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Biomedical subjects

Hideoki Ogawa

Publications and source records attributed to Hideoki Ogawa.

At least 55 records · Page 3Linked to original sources
PubMed2005

Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization.

Mast cells accumulate in tissues undergoing angiogenesis during tumor growth, wound healing, and tissue repair. Mast cells can secrete angiogenic factors such as vascular endothelial growth factor (VEGF). Ionizing irradiation has also been shown to have angiogenic potential in malignant and nonmalignant diseases. We observed that low-dose irradiation fosters mast cell-dependent vascular regeneration in a limb ischemia model. Irradiation promoted VEGF production by mast cells in a matrix metalloproteinase-9 (MMP-9)-dependent manner. Irradiation, through MMP-9 up-regulated by VEGF in stromal and endothelial cells, induced the release of Kit-ligand (KitL). Irradiation-induced VEGF promoted migration of mast cells from the bone marrow to the ischemic site. Irradiation-mediated release of KitL and VEGF was impaired in MMP-9-deficient mice, resulting in a reduced number of tissue mast cells and delayed vessel formation in the ischemic limb. Irradiation-induced vasculogenesis was abrogated in mice deficient in mast cells (steel mutant, Sl/Sl(d) mice) and in mice in which the VEGF pathway was blocked. Irradiation did not induce progenitor mobilization in Sl/Sl(d) mice. We conclude that increased recruitment and activation of mast cells following irradiation alters the ischemic microenvironment and promotes vascular regeneration in an ischemia model. These data show a novel mechanism of neovascularization and suggest that low-dose irradiation may be used for therapeutic angiogenesis to augment vasculogenesis in ischemic tissues.

Animals↗
PubMed2005

The human beta-defensins (-1, -2, -3, -4) and cathelicidin LL-37 induce IL-18 secretion through p38 and ERK MAPK activation in primary human keratinocytes.

In addition to its physical barrier against invading microorganisms, the skin produces antimicrobial peptides, human beta-defensins (hBDs) and cathelicidin LL-37, that participate in the innate host defense. Because IL-18 is produced by keratinocytes and involved in skin diseases in which hBDs and LL-37 are highly expressed, we hypothesized that these peptides would activate keratinocytes to secrete IL-18. We found that hBD-2, -3, and -4 and LL-37, but not hBD-1, activated normal human keratinocytes to secrete IL-18; this secretion reached peak strength at 3 h. In addition, the combination of peptides resulted in a synergistic effect on IL-18 secretion. We also revealed that hBD-2, -3, and -4 and LL-37 increased IL-18 mRNA expression, and that IL-18 secretion was more enhanced in keratinocytes differentiated in vitro with high Ca2+-containing medium. Furthermore, because IL-18 secretion induced by hBDs and LL-37 could not be suppressed by caspase-1 or caspase family inhibitors, and because these peptides failed to increase caspase-1 activity, we suggest that hBD- and LL-37-induced IL-18 secretion is probably via a caspase-1-independent pathway. To determine the molecular mechanism involved, we demonstrated that IL-18 secretion was through p38 and ERK1/2 MAPK pathways, because the inhibitors of p38 and ERK1/2, but not JNK, almost completely nullified IL-18 secretion. Moreover, hBD-2, -3, and -4 and LL-37 could induce the phosphorylation of p38 and ERK1/2, but not JNK. Thus, the ability of hBDs and LL-37 to induce IL-18 secretion by keratinocytes provides a new mechanism for these peptides in innate immunity and an understanding of their role in the pathogenesis of skin disorders.

Amino Acid Sequence↗
PubMed2005

GATA-1 is required for expression of Fc{epsilon}RI on mast cells: analysis of mast cells derived from GATA-1 knockdown mouse bone marrow.

The high-affinity receptor for IgE (FcepsilonRI) that is expressed on the surface of mast cells plays an important role in antigen/IgE-mediated allergic reactions. We have previously found that critical elements in the promoter of the FcepsilonRI alpha- and beta-chain genes are recognized by the transcription factor GATA-1 in electrophoretic mobility shift assays coupled with a transient expression system for the alpha- and beta-chain promoters. To confirm that GATA-1 is involved in the expression of FcepsilonRI definitively, we generated bone marrow-derived mast cells from GATA-1 knockdown (KD) heterozygous mice. FACS analysis showed that the frequency of FcepsilonRI-positive cells was significantly decreased in mast cells derived from bone marrow of GATA-1 KD mice. Reverse transcription-PCR analysis showed that the level of transcripts not only for GATA-1 but also for both the alpha- and beta-chains was significantly lower in KD mast cells, whereas that of the FcepsilonRI gamma-chain was not affected. Degranulation caused by cross-linking of FcepsilonRI on mast cells prepared from KD mice was markedly repressed in comparison with that of wild-type mast cells. We concluded that the transcription factor GATA-1 positively regulates FcepsilonRI alpha- and beta-chain expression and therefore is involved in mast cell development.

Animals↗
PubMed2005

Synergistic effect of antibacterial agents human beta-defensins, cathelicidin LL-37 and lysozyme against Staphylococcus aureus and Escherichia coli.

BACKGROUND: The antimicrobial properties of the skin are attributed to several agents including human beta-defensins (hBDs), cathelicidin LL-37 and skin lysozyme. Although these antibacterial agents reside in the skin to protect it against infection, it is not well known whether the total analysis of all combinations of these agents may result in synergistic effect to enhance their antibacterial activities against invading microorganisms. OBJECTIVE: To elucidate the interactions between keratinocyte-derived antibacterial agents in the extracellular milieu, we investigated the individual and synergistic activities of hBDs, LL-37 and lysozyme against Staphylococcus aureus and Escherichia coli in neutral and acidic milieus. METHODS: The colorimetric method using alamarBlue was employed to assess the antibacterial activities of hBD-1, -2, -3, LL-37 and lysozyme and the viability of bacteria was read spectrophotometrically. RESULTS: In both neutral and acidic pH milieus, hBD-1, -2, -3, LL-37 and lysozyme exhibited antibacterial activity against S. aureus and E. coli in a dose-dependent manner. Interestingly, the antibacterial activity of hBD-1, -2, -3 and lysozyme but not LL-37 was significantly enhanced in acidic milieu (pH 4.6). Furthermore, various combinations of above agents resulted in a synergistic or additive antibacterial effect against S. aureus and E. coli in neutral milieu. The synergistic effect of hBDs, LL-37 and lysozyme against S. aureus was further significantly enhanced in acidic milieu. In contrast, above antibacterial agents exhibited mainly additive rather than synergistic effect on antibacterial activity against E. coli in acidic milieu. CONCLUSION: Taken together, these results provide a novel evidence of antimicrobial mechanism of natural human skin-derived antibacterial agents against bacterial infection, and their involvement in innate immunity.

Antimicrobial Cationic Peptides↗
PubMed2005

Recombinant Der p 1 and Der f 1 with in vitro enzymatic activity to cleave human CD23, CD25 and alpha1-antitrypsin, and in vivo IgE-eliciting activity in mice.

BACKGROUND: The major house dust mite group 1 allergens Der p 1 and Der f 1 are the most potent indoor allergens. Der p 1 cleaves human cell surface molecules, the low-affinity IgE receptor (CD23/FcepsilonRII), the alpha-subunit of the IL-2 receptor (CD25), and a protease inhibitor alpha1-antitrypsin, and in vitro and in vivo studies suggested the importance of its proteolytic activity in the pathogenesis of allergy. Recently, we established an efficient system to prepare correctly folded active recombinant Der p 1 and Der f 1 (Der p 1-N52Q and Der f 1-N53Q) with similar molecular sizes, secondary structures and allergenicities as their natural types. To evaluate whether Der p 1-N52Q and Der f 1-N53Q are suitable for use in future in vitro and in vivo studies as alternatives to the natural types, we investigate their proteolytic activity to cleave the human proteins and IgE-eliciting activity in mice. METHODS: Proteolytic activities of Der p 1-N52Q and Der f 1-N53Q against a short peptide substrate, a collagen substrate Azocoll, human CD23 and CD25 expressed on the cells and human alpha1-antitrypsin were analyzed by kinetic assays for proteolysis of the fluorogenic or colorimetric substrates, flow cytometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Mice were intraperitoneally immunized with Der p 1-N52Q and Der f 1-N53Q adsorbed on Alum, and the serum IgE levels were measured by sandwich ELISA. RESULTS: Der p 1-N52Q and Der f 1-N53Q showed proteolytic specificities against the short peptide substrate, Azocoll, human cell surface CD23 and CD25 and human alpha1-antitrypsin, and elicited significant serum IgE levels in immunized mice. CONCLUSION: The recombinant forms, Der p 1-N52Q and Der f 1-N53Q, will be useful tools as alternatives to the natural Der p 1 and Der f 1 for various in vitro and in vivo analyses.

Animals↗
PubMed2005

Suppression of serum IgE response and systemic anaphylaxis in a food allergy model by orally administered high-dose TGF-beta.

Some epidemiological or association studies suggest that transforming growth factor-beta (TGF-beta) in breast milk may be a decisive factor in diminishing the risk of allergic diseases during infancy. The observations have prompted us to investigate whether TGF-beta, when taken orally, can affect allergic immune responses. Repeated high-dose ovalbumin peptide (OVA) feeding was previously reported to induce OVA-specific IgE production and an anaphylactic reaction after intravenous challenge of OVA in OVA-TCR transgenic mice, which might represent a model for food allergy. By using this model, we showed here that oral administration of high-dose TGF-beta simultaneously with OVA feeding significantly inhibited the OVA-specific IgE elevation and anaphylactic reaction in OVA-TCR transgenic DO11.10 mice. These effects were associated with suppression of OVA-specific IL-4 production and GATA-3 expression and with up-regulation of IFN-gamma production and T-bet expression by splenocytes. Intra-peritoneal injection of anti-TGF-beta-neutralizing antibody abolished the inhibitory effects of orally administered TGF-beta on the serum IgE response and anaphylactic reaction after OVA feeding in DO11.10 mice. Interestingly, oral administration of high-dose TGF-beta suppressed activation-induced T cell death induced by OVA feeding in DO11.10 mice. We thus conclude that TGF-beta, when taken orally at high dose, has the capacity to modulate a food allergy-related reaction, at least in part, through its systemic activity.

Administration, Oral↗
PubMed2005

Smad3 deficiency in mast cells provides efficient host protection against acute septic peritonitis.

Mast cells play an important role in innate immunity as well as in allergic reaction. However, regulatory mechanisms underlying mast cell-mediated innate immune responses remain largely unknown. Here we determined whether Smad3, a major signal transducer of TGF-beta, regulates innate immune response by mast cells against Gram-negative bacteria. Bone marrow-derived mast cells (BMMC) obtained from Smad3 null mutant mice showed augmented capacity to produce proinflammatory cytokines upon stimulation with a Gram-negative bacteria-associated product, LPS. In acute septic peritonitis model induced by cecal ligation and puncture, mast cell-deficient W/W(v) mice reconstituted with Smad3 null BMMC had significantly higher survival rate than W/W(v) mice reconstituted with wild-type BMMC, which was associated with higher production of proinflammatory cytokines in the peritoneal cavity. These in vitro and in vivo results suggest that Smad3 in mast cells functions as inhibitory for mast cell-mediated innate immune response against Gram-negative bacteria. Suppression of Smad3 expression in mast cells may thus have therapeutic potential for Gram-negative bacterial infection such as acute septic peritonitis by augmenting innate immune responses of mast cells.

Acute Disease↗
PubMed2005

Multiple-mutation at a potential ligand-binding region decreased allergenicity of a mite allergen Der f 2 without disrupting global structure.

We assessed the effect of multiple-mutations within one IgE-binding area on allergenicity of Der f 2. The triple-mutant of Der f 2, P34/95/99A, exhibited the most significant reduction of allergenicity and circular dichroism analysis showed that the global structure of Der f 2 was maintained in P34/95/99A. These results indicate that such a strategy is effective when designing allergen-vaccines, which achieve less allergenicity for a broad population of patients without disrupting the global structure. Structurally, Der f 2 is a member of the MD-2 related lipid-recognition proteins. The sites for the triple-mutation located on the characteristically charged entrance of a cavity and corresponded to the regions critical to ligand-binding in the Niemann-Pick type 2 disease protein and MD-2.

Amino Acid Motifs↗
PubMed2005

Recombinant Der p 1 and Der f 1 exhibit cysteine protease activity but no serine protease activity.

Although mite major group 1 allergens, Der p 1 and Der f 1, were first isolated as cysteine proteases, some studies reported that natural Der p 1 exhibits mixed cysteine and serine protease activity. Clarifying whether the serine protease activity originates from Der p 1 or is due to contamination is important for distinguishing between the pathogenic proteolytic activities of group 1 allergens and mite-derived serine proteases. Recombinant mite group 1 allergens would be useful tool for addressing this issue, because they are completely free from contamination by mite serine proteases. Recombinant Der p 1 and Der f 1, and highly purified natural forms exhibited only cysteine protease activity. However, commercially available natural forms exhibited both activities, but the two activities were eluted into different fractions in size-exclusion column chromatography. The substrate specificity associated with the serine protease activity was similar to that of Der f 3. These results indicate that the serine protease activity does not originate from group 1 allergens.

Antigens, Dermatophagoides↗
PubMed2005

Dilution method to refold bacterially expressed recombinant Der f 2 and Der p 2 to exhibit the secondary structure and histamine-releasing activity of natural allergens.

BACKGROUND: Structurally refolded recombinant forms of major house dust mite group 2 allergens, Der f 2 and Der p 2, expressed in Escherichia coli, were prepared by solubilizing the insoluble products with urea and subsequently dialyzing against buffer. In this study, we determined conditions for refolding the urea-denatured recombinant Der f 2 and Der p 2 by one-step dilution as an alternative to dialysis, which requires several steps of handling and much time and cost. METHODS: The insoluble bacterial product containing recombinant Der f 2 was solubilized with a buffer containing 8 M urea, and the solution was diluted to various urea concentrations. The refolding efficiency in each dilution was estimated from the height of the peak corresponding to the folded recombinant Der f 2 and that containing the aggregated form on anion exchange chromatography. The structure and allergenicity of the purified recombinant Der f 2 and Der p 2 refolded using the dilution method were analyzed based on circular dichroism and a basophil histamine-releasing assay, respectively. RESULTS: Although the refolding efficiency decreased as the urea concentration in the dilution increased, experimental conditions whereby the protein and urea concentrations in the dilution were less than 0.5 mg/ml and 0.8 M, respectively, achieved maximum refolding efficiency. The recombinant allergens prepared by the dilution method exhibited the secondary structure and histamine-releasing activity of natural allergens purified from mite culture. CONCLUSIONS: The dilution method established in this study is more convenient in terms of handling, time, and cost than the dialysis method and will be useful for large-scale production and for the preparation of numbers of mutants to analyze IgE epitopes.

Allergens↗
PubMed2005

Analysis of PU.1/ICSBP (IRF-8) complex formation with various PU.1 mutants: molecular cloning of rat Icsbp (Irf-8) cDNA.

We isolated cDNA encoding a full-length Interferon (IFN) consensus sequence binding protein [Icsbp; also called IFN regulatory factor-8 (Irf-8)] from rat and analyzed interaction between ICSBP and PU.1, an Ets-family transcription factor regulating hematopoietic cell-specific promoters. Electrophoretic mobility shift assay indicated that PU.1 with deletion of the PEST domain could not bind to ICSBP, although loss of the PEST domain had no effect on DNA-binding ability of PU.1 itself. An amino-acid replacement of Ser147 by Ala of the PEST domain of PU.1 did not affect DNA-binding ability of PU.1 to form a binary complex, PU.1/DNA, but clearly decreased the ternary complex formation of PU.1/ICSBP/DNA. Phosphatase treatment of the ternary complex markedly decreased PU.1/ICSBP/DNA-complex formation. These results indicated that Ser147 of PU.1 is definitively required for forming a complex with ICSBP and phosphorylation of PU.1 and/or ICSBP is critical for formation of the ternary complex.

Animals↗
PubMed2005

Mast cells acquire monocyte-specific gene expression and monocyte-like morphology by overproduction of PU.1.

PU.1 is a myeloid- and lymphoid-specific transcription factor that belongs to the Ets family. Recently, we found that overproduction of PU.1 in mouse bone marrow-derived hemopoietic progenitor cells induced monocyte-specific gene expression and caused their monocyte-like morphological change. In the present study, PU.1 was overproduced by using retrovirus expression system in differentiated bone marrow-derived mast cells. By overexpression of PU.1, cell surface expression of MHC class II, CD11b, CD11c, and F4/80 was induced, accompanied by reduced expression of c-kit, a mast cell-specific marker. Morphology of PU.1-transfected cells was altered toward monocyte-like one. PU.1-overproducing cells acquired T cell stimulatory ability and showed an increase in response to LPS stimulation, while response through FcepsilonRI was markedly reduced by overproduction of PU.1. These results suggest that the differentiated mast cells still have potential to display monocytic features. When PU.1 was overproduced in a different type of mast cell, peritoneal mast cells, similar monocyte-like morphological change, and the expression of CD11b and F4/80 were induced. However, surface level of CD11c and MHC class II was not affected. These results indicate that the potential capacity to exhibit monocytic features is different between both the mast cells.

Animals↗
PubMed2005

Analysis of the structure and allergenicity of recombinant pro- and mature Der p 1 and Der f 1: major conformational IgE epitopes blocked by prodomains.

BACKGROUND: The major house dust mite group 1 allergens Der p 1 and Der f 1, which belong to the papain-like cysteine protease family, are the most potent of indoor allergens. However, little information is available on the location of IgE epitopes. OBJECTIVE: We investigated the allergenicities of recombinant proforms and mature forms of Der p 1 and Der f 1 to compare them with natural Der p 1 and Der f 1 and to obtain information on the conformational IgE-binding epitopes. METHODS: Secreted pro-Der p 1 and pro-Der f 1 and their mutants without hyperglycosylation expressed in yeast were converted to mature forms. We purified the proforms and mature forms and analyzed their apparent molecular sizes and secondary structures by means of gel-filtration and circular dichroism analysis and their allergenicities by means of assays for IgE binding, IgE-binding inhibition, and basophil histamine release. The tertiary structure of pro-Der f 1 was predicted by molecular modeling. RESULTS: The recombinant mature forms exhibited similar molecular sizes, secondary structures, and allergenicities as their natural types. On the other hand, their proforms exhibited different secondary structures and less allergenicities than the mature forms in all sera and volunteers tested. Molecular modeling revealed that the prosegment is anchored at the prosegment-binding loop and the substrate-binding cleft on the surface of the mature portion. CONCLUSIONS: Our studies indicate that the prodomains of Der p 1 and Der f 1 reduce allergenicity and that the major conformational IgE epitopes commonly found in a broad population of patients exist within the 2 regions blocked by the prosegments. Recombinant Der p 1 and Der f 1 and the findings in the present study will be the basis for allergen standardization and the design of safer and more effective allergen vaccines.

Animals↗
PubMed2005

Cystatin A inhibits IL-8 production by keratinocytes stimulated with Der p 1 and Der f 1: biochemical skin barrier against mite cysteine proteases.

BACKGROUND: Der p 1 and Der f 1 are the most immunodominant allergens produced by house dust mites and are suspected to be involved in the pathogenesis of allergy through their cysteine protease activity. However, stimulation of keratinocytes by these protease allergens and protective systems in the skin against them have not been well investigated. OBJECTIVE: We purified and identified the dominant skin-derived inhibitor against the proteolytic activities of these allergens and analyzed its effect on keratinocyte activation. METHODS: Recombinant allergens were used for the experiments. We analyzed whether human sweat inhibits the enzymatic activities of Der p 1 and Der f 1 and used sweat as the skin-derived material to isolate the inhibitor. The inhibitor was purified by means of column chromatography and subsequently identified by means of protein sequencing and immunoblotting. Keratinocytes were stimulated with the allergens in the absence or presence of the inhibitor, and the concentration of secreted IL-8 was measured. RESULTS: Sweat inhibited the proteolytic activities of Der p 1 and Der f 1. The sweat inhibitor was identified as cystatin A. The stimulation of normal human keratinocytes and the human keratinocyte cell line HaCaT with these protease allergens upregulated IL-8 secretion, and addition of cystatin A blocked this upregulation. Normal human keratinocytes secreted cystatin A into the medium. CONCLUSIONS: The proteolytic activity of Der p 1 and Der f 1 stimulates human keratinocytes in vitro. Cystatin A produced by keratinocytes is the dominant biochemical skin barrier that eliminates the enzymatic activity of these mite cysteine proteases and prevents them from stimulating keratinocytes.

Animals↗
PubMed2005

SB-431542 inhibits TGF-beta-induced contraction of collagen gel by normal and keloid fibroblasts.

BACKGROUND: Transforming growth factor (TGF)-beta induces fibroblast contraction, which is implicated in wound healing and keloid formation. SB-431542 is a novel specific inhibitor of TGF-beta type I receptor kinase activity. OBJECTIVE: We sought to determine whether SB-431542 inhibited TGF-beta-induced fibroblast contraction. METHODS: We used an in vitro type I collagen gel contraction assay with normal or keloid dermal fibroblasts incorporated. RESULTS: TGF-beta induced contraction of collagen gels with normal dermal fibroblasts incorporated, which was efficiently suppressed by SB-431542. Keloid fibroblasts showed higher basal contraction of collagen gels in the absence of TGF-beta than normal fibroblasts, which was enhanced by addition of TGF-beta. SB-431542 suppressed both the basal and TGF-beta-enhanced contraction of collagen gels by keloid fibroblasts. These inhibitory effects of SB-431542 were associated with suppression of TGF-beta-induced alpha-smooth muscle actin (alpha-SMA) expression and phosphorylation of Smad2 in normal and keloid fibroblasts. CONCLUSION: SB-431542 can suppress TGF-beta-induced contraction of collagen gel by normal and keloid dermal fibroblasts. Importantly, SB-431542 can inhibit basal contraction of collagen gel by keloid fibroblasts. These results suggest that an inhibitor of TGF-beta type I receptor kinase activity may have therapeutic potential for excessive skin contraction as observed in keloid.

Actins↗
PubMed2005

Tenascin-C is upregulated in the skin lesions of patients with atopic dermatitis.

BACKGROUND: Tenascin-C is a large, hexameric extracellular matrix glycoprotein that is expressed during embryogenesis, carcinogenesis and wound healing. In normal adult human skin the expression level of tenascin-C is low, but levels are elevated in skin tumors and rise significantly in the dermal compartment during wound healing. Although the expression of tenascin-C could be upregulated by inflammatory cytokines, the role of tenascin-C in atopic dermatitis (AD) is still unclear. OBJECTIVE: To identify genes that plays a role in AD. METHODS: We screened for differentially expressed genes in lesional and non-lesional skin of AD patients using DNA microarray. Then we monitored with quantitative PCR the expression of the novel disease related genes in human keratinocytes or pinnae from NC/Nga mice. RESULTS: We found that tenascin-C gene expression was expressed at higher levels in lesional skin compared to non-lesional skin of the patients, whereas it was not upregulated in the skin of psoriatic patients or healthy controls. In human cultured keratinocytes, tenascin-C was markedly upregulated by IL-4 and IL-13, and moderately upregulated by IFN-gamma. Tenascin-C expression was also upregulated in the AD-like skin lesions induced in NC/Nga mice ears by intradermal injection of mite antigen, and this upregulation was inhibited by prednisolone. CONCLUSION: These results suggest that upregulation of the tenascin-C expression is specific to AD lesions, and that tenascin-C may therefore play a critical role in regulating the underlining inflammatory processes, which are involved in the pathology of AD.

Animals↗