Thymostimulin in malignant pleural effusions

Twelve patients were admitted to the trial: 1 primary pleural tumour (mesothelioma), 7 metastases of breast carcinoma, 1 lung epidermoid, 1 lung adenocarcinoma, 1 uterine carcinoma, and 1 ovarian carcinoma. Thymostimulin (TS) was injected intrapleurally at the mean dose of 140 mg (2 mg/kg). The mesothelioma was the only resistant case. Complete remission was obtained in 4 patients after 3 weeks and in 4 within 8 weeks of treatment. This preliminary trial has shown that intrapleural treatment of neoplastic pleural effusion with a thymic hormone preparation can induce rapid and efficient palliation without subjecting the patients to distressing or toxic procedures.     

Superoxide dismutase 2 as a marker to differentiate tuberculous pleural effusions from malignant pleural effusions

OBJECTIVES:Our previous study demonstrated that superoxide dismutase levels were higher in tuberculous pleural effusions than in malignant pleural effusions, but that this difference could not be used to discriminate between the two. The objective of the present study was to investigate the levels of superoxide dismutase 2 in pleural effusions and to evaluate the diagnostic significance of pleural effusion superoxide dismutase 2.METHODS:Superoxide dismutase 2 concentrations were determined in pleural effusions from 54 patients with tuberculous pleural effusion and 33 with malignant pleural effusion using an enzyme-linked immunosorbent assay (ELISA) kit. Pleural effusion interferon gamma and tumor necrosis factor alpha levels were also analyzed by ELISA. The Mann-Whitney U test was used to evaluate the significance of differences. Associations between superoxide dismutase 2 concentrations and sex, age and smoking habits were assessed using Spearman''s or Pearson''s correlation coefficient analysis. Receiver operator characteristic analysis was performed to evaluate the value of superoxide dismutase 2 levels in the discrimination of tuberculous pleural effusion from malignant pleural effusion.RESULTS:Superoxide dismutase 2 levels were significantly higher in patients with tuberculous pleural effusion compared with those with malignant pleural effusion (p<0.05). When superoxide dismutase 2 was used to differentiate between tuberculous pleural effusions and malignant pleural effusions, the area under the receiver operator characteristic curve was 0.909 (95% confidence interval, 0.827-0.960; p<0.01). With a cut-off value of 54.2 ng/mL, the sensitivity, specificity, positive likelihood ratio and negative likelihood ratio were 75.8% (95%CI: 57.7-88.9%), 98.1% (95%CI: 90.1-99.7%), 40.91 and 0.25, respectively. Furthermore, significant correlations between pleural effusion superoxide dismutase 2 and interferon gamma (r = 0.579, p<0.01) and between pleural effusion superoxide dismutase 2 and tumor necrosis factor alpha (r = 0.396, p<0.01) were observed.CONCLUSION:Pleural effusion superoxide dismutase 2 can serve as a biomarker for differentiating between tuberculous pleural effusions and malignant pleural effusions. Because of the high correlations of superoxide dismutase 2 with pleural effusion interferon gamma and tumor necrosis factor alpha levels, this marker may act as an inflammatory factor that plays an important role in the development of tuberculous pleural effusion.     

Immunoperoxidase staining of malignant cells in pleural effusions

The indirect immunoperoxidase method was used to detect the presence of carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), keratin and alpha-1-antichymotrypsin (alpha 1ACT) in cells of pleural fluid sediments from 30 patients with pleural malignancies (18 mesotheliomas and 12 metastatic carcinomas). CEA was negative in all mesotheliomas and positive in all carcinomas but one (an ovarian serous cystadenocarcinoma); alpha 1ACT was positive in mesotheliomas and negative in carcinomas; EMA and keratin were positive in both types of tumor. These data suggest that the use of immunostaining against CEA and alpha 1ACT seems to improve the cytologic differential diagnosis between malignant mesothelioma and metastatic carcinoma in pleural effusions.     

Proteomic analysis of exosomes isolated from human malignant pleural effusions

Exosomes are membrane vesicles from endosomal origin secreted by various cells such as hematopoietic, epithelial, and tumor cells. Exosomes secreted by tumor cells contain specific antigens potentially useful for immunotherapeutic purposes. Our aim was to determine if exosomes are present in human cancerous pleural effusions and to identify their proteomic content. Exosomes were purified by sucrose gradient ultracentrifugation, and electron microscopy was used to check both concentration and purity of exosomes. Proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and protein bands were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Western blotting. Exosomes were present in pleural fluid obtained from patients suffering from mesothelioma (n = 4), lung cancer (n = 2), breast cancer (n = 2), and ovarian cancer (n = 1). As previously reported by others, antigen-presenting molecules, cytoskeletal proteins, and signal transduction-involved proteins were present. Proteins not previously reported were identified (SNX25, BTG1, PEDF, thrombospondin 2). Different types of immunoglobulins and complement factors were abundantly present in the sucrose fractions containing exosomes. Exosome-directed specificity of these immunoglobulins was not observed. In conclusion, sucrose gradient ultracentrifugation allows isolation of exosomes from malignant pleural effusions. However, pleural fluid proteins and especially immunoglobulins are coisolated and may hamper the use of exosomes isolated from malignant effusion for immunotherapy programs.     

Vascular endothelial growth factor in malignant and tuberculous pleural effusions

The purpose of this study is to assess the usefulness of soluble vascular endothelial growth factor (VEGF) in the effusions of patients with malignant and tuberculous diseases. Using a sandwich enzyme-linked immunoadsorbent assay, VEGF concentration was measured in malignant (n=17) and tuberculous (n=11) pleural effusions. Pleural biopsy, cytology or microbiological methods were used to make final diagnoses. Adenosine deaminase (ADA) levels in tuberculous pleural effusions were significantly higher than those in malignant pleural effusions. The median level of VEGF in patients with malignant effusions (median, 2418 pg/mL; range, 97-62103 pg/mL) was significantly higher than tuberculous effusions (median, 994 pg/mL; range, 44-3552 pg/mL). There were no significant differences in pleural VEGF levels in patients with different histological types of lung cancer. The VEGF level was not correlated with ADA, lactate dehydrogenase and total protein levels of pleural fluid. In conclusion, pleural VEGF levels in patients with malignant effusions were significantly higher than tuberculous effusions, and the measurement of pleural VEGF is helpful in discriminating between malignant and tuberculous effusions. Further studies are needed to determine the clinical value of VEGF as a tumor marker and a prognostic factor.     

Adenoviral gene transfer is inhibited by soluble factors in malignant pleural effusions

Direct in vivo gene delivery is a prerequisite for many gene therapy strategies; however, efficacy has been limited by a lack of therapeutic gene transfer. In studying intrapleural malignancy as a model for the gene therapy of non-small cell lung cancer, we previously identified soluble chondroitin sulfate-proteoglycans/glycosaminoglycans (CS-PG/GAGs) in malignant pleural effusions (MPE) as factors that inhibit retroviral vector (RV) transduction. Similarly, we have observed inhibition to gene transfer in the fluid component of MPE using adenoviral (Ad) vectors. Analyses indicate that the factors responsible for the block are filterable, soluble, titrable, and heat stable (56 degrees C). Passage through microporous membranes fractionates the inhibitory factors into large (> 100 kD) components of the effusions. In contrast to RV transduction, hyaluronic acid or CS-PG/GAGs are not the inhibitors because the block is not reversed by pretreatment of the effusions with mammalian hyaluronidase, and exogenous addition of GAGs into the transduction media does not diminish Ad transduction. In considering the mechanism of action of the inhibitory factors, we observe that Ad entry, and specifically the binding of radiolabeled Ad to its target cell, is inhibited in the presence of MPE. Ad internalization may also be impaired; however, these studies exclude soluble fibronectin in MPE as a competitive inhibitor of Ad transduction. Lastly, sepharose A- mediated immunoglobulin depletion of MPE only partially reverses the block, and significant inhibition to Ad gene transfer persists at lower adenovirus:target cell ratios. Identifying the structural and functional basis for inhibition to Ad gene transfer may yield specific strategies to enable better in vivo translation of gene therapy approaches.     

Measurement of placental alkaline phosphatase activity in benign and malignant pleural effusions.

The usefulness of placental alkaline phosphatase (PLAP) as a diagnostic marker of malignancy was assessed in pleural fluid from 60 patients with effusions. Pleural fluid PLAP activities were measured by an enzyme linked immunoassay (ELISA) using the two monoclonal antibodies H17E2 and H317. Similar values were found in groups of patients with primary bronchial tumours (n = 12), secondary malignancies (n = 23), and "benign" conditions (n = 25). The highest values were found in a small subgroup of patients with metastatic ovarian carcinoma. However, the production of this enzyme by normal lung makes the measurement of PLAP in pleural fluid unhelpful as a diagnostic aid to distinguish "benign" from malignant effusions.     

Cytodiagnosis of rheumatoid pleural effusions

The stained smears of the deposits from one pericardial and 19 pleural effusions complicating rheumatoid arthritis were examined. On the basis of clinical and biochemical evidence it was considered that in six cases the effusions were due to the rheumatoid disease while in a further nine cases the association was considered likely. In the remaining five cases the association was considered to be due to chance as other causes for the effusions were diagnosed.On cytological examination, seven cases showed a characteristic picture of degenerating polymorphs with amorphous extracellular material and epithelioid cells many of which were multinucleate. Five others contained similar amorphous material without epithelioid cells; of these two had many plasma cells and a third numerous macrophages probably containing ;droplets' of rheumatoid factor complex.Thus in 12 of 15 cases a definite diagnosis of rheumatoid effusion could be made. In the remaining five cases cytological examination confirmed that the effusions were unrelated to the rheumatoid disease.The extracellular material gave a non-specific fluorescence with labelled anti-gamma globulin antisera, and since this reaction was not seen in control pleural fluid deposits, or with preparations of fibrin, it may have a confirmatory value.It is concluded that in many cases reliable cytological evidence can be found to confirm or refute a diagnosis of rheumatoid pleural or pericardial effusion. This may be helpful in the management of the rheumatoid disease.     

Fibronectin in exudative pleural effusions.

Fibronectin is a glycoprotein found in body fluids, loose connective tissue matrix and in basement membranes. Fibronectin in pleural effusion was found to be immunologically indistinguishable from the plasma form, as shown by double-diffusion analysis. Fibronectin isolated from pleural fluid by affinity chromatography on gelatin-Sepharose had a polypeptide pattern similar to that of plasma fibronectin in SDS-polyacrylamide gel electrophoresis. In 28 patients with infectious or non-specific pleural effusion fibronectin concentrations in pleural fluid were 335 +/- 104 micrograms/ml (mean +/- SD), in 15 patients with malignant disease the concentrations were 369 +/- 173 micrograms/ml and in 26 patients with tuberculosis 441 +/- 103 micrograms/ml. The highest concentrations, 605 +/- 252 micrograms/ml, of fibronectin in pleural fluid were detected in 14 patients with connective tissue diseases. The results suggest that increased fibronectin concentrations reflect the presence of a pleurisy due to connective tissue disease or tuberculosis rather than other infectious or malignant disease.     

Beta 2 microglobulin in pleural effusions

Beta 2 microglobulin (beta 2m) concentrations in serum and pleural fluid from 64 patients with pleural effusion were studied. The level of beta 2m in pleural fluid was generally twice that in serum. The ratio of pleural fluid beta 2m to serum beta 2m in patients groups defined according to the final diagnosis showed an interestingly high value in tuberculous pleuritis and in patients with rheumatoid arthritis with pleural effusion. There was a positive correlation between the beta 2m and lysozyme contents in pleural fluid, suggesting local and simultaneous activation of different cell lines when the pleura is involved. We suggest that pleural fluid and concomitant serum beta 2m measurements should be taken into consideration when pleural effusion of tuberculous origin is suspected. Furthermore, beta 2m determination might help to differentiate between rheumatoid pleural fluid and pleural involvement due to the other systemic diseases.     

Neurotrophin system activation in pleural effusions

Neurotrophins (NTs) expression was assessed in malignant and non-malignant pleural effusions (inflammatory exudates and transudates). Enzyme-linked immunosorbent assay, in malignant exudates from small and non-small cell lung cancer (SCLC and NSCLC), detected nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their levels are higher as compared with inflammatory and transudative effusions. By immunoblots, in cultured cancer cells coming from malignant pleural effusions, NTs and low- and high-affinity NT receptors were detected in a percentage of SCLC and NSCLC. Proliferation assay demonstrated that BDNF significantly increased cancer cell proliferation in vitro, on the contrary, NT-3 reduced cancer cell growth rate and NGF did not modify cell growth. Moreover, NGF protects cells from death during starvation. These effects are reverted by the addition of NT receptor antagonists. Cultured cancer cells injected into the lung of immunodeficient mice generate lung tumors expressing NTs and NT receptors. These findings suggest that NTs may be able to modulate cancer cell behavior and their growth.     

Cytology of pleural effusions in rheumatoid arthritis

Rheumatoid pleural effusions are relatively uncommon. The cytologic examination of such effusions can be diagnostic of the underlying disease; this is of great clinical significance when the rheumatoid condition has not been diagnosed prior to the pleural involvement. The diagnostic cytologic abnormalities include large elongated and multinucleated giant cells and macrophages in a background of granular and necrotic debris. The cytologic characteristics parallel the histologic features of pleural rheumatoid nodules.     

Macrophages from malignant effusions

Cells from 11 malignant effusions have been examined for macrophage functional attributes and for evidence of macrophage malignancy. Three patients with Hodgkin's disease and one patient with histiocytic medullary reticulosis had atypical macrophages in their serous fluids. This adds further evidence to support the theory that the macrophage is the malignant cell in these tumour types. Two other patients with LRMPS tumours had atypical non-adherent cells in their effusions; these were probably lymphoid in origin. Samples from patients with benign disease and with epithelial tumours did not have atypical LRMPS cells. One patient with Hodgkin's disease lacked the atypical macrophage. In this patient the ascites was not due primarily to tumour involvement. There was no evidence to suggest a Hodgkin's disease-related macrophage deficit as has been reported in tissue samples. It is concluded that cells from malignant effusions are a particularly valuable source of information about the histogenesis of tumours which may be of macrophage origin.     

Diagnostic value of procalcitonin in pleural effusions

This study was to determine the diagnostic value of procalcitonin (PCT) in the differentiation of infectious and non-infectious causes of pleural effusion. From January 2005 to April 2005, we measured the PCT levels of pleural effusion from 76 patients using an immunoluminometric assay. The types of pleural infusions studied were para-pneumonic effusion (n = 26), empyema (n = 7), tuberculous pleurisy (n = 8), malignant pleural effusion (n = 25) and transudative pleural effusion (n = 8). The PCT levels were low in transudative pleural effusions (0.188 ± 0.077 ng/mL) and tuberculous pleurisy (0.130 ± 0.069 ng/mL), but high in empyema (5.147 ± 3.056 ng/mL), para-pneumonic effusion (1.091 ± 0.355 ng/mL), and malignant pleural effusion (0.241 ± 0.071 ng/mL). The receiver-operating characteristic curve analysis for an optimal discrimination between empyema and para-pneumonic effusion from non-para-pneumonic effusion could be performed at a cut-off point of 0.18 ng/mL with area under the curve of 0.776 (sensitivity: 69.7%, specificity: 72.1%). The correlation was found between pleural effusion PCT and serum PCT levels in 16 patients (r ² = 0.967, p < 0.001). In conclusion, a high pleural effusion PCT level suggests the presence of empyema and para-pneumonic effusion.     

IL-6 in malignant pleural effusions and its augmentation by intrapleural instillation of IL-2.

The levels and activities of endogenous IL-6 in malignant pleural effusions due to lung cancer before and during daily intrapleural instillations of recombinant IL-2 were examined by enzyme immunoassay and bioassay using an IL-6-dependent murine hybridoma cell line MH60.BSF2. Before therapy, malignant pleural effusions contained various levels of IL-6. Daily intrapleural instillation of recombinant IL-2 for treatment of malignant pleurisy resulted in significant augmentation of the levels and activities of IL-6 in the pleural effusions. On fractionation of the pleural effusion by chromatography, one major peak of material with a mol. wt of 24 kD showed IL-6 activity. In contrast, no significant level of tumour necrosis factor-alpha or IL-1 beta was detectable in pleural effusions before or during local IL-2 therapy. These data suggest that IL-2 is an important regulatory factor of secondary IL-6 production.