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Background: In the field of xenotransplantation, digital image analysis (DIA) is an asset to quantify heterogeneous cell infiltrates around transplanted encapsulated islets. Materials and Methods: RGD-alginate was used to produce empty capsules or to encapsulate neonatal porcine islets (NPI) with different combinations of human pancreatic extracellular matrix (hpECM), porcine mesenchymal stem cells (pMSC) and a chitosan anti-fouling coating. Capsules were transplanted subcutaneously in rats for one month and then processed for immunohistochemistry. Immunostainings for macrophages (CD68) and lymphocytes (CD3) were quantified by DIA in two concentric regions of interest (ROI) around the capsules. DIA replicability and reproducibility were assessed by two blind operators. Repeatability was evaluated by processing the same biopsies at different time points. DIA was also compared with quantification by point counting (PC). Results: Methodology validation: different sizes of ROIs were highly correlated. Intraclass correlation coefficients confirmed replicability and reproducibility. Repeatability showed a very strong correlation with CD3 stains and moderate/strong for CD68 stains. Group comparisons for CD68 IHC at each time point proved internal consistency. Point counting and DIA were strongly correlated with both CD3 and CD68. Capsule biocompatibility: Macrophage infiltration was higher around capsules containing biomaterials than around empty and RGD-alginate- NPI capsules. Lymphocytic infiltration was comparable among groups containing cells and higher than in empty capsules. Conclusion: We validated a semi-automated quantification methodology to assess cellular infiltrates and successfully applied it to investigate graft biocompatibility, showing that neonatal porcine islets encapsulated in alginate alone triggered less infiltration than capsules containing islets and bioactive materials.

In the spinal cord, ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities including locomotion. Interneurons arise during embryonic development from distinct progenitor domains orderly distributed along the dorso-ventral axis of the neural tube. The p2 progenitor domain generates at least five V2 interneuron populations. However, identification and characterization of all V2 populations remain currently incomplete and the mechanisms that control their development remain only partly understood. In this study, we report the generation of a Vsx1-CreERT2 BAC transgenic mouse line that drives CreERT2 recombinase expression mimicking endogenous Vsx1 expression pattern in the developing spinal cord. We showed that the Vsx1-CreERT2 transgene can mediate recombination in V2 precursors with a high efficacy and specificity. Lineage tracing demonstrated that all the V2 interneurons in the mouse developing spinal cord derive from cells expressing Vsx1. Finally, we confirmed that V2 precursors generate additional V2 populations that are not characterized yet. Thus, the Vsx1-CreERT2 line described here is a useful genetic tool for lineage tracing and for functional studies of the mouse spinal V2 interneurons.

In the spinal cord, ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities including locomotion. Interneurons arise during embryonic development from distinct progenitor domains orderly distributed along the dorso-ventral axis of the neural tube. The p2 progenitor domain generates at least five V2 interneuron populations. However, identification and characterization of all V2 populations remain currently incomplete and the mechanisms that control their development remain only partly understood. In this study, we report the generation of a Vsx1-CreER

PURPOSE: To investigate whether adipose tissue–derived stem cells (ASCs) protect the primordial follicle pool, not only by decreasing direct follicle loss but also by modulating follicle activation pathways. METHODS: Twenty nude mice were grafted with frozen-thawed human ovarian tissue from 5 patients. Ten mice underwent standard ovarian tissue transplantation (OT group), while the remaining ten were transplanted with ASCs and ovarian tissue (2-step/ASCs+OT group). Ovarian grafts were retrieved on days 3 (n = 5) and 10 (n = 5). Analyses included histology (follicle count and classification), immunohistochemistry (c-caspase-3 for apoptosis and LC3B for autophagy), and immunofluorescence (FOXO1 for PI3K/Akt activation and YAP for Hippo pathway disruption). Subcellular localization was determined in primordial follicles on high-resolution images using structured illumination microscopy. RESULTS: The ASCs+OT group showed significantly higher follicle density than the OT group (p = 0.01). Significantly increased follicle atresia (p

Vascular hyperpermeability is a determinant factor in the pathophysiology of sepsis. While, AMP-activated protein kinase (AMPK) is known to play a role in maintaining endothelial barrier function in this condition. Therefore, we investigated the underlying molecular mechanisms of this protective effect. &alpha, 1AMPK expression and/or activity was modulated in human dermal microvascular endothelial cells using either &alpha, 1AMPK-targeting small interfering RNA or the direct pharmacological AMPK activator 991, prior to lipopolysaccharide (LPS) treatment. Western blotting was used to analyze the expression and/or phosphorylation of proteins that compose cellular junctions (zonula occludens-1 (ZO-1), vascular endothelial cadherin (VE-Cad), connexin 43 (Cx43)) or that regulate actin cytoskeleton (p38 MAPK, heat shock protein 27 (HSP27)). Functional endothelial permeability was assessed by in vitro Transwell assays, and quantification of cellular junctions in the plasma membrane was assessed by immunofluorescence. Actin cytoskeleton remodeling was evaluated through actin fluorescent staining. We consequently demonstrate that &alpha, 1AMPK deficiency is associated with reduced expression of CX43, ZO-1, and VE-Cad, and that the drastic loss of CX43 is likely responsible for the subsequent decreased expression and localization of ZO-1 and VE-Cad in the plasma membrane. Moreover, &alpha, 1AMPK activation by 991 protects against LPS-induced endothelial barrier disruption by reinforcing cortical actin cytoskeleton. This is due to a mechanism that involves the phosphorylation of p38 MAPK and HSP27, which is nonetheless independent of the small GTPase Rac1. This results in a drastic decrease of LPS-induced hyperpermeability. We conclude that &alpha, 1AMPK activators that are suitable for clinical use may provide a specific therapeutic intervention that limits sepsis-induced vascular leakage.

Ulipristal acetate (UPA), a selective progesterone receptor modulator (SPRM), offers new therapeutic options for the clinical management of large uterine fibroids associated with heavy menstrual bleeding or with other moderate or severe symptoms (bulk symptoms, pelvic pain, decreased quality of life). SPRM are synthetic compounds that exert an agonist or antagonist effect on target tissues by their binding to progesterone receptors. UPA reduces fibroid size, controls bleeding in a high percentage of women and significantly improves quality of life. The present review aims to provide insights into UPA indications and its mechanism of action.

RESEARCH QUESTION: Does ulipristal acetate (UPA) modify the expression of genes related to apoptosis or the extracellular matrix in uterine myomas and are any modifications associated with a clinical response? DESIGN: Targeted analysis of 176 apoptosis- or extracellular-matrix-related genes was conducted using polymerase chain reaction (PCR) arrays. Relevant results were validated by quantitative PCR. Four groups were established: responsive short-term (one course, n = 9), responsive long-term (two to four courses, n = 9), non-responsive (n = 9), and the control group who was not given any hormone therapy (n = 9). The clinical response was monitored by medical imagery and considered significant when volume reduction was greater than 25%. RESULTS: Compared with untreated myomas, significant changes in expression of four genes were found in UPA-treated myomas. Gene expression of integrin subunit beta 4 was repressed by UPA treatment (fold change [FC] = -12.50, P < 0.001, q < 0.001), tenascin-C expression was downregulated in UPA-responsive patients (FC = -2.50, P = 0.010, q = 0.090), survivin was repressed in short-term UPA-responsive tumours (FC = -7.69, P < 0.001, q = 0.010), and catenin delta 2 gene expression was upregulated in non-responsive myomas (FC = +7.36, P < 0.001, q = 0.010). CONCLUSION: This characterization provides the first molecular distinction between myomas responsive or non-responsive to UPA treatment.

Peroxisomes are ubiquitous organelles formed by peroxisome biogenesis (PB). During PB, peroxisomal matrix proteins harboring a peroxisome targeting signal (PTS) are imported inside peroxisomes by peroxins, encoded by PEX genes. Genetic alterations in PEX genes lead to a spectrum of incurable diseases called Zellweger spectrum disorders (ZSD). In vitro drug screening is part of the quest for a cure in ZSD by restoring PB in ZSD cell models. In vitro PB evaluation is commonly achieved by immunofluorescent staining or transient peroxisome fluorescent reporter expression. Both techniques have several drawbacks (cost, time-consuming technique, etc.) which we overcame by developing a third-generation lentiviral transfer plasmid expressing an enhanced green fluorescent protein fused to PTS1 (eGFP–PTS1). By eGFP–PTS1 lentiviral transduction, we quantified PB and peroxisome motility in ZSD and control mouse and human fibroblasts. We confirmed the stable eGFP–PTS1 expression along cell passages. eGFP signal analysis distinguished ZSD from control eGFP–PTS1-transduced cells. Live eGFP–PTS1 transduced cells imaging quantified peroxisomes motility. In conclusion, we developed a lentiviral transfer plasmid allowing stable eGFP–PTS1 expression to study PB (deposited on Addgene: #133282). This tool meets the needs for in vitro PB evaluation and ZSD drug discovery.

Introduction Vascular hyperpermeability is a major aspect of sepsis pathophysiology. We previously demonstrated that AMPK protects against sepsis induced vascular hyperpermeability by mechanisms involving interendothelial junctions (IEJs) regulation. The SGLT2 inhibitor Canagliflozin, prescribed in diabetes and known for its cardiovascular protective properties, has recently been shown to activate AMPK in different cell types. Objectives Here, we show that Canagliflozin might be considered as a new therapeutic approach against sepsis induced vascular hyperpermeability. We also decipher molecular mechanisms underlying this protective effect. Methods In vivo experiments were performed on endothelial-specific α1AMPK knockout (e-AMPK KO) mice. Canagliflozin administration was achieved by oral gavage (100 mg/kg) and validated by plasmatic dosages. LPS was administered by intraperitoneal injection (10 mg/kg). Myocardial edema was assessed by Evans Blue Dye extravasation measurement. In vitro experiments were performed in Human Dermal Blood Endothelial Cells (HDBEC). α1AMPK was invalidated with a specific siRNA or activated by Canagliflozin (10 μM). Results We show that Canagliflozin activates AMPK in HDBEC at physiological concentrations. Canagliflozin administration strongly protects against LPS induced vascular leakage in WT but not in e-AMPK KO mice. Accordingly, albuminemia is preserved by Canagliflozin treatment in septic WT mice. More interestingly, Canagliflozin treatment increases survival in response to sepsis injury. In vitro, AMPK activation by Canagliflozin preserves the integrity of IEJs (Cx43, VeCad, ZO1). Conclusion Our work demonstrates the protective effects of Canagliflozin on sepsis induced vascular hyperpermeability and mortality, and highlights key molecular mechanisms involved in this protection. This should open the field to consider Canagliflozin as a new therapeutic support of sepsis induced vascular hyperpermeability.

Uterine fibroids are the most common tumors affecting premenopausal women, responsible for bleeding, pain, and reduced quality of life. When symptomatic, their management mainly involves surgery, which is all too often radical (hysterectomy). While surgical options sparing the uterus (hysteroscopic and laparoscopic myomectomy) and other non-surgical approaches do indeed exist, drug-based therapies are associated with lower costs and morbidity rates. Since progesterone is required for fibroid growth, gonadotropin agonists have been used to control bleeding and decrease fibroid volume, but they only represent a temporary remedy due to adverse events. Ulipristal acetate (UPA), a selective progesterone receptor modulator, is indicated for fibroid management. It is safe, provides fast control of bleeding, and causes sustained fibroid volume reduction in the vast majority of cases (80%). Indeed, UPA-treated fibroids shrink by a combination of inhibition of cell proliferation, stimulation of cell death, and fibrosis resorption. In the case of symptom recurrence, repeated intermittent 3-month courses of daily UPA considerably maximize the impact of treatment, sometimes resulting in complete disappearance of treated fibroids. Despite the therapeutic dose of UPA being very well tolerated, patients with liver anomalies or disorders should be excluded at screening according to European Medicines Agency-Pharmacovigilance Risk Assessment Committee (PRAC) recommendations. We therefore propose new algorithms for fibroid management in premenopausal women with symptomatic fibroids, depending on their localization, the patient's wishes, and clinical response, while monitoring liver enzymes and bilirubin, as recommended by the PRAC, in order to minimize the risks of possible liver toxicity.

ContextUlipristal acetate (UPA), a selective progesterone receptor modulator, clinically reduces uterine myoma size in 80% of cases. However, the molecular mechanism of action is still poorly understood, as is the reason why 20% of myomas do not respond to treatment.ObjectiveTo elucidate whether matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are associated with myoma volume shrinkage after UPA therapy.DesignProspective study.SettingAcademic research unit of a university hospital.PatientsUterine biopsies were obtained from 59 patients with symptomatic myomas undergoing myomectomy, 45 of whom were treated preoperatively with either one or greater than or equal to two, 3-month courses of UPA and 14 not given any hormone therapy to serve as controls. Myoma volume was individually monitored during UPA therapy to determine any substantial clinical response (defined as a reduction in volume of >25%). Three groups were established based on the response to treatment: responsive (R) after one course (n = 12); R after two to four courses (n = 15); and nonresponsive (NR; n = 18).InterventionsUPA treatment given as preoperative management for symptomatic myomas.Main Outcome MeasuresMMP and TIMP expression assessed by zymography and immunohistochemistry.ResultsCompared with controls and NR myomas, responders showed significantly higher expression levels for MMP-1 (P < 0.0001) and MMP-2 (P = 0.009) and significantly lower expression levels for TIMP-1 (P = 0.040).ConclusionsThe correlation found between MMP expression and volume fold change supports the notion that MMPs play a key role in UPA-induced myoma shrinkage.

Uterine fibroids are the most commonly encountered benign uterine tumors in women of reproductive age. As progesterone is known to play a key role in promoting myoma growth, the goal of the study was to analyze the efficacy of selective progesterone receptor modulators (SPRMs). From four studies, it was concluded that UPA (ulipristal acetate) treatment was able to control myoma-associated uterine bleeding in over 90% of cases and significantly reduce myoma volume in more than 80% of women. The results of long-term intermittent therapy (PEARL III and PEARL IV studies) (4 courses of 3 months) demonstrated that more than one course of UPA is able to maximize its potential benefits in terms of control of bleeding and fibroid volume reduction. The treatment was considered safe, even at the level of endometrial changes. With the advent of SPRMs, new algorithms should be discussed, as there is no doubt that there is a place for medical therapy with SPRMs in the current armamentarium of fibroid management.