Prevalence and impact of rhinitis in asthma. SACRA, a cross-sectional nation-wide study in Japan.

BACKGROUND: Asthma and rhinitis are common co-morbidities everywhere in the world but nation-wide studies assessing rhinitis in asthmatics using questionnaires based on guidelines are not available. OBJECTIVE: To assess the prevalence, classification, and severity of rhinitis using the Allergic Rhinitis and its Impact on Asthma (ARIA) criteria in Japanese patients with diagnosed and treated asthma. METHODS: The study was performed from March to August 2009. Patients in physicians' waiting rooms, or physicians themselves, filled out questionnaires on rhinitis and asthma based on ARIA and Global Initiative for Asthma (GINA) diagnostic guides. The patients answered questions on the severity of the diseases and a Visual Analog Scale. Their physicians made the diagnosis of rhinitis. RESULTS: In this study, 1910 physicians enrolled 29,518 asthmatics; 15,051 (51.0%) questionnaires were administered by physician, and 26,680 (90.4%) patients were evaluable. Self- and physician-administered questionnaires gave similar results. Rhinitis was diagnosed in 68.5% of patients with self-administered questionnaires and 66.2% with physician-administered questionnaires. In this study, 994 (7.6%) patients with self-administered and 561 (5.2%) patients with physician-administered questionnaires indicated rhinitis symptoms on the questionnaires without a physician's diagnosis of rhinitis. Most patients with the physician's diagnosis of rhinitis had moderate/severe rhinitis. Asthma control was significantly impaired in patients with a physician's diagnosis of rhinitis for all GINA clinical criteria except exacerbations. There were significantly more patients with uncontrolled asthma as defined by GINA in those with a physician's diagnosis of rhinitis (25.4% and 29.7%) by comparison with those without rhinitis (18.0% and 22.8%). CONCLUSION: Rhinitis is common in asthma and impairs asthma control.

[Sarcomatoid carcinoma of the thymus which caused acute epicarditis].

Thymic carcinoma is rare. Particularly sarcomatoid carcinoma of the thymus is a very rare disease it has been reported in only 15 patients to date. The prognosis is very poor and diagnosis and treatment have not yet been established. We report a case of 63-year-old man who was initially diagnosed with acute pericarditis and was finally found to be sarcomatoid carcinoma of the thymus. He underwent surgery and the tumor was completely resected. However, 6 months after surgery, local recurrence was noted. The patient was treated by radiotherapy followed by paclitaxel monotherapy. Partial remission was achieved transiently with paclitaxel, but the tumor again recurred. He died 33 months after surgery. The possibility of diseases like this tumor must be kept in mind for a patient with chest symptoms. Paclitaxel monotherapy is likely to be effective in treating sarcomatoid carcinoma of the thymus.

E-Cadherin gene promoter hypermethylation in primary human gastric carcinomas.

BACKGROUND: E (epithelial)-cadherin, the cell adhesion molecule also considered a potential invasion/metastasis suppressor, is mutationally inactivated in nearly half of all undifferentiated-scattered (diffuse-type) gastric carcinomas. In addition, silencing of E-cadherin by CpG methylation within its promoter region has been reported in several gastric carcinoma cell lines. We investigated the methylation status of the E-cadherin promoter region in 53 primary human gastric carcinomas. METHODS: Hypermethylation of the E-cadherin promoter was determined by utilizing methylation-specific polymerase chain reaction (PCR)-single-strand conformation polymorphism (MSP-SSCP) analysis followed by direct sequencing of PCR products. Expression of E-cadherin was studied by western blot analysis. All statistical tests were two-sided. RESULTS: Hypermethylation of the E-cadherin promoter was evident in 27 (51%) of 53 primary gastric carcinomas examined by MSP-SSCP. It occurred more frequently in carcinomas of the undifferentiated-scattered type (in 15 [83%] of 18) than in other histologic subtypes (in 12 [34%] of 35) (P =.0011, Fisher's exact test), and it was present at similar rates in early (in six [60%] of 10) versus advanced (in 21 [49%] of 43) carcinomas (P =.73, Fisher's exact test). Methylation occurring at all cytosine-guanosine sequences (CpGs) near the transcriptional start site was confirmed in six of six tumors examined by bisulfite-DNA sequencing, including two early gastric carcinomas. In addition, loss or diminished expression of E-cadherin was confirmed by western blotting in four of the six tumor tissues demonstrating hypermethylation. CONCLUSIONS: The E-cadherin promoter frequently undergoes hypermethylation in human gastric cancers, particularly those of the undifferentiated-scattered histologic subtype. E-cadherin promoter hypermethylation is associated with decreased expression and may occur early in gastric carcinogenesis.

Frequent microsatellite alterations on chromosome 3p in esophageal squamous cell carcinoma.

By the microsatellite assay, two types of genetic alterations, loss of heterozygosity (LOH) and replication error (RER), were examined using 7 dinucleotide repeat markers [D3S1317 (3p26); CI3-1169 (3p25); CI3-946 (3p25); D3S1255 (3p24.2-25); CI3-771 (3p21.3); CI3-1413 (3p14.1-14.3); and CI3-373 (3p13)] on the short arm of chromosome 3p as well as 3 markers [D2S123 (2p15-16), IFNA (9p22), and D16S408 (16q12.1-13)] on other chromosomes in 35 patients with esophageal squamous cell carcinoma. On 3p, LOH was detected in 34% (12 of 35) and RER was detected in 60% (21 of 35) at single or multiple loci. RER occurred at a similar frequency in all stages and did not correlate with clinicopathological characteristics. On the other hand, LOH at the 3p25 locus was more frequently detected in carcinomas with lymph node metastasis than in those without it (P < 0.05). The incidences of microsatellite alterations were low on the chromosomes other than 3p, except at D2S123, where the incidence of RER was 20%. These findings suggest that RER on 3p is an early event and that a tumor suppressor gene which is involved in the progression of esophageal squamous cell carcinoma may exist near the 3p25 locus.

Detection of frequent p53 gene mutations in primary gastric cancer by cell sorting and polymerase chain reaction single-strand conformation polymorphism analysis.

Mutations of the p53 gene were investigated after tumor cell enrichment by cell sorting based on differences in DNA content and polymerase chain reaction single-strand conformation polymorphism analysis in 24 surgical specimens of primary gastric cancer. p53 mutations were detected in exons 4-8 in 64% (9 of 14) of aneuploid tumors but in none of 10 diploid tumors examined. Four of five tumors containing two or three aneuploid subpopulations showed the presence of p53 gene mutations. No correlation was found between the presence of p53 mutations and the degree of histological differentiation of tumors. These findings suggest that p53 gene mutations are related to DNA ploidy alterations as relatively late events of carcinogenesis in gastric cancer. The present method is highly sensitive for detection of genetic abnormalities and is applicable even when various kinds of nontumorous cells are present in tumor samples.

Effect of tunicamycin on production by mouse fibroblast L929 cells of the factor-stimulating differentiation of mouse myeloid leukemic cells and the colony-stimulating factor.

Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor), which is suggested to be a glycoprotein. On the other hand, growth and differentiation of normal precursor cells of macrophages and granulocytes can be stimulated by a glycoprotein termed colony-stimulating factor (CSF). Mouse fibroblast L929 cells were found to produce both the D-factor and CSF. The properties of the D-factor and CSF and the roles of carbohydrates in the molecules of these factors were examined using tunicamycin, a specific inhibitor of asparaginase-linked glycosylation. Although both the D-factor and CSF were produced by L-cells in usual medium containing fetal calf serum, production of D-factor, but not CSF, was reduced by omission of serum from the medium. The activity of the D-factor was slightly decreased by treating the L-cells with tunicamycin (0.5 microgram/ml) in the presence of 2% fetal calf serum, without any decrease in CSF activity. Conditioned medium of L-cells incubated with or without tunicamycin was fractionated by gel filtration on a Sephadex G-200 column. Normal D-factor appeared as a single peak with an apparent molecular weight of 67,000. D-factor produced in the presence of tunicamycin had an apparent molecular weight of 25,000. On the other hand, most of the CSF was eluted in the void volume, even when it was produced in the presence of tunicamycin. The D-factor produced in the presence of tunicamycin was more sensitive than normal D-factor was to trypsin or heat treatment at 70 degrees. The CSF produced in the presence of tunicamycin was resistant to these treatments. These results indicate that the D-factor is distinct from CSF. Furthermore, the results suggest that the D-factor produced by L-cells is also a glycoprotein and that, although carbohydrate is not essential for production or activity of the D-factor, it contributes to stabilizing the protein portion of D-factor.

In vitro effects of an antitumor antibiotic, ascofuranone, on the murine immune system.

Effects of an antitumor antibiotic, ascofuranone (AF) on the murine immune system were studied. Unlike lectins, AF did not induce any proliferative response of splenocytes. Furthermore, AF significantly inhibited proliferative response of splenocytes in response to lectins, such as concanavalin A, lipopolysaccharide, or phytohemagglutinin above 5 micrograms/ml. In concanavalin A-induced T-lymphocyte response, AF selectively inhibited the formation of interleukin 2 (IL-2) receptors, which was observed above 0.4 micrograms/ml. On the other hand, the inhibitory effect on the proliferative response to IL-2 of T-lymphocytes, which had already obtained IL-2 receptors, was observed above 10 micrograms/ml. IL-2 production of splenocytes in response to concanavalin A was also suppressed by AF above 2 micrograms/ml and only 3% of IL-2 was produced in the presence of AF, 10 micrograms/ml. However, AF-activated macrophages and their glycolysis was significantly stimulated. Activation of macrophages by AF was also confirmed by stimulation of interleukin 1 production and tumoricidal activity. However, natural killer activity of splenocytes was suppressed at the concentration where significant activation of tumoricidal activity of macrophages was observed. Therefore, AF had a dual effect on the immune system. Macrophages were activated to produce interleukin 1 and to kill tumor cells. On the other hand, functions of lymphocytes were suppressed.

Mutations of the APC gene occur during early stages of gastric adenoma development.

Mutations of the adenomatous polyposis coli (APC) gene have recently been shown to play an important role in colorectal tumorigenesis. We investigated mutations of the APC gene in 30 gastric adenomas obtained endoscopically. Mutations of the APC gene were examined by polymerase chain reaction-single-strand conformation polymorphism analysis followed by sequencing of the polymerase chain reaction products. Mutations were detected in 20% (6 of 30) of gastric adenomas. In addition, deletion of the remaining allele that subsequently led to complete inactivation of the APC gene was confirmed in one-half (3 of 6) of the tumors with APC gene mutations. Sequencing analysis confirmed that the mutations resulted in truncation of the gene products or in an amino acid change. The incidences of mutations of the APC gene remained constant regardless of the size or degree of histological atypia. Our observations suggest that mutations of the APC gene, similarly to those in colorectal tumorigenesis, occur during the early stages of gastric adenoma development.

Microsatellite alterations in adenoma and differentiated adenocarcinoma of the stomach.

In order to elucidate the significance of the adenoma-carcinoma sequence in gastric carcinogenesis from a genetic point of view, we examined microsatellite alterations (replication error and loss of heterozygosity) on chromosomes 2p (D2S123), 3p (D3S1317), 5q (D5S409), 9p (IFNA), and 13q (D13S153) as well as p53 gene mutations in 13 adenomas and 23 differentiated adenocarcinomas including 8 early carcinomas of the stomach. Replication error was detected in only one of the adenomas (8%, 1/13) at the D5S409 locus and in none at the other loci, and loss of heterozygosity was also an infrequent event found in one adenoma (14%, 1/7 informative cases) at D5S409 and in none at the other loci. A p53 gene mutation was detected in one (8%, 1/13) of the adenomas. Thus, microsatellite alterations and p53 gene mutations are rare events in adenomas. In differentiated adenocarcinomas, replication error was detected in 4 (17%, 4/23) at single or multiple loci, and loss of heterozygosity was observed frequently at D3S1317 (25%, 3/12), D5S409 (67%, 6/9), and IFNA (26%, 5/19). Mutations in the p53 gene were detected in 9 (39%, 9/23) of the differentiated adenocarcinomas. Microsatellite alterations on several chromosomes and mutations in the p53 gene were frequent in differentiated adenocarcinomas, even those at an early stage. These results suggest that the adenoma-carcinoma sequence is relatively rare in gastric carcinogenesis, and that the majority of differentiated adenocarcinomas of the stomach may develop through a de novo pathway.

Suppression of hypertriglyceridemia of Ehrlich carcinoma-bearing mice by an antibiotic, ascofuranone.

Ehrlich ascites carcinoma-bearing mice exhibit hypertriglyceridemia. An antitumor antibiotic, ascofuranone, suppressed tumor-induced hypertriglyceridemia when administered i.p. even when no evident antitumor activity was observed without affecting the levels of free fatty acids, phospholipids, cholesterol, glucose, and total protein in plasma. Ascofuranone did not reduce plasma triglycerides of normal mice. Insulin and clofibrate, known modifiers of lipid metabolism, showed no significant suppression. Ascofuranone is also effective on solid tumor-induced hypertriglyceridemia. Another notable change of metabolism affected by tumor-bearing in the early stage where hypertriglyceridemia has not yet fully progressed is hypoglycemia. Although ascofuranone did not affect hypoglycemia, the suppressive effect on hypertriglyceridemia was more evident when ascofuranone was administered in the early stage than in the later stage. These results suggest that ascofuranone suppresses hypertriglyceridemia by specifically affecting the changes of host metabolism which is induced in the early stage of tumor bearing.

Inactivation of the CDKN2 gene by homozygous deletion and de novo methylation is associated with advanced stage esophageal squamous cell carcinoma.

We examined the genomic status of the CDKN2 gene including de novo methylation of 5' CpG islands in primary and metastatic tumor samples from 31 patients with esophageal squamous cell carcinoma. One somatic frame shift mutation (1 of 31; 3.2%) was identified by PCR-single strand conformational polymorphism analysis and DNA sequencing. Homozygous deletion and de novo methylation of the gene were confirmed in 5 (16%) and 6 (19%) of 31 patients, respectively. Homozygous deletion and de novo methylation were significantly associated with silencing of gene expression (P < 0.01). Aberrations of the CDKN2 gene were detected in tumors with lymph node metastasis and muscular invasion (12 of 22; 54%) and in none of stage I tumors (0 of 9.0%; P < 0.05). These results suggest that homozygous deletion and de novo methylation are predominant mechanisms of inactivation of the CDKN2 gene and may be associated with metastatic and invasive phenotypes of esophageal squamous cell carcinoma.

Two distinct regions of deletion on the long arm of chromosome 5 in differentiated adenocarcinomas of the stomach.

Frequent loss of heterozygosity (LOH) on the long arm of chromosome 5 (5q) has been reported in many types of human malignancies, including gastric carcinoma. One of the targets of 5q-LOH in colorectal carcinoma is certainly the adenomatous polyposis coli (APC) gene on 5q21. However, other evidence has suggested the presence of another tumor suppressor gene in this region which may be inactivated in gastric carcinoma. In the present study, to determine the location of the putative tumor suppressor gene on 5q, LOH at nine microsatellite loci on 5q were investigated at 38 differentiated adenocarcinomas of the stomach that probably did not carry APC mutations. LOH at any locus on 5q occurred in 37% (14 of 38) of the tumors. Although many tumors exhibited large interstitial deletions on 5q that included the APC locus (5q21), we have identified minimum regions of deletion as the D5S428 locus and the interferon regulatory factor-1 (IRF-1) locus. Thus, at least two putative tumor suppressor genes, which play a crucial role in the genesis of differentiated adenocarcinoma of the stomach and are distinct from the APC gene, lie on 5q.

Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability.

Human gastric carcinoma shows a higher prevalence of microsatellite instability (MSI) than does any other type of sporadic human cancer. The reasons for this high frequency of MSI are not yet known. In contrast to endometrial and colorectal carcinoma, mutations of the DNA mismatch repair (MMR) genes hMLH1 or hMSH2 have not been described in gastric carcinoma. However, hypermethylation of the hMLH1 MMR gene promoter is quite common in MSI-positive endometrial and colorectal cancers. This hypermethylation has been associated with hMLH1 transcriptional blockade, which is reversible with demethylation, suggesting that an epigenetic mechanism underlies hMLH1 gene inactivation and MMR deficiency. Therefore, we studied the prevalence of hMLH1 promoter hypermethylation in a total of 65 gastric tumors: 18 with frequent MSI (MSI-H), 8 with infrequent MSI (MSI-L), and 39 that were MSI negative. We found a striking association between hMLH1 promoter hypermethylation and MSI; of 18 MSI-H tumors, 14 (77.8%) showed hypermethylation, whereas 6 of 8 MSI-L tumors (75%) were hypermethylated at hMLH1. In contrast, only 1 of 39 (2.6%) MSI-negative tumors demonstrated hMLH1 hypermethylation (P<0.0001 for MSI-H or MSI-L versus MSI-negative). Moreover, hypermethylated cancers demonstrated diminished expression of hMLH1 protein by both immunohistochemistry and Western blotting, whereas nonhypermethylated tumors expressed abundant hMLH1 protein. These data indicate that hypermethylation of hMLH1 is strongly associated with MSI in gastric cancers and suggest an epigenetic mechanism by which defective MMR occurs in this group of cancers.

Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia.

The adenomatous polyposis coli (APC) tumor suppressor gene is mutationally inactivated in both familial and sporadic forms of colorectal cancers. In addition, hypermethylation of CpG islands in the upstream portion of APC, a potential alternative mechanism of tumor suppressor gene inactivation, has been described in colorectal cancer. Because a subset of both gastric and colorectal cancers display the CpG island methylator phenotype, we hypothesized that epigenetic inactivation of APC was likely to occur in at least some gastric cancers. APC exhibits two forms of transcripts from exons 1A and 1B in the stomach. Therefore, we investigated CpG island methylation in the sequences upstream of exons 1A and 1B, i.e., promoters 1A and 1B, respectively. We evaluated DNAs from 10 gastric cancer cell lines, 40 primary gastric cancers, and 40 matching non-cancerous gastric mucosae. Methylated alleles of promoter 1A were present in 10 (100%) of 10 gastric cancer cell lines, 33 (82.5%) of 40 primary gastric cancers, and 39 (97.5%) of 40 noncancerous gastric mucosae. In contrast, promoter 1B was unmethylated in all of these same samples. APC transcripts from exon 1A were not expressed in nine of the 10 methylated gastric cancer cell lines, whereas APC transcripts were expressed from exon 1B. Thus, expression from a given promoter correlated well with its methylation status. We conclude that in contrast to the colon, methylation of promoter 1A is a normal event in the stomach; moreover, promoter 1B is protected from methylation in the stomach and thus probably does not participate in this form of epigenetic APC inactivation.

Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia.

A significant portion of gastric cancers exhibit defective DNA mismatch repair, manifested as microsatellite instability (MSI). High-frequency MSI (MSI-H) is associated with hypermethylation of the human mut-L homologue 1 (hMLH1) mismatch repair gene promoter and diminished hMLH1 expression in advanced gastric cancers. However, the relationship between MSI and hMLH1 hypermethylation has not been studied in early gastric neoplasms. We therefore investigated hMLH1 hypermethylation, hMLH1 expression and MSI in a group of early gastric cancers and gastric adenomas. Sixty-four early gastric neoplasms were evaluated, comprising 28 adenomas, 18 mucosal carcinomas, and 18 carcinomas with superficial submucosal invasion but clear margins. MSI was evaluated using multiplex fluorescent PCR to amplify loci D2S123, D5S346, D17S250, BAT 25 and BAT 26. Methylation-specific PCR was performed to determine the methylation status of hMLH1. In two hypermethylated MSI-H cancers, hMLH1 protein expression was also evaluated by immunohistochemistry. Six of sixty-four early gastric lesions were MSI-H, comprising 1 adenoma, 4 mucosal carcinomas, and 1 carcinoma with superficial submucosal invasion. Two lesions (one adenoma and one mucosal carcinoma) demonstrated low-frequency MSI (MSI-L). The remaining 56 neoplasms were MSI-stable (MSI-S). Six of six MSI-H, one of two MSI-L, and none of thirty MSI-S lesions showed hMLH1 hypermethylation (P<0.001). Diminished hMLH1 protein expression was demonstrated by immunohistochemistry in two of two MSI-H hypermethylated lesions. hMLH1 promoter hypermethylation is significantly associated with MSI and diminished hMLH1 expression in early gastric neoplasms. MSI and hypermethylation-associated inactivation of hMLH1 are more prevalent in early gastric cancers than in gastric adenomas. Thus, hypermethylation-associated inactivation of the hMLH1 gene can occur early in gastric carcinogenesis.

Modification of fatty acid composition in adipocyte plasma membranes by an oral treatment with a new antidiabetic agent, AS-6, in genetically obese diabetic mice, db/db.

Male 12-week-old C57BL/KsJ db/db mice were treated for 1 week with a dietary admixture of an experimental antidiabetic agent, AS-6 (4-O-carboxymethylascochlorin, 0.1%). The fatty acid composition of the adipose tissue and its plasma membranes in the treated mice was compared with that in untreated db/db mice and their lean littermates. The results indicate that, when compared with the lean, the db/db adipose tissue and its plasma membrane are extremely rich in nonessential fatty acids, and AS-6 treatment modifies the fatty acyl composition only in the membranes in which 16:1 and 18:1 increase and C18 decreases.

An ascochlorin derivative, AS-6, reduces insulin resistance in the genetically obese diabetic mouse, db/db.

An ascochlorin derivative, AS-6, is a new hypoglycemic agent orally active in both obese hyperinsulinemic and insulin-deficient diabetic animal models. AS-6, when given as a 0.025-0.2% admixture in the diet, dose-dependently ameliorated polydipsia, polyuria, and glycosuria in the genetically obese diabetic mouse, C57BL/KsJ db/db, while neither insulin nor tolbutamide showed any beneficial effects. The amelioration by AS-6 was associated with a marked decrease in serum glucose and triglyceride. The effects persisted at least 10 wk, accompanied by a steady decrease in drinking water consumption. The chronic treatment prevented pancreatic islet degeneration, e.g., degranulation of the beta-cells, basophilic appearance of the exocrine border around the islets, and small round cell infiltration. The isolated islets from AS-6-treated mice released much more insulin in response to glucose than those from untreated controls. A significant correlation between serum immunoreactive insulin and glucose/triglyceride from both treated and untreated mice suggests that AS-6 restores sensitivity and responsiveness to insulin to the mice. In fact, the combined treatment with insulin synergistically decreased serum glucose by 50% below AS-6 treatment alone. Furthermore, the epididymal fat pad slices from AS-6-treated db/db mice increased CO2 generation and lipogenesis over the untreated controls, and the glucose metabolic rate (CO2 generation plus lipogenesis from U-[14C]-glucose) in the slices and the serum glucose level inversely correlated at r = 0.8799.(ABSTRACT TRUNCATED AT 250 WORDS)

Inducible nitric oxide synthase and tumor necrosis factor-alpha in myocardium in human dilated cardiomyopathy.

OBJECTIVES: We examined the mRNA expression and protein localization of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) in myocardial tissue obtained from patients with dilated cardiomyopathy (DCM). BACKGROUND: The etiology of DCM is unknown, but viral infection or autoimmune abnormalities that induce cytokine expression have been proposed as pathogenetic factors. Nitric oxide (NO), synthesized by nitric oxide synthase (NOS), has negative inotropic and cytotoxic effects on cardiomyocytes. Cytokines such as TNF-alpha are potent stimulators of iNOS expression. Expression of iNOS leads to excessive production of NO in the myocardium and may modulate cardiac contractility and ventricular morphology. METHODS: We examined the mRNA expression and protein localization of iNOS and TNF-alpha in myocardial tissue obtained from 24 patients with DCM, 20 patients with hypertrophic cardiomyopathy (HCM) and 15 control subjects, using the reverse transcriptase-polymerase chain reaction method and immunohistochemical studies. We then compared the differences in clinical characteristics between DCM patient subgroups with and without myocardial iNOS expression. RESULTS: Messenger RNA expression of iNOS and TNF-alpha was observed, respectively, in 13 (54%) and 18 (75%) patients with DCM. Gene expression of TNF-alpha was consistently detected in endomyocardial tissue from patients with DCM and INOS expression. Inducible NOS protein was evident only in cardiomyocytes, whereas TNF-alpha was apparent in both cardiomyocytes and endomyocardial endothelium. Neither mRNA expression nor protein localization of iNOS or TNF-alpha was observed in cardiac tissue obtained from patients with HCM or control subjects. Patients with DCM and iNOS mRNA showed a lower left ventricular ejection fraction (p < 0.01) and a higher left ventricular volume (p < 0.05) than the negative DCM group. CONCLUSIONS: Inducible NOS was consistently coexpressed with TNF-alpha in myocardial tissue obtained from a subgroup of patients with DCM and advanced left ventricular dysfunction.

Production and pharmacologic modulation of the granulocyte-associated allergic responses to ovalbumin in murine skin models induced by injecting ovalbumin-specific Th1 or Th2 cells.

Because interferon-gamma, interleukin-4, and interleukin-5 have been identified at the mRNA and protein levels in the lesional skin of patients with atopic dermatitis, we investigated the roles played by granulocytes as effector cells in allergic inflammation by using two unique murine skin models. In vitro generated Th1 and Th2 cells from naïve splenocytes of antiovalbumin T cell receptor transgenic BALB/C mice were adoptively transferred with ovalbumin into the ear pinnae or air-pouches produced in the back skin of naïve, nontransgenic BALB/C mice. The injection of Th1 cells with ovalbumin induced delayed type ear swelling that peaked at 48 h, whereas that of Th2 resulted in ear swelling that peaked at a much earlier time, 24 h. Histologic study of the swollen ear skin and granulocytes recruited into the air-pouch demonstrated that, although the Th1-induced inflammation caused a neutrophil-predominant infiltrate with few eosinophils, larger numbers of eosinophils accumulated in the Th2-induced inflammation. Using these murine models, we further evaluated the effects of drugs used for the treatment of atopic diseases. The results showed that FK506 administration could effectively reduce skin inflammation induced by either Th cells. Interestingly, the neutrophil elastase inhibitor ONO-6818 efficiently inhibited Th1-induced inflammation. In contrast, a leukotriene receptor antagonist, ONO-1078, specifically suppressed Th2-induced inflammation. We also found that each ONO drug exerted direct influence on specified granulocytes, as neither affected in vitro production of relevant Th cytokines. Thus, we succeeded in developing animal skin inflammation models in which we can evaluate the contribution of protein antigen-specific Th1 or Th2 cells through the action of granulocytic effector cells.

Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae.

The glucoamylase-encoding gene (glaA) from Aspergillus oryzae was cloned using its cDNA as a probe, which had been isolated previously. From comparison of nucleotide (nt) sequences of genomic clones with its cDNA, the glaA gene was found to contain four short putative introns, 45-56 nt in length. The A. oryzae glaA gene shared 62% homology at the nt level with the A. niger glaA gene with the four introns located at the same position. The 5'-flanking region contained a TATA box at nt-72 from the start codon, and two putative CAAT sequences at nt-87 and -331. Genomic Southern analysis and physical mapping showed that the glaA gene is located on the smallest chromosome (3.4 Mb) of six separated bands of chromosomes. Clones containing the glaA gene, when re-introduced intro A. oryzae, resulted in a three- to eightfold increase in glucoamylase activity.