Hematopoietic Stem Cell Requirement for Macrophage Regeneration Is Tissue Specific.

Tissue-resident macrophages (TRMΦ) are important immune sentinels responsible for maintaining tissue and immune homeostasis within their specific niche. Recently, the origins of TRMΦ have undergone intense scrutiny, in which now most TRMΦ are thought to originate early during embryonic development independent of hematopoietic stem cells (HSCs). We previously characterized two distinct subsets of mouse peritoneal cavity macrophages (MΦ) (large and small peritoneal MΦ) whose origins and relationship to both fetal and adult long-term (LT) HSCs have not been fully investigated. In this study, we employ highly purified LT-HSC transplantation and in vivo lineage tracing to show a dual ontogeny for large and small peritoneal MΦ, in which the initial wave of peritoneal MΦ is seeded from yolk sac-derived precursors, which later require LT-HSCs for regeneration. In contrast, transplanted fetal and adult LT-HSCs are not able to regenerate brain-resident microglia. Thus, we demonstrate that LT-HSCs retain the potential to develop into TRMΦ, but their requirement is tissue specific in the peritoneum and brain.

Repurposing dichloroacetate for the treatment of women with endometriosis.

Endometriosis is a chronic pain condition affecting ∼176 million women worldwide. It is defined by the presence of endometrium-like tissue (lesions) outside the uterus, most commonly on the pelvic peritoneum. There is no cure for endometriosis. All endometriosis drug approvals to date have been contraceptive, limiting their use in women of child-bearing age. We have shown that human peritoneal mesothelial cells (HPMCs) recovered from the pelvic peritoneum of women with endometriosis exhibit significantly higher glycolysis, lower mitochondrial respiration, decreased enzymatic activity of pyruvate dehydrogenase (PDH), and increased production of lactate compared to HPMCs from women without disease. Transforming growth factor-ß1 (TGF-ß1) is elevated in the peritoneal fluid from women with endometriosis, and exposure of HPMCs to TGF-ß1 exacerbates this abnormal phenotype. Treatment of endometriosis HPMCs with the pyruvate dehydrogenase kinase (PDK) inhibitor/PDH activator dichloroacetate (DCA) normalizes HPMC metabolism, reduces lactate secretion, and abrogates endometrial stromal cell proliferation in a coculture model. Oral DCA reduced peritoneal fluid lactate concentrations and endometriosis lesion size in a mouse model. These findings provide the rationale for targeting metabolic processes as a noncontraceptive treatment for women with endometriosis either as a primary nonhormonal treatment or to prevent recurrence after surgery.

Higher expression of KCNK10 (TREK-2) K+ channels and their functional upregulation by lipopolysaccharide treatment in mouse peritoneal B1a cells.

Innate-like CD5+ B1a cells localized in serous cavities are activated by innate stimuli, such as lipopolysaccharide (LPS), leading to T cell-independent antibody responses. Although ion channels play crucial roles in the homeostasis and activation of immune cells, the electrophysiological properties of B1a cells have not been investigated to date. Previously, in the mouse B cell lymphoma cells, we found that the voltage-independent two-pore-domain potassium (K2P) channels generate a negative membrane potential and drive Ca2+ influx. Here, we newly compared the expression and activities of K2P channels in mouse splenic follicular B (FoB), marginal zone B (MZB), and peritoneal B1a cells. Next-generation sequencing analysis showed higher levels of transcripts for TREK-2 and TWIK-2 in B1a cells than those in FoB or MZB cells. Electrophysiological analysis, using patch clamp technique, revealed higher activity of TREK-2 with the characteristic large unitary conductance (~ 250 pS) in B1a than that in FoB or MZB cells. TREK-2 activity was further increased by LPS treatment (>2 h), which was more prominent in B1a than that in MZB or FoB cells. The cytosolic Ca2+ concentration of B cells was decreased by high-K+-induced depolarization (ΔRKCl (%)), suggesting the basal Ca2+ influx to be driven by negative membrane potential. The LPS treatment significantly increased the ΔRKCl (%) in B1a, though not in FoB and MZB cells. Our study was the first to compare the K2P channels in mouse primary B cell subsets, elucidating the functional upregulation of TREK-2 and augmentation of Ca2+ influx by the stimulation of Toll-like receptor 4 in B1a cells.

C1 inhibitor mitigates peritoneal injury in zymosan-induced peritonitis.

Peritonitis, due to a fungal or bacterial infection, leads to injury of the peritoneal lining and thereby forms a hazard for the long-term success of peritoneal dialysis (PD) and remains a lethal complication in patients with PD. This study investigated whether C1 inhibitor (C1-INH) could protect against the progression of peritoneal injuries with five daily administrations of zymosan after mechanical scraping of the rat peritoneum to mimic fungal peritonitis. Severe peritoneal injuries were seen in this model, accompanied by fibrinogen/fibrin exudation and peritoneal deposition of complement activation products such as activated C3 and C5b-9. However, intraperitoneal injection of C1-INH decreased peritoneal depositions of activated C3 and C5b-9, ameliorated peritoneal thickening, reduced the influx of inflammatory cells, and prevented the production of peritoneal fibrous layers with both one and two doses of C1-INH each day. Our results suggest that C1-INH might be useful to protect against peritoneal injuries after causes of peritonitis such as fungal infection. This clinically available agent may thus help extend the duration of PD.NEW & NOTEWORTHY Peritoneal injuries associated with peritonitis comprise an important issue to prevent long-term peritoneal dialysis (PD) therapy. Here, we showed that C1 inhibitor (C1-INH), as an anticomplement agent, protected against peritoneal injuries in a peritonitis animal model related to fungal infection. Therefore, C1-INH might be useful to protect against peritoneal injuries after peritonitis due to fungal infection. This clinically available agent may thus help extend the duration of PD.

Mast Cell Degranulation Increases Mouse Mast Cell Protease 4-Dependent Vasopressor Responses to Big Endothelin-1 But Not Angiotensin I.

Mouse mast cell protease 4 (mMCP-4), the murine functional analog to the human chymase, is a serine protease synthesized and stored in mast cell secretory granules. Our previous studies reported physiologic and pathologic roles for mMCP-4 in the maturation and synthesis of the vasoactive peptide endothelin-1 (ET-1) from its precursor, big ET-1. The aim of this study was to investigate the impact of mast cell degranulation or stabilization on mMCP-4-dependent pressor responses after the administration of big ET-1 or angiotensin I (Ang I). In anesthetized mice, mast cell degranulation induced by compound 48/80 (C48/80) or stabilization by cromolyn enhanced or repressed, respectively, the dose-dependent vasopressor responses to big ET-1 in wild-type (WT) mice but not in mMCP-4 knockout mice in a chymase inhibitor (TY-51469)-sensitive fashion. In addition, mMCP-4-dependent hydrolysis of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was depleted or enhanced in peritoneal mast cells isolated from mice pretreated with C48/80 or cromolyn, respectively. Furthermore, C48/80 or cromolyn markedly increased or abolished, respectively, ET-1 (1-31) conversion from exogenous big ET-1 in WT mice peritoneal fluid-isolated mast cells, in vitro. Finally, the vasopressor responses to Ang I were unaffected by mast cell activation or stabilization, whereas those induced by the angiotensin-converting enzyme-resistant Ang I analog, [Pro11, D-Ala12] Ang I, were potentiated by C48/80. Altogether, the present study shows that mast cell activation enhances the mMCP-4-dependent vasoactive properties of big ET-1 but not Ang I in the mouse model. SIGNIFICANCE STATEMENT: The current work demonstrates a significant role for mast cell stability in the cardiovascular pharmacology of big endothelin-1 but not angiotensin I in the murine systemic circulation.

Effects of TGF-ß1 Receptor Inhibitor GW788388 on the Epithelial to Mesenchymal Transition of Peritoneal Mesothelial Cells.

We investigated the effectiveness of the transforming growth factor beta-1 (TGF-ß) receptor inhibitor GW788388 on the epithelial to mesenchymal transition (EMT) using human peritoneal mesothelial cells (HPMCs) and examined the effectiveness of GW788388 on the peritoneal membrane using a peritoneal fibrosis mouse model. HPMCs were treated with TGF-ß with or without GW788388. Animal experiments were conducted on male C57/BL6 mice. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate. GW788388 was administered by once-daily oral gavage. The morphological change, cell migration, and invasion resulted from TGF-ß treatment, but these changes were attenuated by cotreatment with GW788388. TGF-ß-treated HPMCs decreased the level of the epithelial cell marker and increased the levels of the mesenchymal cell markers. Cotreatment with GW788388 reversed these changes. Phosphorylated Smad2 and Smad3 protein levels were stimulated with TGF-ß and the change was attenuated by cotreatment with GW788388. For the peritoneal fibrosis mice, thickness and collagen deposition of parietal peritoneum was increased, but this change was attenuated by cotreatment with GW788388. GW788388, an orally available potent TGF-ß receptor type 1 inhibitor, effectively attenuated TGF-ß-induced EMT in HPMCs. Cotreatment with GW788388 improved peritoneal thickness and fibrosis, and recovered peritoneal membrane function in a peritoneal fibrosis mouse model.

A Possible Inhibitory Role of Sialic Acid on MUC1 in Peritoneal Dissemination of Clear Cell-Type Ovarian Cancer Cells.

The role of sialic acids on MUC1 in peritoneal dissemination of ovarian cancer cells was investigated. A human ovarian carcinoma cell line, ES-2, was transfected with full-length MUC1 containing 22 or 42 tandem repeats. These transfectants were less adherent to monolayers of patient-derived mesothelial cells than ES-2/mock transfectants. When these cells were inoculated into the abdominal cavity of female nude mice, mice that had received the transfectants showed better survival. When the transfectants were mixed with sialidase and injected, the survival was poorer, whereas when they were mixed with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid, a sialidase inhibitor, the survival was significantly prolonged. These behaviors, concerned with peritoneal implantation and dissemination observed in vitro and in vivo, were dependent on the expression of MUC1. Therefore, sialic acid linked to MUC1 in the form, at least in part, of sialyl-T, as shown to be recognized by monoclonal antibody MY.1E12, is responsible for the suppression of adhesion of these cells to mesothelial cells and the suppression of peritoneal implantation and dissemination.

LGR4 maintains HGSOC cell epithelial phenotype and stem-like traits.

OBJECTIVE: High-grade serous ovarian cancer (HGSOC) is lethal mainly due to extensive metastasis. Cancer cell stem-like properties are responsible for HGSOC metastasis. LGR4, a G-protein-coupled receptor, is involved in the maintenance of stem cell self-renewal and activity in some human organs. METHODS: TCGA and CCLE databases were interrogated for gene mRNA in ovarian cancer tissues and cell lines. Gain and loss of functions of LGR4, ELF3, FZD5 and WNT7B were performed to identify their roles in ovarian cancer cell epithelial phenotype and stem-like properties. In vivo experiments were performed to observe the effect of LGR4 on ovarian cancer cell growth and peritoneal seeding. The binding of ELF3 to LGR4 gene promoter was investigated by dual-luciferase reporter assays and ChIP. RESULTS: LGR4 was shown to be overexpressed in HGSOCs and maintain the epithelial phenotype of HGSOC cells. LGR4 knockdown suppressed POU5F1, SOX2, PROM1 (CD133) and ALDH1A2 expression. Furthermore, LGR4 knockdown reduced CD133+ and ALDH+ subpopulations and impaired tumorisphere formation. To the contrary, LGR4 overexpression enhanced POU5F1 and SOX2 expression and tumorisphere formation capacity. LGR4 knockdown inhibited HGSOC cell growth and peritoneal seeding in xenograft models. Mechanistically, LGR4 and ELF3, an epithelium-specific transcription factor, formed a reciprocal regulatory loop, which was positively modulated by WNT7B/FZD5 ligand-receptor pair. Consistently, knockdown of ELF3, WNT7B, and FZD5, respectively, disrupted HGSOC cell epithelial phenotype and stem-like properties. CONCLUSION: Together, these data demonstrate that WNT7B/FZD5-LGR4/ELF3 axis maintains HGSOC cell epithelial phenotype and stem-like traits; targeting this axis may prevent HGSOC metastasis.

Regulation of epithelial-mesenchymal transition via sonic hedgehog/glioma-associated oncogene homolog 1 signaling pathway in peritoneal mesothelial cells.

Sonic hedgehog (Shh) signaling regulating epithelial-mesenchymal transition (EMT) in cultured rat peritoneal mesothelial cells (PMCs) remains an under-investigated topic. The current study aimed to elucidate the role of Shh signaling in the regulation of EMT in PMCs to attenuate peritoneal injury, with the view of enhancing the efficacy of peritoneal dialysis (PD). PMCs were initially extracted from male Wistar rats using pancreatic enzyme digestion. The expression of Shh and glioma-associated oncogene homolog (Gli1) was quantitatively analyzed using the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Migration of PMCs was determined using Transwell assay. The expression of Shh, Gli1, and EMT markers including α-smooth muscle actin (α-SMA), fibronectin, collagen I, snail1, and E-cadherin was examined by RT-qPCR, western blot analysis, and immunofluorescence respectively. High glucose induction was identified to promote cell migration and increase the expression of Shh and Gli1 in a dose- and time-dependent manner in rat PMCs. Cyclopamine (CPN) was observed to block the Shh signaling induced by high glucose, accompanied by cell migration inhibition, decreased expression of α-SMA, fibronectin, collagen I and snail1 as well as increased expression of E-cadherin. Altogether, overexpression of Gli1 by transfected Gli1 plasmid promotes cell migration and upregulates α-SMA, fibronectin, Snail1, and collagen I expression, while downregulating E-cadherin expression. Shh/Gli1 signaling is important in mediating EMT in rat PMCs, which provides a potential novel therapeutic approach for clinical investigation on renal failure treatment.

Prognostic indicators in pancreatic cancer patients undergoing total pancreatectomy.

PURPOSE: To evaluate the long-term outcomes of total pancreatectomy in a modern cohort of pancreatic cancer patients and to establish whether any factors identified prior to pancreatic resection were related to poor survival. METHODS: We analyzed, retrospectively, patients who underwent total pancreatectomy for pancreatic cancer between 2007 and 2016. The short- and long-term outcomes were investigated and Cox regression analysis was used to evaluate the prognostic factors identified before resection. RESULTS: The subjects were 49 patients with a mean age of 65 years, who underwent total pancreatectomy in our hospital during the study period. Peritoneal washing cytology was performed in 48 patients, with positive results in 4 (8.3%). There was no 30-day mortality. The median overall survival was 22.5 months, with a 5-year survival rate of 28.5%. Univariate analyses of the pre-resection variables revealed that overall survival was associated with tumor location, resectability classification, maximum standardized uptake value of positron emission tomography, the preoperative carbohydrate antigen 19-9 level, and peritoneal washing cytology status. Multivariate analysis revealed that positive peritoneal washing cytology status and the maximum standardized uptake value were independent predictors of poor survival. CONCLUSION: Total pancreatectomy for pancreatic cancer is appropriate for selected patients, but peritoneal washing cytology and positron emission tomography should be performed preoperatively.

Reprogramming of Mesothelial-Mesenchymal Transition in Chronic Peritoneal Diseases by Estrogen Receptor Modulation and TGF-ß1 Inhibition.

In chronic peritoneal diseases, mesothelial-mesenchymal transition is determined by cues from the extracellular environment rather than just the cellular genome. The transformation of peritoneal mesothelial cells and other host cells into myofibroblasts is mediated by cell membrane receptors, Transforming Growth Factor ß1 (TGF-ß1), Src and Hypoxia-inducible factor (HIF). This article provides a narrative review of the reprogramming of mesothelial mesenchymal transition in chronic peritoneal diseases, drawing on the similarities in pathophysiology between encapsulating peritoneal sclerosis and peritoneal metastasis, with a particular focus on TGF-ß1 signaling and estrogen receptor modulators. Estrogen receptors act at the cell membrane/cytosol as tyrosine kinases that can phosphorylate Src, in a similar way to other receptor tyrosine kinases; or can activate the estrogen response element via nuclear translocation. Tamoxifen can modulate estrogen membrane receptors, and has been shown to be a potent inhibitor of mesothelial-mesenchymal transition (MMT), peritoneal mesothelial cell migration, stromal fibrosis, and neoangiogenesis in the treatment of encapsulating peritoneal sclerosis, with a known side effect and safety profile. The ability of tamoxifen to inhibit the transduction pathways of TGF-ß1 and HIF and achieve a quiescent peritoneal stroma makes it a potential candidate for use in cancer treatments. This is relevant to tumors that spread to the peritoneum, particularly those with mesenchymal phenotypes, such as colorectal CMS4 and MSS/EMT gastric cancers, and pancreatic cancer with its desmoplastic stroma. Morphological changes observed during mesothelial mesenchymal transition can be treated with estrogen receptor modulation and TGF-ß1 inhibition, which may enable the regression of encapsulating peritoneal sclerosis and peritoneal metastasis.

CX3CL1-CX3CR1 interaction mediates macrophage-mesothelial cross talk and promotes peritoneal fibrosis.

Peritoneal dialysis (PD) is limited by chronic fibrotic remodeling of the peritoneal wall, a transforming growth factor-ß (TGF-ß)-mediated process. The fractalkine (CX3CL1) receptor CX3CR1 is expressed on macrophages and monocytes, where it is a marker of TGFß expression. Detection of its ligand CX3CL1 on the peritoneal mesothelium led us to hypothesize a pathophysiologic role of CX3CL1-CX3CR1 interaction in peritoneal fibrosis. We found that CX3CL1 was expressed on peritoneal mesothelial cells from PD patients and in a murine PD model. CX3CR1, mostly expressed on macrophages in the peritoneal wall, promoted fibrosis induced by chronic dialysate exposure in the mouse model. Our data suggest a positive feedback loop whereby direct interaction with CX3CR1-expressing macrophages promotes mesothelial expression of CX3CL1 and TGFß expression. In turn, TGFß upregulates CX3CR1 in murine and human monocytic cells. Upstream, macrophage cytokines including interleukin-1ß (IL-1ß) promote mesothelial CX3CR1 and TGFß expression, providing a starting point for CX3CL1-CX3CR1 interaction. IL-1ß expression was enhanced by exposure to dialysate both in vitro and in the mouse models. Our data suggest that macrophage-mesothelial cell crosstalk through CX3CR1-CX3CL1 interaction enhances mesothelial TGFß production, promoting peritoneal fibrosis in response to dialysate exposure. This interaction could be a novel therapeutic target in PD-associated chronic peritoneal fibrosis.

Tamoxifen and bone morphogenic protein-7 modulate fibrosis and inflammation in the peritoneal fibrosis model developed in uremic rats.

BACKGROUND: Peritoneal fibrosis (PF) represents a long-term complication of peritoneal dialysis (PD), affecting peritoneal membrane (PM) integrity and function. Understanding the mechanisms underlying PF development in an uremic environment aiming alternative therapeutic strategies for treating this process is of great interest. The aim of this study was to analyze the effects of tamoxifen (TAM) and recombinant BMP7 (rBMP7) in an experimental model of PF developed in uremic rats. METHODS: To mimic the clinical situation of patients on long-term PD, a combo model, characterized by the combination of PF and CKD with severe uremia, was developed in Wistar rats. PF was induced by intraperitoneal (IP) injections of chlorhexidine gluconate (CG), and CKD was induced by an adenine-rich diet. Uremia was confirmed by severe hypertension, increased blood urea nitrogen (BUN> 120 mg/dL) and serum creatinine levels (> 2 mg/dL). Uremic rats with PF were treated with TAM (10 mg/Kg by gavage) or BMP7 (30 µg/Kg, IP). Animals were followed up for 30 days. RESULTS: CG administration in uremic rats induced a striking increase in PM thickness, neoangiogenesis, demonstrated by increased capillary density, and failure of ultrafiltration capacity. These morphological and functional changes were blocked by TAM or rBMP7 treatment. In parallel, TAM and rBMP7 significantly ameliorated the PM fibrotic response by reducing α-SMA, extracellular matrix proteins and TGF-ß expression. TAM or rBMP7 administration significantly inhibited peritoneal Smad3 expression in uremic rats with PF, prevented Smad3 phosphorylation, and induced a remarkable up-regulation of Smad7, an intracellular inhibitor of TGFß/Smad signaling, contributing to a negative modulation of profibrotic genes. Both treatments were also effective in reducing local inflammation, possibly by upregulating IκB-α expression in the PM of uremic rats with PF. In vitro experiments using primary peritoneal fibroblasts activated by TGF-ß confirmed the capacity of TAM or rBMP7 in blocking inflammatory mediators, such as IL-1ß expression. CONCLUSIONS: In conclusion, these findings indicate important roles of TGF-ß/Smad signaling in PF aggravated by uremia, providing data regarding potential therapeutic approaches with TAM or rBMP7 to block this process.

Arctigenin alleviates TGF-ß1-induced epithelial-mesenchymal transition and PAI-1 expression via AMPK/NF-κB pathway in peritoneal mesothelial cells.

Peritoneal fibrosis (PF) caused by long-term peritoneal dialysis is closely associated with the epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs). Moreover, the anti-fibrotic role of Arctigenin (Arc) has been reported in several fibrosis disorders. Therefore, the preventive effect of Arc on transforming growth factor-ß1 (TGF-ß1)-induced EMT and the underlying mechanisms in HPMCs was investigated in this study. Firstly, the PD model was established by TGF-ß1 stimulation in cultured HPMCs in vitro, we found that TGF-ß1 significantly increased the EMT markers (α-SMA, vimentin, and fibronectin) and plasminogen activator inhibitor type 1 (PAI-1) expressions, but decreased epithelial marker (E-cadherin). Co-treatment with Arc (10, 20, 40 µM) ameliorated TGF-ß1-induced EMT in a dose-dependent manner, and the expression of PAI-1 was also inhibited by Arc, which was abrogated by restoration of PAI-1. Moreover, Arc enhanced the phosphorylated AMP-activated protein kinase (AMPK), but inhibited the phosphorylated IκBα level and nuclear translocation of nuclear factor κB (NF-κB) p65 in TGF-ß1-induced HPMCs. ChIP and Luciferase reporter assays verified that the increased binding capacity of NF-κB to the promoter of PAI-1 induced by TGF-ß1 was reversely attenuated by Arc in HPMCs. However, the effect of Arc on TGF-ß1-induced NF-κB activation, PAI-1 expression and EMT in HPMCs was attenuated by AMPK agonist Compound C. In conclusion, these data demonstrated that Arc suppressed TGF-ß1-induced EMT by activating the AMPK/NF-κB pathway to inhibit PAI-1 expression in HPMCs. Therefore, Arc might act as a potential therapeutic agent for PD treatment.

The establishment of a mouse model of deep endometriosis.

STUDY QUESTION: Is it possible to establish a mouse model of deep endometriosis (DE)? SUMMARY ANSWER: A mouse DE model that is macroscopically and microscopically similar to nodular lesions in humans can be constructed in as short as 3 weeks by intraperitoneal injection of uterine fragments along with the infusion of substance P (SP) and/or calcitonin gene-related peptide (CGRP). WHAT IS KNOWN ALREADY: Although a baboon DE model was reported 5 years ago, its prohibitive cost and demand for facilities and expertise associated with the use of non-human primates put its use out of reach for most laboratories. STUDY DESIGN, SIZE, DURATION: A total of 48 female Balb/C mice were used for this study. Among them, 16 were randomly selected as donors that contributed uterine fragments, and the remaining 32 were recipient mice. The mice with induced endometriosis were followed up for 3-4 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: One day before the induction of endometriosis by intraperitoneal injection of uterine fragments, osmotic pumps were inserted into equal groups of recipient mice to infuse either sterile saline, SP, CGRP, or both SP and CGRP. The hotplate test was administrated to all mice at the baseline and before and after induction of endometriosis. Four (3 for the SP+CGRP group) weeks after induction, all mice were sacrificed. Their endometriotic lesions were excised, weighed and processed for histopathologic examination, and histochemistry, immunohistochemistry and immunofluorescence analyses of markers of proliferation, angiogenesis, epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM), mesothelial-mesenchymal transition (MMT) and endothelial-mesenchymal transition (EndoMT) were done. The extent of lesional fibrosis was evaluated by Masson trichrome staining. To further evaluate surrounding organ/tissue invasion, the peritoneal areas adhesive to the lesions were excised for immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Endometriotic lesions in mice treated with SP and/or CGRP satisfied all requirements for DE, i.e. presence of endometrial epithelial and stromal cells, abundance of fibromuscular content, and encapsulation in surrounding tissues or organs. The lesion weight in the CGRP, SP and SP+CGRP groups was 1.62, 2.14 and 2.18-fold, respectively, heavier than that of control group. Concomitantly, the SP, CGRP and SP+CGRP groups had significantly shorter hotplate latency than that of control group. Lesions in mice treated with SP and/or CGRP, especially with SP+CGRP, exhibited characteristics consistent with EMT, FMT, SMM and extensive fibrosis, along with signs of MMT and EndoMT. Lesional invasion into surrounding tissues/organs was found to be 25.0, 75.0 and 87.5% in mice treated with CGRP, SP and SP+CGRP, but none in control mice. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study is limited by the use of histologic and immunohistochemistry analyses only and lacks molecular data. WIDER IMPLICATIONS OF THE FINDINGS: The establishment of a mouse DE model supports the idea that endometriotic lesions are wounds undergoing repeated tissue injury and repair and underscores the importance of microenvironments in shaping the lesions' destiny. In addition, signs consistent with MMT and EndoMT indicate that there may be more culpable factors that still remain unidentified and should be pursued in the future. Moreover, the close correlation between the extent of lesional fibrosis and markers of EMT, MMT, EndoMT, FMT and SMM as shown here should facilitate our understanding of the molecular mechanisms underlying the DE pathophysiology. Since this DE model is based on a biologically plausible and evidence-backed theory, it should shed much needed insight into the molecular mechanisms underlying the pathophysiology of DE. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by Grants 81471434 (S.W.G.), 81530040 (S.W.G.), 81771553 (S.W.G.), 81671436 (X.S.L.) and 81871144 (X.S.L.) from the National Natural Science Foundation of China. None of the authors has any conflict of interest to disclose.

Involvement of STAT3 Signaling in High Glucose-Induced Epithelial Mesenchymal Transition in Human Peritoneal Mesothelial Cell Line HMrSV5.

BACKGROUND/AIMS: Peritoneal fibrosis (PF) is a common complication in patients receiving long-term peritoneal dialysis, which results in damage to peritoneal functions. Epithelial-mesenchymal transition (EMT) is a key step in the early pathogenesis of PF. Increasing evidence has shown that signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved in EMT and tissue fibrosis by interacting with distinct EMT-inducing molecules, including transforming growth factor (TGF)-ß and advanced glycation end products (AGEs). This study investigated the involvement of STAT3 in the PF process. METHODS: We used high glucose-treated human peritoneal mesothelial cell line HMrSV5 as an in vitro model to expose the peritoneal mesothelial cells to high-glucose dialysate. Expression of EMT markers was detected by qRT-PCR. Accumulation of methylglyoxal (MGO) and AGEs in the culture supernatant were measured by enzyme-linked immunosorbent assay. Phosphorylation of STAT3 was assessed by Western blot. RESULTS: Results showed that high glucose upregulated TGF-ß, increased the productions of MGO and AGEs, and induced EMT in HMrSV5 cells. High glucose also activated the STAT3 pathway. STAT3 inhibitor reduced the high glucose-induced EMT, via reducing TGF-ß expression and repressing the accumulation of MGO and AGEs. CONCLUSION: Our results revealed a critical role for STAT3 signaling in high glucose-induced EMT in HMrSV5 cells, and suggested that inhibition of STAT3 might be a treatment for high glucose-induced fibrogenesis in PF.

Ginsenoside Rg3 Reduces Epithelial-Mesenchymal Transition Induced by Transforming Growth Factor-ß1 by Inactivation of AKT in HMrSV5 Peritoneal Mesothelial Cells.

BACKGROUND Ginsenosides, including ginsenoside Rg3, are components of Panax ginseng C.A. Meyer (Araliaceae) used in traditional Chinese medicine. Long-term peritoneal dialysis induces peritoneal fibrosis that impairs ultrafiltration and is associated with epithelial-mesenchymal transition (EMT) of peritoneal cells. This study aimed to investigate the effects of ginsenoside Rg3 on EMT induced by transforming growth factor-ß1 (TGF-ß1) in HMrSV5 human peritoneal mesothelial cells. MATERIAL AND METHODS The cell counting kit-8 (CCK-8) assay measured HMrSV5 cell viability. The expression of EMT markers, E-cadherin, vimentin, and alpha-smooth muscle actin (alpha-SMA) were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The wound-healing assay determined cell migration. The S-phase of the cell cycle was assessed by 5-ethynyl-2'-deoxyuridine (EdU) labeling, and expression of phosphorylated AKT was measured by Western blot. The effect of ginsenoside Rg3 and the AKT activator SC79 on the TGF-ß1-induced EMT of HMrSV5 cells were evaluated. RESULTS Low concentration of ginsenoside Rg3 did not effect cell viability of HMrSV5 cells. TGF-ß1 treatment decreased the expression of E-cadherin, and increased the expression of vimentin and alpha-SMA and promoted cell migration of HMrSV5 cells. However, co-treatment of ginsenoside Rg3 and TGF-ß1 significantly reduced TGF-ß1-induced EMT in HMrSV5 cells. TGF-ß1 increased the phosphorylation of AKT and increased the expression of Smurf2. Ginsenoside Rg3 reduced TGF-ß1-induced activation of AKT and Smurf2. SC79 reversed the effects of ginsenoside Rg3 on TGF-ß1-induced EMT in HMrSV5 cells. CONCLUSIONS Ginsenoside Rg3 inhibited EMT induced by TGF-ß1 in HMrSV5 human peritoneal mesothelial cells by inhibiting the activation of AKT.

B-1 cell response in immunity against parasites.

The peritoneal cavity has a microenvironment capable of promoting proliferation, differentiation, and activation of the resident cells and recruitment of blood cells through the capillary network involved in the peritoneum. Among the cells found in the peritoneal cavity, B-1 cells are a particular cell type that contains features that are not very well defined. These cells differ from conventional B lymphocytes (B-2) by phenotypic, functional, and molecular characteristics. B-1 cells can produce natural antibodies, migrate to the inflammatory focus, and have the ability to phagocytose pathogens. However, the role of B-1 cells in immunity against parasites is still not completely understood. Several experimental models have demonstrated that B-1 cells can affect the susceptibility or resistance to parasite infections depending on the model and species. Here, we review the literature to provide information on the peculiarities of B-1 lymphocytes as well as their interaction with parasites.

[Cytological diagnostics of intraoperative peritoneal washes in gastric cancer.]

Diagnosis of peritoneal microcanceromatosis is the most important task allowing to determine treatment strategy for patients with stomach cancer. Laparoscopy combined with peritoneal flushing and subsequent cytological examination should be performed to detect the peritoneal microcanceromatosis at the preoperative stage. The objective of this work was to improve cytological diagnostics of peritoneal washings using immunocytochemical techniques and the cell block method. The work was carried out on the basis of 276 surgical peritoneal washings in patients with stomach cancer who were on treatment in the department of high-tech surgery of the Moscow Clinical Scientific Centre of the State Budgetary Healthcare Institution named after Loginov A.S. from June 2016 to June 2018. As a result, the optimal panel of monoclonal antibodies (Ber-EP4, CEA, CK20) was chosen, which increased the sensitivity from 52% to 96% and the specificity of cytological diagnosis from 80% to 98%, and the overall accuracy of the method from 67% to 98%.

Targeting Toll-like receptors with soluble Toll-like receptor 2 prevents peritoneal dialysis solution-induced fibrosis.

Peritoneal membrane failure due to fibrosis limits the use of peritoneal dialysis (PD). Peritoneal fibrosis may potentially be induced by sterile inflammation caused by ongoing cellular stress due to prolonged exposure to PD solutions (PDS). Effective therapies to prevent this process remain to be developed. Toll-like receptors (TLRs) mediate sterile inflammation by recognizing damage-associated molecular patterns (DAMPs) released by cellular stress. We evaluated the involvement of TLRs and DAMPs in PDS-induced fibrosis models and the therapeutic potential of TLR-DAMP targeting for preventing fibrosis. A range of PDS elicited pro-inflammatory and fibrotic responses from PD patient peritoneal leukocytes, mesothelial cells and mouse peritoneal leukocytes. TLR2/4 blockade of human peritoneal cells or TLR2/4 knockouts inhibited these effects. PDS did not induce rapid ERK phosphorylation or IκB-α degradation, suggesting that they do not contain components capable of direct TLR activation. However, PDS increased the release of Hsp70 and hyaluronan, both TLR2/4 DAMP ligands, by human and mouse peritoneal cells, and their blockade decreased PDS-driven inflammation. Soluble TLR2, a TLR inhibitor, reduced PDS-induced pro-inflammatory and fibrotic cytokine release ex vivo. Daily catheter infusion of PDS in mice caused peritoneal fibrosis, but co-administration of soluble TLR2 prevented fibrosis, suppressed pro-fibrotic gene expression and pro-inflammatory cytokine production, reduced leukocyte/neutrophil recruitment, recovered Treg cell levels and increased the Treg:Th17 ratio. Thus, TLR2/4, Hsp70 and hyaluronan showed major roles in PDS-induced peritoneal inflammation and fibrosis. The study demonstrates the therapeutic potential of a TLR-DAMP targeting strategy to prevent PDS-induced fibrosis.