A systematic comparison of protocols for recovery of high-quality rna from human islets extracted by laser capture microdissection

The isolation of high-quality RNA from endocrine pancreas sections represents a consider-able challenge largely due to the high ribonuclease levels. Laser capture microdissection (LCM) of mammalian islets, in association with RNA extraction protocols, has emerged as a feasible approach to characterizing their genetic and proteomic profiles. However, a validated protocol to obtain high-quality RNA from LCM-derived human pancreas specimens that is appropriate for next-generation sequencing analysis is still lacking. In this study, we applied four methods (Picopure extraction kit, Qiazol protocol, Qiazol + Clean-up kit, and RNeasy Microkit + Carrier) to extract RNA from human islets obtained from both non-diabetic individuals and patients with type 2 diabetes who had undergone partial pancreatectomy, as well as handpicked islets from both non-diabetic and diabetic organ donors. The yield and purity of total RNA were determined by 260/280 absorbance using Nanodrop 100 and the RNA integrity number with a bioanalyzer. The results indicated that among the four methods, the RNeasy MicroKit + Carrier (Qiagen) provides the highest yield and purity

Phase 2 Study of Pomalidomide (CC-4047) Monotherapy for Children and Young Adults With Recurrent or Progressive Primary Brain Tumors

INTRODUCTION: Treatment of recurrent primary pediatric brain tumors remains a major challenge, with most children succumbing to their disease. We conducted a prospective phase 2 study investigating the safety and efficacy of pomalidomide (POM) in children and young adults with recurrent and progressive primary brain tumors. BACKGROUND: METHODS: Patients with recurrent and progressive high-grade glioma (HGG), diffuse intrinsic pontine glioma (DIPG), ependymoma, or medulloblastoma received POM 2.6 mg/m2/day (the recommended phase 2 dose [RP2D]) on days 1-21 of a 28-day cycle. A Simon’s Optimal 2-stage design was used to determine efficacy. Primary endpoints included objective response (OR) and long-term stable disease (LTSD) rates. Secondary endpoints included duration of response, progression-free survival (PFS), overall survival (OS), and safety. RESULTS: 46 patients were evaluable for response (HGG, n = 19; DIPG, ependymoma, and medulloblastoma, n = 9 each). Two patients with HGG achieved OR or LTSD (10.5% [95% CI, 1.3%-33.1%]; 1 partial response and 1 LTSD) and 1 patient with ependymoma had LTSD (11.1% [95% CI, 0.3%-48.2%]). There were no ORs or LTSD in the DIPG or medulloblastoma cohorts. The median PFS for patients with HGG, DIPG, ependymoma, and medulloblastoma was 7.86, 11.29, 8.43, and 8.43 weeks, respectively. Median OS was 5.06, 3.78, 12.02, and 11.60 months, respectively. Neutropenia was the most common grade 3/4 adverse event. CONCLUSIONS: Treatment with POM monotherapy did not meet the primary measure of success in any cohort. Future studies are needed to evaluate if POM would show efficacy in tumors with specific molecular signatures or in combination with other anticancer agents. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, identifier NCT03257631; EudraCT, identifier 2016-002903-25

Intra-islet insulin synthesis defects are associated with endoplasmic reticulum stress and loss of beta cell identity in human diabetes

Aims/hypothesis: Endoplasmic reticulum (ER) stress and beta cell dedifferentiation both play leading roles in impaired insulin secretion in overt type 2 diabetes. Whether and how these factors are related in the natural history of the disease remains, however, unclear. Methods: In this study, we analysed pancreas biopsies from a cohort of metabolically characterised living donors to identify defects in in situ insulin synthesis and intra-islet expression of ER stress and beta cell phenotype markers. Results: We provide evidence that in situ altered insulin processing is closely connected to in vivo worsening of beta cell function. Further, activation of ER stress genes reflects the alteration of insulin processing in situ. Using a combination of 17 different markers, we characterised individual pancreatic islets from normal glucose tolerant, impaired glucose tolerant and type 2 diabetic participants and reconstructed disease progression. Conclusions/interpretation: Our study suggests that increased beta cell workload is accompanied by a progressive increase in ER stress with defects in insulin synthesis and loss of beta cell identity. Graphical abstract: [Figure not available: see fulltext.

GP-initiated preconception counselling in a randomised controlled trial does not induce anxiety

BACKGROUND: Preconception counselling (PCC) can reduce adverse pregnancy outcome by addressing risk factors prior to pregnancy. This study explores whether anxiety is induced in women either by the offer of PCC or by participation with GP-initiated PCC. METHODS: Randomised trial of usual care versus GP-initiated PCC for women aged 18–40, in 54 GP practices in the Netherlands. Women completed the six-item Spielberger State Trait Anxiety Inventory (STAI) before PCC (STAI-1) and after (STAI-2). After pregnancy women completed a STAI focusing on the first trimester of pregnancy (STAI-3). RESULTS: The mean STAI-1-score (n = 466) was 36.4 (95% CI 35.4 – 37.3). Following PCC there was an average decrease of 3.6 points in anxiety-levels (95% CI, 2.4 – 4.8). Mean scores of the STAI-3 were 38.5 (95% CI 37.7 – 39.3) in the control group (n = 1090) and 38.7 (95% CI 37.9 – 39.5) in the intervention group (n = 1186). CONCLUSION: PCC from one's own GP reduced anxiety after participation, without leading to an increase in anxiety among the intervention group during pregnancy. We therefore conclude that GPs can offer PCC to the general population without fear of causing anxiety. Trial Registration: ISRCTN5394291

Research activity and capability in the European reference network MetabERN

BACKGROUND: MetabERN is one of the 24 European Reference Networks created according to the European Union directive 2011/24/EU on patient's rights in cross border healthcare. MetabERN associates 69 centres in 18 countries, which provide care for patients with Hereditary Metabolic Diseases, and have the mission to reinforce research and provide training for health professionals in this field. MetabERN performed a survey in December 2017 with the aim to produce an overview documenting research activities and potentials within the network. As the centres are multidisciplinary, separated questionnaires were sent to the clinical, university and laboratory teams. Answers were received from 52 out of the 69 centres of the network, covering 16 countries. A descriptive analysis of the information collected is presented. RESULTS: The answers indicate a marked interest of the respondents for research, who expressed high motivation and commitment, and estimated that the conditions to do research in their institution were mostly satisfactory. They are active in research, which according to several indicators, is competitive and satisfies standards of excellence, as well as the education programs offered in the respondent's universities. Research in the centres is primarily performed in genetics, pathophysiology, and epidemiology, and focuses on issues related to diagnosis. Few respondents declared having activity in human and social sciences, including research on patient's quality of life, patient's awareness, or methods for social support. Infrastructures offering services for medical research were rarely known and used by respondents, including national and international biobanking platforms. In contrast, respondents often participate to patient registries, even beyond their specific field of interest. CONCLUSIONS: Taken as a whole, these results provide an encouraging picture of the research capacities and activities in the MetabERN network, which, with respect to the number and representativeness of the investigated centres, gives a comprehensive picture of research on Hereditary Metabolic Diseases in Europe, as well as the priorities for future actions. Marginal activity in human and social sciences points out the limited multidisciplinary constitution of the responding teams with possible consequences on their current capability to participate to patient's empowerment programs and efficiently collaborate with patient's advocacy groups

Diagnosis and severity criteria for sinusoidal obstruction syndrome/veno-occlusive disease in pediatric patients : a new classification from the European society for blood and marrow transplantation

The advances in hematopoietic cell transplantation (HCT) over the last decade have led to a transplant-related mortality below 15%. Hepatic sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD) is a life-threatening complication of HCT that belongs to a group of diseases increasingly identified as transplant-related, systemic endothelial diseases. In most cases, SOS/VOD resolves within weeks; however, severe SOS/VOD results in multi-organ dysfunction/failure with a mortality rate > 80%. A timely diagnosis of SOS/VOD is of critical importance, given the availability of therapeutic options with favorable tolerability. Current diagnostic criteria are used for adults and children. However, over the last decade it has become clear that SOS/VOD is significantly different between the age groups in terms of incidence, genetic predisposition, clinical presentation, prevention, treatment and outcome. Improved understanding of SOS/VOD and the availability of effective treatment questions the use of the Baltimore and Seattle criteria for diagnosing SOS/VOD in children. The aim of this position paper is to propose new diagnostic and severity criteria for SOS/VOD in children on behalf of the European Society for Blood and Marrow Transplantation.Peer reviewe

A systematic comparison of protocols for recovery of high-quality rna from human islets extracted by laser capture microdissection

The isolation of high-quality RNA from endocrine pancreas sections represents a consider-able challenge largely due to the high ribonuclease levels. Laser capture microdissection (LCM) of mammalian islets, in association with RNA extraction protocols, has emerged as a feasible approach to characterizing their genetic and proteomic profiles. However, a validated protocol to obtain high-quality RNA from LCM-derived human pancreas specimens that is appropriate for next-generation sequencing analysis is still lacking. In this study, we applied four methods (Picopure extraction kit, Qiazol protocol, Qiazol + Clean-up kit, and RNeasy Microkit + Carrier) to extract RNA from human islets obtained from both non-diabetic individuals and patients with type 2 diabetes who had undergone partial pancreatectomy, as well as handpicked islets from both non-diabetic and diabetic organ donors. The yield and purity of total RNA were determined by 260/280 absorbance using Nanodrop 100 and the RNA integrity number with a bioanalyzer. The results indicated that among the four methods, the RNeasy MicroKit + Carrier (Qiagen) provides the highest yield and purity