Biomedical subjects

Hiroyuki Kishi

Publications and source records attributed to Hiroyuki Kishi.

12 recordsLinked to original sources

PubMed2006

Suppression of experimental osteoarthritis by adenovirus-mediated double gene transfer.

BACKGROUND: Osteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-alpha receptor type I (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis. METHODS: Adenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 x 10(8) plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 x 10(8) pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 x 10(8) pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated. RESULTS: Intra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-alpha blockade has little effect on joint destruction. CONCLUSION: The enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.

Adenoviridae↗

PubMed2006

Amplification and analysis of cDNA generated from a single cell by 5'-RACE: application to isolation of antibody heavy and light chain variable gene sequences from single B cells.

The technique of 5'-rapid amplification of cDNA ends (5'-RACE) is widely used to amplify unknown sequences at the 5' end of a messenger RNA (mRNA). However, conventional 5'-RACE is inappropriate for producing cDNAs from a single cell due to the small quantity of mRNA present in one cell. In this study, we report an improved 5'-RACE method that is suitable for generating cDNA from a single cell. In this method, the first-strand cDNA was directly synthesized from a single cell, and both the tailing reaction and second-strand cDNA synthesis were performed in the same tube without purifying the cDNA sample. Using this method, we were able to amplify the cDNA of the immunoglobulin (Ig) variable region gene from more than 50% of single B cells. The amplified cDNA fragment contained a full-length Ig variable region including a 5'-untranslated region, a leader sequence, and an initiation codon. This method may thus be applicable for a comprehensive analysis of the Ig variable genes of the lymphocyte repertoire in humans and animals, thereby contributing to the development of antibody-based therapeutics for infectious diseases.

B-Lymphocytes↗

PubMed2006

[Reactivation of Epstein-Barr virus is involved in the pathogenesis of lymphomatoid granulomatosis].

Recent studies point to a role of Epstein-Barr virus in the pathogenesis of lymphomatoid granulomatosis. Little is however known about the association of the pathogenesis of the disease with Epstein-Barr virus reactivation. A 46-year-old man presented with fever and general malaise. He had been admitted with fever of unknown origin 10 years previously. Chest radiographs and CT scans showed multiple nodular shadows in both lung fields. PET/CT showed strong accumulation in lung nodules, femoral muscle, lumbar subcutaneous nodule. He died of rapidly progressive neurological lesions. The definitive diagnosis of lymphomatoid granulomatosis was made from histological findings of the surgical biopsy specimen and at autopsy. Elevation of early antigen-IgG titer in serum was shown in this case. This result suggests that reactivation of Epstein-Barr virus is involved in the development of the disease.

Herpesvirus 4, Human↗

PubMed2005

Single-cell microarray for analyzing cellular response.

Detection of cellular response by measuring intracellular calcium, (Ca2+)i with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30,000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.

Amino Acid Sequence↗

PubMed2005

Characterization of the proximal enhancer element and transcriptional regulatory factors for murine recombination activating gene-2.

Recombination-activating gene (RAG)-1 and RAG-2 are essential for V(D)J recombination and are expressed specifically in lymphoid cells. We previously identified two putative enhancer elements, the proximal and distal enhancers, located at -2.6 and -8 kb, respectively, 5' upstream of mouse RAG-2, and characterized the distal enhancer element in detail. In this study, to characterize the proximal enhancer in vitro as well as in vivo, we first defined a 170-bp core enhancer element within the proximal enhancer (Ep) and determined its activity in various cells. Ep conferred enhancer activity only in B-lymphoid cell lines, but not in T- or non-lymphoid cell lines. Analysis of the transgenic mice carrying an EGFP reporter gene linked with Ep revealed that Ep activated the transcription of the reporter gene in bone marrow and spleen, but not in thymus or non-lymphoid tissues. Ep was active in both B220+IgM- and B220+IgM+ subpopulations in the bone marrow and in the B220+ subpopulation in the spleen. Using electrophoretic mobility shift assays and mutational assays, we found that Ikaros and CCAAT/enhancer binding protein cooperatively bind Ep and function as the transcription factors responsible for B cell-specific enhancer activity. These results demonstrate the role of Ep as a cis-regulatory enhancer element for RAG-2-specific expression in B-lymphoid lineages.

Animals↗

PubMed2004

Activation of mouse RAG-2 promoter by Myc-associated zinc finger protein.

Recombination activating gene-1 (RAG-1) and RAG-2 are expressed specifically in lymphocytes undergoing the antigen receptor gene rearrangement during the lymphocyte development. Our previous study showed that the -41 to -17 nucleotides (nt) 5' -upstream region of mouse RAG-2 were pre-requisite for the core promoter activity and that Pax-5/c-Myb/LEF-1 protein-protein complex was responsible for its activity in immature B cells. In this study, we show that the -65/-42 sequence, the non-conserved sequence between human and mouse RAG-2 promoter, is necessary for the full promoter activity for mouse RAG-2. Electrophoresis mobility shift assay revealed that Myc-associated zinc finger protein (MAZ) as well as SP1/3 binds a GA box in this region. Using chromatin immunoprecipitation, we show that MAZ binds the RAG-2 promoter region in pre-B cells. Furthermore, we show that MAZ synergistically activates the murine RAG-2 promoter with Pax-5/c-Myb/LEF-1 complex. These results first demonstrate that MAZ participates in activation of mouse RAG-2 promoter.

Animals↗

PubMed2003

Heat shock protein 70 binds caspase-activated DNase and enhances its activity in TCR-stimulated T cells.

DNA fragmentation is a hallmark of cells undergoing apoptosis and is mediated mainly by the caspase-activated DNase (CAD or DNA-fragmentation factor 40 [DFF40]), which is activated when released from its inhibitor protein (ICAD or DFF45) upon apoptosis signals. Here we analyzed the effect of heat shock protein 70 (Hsp70) on CAD activity in T-cell receptor (TCR)-induced apoptosis using a T-cell line (TAg-Jurkat). Overexpression of Hsp70 significantly augmented the apoptotic cell death as well as DNA fragmentation in CD3/CD28- or staurosporine-stimulated cells. Following stimulation of cells with CD3/CD28 or staurosporine, Hsp70 was coprecipitated with free CAD, but not with CAD associated with ICAD. Furthermore, the purified Hsp70 dose-dependently augmented DNA-fragmentation activity of caspase-3-activated CAD in a cell-free system. Peptide-binding domain-deleted Hsp70 could neither bind nor augment its activity, while adenosine triphosphate (ATP)-binding domain-deleted Hsp70 or the peptide-binding domain itself bound CAD and augmented its activity. These results indicate that the the binding of Hsp70 to the activated CAD via the peptide-binding domain augments its activity. Although CAD lost its activity in an hour after being released from ICAD in vitro, its activity was retained after an hour of incubation in the presence of Hsp70, suggesting that Hsp70 may be involved in stabilization of CAD activity. Finally, CAD that had been coprecipitated with Hsp70 from the cell lysate of staurosporine-activated 293T cells induced chromatin DNA fragmentation and its activity was not inhibited by ICAD. These results suggest that Hsp70 binds free CAD in TCR-stimulated T cells to stabilize and augment its activity.

Caspases↗

PubMed2003

Possible involvement of glial cell line-derived neurotrophic factor and its receptor, GFRalpha1, in survival and maturation of thymocytes.

The glial cell line-derived neurotrophic factor (GDNF) and its receptors (GFR) play important roles in the promotion of survival and differentiation of central and peripheral neuronal populations. We show that GFRalpha1, a component of GDNF receptor, was expressed in thymocytes at an early stage of thymocyte-development and was involved in the survival of thymocyte precursors. GFRalpha1and GDNF were expressed in thymus, but not in spleen or lymph nodes in adult mice. During embryonic thymocyte development, GFRalpha1 was predominantly expressed on thymocytes from days 14.5 to 16.5 of gestation, and thereafter its expression gradually declined. In adult thymus, GFRalpha1 was expressed only on CD4(-)CD8(-) double-negative (DN) thymocytes, but not on CD4(+)CD8(+) double-positive or single-positive thymocytes. It was strongly expressed on RAG2(-/-) thymocytes arrested at the DN stage, and ist expression was reduced during their differentiation after in vivo anti-CD3 antibody stimulation. Additionally, fetal thymocyte precursors grew in serum-free medium of the fetal thymus organ culture system in the presence of recombinant GDNF (rGDNF), while the cells without rGDNF died. These results suggested that GDNF/GFRalpha1 are involved in the survival of both the nervous system and DN immature thymocytes.

Animals↗

PubMed2002

Lymphoid enhancer-binding factor-1 binds and activates the recombination-activating gene-2 promoter together with c-Myb and Pax-5 in immature B cells.

The recombination-activating gene (RAG)-1 and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of Ag receptor genes. We studied the regulation of murine RAG-2 promoter and revealed that -41/-17 RAG-2 promoter region, which was indispensable for the RAG-2 promoter activity in B cell lines, contained binding sites for lymphoid enhancer-binding factor-1 (LEF-1), c-Myb, and Pax-5. We showed that these three transcription factors bound the promoter region in vitro and in vivo. Cotransfection assays using a human embryonic kidney cell line (293T) showed that LEF-1, c-Myb, and Pax-5 cooperatively activated the RAG-2 promoter, via their synergistic DNA binding. We also showed that LEF-1, c-Myb, and Pax-5 physically interact in the cells. Finally, we demonstrated that a dominant-negative LEF-1 protein, which lacks the binding site for beta-catenin, suppressed the RAG-2 promoter activity as well as the endogenous RAG-2 expression in a pre-B cell line (18.81). These results suggest that LEF-1/beta-catenin complex regulates the RAG-2 promoter activation in concert with c-Myb and Pax-5 in immature B cells. The link between LEF-1/beta-catenin and Wnt signaling in B lineage cells will be discussed.

5' Untranslated Regions↗

PubMed2002

Characterization of chromatin structure and enhancer elements for murine recombination activating gene-2.

Recombination-activating genes (RAGs) play a critical role in V(D)J recombination machinery and their expression is specifically regulated during lymphocyte ontogeny. To elucidate the molecular mechanisms regulating murine RAG-2 expression, we examined a chromatin structure of 25-kb DNA segment adjacent to murine RAG-2 by analyzing DNase I hypersensitive (HS) sites. In a RAG-2-expressing murine pre-B cell line, three lymphoid cell-specific HS sites (HS1, HS2, and HS3) were identified. Among these HS sites, one HS site (HS3) that locates in the RAG-2 promoter was associated only with RAG-2-expressing cell lines. Using the transient enhanced green fluorescence protein reporter gene assays, we identified two enhancer elements in the 5'-upstream region of RAG-2 that corresponded to HS1 and HS2. One of the enhancer elements (D3) exhibited enhancer activity only in the lymphoid cell lines. Analysis of the transgenic mice carrying the enhanced green fluorescence protein-reporter gene linked with D3 revealed that D3 activated the reporter gene-expression in the primary lymphoid tissues, but not in the secondary lymphoid tissues or nonlymphoid tissues. D3 was active in CD4(-)CD8(-), but not in CD4(+)CD8(+) or CD4(+)CD8(-) thymocytes in the thymus, and also active in B220(+)IgM(-), but not in B220(+)IgM(+), cells in the bone marrow. Finally, our data suggested that C/EBP may bind to the D3 enhancer and function as one of the transcription factor(s) responsible for the enhancer activity. These results show that the tissue- and stage-specific expression of murine RAG-2 is regulated by alteration of the chromatin structure as well as cis-regulatory enhancer elements.

3T3 Cells↗

PubMed2002

Cooperative binding of c-Myb and Pax-5 activates the RAG-2 promoter in immature B cells.

The recombination activating gene-1 (RAG-1) and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of antigen receptor genes. The regulation of murine RAG-2 promoter was studied and it was revealed that the -41/-17 RAG-2 promoter region, which is conserved between humans and mice, was indispensable for the RAG-2 promoter activity in B-cell lines. The region contained 2 cis elements that bound c-Myb and Pax-5. Mutation in the c-Myb-binding site in the promoter reduced the promoter activity in B-cell lines. Cooperative activation of the RAG-2 promoter was seen by a combination of c-Myb and Pax-5 in a human embryonic kidney cell line (293T), via their synergistic DNA-binding. Deletion experiments showed that the C-terminus of c-Myb was responsible for their interaction. Furthermore, the dominant-negative c-Myb mutant suppressed the activation of the RAG-2 promoter in a pre-B-cell line as well as in 293T cells. These results suggest that cooperative binding of c-Myb and Pax-5 to the RAG-2 promoter is one of the mechanisms to direct the restricted expression of the RAG-2 in immature B cells.

Animals↗

PubMed2002

The regulation of DNAse activities in subcellular compartments of activated thymocytes.

Thymocytes expressing self-reactive T-cell receptors (TCR) are eliminated in the thymus through a TCR-mediated signal. This cell death signal (negative selection) generates nuclear morphological change and DNA fragmentation in thymocytes. However, the pathway leading to DNA fragmentation of thymocytes following TCR engagement remains obscure. In this study, we investigated the localization and function of caspase-activated DNAse (CAD) and its inhibitor (ICAD) in thymocytes prior to or after in vivo TCR stimulation. We showed that CAD and ICAD are co-localized in microsome, nuclei and cytosol in unstimulated thymocytes. Following in vivo TCR engagement, ICAD located in cytosol and microsome was degraded and the resulting activated CAD induced chromosomal DNA fragmentation. CAD present in cytosol and microsome of unstimulated thymocytes was activated by recombinant caspase-3, and microsomal CAD was released to the cytosol. These results demonstrate that TCR engagement of thymocytes induces caspase-3-dependent activation of CAD localized in both cytosol and microsome, leading to DNA fragmentation in harmony.

Animals↗