Biomedical subjects

S Nagai

Publications and source records attributed to S Nagai.

At least 19 recordsLinked to original sources

PubMed1996

T cell receptor (TCR) V gene segment use in HLA-typed Japanese healthy subjects.

The expression of 13 different alpha and beta V gene segments of the T cell receptor for antigen (TCR) was examined, using V gene-specific MoAbs, on human peripheral blood T lymphocytes from 32 healthy Japanese subjects. In addition, to examine associations between TCR V gene products and HLA alleles, the HLA class I and class II types of all subjects were serologically determined. The reactivities of the anti-TCR V-specific MoAbs were, with some significant exceptions, similar to those previously described in healthy Caucasian subjects. We found a non-random V gene usage as well as a statistically significant bias of the expression of eight V beta gene products towards the CD4+ subpopulation, and a significant skewness in the usage of V alpha 12 towards the CD8+ population. Some subjects showed increased reactivities (above 10%) of certain MoAbs, mainly in the CD8+ subpopulation. We found no distinct correlation between any certain HLA class I or II allele and TCR V gene usage in the CD8+ or CD4+ subpopulations, respectively. In conclusion, the pattern of anti-TCR V-specific MoAb reactivities found in CD4+ and CD8+ subsets of peripheral blood lymphocytes of healthy Japanese subjects was in general found to match that previously described in healthy Caucasian subjects.

Adult↗

PubMed1996

Heterogenous expression of the related MPB70 and MPB83 proteins distinguish various substrains of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv.

MPB70 and MPB83 are homologous cross-reactive secreted mycobacterial proteins with very limited species distribution. The expression of these two proteins was compared between several substrains of Mycobacterium bovis BCG, virulent M. bovis and Mycobacterium tuberculosis H37Rv. A polyclonal antibody specific for MPB70 in Western blotting, and a monoclonal antibody, MBS43, found to be specific for MPB83 in ELISA and Western blotting, were used for the comparison. The previously established pattern of high- and low-producing substrains of BCG for MPB70 is only partially applicable for MPB83. MPB70 low-producing strains are also MPB83 low-producing, but the expression of MPB83 is much more variable than the expression of MPB70 in the MPB70 high-producing strains. Purified MPB83 (23 kDa) was found to be glycosylated. A band in SDS-PAGE at 1-2 kDa lower than that of purified MPB83 may represent unglycosylated MPB83. Furthermore, it was confirmed that purified MPB70 (22 kDa) is unglycosylated. There is cross-reactive antigen at 26 kDa. The MPB83 related antigen at 26 kDa was found to be the most abundant. These findings indicate greater heterogeneity between different substrains of BCG than previously realized. Virulent M. bovis produce and secrete large amounts of MPB70 and MPB83 while both these proteins occur in a far lower concentration in M. tuberculosis.

Antibody Specificity↗

PubMed1996

Different percentages of peripheral blood gamma delta + T cells in healthy individuals from different areas of the world.

The frequency of gamma delta + T cells in the peripheral blood of 26 Turkish, 24 Swedish, 35 Japanese and 14 "Asian' (non-Japanese) healthy blood donors and healthy volunteers were investigated by flow cytometry. In the Turkish group, 9.3% (median value) of the CD3+ peripheral blood T cells expressed the gamma delta T cell receptor. A similar level of gamma delta + T cells was found in the non-Japanese "Asian' healthy volunteers (9.2%), while significantly lower values were detected in the Swedish (4.2%) and Japanese (4.5%) groups. These dramatic differences in normally occurring gamma delta + T cells in different groups of healthy individuals were further reflected by a low incidence of > 10% gamma delta + T cells in the Swedish (0/24) and Japanese (6/35) groups compared to the Turkish (12/26) and "Asian' (5/14) groups. The described gamma delta + T cell differences between distinct ethnic groups are thus likely to be a consequence of environmental factors, but additional genetic influences cannot be ruled out. The present study demonstrates the potential importance of the ethnic origin and environmental history of subjects examined in studies of gamma delta + T cells-disease relations.

Adolescent↗

PubMed1996

[Chest wall reconstruction using polyester mesh].

From January 1987 through December 1994, we performed chest wall reconstruction using the polyester mesh in 15 patients with lung cancer, 11 with empyema after open drainage, 8 with chest wall tumor and 1 with radiation dermatitis and costal chondritis. Twenty five patients were resected 3 or more ribs. Chest wall defects were reconstructed with the polyester mesh covered with Gore-Tex soft tissue patch. Twenty two cases passed more than a year without signs of infection and follow-up averaged 27.6 months. Polyester mesh was removed due to bronchial fistula (3 cases), deformities (3 cases) and abscess formation (1 case). In these cases, polyester mesh was well incorporated and had no foreign body change. In conclusion, the polyester mesh seems to be a dependable prosthetic material for chest wall reconstruction.

Adult↗

PubMed1996

[Flow cytometric analysis of the DNA content of resected non-small cell lung cancer with reference to long-term follow-up].

We measured the cellular DNA content of paraffin-embedded tumor specimens by flow cytometry from 340 cases of resected non-small cell lung cancer, and investigated the correlation of DNA content and prognosis of these cases with long-term follow-up. These 340 cases were divided into some populations according to pathological stage, histologic type, surgical curativity and N factor, and we compared the prognosis of DNA diploidy cases and DNA aneuploidy cases in each population. DNA aneuploidy cases had a significantly less favorable prognosis than DNA diploidy cases in population of stage I adenocarcinoma, stage IIIA non-small cell lung cancer and N2 cases among stage IIIA non-small cell lung cancer, all after curative operation. But in other populations, there was no significant difference in prognosis between DNA diploidy cases and DNA aneuploidy cases. In conclusion, DNA ploidy pattern is a prognostic factor for survival in patients with stage I adenocarcinoma and N2 cases of stage IIIA non-small cell lung cancer.

Adenocarcinoma↗

PubMed1996

[Pulmonary typical carcinoid with metastases to pulmonary hilar, and mediastinal lymph nodes].

A 50-year-old woman was admitted to our hospital complaining of recurrent bloody sputum and hemoptysis. Chest X-ray films showed an infiltrative shadow in the left lower lung field. Chest computed tomograms showed a nodular tumor shadow near the left B8 and this tumor shadow was found between A8 and A9 by pulmonary arteriography. Bronchoscopic examination revealed a red coagulum in the left B8 and cytologic examination of broncho alveolar lavage fluid revealed atypical squamous cells. With a clinical diagnosis of squamous cell carcinoma of the lung, a left lower lobectomy with hilar and mediastinal lymph node dissection was done. A milk-white tumor was found in the lower lobe of the resected lung. The pathological diagnosis of the resected tissue specimen was carcinoid, accompanied by pulmonary, hilar, and mediastinal lymph node metastases. Reduced immunofluorescence by Chromogranin A and serotonin staining were further evidence that the tumor was atypical. This case is very interesting in that the tumor metastasized to the lung and lymph nodes, even-though it was pathologically typical.

Carcinoid Tumor↗

PubMed1995

Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice.

Our study investigates the recall of immunity in the mouse model of memory immunity to tuberculosis infection. The results provide evidence that recall of immunity is expressed as an accelerated accumulation of potent effector cells in the infected target organs. These effector cells were recruited from the resting pool of memory cells and were immediately triggered to exert their effector functions, leading to a massive release of Th1 cytokines detectable both in splenic extracts and in the serum within the first 24 h of infection. During a primary infection, in contrast, a 14-day delay was observed before significant cytokine levels were reached. After the initial effector phase, the cells blasted and entered into clonal expansion, resulting in a rapid increase in the total number of CD4 CD45RBlow cells in the spleen. The recall of memory immunity was highly efficient and controlled an infectious challenge within the first week. The molecules recognized by the memory effector subset were the proteins secreted from Mycobacterium tuberculosis during growth. By separating the CD4 population into CD45RBhigh and CD45RBlow subsets, the memory effector cells were demonstrated to reside predominantly in the activated population of CD45RBlow CD44high LFA-1high L-selectinlow cells. The key antigenic targets recognized by these cells were identified as Ag85B and a secreted 6-kDa protein (ESAT-6) that elicited the release of exceedingly high levels of IFN-gamma. ESAT-6 was biochemically purified, characterized, and the gene encoding the protein was cloned.

Amino Acid Sequence↗

PubMed1995

Pharmacological study of stem-cell-factor-induced mast cell histamine release with kinase inhibitors.

Stem cell factor (SCF) is a ligand for c-kit receptor and has a critical role in the development of mast cells. In this study, we investigated the effect of a panel of kinase inhibitors on SCF-induced histamine release from rat peritoneal mast cells. Genistein, an inhibitor of tyrosine kinases, inhibited SCF-induced histamine release with IC50 of 1.6 x 10(-5) M. Wortmannin, an inhibitor of phosphatidylinositol 3'-kinase (PI3 kinase), inhibited histamine release stimulated with SCF dose-dependently with IC50 of 4 x 10(-9) M. KT5926, an inhibitor of myosin light chain (MLC) kinase, reduced histamine release with IC50 of 1.8 x 10(-7) M. Staurosporine, an inhibitor of protein kinases, also inhibited SCF-induced histamine release with IC50 of 6.5 x 10(-8) M. These results show the early involvement of tyrosine kinase and PI3 kinase and the possible role of MLC kinase in the late secretory phase in the signaling pathway used by SCF.

Alkaloids↗

PubMed1995

Delayed-type hypersensitivity to a recombinant mycobacterial antigen, MPB64, in guinea pigs sensitized to Mycobacterium tuberculosis or Mycobacterium bovis BCG.

Recombinant MPB64 (rMPB64), a mycobacterial antigen, was obtained from an Escherichia coli clone transformed with a recombinant expression vector, pMAL64c. The rMPB64 was examined for the activity to elicit delayed-type hypersensitivity (DTH) in guinea pigs injected with liver Mycobacterium tuberculosis H37Rv or live M. bovis BCG Tokyo. It was found that rMPB64 has the same reactivity as native MPB64 (nMPB64) or MPT64 (nMPT64) and the potency to elicit DTH was 13.4 times higher than that of PPD. Because MPB64 is secreted only by living M. tuberculosis and some strains of BCG, it is possible to use this antigen for the diagnosis of tuberculosis.

Amino Acid Sequence↗

PubMed1995

Establishment and characterization of human gastric and colonic xenograft lines resistant to CPT-11 (a new derivative of camptothecin).

CPT-11-resistant human gastric and colonic xenograft lines were established by direct intratumoral injection of CPT-11 into subcutaneous SC-1-NU and CC-2-NU tumors in nude mice once a week for 10 months. The resistance of these xenograft lines to CPT-11 was confirmed by growth inhibition rate, to be 36.3% and 45.4%, respectively, compared to each parent cell line. DNA topoisomerase I activity of the nuclear extracts of SC-1-NU/CPT-11 and CC-2-NU/CPT-11, as assayed by relaxation of supercoiled DNA Col-E1, was significantly less than those of the parent lines. The cellular levels of topoisomerase I in those resistant lines measured by Western blot analysis were 0.57- and 0.79-fold lower than those of the parental lines, respectively. However, the activity of DNA topoisomerase II of those resistant cell lines assayed by decatenation of kinetoplast DNA was higher than that of the parental lines and the cellular levels of topoisomerase II in the resistant lines measured by Western blot analysis were 10.8- and 8.1-fold higher than those of the parent lines. Intracellular accumulation of CPT-11 in CPT-11-resistant tumors was not changed as compared to that of the parental lines, but hydrolysis of CPT-11 to more active SN-38 was reduced in the resistant tumors.

Animals↗

PubMed1995

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli.

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3'S), which was identified after purification by a variety of spectroscopic methods.

Amino Acid Sequence↗

PubMed1995

CT and pathological correlation of pulmonary sarcoidosis.

In CT the presence of mediastinal or hilar lymphadenopathies and thickened bronchovascular bundles are landmarks for the diagnosis of pulmonary sarcoidosis. The major CT findings for parenchymal involvement are thickened bronchovascular bundles, large parenchymal nodules, pleural or subpleural nodules, ground-glass opacities, local lung volume loss (distortion of the lung parenchyma), and microscopic and macroscopic honeycombing. The thickened bronchovascular bundles correspond histologically to granulomas, either with or without perigranulomatous fibrosis in the connective tissue sheath around the pulmonary vessels and airways. Conglomerate granulomas are represented on CT by high-attenuation nodules, and the ground-glass opacities are caused by the summation of a number of small granulomas in the interstitium.

Adult↗

PubMed1995

Pulmonary sarcoidosis: population differences and pathophysiology.

Sarcoidosis is characterized as a hyperimmune response to an unknown agent(s) at the lesion sites. Regarding pulmonary lesions, macrophage-T lymphocyte alveolitis precedes an epithelioid cell granuloma formation. In the 35 years since the first International Conference on Sarcoidosis, the definition of sarcoidosis has been revised several times, and many new pathophysiologic mechanisms have been proposed. In this review, we focused on four important issues: (1) possible population differences, (2) possible causative agent(s) and the current problems of disease susceptibility or resistance, (3) recent data on pathogenesis (including a genetic approach) and pathophysiologic processes, and (4) monitoring of disease activity.

Adult↗

PubMed1995

Homology between the MPB70 and MPB83 proteins of Mycobacterium bovis BCG.

Isolation of MPB83 from Mycobacterium bovis BCG Tokyo culture fluid is described. MPB70 and MPB83 have similar molecular mass as judged by SDS-PAGE but differ in isoelectric points. Peptides isolated after CNBr cleavage of MPB83 revealed extensive homology as well as distinct differences from corresponding parts of the amino acid sequence deduced from the mpb70 gene cloned by Terasaka et al. Antibodies produced by immunization with MPB70 and MPB83 had distinctly different fine specificity revealing cross-reactivity between the proteins. These findings indicate that two distinct, homologous genes code for these proteins. Sensitization with live BCG Tokyo also induced T cell responses to MPB83 with development of delayed type hypersensitivity in guinea pigs.

Amino Acid Sequence↗

PubMed1995

Secretion of MPB64 antigen by a recombinant clone of Mycobacterium smegmatis: characterization and application for the diagnosis of tuberculosis.

MPB64, a specific antigen to Mycobacterium tuberculosis complex (TB complex), was produced and secreted by a clone of M. smegmatis-MPB64 where the structural gene of MPB64 was inserted using a new mycobacteria, E. coli shuttle plasmid pNIS vector. Antibodies against the recombinant MPB64 (rMPB64) were used for the reverse particle latex agglutination (RPLA) test to detect the MPB64 antigen rapidly. RPLA tests were applied to the shock cultures and the clinical isolates of mycobacteria to identify TB complex. RPLA with anti-MPB64 antibody-coated latex beads completely distinguished TB complex from other mycobacteria. Thus, it is suggested that RPLA with anti-MPB64 antibody would be a new, easy and inexpensive method for the rapid diagnosis of tuberculosis.

Antigens, Bacterial↗

PubMed1995

Identification and functional characterization of thioredoxin of Mycobacterium tuberculosis.

We have previously described a Mycobacterium tuberculosis protein designated MPT46 that was present in culture filtrates. Here we report that the MPT46 protein is thioredoxin of M. tuberculosis. MPT46 is recognized by antibodies to thioredoxin (Trx) of Escherichia coli, and antibodies of MPT46 recognize Mycobacterium leprae Trx. Moreover, MPT46 was shown to have enzymatic activity identical to that of Trx of other species, such as its ability to reduce insulin. These findings identify MPT46 as a functionally active Trx.

Bacterial Proteins↗

PubMed1995

Guinea pig cellular immune responses to proteins secreted by Mycobacterium tuberculosis.

To study the immunological activity of proteins secreted by Mycobacterium tuberculosis, we carried out comparative studies in guinea pigs infected intravenously with 2.5 x 10(3) CFU of this organism or with 2.5 x 10(4) CFU of Mycobacterium bovis BCG. Groups of infected guinea pigs were skin tested with fractions of secreted proteins covering well-defined narrow-molecular-mass regions, or such fractions were used for lymphocyte stimulation experiments. The lymphocyte stimulation experiments showed that the fraction containing proteins with molecular masses below 10 kDa had a superior stimulating capacity in tuberculous guinea pigs whereas the 24- to 30-kDa fraction gave significantly higher skin reactions in this group compared with BCG-vaccinated guinea pigs. A precise mapping within the region from 23 to 35 kDa by using a combination of narrow overlapping fractions and purified proteins enabled the identification of the 24-kDa antigen MPT64 as a molecule specific for tuberculous infection. Thus, MPT64 is a promising candidate for a specific diagnostic skin test reagent for human tuberculosis.

Animals↗

PubMed1995

Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.

Amino Acid Sequence↗