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H Kohsaka

Publications and source records attributed to H Kohsaka.

At least 19 recordsLinked to original sources
PubMed1997

Evidence for self and nonself peptide partial agonists that prolong clonal survival of mature T cells in vitro.

When examining the effects of peptide analogues without proliferation-inducing activity on three human CD4+ T cell clones with distinct TCRbeta recognizing a nonself mycobacterial bacillus Calmette-Guerin a (BCGa) peptide fragment (EEYLILSARDVLAVVSK)/HLA-DR14 complex, we found that 1) stimulation of T cells with a one-residue-substituted analogue or a minimally homologous self peptide fragment derived from human connexin 26 (IMILVVAAKEVWGDEQA) can prolong the in vitro survival of T cells, in a clone specific-manner; 2) this prolongation is associated with the up-regulation of Bcl-xL without proliferation; and 3) these peptide-clone combinations are capable of inducing lymphokine secretion. Thus, peptide partial agonism may play a role in the survival of not only thymocytes but also mature T cells, in the absence of wild-type ligands.

CD4-Positive T-Lymphocytes↗
PubMed1997

Double-negative BV8S3 T cells expanded in MRL lpr/lpr lymph nodes conserve TCRBJ gene usage of single-positive BV8S3 T cells.

Enlarged lymph nodes of mice with lpr mutation consist predominantly of CD4- CD8- (double-negative: DN) T cells. Among them, TCRBV8S3 (V beta 8.3) T cells are overrepresented as compared to those in single-positive (SP) T cells. To address the question of whether the expansion of oligoclonal T cells is responsible for the increase in TCRBV8S3 cells, we examined the TCRBJ gene repertoires of BV8S3 DN and SP T cells from multiple MRL lpr/lpr mice. The BJ repertoires of BV3 (V beta 3), BV8S1 (V beta 8.1) and BV8S2 (V beta 8.2) were studied for comparison with those of BV8S3 T cells. The employed method, which was based on a PCR-ELISA technique, was newly developed and allowed us to make a precise quantitation of TCRBJ gene usage of the multiple lymphocyte samples. The results showed that there were no biases of the BJ gene usage by BV8S3 DN T cells as well as other BV T cells. Furthermore, the BJ gene usage of CD4 and CD8 BV8S3 T cells was conserved by the DN T cells. It is suggested that the BV8S3 DN T cells were not expanded by specific antigens. The expansion may result from aberrant regulation specific to the BV8S3-expressing T cells.

Animals↗
PubMed1997

[A refractory case of adult-onset Still's disease].

We report here a case of adult-onset Still's disease (AOSD), who finally responded to a combination of cyclophosphamide (CPA) and gold sodium thiomalate (GST) after two years of active disease. A 23-year-old man having continuous high fever with skin rash, polyarthralgia and increased serum ferritin, was diagnosed as AOSD, and oral corticosteroid was initially effective. His symptoms recurred one year later without clinical improvement to increased dosage of steroid. He was admitted to our hospital with pericarditis and pleural effusion but did not respond to either intravenous (i.v.) pulse steroid therapy, methotrexate (MTX) or high dose i.v. gamma-globulin. He was partly responsive to monthly i.v. injection of CPA, but clinical symptoms did not completely subside and hyperferritinemia persisted. GST, initiated in combination with CPA, however, was successful to induce complete remission. MTX has recently been reported to be efficacious to steroid-resistant AOSD, but CPA and gold compounds might be useful to refractory case of AOSD.

Adult↗
PubMed1996

The human immunoglobulin V(H) gene repertoire is genetically controlled and unaltered by chronic autoimmune stimulation.

The factors controlling immunoglobulin (Ig) gene repertoire formation are poorly understood. Studies on monozygotic twins have helped discern the contributions of genetic versus environmental factors on expressed traits. In the present experiments, we applied a novel anchored PCR-ELISA system to compare the heavy chain V gene (V(H)) subgroup repertoires of mu and gamma expressing B lymphocytes from ten pairs of adult monozygotic twins, including eight pairs who are concordant or discordant for rheumatoid arthritis. The results disclosed that the relative expression of each Ig V(H) gene subgroup is not precisely proportional to its relative genomic size. The monozygotic twins had more similar IgM V(H) gene repertoires than did unrelated subjects. Moreover, monozygotic twins who are discordant for RA also use highly similar IgM V(H) gene-subgroup repertoires. Finally, the V(H) gene repertoire remained stable over time. Collectively, these data reveal that genetic factors predominantly control V(H) gene repertoire formation.

Adult↗
PubMed1996

Ceramide induces apoptosis via CPP32 activation.

Although both ceramide and interleukin-1beta converting enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report here that cell-permeable ceramide induced cleavage and activation of CPP32, a Ced-3/ICE-like protease, but not ICE. Ceramide-induced apoptosis of Jurkat cells was blocked by the CPP32-specific tetrapeptide inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO. Furthermore, variant Jurkat cells with defective CPP32 activation were resistant to both anti-Fas- and ceramide-induced apoptosis. These results indicate that CPP32 activation is required for ceramide-induced apoptosis, and suggest sphingomyelin-ceramide pathway functions upstream of CPP32.

Apoptosis↗
PubMed1996

Genetic control of T cell receptor BJ gene expression in peripheral lymphocytes of normal and rheumatoid arthritis monozygotic twins.

The amino acids encoded at the junctions of T cell receptor (TCR) V and J genes directly interact with MHC bound peptides. However, the regulation of the human TCRBJ gene repertoire has been difficult to analyze, because of the potentially complex number of BJ gene rearrangements. To overcome this problem, we developed a PCR-ELISA method to study BJ gene expression, and compared peripheral T lymphocytes from 12 pairs of monozygotic twins, including 6 rheumatoid arthritis (RA) discordant pairs, and 5 normals. Analyses of the TCRBV5, 13 and 17 gene families, which have been reported to be increased in RA patients, showed: (a) the three TCRBV transcripts have common features of BJ gene usage; (b) TCR transcripts from each TCRBV family display a distinctive BJ gene profile, which is displayed better by CD4+ than CD8+ lymphocytes; (c) the BJ gene repertoires of monozygotic twins are more similar than those of unrelated individuals; and (d) the inflammation of RA does not induce specific changes in the genetically determined pattern of BJ expression. These results indicate that the frequency of expression particular TCRBV-TCRBJ recombinants in human lymphocytes is controlled genetically, and is maintained despite the presence of a chronic inflammatory disease.

Adult↗
PubMed1996

The presence of costimulatory molecules CD86 and CD28 in rheumatoid arthritis synovium.

OBJECTIVE: To investigate the expressions of co-stimulatory molecules CD86 (B7-2, B70) and CD28 by cells obtained from the synovial tissues (ST) and synovial fluids (SF) of patients with rheumatoid arthritis (RA). METHODS: Monoclonal antibodies (MAb) against CD86 and CD28 were used for immunochemical study of synovia from 18 RA patients, 4 osteoarthritis (OA) patients, and 4 normal subjects. These MAb were also used for flow cytometry of isolated ST cells from 8 RA and 5 OA patients and of SF mononuclear cells from 5 RA and 5 OA patients. RESULTS: Immunohistochemical examination revealed that CD86+ cells occurred in 11 of the 18 RA synovia, but in none of the 4 OA or 4 normal synovia. Most of the positive cells had macrophage-like morphology, and surrounded lymphoid aggregates. Most cells within lymphoid aggregates were stained positively for CD28. Flow cytometry showed that CD86+ cells comprised 2.9-33.4% (average 14.3%) of the total ST cells and 2.1-14.9% (average 6.1%) of the total SF mononuclear cells from RA patients. Approximately 40% of the CD86+ cells expressed CD14. A majority (mean 72%, range 57-89%) of the T cells in ST and SF expressed CD28. RA synovia expressed more CD86 molecules than did OA synovia (mean frequency of positive cells 14.3% versus 2.8%; mean fluorescence intensity 104.6 versus 40.9).

Antigens, CD↗
PubMed1996

Shared amino acid motifs in T-cell receptor beta junctional regions of bronchoalveolar T cells in patients with pulmonary sarcoidosis.

To clarify the nature of the accumulated lung T cells of pulmonary sarcoidosis, we studied bronchoalveolar and peripheral blood (PB) lymphocytes from Japanese patients for expressed T-cell receptor beta-chain variable (TCR V beta) gene usage. Quantitative polymerase chain reaction technique revealed that the V beta repertoires of bronchoalveolar and PB T cells were heterogeneous. However, the bronchoalveolar lymphocytes from five out of 10 patients showed overrepresentation of T cells, with a few TCR V beta families, which varied among individuals, possibly reflecting a difference in the human leukocyte antigen (HLA) backgrounds. Nucleotide sequence analysis of third complementarity determining region (CDR3) segments in overrepresented V beta families demonstrated limited diversity of the transcripts. Two HLA-DR 12(5)+ individuals with overrepresented V beta 6 gene transcripts in the lungs shared a common amino acid motif within their CDR3. By contrast, non-overrepresented TCR V beta transcripts did not show clonal expansion, and the oligoclonal clones in the lung were not found in PB of the corresponding patient. These results showed that, at least in subsets of Japanese patients with pulmonary sarcoidosis, a limited number of T-cell clones are accumulated in the lung in response to a specific antigen. They may mediate an immune response to shape pathology of the disease.

Adult↗
PubMed1996

[Two cases of lupus nephritis having a high titer of anti-(histone-DNA) complex antibody without IgG anti-dsDNA antibody].

IgG antibody specific to double-stranded DNA (dsDNA) has been recognized as a predictive indicator of the renal involvement in systemic lupus erythematosus. However, we recently experienced two cases of overt lupus nephritis without IgG anti-dsDNA antibody. In both cases, high titers of antibody to the histone dimer (H2A-H2B)-dsDNA complex were detected. They responded well to the corticosteroid therapy, with a cytotoxic agent in one case, and the titers of anti-(histone-DNA) antibody was decreased along with the improvement of proteinuria. Based on the recent reports suggesting a pathogenic role of this antibody in lupus nephritis, we suggest that measurement of the anti-(histone-DNA) antibody is necessary in lupus nephritis patients, especially when IgG anti-dsDNA antibody is not detectable.

Adult↗
PubMed1996

Influence of the rheumatoid arthritis-associated shared epitope on T-cell receptor repertoire formation.

Rheumatoid arthritis is associated with several human leukocyte antigen DRB1 types that express a common five-amino acid sequence called the shared epitope. Here we show that the human leukocyte antigen DRB1 shared epitope expands naive T lymphocytes that express the same T-cell-receptor variable region-joining region combinations that are prevalent in the synovia of rheumatoid arthritis patients. Thus, the shared epitope could affect rheumatoid arthritis disease susceptibility by selecting in the premorbid state specific T-cell subsets that contribute to synovial inflammation.

Arthritis, Rheumatoid↗
PubMed1995

Analysis of immunoglobulin VH gene repertoire by an anchored PCR-ELISA.

We developed a novel technique to analyze the relative concentration of the expressed immunoglobulin (Ig) VH genes using an enzyme-linked immunosorbent assay (ELISA). Expressed Ig cDNA are amplified via anchored PCR and then subjected to a "nested PCR" reaction that attaches biotin to the 5' end of the antisense strand. This allows us to tether the antisense strand of PCR products onto avidin-coated ELISA plates. Digoxigenin-labeled oligonucleotides specific for the leader sequence sense strand of each major Ig VH gene subgroup are used to probe the plate-tethered, alkaline-denatured, and single-stranded antisense cDNA. Bound probes then are detected with alkaline-phosphatase-conjugated anti-digoxigenin antibodies. Using this method, we assessed the distribution of Ig VH genes used by IgM-expressing blood B cells of normal adults. We found the predominant subgroup is VH3, representing approximately half (range 41-59%) of the expressed IgM repertoire. The next largest subgroups used are VH4 (19-23%), VH1 (15-17%), and VH5 (7-11%). The VH2, VH6, and VH7 subgroups each constitute less than 3% of the expressed IgM repertoire. These results agree with those obtained using traditional and more laborious methods that analyze the distribution of Ig clones in cDNA libraries. In addition, we find that this method compares favorably in sensitivity and specificity to more conventional techniques for assessing the clonality of blood or tissue B-cell populations. This rapid and nonradioactive method should have utility for assessing the Ig repertoires expressed by normal, autoimmune, or neoplastic B-cell populations.

Adult↗
PubMed1995

Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy.

During the course of gene therapy experiments in rodents, using intramuscular injections of plasmid DNA derived from Escherichia coli, we noted dose-related toxicity. This observation prompted a search for possible contaminants of DNA samples. We used the highly specific and sensitive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioactivity in DNA samples, and found plasmid DNA derived from standard E. coli bacterial strains, using traditional DNA isolation protocols, to be heavily contaminated with endotoxin, or lipopolysaccharide (LPA). Standard DNA isolation procedures resulted in the copurification of up to 500 micrograms/ml of LPS. LPS is a potent inducer of cytokines and other inflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has important implications for in vitro transfection studies and microinjection of DNA into embryos. A simple and efficient protocol to reduce LPS contamination of plasmid DNA was developed. The conversion of intact bacteria to spheroplasts prior to the isolation of plasmid DNA, incubation with lysozyme, treatment with the detergent n-octyl-beta-D-thioglucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This represents a 10,000-fold reduction in LPS contamination, compared to conventional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plasmid DNA.

Animals↗
PubMed1995

Diverse T cell receptor beta gene usage by infiltrating T cells in the lacrimal glands of Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease characterized by T cell infiltration into the salivary and lacrimal glands (LG). Previous studies on T cell receptor (TCR) usage in the minor salivary glands (SG) have yielded controversial results. We studied TCR beta gene usage of the T cells infiltrating to LG, which is the other major target organ of SS. Total RNA was extracted from fresh LG and SG biopsy samples, and peripheral blood mononuclear cells from five SS patients, and converted to cDNA. TCR V beta gene repertoire was then assessed with quantitative polymerase chain reaction (PCR) assay. Oligoclonality was studied by sequencing V-D-J junctional regions of the PCR products. The TCR V beta gene usage in LG was diverse in every patient irrespective of disease duration, and similar to that of peripheral lymphocytes from a corresponding patient. The junctional region sequences of over-expressed V beta families in LG T cells were heterogeneous. We did not find any identical clones shared by LG, SG and peripheral blood. These results showed that the infiltrating T cells in LG of SS patients are polyclonal, and LG and SG do not share the same dominant T cell clonotypes. These suggest that TCR-targeted disease manipulation may have a limited effect on SS.

Aged↗
PubMed1995

Toxocara canis adult worm antigen induces proliferative response of healthy human peripheral blood mononuclear cells.

The proliferative response of human peripheral blood mononuclear cells (PBMC) from healthy donors to Toxocara canis adult worm antigens (TcA) was examined. PBMC from all donors examined (n = 7) strongly responded to TcA in a dose-dependent fashion after six days of culture, irrespective of their serological reactivity. In contrast, cord blood mononuclear cells did not react to TcA. The proliferation of PBMC in response to TcA was completely inhibited by anti-HLA-DR antibody. Purified CD4+ T cells reconstituted with autologous irradiated antigen presenting cells (APC) vigorously proliferated in response to TcA, but this was abrogated by pretreatment of APC with paraformaldehyde. Significant IL-2, IL-3, IL-4, IL-5 and IFN-gamma mRNA expression was detected in PBMC stimulated with TcA, with expression peaking at 72 h after stimulation. IL-1 beta, IL-6, IL-10 and GM-CSF mRNA expression was also upregulated, peaking at 24 h after stimulation. Taken together, these results suggest that adult T. canis-derived antigens have the ability to activate human PBMC as conventional antigens, possibly due to their cross-reactivity, which may be involved in the host defence against helminth infection.

Adult↗
PubMed1995

Nitric oxide production and inducible nitric oxide synthase expression in inflammatory arthritides.

In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P < 0.05). Immunohistochemical analysis as well as in situ hybridization showed that inducible NO synthase was predominantly expressed in synovial lining cells, endothelial cells, chondrocytes, and to a lesser extent, in infiltrating mononuclear cells and synovial fibroblasts. The synovial lining cells and the infiltrating cells expressing inducible NO synthase were identified where CD14+ cells were located. Together with morphological features, this suggests that they are type A synoviocytes. NO production from freshly isolated synoviocytes and chondrocytes was up-regulated by in vitro stimulation with a combination of IL-TNF-beta, TNF-alpha, and LPS. In summary, the present results suggest that NO is produced primarily by CD14+ synoviocytes, chondrocytes, and endothelial cells in inflammatory joints of arthritides. NO production can be upregulated by cytokines present in inflamed joints. The increased NO production may thus contribute to the pathological features in inflammatory arthritides.

Arthritis, Rheumatoid↗
PubMed1994

Competitive RT-PCR ELISA: a rapid, sensitive and non-radioactive method to quantitate cytokine mRNA.

We have developed a non-radioactive method to quantitate precisely levels of gene expression. This method is based on RT-PCR (reverse transcriptase-polymerase chain reaction) with an RNA competitor, followed by the covalent capture of the amplified DNA onto the wells of microtiter plates, and the quantitation of the PCR product by oligonucleotide hybridization and ELISA (enzyme-linked immunosorbent assay). The assay can reproducibly detect 1 zeptomole mRNA. The assay was successfully used to quantitate mRNA levels of the T cell derived cytokines interleukin-2, interleukin-4 and interferon-gamma in resting and stimulated human lymphocytes. Because it is performed in a microtiter ELISA format, this rapid, sensitive and non-radioactive method should facilitate measurements of gene expression, particularly in large clinical studies.

Base Sequence↗