-
Current Protocols in Immunology Aug 2015Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative...
Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments.
Topics: Animals; Ascitic Fluid; Bone Marrow Cells; Cell Separation; Mice; Neutrophils
PubMed: 26237011
DOI: 10.1002/0471142735.im0320s110 -
The Netherlands Journal of Medicine Oct 2016Accumulation of fluid in the peritoneal cavity - ascites - is commonly encountered in clinical practice. Ascites can originate from hepatic, malignant, cardiac, renal,... (Review)
Review
Accumulation of fluid in the peritoneal cavity - ascites - is commonly encountered in clinical practice. Ascites can originate from hepatic, malignant, cardiac, renal, and infectious diseases. This review discusses the current recommended diagnostic approach towards the patient with ascites and summarises future diagnostic targets.
Topics: Ascites; Ascitic Fluid; Culture Techniques; Diagnosis, Differential; Heart Failure; Humans; Laparoscopy; Liver Cirrhosis; Neoplasms; Pancreatic Diseases; Paracentesis; Polymerase Chain Reaction; Practice Guidelines as Topic; Tuberculosis; Ultrasonography
PubMed: 27762220
DOI: No ID Found -
Methods in Molecular Biology (Clifton,... 2022The accumulation of peritoneal fluid, referred to as ascites, is common in ovarian cancer. This fluid is a complex mixture that may include cells as well as a diverse...
The accumulation of peritoneal fluid, referred to as ascites, is common in ovarian cancer. This fluid is a complex mixture that may include cells as well as a diverse array of cytokines and growth factors. Here we describe a comprehensive method to process ascites to maximize data collection. The cellular fraction and fluid are first separated by centrifugation. The fluid can be frozen for later analysis of soluble factors or for use in in vitro experiments. The cellular fraction can be processed to analyze its composition or stored for future use.
Topics: Ascites; Ascitic Fluid; Cytokines; Female; Humans; Intercellular Signaling Peptides and Proteins; Ovarian Neoplasms
PubMed: 34918288
DOI: 10.1007/978-1-0716-1956-8_5 -
International Journal of Surgery... Nov 2023Staging laparoscopy for gastric cancer is recommended to assess the tumor's locoregional extension and exclude peritoneal disease. As there is no consensus on optimizing...
BACKGROUND
Staging laparoscopy for gastric cancer is recommended to assess the tumor's locoregional extension and exclude peritoneal disease. As there is no consensus on optimizing the procedure's diagnostic accuracy, we aimed to systematically review the literature on operative techniques, followed by peritoneal lavage fluid assessment in gastric cancer patients. Specifically, we sought to indicate the most common characteristics of the procedure and cytological evaluation.
METHODS
This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The protocol for this systematic review was registered on PROSPERO database (CRD: 42022306746). On September 2022, a search was carried out using Embase, Medline ALL, Cochrane Central Register of Controlled Trials, and Web of Science Core Collection.
RESULTS
The search identified 1632 studies on staging laparoscopy and 2190 studies on peritoneal fluid assessment. Some 212 studies were included. Open Hasson was the method of choice in accessing the peritoneal cavity in 65% of the studies, followed by establishing a pneumoperitoneum at 10-12 mmHg in 52% of reports. Most frequently, the patient was positioned supine (70%), while a 30° scope and three ports were used to assess the peritoneal cavity clockwise (72%, 77%, and 85%, respectively). Right and left upper abdomen quadrants were the predominant area of laparoscopic exploration (both 65%), followed by the primary tumor region (54%), liver and pelvis (both 30%), and small bowel and spleen (19% and 17%, respectively). Regions of peritoneal lavage and aspiration were limited to the pelvis (50%), followed by right and left upper abdomen quadrants (37.5% and 50%, respectively). No studies compared different methods of operative techniques or analysis of ascites/fluid.
CONCLUSIONS
This study indicates a high heterogeneity in the technique of staging laparoscopy and peritoneal fluid assessment in gastric cancer patients. Further research and initiatives to reach a consensus on the standardization of the procedure are warranted.
Topics: Humans; Stomach Neoplasms; Ascitic Fluid; Neoplasm Staging; Laparoscopy; Peritoneal Lavage
PubMed: 37581636
DOI: 10.1097/JS9.0000000000000632 -
International Journal of Medical... 2022Interleukin-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... (Review)
Review
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.
Topics: Ascitic Fluid; Endometriosis; Female; Humans; Interleukin-17; Th17 Cells
PubMed: 35582411
DOI: 10.7150/ijms.71972 -
Molecular Cancer Nov 2023Knowing the homologous recombination deficiency (HRD) status in advanced epithelial ovarian cancer (EOC) is vital for patient management. HRD is determined by...
BACKGROUND
Knowing the homologous recombination deficiency (HRD) status in advanced epithelial ovarian cancer (EOC) is vital for patient management. HRD is determined by BRCA1/BRCA2 pathogenic variants or genomic instability. However, tumor DNA analysis is inconclusive in 15-19% of cases. Peritoneal fluid, available in > 95% of advanced EOC cases, could serve as an alternative source of cell-free tumor DNA (cftDNA) for HRD testing. Limited data show the feasibility of cancer panel gene testing on ascites cfDNA but no study, to date, has investigated HRD testing.
METHODS
We collected ascites/peritoneal washings from 53 EOC patients (19 from retrospective cohort and 34 from prospective cohort) and performed a Cancer Gene Panel (CGP) using NGS for TP53/HR genes and shallow Whole Genome Sequencing (sWGS) for genomic instability on cfDNA.
RESULTS
cfDNA was detectable in 49 out of 53 patients (92.5%), including those with limited peritoneal fluid. Median cfDNA was 3700 ng/ml, with a turnaround time of 21 days. TP53 pathogenic variants were detected in 86% (42/49) of patients, all with HGSOC. BRCA1 and BRCA2 pathogenic variants were found in 14% (7/49) and 10% (5/49) of cases, respectively. Peritoneal cftDNA showed high sensitivity (97%), specificity (83%), and concordance (95%) with tumor-based TP53 variant detection. NGS CGP on cftDNA identified BRCA2 pathogenic variants in one case where tumor-based testing failed. sWGS on cftDNA provided informative results even when tumor-based genomic instability testing failed.
CONCLUSION
Profiling cftDNA from peritoneal fluid is feasible, providing a significant amount of tumor DNA. This fast and reliable approach enables HRD testing, including BRCA1/2 mutations and genomic instability assessment. HRD testing on cfDNA from peritoneal fluid should be offered to all primary laparoscopy patients.
Topics: Female; Humans; BRCA1 Protein; BRCA2 Protein; Mutation; Ovarian Neoplasms; Circulating Tumor DNA; Homologous Recombination; Ascitic Fluid; Ascites; Prospective Studies; Retrospective Studies; Carcinoma, Ovarian Epithelial; Genomic Instability
PubMed: 37932736
DOI: 10.1186/s12943-023-01864-1 -
International Journal of Molecular... Jul 2021Endometriosis is a common gynaecological disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. It is associated with chronic...
Endometriosis is a common gynaecological disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. It is associated with chronic pelvic inflammation and autoimmune reactivity manifesting by autoantibody production and abrogated cellular immune responses. Endometriotic peritoneal fluid contains various infiltrating leucocyte populations and a bulk of proinflammatory and immunoregulatory cytokines. However, the nature and significance of the peritoneal milieu in women with endometriosis still remains obscure. Therefore, the aim of the present study was to investigate the immunoregulatory activity of the peritoneal fluid (PF) from women with endometriosis. The peritoneal fluid samples were collected during laparoscopic surgery from 30 women with and without endometriosis. Immunoregulatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) and chemokines (CCL2, CCL5, CXCL8 and CXCL9) were evaluated in PF and culture supernatants generated by unstimulated and CD3/CD28/IL-2-stimulated CD4 T cells cultured in the presence of PF. The effect of PF on the generation of Treg and Th17 cells in CD4 T cell cultures, as well as the natural cytotoxic activity of peripheral blood mononuclear cells, was also investigated. Concentrations of IL-6, IL-10, CCL2, CXCL8 and CXCL9 were significantly upregulated in the PF from women with endometriosis when compared to control women, whereas concentrations of other cytokines and chemokines were unaffected. The culturing of unstimulated and CD3/CD28/IL-2-stimulated CD4 T cells in the presence of endometriotic PF resulted in the downregulation of their IL-2, IFN-γ, IL-17A and TNF production as compared to culture medium alone. On the other side, endometriotic PF significantly stimulated the production of IL-4 and IL-10. Endometriotic PF also stimulated the release of CCL2 and CXCL8, whereas the production of CCL5 and CXCL9 was downregulated. Endometriotic PF stimulated the generation of Treg cells and had an inhibitory effect on the generation of Th17 cells in cultures of CD4 T cells. It also inhibited the NK cell cytotoxic activity of the peripheral blood lymphocytes. These results strongly imply that the PF from patients with endometriosis has immunoregulatory/immunosuppressive activity and shifts the Th1/Th2 cytokine balance toward the Th2 response, which may account for deviation of local and systemic immune responses. However, a similar trend, albeit not a statistically significant one, was also observed in case of PF from women without endometriosis, thus suggesting that peritoneal milieu may in general display some immunoregulatory/immunosuppressive properties. It should be stressed, however, that our present observations were made on a relatively small number of PF samples and further studies are needed to reveal possible mechanism(s) responsible for this phenomenon.
Topics: Adult; Ascitic Fluid; Case-Control Studies; Cells, Cultured; Chemokines; Endometriosis; Female; Humans; Immune Tolerance; Middle Aged; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Th2 Cells; Up-Regulation; Young Adult
PubMed: 34360900
DOI: 10.3390/ijms22158134 -
Iranian Journal of Immunology : IJI Jun 2019Endometriosis is a chronic inflammatory disease with the growth of endometrial cells out of uterus and in the peritoneal cavity. T cell subsets participate in the...
BACKGROUND
Endometriosis is a chronic inflammatory disease with the growth of endometrial cells out of uterus and in the peritoneal cavity. T cell subsets participate in the establishment and progress of the disease by producing different cytokines.
OBJECTIVE
To investigate a group of cytokines related to Th1/Th2/Th17/Treg subsets within both peripheral blood and peritoneal fluid (PF) samples from infertile endometriosis women.
METHODS
Peripheral blood and PF samples were collected from 30 infertile endometriosis and 30 non-endometriosis fertile women during laparoscopy. Concentration of cytokines, including TNF-α, IFN-γ, TGF-β1, IL-4, IL-10, IL-17 and IL-23 were evaluated using ELISA method.
RESULTS
Results indicated that the concentration of IFN-γ within serum was significantly reduced in endometriosis group (p=0.001). Regarding PF cytokines, TGF-β1 was increased in endometriosis group (p=0.030). Furthermore, the ratios of IFN-γ/TGF-β1 and IL-17/IL-23 were significantly different between endometriosis and non-endometriosis women in serum samples (p<0.001 and p<0.01 respectively). The ratios of TNF-α/IL-10 and IL-17/IL-10 were also significantly different regarding PF samples between the two studied groups (p<0.04 and p<0.03 respectively). Finally, significant correlations were observed between the levels of IL-17 and IL-23, inflammatory and anti-inflammatory cytokines, in both samples and serum to PF inflammatory cytokines.
CONCLUSION
Based on the results of the present study, in women with endometriosis, the disturbance of cytokines network might gradually activate the inflammatory responses and tissue repair, resulting in endometriosis development after several years.
Topics: Adult; Ascitic Fluid; Cytokines; Endometriosis; Endometrium; Female; Humans; Infertility, Female; Middle Aged; T-Lymphocyte Subsets; Young Adult
PubMed: 31182689
DOI: 10.22034/IJI.2019.80258 -
International Journal of Molecular... Oct 2021Most patients with ovarian cancer (OvCA) present peritoneal disseminated disease at the time of diagnosis. During peritoneal metastasis, cancer cells detach from the... (Review)
Review
Most patients with ovarian cancer (OvCA) present peritoneal disseminated disease at the time of diagnosis. During peritoneal metastasis, cancer cells detach from the primary tumor and disseminate through the intraperitoneal fluid. The peritoneal mesothelial cell (PMC) monolayer that lines the abdominal cavity is the first barrier encountered by OvCA cells. Subsequent progression of tumors through the peritoneum leads to the accumulation into the peritoneal stroma of a sizeable population of carcinoma-associated fibroblasts (CAFs), which is mainly originated from a mesothelial-to-mesenchymal transition (MMT) process. A common characteristic of OvCA patients is the intraperitoneal accumulation of ascitic fluid, which is composed of cytokines, chemokines, growth factors, miRNAs, and proteins contained in exosomes, as well as tumor and mesothelial suspended cells, among other components that vary in proportion between patients. Exosomes are small extracellular vesicles that have been shown to mediate peritoneal metastasis by educating a pre-metastatic niche, promoting the accumulation of CAFs via MMT, and inducing tumor growth and chemoresistance. This review summarizes and discusses the pivotal role of exosomes and MMT as mediators of OvCA peritoneal colonization and as emerging diagnostic and therapeutic targets.
Topics: Ascitic Fluid; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cytokines; Epithelial-Mesenchymal Transition; Epithelium; Exosomes; Female; Humans; Intercellular Signaling Peptides and Proteins; Ovarian Neoplasms; Peritoneal Neoplasms; Peritoneum
PubMed: 34768926
DOI: 10.3390/ijms222111496 -
International Journal of Molecular... Dec 2022Laparoscopy as a diagnostic tool for patients with suspected endometriosis is associated with several potentially life-threatening complications. Therefore, it is...
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 ( = 0.874, = 0.469). No significant differences were observed in collagen IV levels ( = 0.385, = 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.
Topics: Humans; Female; Endometriosis; Ascitic Fluid; Fibronectins; Collagen Type IV; Biomarkers
PubMed: 36555313
DOI: 10.3390/ijms232415669