[Respiratory tract diseases and apoptosis].
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Apoptosis↗
Biomedical subjects
K Kuwano
Publications and source records attributed to K Kuwano.
An anergic cytotoxic T lymphocyte clone exhibits granule exocytosis-mediated cytotoxicity.
T cell receptor (TCR) occupancy in the absence of a costimulatory signal transforms T helper (Th) cells or cytotoxic T lymphocytes (CTL) into a state of anergy. The anergic T cells are unable to produce cytokines; nevertheless, they maintain their killing activity. We investigated the mechanisms through which anergic CTL causes lysis of target cells. Treatment of a CTL clone with phorbol myristate acetate and calcium ionophore A23187 (P/A) transformed these cells to anergic cells. While the anergic CTL clones failed to secrete TNF-alpha in the culture supernatant, they were still able to kill antigen-specific target cells via a granule exocytosis-mediated pathway. This was evident by the synthesis of perforin mRNA and release of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester esterase by these cells. The anergic CTL clone also showed a low degree of Fas-mediated lysis of normal target cells. In addition, we generated anergic bulk CTL by treatment with P/A and observed that the anergic bulk CTL failed to produce TNF upon antigen stimulation, but retained target killing activity via a granule exocytosis mechanism. Our results suggest that the killing mechanisms of anergic CTL are mediated to a large extent by a granule exocytosis-mediated pathway.
Animals↗
Immunohistochemically detected p53 and P-glycoprotein predict the response to chemotherapy in lung cancer.
While resistance to chemotherapy is a major problem in lung cancer treatment, there is no useful predictor of treatment response. We thus designed this study to determine the utility of p53 and P-glycoprotein expression in predicting the response to chemotherapy in patients with primary lung cancer, retrospectively. We evaluated transbronchial biopsy (TBB) specimens from 60 patients with lung cancer, who were previously untreated. Formalin-fixed, paraffin-embedded TBB specimens were immunostained using anti-p53 antibody (DO-1) and anti-P-glycoprotein antibody (JSB-1). The positivity of p53 was 63%, and that of P-glycoprotein was 17%. No correlation was observed between p53 and P-glycoprotein immunostaining. Positivity of p53 correlated significantly (P = 0.004) with a lack of response to chemotherapy in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC). In contrast, positivity of P-glycoprotein was correlated with chemotherapy resistance in SCLC (P = 0.003), but not in NSCLC. Multiple logistic regression analysis revealed that positive immunostaining for p53 was a significant risk factor for chemotherapy resistance in NSCLC. These results suggest that immunostaining of p53 and P-glycoprotein for TBB specimens may help to predict response to chemotherapy in NSCLC and SCLC, although the results should be confirmed in a larger, more homogeneous series.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Definition of amino acid residues on the epitope responsible for recognition by influenza A virus H1-specific, H2-specific, and H1- and H2-cross-reactive murine cytotoxic T-lymphocyte clones.
We defined the epitopes recognized by three influenza A virus-specific, H-2Kd-restricted CD8(+) cytotoxic T-lymphocyte (CTL) clones: H1-specific clone A-12, H2-specific clone F-4, and H1- and H2-cross-reactive clone B7-B7. The A-12 and B7-B7 clones recognized the same peptide, which comprises amino acids 533 to 541 (IYSTVASSL) of A/PR/8 hemagglutinin (HA). The F-4 and B7-B7 clones both recognized the peptide which comprise amino acids 529 to 537 (IYATVAGSL) of A/Jap HA. Amino acids 533 to 541 of A/PR/8 HA are compatible with amino acids 529 to 537 of A/Jap HA. Amino acid S at positions 3 and 7 was responsible for recognition by H1-specific clone A-12, while amino acid G at position 7 was responsible for recognition by H2-specific clone F-4. Two conserved amino acids, T at position 4 and A at position 6, were responsible for recognition by H1-, and H2-cross-reactive clone B7-B7. These results indicate that a single nine-amino-acid region is recognized by HA-specific CTL clones of three different subtype specificities and that the amino acids responsible for the recognition by the CTL clones are different.
Amino Acid Sequence↗
Expression of B7-1, B7-2, and interleukin 12 in bleomycin-induced pneumopathy in mice.
BACKGROUND: The bleomycin-induced pneumopathy involves a T cell-mediated immune response. T cell activation requires both antigen/MHC recognition and costimulatory signals. The CD28 receptor on T cells with its ligand B7 represents one of the most important examples of this costimulation. Interleukin 12 (IL-12) has a strong synergistic effect with the B7-1/CD28 interaction on inducing proliferation and cytokine production in T cells. METHODS: In this study, we investigated the expression of B7-1, B7-2, and IL-12 in bleomycin-induced pneumopathy in mice using reverse transcription polymerase chain reaction (RT-PCR), RT in situ PCR, and immunohistochemistry. RESULTS: We observed concurrent upregulation of B7-1, B7-2, and IL-12p40 mRNA in the lung tissues at 1 h to 7 days after bleomycin instillation into the trachea. B7-1 mRNA and protein were found in bronchiolar epithelial cells as well as macrophages, B7-2 and IL-12p40 mRNA appeared to be expressed in mononuclear cells. CONCLUSIONS: These findings indicate that T cell-mediated immune response in this model involves the upregulation of B7-1, B7-2, and IL-12p40 mRNA, and also demonstrate the aberrant expression of B7-1 in bronchiolar epithelial cells.
Animals↗
Endothelial cell apoptosis in lipopolysaccharide-induced lung injury in mice.
BACKGROUND: Studies have shown the importance of apoptosis in vascular injury in vitro. We postulated that apoptosis of the endothelium contributes to vascular injury in vivo and may be involved in acute lung injury. METHODS: To test this hypothesis, we investigated the incidence of endothelial cell apoptosis in acute lung injury induced in mice by the administration of lipopolysaccharide (LPS). Male ICR mice were administered LPS (20 mg/kg body weight) intravenously and sacrificed at specified times thereafter. RESULTS: Histologic findings were consistent with acute lung injury which increased with time from 3 to 48 h after injection. Electrophoretic analysis of DNA that was extracted from lung tissue and 3'-end-labeled with digoxigenin demonstrated a fragmentation of DNA starting at 6 h. In situ terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) demonstrated DNA strand breaks in the endothelial cells. TUNEL also revealed DNA strand breaks in bronchial and alveolar epithelial cells as well as inflammatory cells in the interstitium. These TUNEL-positive cells appeared 6 h after injection. Electron-microscopic examination of the endothelium strongly suggested the morphological characteristics of apoptosis. CONCLUSION: Apoptosis was induced by LPS administration in endothelial cells in vivo. A role for such apoptosis is suggested in acute lung injury.
Animals↗
Expression of p53, p21 (Waf1/Cip1/Sdi1) and Fas antigen in collagen vascular and granulomatous lung diseases.
Fas is expressed in various cells and transduces the cell death signal. p21 is a mediator of p53-dependent G1 arrest associated with deoxyribonucleic acid (DNA) damage. The upregulation of p53 and p21 associated with DNA damage in idiopathic pulmonary fibrosis has been described previously. In this study, p53, p21, and Fas expression and DNA damage were examined in interstitial pneumonia associated with collagen vascular diseases (CVD-IP). DNA damage was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labelling (TUNEL) and p53, p21 and Fas proteins were detected by immunohistochemistry in 13 cases of CVD-IP, 13 of sarcoidosis, seven of hypersensitivity pneumonitis (HP) and eight control patients with normal lung parenchyma. TUNEL-positive signals were found in bronchiolar or alveolar epithelial cells in 11 of 13 (85%) specimens of CVD-IP, but not in sarcoidosis, HP or controls, except for a case of chronic HP with pulmonary fibrosis. p53, p21 and Fas were detected in bronchiolar or alveolar epithelial cells in nine (69%), 10 (77%) and 12 (92%) of 13 specimens of CVD-IP, respectively, but not in sarcoidosis, HP or controls, except for a case of chronic HP. These results suggest that the upregulation of p53, p21 and Fas in bronchiolar and alveolar epithelial cells associated with deoxyribonucleic acid damage may participate in the process of pulmonary fibrosis in interstitial pneumonia associated with collagen vascular diseases and chronic hypersensitivity pneumonitis.
Adult↗
Cryopreservation of Eisenia bicyclis (Laminariales, Phaeophyta) in liquid nitrogen.
Gametophytic cells of Eisenia bicyclis (Kjellman) Setchell were cryopreserved in liquid nitrogen (LN). Ethylene glycol was the most effective cryoprotectant when used alone, and in combination with 10% (w/v) proline improved the survival. Viability of female and male cryopreserved cells after thawing reached 62.0% and 52.6% immediately after thawing, but these levels decreased to 31.1% and 27.2% after 4 days postthawing culture. The optimal prefreezing temperature was -40 degreesC, and cells prefrozen to temperatures >-40 degreesC were damaged mainly by intracellular ice crystal formation during immersion in LN, while those prefrozen to temperatures <-40 degreesC were damaged mainly by excessive dehydration during prefreezing. The survival rates were not affected after storage in LN for at least 200 days.
Journal Article↗
[The roles of apoptosis in lung injury and fibrosis].
Apoptosis serves important roles in organ development, cell differentiation, and the maintenance of homeostasis. Lung injury studies have underlined the role of fibroblast and endothelial cell apoptosis during lung repair from acute lung injuries, and demonstrated apoptosis of alveolar epithelial cells in association with diffuse alveolar damage. Pulmonary fibrosis is characterized by the loss of lung epithelial cells and the proliferation of fibroblasts. It is possible that p53, p21, Fas, Fas ligand, and other apoptosis-regulating proteins play an important role in the pathophysiology of lung injury and fibrosis.
Animals↗
Effects of monoclonal antibody to P-selectin and analogue of sialyl Lewis X on cyclic flow variations in stenosed and endothelium-injured canine coronary arteries.
BACKGROUND: A fundamental role of cell adhesion molecules is implicated in the disease processes of acute coronary syndromes. We have shown an increase in the soluble form of P-selectin in these syndromes, suggesting the important interaction between P-selectin and sialyl Lewis X (SLe(x)) for the pathophysiology of these syndromes. To further test this, we examined the effects of a monoclonal antibody against P-selectin (PB1.3) and a carbohydrate analogue of SLe(x) (SLe(x)-OS) on cyclic flow variations (CFVs) in stenosed and endothelium-injured canine coronary arteries. METHODS AND RESULTS: Anesthetized, open-chest dogs (n = 48) were divided into six groups after CFVs were established. Dogs received intravenous normal saline, PB1.3 (1 mg/kg bolus), a low dose (5 mg/kg bolus) or a high dose (40 mg/kg bolus) of SLe(x)-OS followed by an infusion (5 mg.kg-1.h-1) for 60 minutes, a combination of PB1.3 and SLe(x)-OS (low dose), or a combination of a nonblocking antibody against P-selectin (PNB1.6, 1 mg/kg) and SLe(x)-OS (low dose). Although saline, PB1.3, SLe(x)-OS (low dose), and the combination of PNB1.6 and SLe(x)-OS (low dose) did not affect CFVs, the high dose of SLe(x)-OS and the combination of PB1.3 and SLe(x)-OS (low dose) significantly reduced CFVs. CONCLUSIONS: These findings indicate that the high dose of SLe(x)-OS and the combination of PB1.3 and the low dose of SLe(x)-OS provide protection against CFVs. Thus, the adhesive interaction between P-selectin and SLe(x) may play an important role in mediating CFVs in this model.
Animals↗
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.
Adenoviridae↗
Production of a T-cell clone which reacts with membrane proteins of Acholeplasma laidlawii.
The role of cellular immunity in mycoplasma infection is not completely understood. In this study, we established mycoplasma-specific T-cell clones to evaluate cellular immunity in mycoplasma infection. We developed a T-cell clone (G-10) which was stimulated with Acholeplasma laidlawii. The T-cell clone G-10, CD4+ and T-cell receptor (TCR) alpha beta- recognized the 42- and 65-kilodalton (kDa) membrane proteins of A. laidlawii and responded to A. hippikon. Hence, the application of mycoplasma-specific T cells such as G-10 in animal models may allow the assessment of cellular immune response to mycoplasma infection.
Acholeplasma laidlawii↗
Aspergillus fumigatus Asp fI DNA is prevalent in sputum from patients with coal workers' pneumoconiosis.
Aspergillus fumigatus is an apportunistic nosocomial pathogen in immunosuppressed patients or in the lesion where the local defense mechanism was impaired. Patients with pneumoconiosis are known to be susceptible to chronic necrotizing pulmonary aspergillosis. Therefore, we hypothesized that A. fumigatus might be prevalent in sputum from patients with coal workers' pneumoconiosis, and also that asthmatic symptoms in patients with coal workers' pneumoconiosis may be associated with the presence of A. fumigatus. We tested for A. fumigatus in the sputum from patients with coal workers' pneumoconiosis by nested polymerase chain reaction amplification of the Asp fI gene. Sequences specific for this gene were detectable in 5 of 11 (45.5%) patients with coal workers' pneumoconiosis with asthmatic symptoms (group A), 5 of 10 (50.0%) patients with coal workers' pneumoconiosis without asthmatic symptoms (group B) and only 1 of 9 (11.1%) patients with chronic airflow obstruction without pneumoconiosis (group C). The frequency of the Asp fI gene detection was significantly higher in groups A and B than in group C (p < 0.05). The prevalence of A. fumigatus was not associated with asthmatic symptoms. These results demonstrated that A. fumigatus was prevalent in patients with coal workers' pneumoconiosis. We speculate that colonization with A. fumigatus may be associated with this disease.
Aged↗
Apoptosis and expression of Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice.
The incidence of apoptosis and the expression of Fas antigen (Fas)/Fas ligand (FasL) mRNA in bleomycin-induced pulmonary fibrosis in mice were examined. Male ICR mice were intratracheally instilled with bleomycin (5 U/kg of body weight). The controls were injected with sterile saline. The animals were anesthetized and killed at 1, 6, and 12 h, and 1, 3, 5, 7, 9, and 14 days after bleomycin instillation. We assessed the incidence of apoptosis in lung tissues by DNA fragmentation on agarose gel electrophoresis, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling, and electron microscopy. The expression of Fas and FasL mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). The localization of Fas mRNA was analyzed by in situ hybridization and that of FasL mRNA was analyzed by RT in situ PCR. The results showed that (1) a single instillation of bleomycin leads to the rapid appearance of apoptosis in bronchial and alveolar epithelial cells, which resolves within 1 day, and (2) apoptosis reappears on day 7 and continues for over 14 days after bleomycin instillation. This was accompanied with a progression of fibrosis. Corticosteroid administration completely blocked both apoptosis and fibrosis. The expression of Fas mRNA was upregulated in the alveolar epithelial cells by the bleomycin instillation. FasL mRNA was also upregulated in infiltrating lymphocytes after bleomycin treatment, but not in the control mice. The administration of corticosteroids suppressed the expression of Fas and FasL mRNA as well as apoptosis and fibrosis. Although these results do not show that apoptosis mediated by the Fas/FasL system is directly linked to bleomycin-induced fibrosis, we speculate that excessive apoptosis and the Fas/FasL system play a role in the pathogenesis of bleomycin-induced lung injury.
Animals↗
Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of Fas antigen.
Fas antigen is a cell surface protein that mediates apoptosis, and it is expressed in various cells and tissues. Fas ligand binds to its receptor Fas, thus inducing apoptosis of Fas-bearing cells. Malfunction of the Fas-Fas ligand system causes lymphoproliferative disorders and autoimmune diseases, whereas its exacerbation may cause tissue destruction. We hypothesize that excessive apoptosis mediated by Fas-Fas ligand interaction may damage alveolar epithelial cells and result in pulmonary fibrosis. Mice were allowed to inhale repeatedly an aerosolized anti-Fas antibody for 14 days. The nuclei of bronchial and alveolar epithelial cells were positively stained by in situ DNA nick end labeling. Electron microscopy demonstrated apoptotic changes in bronchial and alveolar epithelial cells. Histologic findings and hydroxyproline content showed the development of pulmonary fibrosis, which was dependent on the dose of anti-Fas antibody. The repeated inhalation of control antibody (isotype-matched control hamster IgG) did not induce apoptosis of epithelial cells or pulmonary fibrosis. The expression of TGF-beta mRNA was upregulated from day 7 to day 28 in lung tissues of anti-Fas antibody-treated mice but not in those of control mice. In this report, we present the evidence that repeated inhalation of anti-Fas antibody mimicking Fas-Fas ligand crosslinking induces excessive apoptosis and inflammation, which results in pulmonary fibrosis in mice.
Animals↗
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.
Adenovirus Infections, Human↗
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.
Acute Disease↗
P53 and p21 (Waf1/Cip1) mRNA expression associated with DNA damage and repair in acute immune complex alveolitis in mice.
The tumor suppresser p53 is a cell cycle checkpoint protein that contributes to the preservation of genetic stability by mediating either a G1 arrest or apoptosis in response to DNA damage. p53 causes growth arrest through transcriptional activation of the cyclin-dependent kinase inhibitor p21. During p53-mediated suppression of cell proliferation, p21 is important for coordinating cell cycle progression, DNA replication, and repair of damaged DNA. The purpose of this study is to investigate the expression of p53 and p21 mRNA in association with DNA damage and normal repair in acute immune complex alveolitis in mice. Male ICR mice were injected intravenously with IgG antibodies against oval albumin, aerosolized with oval albumin solution, and killed at 4, 6, 12, 24, and 48 hours and 1 week after aerosolization. We assessed the expression of p53 and p21 mRNA by reverse transcriptase (RT)-PCR and by RT in situ PCR. We also assessed DNA damage by terminal deoxynucleotidyl transferase mediated biotin-dUTP nick-end-labeling (TUNEL) and by gel electrophoresis of DNA extracted from lung tissues. The results of RT-PCR and RT in situ PCR showed that p53 and p21 mRNA were concurrently up-regulated at 4 to 48 hours after aerosolization in alveolar epithelial cells. Bronchial and alveolar epithelial cells were positively stained by TUNEL in this period but not at 1 week after aerosolization or in control mice. The result of electrophoretic analysis of DNA was compatible with that of TUNEL. These studies suggest that the responses of p53 and p21 mRNA are associated with physiologic processes of DNA damage and repair in acute immune complex alveolitis in mice.
Animals↗