Overexpression of Fut 2, 4, and 8, and nuclear localization of Fut 4 in ovarian cancer cell lines induced by ascitic fluids from epithelial ovarian cancer patients.

Fucosyltransferases (Fut) regulate the fucosylation process associated with tumorogenesis in different cancer types. Ascitic fluid (AF) from patients diagnosed with advanced stage of epithelial ovarian cancer (EOC) is considered as a dynamic tumor microenvironment associated with poor prognosis. Previous studies from our laboratory showed increased fucosylation in SKOV-3 and OVCAR-3, cancer-derived cell lines, when these cells were incubated with AFs derived from patients diagnosed with EOC. In the present work we studied three fucosyltransferases (Fut 2, Fut 4, and Fut 8) in SKOV-3, OVCAR-3 and CAOV-3 cell lines in combination with five different AFs from patients diagnosed with this disease, confirming that all tested AFs increased fucosylation. Then, we demonstrate that mRNAs of these three enzymes were overexpressed in the three cell lines under treatment with AFs. SKOV-3 showed the higher overexpression of Fut 2, Fut 4, and Fut 8 in comparison with the control condition. We further confirmed, in the SKOV-3 cell line, by endpoint PCR, WB, and confocal microscopy, that the three enzymes were overexpressed, being Fut 4 the most overexpressed enzyme compared to Fut 2 and Fut 8. These enzymes were concentrated in vesicular structures with a homogeneous distribution pattern throughout the cytoplasm. Moreover, we found that among the three enzymes, only Fut 4 was located inside the nuclei. The nuclear location of Fut 4 was confirmed for the three cell lines. These results allow to propose Fut 2, Fut 4, and Fut 8 as potential targets for EOC treatment or as diagnostic tools for this disease.

Comparison of microbial abundance and diversity in uterine and peritoneal fluid in infertile patients with or without endometriosis.

INTRODUCTION: Endometriosis (EM) is a multifactorial disease that affects 10 - 15% of women of reproductive age. Additionally, 30-50% of women with EM suffer from infertility. The mechanism of infertility caused by EM has not yet been consistently explained. In recent years, studies have shown a link between infertility associated with EM and changes in the reproductive tract microbiota. METHODS: In this study, we involved 26 EM patients (8 cases of stage I-II and 18 cases of stage III-IV) and 31 control subjects who were tubal obstruction-related infertility (TORI). The samples from peritoneal fluid (PF) and uterine fluid (UF) were collected and sequenced by 16 S rRNA amplicon. RESULTS: In the comparison of microbial diversity, we found no significant differences in the microbial diversity of PF and UF between patients with stage I-II EM and those with TORI. However, there was a significant difference in microbial diversity among patients with stage III-IV EM compared to the previous two groups. Lactobacillus decreased in PF of EM compared to the control group, while it increased in UF. In PF, the abundance of Pseudomonas, Enterococcus, Dubosiella and Klebsiella was significantly higher in patients with stage III-IV compared to TORI patients. And in UF, the main differences existed between stage I-II EM compared to the other two groups. The abundance of pontibacter, aquabacterium, Rikenellaceae and so on at the genus level was significantly enriched in the EM patients with stage I-II. In the analysis based on KEGG database, EM may affect the receptivity related pathways of the endometrium by influencing changes in the uterine microbiota. CONCLUSION: Our results indicated that as EM progresses, the microorganisms in UF and PF keep changing. These changes in the microbiota, as well as the resulting alternations in gene functional classification, may play an important role in the infertility associated with EM.

Autoantibody screening of plasma and peritoneal fluid of patients with endometriosis.

STUDY QUESTION: Are there specific autoantibody profiles in patients with endometriosis that are different from those in controls? SUMMARY ANSWER: This study did not reveal a significantly higher prevalence of autoantibodies in the studied groups of patients. WHAT IS KNOWN ALREADY: Various inflammatory factors are postulated to be involved in the pathomechanisms of endometriosis, and a potential link exists with autoimmune diseases, which may also play an important role. As the diagnosis of endometriosis remains invasive, it can only be confirmed using laparoscopy with histopathological examination of tissues. Numerous studies have focused on identifying useful biomarkers to confirm the disease, but without unequivocal effects. Autoantibodies are promising molecules that serve as potential prognostic factors. STUDY DESIGN, SIZE, DURATION: A multicentre, cross-sectional study was conducted over 18 months (between 2018 and 2019), at eight Departments of Obstetrics and Gynaecology in several cities across Poland on 137 patients undergoing laparoscopic examination for the diagnosis of endometriosis. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: During laparoscopy, we obtained plasma samples from 137 patients and peritoneal fluid (PF) samples from 98 patients. Patients with autoimmune diseases were excluded from the study. Autoantibody profiling was performed using HuProt v3.1 human proteome microarrays. MAIN RESULTS AND THE ROLE OF CHANCE: We observed no significant differences in the expression of autoantibodies in the plasma or PF between the endometriosis and control groups. The study revealed that in the PF of women with Stage II endometriosis, compared with other stages, there were significantly higher reactivity signals for ANAPC15 and GABPB1 (adj. P < 0.016 and adj. P < 0.026, respectively; logFC > 1 in both cases). Comparison of the luteal and follicular phases in endometriosis patients revealed that levels of NEIL1 (adj. P < 0.029), MAGEB4 (adj. P < 0.029), and TNIP2 (adj. P < 0.042) autoantibody signals were significantly higher in the luteal phase than in the follicular phase in PF samples of patients with endometriosis. No differences were observed between the two phases of the cycle in plasma or between women with endometriosis and controls. Clustering of PF and plasma samples did not reveal unique autoantibody profiles for endometriosis; however, comparison of PF and plasma in the same patient showed a high degree of concordance. LIMITATIONS, REASONS FOR CAUTION: Although this study was performed using the highest-throughput protein array available, it does not cover the entire human proteome and cannot be used to study potentially promising post-translational modifications. Autoantibody levels depend on numerous factors, such as infections; therefore the autoantibody tests should be repeated for more objective results. WIDER IMPLICATIONS OF THE FINDINGS: Although endometriosis has been linked to different autoimmune diseases, it is unlikely that autoimmune responses mediated by specific autoantibodies play a pivotal role in the pathogenesis of this inflammatory disease. Our study shows that in searching for biomarkers of endometriosis, it may be more efficient to use higher-throughput proteomic microarrays, which may allow the detection of potentially new biomarkers. Only research on such a scale, and possibly with different technologies, can help discover biomarkers that will change the method of endometriosis diagnosis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by a grant from the Polish Ministry of Health (grant no. 6/6/4/1/NPZ/2017/1210/1352). It was also funded by the Estonian Research Council (grant PRG1076) and the Horizon 2020 Innovation Grant (ERIN; grant no. EU952516), Enterprise Estonia (grant no. EU48695), and MSCA-RISE-2020 project TRENDO (grant no. 101008193). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Validation of a new simple scoring system to predict spontaneous bacterial peritonitis in patients with cirrhosis and ascites.

BACKGROUND: Recently, a simple scoring system named the Mansoura scoring system was developed to predict spontaneous bacterial peritonitis (SBP) in patients with cirrhosis and ascites. However, the efficacy of this newly developed system has not been extensively investigated. We aimed to validate a new simple scoring system for the rapid diagnosis or exclusion of SBP without paracentesis. METHODS: Adult patients with cirrhosis and ascites admitted to Cho Ray Hospital between November 2021 and May 2022 were included. The area under the receiver operating characteristic (AUROC) curve of the Mansoura simple scoring system for predicting SBP was calculated using the Stata software. Other independent laboratory tests for predicting SBP (C-reactive protein [CRP], neutrophil-to-lymphocyte ratio [NLR], and mean platelet volume [MPV]) were assessed and compared using the Mansoura scoring system. RESULTS: A total of 121 patients were included in this study. The Mansoura scoring system showed good performance in predicting SBP in patients with cirrhosis and ascites (AUROC:0.89). At the cut-off ≥ 4 points, the scoring system achieved a specificity of 97.7% with a positive predictive value for the diagnosis of SBP of 93.5%. Multivariate analysis was performed using our data and showed that NLR, CRP level, and MPV were independent factors related to SBP. CONCLUSION: The Mansoura scoring system demonstrated good performance in predicting SBP in patients with cirrhosis and ascites and may help guide management decisions.

Investigation of the Changes in Concentrations of Vitamin D-Binding Protein and Lactoferin in Plasma and Peritoneal Fluid of Patients with Endometriosis.

An evaluation of the association between the concentrations of vitamin D-binding protein and lactoferrin in the plasma and peritoneal fluid may facilitate the elucidation of molecular mechanisms in endometriosis. Vitamin D-binding protein and lactoferrin concentrations were measured by ELISA in plasma and peritoneal fluid samples from 95 women with suspected endometriosis as classified by laparoscopy into groups with (n = 59) and without endometriosis (n = 36). There were no differences (p > 0.05) in the plasma and peritoneal fluid concentrations of vitamin D-binding protein and lactoferrin between women with and without endometriosis. In women with endometriosis, there was a significant correlation between plasma and peritoneal fluid vitamin D-binding protein concentrations (r = 0.821; p = 0.000), but there was no correlation between lactoferrin concentrations in those compartments (r = 0.049; p > 0.05). Furthermore, in endometriosis, lactoferrin was found to correlate poorly with vitamin D-binding protein (r= -0.236; p > 0.05) in plasma, while in the peritoneal fluid, the correlation between those proteins was significant (r = 0.399; p = 0.002). The characteristic properties of vitamin D-binding protein and lactoferrin and the associations between their plasma and peritoneal fluid concentrations found in women with endometriosis may provide a novel panel of markers to identify high-risk patients in need of further diagnostic measures.

Does the Use of the "Proseek® Multiplex Inflammation I Panel" Demonstrate a Difference in Local and Systemic Immune Responses in Endometriosis Patients with or without Deep-Infiltrating Lesions?

Endometriotic lesions are able to infiltrate surrounding tissue. This is made possible partly by an altered local and systemic immune response that helps achieve neoangiogenesis, cell proliferation and immune escape. Deep-infiltrating endometriosis (DIE) differs from other subtypes through the invasion of its lesions over 5 mm into affected tissue. Despite the invasive nature of these lesions and the wider range of symptoms they can trigger, DIE is described as a stable disease. This elicits the need for a better understanding of the underlying pathogenesis. We used the "Proseek® Multiplex Inflammation I Panel" in order to simultaneously detect 92 inflammatory proteins in plasma and peritoneal fluid (PF) of controls and patients with endometriosis, as well as in particular patients with DIE, in order to gain a better insight into the systemically and locally involved immune response. Extracellular newly identified receptor for advanced gycation end-products binding protein (EN-RAGE), C-C motif Chemokine ligand 23 (CCL23), Eukaryotic translation initiation factor 4-binding protein 1 (4E-BP1) and human glial cell-line derived neurotrophic factor (hGDNF) were significantly increased in plasma of endometriosis patients compared to controls, whereas Hepatocyte Growth factor (HGF) and TNF-related apoptosis inducing ligand (TRAIL) were decreased. In PF of endometriosis patients, we found Interleukin 18 (IL-18) to be decreased, yet Interleukin 8 (IL-8) and Interleukin 6 (IL-6) to be increased. TNF-related activation-induced cytokine (TRANCE) and C-C motif Chemokine ligand 11 (CCL11) were significantly decreased in plasma, whereas C-C motif Chemokine ligand 23 (CCL23), Stem Cell Factor (SCF) and C-X-C motif chemokine 5 (CXCL5) were significantly increased in PF of patients with DIE compared to endometriosis patients without DIE. Although DIE lesions are characterized by increased angiogenetic and pro-inflammatory properties, our current study seems to support the theory that the systemic immune system does not play a major role in the pathogenesis of these lesions.

The Influence of Lactoferrin in Plasma and Peritoneal Fluid on Iron Metabolism in Women with Endometriosis.

The aim of this study was to investigate the relationship between lactoferrin and iron and its binding proteins in women with endometriosis by simultaneously measuring these parameters in plasma and peritoneal fluid. Ninety women were evaluated, of whom 57 were confirmed as having endometriosis. Lactoferrin was measured by ELISA, transferrin, ferritin and iron on a Cobas 8000 analyser. Lactoferrin and transferrin in peritoneal fluid were lower compared to plasma, in contrast to ferritin and iron. In plasma, lactoferrin showeds associations with iron and transferrin in endometriosis and with ferritin in the group without endometriosis. Lactoferrin in peritoneal fluid correlated with lactoferrin, iron and transferrin of plasma in patients without endometriosis. The ratio of lactoferrin concentration in peritoneal fluid to plasma differentiated stage I versus IV of endometriosis and was negatively correlated with the iron ratio in patients without endometriosis. The ferritin ratio differentiated women with and without endometriosis. The very high ferritin ratios, especially in advanced stages of endometriosis, suggest the protective involvement of this protein in peritoneal fluid and the loss of this role by lactoferrin. The results demonstrate the validity of assessing iron metabolism in women with endometriosis, which may be useful as a marker of the disease and its progression.

Ascitic Fluid Calprotectin And Ratio Of Calprotectin To Total Protein In Spontaneous Bacterial Peritonitis.

Objectives: To evaluate the diagnostic and prognostic role of ascitic fluid calprotectin and its ratio to total protein in spontaneous bacterial peritonitis cases. Method: The prospective study was conducted at Kafrelsheikh University Hospital, Egypt, from November 2019 to December 2020, and comprised cirrhotic patients of either gender with ascites. Diagnostic abdominal paracentesis was performed for all patients and ascetic fluid calprotectin was measured. Patients were followed for development of spontaneous bacterial peritonitis or mortality. Data was analysed using SPSS 20. RESULTS: Of the 90 patients, 61(67.7%) were males and 29(32.2%) were females. There were 67(74.4%) patients with spontaneous bacterial peritonitis; 48(71.6%) males and 19(28.3%) females with mean age 60.42±8.3 years. The remaining 23(25.5%) did not have spontaneous bacterial peritonitis; 13(56.5%) males and 10(43.4%) females with mean age 59.7±7.4 years. The patients had significantly higher calprotectin, and calprotectin/total protein ratio (p<0.05). Logistic regression identified ascitic fluid calprotectin as a significant predictor of mortality (p=0.05). The non-survivors had significantly higher ascitic fluid calprotectin and calprotectin/total protein ratio compared to the survivors (p<0.05). CONCLUSIONS: Ascites calprotectin level and itsratio to total protein wasfound to be accurate diagnostic and predictive biomarkers for spontaneous bacterial peritonitis.

Changes in MCP-1, HGF, and IGF-1 expression in endometrial stromal cells, PBMCs, and PFMCs of endometriotic women following 1,25(OH)2D3 treatment.

1,25(OH)2D3 has anti-inflammatory and growth inhibitory effects. Our study explored the effect of 1,25(OH)2D3 treatment on the expression of monocyte chemotactic protein-1 (MCP-1), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) by peripheral blood mononuclear cells (PBMCs), peritoneal fluid mononuclear cells (PFMCs), endometrial stromal cells (ESCs), and its effect on the proliferation of PBMCs and PFMCs of patients with endometriosis compared with controls. PBMCs, PFMCs, and ESCs were obtained from 10 endometriosis patients and 10 non-endometriotic individuals. After treating cells with 0.1 µM of 1,25(OH)2D3 for 6, 24, and 48 h, the gene and protein expression of mentioned factors were evaluated by real-time PCR and ELISA methods, respectively. 1,25(OH)2D3 treatment significantly reduced the protein expression of MCP-1, HGF, and IGF-1 in PBMCs and PFMCs of endometriotic patients at 48 h (p < 0.05-<0.01). Also, this treatment significantly reduced MCP-1, HGF, and IGF-1 gene and/or protein expression in EESCs and EuESCs at 24 and 48 h (p < 0.05-<0.01). 1,25(OH)2D3 treatment also reduced the proliferation of PBMCs and PFMCs of endometriotic patients compared with controls (p < 0.01). 1,25(OH)2D3 can be considered as a potentially effective agent in the prevention and treatment of endometriosis along with other therapies.

CSF-1-induced DC-SIGN+ macrophages are present in the ovarian endometriosis.

BACKGROUND: Researchers have found that macrophages are the predominant cells in the peritoneal fluid (PF) of endometriosis patients. CSF-1 has been found to accumulate in the lesions and PF of endometriosis patients, and CSF-1 induces THP-1-derived macrophages to polarize toward a CD169+ DC-SIGN+ phenotype. Does the cytokine CSF-1 induce monocytes to differentiate into macrophages with a DC-SIGN+ phenotype in endometriosis? METHODS: The level of CSF-1 in the endometrium of control subjects, and the eutopic, and ectopic endometrium of endometriosis patients was evaluated by real-time polymerase chain reaction (qRT-PCR) and was determined by enzyme-linked immunosorbent assay (ELISA) in the PF of control and endometriosis patients. CSF-1 expression was examined with a MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel. DC-SIGN+ macrophages were detected by immunohistochemical staining of tissues and flow cytometric analysis of the PF of control subjects (N = 25) and endometriosis (N = 35) patients. The phenotypes and biological activities of CSF-1 -induced macrophages were compared in an in vitro coculture system with peripheral blood lymphocytes from control subjects. RESULTS: In this study, we found that the proportion of DC-SIGN+ CD169+ macrophages was higher in the abdominal immune microenvironment of endometriosis patients. CSF-1 was primarily secreted from ectopic lesions and peritoneum in mice with endometriosis. In addition, CSF-1 induced the polarization of macrophages toward a DC-SIGN+ CD169+ phenotype; this effect was abolished by the addition of an anti-CSF-1R antibody. CSF-1 induced the generation of DC-SIGN+ macrophages, leading to a depressed status of peripheral blood lymphocytes, including a high percentage of Treg cells and a low percentage of CD8+ T cells. Similarly, blockade with the anti-CSF-1R antibody abrogated this biological effect. CONCLUSIONS: This is the first study on the role of DC-SIGN+ macrophages in the immune microenvironment of endometriosis. Further study of the mechanism and biological activities of CSF-1-induced DC-SIGN+ macrophages will enhance our understanding of the physiology of endometriosis.

Clinical significance of lipid droplets formed in the peritoneal fluid after laparoscopic surgery for gastric cancer.

BACKGROUND: Several studies have previously reported that laparoscopic surgery using an energy sealing device generates hazardous surgical smoke. However, the droplets appearing on the surface of peritoneal fluid irrigated with saline, after dissection phase of laparoscopic gastrectomy were ignored for a long time. This study aimed to investigate the composition and clinical significance of these droplet particles. METHODS: This study prospectively enrolled 15 patients with early gastric cancer (cT1NanyM0) who were scheduled for laparoscopic gastrectomy. Floating phases of peritoneal irrigation fluid containing droplets in dissected area were retrieved before and after surgical dissection. Using gas chromatography analysis, the areas under the peak were compared between the samples retrieved before and after surgical dissection. We also analyzed if the area value with significant change was related to the inflammatory response. RESULTS: In gas chromatography, the area values after laparoscopic surgical dissection were significantly increased in 10 out of 37 kinds of fatty acids, compared to those before surgical dissection. The significant increase in area value of α-linoleic and eicosadienoic acids were positively correlated with the elevated level of C-reactive protein at postoperative day 2 (Spearman's ρ = 0.843, P < 0.001; Spearman's ρ = 0.785, P = 0.001). CONCLUSIONS: The lipid droplets, generated after laparoscopic lymphadenectomy during gastric cancer surgery, contained various types of fatty acids, and some of them have been found to be associated with inflammatory response.

IL-17: an important pathogenic factor in endometriosis.

Interleukin-17 (IL-17) is known as a Th17-cell-derived proinflammatory cytokine, which plays a pivotal role in several inflammatory and autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, and psoriasis. Emerging evidence has shown that IL-17 is linked to endometriosis, although the etiology of endometriosis is still unknown. The IL-17 expression is up-regulated in serum, peritoneal fluid (PF) and endometriotic lesions from patients with endometriosis but the related regulation mechanisms are complex and obscure. Meanwhile, the specific roles of IL-17 in endometriosis are also worthy of further exploration. Through the integration and summary of literature, we conclude that the secretion of IL-17 increases under the regulation of ectopic microenvironment and other factors, and then IL-17 is deeply involved in endometriosis in the regulation of immune microenvironment, the invasion and growth of ectopic lesions, and so on, which implies its therapeutic value in this disorder.

Comparison of Methods for MicroRNA Isolation from Extracellular Vesicles Obtained from Ascitic Fluids.

Secreted extracellular vesicles (EVs) contain active biomolecules, including miRNAs, composition of which reflects epigenetic changes occurring in cells during pathological processes, in particular, malignant transformation. The accumulated pool of data on the role of EVs in carcinogenesis has stimulated investigations of the EV-derived cancer markers. The most important factor limiting development of this scientific direction is lack of "gold standards" both for methods of EV isolation from biological fluids and for analyzing their molecular content, including composition of miRNAs. Here we first examined efficacy of various methods for small RNA isolation from EVs contained in ascitic fluid for subsequent miRNA analysis. Comparison of different commercial kits showed advantages of the methods based on phenol-chloroform extraction: Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit. Analysis of the small RNA transcriptome showed presence of various classes of molecules in the EVs, among which proportion of miRNAs averaged 6% and reaching 10% with the Total Exosome RNA & Protein Isolation Kit. The PureLink miRNA Isolation Kit demonstrated the lowest efficiency. The miRNeasy Advanced Serum/Plasma Kit showed the highest concentration of the small RNA fraction, miRNA proportion of which, however, did not exceed that obtained with the miRNeasy Serum/Plasma Kit and Total Exosome RNA & Protein Isolation Kit. Moreover, RT-PCR analysis of the individual molecules showed lower levels of each of investigated miRNAs (miR-1246, miR-200b-5p, miR-200c-3p, and miR-23a-3p) when using the miRNeasy Advanced Serum/Plasma Kit. In conclusion, Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit can be considered as optimal kits in terms of performance based on combination of the studied characteristics, including small RNA concentration, percentage of microRNA according to bioanalyzer and sequencing results, and levels of individual miRNAs detected by RT-PCR.

Plasma and Peritoneal Fluid Fibronectin and Collagen IV Levels as Potential Biomarkers of Endometriosis.

Laparoscopy as a diagnostic tool for patients with suspected endometriosis is associated with several potentially life-threatening complications. Therefore, it is imperative to identify reliable, non-invasive biomarkers of the disease. The aim of this study was to analyse the concentrations of fibronectin and type IV collagen in peritoneal fluid and plasma to assess their role as potential biomarkers in the diagnosis of endometriosis. Fibronectin and collagen IV protein levels were assessed by surface plasmon resonance imaging (SPRi) biosensors with the usage of monoclonal antibodies. All patients enrolled in the study were referred for laparoscopy for the diagnosis of infertility or chronic pelvic pain (n = 84). The study group included patients with endometriosis confirmed during surgery (n = 49). The concentration of fibronectin in the plasma (329.3 ± 98.5 mg/L) and peritoneal fluid (26.8 ± 11.1 µg/L) in women with endometriosis was significantly higher than in the control group (251.2 ± 84.0 mg/L, 7.0 ± 5.9 µg/L). Fibronectin levels were independent of endometriosis stage (p = 0.874, p = 0.469). No significant differences were observed in collagen IV levels (p = 0.385, p = 0.465). The presence of elevated levels of fibronectin may indicate abnormalities in cell-ECM signalling during the course of endometriosis, and may be a potential biomarker for early detection.

Comparative Analysis of Tumor-Associated microRNAs and Tetraspanines from Exosomes of Plasma and Ascitic Fluids of Ovarian Cancer Patients.

Ovarian cancer (OC) is one of the most common and fatal types of gynecological cancer. In the early phase of OC detection, the current treatment and diagnostic methods are not efficient and sensitive enough. Therefore, it is crucial to explore the mechanisms of OC metastasis and discover valuable factors for early diagnosis of female cancers and novel therapeutic strategies for metastasis. Exosomes are known to be involved in the development, migration, and invasion of cancer cells, and their cargo could be useful for the non-invasive biopsy development. CD151- and Tspan8-positive exosomes are known to support the degradation of the extracellular matrix, and are involved in stroma remodeling, angiogenesis and cell motility, as well as the association of miR-24 and miR-101 with these processes. The objective of this study was to explore the relationship of these components of exosomal cargo, in patients with OC, to clarify the clinical significance of these markers in liquid biopsies. The levels of tetraspanins Tspan8+ and CD151+ exosomes were significantly higher in plasma exosomes of OC patients compared with healthy females (HFs). The relative levels of miR-24 and miR-101 in plasma exosomes of HFs were significantly higher than in plasma exosomes of OC patients, while the levels of these microRNAs in exosomes from plasma and ascites of ill females showed no difference. Our study revealed a strong direct correlation between the change in the ascites exosomes CD151+Tspan8+ subpopulation level and the expression levels of the ascites (R = 0.81, p < 0.05) and plasma exosomal miR-24 (R = 0.74, p < 0.05) in OC patients, which confirms the assumption that exosomal cargo act synergistically to increase cellular motility, affecting cellular processes and signaling. Bioinformatics analysis confirmed the involvement of CD151 and Tspan8 tetraspanins and genes controlled by miR-24-3p and miR-101 in signaling pathways, which are crucial for carcinogenesis, demonstrating that these tetraspanins and microRNAs are potential biomarkers for OC screening, and predictors of poor clinicopathological behavior in tumors.

The Levels of Ghrelin, Glucagon, Visfatin and Glp-1 Are Decreased in the Peritoneal Fluid of Women with Endometriosis along with the Increased Expression of the CD10 Protease by the Macrophages.

The aim of this study was to evaluate the levels of ten energy metabolism factors: C-peptide, ghrelin, GIP, GLP-1, glucagon, insulin, leptin, PAI-1 (total), resistin, and visfatin, and to determine the expression of GLP1R receptors, CD10, CD26 proteases, and pro-inflammatory marker CD86 by macrophages in the peritoneal fluid (PF) in patients with endometriosis. The study included 54 women with endometriosis and a control group of 30 women with uterine myoma without signs of endometriosis. The levels of factors in PF were assessed by a multiplex method. Expression of GLP1R receptors, CD10, CD26 proteases, and CD86 by macrophages was evaluated using flow cytometry. It was found that in women with endometriosis, the concentrations of ghrelin, GLP-1, glucagon, and visfatin in PF were reduced (p = 0.007, p = 0.009, p = 0.002, p = 0.008, respectively). At the same time, there was a noted increase in the CD10 protease expression by peritoneal macrophages (p = 0.044). Correlation analysis showed a positive correlation of ghrelin and GLP-1 levels with CD86 macrophage expression (p = 0.044, p = 0.022, respectively) in the study group; a positive correlation was also found between the levels of GLP-1, glucagon, and visfatin with CD26 macrophage expression (p = 0.041, p = 0.048, p = 0.015, respectively) in PF. No correlations were found in the control group. These results indicate that a decrease in the levels of ghrelin, GLP-1, glucagon, and visfatin in PF may contribute to endometriosis development through their impact on the expression of pro-inflammatory markers of PF macrophages.

The Significance of Blood and Peritoneal Fluid Biochemical Markers in Identifying Early Anastomotic Leak following Colorectal Resection-Findings from a Single-Center Study.

Background and Objectives: The aim of our study was to evaluate the value of leukocyte, C reactive protein (CRP), procalcitonin, lactate, and carcinoembryonic antigen (CEA) in blood and peritoneal fluid in early recognition of anastomotic leak (AL) after colorectal resections. Materials and Methods: Our pilot prospective cohort study was conducted at the abdominal surgery department at University Medical Center Ljubljana. A total of 43 patients who underwent open or laparoscopic colorectal resection because of benign or malignant etiology were enrolled. All of the patients had primary anastomosis without stoma formation. Results: Three patients in our patient group developed AL (7%). We found a statistically significant elevation of serum lactate levels in patients that developed AL compared to those who did not but noted no statistically relevant difference in the blood or peritoneal fluid levels of other biochemical markers. Conclusions: Elevated lactate levels may be considered a promising biomarker for the early diagnosis of AL, but more research on bigger patient groups is warranted.

Histological sequence of the development of rat mesothelioma by MWCNT, with the involvement of apolipoproteins.

A rat model of mesothelioma development by peritoneal injection of multiwalled carbon nanotube (MWCNT) has been established and found to be useful to understand the mechanisms underlying fibrous particles-associated carcinogenesis. Its detailed histological sequence, however, remains largely obscure. We therefore aimed to assess the time-course of mesothelioma development by MWCNT and evaluate a set of lipoprotein-related molecules as potential mechanism-based biomarkers for the phenomenon. Male Fischer 344 rats were injected intraperitoneally (ip) with MWCNT (MWNT-7) at 1 mg/kg body weight, and necropsied at 8, 16, 24, 32, or 42 wk after injection. For biochemical analyses of the lipoprotein-related molecules, more samples, including severe mesothelioma cases, were obtained from 2 other carcinogenicity tests. Histologically, in association with chronic inflammation, mesothelial proliferative lesions appeared at c. Wk-24. Before and at the beginning of the tumor development, a prominent infiltration of CD163-positive cells was seen near mesothelial cells. The histological pattern of early mesothelioma was not a papillary structure, but was a characteristic structure with a spherical appearance, composed of the mesothelioma cells in the surface area that were underlain by connective tissue-like cells. Along with the progression, mesotheliomas started to show versatile histological subtypes. Serum levels of apolipoprotein A-I and A-IV, and a ratio of HDL cholesterol to total cholesterol were inversely correlated with mesothelioma severity. Overall, the detailed histological sequence of mesotheliomagenesis by MWCNT is demonstrated, and indicated that the altered profile of apolipoproteins may be involved in its underlying mechanisms.

Extracellular CIRP decreases Siglec-G expression on B-1a cells skewing them towards a pro-inflammatory phenotype in sepsis.

BACKGROUND: Sepsis is a life-threatening disease syndrome caused by a dysregulated host response to infection and injury. Extracellular cold-inducible RNA-binding protein (eCIRP) acts as a damage-associated molecular pattern. Peritoneal cavity (PerC) B-1a cells attenuate inflammation and tissue injury by spontaneous releasing natural IgM and IL-10. Sialic acid-binding immunoglobulin-type lectin-G (Siglec-G) is a CD33-related receptor highly expressed in B-1a cells to serve critical immunoregulatory functions. In sepsis, B-1a cell numbers in PerC are decreased. We hypothesized that eCIRP causes the reduction of PerC B-1a cells and alters their function during sepsis. METHODS: Sepsis was induced in WT and CIRP-/- mice by cecal ligation and puncture (CLP). PerC washout cells were collected and B-1a cells and Siglec-G were assessed by flow cytometry. Mice were i.p. injected with recombinant murine (rm) CIRP and after 20 h, Siglec-G expression in PerC B-1a cells were assessed. PerC B-1a cells were treated with rmCIRP for 4 h and Siglec-G expression was assessed. PerC B-1a cells were pre-treated with anti-Siglec-G Ab and then after stimulated with rmCIRP for 24 h, IL-6 levels in the culture supernatants were assessed. RESULTS: eCIRP levels in the PerC were elevated in septic mice. In WT mice, the frequencies and numbers of total and Siglec-G+ B-1a cells in the PerC were significantly decreased in the CLP group compared to sham group, whereas in CIRP-/- mice, their frequencies and numbers in sepsis were significantly rescued compared to WT septic mice. Mice injected with rmCIRP showed decreased frequencies and numbers of total and Siglec-G+ PerC B-1a cells compared to PBS-injected mice. In vitro treatment of PerC B-1a cells with rmCIRP demonstrated significant reduction in Siglec-G mRNA and protein compared to PBS group. PerC B-1a cells treated with anti-Siglec-G Ab had significantly higher production of IL-6 in response to rmCIRP compared to IgG control. Anti-Siglec-G Ab treated B-1a cells co-cultured with macrophages produced significantly higher levels of IL-6, and TNF-α, and lower levels of IL-10 compared to IgG-treated B-1a cells and macrophage co-cultures stimulated with rmCIRP. CONCLUSION: eCIRP reduces PerC B-1a cell pool and skews them to a pro-inflammatory phenotype by downregulating Siglec-G expression. Targeting eCIRP will retain Siglec-G expressing B-1a cells in the PerC and preserve their anti-inflammatory function in sepsis.

Peritoneal Level of CD206 Associates With Mortality and an Inflammatory Macrophage Phenotype in Patients With Decompensated Cirrhosis and Spontaneous Bacterial Peritonitis.

BACKGROUND & AIMS: Peritoneal macrophages (PMs) regulate inflammation and control bacterial infections in patients with decompensated cirrhosis. We aimed to characterize PMs and associate their activation with outcomes of patients with spontaneous bacterial peritonitis (SBP). METHODS: We isolated PMs from ascites samples of 66 patients with decompensated cirrhosis (19 with SBP) and analyzed them by flow cytometry, quantitative real-time polymerase chain reaction, functional analysis, and RNA microarrays. We used ascites samples of a separate cohort of 111 patients with decompensated cirrhosis (67 with SBP) and quantified the soluble form of the mannose receptor (CD206) and tumor necrosis factor by enzyme-linked immunosorbent assay (test cohort). We performed logistic regression analysis to identify factors associated with 90-day mortality. We validated our findings using data from 71 patients with cirrhosis and SBP. Data from 14 patients undergoing peritoneal dialysis for end-stage renal disease but without cirrhosis were included as controls. RESULTS: We used surface levels of CD206 to identify subsets of large PMs (LPM) and small PMs (SPM), which differed in granularity and maturation markers, in ascites samples from patients with cirrhosis. LPMs vs SPMs from patients with cirrhosis had different transcriptomes; we identified more than 4000 genes that were differentially regulated in LPMs vs SPMs, including those that regulate the cycle, metabolism, self-renewal, and immune cell signaling. LPMs had an inflammatory phenotype, were less susceptible to tolerance induction, and released more tumor necrosis factor than SPMs. LPMs from patients with cirrhosis produced more inflammatory cytokines than LPMs from controls. Activation of PMs by Toll-like receptor agonists and live bacteria altered levels of CD206 on the surface of LPMs and release of soluble CD206. Analysis of serial ascites fluid from patients with SBP revealed loss of LPMs in the early phase of SBP, but levels increased after treatment. In the test and validation cohorts, patients with SBP and higher concentrations of soluble CD206 in ascites fluid (>0.53 mg/L) were less likely to survive for 90 days than those with lower levels. CONCLUSIONS: Surface level of CD206 can be used to identify mature, resident, inflammatory PMs in patients with cirrhosis. Soluble CD206 is released from activated LPMs and increased concentrations in patients with cirrhosis and SBP indicate reduced odds of surviving for 90 days.