To investigate the clinical significance of determination of plasma tissue factor (TF) antigen, we have developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for plasma TF, using two different monoclonal antibodies against TF apoprotein, 6B4 (catching antibody) and 5G9 (detecting antibody), and tetramethyl benzidine/H2O2 as substrates. Titration curves of recombinant human TF in buffer containing Triton X-100 were linear within the range from 50 to 2000 pg/ml. The total assay time was 3 h. Ultracentrifugation and immunoblot analysis indicated that human plasma and urine contained 50,000 g sedimentable and non-sedimentable forms of TF, both of which were detected by our ELISA method. Plasma and urine concentrations of TF in healthy subjects and patients with various diseases were measured by the ELISA method. In healthy subjects, plasma and urinary TF levels were found to be 149 +/- 72 pg/ml (n = 30) and 175 +/- 60 pg TF/urine creatinine mg (n = 95), respectively. TF was increased in plasma of patients with disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura, vasculitis associated with collagen diseases, diabetic microangiopathy and chronic renal failure receiving haemodialysis, but not in the plasma of endotoxaemic patients without DIC. The plasma TF/serum creatinine ratio did not show a positive correlation. Measurement of TF antigen in plasma may be useful for evaluating the endothelial damage and cell destruction in TF-containing tissues.

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Summary Tissue factor (TF) is an initiator of the extrinsic cascade of blood coagulation. Although recent studies have revealed a relationship between metastatic properties and TF expression in some neoplastic cells, the significance of TF in lung cancer, especially in non-small-cell lung cancer (NSCLC), is still unclear. In this study, TF was detected in NSCLC cell lines by functional study, Western blot analysis and immunocytochemical staining. TF levels in eight NSCLC cell lines were also quantitated by enzyme-linked immunosorbent assay (ELISA), and TF expression was evaluated in 55 specimens of surgically resected NSCLCs. NSCLC cell lines derived from metastatic lesions produced high levels of TF (48.3 ± 23.5 ng 10 -6 cells, mean ± s.e.m.), whereas those derived from primary lesions produced low levels of TF (0.2 ± 0.1 ng 10 -6 cells). Immunohistochemical studies disclosed significantly stronger staining for TF in cells from NSCLC patients with metastasis than in those without metastasis. Among the 28 patients with metastasis, ten were strongly positive, 16 were moderately positive and two were negative for TF. In contrast, among the 27 patients without metastasis, only two were strongly positive, 18 were moderately positive and seven were negative for TF. Therefore, malignant cells from patients with lung cancer produce various levels of TF, and TF may play an important role in the metastatic process.Keywords: tissue factor; lung cancer; non-small-cell lung cancer; metastasis 472British Journal of Cancer (1999) 79(3/4), 472-477 © 1999 Cancer Research Campaign Article no. bjoc.1998 Received 1 September 1997 Revised 8 April 1998 Accepted 16 April 1998 Correspondence to: Y Yoshizawa, Department of Pulmonary Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan TF in non-small-cell lung cancer 473

(1,000 mg per day for 3 days), followed by oral prednisolone (40-60 mgper day). Seven patients were received CsA (1.0-2.0 mg/kg per day) after the treatment with corticosteroids. We attempted to keep the blood trough level of CsA between 100 and 150 ng/ml.Results Amongthe 7 patients treated with CsA, 4 patients have survived for 60, 120, 276 and 208 weeks, respectively; 2 did not respond to pulse-therapy with methylprednisolone and died within 8 weeks after the start of CsAtreatment. The other patient experienced reexacerbation and died 87 weeks after the discontinuation of CsA due to the development of viral encephalitis. In contrast, all 6 patients treated without CsA died within 66 weeks after the onset of acute exacerbation. Four of these patients responded to pulse-therapy with methylprednisolone, but their condition deteriorated again while the subsequent prednisolone was being tapered. Conclusion CsAseems to prevent re-exacerbation of IPF and improve the patients' chances for long-term survival.

The aim of this study was to examine the effect of ONO-5046, a neutrophil elastase (NE) inhibitor, on a model of acute lung injury induced by tumor necrosis factor alpha (TNFalpha) and phorbol myristate acetate (PMA)-activated neutrophils in isolated perfused rabbit lungs. 120 min after TNFalpha (4,000 JRU/ml) was injected into the pulmonary artery (PA), 5 x 10(7) PMA-stimulated neutrophils were infused into the PA together with 1251-rabbit serum albumin (RSA). In the ONO-5046-treated group (ONO), ONO-5046 (20 mg/kg/h) was continuously infused during the experimental period from 30 min prior to neutrophil administration. Saline, the ONO-5046 vehicle, was infused instead of ONO-5046 in the positive control group (ALD) and nonactivated neutrophils were infused without TNFalpha in the negative control group (Cont). PA pressure was monitored over a 240 min period, and bronchoalveolar lavage (BAL) was performed at the end of the experiment. Lung tissues were examined immunohistochemically for the expression of thrombomodulin (TM). The levels of TM in the perfusate were also measured by ELISA and the radioactivities in the BAL fluid, lung tissue and perfusate were determined to calculate the permeability index (PI) as an indicator of alveolar septal or vascular endothelial damage. The rabbit lungs infused with ONO-5046 showed slower and less increases in PA pressure compared with ALD group. The PI was significantly higher in ALD group (PI[BAL] = 0.028 +/- 0.014, PI[LUNG] = 0.04 +/- 0.003) than Cont (PI[BAL] = 0.002 +/- 0.001, PI[LUNG] = 0.015 +/- 0.003) and ONO group (PI[BAL] = 0.004 +/- 0.003, PI[LUNG] = 0.028 +/- 0.003 (p < 0.05). ALD group had higher TM levels in the perfusate and showed decreased expression of TM on the vascular endothelium compared to Cont and ONO group, suggesting that there was shedding of TM on endothelium and ONO-5046 attenuated a shedding of TM. In conclusion, ONO-5046 attenuated acute lung injury by inhibiting the alveolar epithelial and vascular endothelial injury triggered by activated neutrophils. NE appears to play an important role in the neutrophil-induced increase of pulmonary epithelial and microvascular permeability observed in acute lung injury.