Effusion Fluid Of Patients Research Articles

Increased expression of PRRs and NOS in patients with endometriosis (P3143)

Abstract Objective: To assess these immune responses, we evaluated the levels of expression of Toll-like receptors (TLR)-1, -2, -4, -5, and -9; nucleotide-binding oligomerization domains (NOD)-1 and -2; interleukins-1β, -6, -8, -10, and -12; interferon-γ; tumor necrosis factor-α; inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS); and immunoglobulins (Igs) in patients with endometriosis. Methods: The levels of TLRs, NODs, cytokines, and NOS mRNAs in peritoneal effusion were assessed by real time polymerase chain reaction, and IgG, IgA and IgM concentrations were measured by enzyme-linked immunosorbent assays (ELISA) in 40 patients with and 40 without endometriosis. Findings from the two groups were compared. Results: We observed expression of all Pattern Recognition Receptors (PRRs), cytokines, and NOS mRNAs and immunoglobulins in the effusion fluid of patients with and without endometriosis. The levels of TLR-2 and -9; NOD-1 and -2; iNOS and eNOS mRNA and CA 125 were significantly higher in the endometriosis than in the non-endometriosis group (p<0.05 each). Moreover, PRRs, cytokine, and NOS showed significant correlations (p<0.05). Conclusions: PRRs, cytokines, and NOS, which act cooperatively in the innate immune response, are closely associated with endometriosis. Increased expression of PRRs and NOS in peritoneal fluid may be associated with endometriosis.

Increased Expression of Pattern Recognition Receptors and Nitric Oxide Synthase in Patients with Endometriosis

Objective: Endometriosis is characterized by repeated inflammatory changes and serious adhesions, inducing innate and adaptive immune responses within the abdominal cavity. To assess these immune responses, we evaluated the levels of expression of Toll-like receptors (TLR)-1, -2, -4, -5, and -9; nucleotide-binding oligomerization domains (NOD)-1 and -2; interleukins-1β, -6, -8, -10, and -12; interferon-γ; tumor necrosis factor-α; inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS); and immunoglobulins (Igs) in patients with endometriosis.Methods: The levels of TLRs, NODs, cytokines, and NOS mRNAs in peritoneal effusions were assessed by real time reverse transcription-polymerase chain reaction; and IgG, IgA and IgM concentrations were measured by enzyme-linked immunosorbent assays (ELISA) in 40 patients with and 40 without endometriosis. Findings from the two groups were compared.Results: We observed expression of all pattern recognition receptors (PRRs), cytokines, and NOS mRNAs and Igs in the effusion fluid of patients with and without endometriosis. The levels of TLR-2 and -9; NOD-1 and -2; iNOS and eNOS mRNAs and CA 125 were significantly higher in the endometriosis than in the non-endometriosis group (p<0.05 each). Moreover, PRR, cytokine, and NOS expression showed significant correlations (p<0.05).Conclusions: PRRs, cytokines, and NOS, which act cooperatively in the innate immune response, are closely associated with endometriosis. Increased expression of TLR-2, TLR -9, NOD-1, NOD-2, and NOS mRNA in peritoneal fluid may be associated with endometriosis.

The pan-HDAC Inhibitor Suberoylanilide Hydroxamic Acid Targets Self Renewal of Breast Cancer Stem Cells.

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC often lack estrogen receptor and present with skin metastasis characterized by the presence of tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly over-express E-cadherin and have characteristics of stem cells. Based on our initial observations that IBC cells express multiple forms of histone deacetylase (HDAC) enzymes involved in epigenetic modulation of multiple genes associated with critical functions of both embryonic stem cells and cancer cells, the present studies evaluated the effects of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on self-renewal, expression of transcription factors associated with self renewal, homotypic aggregation, and E-cadherin expression by mammospheres of SUM149 and SUM190 established IBC cell lines and of tumor cells isolated from pleural effusion fluid of patients with IBC and LABC.Methods: Low adherence culture conditions that promote mammosphere formation and clonogenic assays were used to assess effects of SAHA on self renewal. Real time PCR arrays and protein arrays were used to evaluate the effects of SAHA on transcription factors Oct4, Nanog and Sox-2 and breast cancer associated genes including E-cadherin and ER/PR. Flow cytometry was used to document stem cell markers present on breast cancer cells grown under low adherence conditions. Confocal microscopy was used to document the effects of SAHA on E-cadherin localization and homotypic aggregation by mammospheres.Results: SAHA inhibited self-renewal of second and third passage mammospheres as assessed by clonogenic assays, which was associated with loss of Oct4, Nanog and Sox-2 transcription factors associated with stem cell self renewal an pluripotency. SAHA blocked E-cadherin mRNA and protein assessed by PCR and Western blotting and visualized by confocal imaging which demonstrated that loss of E-cadherin between cells comprising mammospheres blocked homotypic aggregation, leading to lack of integrity of the 3-dimensional spheroids. Moreover, SAHA induced ER-/low mammospheres to re-express ER mRNA and protein which was associated with responsiveness to tamoxifen and conversely, SAHA inhibited ER expression in ER+breast tumor cells. The effective inhibitory concentration50 (IC50) of SAHA to induce these effects in both established cell lines and in mammospheres derived from tumor cells isolated from pleural effusion fluid of patients with IBC and LABC was in the range of 0.37-3.31 uM, which is achievable by oral administration of SAHA. Initial in vivo studies indicate that intraperitoneal injection of SAHA effectively inhibits SUM149 breast tumor xenograft growth and formation of metastatic lesions.Conclusions: SAHA promotes differentiation of IBC stem cells and blocks E-cadherin expression, which is a hallmark of IBC skin metastasis. Taken together with the observations that SAHA can re-program ER function depending upon the ER status of the breast cancer cells, this observations suggest that SAHA may provide additional therapeutic opportunities for patients with LABC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3141.

Leukocyte esterase activity in effusion fluid of patients with otitis media.

Fluid obtained during myringotomy and tube placement in 20 patients with middle ear effusions was assayed for leukocyte esterase activity using a quantitative spectrophotometric assay. This quantitative assay used the synthetic substrate, N-tosyl indoxyl alaninate. Seven of the 20 samples showed no measurable enzyme activity (8 U/ml or less). The remaining samples demonstrated activity ranging from 20 to 1600 units. Although enzyme activity did not correlate well with the physical appearance of the fluid, it did correlate with clinical history, suggesting the presence of a purulent exudate rather than serous effusion. Leukocyte esterase activity in the fluid appears to hold promise as an indicator for the presence of chronic middle ear infection. The enzyme can be assayed by a simple and fast diagnostic strip test, with results available almost immediately.

Immunosuppressive Factors in Human Cancer

This chapter reviews the factors on immunosuppressive molecules that are elaborated by tumor cells or found in sera and effusions. Immunosuppressive factors can be found in normal sera, but their level is increased in cancer sera. These factors may contribute to the defective cellular responses of patients. Some well-characterized immunosuppressive molecules are transforming growth factor β (TGF-β), lymphocyte blastogenesis inhibitory factor (LBIF), p15E, and suppressive E-receptor (SER). TGF-β is produced by most cells and has an extraordinarily wide range of biological activities. p15E protein possesses a remarkable range of immunosuppressive activities. The SER is isolated from malignant effusions derived from patients with several types of cancer and inhibited several cellular immune responses. The exact mechanism of the immunosuppression is still uncertain, but a multiplicity of factors contributes to its occurrence: suppressor T cells and suppressor macrophages, immune complexes, acute phase proteins, tumor-derived suppressor molecules, suppressor substances in sera and effusion fluids of patients, and chemotherapy and irradiation. The colony-stimulating factors, acute-phase proteins, and miscellaneous molecules are some of the immunosuppressive factors in human cancer.

Elevated serum CA 125 levels in patients with benign ascitic or pleural effusions.

The tumor-associated antigen CA 125 is widely used in monitoring of ovarian carcinomas. As this determinant is also expressed in proliferating mesothelia, we studied CA 125 levels in serum and effusion fluid of patients with ascitic and pleural effusions. Patients with benign and malignant disease were included. There was no statistically significant or diagnostically useful difference between 26 benign and 44 malignant cases in the CA 125 levels in effusions or sera. In 77% of benign cases, serum levels exceeding 105 U/ml (3 times the recommended upper limit of normal values) were found. Thus, in patients with serous effusions, elevated serum CA 125 values alone do not provide evidence for the presence of an (ovarian) malignancy, but may also indicate a proliferation of mesothelium due to benign disease.

Diagnostic Value of Carcinoembryonic Antigen (CEA) Assay in Pleural Effusions

Carcinoembryonic antigen (CEA) levels were evaluated by a direct radioimmunoassay in the effusion fluids and plasma samples from 107 patients with pleural effusions of unknown etiology. CEA levels higher than 20 ng/ml were detected in 55% of pleural effusions from patients with malignant disease: 40% of the patients in this group had values of plasmatic CEA lower than 20 ng/ml. With the only exception of 4 patients with empyema, CEA levels were uniformly lower than 20 ng/ml in the effusion fluid of patients with benign disease. Moreover, CEA levels higher than 20 ng/ml were observed in 81.2% of pleural effusions and 31.2% of plasma samples from patients with cytology positive for malignancy, whereas increased CEA levels were observed in 37.5% of effusion fluids and 33.3% of plasma samples from patients with malignant disease but negative cytology in the pleural effusion. CEA assay in the effusion fluid represents a useful tool of high specificity and good sensitivity to discriminate between benign and malignant etiology of pleural effusions.

Levels of fibrinogen/fibrin degradation fragment E and related substances in sera and effusions of patients with malignant disease.

A radioimmunoassay (RIA) was developed and used to determine the level of fragment E [a fibrinogen/fibrin degradation product (FDP)] and of fragment-E-containing substances (FES) in sera and effusion fluids of patients with malignant diseases. Sera of patients with other diseases and sera of healthy individuals served as controls. Results were expressed as units/ml (U/ml), one unit being equivalent to 40 ng pure fragment E. Effusion fluids of both malignant and nonmalignant origin contained relatively high levels of fragment-E-containing substances, up to 7,500 U/ml. Normal sera had less than 30 U/ml, while sera of patients with a variety of neoplastic or nonneoplastic conditions contained larger amounts, reaching to hundreds and, in rare cases (some patients with rheumatoid arthritis), even thousands of U/ml. Some of the highest levels in the malignant sera were found in samples from patients with Burkitt's lymphoma and stomach cancer. About 10%-20% of the reactive material in effusions and 20%-40% in the sera consisted of fragment E. These results confirm earlier findings of high FDP levels in neoplasia. Given the higher accuracy of the radioimmunoassay and its suitability for large scale testing, it would appear worthwhile to continue such studies to explore the clinical usefulness of the RIA for fragment E.

Antigens in immune complexes from patients with breast cancer. Identification of autoantigens in immune complexes isolated from breast cancer effusions.

Sera and effusion fluids of patients with breast cancer (BC) contain immune complexes (IC). Antigens present in these complexes were isolated as follows: a pool of effusions from patients with BC was fractionated with ammonium sulfate. The proteins precipitating at 40% saturation were further fractionated by filtration through a Sephadex G-200 column. The material recovered in the first peak (molecules larger than monomeric IgG) was brought to pH 3.0 to dissociate the IC, and the mixture was filtered through a column of Sephacryl S-300 at pH 3.0. Proteins smaller than monomeric IgG were collected, radioiodinated, and used as antigens (125Ag) to search for corresponding antibodies in sera of patients with BC (BCS) and of healthy individuals (NHS). 125Ag was reacted with the sera and the immune complexes obtained were precipitated with an antiserum to human Ig and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Both NHS and BCS contained antibodies against two antigens; one of these appeared as a strong band of 17KD, the other as a doublet of approximately 25KD. It is concluded that some of the proteins in the IC from patients with BC are auto-antigens. No BC-specific antigens were identified.

Cancer-associated proteins in effusion fluids.

Two cancer-associated proteins, carcinoembryonic antigen (CEA) and pregnancy-associated alpha2 glycoprotein (PAG), together with 13 normal serum proteins were measured in the serum and effusion fluid of patients with ascites and pleural effusions. The results indicate that CEA measurement in effusion fluid is more effective than serum measurement in distinguishing cancerous from congestive or inflammatory effusions. Comparisons with the results of cytological examination suggest that fluid CEA estimation may prove a useful clinical tool. Serum PAG levels were higher in patients with cancer, but fluid determination offers no advantage in separating the disease groups. Similarly, the estimation of individual normal serum proteins in effusion fluids is unlikely to be of diagnostic value.