Biomedical subjects

H Kohsaka

Publications and source records attributed to H Kohsaka.

At least 19 recordsLinked to original sources

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↗

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↗

PubMed1994

Solid-phase polymerase chain reaction.

The polymerase chain reaction (PCR) has facilitated the diagnosis of infectious diseases and genetic disorders, because of its ability to amplify minute amounts of nucleic acids. To distinguish genes of interest from nonspecifically amplified DNA, PCR products commonly are fractionated by electrophoresis, transferred to membranes, and then probed with a labeled internal sequence-specific oligonucleotide. Alternatively, the PCR products have been labeled directly, and hybridized to immobilized oligonucleotide probes. These methods require the tedious physical transfer of the PCR products. In order to amplify and immobilize genes simultaneously, we have developed a simple solid-phase PCR method. The technique enabled us to detect the HIV envelope gene readily without any transfer of amplified DNA.

Base Sequence↗

PubMed1993

Induction of antibodies to a kappa V region by gene immunization.

Direct gene transfer into muscle can lead to sustained gene expression at the injected sites. Here we tested the ability of in vivo gene transfection to immunize mice against an isolated human IgV region. The Humkv325 germline kappa L chain V gene encodes the kappa L chain in V regions of several human IgM autoantibodies and is used frequently in chronic lymphocytic leukemia. This gene was inserted into a mammalian expression vector, pREP7, to produce pREVk3. Mice injected i.m. with pREVk3 produced antibodies against the V region of Glo, a human monoclonal IgM paraprotein whose kappa L chain is encoded by Humkv325. Co-injection of an expression vector encoding IL-2 enhanced anti-Glo antibody production fivefold and induced a localized delayed hypersensitivity reaction. Antibody production also was induced when vectors encoding Humkv325 and IL-2 were injected s.c. These experiments demonstrate that gene immunization vectors can stimulate immune responses to antibody V region determinants.

Animals↗

PubMed1993

Microtiter format gene quantification by covalent capture of competitive PCR products: application to HIV-1 detection.

We have developed a simple gene quantification system using the competitive polymerase chain reaction (CPCR) followed by microtiter format analysis. CPCR is carried out using a mutant competitor with the same size as the target DNA product, and a minimal base exchange to insure the same amplification kinetics. One primer is aminated at the 5' end to produce PCR products that are captured onto carboxylated wells of microtiter plates through peptide bond formation. The non-aminated DNA strands are stripped off from the wells by alkali washing, and the remaining aminated strands are hybridized with either a digoxigenin-labeled wild type-specific oligonucleotide probe or a competitor-specific probe. To standardize the hybridization conditions of the probes, a DNA construct containing wild type and mutant competitor sequences in tandem is captured at different concentrations, hybridized with the probes, and used to generate a standard curve. Bound probes are detected by anti-digoxigenin antibody conjugated with peroxidase and chromogen. Optical densities are recorded with a conventional microtiter plate reader and converted to concentrations according to the standard curves. The ratios of wild type DNA to mutant competitor are used to determine the initial amounts of wild type DNA in the samples. This method was used successfully to quantify human immunodeficiency virus type 1 (HIV-1) env gene in human lymphocytes. It only requires a thermal cycler and a conventional microtiter plate reader, and can be readily done on a large scale. Potential applications include detection of other pathogens, diagnosis of genetic disorders and studies of gene expression.

Base Sequence↗

PubMed1993

Divergent T cell receptor gamma repertoires in rheumatoid arthritis monozygotic twins.

OBJECTIVE: To determine if the expressed T cell receptor (TCR) gamma repertoire is altered in rheumatoid arthritis (RA). METHODS: Peripheral blood lymphocytes were collected from monozygotic twins who were either concordant or discordant for RA, or from a normal twin pair. TCR gamma-specific complementary DNA libraries were constructed using the anchored polymerase chain reaction. Gene usage was analyzed by plaque hybridization and sequencing. RESULTS: The expressed TCR V gamma repertoires both in RA patients and normal subjects were extremely diverse. Monozygotic twins who were concordant for RA expressed very different frequencies of TCR V gamma genes. CONCLUSION: RA does not lead to a specific clonal expansion or deletion of TCR V gamma genes in peripheral blood.

Adult↗

PubMed1993

The expressed T cell receptor V gene repertoire of rheumatoid arthritis monozygotic twins: rapid analysis by anchored polymerase chain reaction and enzyme-linked immunosorbent assay.

Because of heterogeneity in the outbred human population, it has been difficult to determine the genetic factors that influence the expressed T cell receptor (TcR) repertoire in autoimmune diseases. To overcome this problem, we have developed a combination of anchored polymerase chain reaction (APCR) and enzyme-linked immunosorbent assay (ELISA) that can accurately assess TcR V gene frequencies in numerous clinical samples. The results are independent of amplification efficiency, and V gene usage can be readily analyzed with an ELISA plate reader and associated software. Using this method, the TcR V beta gene repertoires in peripheral lymphocytes from nine sets of identical twins, normal, concordant or discordant for rheumatoid arthritis (RA), were studied. The TcR V beta results were compared with TcR V gamma frequencies in the same specimens as determined by APCR-ELISA and cDNA sequence analysis. The results showed a marked similarity in the TcR V beta gene repertoires between identical twins, compared to unrelated subjects (p < 0.05) whether or not they were concordant or discordant for RA. In contrast, the TcR V gamma gene repertoires in the monozygotic twins differed as much as in controls. The data imply that (a) the human TcR V beta gene repertoire in peripheral blood is genetically controlled, whereas (b) the TcR V gamma gene repertoire is primarily influenced by environmental stimuli, and (c) RA causes no consistent change in TcR V beta repertoire of peripheral blood. The APCR-ELISA method, in the context of large-scale family and population studies, should facilitate a more precise delineation of the genetic factors that regulate human TcR V beta expression.

Adult↗

PubMed1993

Regulation of the mature human T cell receptor gamma repertoire by biased V-J gene rearrangement.

To delineate how gene rearrangement influences the expressed human gamma delta T cell repertoire, we generated T cell receptor gamma (TCR gamma) V domain-specific cDNA libraries from the peripheral lymphocytes of eight donors and sequenced a total of 232 TCR gamma gene transcripts. The libraries consisted of both in-frame and out-of-frame rearranged TCR gamma genes. The in-frame TCR gamma gene transcripts were used to determine the diversity of functional T cells, whereas the out-of-frame transcripts, primarily derived from alpha beta T cells, were used to assess the frequencies of TCR V gamma-J gamma rearrangements in progenitor T lymphocytes. The results showed that both sets of transcripts exhibited strikingly restricted V gamma-J gamma combinations. Only 11 of 40 potential V gamma-J gamma rearrangements were common ( > or = 3% of total). The pattern of gene usage in the functional and nonfunctional transcripts was similar and did not differ markedly among donors. The only exception was the predominance of V gamma 9-JP in potentially functional transcripts from seven of eight individuals. These results show that V gamma-J gamma rearrangement is nonrandom and suggest that the diversity of TCR gamma genes in the functional gamma delta T cell repertoire partly depends upon preferentially rearranged V gamma-J gamma gene combinations. However, the expansion of V gamma 9/V gamma 2 T cells in adult peripheral blood can only be explained by antigenic selection of relatively rare V gamma 9-JP recombinants.

Adult↗

PubMed1991

Production of interleukin-1 beta-like factor with synovial cell growth promoting activity from adult T-cell leukemia cells.

We observed a case of adult T-cell leukemia (ATL) with proliferative synovitis. Culture supernatants from ATL cells (ATL-SN) obtained from the peripheral blood constitutively produced an interleukin-1 (IL-1)-like factor in vitro, as shown by the growth inhibition factor (GIF) assay using the A375 melanoma cell line and the lymphocyte activating factor (LAF) assay using C3H/HeJ thymocytes. Neutralization studies indicated that polyclonal antibodies against IL-1 beta blocked most (80%) of the activity in ATL-SN. In addition, increased amounts of IL-1 beta mRNA were found in the ATL cells by dot blot analysis. Sephacryl S-200 chromatography showed that the molecular weight of this factor was approximately 17.5 kDa, and Western blot analysis revealed that this factor reacted with polyclonal anti-IL-1 beta antibody under the reduced condition. The isoelectric point was 7.5. Furthermore, ATL-SN showed significant activity in promoting the growth of synovial cells in parallel with IL-1 activity. These data suggest that the constitutive production of this IL-1 beta-like factor might be responsible for proliferative synovitis in this case.

Aged↗

PubMed1990

Fine epitope mapping of the human SS-B/La protein. Identification of a distinct autoepitope homologous to a viral gag polyprotein.

To analyze the autoepitopes on the SS-B/La protein, a cDNA covering the entire region coding the protein was isolated from a human cDNA library. The cDNA was subcloned into an expression plasmid vector, pEX, to express its protein product as a fusion protein with cro-beta-galactosidase in Escherichia coli. A recombinant pEX plasmid expressing three-fourths of the protein (amino acid 112-408) was also constructed. The antigenicities of these recombinant proteins were confirmed with a patient's serum. Their various deletion mutants were produced with exonuclease III treatment from the 3' ends of the cDNAs without changing the proper translational frame. Immunoblot analysis and enzyme-linked immunosorbent assay were used to evaluate the reactivities of the recombinant proteins with patients' sera to determine the autoepitopes. A narrow segment (amino acid 88-101) and the region where several epitopes were located (amino acid 283-338) on the SS-B/La protein were universally recognized by all the sera with anti-SS-B/La antibodies examined. An additional epitope region (amino acid 179-220) was recognized by some patients' sera. Computer analysis revealed that the most distinct autoepitope, amino acid 88-101, had a striking homology to a retroviral gag polyprotein. These findings indicate that exogenous or endogenous retroviruses may play a role in initiation of the anti-SS-B/La autoimmunity.

Amino Acid Sequence↗

PubMed1989

Constitutive production of interleukin 6/B cell stimulatory factor-2 from inflammatory synovium.

Interleukin 6 (IL-6) production from synovial tissues of various diseases was examined. Augmented IL-6 production was found in inflammatory synovium not only in RA but also in other kinds of synovitis, including psoriatic arthritis and Behçet's disease. Increased amounts of IL-6-mRNA were detected in rheumatoid synovium using a dot blot hydridization technique. Furthermore, there was a positive correlation between IL-6 production and accumulation of plasma cells in the synovium. These findings suggest that IL-6 plays an important role not only in immune response but also in active inflammation in various kinds of synovitis.

Adult↗