Sheep-Specific Immunohistochemical Panel for the Evaluation of Regenerative and Inflammatory Processes in Tissue-Engineered Heart Valves

The creation of living heart valve replacements via tissue engineering is actively being pursued by many research groups. Numerous strategies have been described, aimed either at culturing autologous living valves in a bioreactor (in vitro) or inducing endogenous regeneration by the host via resorbable scaffolds (in situ). Whereas a lot of effort is being invested in the optimization of heart valve scaffold parameters and culturing conditions, the pathophysiological in vivo remodeling processes to which tissue-engineered heart valves are subjected upon implantation have been largely under-investigated. This is partly due to the unavailability of suitable immunohistochemical tools specific to sheep, which serves as the gold standard animal model in translational research on heart valve replacements. Therefore, the goal of this study was to comprise and validate a comprehensive sheep-specific panel of antibodies for the immunohistochemical analysis of tissue-engineered heart valve explants. For the selection of our panel we took inspiration from previous histopathological studies describing the morphology, extracellular matrix composition and cellular composition of native human heart valves throughout development and adult stages. Moreover, we included a range of immunological markers, which are particularly relevant to assess the host inflammatory response evoked by the implanted heart valve. The markers specifically identifying extracellular matrix components and cell phenotypes were tested on formalin-fixed paraffin-embedded sections of native sheep aortic valves. Markers for inflammation and apoptosis were tested on ovine spleen and kidney tissues. Taken together, this panel of antibodies could serve as a tool to study the spatiotemporal expression of proteins in remodeling tissue-engineered heart valves after implantation in a sheep model, thereby contributing to our understanding of the in vivo processes which ultimately determine long-term success or failure of tissue-engineered heart valves

Impact of Complications After Pancreatoduodenectomy on Mortality, Organ Failure, Hospital Stay, and Readmission Analysis of a Nationwide Audit:Analysis of a Nationwide Audit

OBJECTIVE: To quantify the impact of individual complications on mortality, organ failure, hospital stay, and readmission after pancreatoduodenectomy. SUMMARY OF BACKGROUND DATA: An initial complication may provoke a sequence of adverse events potentially leading to mortality after pancreatoduodenectomy. This study was conducted to aid prioritization of quality improvement initiatives. METHODS: Data from consecutive patients undergoing pancreatoduodenectomy (2014-2017) were extracted from the Dutch Pancreatic Cancer Audit. Population attributable fractions (PAF) were calculated for the association of each complication (ie, postoperative pancreatic fistula, postpancreatectomy hemorrhage, bile leakage, delayed gastric emptying, wound infection, and pneumonia) with each unfavorable outcome [ie, in-hospital mortality, organ failure, prolonged hospital stay (>75th percentile), and unplanned readmission), whereas adjusting for confounders and other complications. The PAF represents the proportion of an outcome that could be prevented if a complication would be eliminated completely. RESULTS: Overall, 2620 patients were analyzed. In-hospital mortality occurred in 95 patients (3.6%), organ failure in 198 patients (7.6%), and readmission in 427 patients (16.2%). Postoperative pancreatic fistula and postpancreatectomy hemorrhage had the greatest independent impact on mortality [PAF 25.7% (95% CI 13.4-37.9) and 32.8% (21.9-43.8), respectively] and organ failure [PAF 21.8% (95% CI 12.9-30.6) and 22.1% (15.0-29.1), respectively]. Delayed gastric emptying had the greatest independent impact on prolonged hospital stay [PAF 27.6% (95% CI 23.5-31.8)]. The impact of individual complications on unplanned readmission was smaller than 11%. CONCLUSION: Interventions focusing on postoperative pancreatic fistula and postpancreatectomy hemorrhage may have the greatest impact on in-hospital mortality and organ failure. To prevent prolonged hospital stay, initiatives should in addition focus on delayed gastric emptying

Season of Birth and Dopamine Receptor Gene Associations with Impulsivity, Sensation Seeking and Reproductive Behaviors

Season of birth (SOB) has been associated with many physiological and psychological traits including novelty seeking and sensation seeking. Similar traits have been associated with genetic polymorphisms in the dopamine system. SOB and dopamine receptor genetic polymorphisms may independently and interactively influence similar behaviors through their common effects on the dopaminergic system.Based on a sample of 195 subjects, we examined whether SOB was associated with impulsivity, sensation seeking and reproductive behaviors. Additionally we examined potential interactions of dopamine receptor genes with SOB for the same set of traits. Phenotypes were evaluated using the Sociosexual Orientation Inventory, the Barratt Impulsivity Scale, the Eysenck Impulsivity Questionnaire, the Sensation Seeking Scale, and the Delay Discounting Task. Subjects were also asked about their age at first sex as well as their desired age at the birth of their first child. The dopamine gene polymorphisms examined were Dopamine Receptor D2 (DRD2) TaqI A and D4 (DRD4) 48 bp VNTR. Primary analyses included factorial genderxSOB ANOVAs or binary logistic regression models for each dependent trait. Secondary analysis extended the factorial models by also including DRD2 and DRD4 genotypes as independent variables. Winter-born males were more sensation seeking than non-winter born males. In factorial models including both genotype and season of birth as variables, two previously unobserved effects were discovered: (1) a SOBxDRD4 interaction effect on venturesomeness and (2) a DRD2xDRD4 interaction effect on sensation seeking.These results are consistent with past findings that SOB is related to sensation seeking. Additionally, these results provide tentative support for the hypothesis that SOB modifies the behavioral expression of dopaminergic genetic polymorphism. These findings suggest that SOB should be included in future studies of risky behaviors and behavioral genetic studies of the dopamine system

Sheep-Specific Immunohistochemical Panel for the Evaluation of Regenerative and Inflammatory Processes in Tissue-Engineered Heart Valves

The creation of living heart valve replacements via tissue engineering is actively being pursued by many research groups. Numerous strategies have been described, aimed either at culturing autologous living valves in a bioreactor (in vitro) or inducing endogenous regeneration by the host via resorbable scaffolds (in situ). Whereas a lot of effort is being invested in the optimization of heart valve scaffold parameters and culturing conditions, the pathophysiological in vivo remodeling processes to which tissue-engineered heart valves are subjected upon implantation have been largely under-investigated. This is partly due to the unavailability of suitable immunohistochemical tools specific to sheep, which serves as the gold standard animal model in translational research on heart valve replacements. Therefore, the goal of this study was to comprise and validate a comprehensive sheep-specific panel of antibodies for the immunohistochemical analysis of tissue-engineered heart valve explants. For the selection of our panel we took inspiration from previous histopathological studies describing the morphology, extracellular matrix composition and cellular composition of native human heart valves throughout development and adult stages. Moreover, we included a range of immunological markers, which are particularly relevant to assess the host inflammatory response evoked by the implanted heart valve. The markers specifically identifying extracellular matrix components and cell phenotypes were tested on formalin-fixed paraffin-embedded sections of native sheep aortic valves. Markers for inflammation and apoptosis were tested on ovine spleen and kidney tissues. Taken together, this panel of antibodies could serve as a tool to study the spatiotemporal expression of proteins in remodeling tissue-engineered heart valves after implantation in a sheep model, thereby contributing to our understanding of the in vivo processes which ultimately determine long-term success or failure of tissue-engineered heart valves

Sheep-specific immunohistochemical panel for the evaluation of regenerative and inflammatory processes in tissue-engineered heart valves

\u3cp\u3eThe creation of living heart valve replacements via tissue engineering is actively being pursued by many research groups. Numerous strategies have been described, aimed either at culturing autologous living valves in a bioreactor (in vitro) or inducing endogenous regeneration by the host via resorbable scaffolds (in situ). Whereas a lot of effort is being invested in the optimization of heart valve scaffold parameters and culturing conditions, the pathophysiological in vivo remodeling processes to which tissue-engineered heart valves are subjected upon implantation have been largely under-investigated. This is partly due to the unavailability of suitable immunohistochemical tools specific to sheep, which serves as the gold standard animal model in translational research on heart valve replacements. Therefore, the goal of this study was to comprise and validate a comprehensive sheep-specific panel of antibodies for the immunohistochemical analysis of tissue-engineered heart valve explants. For the selection of our panel we took inspiration from previous histopathological studies describing the morphology, extracellular matrix composition and cellular composition of native human heart valves throughout development and adult stages. Moreover, we included a range of immunological markers, which are particularly relevant to assess the host inflammatory response evoked by the implanted heart valve. The markers specifically identifying extracellular matrix components and cell phenotypes were tested on formalin-fixed paraffin-embedded sections of native sheep aortic valves. Markers for inflammation and apoptosis were tested on ovine spleen and kidney tissues. Taken together, this panel of antibodies could serve as a tool to study the spatiotemporal expression of proteins in remodeling tissue-engineered heart valves after implantation in a sheep model, thereby contributing to our understanding of the in vivo processes which ultimately determine long-term success or failure of tissue-engineered heart valves.\u3c/p\u3

CLEC16A interacts with retromer and TRIM27, and its loss impairs endosomal trafficking and neurodevelopment

CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR–Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development

Risk Models for Developing Pancreatic Fistula after Pancreatoduodenectomy:Validation in a Nationwide Prospective Cohort

OBJECTIVE: To evaluate the performance of published fistula risk models by external validation, and to identify independent risk factors for postoperative pancreatic fistula (POPF).SUMMARY BACKGROUND DATA: Multiple risk models have been developed to predict POPF after pancreatoduodenectomy. External validation in high-quality prospective cohorts is, however, lacking or only performed for individual models.METHODS: A post-hoc analysis of data from the stepped-wedge cluster randomized PORSCH trial was performed. Included were all patients undergoing pancreatoduodenectomy in the Netherlands (January 2018-November 2019). Risk models on POPF were identified by a systematic literature search. Model performance was evaluated by calculating the area under the receiver operating curves (AUC) and calibration plots. Multivariable logistic regression was performed to identify independent risk factors associated with clinically relevant POPF.RESULTS: Overall, 1358 patients undergoing pancreatoduodenectomy were included, of whom 341 patients (25%) developed clinically relevant POPF. Fourteen risk models for POPF were evaluated, with AUCs ranging from 0.62-0.70. The updated alternative fistula risk score had an AUC of 0.70 (95% CI 0.69-0.72). The alternative fistula risk score demonstrated an AUC of 0.70 (95% CI 0.68-0.71), whilst an AUC of 0.70 (95% CI 0.69-0.71) was also found for the model by Petrova et al Soft pancreatic texture, pathology other than pancreatic ductal adenocarcinoma or chronic pancreatitis, small pancreatic duct diameter, higher body-mass index, minimally invasive resection and male sex were identified as independent predictors of POPF.CONCLUSION: Published risk models predicting clinically relevant POPF after pancreatoduodenectomy have a moderate predictive accuracy. Their clinical applicability to identify high-risk patients and guide treatment strategies is therefore questionable.</p

Multidisciplinary interaction and MCD gene discovery. The perspective of the clinical geneticist

The increasing pace of gene discovery in the last decade has brought a major change in the way the genetic causes of brain malformations are being diagnosed. Unbiased genomic screening has gained the first place in the diagnostic protocol of a child with congenital (brain) anomalies and the detected variants are matched with the phenotypic presentation afterwards. This process is defined as “reverse phenotyping”. Screening of DNA, through copy number variant analysis of microarrays and analysis of exome data on different platforms, obtained from the index patient and both parents has become a routine approach in many centers worldwide. Clinicians are used to multidisciplinary team interaction in patient care and disease management and this explains why the majority of research that has led to the discovery of new genetic disorders nowadays proceeds from clinical observations to genomic analysis and to data exchange facilitated by open access sharing databases. However, the relevance of multidisciplinary team interaction has not been object of systematic research in the field of brain malformations. This review will illustrate some examples of how diagnostically driven questions through multidisciplinary interaction, among clinical and preclinical disciplines, can be successful in the discovery of new genes related to brain malformations. The first example illustrates the setting of interaction among neurologists, geneticists and neuro-radiologists. The second illustrates the importance of interaction among clinical dysmorphologists for pattern recognition of syndromes with multiple congenital anomalies. The third example shows how fruitful it can be to step out of the “clinical comfort zone”, and interact with basic scientists in applying emerging technologies to solve the diagnostic puzzles