Calretinin. A selective marker of normal and neoplastic mesothelial cells in serous effusions

OBJECTIVE: To document that a polyclonal antiserum to calretinin, a 29-kd calcium-binding protein, consistently decorates normal and tumor mesothelial cells in cytologic preparations. STUDY DESIGN: Thirty-three archival cytologic specimens from eight patients with histologically confirmed malignant mesothelioma and 13 from patients with metastatic serous effusions were destained and then immunostained with anticalretinin antiserum. For investigation of cell suspensions, four pleural fluids were incubated with anticalretinin antiserum. After cytocentrifugation the specimens were stained in accordance with the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. For electron microscopic examination the cell suspensions were then incubated with gold-labeled antirabbit antibody. RESULTS: The diagnostic sensitivity of this new immunocytochemical approach reached 100% for the eight malignant mesotheliomas investigated. Only 3 of the 13 adenocarcinomas metastatic to the serous membranes included in this study were weakly reactive, accounting for 81% specificity. Binding of anticalretinin antiserum to living mesothelial cells was consistently documented in all four cases investigated. CONCLUSION: Calretinin is a very useful marker for positive identification of normal and tumor mesothelial cells in serous effusions.     

Immunohistochemical panel for distinguishing between carcinoma and reactive mesothelial cells in serious effusions

OBJECTIVE: This study was designed to assess whether a new panel of antibodies is a useful adjunct in the differential diagnosis of carcinoma and reactive mesothelial cells. STUDY DESIGN: Complete, one-hour immunohistochemistry using antibodies against cytokeratin (CK), carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and fibronectin was applied to cell blocks from 76 pleural and peritoneal fluid specimens. Fifty patients with histologically diagnosed primary carcinomas and 26 without evidence of malignancy were included. The results were correlated with routine cytologic results. RESULTS: The final cytologic diagnoses were 28 malignant effusions and 48 benign effusions. CEA and EMA were present in 25 (89%) and 24 (86%) of 28 carcinoma cases, respectively. These determinants were absent from reactive mesothelial cells. Fibronectin strongly labeled reactive mesothelial cells, with no staining of carcinoma cells. Carcinoma cells expressed at least two antibodies to CK, CEA and EMA and were negative to fibronectin. Reactive mesothelial cells expressed both CK and fibronectin. In 6 of 28 carcinoma cases (21%) the immunohistochemical panel identified carcinoma cells that were not recognized initially on routine cytologic examination. CONCLUSION: A panel of CEA, EMA and fibronectin monoclonal antibodies appears to be suitable for distinguishing between carcinoma cells and reactive mesothelial cells in serous effusions.     

Multiparameter flow cytometric DNA analysis of effusions: a prospective study of 36 cases compared with routine cytology and immunohistochemistry

Single-parameter flow cytometry (SFCM) is limited in its ability to detect aneuploid and diploid malignant cells or accurately estimate S-phase fractions (SPF) in effusions because of the high degree of contamination by benign mesothelial cells and inflammatory cells. We examined 36 pleural and peritoneal fluids by conventional cytology and multiparameter FCM (MFCM) to analyze the DNA content of cells expressing epithelial markers cytokeratin, epithelial membrane antigen, carcinoembryonic antigen, BRST-1, or BRST-3 (B72.3) and compared the results to those found with SCFM. The cases were also studied by immunohistochemistry using the same antibody panel. By routine cytology, 14 of the 36 cases were classified as carcinomas, 11 as reactive, 1 as mesothelioma, and 10 as suspicious. MFCM allowed reclassification of 5 of the 10 suspicious cases as carcinomas and the remaining 5 as reactive cases based on ploidy and marker expression. Whereas SFCM detected only 13 nondiploid carcinomas, MFCM detected 4 diploid and 15 nondiploid carcinomas. All reactive cases were diploid by SFCM or MFCM. The mesothelioma case showed were distinct peaks by MFCM, a diploid peak with SPF of 13.4% and a tetraploid peak with SPF of 36.1%. The SPF of the nondiploid carcinomas ranged from 5.9 to 50.4% and diploid carcinomas, from 3.5 to 14.5% when gated on epithelial cells. The reactive cases had SPF ranging from 0.4 to 4.4%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Athletes as health testing examinees

AIMS AND BACKGROUND: The usefulness of monoclonal antibodies that recognize markers of neoplastic lesions in complementing conventional cytology was evaluated by the avidin-biotin-peroxidase complex, indirect immunoperoxidase technique. METHODS: In order to enhance the sensitivity of the traditional method, a pool of seven combined monoclonal antibodies (Pool C7), which reacts specifically with cells of epithelial origin and is able to distinguish between mesothelial and malignant cells, was tested on cytologic smears of 262 serous effusions. The effusions were benign or neoplastic, mainly from breast, ovary and lung cancers. RESULTS: Immunocytochemical method showed an 100% specificity and 100% of predictivity whereas the sensitivity was 98%, 96% and 95% for breast ovarian and lung carcinomas, respectively. CONCLUSIONS: The results demonstrated that the pool when used together with conventional methods, is useful in analysis of serous effusions in diagnostic investigations.     

Fluorotyping of HLA-A by sequence-specific priming and fluorogenic probing

AIMS: To determine the value of immunocytochemistry in differentiation of malignant pleural mesothelioma from carcinoma in a pleural biopsy using commercially available monoclonal antibodies. METHODS AND RESULTS: A panel of monoclonal antibodies against keratins, epithelial membrane antigen (EMA), epithelial antigen Ber-EP4, carcinoembryonic antigen (CEA), tumour-associated glycoprotein (B72.3), Leu-M1, CD30 (Ber-H2), vimentin and desmin, was applied to 40 cases of malignant pleural mesothelioma and 23 cases of carcinoma metastatic to the pleura (16 pulmonary and seven extrapulmonary). Positivities for Ber-EP4, CEA, B72.3 and Leu-M1 were found to have the highest nosologic sensitivities (87.0%, 65.2%, 52.5% and 43.5%, respectively) and specificities (97.5%, 97.5%, 100% and 95%, respectively) for carcinoma. Positive staining for vimentin had the highest sensitivity (87.5%) with 95.7% specificity for mesothelioma. Positive staining for desmin was found in 45% of mesotheliomas and 0% of carcinomas. Diagnostic sensitivity and diagnostic specificity (P-values) were calculated for these markers. In respect to the diagnostic power defined by the clinically relevant predictive values of positive and negative tests, we found that a two-marker panel of antibodies including vimentin and Ber-EP4 is most useful for the histopathological distinction between carcinoma (pulmonary or extrapulmonary) and malignant pleural mesothelioma. CONCLUSIONS: A combination of Ber-EP4 and vimentin provides the most sensitive and specific pair of markers for distinguishing between malignant pleural mesothelioma and carcinoma metastatic to the pleura. The prevalence of the tested tumours should be taken into account when evaluating the clinical value of ancillary techniques in pathology.     

Allergic eye disease mechanisms

The histologic distinction between epithelial peritoneal mesothelioma and papillary serous carcinoma diffusely involving the peritoneum may be difficult. Although some investigators have indicated that immunohistochemistry can facilitate this differential diagnosis. only a few studies using a limited number of markers have been published. In this study, the immunoreactivity of keratin 5/6, vimentin, epithelial membrane antigen, thrombomodulin, calretinin, MOC-31, Ber-EP4, carcinoembryonic antigen, TAG-72 (B72.3), CD15 (Leu-M1), placental alkaline phosphatase, CA19-9, CA-125, HBME-1, 44-3A6, and S-100 protein was investigated in 35 epithelial peritoneal mesotheliomas, and 45 papillary serous carcinomas [30 ovarian (10 primary and 20 metastatic to the peritoneum) and 15 papillary serous carcinomas of the peritoneum]. After analyzing the results, it is concluded that calretinin, thrombomodulin, and keratin 5/6 are the best positive markers for differentiating epithelial malignant mesotheliomas from papillary serous carcinomas diffusely involving the peritoneum. The best diagnostic discriminators among the antibodies considered to be negative markers for mesothelioma are MOC-31, B72.3, Ber-EP4, CA19-9, and Leu-M1. Immunostaining for carcinoembryonic antigen, placental alkaline phosphatase, epithelial membrane antigen, vimentin, HBME-1, 44-3A6, CA-125, or S-100 have little or no diagnostic utility in establishing the differential diagnosis between these conditions. The results of this study also confirm previous observations indicating that both papillary serous carcinomas of the peritoneum and serous carcinomas of the ovary have a similar phenotype and, therefore, immunohistochemical studies are not useful in separating these entities.     

CEA and CA 15-3 in pleural effusion of advanced breast cancer patients: clinical relevance and diagnostic value

Serum and pleural effusion fluid were tested for CEA concentration in 83 advanced breast cancer patients, in 43 of whom CA 15-3 was also determined. All pleural effusions were clinically malignant. The sensitivity of the CEA test for the presence of pleural metastases was closer to that of the CA 15-3 test in effusion (0.59 and 0.79, respectively) than the sensitivity of CEA compared to CA 15-3 in serum (0.43 vs. 0.79). The use of two markers combined with cytology increased the diagnostic rate from 48% (cytologically positive) to 88% (cytologically positive and/or with one or both markers increased in effusion). A high diagnostic rate in cytologically negative effusions (65%), and in effusions presented as the sole metastatic involvement (100%), points to the clinical value of these two markers. Our results show that markers produced by pleural metastases may be secreted either into the effusion fluid or into serum, or both. This finding, as well as some other observations, are discussed in the present paper.     

Varicella in pregnancy

We report six cases of hyperplastic mesothelial cells located in the sinuses of lymph nodes. All patients but one had a concurrent serosal fluid collection (two pericardial, two pleural, one abdominal) at the time of the lymph node biopsy. All effusions cleared with treatment of the underlying disorder, which included lymphoproliferative processes, congestive heart failure, and inflammatory diseases (Dressler syndrome, vasculitis, and glomerulonephritis). Four cases were associated with vascular prominence of the involved nodal sinuses, a feature that may reflect the cause of the underlying effusion or support the transient persistence of benign mesothelial cells in lymph nodes. Two cases were characterized by distention of the nodal sinuses by sheets of mitotically active mesothelial cells. The differential diagnosis includes metastatic carcinoma, keratin-positive dendritic cells native to lymph nodes, and metastatic malignant mesothelioma. Because the latter shares both clinical and morphological features with cases of benign mesothelial cells in lymph nodes, we believe that this distinction may not always be possible in a given biopsy specimen and therefore that careful clinical follow-up is required in such cases.     

Cytologically proved malignant pleural effusions: distribution of transudates and exudates

PURPOSE: This study attempts to determine the distribution of transudates vs exudates in pathologically proved malignant pleural effusions and the necessity for cytologic studies in patients with a transudative effusion. MATERIALS AND METHODS: A retrospective review of all cytologically positive malignant pleural effusions was performed at Duke University Medical Center over an 18-month period. All effusions were characterized as a transudate or an exudate based on standard criteria, including lactate dehydrogenase and protein values. RESULTS: Ninety-eight patients with a mean age of 62 years were identified as having a cytologically positive malignant pleural effusion and blood chemistry values available to distinguish an exudate from transudate. Ninety-seven patients (99%, 95% confidence interval; 0.94 to 0.99) had criteria for an exudative effusion. One patient (1%) with diffuse metastatic lung cancer had a borderline transudate and was in congestive heart failure at the time of thoracentesis. CONCLUSIONS: Cytologically positive pleural effusions for malignancy are almost always exudates. Cytologic evaluation for malignant cells of a transudative pleural effusion is not recommended.     

Benign and malignant cells in effusions: diagnostic value of image DNA cytometry in comparison to cytological analysis

Identifying tumor cells in body cavity fluids reliably is a well-known diagnostic problem. Since cytometric quantitation of nuclear DNA content appears to be a promising new tool in the diagnosis and prognostic evaluation of many solid human tumors, we examined its validity in detecting malignant cells in cytologically positive effusions. For this purpose, image DNA cytometric measurements, including the evaluation of DNA-ploidy and the calculation of the DNA index (DI), were performed in 80 body cavity fluids. The results were correlated with cytology, clinical course and final histological diagnoses. We used aneuploidy, as shown by interactive image DNA cytometry, as a marker for the malignancy of cells that occur in body cavity fluids with a 100% specificity and 94.8% sensitivity. Cytological investigation showed a 92.3% specificity and 95.4% sensitivity. Combining both methods raised the specificity to 100% and the sensitivity to 98.5% and had a positive predictive value of 100% and a negative predictive value of 93.8%. The DNA-index (DI) was significantly higher in malignant effusions than in benign effusions: 1.5 +/- 0.74 (mean +/- SD) versus 1.11 +/- 0.26 (p < 0.05). Along with the difficult cytological evaluation of malignant cells in body cavity fluids, image DNA cytometry can be a helpful additional method for evaluating these cells. Combining the two techniques results in a highly specific and sensitive prediction of malignant cells. We, therefore, suggest using these methods for the reliable identification of tumor cells in effusions.     

The significance of colour velocity and spectral Doppler ultrasound in the differentiation of liver tumours

BACKGROUND: The distinction between malignant mesothelioma (MM) and adenocarcinoma (ACA) in cytologic specimens frequently is difficult, often requiring immunocytochemistry to support the diagnosis. Recent reports have proposed the utilization of antibodies to mesothelial cell clone HBME-1 and thrombomodulin (TM), because they are immunoreactive in MM and less commonly reactive in ACA. Immunoreactivity for the monoclonal antibody CA 19-9 has been observed in many ACAs and reportedly is absent in MM. METHODS: In this study, immunostaining was performed on formalin fixed, paraffin embedded cell blocks from effusions or fine-needle aspirations using the avidin-biotin-peroxidase method. Thirty-eight MMs and 49 ACAs were tested using antibodies to CA 19-9, HBME-1, and TM. RESULTS: Anti-CA 19-9 stained only 1 of the 37 cases of MM tested (3%), but stained 24 of the 49 cases of ACA (49%). Anti-HBME-1 stained 34 of 38 cases of MM (89%), and 28 of 43 cases of ACA tested (65%). Anti-TM stained 24 of 36 cases of MM (67%), and 21 of 40 cases of ACA tested (53%). CONCLUSIONS: CA 19-9 has utility as part of an immunocytochemical panel for distinguishing ACA from MM, because a positive staining reaction would make the diagnosis of MM unlikely. Although HBME-1 and TM can identify MM positively, each frequently is detected in ACA, thus limiting the utility of these antibodies in cytologic specimens.     

Effects of flutamide on pituitary and adrenal responsiveness to corticotrophin releasing factor (CRF)

Although most studies have indicated that Ber-EP4 immunostaining can assist in differentiating epithelial pleural mesotheliomas from adenocarcinomas that metastasize to the pleura, the percentage of positive cases has varied greatly among different studies. Authors of a recent publication concluded that Ber-EP4 has no diagnostic utility in separating these conditions. To determine whether Ber-EP4 has any value in distinguishing mesothelioma from adenocarcinoma, 70 formalin-fixed epithelial pleural mesotheliomas, 20 pulmonary adenocarcinomas, 59 nonpulmonary adenocarcinomas, 4 squamous cell carcinomas of the lung, 6 transitional cell carcinomas, and 31 adenocarcinomas of unknown origin that metastasized to the pleura were stained with this antibody. Reactivity was observed in 18 (26%) of 70 mesotheliomas and in all 20 (100%) of the pulmonary adenocarcinomas, in 55 (93%) of the 59 nonpulmonary adenocarcinomas, in 4 (100%) of 4 squamous cell carcinomas of the lung, in 4 (67%) of 6 transitional cell carcinomas, and in 26 (84%) of 31 adenocarcinomas of unknown origin that metastasized to the pleura. The staining in the mesotheliomas was focal and restricted to a limited number of cells, in contrast with staining in the pulmonary adenocarcinomas in which it was invariably diffuse. The extent of the staining in the nonpulmonary adenocarcinomas and the metastatic adenocarcinomas of unknown origin was less consistent--negative or focal in some cases and diffuse in others. Therefore, while Ber-EP4 seems to be helpful in separating epithelial pleural mesotheliomas from lung adenocarcinomas, its value in distinguishing mesotheliomas from other tumors metastatic to the pleura is more limited and depends largely on the site of origin of the metastatic tumor.     

Anatomic bases of a vascularized allogenic knee joint transplantation: arterial blood supply of the human knee joint

The reliable identification of tumor cells in populations of atypical cells occurring in body cavity effusions is a well-known diagnostic problem. In order to improve tumor cell detection and to predict disease progression, we developed a cell scoring strategy based on a combination of DNA cytophotometry and immunocytochemistry. For this purpose, morphologically atypical cells obtained from 33 effusion samples were submitted to DNA content analysis and tested for Ber-EP4 immunoreactivity. It turned out that elevated DNA content alone has a low specificity (true negative ratio) and sensitivity (true positive ratio) in predicting disease outcome, whereas Ber-EP4 immunoreactivity alone has a high specificity (100%) but a low sensitivity (56%). In contrast, the use of a scoring system combining the two techniques and relating scores to the previous disease state and the cytomorphology of the atypical cells results in highly specific and sensitive prediction of the disease outcome. We therefore suggest that this approach is a valuable tool for reliably identifying tumor cells in effusions containing populations of cytologically suspect cells.     

Without prior stimulation, tumor-associated lymphocytes from malignant effusions lyse autologous tumor cells in the presence of bispecific antibody HEA125xOKT3

Women suffering from advanced stage ovarian or mammary carcinoma frequently develop malignant ascites or pleural effusions consisting of tumor cells and tumor-associated lymphocytes (TALs). Locoregional immunotherapy with bispecific antibodies (bsAbs), which retarget T cells to tumor cells and induce their lysis, has been applied as an adjuvant treatment in the late stage of the disease. Until now, most of these therapies use peripheral blood mononuclear cells (PBMCs) as effector cells that have been stimulated and expanded ex vivo and loaded with bsAb before reinjection. Here we investigated whether TALs derived from malignant ascites or pleural effusions can be used as bsAb-guided effector cells without prior in vitro stimulation. For this we established a bsAb, HEA125xOKT3, which recognizes the epithelial antigen Egp34 on carcinoma cells and the CD3 molecule on T cells. BsAb HEA125xOKT3 induced lysis of various Egp34-expressing carcinoma lines by stimulated PBMCs. Optimal cytotoxicity was achieved at a bsAb concentration of 1 microg/ml. In three ovarian and two mammary carcinoma patients, we demonstrated efficient lytic activity of lymphocytes, isolated from malignant ascites or pleural effusion. Without prior stimulation, they lysed autologous tumor cells in the presence of bsAb HEA125xOKT3, indicating that they are already activated. Along this line, a subset of CD4+ and CD8+ unstimulated TALs expressed the early activation marker CD69. They are, however, negative for CD95, and only a small subpopulation of CD4+ TALs expresses CD25. OKT3/interleukin 2 stimulation of TALs increased the expression of activation markers on the CD4+ and CD8+ T-cell compartment. The activation markers CD69, CD25, CD95, and DR molecules are up-regulated on both T-cell types. However, lysis of autologous tumor cells by stimulated TALs is not significantly enhanced compared with unstimulated TALs. Our results may offer a novel and promising concept of adjuvant immunotherapy for ovarian and mammary carcinoma patients. Preactivation and expansion of PBMCs can be circumvented by exploring the cytotoxic capacity of unstimulated TALs in the presence of bsAbs in a locoregional therapeutic approach.     

Experience with closed needle biopsy and pleural fluid cytology in the diagnosis of malignant pleural effusions in Yaounde, Cameroon

To determine and compare the efficacy of pleural fluid cytology and closed needle biopsy of the pleura in establishing the diagnosis of malignant pleural effusions in Yaounde, we reviewed the medical records of all consecutive patients with a pleural effusion admitted in unit B of the Chest Clinic of the Jamot Hospital between January 1990 and December 1994. Fifty four cases of malignant pleural effusion were diagnosed over this period. Closed needle biopsy of the pleura alone permitted a diagnosis of malignancy involving the pleura in 32 instances while cytological studies of pleural fluid provided a diagnosis in thirty six cases. A combination of both techniques was diagnostic in 48 (88.9%) patients. We recommend that both pleural fluid cytology and closed needle biopsy of the pleura be used concomitantly in the evaluation of pleural effusion for which malignancy is suspected.     

Doppler flowmetry in the planning of perforator flaps

Parapneumonic pleural effusions are associated with the presence of a variety of inflammatory cells whose influx into the pleural space is attributed to the presence of inflammatory cytokines. Macrophage inflammatory protein-1alpha (MIP-1alpha), an important mononuclear chemokine, plays a critical role in pulmonary parenchymal inflammatory disease, but its role in the recruitment and activation of mononuclear phagocytes in the pleural space is unknown. In this study we demonstrate that complicated parapneumonic pleural effusions (empyema) and uncomplicated parapneumonic pleural effusions contain significantly (P < .001) higher levels of MIP-1alpha with higher numbers of mononuclear cells when compared with effusions resulting from malignancy and congestive heart failure. The MIP- 1alpha was biologically active and contributed 43% and 37% of the mononuclear chemotactic activity of complicated and uncomplicated parapneumonic pleural fluids, respectively. In vitro, human mesothelial cells, when stimulated with interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or bacterial lipopolysaccharide (LPS), produced MIP-1alpha. Northern blot analysis confirmed that both endogenous (IL-1beta or TNF-alpha) and exogenous (LPS) factors induce MIP-1alpha expression in mesothelial cells. Supernatants from activated mesothelial cells demonstrated chemotactic activity for mononuclear cells. This activity was blocked by MIP-1alpha antibody, indicating that the MIP-1alpha released was biologically active. We conclude that in parapneumonic pleural effusions, MIP-1alpha plays a major but not exclusive role in the recruitment of mononuclear leukocytes from the vascular compartment to the pleural space, and pleural mesothelial cells by production of MIP-1alpha actively participate in this process.     

Rheumatic fever in the Kimberley region of Western Australia

BACKGROUND: Effusions in patients with renal cell carcinoma are rare; the clinicopathologic features of these patients have not been described fully. METHODS: All effusions from patients with renal cell carcinoma obtained between 1986 and 1997 at the study institution were reviewed. RESULTS: Twelve effusions from 9 patients were benign, and 8 effusions from 7 patients were malignant. Patients with sarcomatoid tumors presented early with benign effusions, and patients with papillary tumors presented later with malignant effusions. Patients with clear cell tumors were intermediate. The majority of patients who developed malignant effusions had tumors that were classified as T3 or higher (according to the American Joint Committee on Cancer) at the time of resection. Tumor cells had abundant clear to vacuolated cytoplasm, large nuclei, and prominent nucleoli. Cells from clear cell and papillary tumors could not be distinguished in effusion specimens unless papillae were present. At last follow-up 13 of 15 patients were dead of disease within 2 years of the onset of effusion (median 24 weeks; range, 1-93 weeks), including 7 of 9 patients with benign effusions. CONCLUSIONS: Malignant effusions due to renal cell carcinoma most commonly occur in patients with papillary and clear cell tumors. Malignant effusions from these two tumor types are difficult to distinguish unless papillae are present. Effusions associated with renal cell carcinoma confer a poor prognosis.     

Diagnostic value of p53 protein and flow cytometric DNA analysis in the study of serous effusions

OBJECTIVE: To investigate the diagnostic value of p53 protein and DNA analysis in the study of serous effusions. STUDY DESIGN: A total of 76 samples of serous effusions were studied by immunohistochemistry for p53 protein and flow cytometric (FCM) DNA analysis. The results were correlated with final cytologic diagnoses, which were confirmed by immunohistochemistry using antibodies against cytokeratin, carcinoembryonic antigen, epithelial membrane antigen and fibronectin. RESULTS: Final cytologic diagnoses included 28 malignant effusions and 48 benign effusions. No expression of p53 protein was seen in benign effusions. In contrast, p53 protein expression was seen in 19/28 (sensitivity 68%) malignant effusions. FCM detected aneuploid cells in 12/28 (43% sensitivity) of malignant and 0/46 of benign effusions. Immunohistochemical determination of p53 protein combined with FCM DNA analysis increased sensitivity to 79%. CONCLUSION: Immunohistochemical determination of p53 protein and FCM DNA analysis can aid in making an accurate and specific diagnosis of serous effusions, but the principal limitation of these tests is their relatively low sensitivity.     

Retroviral gene transfer is inhibited by chondroitin sulfate proteoglycans/glycosaminoglycans in malignant pleural effusions

Gene therapy may be an important adjuvant for treating cancer in the pleural space. The initial results of retroviral gene transfer to cancer cells in malignant pleural effusions revealed that transduction was markedly inhibited, and studies to characterize the inhibitory factor(s) were performed. The inhibition was contained within the soluble, rather than cellular, components of the effusions and was demonstrated with amphotropic, gibbon ape leukemia virus, and vesicular stomatitis virus-glycoprotein pseudotyped retroviral vectors. After excluding complement proteins, a series of studies identified chondroitin sulfates (CSs) as the inhibitory substances. First, treatment of the effusions with mammalian hyaluronidase or chondroitinases, but not Streptomyces hyaluronidase, abolished the inhibitory activity. Second, addition of exogenous CS glycosaminoglycans mimicked the inhibition observed with pleural effusions. Third, immunoassays and biochemical analyses of malignant pleural effusion specimens revealed CS in relevant concentrations within pleural fluid. Fourth, proteoglycans/glycosaminoglycans isolated from the effusions inhibited retroviral gene transfer. Analyses of the mechanism of inhibition indicate that the chondroitin sulfates interact with vector in solution rather than at the target cell surface. These results suggest that drainage of the malignant pleural effusion, and perhaps enzymatic pretreatment of the pleural cavity, will be necessary for efficient retroviral vector mediated gene delivery to pleural metastases.     

Neoplastic human tissue mast cells express the adhesion molecule CD44/HCAM

Expression of the homing-associated cell adhesion molecule/HCAM (CD44) in normal/reactive and neoplastic human tissue mast cells (TMC) was determined immunohistochemically using the antibody DAKO-DF1485, which detects all isoforms of CD44. Studies were performed on 30 routinely processed specimens. Twenty of these, from bone marrow, skin, spleen, liver, lymph node and jejunal mucosa, contained infiltrates of TMC. These represented various types of generalized mastocytosis/systemic mast cell disease, including benign systemic mastocytosis. Ten specimens consisted of tissue with a marked reactive increase in TMC; most of these were lymph nodes with chronic nonspecific lymphadenitis and benign or malignant solid tumours. In all 30 specimens TMC exhibited an annular pattern of immunostaining, which was usually very strong. Both normal/reactive and neoplastic TMC exhibited consistent immunoreactivity with the antibody DAKO-DF1485, and this antibody may be of diagnostic value in the detection of atypical TMC associated with malignant mastocytosis. TMC and their neoplastic derivatives belong to a large family of mesenchymal and epithelial cells containing the principal surface receptor for hyaluronan.