Search Results (182)

Human leukocyte extract (HLE), a non-immunogenic dialyzable leukocyte preparation (<10 kDa), may serve as a safe adjuvant in immunotherapy. We investigated the effects of albendazole (ABZ), HLE, and their combination in Mesocestoides vogae infected mice, focusing on lymphoid cells in the peritoneal cavity, the site of larval proliferation and parasite-induced immunosuppression. Peritoneal lymphoid cells were analysed by flow cytometry and qPCR. Cells proliferative responses to ConA, LPS, and parasite excretory/secretory (E/S) antigens, cytokine production (ELISA), IgM and IgG isotypes in exudates and parasite antigen recognition (Western blot) were assessed. Efficacy was measured by larval burden and 14-3-3 gene expression in larvae. HLE combined with ABZ enhanced larval clearance and suppressed 14-3-3 gene expression in larvae. HLE and combination therapy increased CD3⁺ T cell frequencies, especially CD3^+high, reduced regulatory CD3+/IL-10 Tregs and expression of Foxp3⁺. All treatments diminished CD19⁺/IL-10⁺ Bregs, correlating with lower CD9 and Atf3 mRNA levels compared to infected mice. Transcription factors T-bet expression was strongly upregulated, while GATA3 was moderately elevated. IFN-γ production and T/B cell proliferation were restored after HLE and combination therapy, partially, even in the presence of E/S antigens. IgM and total IgG levels against parasite antigens declined, while Th1-associated IgG2a increased in ABZ+HLE and HLE-treated groups. Albendazole failed to reverse the immunosuppressive Treg-type immunity but was more effective in reducing Breg populations and their functions. HLE enhanced ABZ efficacy by restoring Th1 responsiveness, reducing Treg/Breg activity, and modulating antibody profiles. It represents a promising immunomodulatory adjuvant in the treatment of the infections associated with Th2/Treg-driven immunosuppression. Full article

Background/Objectives: Endometriosis and high-grade serous ovarian cancer (HGS-OC) share common features within the peritoneal immune microenvironment, yet they exhibit divergent clinical outcomes. This study aimed to dissect the immune–metabolic landscape of the peritoneal cavity in patients with endometriosis and ovarian cancer by evaluating macrophage polarization, intracellular signaling pathways, and iron-driven oxidative stress. Methods: A prospective cohort study enrolled 40 patients with endometriosis, 198 with ascitic ovarian cancer (178 HGS-OC), and 200 controls with benign gynecological conditions. Peritoneal and peripheral blood samples were analyzed via flow cytometry for macrophage (M1/M2) polarization markers, mTOR/AKT expression, and glucose uptake. Inflammatory markers (IL-6, CRP), oxidative stress (ROS), and iron metabolism parameters (hepcidin, ferritin, transferrin, serum/free iron) were quantified. Results: HGS-OC displayed a predominance of M1-polarized tumor-associated macrophages (TAMs) (CD14⁺/CD80⁺/Glut1⁺) and a high M1/M2 ratio (2.5 vs. 0.8 and 0.9; p = 0.019), correlating positively with IL-6 (p = 0.015), ROS (p = 0.023), hepcidin (p = 0.038), and ferritin (p = 0.043). Conversely, endometriosis showed a dominant M2 profile (CD14⁺/CD163⁺), elevated intracellular mTOR and AKT expression in both TAMs and epithelial cells (p < 0.01), and significantly higher ascitic ROS and free iron levels (p = 0.047 and p < 0.0001, respectively). In endometriosis, the M1/M2 ratio correlated inversely with free iron (p = 0.041), while ROS levels were directly associated with iron overload (p = 0.0034). Conclusions: Endometriosis exhibits a distinct immune–metabolic phenotype characterized by M2 macrophage predominance and iron-induced oxidative stress, contrasting with the inflammatory, M1-rich profile of HGS-OC. These findings suggest that iron metabolism and macrophage plasticity contribute to disease persistence in endometriosis and may inform future immunomodulatory strategies. Full article

Cancer cell spheroids autonomously form in the ascites fluid and are considered a conduit for epithelial ovarian cancer metastasis within the peritoneal cavity. Spheroids are homotypic, avascular 3D structures that acquire resistance to anoikis to remain viable after cellular detachment. We used in vitro spheroid model systems to interrogate pathways critical for spheroid cell proliferation, distinct from those driving monolayer cancer cell proliferation. Using the 105C and KOC-7c human ovarian clear cell carcinoma (OCCC) cell lines, which have distinct proliferative phenotypes as spheroids but the same prototypical OCCC gene mutation profile of constitutively activated AKT signaling with the loss of ARID1A, we revealed therapeutic targets that efficiently kill cells in spheroids. RNA-seq analyses compared the transcriptome of 3-day monolayer and spheroid cells from these lines and identified the characteristics of dormant spheroid cell survival, which included the G2/M checkpoint, autophagy, and other stress pathways induced in 105C spheroids, in sharp contrast to the proliferating spheroid cells of the KOC-7c cell line. Next, we assessed levels of various G2/M checkpoint regulators and found a consistent reduction in steady-state levels of checkpoint regulators in dormant spheroid cells, but not proliferative spheroids. Our studies showed that proliferative spheroid cells were sensitive to Wee1 inhibition by AZD1775, but the dormant spheroid cells showed a degree of resistance to AZD1775, both in terms of EC50 values and spheroid reattachment abilities. Thus, we identified biomarkers of dormant spheroids, including the G2/M checkpoint regulators Wee1, Cdc25c, and PLK1, and showed that, when compared to proliferating spheroid cells, the transcriptome of dormant OCCC spheroids is a source of therapeutic targets. Full article

Endometriosis is a gynecological disease characterized by the presence of endometrium-like cells located outside the uterus. The most widely accepted theory for endometriosis development, retrograde menstruation, does not account for extra-pelvic lesions or ones found on other organs in the peritoneal cavity. Similar to ovarian cancer, endometriosis cells can interact with the mesothelial cells of the peritoneal cavity. In ovarian cancer metastasis, ovarian cancer cell spheroids attach and push away the mesothelial cells lining the peritoneal cavity, clearing the mesothelial layer. Since endometriosis cells are known to interact with the mesothelium, we hypothesized that endometriosis cells would be able to form spheroids capable of undergoing mesothelial clearance. To test this, we designed an in vitro mesothelial clearance assay using endometriosis spheroids and a mesothelial cell monolayer. Our results demonstrate that normal and endometriotic epithelial cell spheroids can perform mesothelial clearance similar to ovarian cancer spheroids, though normal endometrial cells do not clear as well as endometriosis cells. Additionally, we demonstrated that our mesothelial clearance assay can test potential pharmacological therapies for endometriosis prior to clinical trials. These results give insight into the development of endometriosis lesions, but further research is needed to determine the mechanisms behind mesothelial clearance in endometriosis. Full article

In adults, expressed in renal cancer (ERC)/mesothelin is exclusively expressed in the mesothelial cells lining the pleural, pericardial, and peritoneal cavities, yet its function under physiological conditions is unknown. To explore this, we studied ERC expression in wild-type (WT) mice at different developmental stages by immunohistochemistry and analyzed the ultrastructure of the mesothelium in WT and Erc-knockout (KO) mice via electron microscopy. Additionally, cardiopulmonary function in adult WT and Erc-KO mice was assessed using echocardiography and the forced oscillation technique (FOT). During embryonic development in WT mice, ERC expression was detected in the epicardium as early as embryonic day (E)12.5 but was absent in the pleura until E18.5. The timing of expression appeared to coincide with the active maturation of these organs, which implied a potential role in cardiopulmonary development. Electron microscopy revealed that microvilli on the mesothelium of Erc-KO mice were immature compared to those of WT mice. Based on these findings, we hypothesized that ERC might contribute to cardiopulmonary function; however, echocardiography and FOT did not reveal any functional differences between WT and Erc-KO mice. This suggests that ERC has limited functional relevance under physiological conditions. Full article

Ascites, a common complication of portal hypertension in cirrhosis, is characterized by the accumulation of fluid within the peritoneal cavity. While traditional theories focus on hemodynamic alterations and renin–angiotensin–aldosterone system (RAAS) activation, recent research highlights the intricate interplay of molecular and cellular mechanisms. Inflammation, mediated by cytokines (interleukin-1, interleukin-4, interleukin-6, tumor necrosis factor-α), chemokines (chemokine ligand 21, C-X-C motif chemokine ligand 12), and reactive oxygen species (ROS), plays a pivotal role. Besides pro-inflammatory cytokines, hepatic stellate cells (HSCs), sinusoidal endothelial cells (SECs), and smooth muscle cells (SMCs) contribute to the process through their activation and altered functions. Once activated, these cell types can worsen ascites accumulationthrough extracellular matrix (ECM) deposition and paracrine signals. Besides this, macrophages, both resident and infiltrating, through their plasticity, participate in this complex crosstalk by promoting inflammation and dysregulating lymphatic system reabsorption. Indeed, the lymphatic system and lymphangiogenesis, essential for fluid reabsorption, is dysregulated in cirrhosis, exacerbating ascites. The gut microbiota and intestinal barrier alterations which occur in cirrhosis and portal hypertension also play a role by inducing inflammation, creating a vicious circle which worsens portal hypertension and fluid accumulation. This review aims to gather these aspects of ascites pathophysiology which are usually less considered and to date have not been addressed using specific therapy. Nonetheless, it emphasizes the need for further research to understand the complex interactions among these mechanisms, ultimately leading to targeted interventions in specific molecular pathways, aiming towards the development of new therapeutic strategies. Full article

Endometriosis is common and poses significant morbidity of lasting impact to young, pre-menopausal women, while ovarian cancer is a lethal gynecologic condition. Both conditions need better treatment. The human omentum is an apron of adipose tissue in the abdominopelvic cavity, the same space in which endometriosis and ovarian cancer manifest. We aim to determine molecular cues emitted by the omentum that aid the trans-coelomic spread of endometriosis and ovarian cancer in the abdomen–pelvic/peritoneal space. Endometriosis and ovarian cancer patients were prospectively recruited. Primary cell cultures of surgically-resected omentum, endometriosis and ovarian cancer were generated, and conditioned media (CM) from the omentum was derived. They were used for in vitro assays to evaluate the effect of the omentum on cell migration, angiogenesis and proliferation in endometriosis and ovarian cancer. Omental CM promoted cell migration in primary cultures of endometriosis and ovarian cancer. Omental CM contained high levels of HGF, SDF-1a, MCP-1, VEGF-A, IL-6 and IL-8. The observed cell migration was blocked by c-MET inhibition, suggesting that HGF/c-MET signaling mediates cell migration in endometriosis and ovarian cancer. Furthermore, PTTG1 was consistently upregulated in the migrated cells in both endometriosis and ovarian cancer. The omentum provides a favorable environment for trans-coelomic spread of endometriosis and ovarian cancer. HGF, c-MET and PTTG1 are potential therapeutic targets for inhibiting the abdomen–pelvic/peritoneal spread of endometriosis and ovarian cancer. Full article

Late-stage epithelial ovarian cancer (EOC) involves the widespread dissemination of malignant disease throughout the peritoneal cavity, often accompanied by ascites. EOC metastasis relies on the formation of multicellular aggregates, called spheroids. Given that Liver Kinase B1 (LKB1) is required for EOC spheroid viability and LKB1 loss in EOC cells decreases tumor burden in mice, we investigated whether the LKB1 complex controls the invasive properties of human EOC spheroids. LKB1 signalling was antagonized through the CRISPR/Cas9 genetic knockout of LKB1 and/or the RNAi-dependent targeting of STE20-related kinase adaptor protein (STRAD, an LKB1 activator). EOC spheroids expressing nuclear GFP (green) or mKate2 (red) constructs were embedded in Matrigel for real-time live-cell invasion monitoring. Migration and invasion were also assessed in spheroid culture using Transwell chambers, spheroid reattachment, and mesothelial clearance assays. The loss of LKB1 and STRAD signalling decreased cell invasion through Matrigel and Transwell membranes, as well as mesothelial cell clearance. In the absence of LKB1, zymographic assays identified a loss of matrix metalloproteinase (MMP) activity, whereas spheroid reattachment assays found that coating plates with fibronectin restored their invasive potential. A three-dimensional EOC organoid model demonstrated that organoid area was greatly reduced by LKB1 loss. Overall, our data indicated that LKB1 and STRAD facilitated EOC metastasis by promoting MMP activity and fibronectin expression. Given that LKB1 and STRAD are crucial for EOC metastasis, targeting LKB1 and/or STRAD could disrupt the dissemination of EOC, making inhibitors of the LKB1 pathway an alternative therapeutic strategy for EOC patients. Full article

Endometriosis is a common chronic disorder characterized by the growth of endometrium-like tissue outside the uterine cavity. The disease is associated with chronic inflammation and pelvic pain and may have an impact on the patient’s fertility. The causative factors and pathophysiology of the disease are still poorly recognized. The dysregulation of the immune system, aberrant tissue remodeling, and angiogenesis contribute to the disease progression. In endometriosis patients, the proteins regulating the breakdown and reorganization of the connective tissue, e.g., collagenases, and other proteases, as well as their inhibitors, show an incorrect pattern of expression. Here, we report that the expression of reversion-inducing cysteine-rich protein with Kazal motifs (RECK), one of the inhibitors of connective tissue proteases, is elevated in endometrioma cysts as compared to normal endometrium from unaffected women. We also demonstrate a reduced level of miR200b in endometriotic tissue that correlates with RECK mRNA levels. Furthermore, we employ the 12Z cell line, derived from a peritoneal endometriotic lesion, and the Ishikawa cell line, originating from endometrial adenocarcinoma to identify RECK as a direct target of miR200b. The described effect of miR200b on RECK, together with the aberrant expression of both genes in endometrioma, may help to understand the role played by the tissue remodeling system in the pathogenesis of endometriosis. Full article

Obesity causes insulin resistance (IR) through systemic low-grade inflammation and can lead to type 2 diabetes mellitus (T2DM). However, the mechanisms that cause IR and T2DM in non-obese individuals are unclear. The Goto-Kakizaki (GK) rat develops IR spontaneously and is a model of non-obese T2DM. These rats exhibit hyperglycemia beginning at weaning and exhibit lower body mass than control Wistar rats. Herein, we tested the hypothesis that macrophages of GK rats are permanently in a pro-inflammatory state, which may be associated with a systemic inflammation condition that mimics the pathogenesis of obesity-induced T2DM. Using eighteen-week-old GK and control Wistar rats, we investigated the proportions of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages isolated from the peritoneal cavity. Additionally, the production of inflammatory cytokines and reactive oxygen species (ROS) in cultured macrophages under basal and stimulated conditions was assessed. It was found that phorbol myristate acetate (PMA) stimulation increased GK rat macrophage ROS production 90-fold compared to basal levels. This response was also three times more pronounced than in control cells (36-fold). The production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), tended to be upregulated in cultured macrophages from GK rats under basal conditions. Macrophages from GK rats produced 1.6 times more granulocyte-macrophage colony-stimulating factor (GM-CSF), 1.5 times more monocyte chemoattractant protein-1 (MCP-1) and 3.3 times more TNF-α than control cells when stimulated with lipopolysaccharide (LPS) (p = 0.0033; p = 0.049; p = 0.002, respectively). Moreover, compared to control cells, GK rats had 60% more M1 (p = 0.0008) and 23% less M2 (p = 0.038) macrophages. This study is the first to report macrophage inflammatory reprogramming towards a pro-inflammatory state in GK rats. Full article

Ovarian cancer remains a leading cause of death among gynecological cancers, largely due to its propensity for peritoneal metastasis and the development of drug resistance. This review concentrates on the molecular underpinnings of these two critical challenges. We delve into the role of exosomes, the nano-sized vesicles integral to cellular communication, in orchestrating the complex interactions within the tumor microenvironment that facilitate metastatic spread and thwart therapeutic efforts. Specifically, we explore how exosomes drive peritoneal metastasis by promoting epithelial–mesenchymal transition in peritoneal mesothelial cells, altering the extracellular matrix, and supporting angiogenesis, which collectively enable the dissemination of cancer cells across the peritoneal cavity. Furthermore, we dissect the mechanisms by which exosomes contribute to the emergence of drug resistance, including the sequestration and expulsion of chemotherapeutic agents, the horizontal transfer of drug resistance genes, and the modulation of critical DNA repair and apoptotic pathways. By shedding light on these exosome-mediated processes, we underscore the potential of exosomal pathways as novel therapeutic targets, offering hope for more effective interventions against ovarian cancer’s relentless progression. Full article

Endometriosis is one of the most common causes of chronic pelvic pain and infertility that affects 10% of women of reproductive age. It is currently defined as the presence of endometrial epithelial and stromal cells at ectopic sites; however, advances in endometriosis research have some authors believing that endometriosis should be re-defined as “a fibrotic condition in which endometrial stroma and epithelium can be identified”. microRNAs (miRNAs) are regulatory molecules that potentially play a role in endometriotic lesion development. There is evidence that suggests that miRNAs, including microRNA-21 (miR-21), participate in fibrotic processes in different organs, including the heart, kidney, liver and lungs. The objective of this study was to understand the role of miR-21 and the mechanisms that can contribute to the development of fibrosis by determining how IL-6 regulates miR-21 expression and how this miRNA regulates the transforming growth factor beta (TGF-β) signaling pathway to promote fibrosis. We investigated the expression of miR-21 in the baboon and mouse model of endometriosis and its correlation with fibrosis. We demonstrated that inflammation and fibrosis are present at a very early stage of endometriosis and that the inflammatory environment in the peritoneal cavity, which includes interleukin 6 (IL-6), can regulate the expression of miR-21 in vitro and in vivo. Full article

Our study explores the role of cancer-derived extracellular exosomes (EXs), particularly focusing on collagen alpha-3 (VI; COL6A3), in facilitating tumor dissemination and metastasis in epithelial ovarian cancer (EOC). We found that COL6A3 is expressed in aggressive ES2 derivatives, SKOV3 overexpressing COL6A3 (SKOV3/COL6A3), and mesenchymal-type ovarian carcinoma stromal progenitor cells (MSC-OCSPCs), as well as their EXs, but not in less aggressive SKOV3 cells or ES2 cells with COL6A3 knockdown (ES2/shCOL6A3). High COL6A3 expression correlates with worse overall survival among EOC patients, as evidenced by TCGA and GEO data analysis. In vitro experiments showed that EXs from MSC-OCSPCs or SKOV3/COL6A3 cells significantly enhance invasion ability in ES2 or SKOV3/COL6A3 cells, respectively (both, p <0.001). In contrast, ES2 cells with ES2/shCOL6A3 EXs exhibited reduced invasion ability (p < 0.001). In vivo, the average disseminated tumor numbers in the peritoneal cavity were significantly greater in mice receiving intraperitoneally injected SKOV3/COL6A3 cells than in SKOV3 cells (p < 0.001). Furthermore, mice intravenously (IV) injected with SKOV3/COL6A3 cells and SKOV3/COL6A3-EXs showed increased lung colonization compared to mice injected with SKOV3 cells and PBS (p = 0.007) or SKOV3/COL6A3 cells and PBS (p = 0.039). Knockdown of COL6A3 or treatment with EX inhibitor GW4869 or rapamycin-abolished COL6A3-EXs may suppress the aggressiveness of EOC. Full article

Peritonitis is a common and life-threatening inflammatory disease. Myeloid cells are elevated in the peripheral blood and contribute to peritonitis, but their circulating dynamics are not clear. In vivo flow cytometry (IVFC) is a noninvasive technique for monitoring the dynamics of circulating cells in live animals. It has been extensively used to detect circulating tumor cells, but rarely for monitoring immune cells. Here, we describe a method adapting an intravital microscope for IVFC so that we can monitor LysM-EGFP-labeled circulating myeloid cells in a tumor necrosis factor (TNF) α-induced peritonitis mouse model. Using this IVFC method, we quantified the blood flow velocity and cell concentration in circulation. We observed a significant increase in LysM-EGFP+ cells in circulation after TNFα intraperitoneal (i.p.) injection, which reached a plateau in ~20 min. Conventional cytometry analysis showed that most LysM-EGFP+ cells were neutrophils. Increasing blood neutrophils were accompanied by neutrophil recruitment to the peritoneal cavity and neutrophil emigration from the bone marrow. We then monitored neutrophil CD64 expression in vivo and found a significant increase in TNFα-induced peritonitis. We also found that CD18 blockade doubled the circulating neutrophil number in TNFα-induced peritonitis, suggesting that CD18 is critical for neutrophil recruitment in peritonitis. Overall, we demonstrate that IVFC techniques are useful for studying the circulating dynamics of immune cells during inflammatory diseases. Full article

Selenium, zinc, copper, and manganese are essential components of antioxidant enzymes involved in the elimination of reactive oxygen species (ROS). Given that cancer cells produce high levels of ROS and the accumulation of ROS can lead to cell death, cancer cells may be susceptible to strategies that reduce ROS elimination. In this work, we prepared several artificial diets that contained normal carbohydrate, protein, and lipid levels but lacked selenium, zinc, copper, or manganese. The anticancer activity of these diets was examined in a metastatic ovarian cancer model, established by injecting ID8 Trp53^−/− murine ovarian cancer cells into the peritoneal cavity of C57BL/6JRj mice. Treatments started 15 days later and consisted of replacing a normal diet with one of the artificial diets for several weeks. A significant improvement in mice survival was observed when the normal diet was replaced with the selenium-free diet. Diets lacking zinc, copper, or manganese showed no significant impact on mice survival. All diets were very well tolerated. The anticancer efficacy of a diet lacking selenium was confirmed in mice with metastatic colon cancer and in mice with metastatic triple-negative breast cancer. These results suggest that diets lacking selenium hold potential for the treatment of metastatic cancers. Full article