Miniaturized engineered heart tissues from hiPSC-derived triple cell type co-cultures to study human cardiac function.

Human heart tissues grown as three-dimensional spheroids and consisting of different cardiac cell types derived from pluripotent stem cells (hiPSCs) recapitulate aspects of human physiology better than standard two-dimensional models in vitro. They typically consist of less than 5000 cells and are used to measure contraction kinetics although not contraction force. By contrast, engineered heart tissues (EHTs) formed around two flexible pillars, can measure contraction force but conventional EHTs often require between 0.5 and 2 million cells. This makes large-scale screening of many EHTs costly. Our goals here were (i) to create a physiologically relevant model that required fewer cells than standard EHTs making them less expensive, and (ii) to ensure that this miniaturized model retained correct functionality. We demonstrated that fully functional EHTs could be generated from physiologically relevant combinations of hiPSC-derived cardiomyocytes (70%), cardiac fibroblasts (15%) and cardiac endothelial cells (15%), using as few as 1.6 × 104 cells. Our results showed that these EHTs were viable and functional up to 14 days after formation. The EHTs could be electrically paced in the frequency range between 0.6 and 3 Hz, with the optimum between 0.6 and 2 Hz. This was consistent across three downscaled EHT sizes tested. These findings suggest that miniaturized EHTs could represent a cost-effective microphysiological system for disease modelling and examining drug responses particularly in secondary screens for drug discovery.

"Triple low" free-breathing CTPA protocol for patients with dyspnoea.

AIM: To assess the performance of a "triple-low" free-breathing protocol for computed tomography pulmonary angiography (CTPA) evaluated on patients with dyspnoea and suspected pulmonary embolism and discuss its application in routine clinical practice for the study of the pulmonary parenchyma and vasculature. MATERIAL AND METHODS: This study was conducted on a selected group of dyspnoeic patients referred for CTPA. The protocol was designed using fast free-breathing acquisition and a small, fixed volume (35 ml) of contrast agent in order to achieve a low-exposure dose. For each examination, radiodensity of the pulmonary trunk and ascending aorta, and the dose-length product (DLP) were recorded. A qualitative analysis was performed of pulmonary arterial enhancement and the pulmonary parenchyma. RESULTS: This study included 134 patients. Contrast enhancement of the pulmonary arteries (409 ± 159 HU) was systematically >250 HU. The duration of acquisition ranged from 0.9 to 1.3 seconds for free-breathing imaging. The mean DLP was in the range of low-dose chest CT acquisitions (145 ± 73 mGy·cm). The analysis was deemed optimal in 90% (120/134) of cases for the pulmonary parenchyma. Sixty-nine per cent (92/134) of cases demonstrated homogeneous enhancement of the pulmonary arteries to the subsegmental level. Only 6% (8/134) of examinations were considered uninterpretable. CONCLUSION: The present "triple-low" CTPA protocol allows convenient analysis of the pulmonary parenchyma and arteries without hindrance by respiratory motion artefacts in dyspnoeic patients.

Elective tracheostomy during COVID-19 outbreak: to whom, when, how? Early experience from Venice, Italy.

PURPOSE: The need for prolonged invasive mechanical ventilation in COVID-19 patients is placing the otorhinolaryngologist in front of an increasing request for tracheostomy. Nowadays, there is uncertainty regarding the timing of tracheostomy, the prognosis of these patients and the safety of healthcare workers. The aim of this study is to evaluate the efficacy and safety of tracheostomy placement in patients with COVID-19. METHODS: A retrospective cohort study on 23 COVID 19 patients, to analyse the timing of tracheostomy, the risk factors associated with in-hospital death and the infection of the involved health care workers. Early tracheostomy was defined as ≤ 10 days and late ones > 10 days. RESULTS: The mortality rate of COVID-19 patients admitted to ICU that underwent tracheostomy was 18%. The overall mortality of patients admitted to ICU was 53%. The univariate analysis revealed that early tracheostomy, SOFA score > 6, and D-dimer level > 4 were significantly associated with a greater risk of death. At the multivariate analysis SOFA score > 6 and D-dimer level > 4 resulted as significant factors for a higher risk of death. No health care workers associated with tracheostomy are confirmed to be infected by SARS-CoV2. CONCLUSION: We suggest to wait at least 14 days to perform tracheostomy. In patients with SOFA score > 6 and D dimer > 4, tracheostomy should not be performed or should be postponed. Optimized procedures and enhanced personal protective equipment can make the tracheostomy safe and beneficial in COVID-19 patients.

Eco-responsibility in the operating theater: An urgent need for organizational transformation.

Factors associating environmental degradation with human health have shown that air pollution is a source of morbi-mortality throughout the world. Unfortunately, hospitals are themselves "silent polluters". As healthcare professionals, we are the guarantors not only of quality of patient care, but also of proper hospital conduct. The aim of this attempt at clarification is to outline what can be done in the operating theater to reduce the environmental impact of the treatments we administer. Our recommendations will go above and beyond regulatory frameworks and draw upon daily practice concerning waste management, energy consumption, utilization of anesthetic agents and multiple forms of waste. A number of French and international pilot experimentations have been carried out and could strongly contribute to the modification of clinical practices with a societal impact, at a time when ecology has become one of the main preoccupations of our fellow citizens.

Kidney cortex segmentation in 2D CT with U-Nets ensemble aggregation.

PURPOSE: This work presents our contribution to one of the data challenges organized by the French Radiology Society during the Journées Francophones de Radiologie. This challenge consisted in segmenting the kidney cortex from coronal computed tomography (CT) images, cropped around the cortex. MATERIALS AND METHODS: We chose to train an ensemble of fully-convolutional networks and to aggregate their prediction at test time to perform the segmentation. An image database was made available in 3 batches. A first training batch of 250 images with segmentation masks was provided by the challenge organizers one month before the conference. An additional training batch of 247 pairs was shared when the conference began. Participants were ranked using a Dice score. RESULTS: The segmentation results of our algorithm match the renal cortex with a good precision. Our strategy yielded a Dice score of 0.867, ranking us first in the data challenge. CONCLUSION: The proposed solution provides robust and accurate automatic segmentations of the renal cortex in CT images although the precision of the provided reference segmentations seemed to set a low upper bound on the numerical performance. However, this process should be applied in 3D to quantify the renal cortex volume, which would require a marked labelling effort to train the networks.

The prognostic significance of glutamic acid decarboxylase antibodies in patients with chronic pancreatitis undergoing total pancreatectomy with islet autotransplantation.

AIM: Islet autotransplantation (IAT) is considered a 'non-immune' model of islet transplant, with no risk for autoimmune-mediated beta cell loss, but we have previously observed de novo type 1 diabetes in one total pancreatectomy with islet autotransplantation (TPIAT) recipient. We aimed to investigate the clinical significance of glutamic acid decarboxylase antibodies (GADA), as a sensitive marker for autoimmune diabetes mellitus (DM), in patients with chronic pancreatitis undergoing TPIAT. METHODS: We identified 9 patients undergoing TPIAT with elevated GADA pre-TPIAT (8 non-diabetic and 1 with C-peptide positive DM), otherwise demographically similar to GADA negative TPIAT recipients (n=341). Metabolic and clinical measures related to islet cell function were recorded both before and after TPIAT. RESULTS: None of the 9 TPIAT patients achieved insulin independence after surgery, vs. 33% of GADA negative patients (n=318 with 1-yr follow-up). The two patients with the highest titters of GADA (>250 IU/mL) both experienced islet graft failure, despite normoglycaemia pre-TPIAT and high islet mass transplanted (5276 and 9378 IEQ per kg), with elevated HbA1c levels post-TPIAT (8.3%, 9.6%). The remaining 7 seven were insulin dependent with partial graft function and HbA1c levels <7%. CONCLUSION: Insulin dependence was more frequent in 9 patients with elevated GADA prior to TPIAT than in GADA negative TPIAT recipients, with graft failure in 2 cases. We speculate that beta-cell autoimmunity may occur in a small subset of TPIAT recipients and that beta cell antibody testing prior to TPIAT may be warranted to identify individuals at higher risk for insulin dependence.

Qualitative and quantitative contrast-enhanced ultrasonography for the characterisation of non-palpable testicular tumours.

AIM: To assess the diagnostic performance of conventional ultrasound (US) and contrast-enhanced ultrasonography (CEUS) in the differential diagnosis of non-palpable intratesticular tumours. MATERIALS AND METHODS: The local ethics review board approved the protocol, and all of the patients provided written informed consent. Between December 2011 and February 2014, men with non-palpable testicular tumours and normal tumour markers who were referred for surgery were included. The tumours were analysed by conventional US, including B-mode and colour Doppler US (CDUS) as well as by CEUS. Morphological aspects and qualitative and quantitative CEUS criteria, based on visual enhancement and time-intensity curves, were assessed for each lesion. RESULTS: Forty patients were ultimately included. Based on histopathological results, the tumours were classified into three groups: benign tumours (n=16), malignant tumours (n=15), and burned-out tumours (n=9). In B-mode, the morphological aspects were significantly different between benign and malignant tumours (p-values from 0.0002 to 0.008). Qualitative and quantitative analyses of the CEUS images revealed that burned-out tumours exhibited significantly less enhancement than malignant and benign tumours: in burned-out tumours, time-intensity curves were flat, whereas in both benign and malignant tumours the curves had a bell-shaped pattern. All intensity parameters were lower for burned-out tumours compared to benign and malignant tumours (p-value from 0.0001 to 0.026). Both benign and malignant tumours enhanced strongly, however, and no significant difference between the two was noted (p-value from 0.0721 to 0.0953). CONCLUSION: Unlike conventional US, which enable benign lesions to be differentiated from malignant or burned-out tumours, CEUS failed to enabled differentiation between benign lesions and malignant vascularised testicular tumours. CEUS appears to have the potential, however, to differentiate burned-out tumours from vascularised testicular tumours.

Human heart disease: lessons from human pluripotent stem cell-derived cardiomyocytes.

Technical advances in generating and phenotyping cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are now driving their wider acceptance as in vitro models to understand human heart disease and discover therapeutic targets that may lead to new compounds for clinical use. Current literature clearly shows that hPSC-CMs recapitulate many molecular, cellular, and functional aspects of human heart pathophysiology and their responses to cardioactive drugs. Here, we provide a comprehensive overview of hPSC-CMs models that have been described to date and highlight their most recent and remarkable contributions to research on cardiovascular diseases and disorders with cardiac traits. We conclude discussing immediate challenges, limitations, and emerging solutions.

Unmethylated Insulin DNA Is Elevated After Total Pancreatectomy With Islet Autotransplantation: Assessment of a Novel Beta Cell Marker.

Beta cell death may occur both after islet isolation and during infusion back into recipients undergoing total pancreatectomy with islet autotransplantation (TPIAT) for chronic pancreatitis. We measured the novel beta cell death marker unmethylated insulin (INS) DNA in TPIAT recipients before and immediately after islet infusion (n = 21) and again 90 days after TPIAT, concurrent with metabolic functional assessments (n = 25). As expected, INS DNA decreased after pancreatectomy (p = 0.0002). All TPIAT recipients had an elevated unmethylated INS DNA ratio in the first hours following islet infusion. In four samples (three patients), INS DNA was also assessed immediately after islet isolation and again before islet infusion to assess the impact of the isolation process: Unmethylated and methylated INS DNA fractions both increased over this interval, suggesting death of beta cells and exocrine tissue before islet infusion. Higher glucose excursion with mixed-meal tolerance testing was associated with persistently elevated INS DNA at day 90. In conclusion, we observed universal early elevations in the beta cell death marker INS DNA after TPIAT, with pronounced elevations in the islet supernatant before infusion, likely reflecting beta cell death induced by islet isolation. Persistent posttransplant elevation of INS DNA predicted greater hyperglycemia at 90 days.

Sitagliptin Treatment After Total Pancreatectomy With Islet Autotransplantation: A Randomized, Placebo-Controlled Study.

Insulin independence after total pancreatectomy and islet autotransplant (TPIAT) for chronic pancreatitis is limited by a high rate of postprocedure beta cell apoptosis. Endogenous glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are increased by dipeptidyl peptidase 4 inhibitor therapy (sitagliptin) may protect against beta cell apoptosis. To determine the effect of sitagliptin after TPIAT, 83 adult TPIAT recipients were randomized to receive sitagliptin (n = 54) or placebo (n = 29) for 12 months after TPIAT. At 12 and 18 months after TPIAT, participants were assessed for insulin independence; metabolic testing was performed with mixed meal tolerance testing and frequent sample intravenous glucose tolerance testing. Insulin independence did not differ between the sitagliptin and placebo groups at 12 months (42% vs. 45%, p = 0.82) or 18 months (36% vs. 44%, p = 0.48). At 12 months, insulin dose was 9.0 (standard error 1.7) units/day and 7.9 (2.2) units/day in the sitagliptin and placebo groups, respectively (p = 0.67) and at 18 months 10.3 (1.9) and 7.1 (2.6) units/day, respectively (p = 0.32). Hemoglobin A1c levels and insulin secretory measures were similar in the two groups, as were adverse events. In conclusion, sitagliptin could be safely administered but did not improve metabolic outcomes after TPIAT.

Metastatic Pancreatic Adenocarcinoma After Total Pancreatectomy Islet Autotransplantation for Chronic Pancreatitis.

Total pancreatectomy with islet autotransplantation (TPIAT) is being used increasingly as a definitive treatment for chronic pancreatitis. Patients with chronic pancreatitis have an elevated risk of pancreatic cancer, which can also masquerade as acute or chronic pancreatitis, making the diagnosis challenging. We describe here the first case of pancreatic ductal adenocarcinoma developing in the liver of a patient after TPIAT for presumed benign chronic pancreatitis. Retrospective analysis of the patient's preoperative serum revealed normal carbohydrate antigen 19-9 and carcinoembryonic antigen levels but elevated levels of microRNAs -10b, -30c, and -106b compared with controls. Screening guidelines are important to reduce the risk of transplantation of malignant tissue. More sensitive screening tools, including the potential use of microRNAs, are needed to detect early preclinical disease, given the highly malignant nature of pancreatic cancer.

[One day diagnosis for breast lesions: Medical and psychological assessment--EVADIASEIN study]. / Diagnostic en un jour des lésions du sein: évaluation médicale et psychologique de la prise en charge--étude EVADIASEIN.

INTRODUCTION: Breast cancer is the most frequent feminine cancer in France and its incidence increases steadily. The time of access to medical care is an indicator of the quality of the treatments recommended by the Plan Cancer 2009-2013, as it influences the diagnosis and reduces psychological morbidity during the pre-diagnosis phase. The one-day diagnosis is a recently initiated concept, which offers to get the results of the biopsy on the day it is performed and facilitates the setting-up of therapeutic care with the surgeon met during the one-day medical consultations. The aim of this study is to evaluate the satisfaction of patients who benefited from a one-day breast lesion diagnosis, as well as confirm the decrease of time of access to medical treatment. METHODS: This is an observational, non-interventional and single-centre study based on 27 patients who benefited from one-day breast lesions diagnosis over two years. The patients were only included who had a classified lesion ACR 4 or 5 and visible in the ultrasound. We analyzed the histological concordance between the biopsy and the definitive histology, the time of access to medical care, and the therapeutic treatments We analyzed the psychological impact of such an organization by sending to the patients a questionnaire including the Psychological Consequence Questionnaire (PCQ) and the Breast Cancer Anxiety Indicator (BCA) allowing to estimate the anxiety generated by the pre-diagnostic phase, the DC-Sat allowing to estimate the satisfaction of the consultation of announcement, as well as the same day diagnosis benefit. RESULTS: The patients were 59.8 years old in average [33-87]. The average time between the date of the mammography and the one-day diagnosis consultation (including the biopsy) was 15.0 days [0-60]. Fifty-seven percent of the patients considered this time as short. The average time between the biopsy date and the start of the treatment was 15.9 days [4-30]. The one-day diagnosis took an average of 1.6 days [1-5]. The results of the PCQ showed an important emotional impact during the diagnosis phase, and the average BCA score reached an average of 3.9 on a scale of 5. However, the patients were very satisfied with the diagnosis consultation with an average of 8.7 on a scale of 10, and 95% think the one-day diagnosis is beneficial to the patients. DISCUSSION: This study shows that the one-day breast-damage diagnosis enables to improve the time of access to care, and meets the current recommendations. Even though faster access to treatment does not reduce the psychological morbidity of awaiting diagnosis, the patients express their satisfaction and find the rapidity of the pre-diagnosis phase beneficial. CONCLUSION: In view of this study, the one-day breast-damage diagnosis appears to be a quality feature in the process of access to care and treatment of the patients.

Successful Application of Closed-Loop Artificial Pancreas Therapy After Islet Autotransplantation.

Total pancreatectomy with islet autotransplantation (TPIAT) may relieve the pain of chronic pancreatitis while avoiding postsurgical diabetes. Minimizing hyperglycemia after TPIAT limits beta cell apoptosis during islet engraftment. Closed-loop (CL) therapy combining an insulin pump with a continuous glucose monitor (CGM) has not been investigated previously in islet transplant recipients. Our objective was to determine the feasibility and efficacy of CL therapy to maintain glucose profiles close to normoglycemia following TPIAT. Fourteen adult subjects (36% male; aged 35.9 ± 11.4 years) were randomized to subcutaneous insulin via CL pump (n = 7) or multiple daily injections with blinded CGM (n = 7) for 72 h at transition from intravenous to subcutaneous insulin. Mean serum glucose values were significantly lower in the CL pump group than in the control group (111 ± 4 vs. 130 ± 13 mg/dL; p = 0.003) without increased risk of hypoglycemia (percentage of time <70 mg/dL: CL pump 1.9%, control 4.8%; p = 0.46). Results from this pilot study suggest that CL therapy is superior to conventional therapy in maintaining euglycemia without increased hypoglycemia. This technology shows significant promise to safely maintain euglycemic targets during the period of islet engraftment following islet transplantation.

Development of Autoimmune-Mediated ß Cell Failure After Total Pancreatectomy With Autologous Islet Transplantation.

Total pancreatectomy with islet autotransplantation (TPIAT) is performed for definitive treatment of chronic pancreatitis; patients are not diabetic before surgery, or have C-peptide positive pancreatogenous diabetes. Thus, TPIAT recipients are not traditionally considered at risk for autoimmune loss of the islet graft. We describe a 43-year-old female who underwent TPIAT with high mass islet graft of 6031 IEQ/kg, with no evidence of presurgical ß cell autoimmunity who developed type 1 diabetes within the first year after TPIAT, resulting in complete loss of beta cell function. The patient had positive GAD and insulin autoantibodies at 1 year and 18 months after TPIAT, not present prior, and undetectable C-peptide after mixed meal and intravenous glucose tolerance testing at 18 months. Glucagon secretion was preserved, suggesting the transplanted alpha cell mass was intact. HLA typing revealed a DR3/DR4 class II haplotype. This case highlights the need to consider de novo type 1 diabetes in patients with unexpected islet graft failure after TPIAT.

Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation.

BACKGROUND: Replacement of ß-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36-72-hour culture period before IT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability. METHODS: After standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C, whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IEQ)/cm(2) adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content [OCR/DNA] and membrane integrity). RESULTS: Results indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin in the culture media. CONCLUSIONS: Current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability before transplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations.

Defective glucagon secretion during hypoglycemia after intrahepatic but not nonhepatic islet autotransplantation.

Defective glucagon secretion during hypoglycemia after islet transplantation has been reported in animals and humans with type 1 diabetes. To ascertain whether this is true of islets from nondiabetic humans, subjects with autoislet transplantation in the intrahepatic site only (TP/IAT-H) or in intrahepatic plus nonhepatic (TP/IAT-H+NH) sites were studied. Glucagon responses were examined during stepped hypoglycemic clamps. Glucagon and symptom responses during hypoglycemia were virtually absent in subjects who received islets in the hepatic site only (glucagon increment over baseline = 1 ± 6, pg/mL, mean ± SE, n = 9, p = ns; symptom score = 1 ± 1, p = ns). When islets were transplanted in both intrahepatic + nonhepatic sites, glucagon and symptom responses were not significantly different than Control Subjects (TP/IAT-H + NH: glucagon increment = 54 ± 14, n = 5; symptom score = 7 ± 3; control glucagon increment = 67 ± 15, n = 5; symptom score = 8 ± 1). In contrast, glucagon responses to intravenous arginine were present in TP/IAT-H recipients (TP/IAT: glucagon response = 37 ± 8, n = 7). Transplantation of a portion of the islets into a nonhepatic site should be seriously considered in TP/IAT to avoid posttransplant abnormalities in glucagon and symptom responses to hypoglycemia.

Comprehensive health assessment and five-yr follow-up of allogeneic islet transplant recipients.

In patients with type 1 diabetes mellitus (T1DM) complicated by severe hypoglycemic episodes, fear of hypoglycemia can significantly impact daily life. We evaluated whether restoration of glycemic awareness and prevention of hypoglycemia by islet allotransplant could reduce fear and improve health status. We conducted a comprehensive evaluation of patient-based outcomes in 48 T1DM subjects screened for allogeneic islet transplant alone (ITA) and 27 subjects who received an ITA. A battery of generic health status and diabetes-specific measures were used to assess ITA at evaluation, six months, and then annually after ITA. Allogeneic islet transplant was associated with a reduction in behaviors adopted in avoiding hypoglycemia (p Value < 0.001) and attenuation in concerns about hypoglycemic episodes (p Value < 0.001). Changes in hypoglycemia fear tracked most closely with insulin use. While there was a trend toward global improvement in health as measured by the EQ-5D (p Value = 0.002) and in depression symptoms as measured by the Beck (p Value = 0.003), physical health remained unchanged following ITA. Our findings support the socioemotional benefits of ITA during the five years after ITA, which to some extent remains dependent on preservation of islet graft function.