Ezh2 emerges as an epigenetic checkpoint regulator during monocyte differentiation limiting cardiac dysfunction post-MI.

Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative immunoregulatory M2-like macrophage polarization; known to impact cardiac repair after Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time an ectopic EZH2, and putative, cytoplasmic inactive localization of the epigenetic enzyme, during monocyte differentiation into M2 macrophages in vitro as well as in immunomodulatory cardiac macrophages in vivo in the post-MI acute inflammatory phase. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation of bivalent gene promoters, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction in female mice post-MI in vivo. In conclusion, our study reveals that pharmacological epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.

Role of protective stoma after primary anastomosis for generalized peritonitis due to perforated diverticulitis-DIVERTI 2 (a prospective multicenter randomized trial): rationale and design (nct04604730).

BACKGROUND: Traditionally, patients with peritonitis Hinchey III and IV due to perforated diverticulitis were treated with Hartmann's procedure. In the past decade, resection and primary anastomosis have gained popularity over Hartmann's procedure and recent guidelines recommend Hartmann's procedure in two situations only: critically ill patients and in selected patients with multiple comorbidity (at high risk of complications). The protective stoma (PS) is recommended after resection with primary anastomosis, however its interest has never been studied. The aim of this trial is to define the role of systematic PS after resection and primary anastomosis for peritonitis Hinchey III and IV due to perforated diverticulitis. METHODS/DESIGN: This DIVERTI 2 trial is a multicenter, randomized, controlled, superiority trial comparing resection and primary anastomosis with (control group) or without (experimental group) PS in patients with peritonitis Hinchey III and IV due to perforated diverticulitis. Primary endpoint is the overall 1 year morbidity according to the Clavien-Dindo classification of surgical complications. All complications occurring during hospitalization will be collected. Late complications occurring after hospitalization will be collected during follow-up. In order to obtain 80% power for a difference given by respective main probabilities of 67% and 47% in the protective stoma and no protective stoma groups respectively, with a two-sided type I error of 5%, 96 patients will have to be included in each group, hence 192 patients overall. Expecting a 5% rate of patients not assessable for the primary end point (lost to follow-up), 204 patients will be enrolled. Secondary endpoints are postoperative mortality, unplanned reinterventions, incisional surgical site infection (SSI), organ/space SSI, wound disruption, anastomotic leak, operating time, length of hospital stay, stoma at 1 year after initial surgery, quality of life, costs and quality-adjusted life years (QALYs). DISCUSSION: The DIVERTI 2 trial is a prospective, multicenter, randomized, study to define the best strategy between PS and no PS in resection and primary anastomosis for patients presenting with peritonitis due to perforated diverticulitis. TRIAL REGISTRATION: ClinicalTrial.gov: NCT04604730 date of registration October 27, 2020. https://clinicaltrials.gov/ct2/show/NCT04604730?recrs=a&cond=Diverticulitis&draw=2&rank=12 .

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction.

OBJECTIVE: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. CONCLUSIONS: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Plasma Peptide Concentrations and Peptide-Reactive Immunoglobulins in Patients with Eating Disorders at Inclusion in the French EDILS Cohort (Eating Disorders Inventory and Longitudinal Survey).

Eating disorders (EDs) are increasingly frequent. Their pathophysiology involves disturbance of peptide signaling and the microbiota-gut-brain axis. This study analyzed peptides and corresponding immunoglobulin (Ig) concentrations in groups of ED. In 120 patients with restrictive (R), bulimic (B), and compulsive (C) ED, the plasma concentrations of leptin, glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and insulin were analyzed by Milliplex and those of acyl ghrelin (AG), des-acyl ghrelin (DAG), and α-melanocyte-stimulating hormone (α-MSH) by ELISA kits. Immunoglobulin G (in response to an antigen) concentrations were analyzed by ELISA, and their affinity for the respective peptide was measured by surface plasmon resonance. The concentrations of leptin, insulin, GLP-1, and PYY were higher in C patients than in R patients. On the contrary, α-MSH, DAG, and AG concentrations were higher in R than in C patients. After adjustment for body mass index (BMI), differences among peptide concentrations were no longer different. No difference in the concentrations of the IgG was found, but the IgG concentrations were correlated with each other. Although differences of peptide concentrations exist among ED subtypes, they may be due to differences in BMI. Changes in the concentration and/or affinity of several anti-peptide IgG may contribute to the physiopathology of ED or may be related to fat mass.

Commensal Hafnia alvei strain reduces food intake and fat mass in obese mice-a new potential probiotic for appetite and body weight management.

BACKGROUND/OBJECTIVES: Based on the recent identification of E.coli heat shock protein ClpB as a mimetic of the anorexigenic α-melanocyte stimulating hormone (α-MSH), the objective of this study was to preclinically validate Hafnia alvei, a ClpB-producing commensal bacterium as a potential probiotic for appetite and body weight management in overweight and obesity. METHODS: The involvement of enterobacterial ClpB in the putative anti-obesity effects was studied using ClpB-deficient E.coli. A food-grade H. alvei HA4597 strain synthetizing the ClpB protein with an α-MSH-like motif was selected as a candidate probiotic to be tested in ob/ob and high-fat diet (HFD)-fed obese and overweight mice. The relevance of the enterobacterial ClpB gene to human obesity was studied by in silico analysis of fecal metagenomes of 569 healthy individuals from the "MetaHIT" database. RESULTS: Chronic per os administration of native but not ClpB-deficient E.coli strain reduced body weight gain (p < 0.05) and daily meal frequency (p < 0.001) in ob/ob mice. Oral gavage of H.alvei for 18 and 46 days in ob/ob and HFD-fed obese mice, respectively, was well tolerated, reduced body weight gain and fat mass in both obesity models (p < 0.05) and decreased food intake in hyperphagic ob/ob mice (p < 0.001). Elevated fat tissue levels of phosphorylated hormone-sensitive lipase were detected in H.alvei -treated ob/ob mice (p < 0.01). Enterobacterial ClpB gene richness was lower in obese vs. non-obese humans (p < 0.0001) and correlated negatively with BMI in genera of Enterobacter, Klebsiella and Hafnia. CONCLUSIONS: H.alvei HA4597 strain reduces food intake, body weight and fat mass gain in hyperphagic and obese mice. These data combined with low enterobacterial ClpB gene abundance in the microbiota of obese humans provide the rationale for using H.alvei as a probiotic for appetite and body weight management in overweight and obesity.

Discovery of the First in Vivo Active Inhibitors of the Soluble Epoxide Hydrolase Phosphatase Domain.

The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme exhibiting two different catalytic activities that are located in two distinct domains. Although the physiological role of the C-terminal hydrolase domain is well-investigated, little is known about its phosphatase activity, located in the N-terminal phosphatase domain of sEH (sEH-P). Herein we report the discovery and optimization of the first inhibitor of human and rat sEH-P that is applicable in vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to rationalize the structure-activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-yl)butanoic acid (22b, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in vivo.

DNA methylation and histone post-translational modifications in the mouse germline following in-vitro maturation of fresh or cryopreserved prepubertal testicular tissue.

RESEARCH QUESTION: Do cryopreservation and in-vitro culture procedures affect the expression of DNA methyltransferases (DNMT) and histone-modifying enzymes, as well as the establishment of DNA methylation and histone post-translational modifications (PTM) in germ cells in prepubertal mouse testicular tissue? DESIGN: This study investigated the expression of epigenetic modification enzymes, DNA methylation and histone PTM, and the spermatogenic progression after in-vitro maturation of fresh or cryopreserved mouse prepubertal testicular tissue. Fresh or cryopreserved testicular fragments from 6-7 days post-partum mice were cultured for 30 days in the presence of retinol with or without FSH. RESULTS: The in-vitro maturation of fresh or cryopreserved tissue allowed the differentiation of spermatogonia into spermatozoa. Differences in the levels of transcripts encoding epigenetic modification enzymes (Dnmt1, Dnmt3a, Jarid1b, Src1, Sirt1, Hdac1) were found between 30-day tissue cultures and age-matched in-vivo controls. DNMT1/DNMT3a expression and the presence of 5-methylcytosine (5mC) were detected in spermatogonia and leptotene/zygotene spermatocytes in cultures. The relative 5mC fluorescence intensity was similar in spermatozoa produced in cultures of cryopreserved tissues or in vivo. H3K4me3, H3K9ac and H4K8ac were present in all germ cell types but differences in the proportion of germ cells containing these epigenetic marks were found after cultures. CONCLUSIONS: Despite differences with the in-vivo situation, DNA methylation and histone methylation and acetylation occur in the mouse germline in in-vitro matured fresh or cryopreserved mouse prepubertal testicular tissue, and the expression of the enzymes catalysing these epigenetic modifications are maintained in vitro.