Yoshitsugu Nomoto

Detection of adenovirus E1A DNA in pulmonary fibrosis using nested polymerase chain reaction

The history of patients with idiopathic pulmonary fibrosis (IPF) shows that the disease may be preceded by a viral-like illness. Although viruses have not been demonstrated, it is possible that viruses were not detected in culture because they do not replicate during latency. We investigated the presence of adenovirus in IPF and interstitial pneumonia associated with collagen vascular disease (CVD-IP), using the nested polymerase chain reaction (PCR) and in situ hybridization (ISH) for the E1A region of the adenovirus genome. Studies were performed on lung tissues obtained by transbronchial lung biopsy from 19 patients with IPF, 10 patients with CVD-IP and, for comparison, from 20 patients with sarcoidosis. The E1A DNA was present in 3 out of 19 (16%) cases of IPF, in 5 of 10 (50%) cases of CVD-IP, and in 2 of 20 (10%) cases of sarcoidosis. The incidence of E1A DNA in CVD-IP was significantly higher than that in sarcoidosis (p<0.05). In patients with IPF and CVD-IP, E1A DNA was more prevalent in patients treated with corticosteroids (6 out of 9 cases; 67%) than in those without it (2 out of 20 cases; 10%) (p<0.01). ISH studies showed that 1 out of 8 cases of IPF and CVD-IP, in which E1A DNA was detected by PCR, was positive for E1A DNA. We conclude that adenovirus E1A is unlikely to be aetiologically involved in the pathogenesis of idiopathic pulmonary fibrosis or interstitial pneumonia associated with collagen vascular disease. However, a latent adenovirus infection may be reactivated or may newly infect the host following corticosteroid administration.

Apoptosis in the course of granulomatous inflammation in pulmonary sarcoidosis.

Sarcoidosis is a chronic inflammatory disease of unknown aetiology characterized by the formation of non-necrotizing granulomas. The course of disease is usually self-limiting with the spontaneous resolution of granuloma. In the immune system, Fas antigen (Fas) and Fas ligand (FasL) are involved in the down regulation of immune reactions by inducing apoptosis. Therefore, it was hypothesized that the Fas/FasL pathway and apoptosis may be associated with the course of granulomatous inflammation in sarcoidosis. Terminal deoxynucleotidyl transferase-mediated biotin nick end-labelling (TUNEL) was performed to assess deoxyribonucleic acid strand breakages as a characteristic of apoptosis. Immunohistochemistry was also performed to detect Fas and FasL protein, and reverse transcriptase polymerase chain reaction (RT-PCR) and RT in situ PCR to detect FasL messenger ribonucleic acid (mRNA). Positive signals for TUNEL were detected in epithelioid histiocytes and lymphocytes within granulomas and in bronchoalveolar lavage (BAL) lymphocytes from patients with sarcoidosis. Positive signals for Fas were also detected in these cells. FasL mRNA was expressed in BAL lymphocytes from 15 of 20 patients with sarcoidosis, but from only one of 10 patients with normal lung parenchyma. FasL protein was expressed in lymphocytes surrounding and within the granuloma. There was a significant correlation between the result of TUNEL and clinical course in patients with sarcoidosis. Apoptosis in epithelioid histiocytes and inflammatory cells seems to participate in the course of granulomatous inflammation. Further studies are needed to determine the role of Fas, FasL and other regulatory factors in apoptosis in the granulomatous inflammation in pulmonary sarcoidosis.

Apoptosis and Fas/Fas ligand mRNA expression in acute immune complex alveolitis in mice

Deoxyribonucleic acid (DNA) strand breaks as a characteristic of apoptosis, and Fas antigen (Fas)/Fas ligand (FasL) expression may participate in acute immune complex alveolitis in mice. Male Institute for Cancer Research (ICR) mice were injected intravenously with immunoglobulin G (IgG) antibodies against ovalbumin and inhaled an aerosolized oval albumin (OA) solution. They were killed at 4, 6, 12, 24, 48 h and 7 days after aerosolization. We assessed DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labelling (TUNEL). The expression of Fas and FasL messenger ribonucleic acid (mRNA) in lung tissues was assessed by reverse transcriptase (RT) polymerase chain reaction, and by in situ hybridization (ISH) to localize Fas mRNA, and RT in situ polymerase chain reaction to localize FasL mRNA. The fragmentation of DNA extracted from lung tissue was found 6-24 h after OA inhalation. TUNEL detected positive signals in bronchial and alveolar epithelial, endothelial and inflammatory cells in the lung tissue. These positive signals had disappeared 7 days after OA inhalation. TUNEL also detected positive signals in apoptotic neutrophils in bronchoalveolar lavage fluid at 6-12 h. Fas mRNA was expressed in the alveolar epithelial and inflammatory cells, while the expression of FasL mRNA appeared to be upregulated in infiltrating inflammatory cells at 6-24 h. These results suggest that apoptosis may be associated with the resolution of inflammation and with tissue repair and also suggest the involvement of the Fas antigen/Fas ligand pathway in acute immune complex alveolitis in mice.

Detection of group C adenovirus DNA in small-cell lung cancer with the nested polymerase chain reaction.

Group C adenovirus is latent in human tissues and can malignantly transform cells. The purpose of this study was to investigate the association between this virus and lung cancer. We investigated latent adenoviral infection using the nested polymerase chain reaction and in situ hybridization in transbronchial biopsy specimens from patients with small-cell lung cancer and non-small-cell lung cancer. The polymerase chain reaction was performed on DNA extracts with two sets of primers directed at a 261-base-pair target sequence of the E1A region of the adenoviral genome. In situ hybridization was performed on histological sections using DNA representing the entire adenovirus type 5 genome. E1A target DNA was present in 11 (31%) of 35 cases of small-cell lung cancer but in none of the 40 cases of non-small-cell lung cancer (P<0.01). Of the 11 cases found positive by PCR, 8 were positive for adenovirus DNA by in situ hybridization. Adenovirus was prominent in tumor cells in 5 of the 8 cases, and in normal epithelial cells in the 3 remaining cases. Adenovirus DNA was not detected by in situ hybridization in specimens in which E1A DNA was not detected by the polymerase chain reaction. Small-cell lung cancer has mutations or deletions in the p53 and retinoblastoma genes more frequently than are found in non-small-cell lung cancer. Therefore, we speculate that adenovirus infection might participate in the pathogenesis of SCLC by producing mutation in these genes, rather than by inhibiting the function of these proteins.

Tyrosine-Specific Protein Kinase Participates in the Pathogenesis of Acute Immune Complex Alveolitis in Rats

Rabbit IgG antibodies against ovalbumin (OA) was injected intravenously into Wistar rats. When the animals were challenged with OA aerosolized by ultrasonic nebulization, acute lung injury occurred as reflected by increased recovery of bronchoalveolar cells, especially polymorphonuclear leukocytes (PMN) in bronchoalveolar lavage fluid (BALF). Lung morphology demonstrated cellular infiltration in the alveolar septa and intra-alveolar hemorrhage. When the rats were administered ST-638, a novel tyrosine kinase inhibitor, intraperitoneally prior to nebulization, the number of PMN in BALF decreased in a dose-dependent manner and superoxide anion (O2-)-producing activity in peripheral leukocytes was significantly suppressed. Furthermore, the reagent inhibited migration of human peripheral blood neutrophils induced by the chemotactic peptide f-met-leu-phe in vitro. These studies strongly indicate that tyrosine kinase plays an important role in immune complex-triggered neutrophil-related lung disorders, and the novel tyrosine kinase inhibitor ST-638 attenuates lung injury by preventing superoxide production and neutrophil migration.