[Ascites CD100 levels and immunomodulation effects in the peripheral blood of patients with liver cirrhosis combined with spontaneous bacterial peritonitis].

Objective: To observe the level and detection of ascites CD100 on the activity of CD4(+) and CD8(+) T lymphocytes in vitro in the peripheral blood of patients with liver cirrhosis combined with spontaneous bacterial peritonitis. Methods: Peripheral blood and ascites were collected from 77 cases of liver cirrhosis (49 patients with liver cirrhosis combined with simple ascites and 28 patients with liver cirrhosis combined with SBP), and peripheral blood was collected from 22 controls. Soluble CD100 (sCD100) in peripheral blood and ascites was detected by an enzyme-linked immunosorbent assay. Flow cytometry was used to detect membrane-bound CD100 (mCD100) on the surface of CD4(+) and CD8(+)T lymphocytes. CD4(+) and CD8(+)T lymphocytes in ascites were sorted. CD4(+)T lymphocyte proliferation, key transcription factor mRNA, and secreted cytokine changes, as well as CD8(+)T lymphocyte proliferation, important toxic molecule mRNA, and secreted cytokine changes, were detected after CD100 stimulation. The killing activity of CD8(+)T cells was detected by direct contact and indirect contact culture systems. Data conforming to normality were compared using one-way ANOVA, a student's t-test, or a paired t-test. Data that did not conform to a normal distribution were compared using either the Krusal-Willis test or the Mann-Whitney test. Results: There was no statistically significant difference in plasma sCD100 level between patients with liver cirrhosis combined simple ascites (1 415 ± 434.1) pg/ml, patients with liver cirrhosis combined with SBP (1 465 ± 386.8) pg/ml, and controls (1 355 ± 428.0) pg/ml (P = 0.655). The ascites sCD100 level was lower in patients with liver cirrhosis combined with SBP than that of patients with simple ascites [(2 409 ± 743.0) pg/ml vs. (2825±664.2) pg/ml, P=0.014]. There was no statistically significant difference in the level of mCD100 in peripheral blood CD4(+) and CD8(+) T lymphocytes among the three groups (P > 0.05). The levels of mCD100 in ascites CD4(+) and CD8(+) T lymphocytes were higher in patients with liver cirrhosis combined with SBP than those in patients with simple ascites (P < 0.05). CD100 stimulation had no significant effect on the proliferation of CD4(+) and CD8(+)T lymphocytes in the ascites of patients with liver cirrhosis combined with SBP (P > 0.05). There were no significant effects on the expression of transcription factors in effector CD4(+)T lymphocytes (T-bet, retinoic acid associated solitary nuclear receptor γt, aromatic hydrocarbon receptor) or secretion of cytokines (interferon-γ, 17, and 22) (P > 0.05). CD100 stimulation had increased the relative expression of perforin, granzyme B, and granlysin mRNA and the levels of secreted interferon-γ and tumor necrosis factor-α, killing activity in ascites CD8+ T lymphocytes of patients with liver cirrhosis combined with SBP (P < 0.05). Conclusion: The active form of CD100 is sCD100 instead of mCD100. There is an imbalance between the expression of sCD100 and mCD100 in the ascites of patients with cirrhosis combined with SBP. sCD100 can enhance the function of CD8(+)T lymphocytes in the ascites of patients with cirrhosis combined with SBP and thus is one of the potential therapeutic targets.

MAIT Cells Are Enriched and Highly Functional in Ascites of Patients With Decompensated Liver Cirrhosis.

BACKGROUND AND AIMS: Patients with advanced liver cirrhosis have an increased susceptibility to infections. As part of the cirrhosis-associated immune dysfunction, mucosal-associated invariant T (MAIT) cells, which have the capacity to respond to bacteria, are severely diminished in circulation and liver tissue. However, MAIT cell presence and function in the peritoneal cavity, a common anatomical site for infections in cirrhosis, remain elusive. In this study, we deliver a comprehensive investigation of the immune compartment present in ascites of patients with decompensated liver cirrhosis, and focus especially on MAIT cells. APPROACH AND RESULTS: To study this, matched peripheral blood and ascites fluid were collected from 35 patients with decompensated cirrhosis, with or without spontaneous bacterial peritonitis (SBP). MAIT cell phenotype and function were analyzed using high-dimensional flow cytometry, and the obtained data were compared with the blood samples of healthy controls (n = 24) and patients with compensated cirrhosis (n = 11). We found circulating MAIT cells to be severely decreased in patients with cirrhosis as compared with controls. In contrast, in ascites fluid, MAIT cells were significantly increased together with CD14+ CD16+ monocytes, innate lymphoid cells, and natural killer cells. This was paralleled by elevated levels of several pro-inflammatory cytokines and chemokines in ascites fluid as compared with plasma. Peritoneal MAIT cells displayed an activated tissue-resident phenotype, and this was corroborated by increased functional responses following stimulation with E. coli or interleukin (lL)-12 + IL-18 as compared with circulating MAIT cells. During SBP, peritoneal MAIT cell frequencies increased most among all major immune cell subsets, suggestive of active homing of MAIT cells to the site of infection. CONCLUSIONS: Despite severely diminished MAIT cell numbers and impaired phenotype in circulation, peritoneal MAIT cells remain abundant, activated, and highly functional in decompensated cirrhosis and are further enriched in SBP. This suggests that peritoneal MAIT cells could be of interest for immune-intervention strategies in patients with decompensated liver cirrhosis and SBP.

Intranodal Administration of Neoantigen Peptide-loaded Dendritic Cell Vaccine Elicits Epitope-specific T Cell Responses and Clinical Effects in a Patient with Chemorefractory Ovarian Cancer with Malignant Ascites.

Chemorefractory ovarian cancer has limited therapeutic options. Hence, new types of treatment including neoantigen-specific immunotherapy need to be investigated. Neoantigens represent promising targets for personalized cancer immunotherapy. We here describe the clinical and immunological effects of a neoantigen peptide-loaded DC-based immunotherapy in a patient with recurrent and chemoresistant ovarian cancer. A 71-year-old female patient with chemorefractory ovarian cancer and malignant ascites received intranodal vaccination of DCs loaded with four neoantigen peptides that were predicted by our immunogenomic pipeline. Following four rounds of vaccinations with this therapy, CA-125 levels were remarkably declined and tumor cells in the ascites were also decreased. Concordantly, the tumor-related symptoms such as respiratory discomfort improved without any adverse reactions. The reactivity against one HLA-A2402-restricted neoantigen peptide derived from a mutated PPM1 F protein was detected in lymphocytes from peripheral blood by IFN-γ ELISPOT assay. Furthermore, the neoantigen (PPM1 F mutant)-specific TCRs were detected in the tumor-infiltrating T lymphocytes post-vaccination. Our results showed that vaccination with intranodal injection of neoantigen peptide-loaded DCs may have clinical and immunological impacts on cancer treatment.

Feasibility, efficacy, and safety of cell-free and concentrated ascites reinfusion therapy (KM-CART) for malignant ascites.

Efficacy for alleviating signs/symptoms of malignant ascites of a renovated CART (cell-free and concentrated ascites reinfusion therapy) system, called KM-CART, was evaluated. A total of 4781 KM-CART procedures were performed in 2109 patients. All patients were accepted unless hemodynamically unstable or consciousness impaired. The ascites were processed and drip-infused into the patient. There were no major complications or deaths. The mean drainage volume was 6.2 L (maximum: 27.7 L), patient symptoms (numerical scale system) were significantly alleviated (45.1 ± 19.0 reduced to 21.2 ± 14.2, P < .001), and patient leg circumference significantly decreased (33.3 ± 4.4 cm reduced to 30.5 ± 4.4 cm, P < .001) without exacerbation of renal function. Collected cancer cells could be utilized for immune therapy. KM-CART is capable of improving the "quality of best supportive care" and can be beneficial in conjunction with medication for alleviating malignant pain.

PD-1+ TIM-3+ T cells in malignant ascites predict prognosis of gastrointestinal cancer.

The liquid biopsy of ascites fluid could be an excellent source of tumor and microenvironment for the study of prognostic biomarkers because of its accessibility. Tumor-infiltrating lymphocytes (TILs) can predict prognosis in multiple malignancies, including the response to immune checkpoint inhibitors, a breakthrough cancer therapy. However, TILs' profiles from malignant ascites have not been extensively studied. Using flow cytometric analysis, we quantified the proportion of exhausted T cells and memory/naive/effector T-cell subsets, among the CD4+ and CD8+ T-cell populations of paired TILs and peripheral blood T cell samples (n = 22). The correlation between CD4+ and CD8+ subset profiles suggested that the combined analysis of CD4+ and CD8+ cells in malignant ascites was clinically significant. We found that cells positive for the exhaustion markers programmed cell death-1 (PD-1), and T-cell immunoglobulin and mucin domain 3 (TIM-3), and cells coexpressing PD-1 and TIM-3 abundantly exist among malignant ascites TILs. Furthermore, patients with high frequency of PD-1+ TIM-3+ cells among the CD4+ and CD8+ T-cell population showed worse clinical outcome in multivariate analysis (n = 27). We propose that exhausted ascites TILs represent a clinically significant prognostic biomarker in advanced gastrointestinal cancer and represent an important target for immune checkpoint inhibitors.

Cross-talk between ovarian cancer cells and macrophages through periostin promotes macrophage recruitment.

Tumor-associated macrophages (TAMs) contribute to tumor progression, but it is not clear how they are recruited to tumor sites. Here we showed that periostin (POSTN) was present at high levels in ovarian cancer ascetic fluids and was correlated with CD163+ TAMs. The high POSTN level and macrophage infiltration were inversely associated with relapse-free survival for ovarian cancer patients. In vitro studies showed that coculture with macrophages significantly increased POSTN production in ovarian cancer cells. Further investigation found that POSTN production in ovarian cancer cells was promoted by transforming growth factor-ß generated by macrophages. Moreover, siRNA of POSTN and POSTN neutralizing antibody treatment showed that ovarian cancer cell-derived POSTN promoted the recruitment of macrophages and modulated their cytokine secretion profile. Collectively, these data indicated that POSTN was an important factor for macrophage recruitment in the tumor microenvironment and is involved in the interactions between macrophages and ovarian cancer cells.

Ex vivo-expanded NK cells from blood and ascites of ovarian cancer patients are cytotoxic against autologous primary ovarian cancer cells.

Ovarian cancer (OC) is the leading cause of gynecological cancer-related death in North America. Most ovarian cancer patients (OCPs) experience disease recurrence after first-line surgery and chemotherapy; thus, there is a need for novel second-line treatments to improve the prognosis of OC. Although peripheral blood-derived NK cells are known for their ability to spontaneously lyse tumour cells without prior sensitization, ascites-derived NK cells (ascites-NK cells) isolated from OCPs exhibit inhibitory phenotypes, impaired cytotoxicity and may play a pro-tumourigenic role in cancer progression. Therefore, it is of interest to improve the cytotoxic effector function of impaired OCP ascites-NK cells at the tumour environment. We investigated the efficacy of using an artificial APC-based ex vivo expansion technique to generate cytotoxic, expanded NK cells from previously impaired OCP ascites-NK cells, for use in an autologous model of NK cell immunotherapy. We are the first to obtain a log-scale expansion of OCP ascites-NK cells that upregulate the surface expression of activating receptors NKG2D, NKp30, NKp44, produce robust amounts of anti-tumour cytokines in the presence of OC cells and mediate direct tumour cytotoxicity against ascites-derived, primary OC cells obtained from autologous patients. Our findings demonstrate that it is possible to generate cytotoxic OCP ascites-NK cells from previously impaired OCP ascites-NK cells, which presents a promising immunotherapeutic target for the second-line treatment of OC. Future work should focus on evaluating the in vivo efficacy of autologous NK cell immunotherapy through the intraperitoneal delivery of NK cell expansion factors to a preclinical xenograft mouse model of human OC.

Activation and propagation of tumor-infiltrating lymphocytes from malignant pleural effusion and ascites with engineered cells for costimulatory enhancement.

Adoptive cell therapy (ACT) of autologous tumor-infiltrating lymphocytes (TILs) has shown an effect on mediating tumor regression in some patients with highly advanced, refractory metastatic malignancy. Here, the in vitro generation of TILs isolated from malignant pleural effusion and ascites was compared with which using engineered cells for costimulatory enhancement (ECCE) and 3 common γ-chain cytokines, interleukin (IL)-2, IL-7, and IL-15, alone or in combination. We showed the robust clinical-scale production of TILs with a less differentiated 'young' phenotype by expansion in the presence of ECCE combined with IL-2/7/15. Furthermore, a major fraction of the TILs generated in this fashion was shown to produce much more IFN-γ and TNF-α, and displayed cytolytic activity against target cells expressing the relevant antigens. To our knowledge, this is the first time that the combination of ECCE and IL-2/7/15 has been applied for the generation of TILs isolated from malignant pleural effusion and ascites.

Impaired neutrophil extracellular traps and inflammatory responses in the peritoneal fluid of patients with liver cirrhosis.

Liver cirrhosis (LC) is an inflammatory process associated with impaired functions in adaptive and innate immune responses at both systemic and local levels, also referred as Cirrhosis-Associated Immune Dysfunction. In this study, we evaluated the functionality of neutrophils from ascitic fluid (AF) of patients with hepatic cirrhosis by testing their ability to generate neutrophil extracellular traps (NETs) in vitro. To further determine the activation state of neutrophils, expression of the activation markers CD66b, CD69, and CD80 on these cells was analysed by flow cytometry. The inflammatory environment in AF was assessed by measured concentration of pro- and anti-inflammatory cytokines. Samples were collected from 40 patients with LC, 20 of them with uncomplicated ascites (ASC) and 20 with spontaneous bacterial peritonitis (SBP). Peripheral blood (PB) neutrophils from healthy individuals were used as control (HC). Our results revealed a significant decrease in the release of NETs in neutrophils from the SBP group compared with HC. Low expression of CD69 and CD80 on neutrophils from AF of SBP patients was also observed. Comparisons of inflammatory cytokine levels in AF from the different study groups (SBP and ASC) revealed significant differences. In conclusion, we demonstrate that the development of complications, such as SBP, increases initially the inflammatory status, but chronically results in impaired neutrophil function as demonstrated by the decreased capability of NETs formation. There is also an increase in both pro-inflammatory and anti-inflammatory cytokines, thus predisposing for new episodes of SPB and increasing morbidity and mortality in cirrhotic patients.

Different Lipid Regulation in Ovarian Cancer: Inhibition of the Immune System.

Lipid metabolism is altered in several cancer settings leading to different ratios of intermediates. Ovarian cancer is the most lethal gynecological malignancy. Cancer cells disperse in the abdominal space and ascites occurs. T cells obtained from ascites are unable to proliferate after an antigenic stimulus. The proliferation of ascites-derived T cells can be restored after culturing the cells for ten days in normal culture medium. No pathway aberrancies were detected. The acellular fraction of ascites can inhibit the proliferation of autologous as well as allogeneic peripheral blood lymphocytes, indicating the presence of soluble factors that interfere with T cell functionality. Therefore, we analyzed 109 lipid mediators and found differentially regulated lipids in suppressive ascitic fluid compared to normal abdominal fluid. Our study indicates the presence of lipid intermediates in ascites of ovarian cancer patients, which coincidences with T cell dysfunctionality. Since the immune system in the abdominal cavity is compromised, this may explain the high seeding efficiency of disseminated tumor cells. Further research is needed to fully understand the correlation between the various lipids and T cell proliferation, which could lead to new treatment options.

IL-15 super-agonist (ALT-803) enhances natural killer (NK) cell function against ovarian cancer.

OBJECTIVE: Natural killer (NK) cells represent a powerful immunotherapeutic target as they lyse tumors directly, do not require differentiation, and can elicit potent inflammatory responses. The objective of these studies was to use an IL-15 super-agonist complex, ALT-803 (Altor BioScience Corporation), to enhance the function of both normal and ovarian cancer patient derived NK cells by increasing cytotoxicity and cytokine production. METHODS: NK cell function from normal donor peripheral blood mononuclear cells (PBMCs) and ovarian cancer patient ascites was assessed using flow cytometry and chromium release assays ±ALT-803 stimulation. To evaluate the ability of ALT-803 to enhance NK cell function in vivo against ovarian cancer, we used a MA148-luc ovarian cancer NOD scid gamma (NSG) xenogeneic mouse model with transferred human NK cells. RESULTS: ALT-803 potently enhanced functionality of NK cells against all ovarian cancer cell lines with significant increases seen in CD107a, IFNγ and TNFα expression depending on target cell line. Function was also rescued in NK cells derived from ovarian cancer patient ascites. Finally, only animals treated with intraperitoneal ALT-803 displayed an NK dependent significant decrease in tumor. CONCLUSIONS: ALT-803 enhances NK cell cytotoxicity against ovarian cancer in vitro and in vivo and is able to rescue functionality of NK cells derived from ovarian cancer patient ascites. These findings suggest that ALT-803 has the potential to enhance NK cell-based immunotherapeutic approaches for the treatment of ovarian cancer.

Predictors of fifty days in-hospital mortality in patients with culture negative neutrocytic ascites.

BACKGROUND: Culture negative neutrocytic ascites is a variant of spontaneous bacterial peritonitis. But there are conflicting reports regarding the mortality associated with culture negativeneutrocytic ascites. Therefore we aim to determine the predictors of mortality associated with culture negativeneutrocytic ascites in a larger sample population. METHODS: We analysed 170 patients consecutively admitted to intensive care unit with diagnosis of culture negative neutrocytic ascites. The clinical, laboratory parameters, etiology of liver cirrhosis was determined along with the scores like model for end stage liver disease, child turcotte pugh were recorded. RESULTS: The 50 day in-hospital mortality rate in culture negative neutrocytic ascites was 39.41% (n = 67). In univariate analysis, means of parameters like total leucocyte count, urea, bilirubin, alanine transaminase, aspartate transaminase, international normalized ratio, acute kidney injury, septic shock, hepatic encephalopathy and model for end stage liver disease were significantly different among survived and those who died (P value ≤0.05). Cox proportional regression model showed the hazard ratio (HR) of acute kidney injury was 2.212 (95% CI: 1.334-3.667), septic shock (HR = 1.895, 95% CI: 1.081-3.323) and model for end stage liver disease (HR = 1.054, 95% CI: 1.020-1.090). Receiver operating characteristics curve showed aspartate aminotransferase (AST) had highest area under the curve 0.761 (95% CI: 0.625-0.785). CONCLUSION: Patients with culture negative neutrocytic ascites have a mortality rate comparable to spontaneous bacterial peritonitis. aspartate aminotransferase, alanine aminotransferase (ALT), acute kidney injury (AKI), model for end stage liver disease (MELD) and septic shock are the independent predictors of 50 days in-hospital mortality in culture negative neutrocytic ascites.

Relative Ascites Polymorphonuclear Cell Count Indicates Bacterascites and Risk of Spontaneous Bacterial Peritonitis.

BACKGROUND AND AIMS: Absolute polymorphonuclear (PMN) counts in ascites define spontaneous bacterial peritonitis (SBP), a severe form of bacterial infection in liver cirrhosis. Bacterascites, another form of ascites infection, can progress to SBP or may resolve spontaneously but is not reflected by absolute PMN counts. We investigated whether the relative ascites PMN count (the absolute PMN count divided by the absolute leukocyte count) provides additional information to detect bacterascites or predict SBP. METHODS: Hospitalized patients with liver cirrhosis requiring paracentesis were stratified with respect to a diagnosis of bacterascites and SBP with a prospective follow-up for 1 year. Diagnostic power of relative PMN counts in ascites was evaluated by receiver operating characteristics curves. RESULTS: At inclusion, we observed 28/269 (10%) and 43/269 (16%) episodes of BA and SBP, respectively. Unlike absolute PMN counts, relative PMN counts in ascites were significantly elevated in bacterascites (p = 0.001). During follow-up, 16 and 30 further episodes of BA and SBP were detected, respectively. Relative PMN counts increased significantly once patients developed BA (p = 0.001). At a threshold of 0.20 for the relative PMN count, sensitivity, specificity, positive and negative predictive values for bacterascites which required antibiotic treatment were 83, 75, 26 and 98%, respectively (p < 0.001). Furthermore, a relative PMN count in ascites ≥0.13 and MELD score >17 was independent factors associated with occurrence of SBP during follow-up. CONCLUSION: The relative PMN count is a cheap immunological marker linked to bacterascites and future SBP, which may help to stratify patients according to their risk of infection.

Natural Killer Cells Response to IL-2 Stimulation Is Distinct between Ascites with the Presence or Absence of Malignant Cells in Ovarian Cancer Patients.

Peritoneal ascites are a distinguishable feature of patients with advanced epithelial ovarian cancer (EOC). The presence of different lymphocyte subsets has been reported in EOC-associated ascites, which also can or not contain malignant cells. The goal of this study was to analyze the functional characteristics of natural killer (NK) cells from EOC-associated ascites in terms of their expression of activating receptors and ascites' contents of lymphocyte subtypes, cytokine profile and presence of EOC cells. NK cell function was evaluated by the expression of the degranulation marker CD107a in resting and interleukin (IL)-2 stimulated NK cells from ascites and blood. Degranulation of NK cells from EOC cell-free ascites was significantly (p < 0.05) higher than all the other groups, either in their resting state or after IL-2 stimulation, suggesting a previous local stimulation. In contrast, treatment with IL-2 had no effect on NK cells from ascites with EOC cells. The amount of regulatory T cells was significantly higher in ascites with EOC cells compared to EOC cell-free ascites. Ascites with EOC cells also had higher levels of tumor necrosis factor (TNF)-α, suggesting inflammation related to the malignancy. In conclusion, the functional performance of NK cells was distinct between EOC cell-free ascites and ascites with EOC cells. The impairment of NK cell response to IL-2 in ascites with EOC cells was consistent with an immunosuppressive tumor microenvironment.

Structural Basis for Antibody Recognition of Lipid A: INSIGHTS TO POLYSPECIFICITY TOWARD SINGLE-STRANDED DNA.

Septic shock is a leading cause of death, and it results from an inflammatory cascade triggered by the presence of microbial products in the blood. Certain LPS from Gram-negative bacteria are very potent inducers and are responsible for a high percentage of septic shock cases. Despite decades of research, mAbs specific for lipid A (the endotoxic principle of LPS) have not been successfully developed into a clinical treatment for sepsis. To understand the molecular basis for the observed inability to translate in vitro specificity for lipid A into clinical potential, the structures of antigen-binding fragments of mAbs S1-15 and A6 have been determined both in complex with lipid A carbohydrate backbone and in the unliganded form. The two antibodies have separate germ line origins that generate two markedly different combining-site pockets that are complementary both in shape and charge to the antigen. mAb A6 binds lipid A through both variable light and heavy chain residues, whereas S1-15 utilizes exclusively the variable heavy chain. Both antibodies bind lipid A such that the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains the lack of LPS recognition. Longstanding reports of polyspecificity of anti-lipid A antibodies toward single-stranded DNA combined with observed homology of S1-15 and A6 and the reports of several single-stranded DNA-specific mAbs prompted the determination of the structure of S1-15 in complex with single-stranded DNA fragments, which may provide clues about the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases.

Grade of soluble inflammatory response is mainly affected by circulating bacterial DNA concentrations in cirrhosis.

BACKGROUND & AIMS: Patients with decompensated cirrhosis show a marked innate immune response that shows a wide variability. The reasons for this fact have not been previously evaluated. This investigation was undertaken to study factors influencing the immune response intensity in both serum and ascitic fluid in patients with cirrhosis and ascites with presence of bactDNA. METHODS: 77 patients with cirrhosis and presence of bactDNA fragments in blood and ascitic fluid were included. Identification of bactDNA was evaluated by 16SrRNA gene PCR followed by nucleotide sequencing and by species-specific PCR. Concentration of amplified bacterial-DNA, bacteria identification, LPS, TNF-alpha, IFN-gamma, Interleukin 12 and nitric oxide in serum and ascitic fluid were evaluated as factors related to intensity of the immune response. RESULTS: Serum and AF levels of bactDNA, TNF-α, IFN-γ and nitric oxide concentration were higher in patients with presence of bactDNA from gram negative bacteria. Serum TNF-α levels showed a significant correlation with concentrations of bactDNA (r = 0.88; P = 0.001) and LPS (r = 0.28; P = 0.016). Serum nitric oxide levels were also significantly correlated with concentrations of bactDNA (r = 0.761; P = 0.001) but not with LPS levels. Levels of INF-γ and IL-12 were not significantly correlated with either bactDNA nor LPS levels. Plasmatic concentration of bactDNA was the most accurately correlated factor with the inflammatory response (ancova model included only levels of bactDNA (r(2) = 0.87, P = 0.047 for TNF-α; r(2) = 0.45, P = 0.03 for NOx). CONCLUSIONS: Bacterial-DNA concentration is the most influencing variable associated with serum TNF-α and nitric oxide response.

Activated regulatory and memory T-cells accumulate in malignant ascites from ovarian carcinoma patients.

Invasive ovarian cancer is associated with poor outcome. The presence of infiltrating regulatory T-cells (Tregs) suppresses protective anti-tumor immune responses, and their accumulation into the tumor microenvironment correlates with reduced survival in ovarian cancer patients. Here, we conducted a detailed characterization of CD4(+) T-cells, CD8(+) T-cells and Treg subsets in the peripheral blood and malignant ascites fluid from seventeen patients with ovarian carcinoma of epithelial origin. Cell distribution, activation status and proliferation status were assessed by multi-color flow cytometry. In ascites fluid, a significant accumulation of CD8(+) cytotoxic T-cells and Tregs was observed compared to peripheral blood. Furthermore, a skewing toward the CD45RA(-) effector/memory compartment was observed in all T-cell subsets in the ascites fluid, but was most pronounced in the Treg population. Regulatory T-cells in the malignant ascites were more activated and had a higher proliferation rate compared to blood-derived cells from the same patient, and their number in ascites was positively correlated with the number of epithelial cells in effusion. In summary, we demonstrate an accumulation of activated CD4(+), CD8(+) and regulatory T-cells in the cancer microenvironment of ovarian carcinoma.

Cytokine profiling of ascites at primary surgery identifies an interaction of tumor necrosis factor-α and interleukin-6 in predicting reduced progression-free survival in epithelial ovarian cancer.

OBJECTIVES: Epithelial ovarian cancer (EOC) typically presents with advanced disease. Even with optimal debulking and response to adjuvant chemotherapy, the majority of patients will have disease relapse. We evaluated cytokine and chemokine profiles in ascites at primary surgery as biomarkers for progression-free survival (PFS) and overall survival (OS) in patients with advanced EOC. METHODS: Retrospective analysis of patients (n =70) who underwent surgery at Roswell Park Cancer Institute between 2002 and 2012, followed by platinum-based chemotherapy. RESULTS: The mean age at diagnosis was 61.8 years, 85.3% had serous EOC, and 95.7% had stage IIIB, IIIC, or IV disease. Univariate analysis showed that ascites levels of tumor necrosis factor (TNF)-α were associated with reduced PFS after primary surgery. Although the ascites concentration of interleukin (IL)-6 was not by itself predictive of PFS, we found that stratifying patients by high TNF-α and high IL-6 levels identified a sub-group of patients at high risk for rapid disease relapse. This effect was largely independent of clinical prognostic variables. CONCLUSIONS: The combination of high TNF-α and high IL-6 ascites levels at primary surgery predicts worse PFS in patients with advanced EOC. These results suggest an interaction between ascites TNF-α and IL-6 in driving tumor progression and resistance to chemotherapy in advanced EOC, and raise the potential for pre-treatment ascites levels of these cytokines as prognostic biomarkers. This study involved a small sample of patients and was an exploratory analysis; therefore, findings require validation in a larger independent cohort.