1D. Samenvattend geeft dit proefschrift ten eerste een overzicht van recente CLEM ontwikkelingen inclusief een praktische handleiding. Daarnaast werd efficiënte labeling op EM materiaal geoptimaliseerd in combinatie met verbeterde detectie methodes, wat vervolgens tot nieuwe inzichten in de T1D pathogenese heeft geleid

TLR3 agonists induce fibronectin aggregation by activated astrocytes:a role of pro-inflammatory cytokines and fibronectin splice variants

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system which eventually results in axonal loss mainly due to failure of remyelination. Previously we have shown that the persistent presence of stable astrocyte-derived fibronectin aggregates in MS lesions impairs OPC differentiation, and thereby remyelination. Here we set out to discern whether and, if so, how inflammatory mediators as present in MS lesions trigger astrocytes to form fibronectin aggregates. Our findings revealed that in slice cultures only upon demyelination, the TLR3 agonist Poly(I:C) evoked astrocytes to form fibronectin aggregates. Consistently, pro-inflammatory cytokine-pretreated astrocytes were more susceptible to Poly(I:C)-induced fibronectin aggregation, indicating that astrocytes form fibronectin aggregates upon a double hit by inflammatory mediators. The underlying mechanism involves disrupted fibronectin fibrillogenesis at the cell surface as a result of a cytokineinduced increase in relative mRNA levels of EIIIA(pos)-Fn over EIIIBpos-Fn and a Poly(I:C)-mediated decrease in integrin affinity. Remarkably, fibronectin aggregation is exacerbated by white matter astrocytes compared to grey matter astrocytes, which may be a reflection of higher expression levels of EIIIA(pos)-fibronectin in white matter astrocytes. Hence, interfering with alternative fibronectin splicing and/or TLR3-mediated signaling may prevent fibronectin aggregation and overcome remyelination failure in MS lesions

Pancreatic beta cell autophagy is impaired in type 1 diabetes

Aims/hypothesis Pancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development. Methods Pancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5). Results We observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3-LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3-LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002). Conclusions/interpretation We provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes

Cost-effectiveness of remote haemodynamic monitoring by an implantable pulmonary artery pressure monitoring sensor (CardioMEMS-HF system) in chronic heart failure in the Netherlands

Aims: Remote haemodynamic monitoring with an implantable pulmonary artery (PA) sensor has been shown to reduce heart failure (HF) hospitalizations and improve quality of life. Cost-effectiveness analyses studying the value of remote haemodynamic monitoring in a European healthcare system with a contemporary standard care group are lacking. Methods and results: A Markov model was developed to estimate the cost-effectiveness of PA-guided therapy compared to the standard of care based upon patient-level data of the MONITOR-HF trial performed in the Netherlands in patients with chronic HF (New York Heart Association class III and at least one previous HF hospitalization). Cost-effectiveness was measured as the incremental cost per quality-adjusted life year (QALY) gained from the Dutch societal perspective with a lifetime horizon which encompasses a wide variety of costs including costs of hospitalizations, monitoring time, telephone contacts, laboratory assessments, and drug changes in both treatment groups. In the base-case analysis, PA-guided therapy increased costs compared to standard of care by €12 121. The QALYs per patient for PA-guided therapy and standard of care was 4.07 and 3.481, respectively, reflecting a gain of 0.58 QALYs. The resulting incremental cost-effectiveness ratio was €20 753 per QALY, which is below the Dutch willingness-to-pay threshold of €50 000 per QALY gained for HF. Conclusions: The current cost-effectiveness study suggests that remote haemodynamic monitoring with PA-guided therapy on top of standard care is likely to be cost-effective for patients with symptomatic moderate-to-severe HF in the Netherlands.</p

Analysing outcome variables with floor effects due to censoring: a simulation study with longitudinal trial data

ackground: Randomised controlled trials (RCTs) are the gold standard to estimate treatment effects. When patients receive effective treatment over time they may reach the limit of a certain measurement scale. This phenomenon is known as censoring and lead to skewed distributions of the outcome variable with an excess of either low (floor effect) or high values (ceiling effect). Applying traditional methods such as linear mixed models to analyse this kind of longitudinal RCT data may result in bias of the regression parameters. To deal with floor effects due to censoring,&nbsp; a tobit mixed model can be used. The objective of this study was to compare the results of longitudinal linear mixed model analyses with longitudinal tobit mixed model analyses.Methods: First, a simulation study was performed in which several situations of RCTs with floor effects were simulated. Second, data from an empirical RCT was analysed with both methods.Results: Although all analyses underestimated the intervention effects, the tobit mixed model performed much better than the linear mixed model in handling floor effects. However, with an increasing number of follow-up measurements in combination with a strong floor effect estimates from the tobit mixed model were also not accurate.Conclusion: tobit mixed model analysis should be used to estimate treatments effects in longitudinal RCTs with floor effects due to censoring.&nbsp

Large-scale electron microscopy database for human type 1 diabetes

Autoimmune β-cell destruction leads to type 1 diabetes, but the pathophysiological mechanisms remain unclear. To help address this void, we created an open-access online repository, unprecedented in its size, composed of large-scale electron microscopy images ('nanotomy') of human pancreas tissue obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD; www.nanotomy.org). Nanotomy allows analyses of complete donor islets with up to macromolecular resolution. Anomalies we found in type 1 diabetes included (i) an increase of 'intermediate cells' containing granules resembling those of exocrine zymogen and endocrine hormone secreting cells; and (ii) elevated presence of innate immune cells. These are our first results of mining the database and support recent findings that suggest that type 1 diabetes includes abnormalities in the exocrine pancreas that may induce endocrine cellular stress as a trigger for autoimmunity

Efficacy of pulmonary artery pressure monitoring in patients with chronic heart failure:a meta-analysis of three randomized controlled trials

Aims Adjustment of treatment based on remote monitoring of pulmonary artery (PA) pressure may reduce the risk of hospital admission for heart failure (HF). We have conducted a meta-analysis of large randomized trials investigating this question. Methods A systematic literature search was performed for randomized clinical trials with PA pressure monitoring devices in patients and results with HF. The primary outcome of interest was the total number of HF hospitalizations. Other outcomes assessed were urgent visits leading to treatment with intravenous diuretics, all-cause mortality, and composites. Treatment effects are expressed as hazard ratios, and pooled effect estimates were obtained applying random effects meta-analyses. Three eligible randomized clinical trials were identified that included 1898 outpatients in New York Heart Association functional classes II–IV, either hospitalized for HF in the prior 12 months or with elevated plasma NT-proBNP concentrations. The mean followup was 14.7 months, 67.8% of the patients were men, and 65.8% had an ejection fraction ≤40%. Compared to patients in the control group, the hazard ratio (95% confidence interval) for total HF hospitalizations in those randomized to PA pressure monitoring was 0.70 (0.58–0.86) (P = .0005). The corresponding hazard ratio for the composite of total HF hospitalizations, urgent visits and all-cause mortality was 0.75 (0.61–0.91; P = .0037) and for all-cause mortality 0.92 (0.73–1.16). Subgroup analyses, including ejection fraction phenotype, revealed no evidence of heterogeneity in the treatment effect. Conclusion The use of remote PA pressure monitoring to guide treatment of patients with HF reduces episodes of worsening HF and subsequent hospitalizations.</p

The status of forensic anthropology in Europe and South Africa:Results of the 2016 FASE questionnaire on forensic anthropology

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