(2S,3S,4R)-1-O-(alpha-D-galactopyranosyl)-2-(N-hexacosanoylamino)- 1,3, 4-octadecanetriol (KRN7000), with an alpha-galactosylceramide structure, is an immunomodulator showing potent antimetastatic activities. In an in vitro study, KRN7000 potently stimulated proliferation and enhanced natural killer activity in spleen cells from BALB/c euthymic mice, but not BALB/c athymic (nu/nu) mice, suggesting that T cells play an important role in these activities. To study the detailed immunostimulatory mechanisms of KRN7000, syngeneic mixed leukocyte reactions were then performed using dendritic cell-enriched fractions (DCEFs) from spleens of BALB/c euthymic and athymic mice. In these studies, KRN7000 enhanced the antigen-presenting cell activities of the DCEFs. In addition, marked production of interleukin-2 and interferon-gamma in BALB/c euthymic mice was observed following treatment with KRN7000 in vitro and in vivo. Because of these strong immunostimulating effects of KRN7000, its antimetastatic effects were also evaluated. Adoptively transferred KRN7000-treated DCEF markedly prolonged the survival time of mice bearing B16 pulmonary metastases. These results demonstrate that KRN7000 may be useful for cancer therapy not only as an antitumor agent but also as an activator of antigen-presenting cells such as dendritic cells, which can be used for adoptive cellular therapy.

Adjuvants, Immunologic↗

PubMed1996

[Relation between the sialic acid/fucose ratio in airway secretions and the degree of airway remodeling in bronchial asthma--reversibility of airflow limitation by beta-agonists and airway remodeling in bronchial asthma].

Airway remodeling caused by inflammation is believed to affect both airway hyperresponsiveness and the reversibility of airflow limitation. Airway remodeling entails changes in the bronchial secretion system. Mucin, one of the glycoprotein components of airway secretions, is a major product of submucosal glands and goblet cells. Airway remodeling causes both qualitative and quantitative changes in much production. We evaluated the relationship of airway remodeling, estimated by the change in glycoprotein in sputum, to airway hyperresponsiveness and to reversibility in patients with bronchial asthma. The %FEV1 after inhalation of a beta agonist was less in patients with severe asthma than in those with mild asthma. The sialic acid/fucose ratio in sputum correlated significantly with the slope of the dose-response curve during inhalation challenge, and with the %FEV1 after beta-agonist inhalation. These results suggest that the sialic acid/ fucose ratio in airway secretions reflects the degree of the airway remodeling in bronchial asthma.

Administration, Inhalation↗

PubMed1996

[Clinical usefulness of helical computed tomography in evaluating chronic obstructive pulmonary diseases].

To assess the usefulness of healical computed tomography (CT) in evaluating pathophysiological abnormalities in chronic obstructive pulmonary disease, we compared the results of helical CT with those of pulmonary-function tests in 46 subjects with pulmonary emphysema. We obtained 3-D images of emphysematous lung tissue by choosing voxels with values less than -930 HU because the mean CT score in 10 normal subjects was -869 +/- 29 HU. For each patient the total lung volume (TLV) was computed from the 3-D image of the entire lung with CT score less than -600 HU, and the volume of emphysematous lung tissue (ELV) was computed from the lung with CT score less than -930 HU. TLV measured on inspiration correlated significantly with TLC (r = 0.601, p < 0.001), and TLV measured on expiration correlated significantly with RV (r = 0.836, p < 0.0001). The difference between inspiratory TLV and expiratory TLV correlated significantly with VC (r = 0.781, p < 0.001). ELV both on inspiration and on expiration correlated significantly with FEV1/FVC (p < 0.001) and with RV (p < 0.01, p < 0.001, respectively). These results suggest that the volume data obtained by helical CT reflect functional abnormalities of the lung. In addition to volume data, the distribution of abnormal lung tissue is easily assessed by helical CT. We conclude that helical CT is useful in evaluating pathophysiological abnormalities in chronic obstructive pulmonary disease.

Humans↗

PubMed1996

[Interactions between bronchial epithelial cells and extracellular matrix proteins].

Attachment and migration of bronchial epithelial cells are important in re-epithelialization after tissue injury. We hypothesized that inflammatory cytokines alter bronchial epithelial cell attachment and migration. To test this hypothesis, we studied the effects of mononuclear-cell-conditioned medium on attachment and migration of bronchial epithelial cells in response to fibronectin in vitro. This medium was prepared from bovine blood mononuclear cells that were stimulated with concanavalin A; it stimulated bronchial epithelial cell migration but inhibited attachment to fibronectin. Sephadex G-75 column chromatography of the medium revealed two peaks of activity for stimulation of migration. Activity in the higher molecular weight peak was partially inhibited by anti-TNF-alpha antibodies. Activity in the low-molecular-weight peak was lipid-extractable, which suggests that it reflected an arachidonate metabolite. We also studied the effect of bovine herpes virus-1 infection on migration of bronchial epithelial cells. Infection with this virus reduced the migration of bronchial epithelial cells; by 6 hours after infection, staining of alpha v beta 3 integrins had become more diffuse and was not localized. Thus, mononuclear cells produce inflammatory cytokines with important effects on the migration of bronchial epithelial cells. Viral infection affects the interactions of bronchial epithelial cells with the extracellular matrix.

Animals↗

PubMed1996

[Video-assisted bedside pleuroscopy under local anesthesia: use of a rigid cystoureteroscope in patients with undiagnosed pleural effusion].

Up to 20% of pleural effusions remain undiagnosed despite history-taking, physical examination, thoracentesis, and percutaneous closed pleural biopsy. The next diagnostic procedure used is often thoracoscopy under general anesthesia in an operating room. We report a technique for beside pleuroscopy and pleural biopsy that can be done without assistance of surgeons. We performed video-assisted pleuroscopy with a rigid cysto-ureteroscope in seven patients with pleural effusion that remained undiagnosed despite extensive clinical evaluation. A sterile 19.8 Fr. rigid cysto-ureteroscope was placed into the pleural space under local anesthesia. Pneumothorax was induced to enhance visualization of the surfaces. Forceps-biopsy specimens were taken of suspicious lesions on the parietal pleural. In three patients the pleural surface appeared smooth and in two the parietal pleural surface was studded. A localized coin-like lesion was seen in one patient, and extensive fibrinogenic adhesions and diffuse opacity of the parietal pleura was seen in another. Using this bedside procedure, we diagnosed pleural tuberculosis in three patients and pleural metastases of adenocarcinoma in one. When done under local anesthesia with a rigid cyst-ureteroscopy, video-assisted pleuroscopy can be a safe and useful diagnostic aid in patients with undiagnosed pleural effusion.

Anesthesia, Local↗

PubMed1995

Loading sequence plays an important role in enhanced load sensitivity of left ventricular relaxation in conscious dogs with tachycardia-induced cardiomyopathy.

BACKGROUND: Left ventricular relaxation rate in the failing heart depends more on the systolic load than in the normal heart. To elucidate the mechanisms for the enhanced load sensitivity of left ventricular relaxation in heart failure, we examined the relative contributions of changes in end-systolic volume and loading sequence to the left ventricular relaxation rate. METHODS AND RESULTS: In seven conscious dogs, the time constant (Td) of left ventricular pressure decay, end-systolic volume, systolic circumferential force, and time to peak force during caval occlusion were compared before and after development of tachycardia-induced heart failure. Rapid ventricular pacing decreased the slope of the end-systolic pressure-volume relation from 4.5 to 2.8 mm Hg/mL (P < .01) and prolonged Td from 33 to 49 ms (P < .01). In normal conditions, caval occlusion reduced end-systolic force (-580 g, P < .01) and end-systolic volume (-7 mL, P < .01) but did not change Td or time to peak force. In heart failure, however, caval occlusion shortened Td (-11 ms, P < .01), with a concomitant decrease in the time to peak force (-30 ms, P < .01), while end-systolic volume and force declined slightly. Consequently, for a comparable reduction in end-systolic force, Td decreased more in heart failure than in normal hearts, suggesting enhanced load sensitivity. Moreover, changes in Td correlated well with those in the time to peak force (r = .79, P < .01) but not with those in end-systolic volume. CONCLUSIONS: Loading sequence rather than elastic recoil seems to play the predominant role in the enhanced load sensitivity of left ventricular relaxation in heart failure.

Animals↗

PubMed1995

Identification and expression of the Neurospora crassa mei-3 gene which encodes a protein homologous to Rad51 of Saccharomyces cerevisiae.

The mei-3 gene of Neurospora crassa encodes a homolog of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins, which are required for recombination and repair of DNA double-strand breaks. To determine the molecular function of MEI3 protein, anti-MEI3 antibody was prepared and used in Western blot analysis. The antibody cross-reacted only with crude extracts prepared from perithecia, the fruiting bodies of Neurospora. The molecular weight of the MEI3 protein was estimated to be 38 kDa. Transformation experiments showed that a DNA fragment longer than previously reported was needed to complement the mei-3 mutation. On sequencing cDNA and genomic DNA, one open reading frame (ORF) was found, which consists of three exons interrupted by two small introns. This ORF encoded a MEI3 protein of 353 amino acids, and the inferred MW of 38 kDa is in good agreement with the results from Western blot analysis. Comparisons of MEI3 with other Rad51 homologs indicated that MEI3 protein contains the two conserved core domains (I and II) generally observed in Rad51 homologs in eukaryotes. Northern blot analysis showed that expression of mei-3 was raised remarkably after UV-irradiation or methyl methanesulfonate (MMS)-treatment. The transcript size was 1.6 kb and this was also larger than was reported previously.

Amino Acid Sequence↗

PubMed1995

Overexpression of prostacyclin synthase inhibits growth of vascular smooth muscle cells.

To define the local effects of prostacyclin (PGI2) on the growth of vascular smooth muscle cells (VSMC), we transfected VSMC with an expression vector harboring the cDNA for PGI2 synthase (PGIS), which catalyzes the rearrangement of prostaglandin H2 to PGI2. Transfection of the human PGIS cDNA into rat VSMC did not affect DNA synthesis under serum-free basal conditions, but it increased PGI2 synthesis and decreased DNA synthesis under serum-stimulated conditions (in the presence of 1 or 5% fetal calf serum). These results demonstrated that locally synthesized PGI2 can exert autocrine and/or paracrine inhibitory effects on VSMC growth. It was also suggested that in vivo transfer of PGIS gene may be useful for the gene therapy for vascular disease such as neointimal hyperplasia.

Animals↗

PubMed1995

Human esophageal carcinomas frequently express the tumor-rejection antigens of MAGE genes.

The human genes MAGE-1 and -3 encode melanoma peptide antigens that are recognized by autologous cytotoxic T lymphocytes. Tumors expressing MAGE genes are potential targets for cancer immunotherapy, because MAGE genes are expressed only in tumor tissue and not in any normal tissue except testis and placenta. However, little is known about MAGE gene expression in human esophageal carcinoma. The purpose of this study was therefore to analyze MAGE gene status in human esophageal carcinoma. We studied the expression status of these genes in 42 surgical samples and in 12 cell lines of human esophageal carcinoma using the reverse transcription polymerase chain reaction (RT-PCR). Various clinicopathological factors were also analyzed. No MAGE gene expression was seen in any of the 42 normal esophageal tissue specimens. In contrast, tumor tissue expressed MAGE-1, -2, and -3 in 26, 18 and 24 specimens, respectively. Thirty-three of 42 tumors expressed at least one MAGE gene. Significant clinicopathologic differences between the tumors were not observed, regardless of the presence or absence of MAGE gene expression. In cell lines, MAGE-1, -2, and -3 gene expression was recognized in 5, 4 and 4 cell lines, respectively. This study demonstrates that MAGE genes are frequently expressed in clinical samples as well as in cell lines of esophageal carcinoma. The identification of MAGE genes, therefore, may open up a new modality of treatment, namely specific immunotherapy, for patients with esophageal carcinoma.

Aged↗

PubMed1995

[Emergency drug therapy of bronchial asthma].

Explore the source record for details and available documents.

Adrenergic beta-Agonists↗

PubMed1995

A non-radioactive DNA sequencing method using biotinylated dideoxynucleoside triphosphates and delta Tth DNA polymerase.

We synthesized a set of four biotinylated dideoxynucleoside triphosphates (biotin-9-ddNTPs) and optimized the reaction conditions for non-radioactive cycle sequencing using modified Tth DNA polymerase (delta Tth) and a chemiluminescent detection system. The resulting sequencing ladders showed lower background compared to those with the conventional non-radioactive sequencing method which uses 5'-biotinylated primers, especially when PCR products were analysed. With our method, DNA sequences can be determined at any primer positions without preparing 5'-biotinylated primers for dideoxy chain-termination.

Biotin↗

PubMed1995

Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element.

There exist two distinct isozymes of prostaglandin-endoperoxide synthase (PES). PES-2 mRNA is synergistically induced by lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol-13-acetate (TPA) in bovine arterial endothelial cells. On the other hand, PES-1 mRNA is constitutively expressed under these conditions. Therefore, the promoter activities of the human genes for PES-1 and -2 in bovine arterial endothelial cells were examined. The 5'-flanking region of the human PES-2 gene (nucleotides -327 to +59) showed promoter activity inducible by LPS and TPA using transient transfection analysis, whereas that of the PES-1 gene (nucleotides -1010 to +69) showed constitutive promoter activity. Destruction of both consensus sequences for the nuclear factor responsible for the interleukin-6 expression (NF-IL6) site (nucleotides -132 to -124) and the cyclic AMP response element (CRE) (nucleotides -59 to -53) of the human PES-2 gene markedly reduced the promoter activity (25%) of the PES-2 gene after combined treatment with LPS and TPA, although single destruction of the NF-IL6 site or the CRE slightly reduced the promoter activity (60 or 90%, respectively). Moreover, cotransfection experiments showed that a trans-acting factor, CCAAT enhancer binding protein delta (C/EBP delta), which binds to both the NF-IL6 site and the CRE, increased the promoter activity of the PES-2 gene mainly through the CRE. C/EBP delta mRNA was rapidly induced by LPS. Collectively, these results suggest that transcription of the PES-2 gene in vascular endothelial cells is regulated through combination of the NF-IL6 site and the CRE and that C/EBP delta functions as one of the trans-acting factors.

Animals↗

PubMed1995

Characterization of the binding sites for [3H]glibenclamide in rat liver membranes.

The specific binding sites for sulfonylureas in the rat liver membrane fraction were demonstrated and characterized. [3H]Glibenclamide binding to the liver membrane was specific, time- and temperature-dependent, and reversible. Scatchard analysis showed a single class binding site. The dissociation constant (Kd) for glibenclamide was 1.1 microM and the binding capacity (Bmax) was 50 pmol/mg protein. [3H]Glibenclamide binding could be displaced by other sulfonylureas. Half-maximal inhibition of binding (IC50) for glimepiride, gliclazide, acetohexamide, tolbutamide and chlorpropamide was 4.2 microM, 74 microM, 0.33 mM, 0.60 mM, 1.2 mM, respectively. Each value is close to the reported blood concentration when a therapeutic dose of each drug is administered orally. The order of IC50 values is coincident with the order of potency of the clinical hypoglycemic effect of these drugs. We had shown that these concentrations of sulfonylureas stimulate 6-phosphofructo-2-kinase in the liver or hepatocytes and inhibit phosphoenolpyruvate carboxykinase in the hepatoma cells. The specific binding sites demonstrated here may play some roles when sulfonylureas affect carbohydrate metabolism in the liver.

Animals↗

PubMed1995

Tumorigenicity of EBNA2-transfected cells.

The Epstein-Barr virus nuclear antigen 2 (EBNA2) gene is thought to be important for transformation by Epstein-Barr virus (EBV), but the mechanism of this transformation is little understood. Here, to examine the transforming ability of EBNA2, we transfected a rat fibroblast cell line F2408 with a recombinant EBNA2 expression plasmid and examined cell morphology, colony formation in soft agar, and tumorigenicity in nude mice. The morphology of transfected clones was similar to those of untransfected cells, but two of seven clones grew in soft agar, and four clones of seven clones reproducibly formed tumors in nude mice. These four clones showed EBNA2 expression, but non-tumorigenic clones did not. These results indicate that the expression of EBNA2 is correlated with tumorigenicity.

Animals↗