Ovarian Cancer-Associated Ascites Have High Proportions of Cytokine-Responsive CD56bright NK Cells.

Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients' blood, as well as significantly (p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher (p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly (p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly (p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive (p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.

[Laboratorial alteration preceding staphylococcal infection clinical manifestations after intestinal transplantation]. / Alteración laboratorial que precede a las manifestaciones clínicas de infección estafilocócica postrasplante intestinal.

BACKGROUND: Hospital-acquired infection, often with Staphylococcus aureus, is an important complication in intestinal transplant. CLINICAL CASE: A 2-year-old girl underwent small bowel transplantation owing to a small bowel volvulus. On the first postoperative day, lymphocyte phenotypes, serum immunoglobulins and chemotactic and phagocytic activity of neutrophils were assessed in peripheral blood. A decrease in the ingestion phase of phagocytosis by neutrophils was identified, in comparison with the results of 20 healthy children. On the second day, the patient had low fever and, on the third, abdominal pain. In view of this, she underwent a laparotomy that revealed purulent ascites due to Staphylococcus aureus. Specific treatment resulted in rapid regression of the infectious condition and good evolution of the patient. CONCLUSIONS: A decrease in the ingestion stage of phagocytosis by neutrophils preceded staphylococcal purulent ascites clinical manifestations, and immunologic assessment contributed to early diagnosis and treatment of the infection. We believe evaluation of neutrophilic activity is important in patients undergoing intestinal transplantation in order for possible hospital-acquired infections to be early diagnosed.

Antecedentes: La infección hospitalaria, frecuentemente por Staphylococcus aureus, es una complicación importante en los pacientes con trasplante intestinal. Caso clínico: Niña de 2 años de edad sometida a trasplante de intestino delgado debido a vólvulo yeyunal. En el primer día del posoperatorio, en la sangre periférica fueron evaluados fenotipo de linfocitos, inmunoglobulinas séricas, actividad quimiotáctica y fagocitaria de neutrófilos. Se identificó disminución de la etapa de ingestión de fagocitosis neutrofílica, en comparación con los resultados de 20 niños saludables. En el segundo día, la paciente presentó fiebre baja y en el tercero, dolor abdominal. Debido a lo anterior fue sometida a laparotomía que reveló ascitis purulenta por Staphylococcus aureus. El tratamiento específico derivó en regresión rápida del cuadro infeccioso y buena evolución. Conclusiones: La disminución de la etapa de ingestión de la fagocitosis neutrofílica precedió a las manifestaciones clínicas de ascitis purulenta estafilocócica; la evaluación inmunológica contribuyó al diagnóstico y tratamiento precoces de la infección. Creemos que es importante la evaluación de la actividad neutrofílica en pacientes sometidos a trasplante intestinal, con la finalidad de diagnosticar tempranamente posibles infecciones hospitalarias.

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.

Isolation of 1E4 IgM Anti-Fasciola hepatica Rediae Monoclonal Antibody from Ascites: Comparison of Two Purification Protocols.

Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties.

Circulating cytokines and chemokines associated with plasma leakage and hepatic dysfunction in Brazilian children with dengue fever.

Dengue fever is usually a benign acute viral infection transmitted by arthropods but may evolve to severe clinical manifestations such as coagulation and/or hemodynamic disorders, caused mainly by an increase of vascular permeability. Deregulated circulating immunological factors have been associated with severity. In Brazil severe cases appeared in children only recently and we evaluated the profile of cytokine/chemokine kinetics in 134 hospitalized young patients during the epidemic in Rio de Janeiro in 2008. Inflammatory cytokines TNF and IFNγ were found elevated during the acute phase in children as well as the anti-inflammatory IL10 and chemokines MIF and CXCL10/IP10, all last three persisting longer during the recovery phase. Severe disease fitting the dengue hemorrhagic fever pattern (WHO, 1997) was associated with higher IL10 and CXCL10/IP10 circulating levels (peak levels at seven days with P<0.01 and P<0.001 respectively as compared to DF). These factors were higher in patients pulmonary effusion or ascites (P<0.05 for IL10 and P<0.01 for CXCL10/IP10). Both factors were also associated with liver changes such as AST increase correlated with CXCL10/IP10 (r=0.4300 with P<0.0001) and patients presenting painful hepatomegaly showed higher circulating levels of IL10 (P<0.01, at 7-9 days) and of CXCL10/IP10 (P<0.05, 4-6 days and P<0.001, 7-9 days) when compared to patients without apparent liver alterations. Most cases presented a history of prior infection (93%). This is the first study demonstrating cytokine and chemokine association with severity during dengue fever in Brazilian children. IL10 and CXCL10/IP10 play a role in the disease severity associated with induction of vascular leakage and a novel association with changes in liver dysfunction.

Ovarian tumor ascites CD14+ cells suppress dendritic cell-activated CD4+ T-cell responses through IL-10 secretion and indoleamine 2,3-dioxygenase.

The observation that Th17 infiltration in ovarian cancer correlates with markedly improved survival has prompted the question of whether ovarian tumor antigen-specific Th17 responses could be stimulated by tumor vaccination. Dendritic cells (DCs) treated with IL-15 and an inhibitor of p38 MAPK signaling (DC(IL-15/p38inhib)) bias T-cell responses toward a Th1/Th17 phenotype, raising the prospect of therapeutic vaccination; however, significant barriers remain. Tumor vaccines, including DC vaccination, usually stimulate immune responses, but the lack of clinical responses in cancer patients has been disappointing. Possible reasons may include an inability of antitumor T cells to migrate into the tumor microenvironment, and an inability of T cells to retain effector function in the face of tumor-associated immune suppression. We found that ovarian tumor antigen-specific CD4(+) T cells induced by DC(IL-15/p38inhib) migrated in response to CXCL12 and CCL22 (both highly expressed in ovarian cancer) and to ascites CD14(+) myeloid cells. Cocultures showed that ascites CD14(+) cells markedly suppressed antigen-specific CD4(+) T responses, but suppression could be alleviated by treatment with anti-IL-10 or inhibition of indoleamine 2,3-dioxygenase. These results suggest that the efficacy of DC vaccination against ovarian cancer may be boosted by agents that inhibit tumor-associated CD14(+) myeloid cell suppression or indoleamine 2,3-dioxygenase activity.

CO2 abdominal insufflation decreases local and systemic inflammatory response in experimental acute pancreatitis.

OBJECTIVES: Acute pancreatitis (AP) is a serious disease that is amplified by an associated systemic inflammatory response. We investigated the effect of CO2 pneumoperitoneum on the local and systemic inflammatory response in AP. METHODS: Acute pancreatitis was induced in Wistar rats by 5% taurocholate intraductal injection. Carbon dioxide pneumoperitoneum was applied for 30 minutes before the induction of AP. Inflammatory parameters were evaluated in the peritoneum (ascites, cell number, and tumor necrosis factor alpha [TNF-alpha]), serum (amylase, TNF-alpha, interleukin-6 [IL-6], and IL-10), pancreas (myeloperoxidase [MPO] activity, cyclo-oxygenase 2 and inducible nitric oxide synthase expression, and histological diagnosis), liver, and lung (mitochondria dysfunction and MPO activity). RESULTS: Abdominal insufflation with CO2 before induction of AP caused a significant decrease in ascites volume, cells, and TNF-alpha in the peritoneal cavity and in serum TNF-alpha and IL-6 but not IL-10 levels. In the pancreas, this treatment reduced MPO activity, acinar and fat necrosis, and the expression of inducible nitric oxide synthase and cyclo-oxygenase 2. There were no significant differences on serum amylase levels, liver mitochondrial function, and pulmonary MPO between groups. CONCLUSIONS: Our data demonstrated that CO2 pneumoperitoneum reduced pancreatic inflammation and attenuated systemic inflammatory response in AP. This article suggests that CO2 pneumoperitoneum plays a critical role on the better outcome in patients undergoing laparoscopic pancreatic surgery.

Combined carcinoembryonic antigen and cytopathologic examination in ascites.

OBJECTIVE: To investigate use of the combined carcinoembryonic antigen (CEA) test and cytopathologic examination to improve the diagnosis of neoplastic vs. nonneoplastic ascites. STUDY DESIGN: The tests were performed prospectively on 130 patients with ascites whose effusions were submitted for cytologic examination. RESULTS: Sixty-seven patients had epithelial tumors, and the cytologic examination was positive in 39 (58.2%). The CEA level was > or = 11.0 ng/mL in 36 patients (53.73%). CEA was helpful in the diagnosis in 18 cases, increasing to 57 (85.07%) the number of positive diagnoses. Eight samples of nonepithelial tumors had low levels of CEA. In 55 patients with nonneoplasic ascites the cytopathologic examination was negative, but the CEA assay was > 11.0 ng/mL in 3 patients. CONCLUSION: The cytopathologic examination should be performed in all cases, and the CEA assay should be done in suspected cases of epithelial neoplasia in which the cytologic examination was negative, there was uncertainty about the histologic type of neoplasia, or a diagnosis of nonepithelial neoplasia was made. When ascitic leukocytosis or hepatic failure is present, one should be cautious in interpreting the CEA assay because false positivity can occur.

[Binding of the antileukemia drug Escherichia coli L-asparaginase to the plasma membrane of normal human mononuclear cells]. / Asociación de la droga antileucémica L-asparraginasa de Escherichia coli a la membrana plasmática de células mononucleares humanas normales.

OBJECTIVE: To demonstrate that the enzyme L-asparaginase from Escherichia coli (EcA) binds to the plasma membranes of normal human lymphocytes and monocytes. MATERIAL AND METHODS: Lymphocytes and monocytes were isolated from heparinized blood samples which came from healthy volunteer donors. The cells were incubated with EcA to detect a possible binding of the enzyme to the mononuclear cells by indirect immunofluorescence using confocal microscopy. Meanwhile, ultracentrifugation was used to obtain the erythrocyte ghost microsomal fraction (P100) which was then analyzed by Western blotting to determine if EcA binds the lipid bilayer unspecifically. For the immunoassays, monospecific polyclonal antibodies were obtained from ascitic tumors developed in mice immunized with commercial L-asparaginase. RESULTS: EcA bins the lymphocyte and monocyte plasma membranes. In monocytes, there occurs a capping phenomenon, that is, the accumulation of fluorescent marker in one region. The image analyzer highlights it clearly at a depth of 3.8 microns. This binding would be unspecific, that is, there is no mediation of a specific receptor that binds EcA. This arises from the ability of the enzyme to bind to the membranes of erythrocyte ghost, as evidenced by the ability of the molecule to associate with a hydrophobic medium. The antibodies against EcA obtained from ascitic tumours developed in mice do not show cross reactivity with Na+/K+ ATPase, aspartate aminotransferase, nor with extracts of blood cells, which would make it a specific tool for the detection of EcA in whole cells and in homogenates electrotransfered to nitrocellulose membranes. CONCLUSION: L-asparaginase from E. coli behaves as a lipoprotein due to its ability to insert itself into hydrophobic environments, in which it resembles an isozyme present in T. pyriformis. The binding of this enzyme to lymphocytes and monocytes, demonstrated in this work, would permit the modification of the antileukemic treatment injecting doses of EcA bound to patient's own isolated immune cells.

Cytologic diagnosis of melanoma in serous effusions. A morphologic and immunocytochemical study.

The diversity of melanoma patterns greatly impairs the interpretation of malignant cells in effusion samples. The presence of melanin pigments greatly helps determine the histogenetic origin of the tumor, but unfortunately many cases do not exhibit this feature. We reviewed cases with a definitive diagnosis of melanoma in order to identify some useful characteristics of the morphologic examination of effusions. We also subjected the effusions to the HMB45 immunoreaction to determine the diagnostic usefulness of this monoclonal antibody. The study was performed on 21 effusion samples containing malignant cells, and the main cytologic findings were similar to those on other neoplasms except for the presence of melanin pigment. The HMB45 immunoreaction was very sensitive, confirming the diagnosis in 14 of 18 cases (77.8%). Melanin pigments seem to be useful markers for melanoma in effusions, and HMB45 can be used as an ancillary method in the differential diagnosis.

Inhibitory activity of soluble IL-2R in sera, ascites and culture supernatants from murine leukaemic cells.

The effect of sera from mice bearing a T cell lymphoid leukaemia (LB) and the supernatants from short term cultures of the tumour cells were studied on cell proliferation using syngeneic and allogeneic normal and tumour cells. An inhibitory activity was demonstrated in 24-48 h supernatants of LB cells in culture and disappeared after 4 days of culture. Inhibitory activity was cytostatic but not cytotoxic and was non-specific since it inhibited the growth of both syngeneic and allogeneic normal and tumour cells. Such activity was found in the 10(5)-1.3 x 10(5) M(r) serum fraction after a Sephacryl S200 chromatography. Though sensitive to protease, trypsin or neuraminidase treatment, which indicated its glycoprotein nature, it remained stable after heating or freezing-thawing cycles as well as after alkaline, acid or hyaluronidase treatment. Addition of exogenous IL-2 abrogated inhibitory activity. ELISA showed the presence of soluble IL-2R both in LB conditioned medium and in above serum fraction. It is demonstrated that the inhibitory factor, soluble IL-2R, is produced by LB leukaemia cells, then secreted into blood and ascitic fluid or released into culture supernatants. Soluble IL-2R exerts inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2.