Recent advance in investigation of gene polymorphisms in Japanese patients with aspirin-exacerbated respiratory disease.

Aspirin-exacerbated respiratory disease (AERD) is a complex clinical syndrome characterised by severe asthmatic attack upon treatment with aspirin and/or non-steroidal anti-inflammatory drugs (NSAIDs). Genetic predisposition has been considered as a crucial determinant and candidate genes have concentrated especially on cysteinyl leukotrienes (LTs)-related genes as the inhibitory action of aspirin and NSAIDs on cyclooxygenase activity may cause overproduction of cysteinyl LTs. However, conflicting results have been reported, in parallel with replication studies in different ethnic groups. Thus, future areas of investigations need to focus on comprehensive approaches towards the discovery of other genetic biomarkers. Unfortunately, few papers have been reported about gene polymorphisms in Japanese patients with AERD. Here, we described on our recent genetic investigations on B2ADR, IL-13, IL-17A, CYP2C19, TBXA2R, CRTH2 and HSP70. This review indicates potential genetic biomarkers contributing to the early diagnosis of AERD, which may include CYP2C19 and HSP70 gene polymorphisms, and future validation studies in independent population are required to provide reassurance about our findings.

GLCCI1 variant accelerates pulmonary function decline in patients with asthma receiving inhaled corticosteroids.

BACKGROUND: In steroid-naive patients with asthma, several gene variants are associated with a short-term response to inhaled corticosteroid (ICS) treatment; this has mostly been observed in Caucasians. However, not many studies have been conducted for other ethnicities. Here, we aimed to determine the relationship between the annual decline in forced expiratory flow volume in one second (FEV1 ) and the variant of the glucocorticoid-induced transcript 1 gene (GLCCI1) in Japanese patients with asthma receiving long-term ICS treatment, taking into account the effect of high serum periostin levels, a known association factor of pulmonary function decline and a marker of refractory eosinophilic/Th2 inflammation. METHODS: In this study, 224 patients with asthma receiving ICS treatment for at least 4 years were enrolled. The effects of single-nucleotide polymorphisms (SNPs) in GLCCI1, stress-induced phosphoprotein 1 (STIP1), and T gene on the decline in FEV1 of 30 ml/year or greater were determined. RESULTS: Besides the known contributing factors, that is, the most intensive treatment step, ex-smoking, and high serum periostin levels (≥95 ng/ml), the GG genotype of GLCCI1 rs37973, and not other SNPs, was independently associated with a decline in FEV1 of 30 ml/year or greater. When patients were stratified according to their serum periostin levels, the GG genotype of rs37973 was significantly associated with blood eosinophilia (≥250/µl) in the high serum periostin group. CONCLUSIONS: A GLCCI1 variant is a risk factor of pulmonary function decline in Japanese patients with asthma receiving long-term ICS treatment. Thus, GLCCI1 may be associated with response to ICS across ethnicities.

Multiple logistic regression analysis of risk factors for the development of steroid-dependent asthma in the elderly: a comparison with younger asthmatics.

BACKGROUND: The percentage of the aged among all patients with bronchial asthma is increasing. OBJECTIVE: To investigate the risk factors for the development of steroid-dependent asthma in the elderly. METHODS: A multiple logistic regression analysis involving various clinical factors between steroid-dependent and -independent asthma was carried out for 59 asthmatics aged over 60 years, including 16 patients with steroid-dependent asthma. The calculated risk for each factor was compared with that obtained from 122 younger asthmatics aged 20-59 years. RESULTS: Among the factors examined (sex, age, period from onset of asthma, type of asthma and family history of asthma, plus history of smoking, atopic dermatitis, allergic rhinitis, chronic sinusitis and nasal polyps), the significant risk factors for the development of steroid dependency in the elderly asthmatics were only family history of bronchial asthma (relative risk 3.6) and smoking history (relative risk 6.9). CONCLUSIONS: Some risk factors for steroid-dependent asthma in younger individuals were not significant in the elderly. Since the smoking history was most closely associated with the development of steroid dependency in the elderly, even though most of them had quit smoking, it is important for patients with asthma to avoid smoking.

Effects of pranlukast, a cysteinyl leukotriene antagonist, on bronchial responsiveness to methacholine in aspirin-intolerant asthmatics treated with corticosteroids.

Cysteinyl leukotrienes (cysLTs) are considered to be the most important mediator involved in the pathogenesis of aspirin-intolerant asthma (AIA). However, the role of cysLTs in the baseline condition of the pathophysiology of AIA when not exposed to non-steroidal antiinflammatory drugs (NSAIDs) as well as that in the pathophysiology of aspirin-tolerant asthma remains to be elucidated. Therefore, we evaluated the effect of pranlukast, a potent, selective cysLT receptor antagonist, on bronchial responsiveness to methacholine, a non-specific stimulus, in 7 well-controlled aspirin-intolerant asthmatics receiving oral or inhaled corticosteroid treatment. Pranlukast was orally administered at a dose of 225 mg twice daily to all patients for 4 weeks, and the methacholine challenge test was performed before and after pranlukast treatment. The methacholine provocative concentration producing a 20% fall in forced expiratory volume in 1 second (PC20-FEV1) was calculated as an index of bronchial hyperresponsiveness (BHR). The geometric mean values of PC20-FEV1 significantly (p = 0.028) increased from 0.34 mg/dl to 0.61 mg/dl after pranlukast treatment. No significant differences were observed in the baseline values of forced vital capacity (FVC) or FEV1 before and after pranlukast treatment. These findings suggest that antagonism of endogenous cysLT by pranlukast may be responsible for the improvement of BHR to methacholine.

Interferon-inducible gene family 1-8U expression in colitis-associated colon cancer and severely inflamed mucosa in ulcerative colitis.

Persistent severe inflammation in colonic mucosa is thought to cause the development of colon cancer in patients with ulcerative colitis (UC). However, predisposing genetic abnormalities have not been identified in this sequence. Using differential display PCR, we isolated cDNA fragments corresponding to mRNAs that were differentially expressed in colitis-associated cancer tissues and mucosa with mild inflammation in the colon of five UC patients. This molecular screening approach identified 60 cDNA fragments, and we sequenced 34 fragments. One cDNA fragment, which is identical to IFN-inducible gene family 1-8U, was strongly expressed in all five UC-associated cancers. 1-8U was also expressed in sporadic colon cancer tissues and colon cancer cell lines, but not in normal mucosa. This gene was strongly expressed in severely inflamed colonic mucosa of UC without colitis-associated colon cancer, although 1-8U expression was not related to the extent and duration of the disease. However, 1-8U was expressed in the colonic mucosa of all patients with chronic, continuously severe inflammation. These results indicated that IFN-inducible gene family 1-8U expression in inflamed colonic mucosa might be used as a preferential marker of colitis-associated colon cancer in UC.

Activation of telomerase is induced by a natural antigen in allergen-specific memory T lymphocytes in bronchial asthma.

The function of the immune system is known to be dependent on the cellular differentiation and clonal expansion of allergen-specific lymphocytes. Telomerase, a ribonucleoprotein enzyme, is believed to be essential for the indefinite proliferation of human cells. To clarify whether telomerase is involved in the pathogenesis of immune diseases as well as of malignancies, we investigated the upregulation of telomerase activity in allergen-specific T lymphocytes. Upregulation of telomerase in allergen-sensitized lymphocytes was induced not only by artificial mitogenic stimulations but also by the natural antigen, house dust mite, which causes allergic diseases. Moreover, the upregulation of telomerase activity in memory T cells activated during allergen-specific immune responses might be associated with the enduring allergen-specific atopic propensity in asthmatics.

Pranlukast, a cysteinyl leukotriene antagonist, reduces serum eosinophil cationic protein levels in patients with asthma.

Cysteinyl leukotrienes (cysLTs) are considered to be important mediators involved in bronchial asthma and the ensuing eosinophilic inflammation. We evaluated the effects of pranlukast, a potent and selective cysLT receptor antagonist, on the clinical course and serum eosinophil cationic protein (ECP) levels of 10 asthmatic patients. A four-week administration of pranlukast increased the morning peak expiratory flow (PEF) (p = 0.007) and decreased as-needed beta 2-agonist use (p = 0.021). Changes in the morning PEF inversely correlated with those in the serum ECP levels (r = -0.80, p = 0.0057). These results suggest that cysLTs are important mediators involved in eosinophilic inflammation, a major pathophysiologic feature of bronchial asthma in humans.

Increased expression of tumor necrosis factor-alpha, interleukin-6, platelet-derived growth factor-B and granulocyte-macrophage colony-stimulating factor mRNA in cells of bronchoalveolar lavage fluids from patients with sarcoidosis.

Cytokines released from activated alveolar macrophages and T-lymphocytes affect the accumulation of monocyte-macrophage-lineage cells and therefore play an important role in the formation of sarcoid granuloma. Although it is likely that certain monokines and lymphokines are involved in the development of sarcoid granulomas, the evidence for this is not unequivocal. In an attempt to clear critical cytokines in the development and maintenance of sarcoid granuloma, we have measured the level of seven cytokine mRNA (TNF-alpha, IL-6, IL-8, TGF-beta, PDGF-B, IFN-gamma, and GM-CSF) in cells obtained by BAL from sarcoidosis patients and normal subjects. To detect cytokine mRNA, we employed a reverse transcription-polymerase chain reaction. We report that the levels of TNF-alpha, IL-6, PDGF-B and GM-CSF mRNA were significantly increased in BAL cells from the patients with pulmonary sarcoidosis compared to controls. No significant differences were observed in the mRNA expression of IL-8, TGF-beta and IFN-gamma. A significant correlation of the expression of the mRNA levels of seven cytokines in the same patients with sarcoidosis was observed between IL-8 and TNF-alpha, PDGF-B, and IL-6, IL-8 and IL-6 and TFN-alpha and PDGF-B and IL-8. This finding indicates that at least these four cytokines are involved in the cytokine network at the local alveolar site of chronic granulomatous inflammation. This study adds a report to the literature that supports a role for cytokine, TNF-alpha, IL-6, PDGF and GM-CSF in particular, in the promotion and maintenance of sarcoid granulomatous inflammation.

cAMP motifs regulating transcription in the aquaporin 2 gene.

Genomic clones including the 5' flanking regions of the AQP2 (aquaporin 2) gene were isolated, and the promoter region was examined by transiently transfecting a promoter-luciferase reporter fusion gene into renal cultured epithelial cells. An orientation specific promoter for the AQP2 gene was found within the proximal 3 kb of 5'-flanking region. Minimal basal promoter activity of the AQP2 gene was found within 198 bp upstream from the transcription start site by deletion analysis. Sequencing the transcriptionally active region revealed a typical TATA box, adenosine 3',5'-cyclic monophosphate (cAMP) responsive element (CRE) and three putative CCAAT boxes in the proximal 1.2-kb region. Significantly, a GATA motif, AP1, AP2, and SP1 transcriptional factor consensus sites were also found in this region. Exposure to cAMP-enhancing agents (1 nM vasopressin or 20 mM forskolin and 250 mM 3-isobutyl-1-methylxanthine) showed that these agents increased luciferase activity in a parallel fashion, suggesting that vasopressin-induced AQP2 gene transcription is mediated through increases in intracellular cAMP in at least one renal cell type, the LLC-PK1 cells. The mechanism of cAMP responsiveness of AQP2 gene transcription was further studied using a series of deletion mutants in renal epithelial cells and other cell types. The cAMP regulatory motifs were shown to exist in a 50-bp sequence between -340 and -290 (containing CRE) and a 65-bp sequence (containing an AP2 site) between -150 and the ATG start site in LLC-PK1 cells. In rat inner medullary collecting duct (IMCD) cells, the cAMP regulatory motifs also exist in a 50-bp sequence between -340 and -290 (containing CRE) and in a 10-bp sequence between -160 and -150 (containing an SP1 site). These separate regions may cooperate to confer full cAMP inducibility to the AQP2 gene in a cell-specific manner.

Effect of Nocardia rubra cell-wall skeleton on cancer prevention in humans.

The retired workers at the chemical weapons plant in Japan are regarded as a high-risk group for cancers. Under the Cancer Preventive Program, Nocardia rubra cell-wall skeleton (N-CWS) was administered to 80 workers directly involved in the production of sulfur mustard and 66 workers engaged in work related to sulfur mustard production. Untreated workers whose age, sex, duties and duration of work at this factory were individually matched to the N-CWS-treated workers were used as controls. During a 4.5-year observation period, development of cancers was found in 7 treated workers and 17 untreated controls. After elimination of the influence of the difference in smoking level, the incidence of subjects who developed cancers was compared statistically between the N-CWS-treated workers and the untreated controls and a significant suppression of development of cancers was noted in the N-CWS-treated workers. Thus, it was concluded that the administration of N-CWS could prevent cancer development in humans.

[A statistical investigation of the influence of allergic factors on intractable asthma by multiple factor analysis].

The relationships of the development of intractability in bronchial asthma with 11 factors, namely 1) sex, 2) age of onset, 3) duration of the disease, 4) severity of the disease, 5) disease type, 6) family history within the third degree of consanguinity, 7) history of smoking, 8) history of atopic dermatitis, 9) history of allergic rhinitis, 10) history of chronic sinusitis and 11) history of nasal polyp were studied by multiple factor analysis in 95 patients. The severity of the disease was shown to be the most important factor in whether the disease becomes intractable or not, followed by the age of onset. The history of atopic dermatitis had the least influence, and the influences of the other factors were not markedly different from one another. Evaluation of each factor according to the category score suggested that severe or moderate non-atopic bronchial asthma in males with a positive family history and positive histories of smoking, chronic sinusitis, nasal polyp and atopic dermatitis within a short period after the onset in the second decade or fifth decade or later tend to become intractable.

Effects of periodic administration of Nocardia rubra cell-wall skeleton on immunoglobulin production and B-cell-stimulatory factor activity in vitro in workers at a poison gas factory.

The former workers at the Okunojima poison gas factory (poison gas workers) are a high-risk group for malignant neoplasms and show abnormalities in cellular immunity. At the same time, poison gas workers often have chronic respiratory diseases, such as chronic bronchitis, and are highly susceptible to respiratory infections. To explore the possibility of immunological cancer prevention, we have periodically administered 200 micrograms Nocardia rubra cell-wall skeleton (N-CWS) to poison gas workers once every 3 months since December 1978. During this period, we noted a significantly lower incidence of influenza among poison gas workers receiving N-CWS than in those not receiving the drug during the influenza epidemic. This finding suggested that the administration of N-CWS enhanced the resistance of these workers to infections. Therefore, periodical administration of N-CWS to poison gas workers was considered to enhance the reduced T-cell function of normalizing antibody production by stimulating the production of B-cell-stimulatory factor (BSF). In the present study, to clarify the mechanism of immunosuppression in the poison gas workers and to examine the effects of continual administration of N-CWS on this condition, we compared the immunoglobulin production and the proliferative and differentiative activities of B-cell-stimulatory factor (BSF) of peripheral blood mononuclear cells (PBMC), in poison gas workers treated or not treated with N-CWS. Comparisons were also made with age-matched healthy controls. In the untreated poison gas workers, immunoglobulin and BSF production of PBMC were reduced as compared with the control group. On the other hand, in the poison gas workers receiving N-CWS, immunoglobulin and BSF production of PBMC were restored nearly to the control level. These results show that in vitro antibody production in the poison gas workers was reduced and that a reduction in BSF production of T cells was one of its causes.

Somatic mutation in peripheral lymphocytes of former workers at the Okunojima poison gas factory.

The former workers at the Okunojima poison gas factory comprise a high risk group for malignant tumors such as respiratory tract cancer. Demonstration of injury to somatic cell genes in this group may provide important data for evaluating the association between mustard gas and malignant tumors. So we measured the frequency of T lymphocytes lacking the hypoxanthine guanine phosphoribosyl transferase (HGPRT) activity, by cloning with interleukin 2 (IL2). In this study, we performed cloning of T lymphocytes lacking the HGPRT activity using recombinant IL2 (rIL2) and observed an increased frequency of somatic mutation in poison gas workers who had had more chances to be exposed to mustard gas and those who had worked for a longer period. This result suggested that inhalation of small amounts of mustard gas damaged somatic cell genes, resulting in carcinogenesis.

Effects of Nocardia rubra cell wall skeleton on interleukin 2 production and lymphocyte proliferation in former poison gas factory workers.

Phytohemagglutinin (PHA)-induced interleukin 2 (IL-2) production and lymphocyte proliferation were measured in former workers of the Okunojima Poison Gas Factory (poison gas workers), who have a high incidence of lung cancer, and the efficacy of administration of Nocardia rubra cell wall skeleton (N-CWS) was studied. In comparison with normal controls and poison gas workers receiving N-CWS, lymphocyte proliferation in poison gas workers not receiving N-CWS showed a significant decrease, while IL-2 production showed a slight though not statistically significant decrease. When N-CWS was administered to poison gas workers, IL-2 production and lymphocyte proliferation were significantly elevated, with a peak two weeks after administration. N-CWS, by elevating IL-2 production of lymphocytes, is considered to have improved the depression of lymphocyte proliferation.

Effects of Nocardia rubra cell wall skeleton on lymphocyte subsets in former poison gas factory workers.

Peripheral blood lymphocyte subsets were determined in former workers of the Okunojima Poison Gas Factory (poison gas workers) having a high incidence of lung cancer, and the effect of administration of Nocardia rubra cell wall skeleton (N-CWS) was studied. In poison gas workers not receiving N-CWS, both the percentage and absolute number of Leu-2a+ cells were significantly increased as compared with normal controls, while both the percentage and absolute number of Leu-7+ cells as well as the Leu-3a/Leu-2a ratio were significantly decreased. In poison gas workers receiving N-CWS, the percentage of Leu-2a+ cells was significantly decreased when compared with poison gas workers not receiving N-CWS, while the percentage of Leu-1+ cells and both the percentage and absolute number of Leu-7+ cells as well as the Leu-3a/Leu-2a ratio were significantly increased. When N-CWS was administered to poison gas workers, the percentage of Leu-2a+ cells significantly decreased, with a minimum two weeks after administration, while the percentage of Leu-3a+ and the Leu-3a/Leu-2a ratio significantly increased and the percentages of Leu-1+ and Leu-7+ cells also increased. Furthermore, the absolute number of Leu-3a+ cells increased markedly with a peak two weeks after administration. The absolute numbers of both Leu-1+ cells and Leu-7+ cells as well as total lymphocytes also significantly increased, but that of Leu-2a+ cells showed hardly any change. It is considered that a single administration of N-CWS transiently corrects the abnormality of lymphocyte subsets, and that repeated administration of N-CWS once every three months may maintain the lymphocyte subsets of poison gas workers at normal levels.