[HHV-6 and drug-induced hypersensitivity syndrome].
Explore the source record for details and available documents.
DNA, Viral↗
Biomedical subjects
Koji Hashimoto
Publications and source records attributed to Koji Hashimoto.
Enzyme-linked immunosorbent assay using bacterial recombinant proteins of human BP230 as a diagnostic tool for bullous pemphigoid.
BACKGROUND: By immunoblot analyses of normal human epidermal extracts, the 230kDa bullous pemphigoid antigen (BP230) is recognized by most bullous pemphigoid (BP) sera. We produced different recombinant glutathione-S-transferase-fusion proteins, which roughly presented N-terminal domain, central rod domain and C-terminal domain of human BP230. OBJECTIVE: In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) using the recombinant proteins for detection of anti-BP230 IgG antibodies and assessed the usefulness of this assay in conjunction with an anti-BP180 ELISA to establish the diagnosis of BP. METHODS: Using the bacterial recombinant proteins of N-terminal and C-terminal domains, we developed an ELISA. A receiver-operating-characteristic (ROC) analysis was performed to determine a cut-off value for the BP230 ELISA. RESULTS: By this BP230 ELISA, 173 (72.4%) of 239 BP sera were positive, while only one (1.1%) of 94 sera from pemphigus vulgaris and pemphigus foliaceus patients was positive and all the 109 normal control sera were negative. Thus, the sensitivity and specificity of the BP230 ELISA were 72.4 and 99.5%, respectively. Interestingly, while 54 (84.4%) of 64 BP sera in active stage and 113 (64.6%) of 175 BP sera in remission were positive in BP180 ELISA, 37 (57.8%) of 64 BP sera in active stage and 136 (77.7%) of 175 BP sera in remission were positive in BP230 ELISA. These results indicate that the titer of anti-BP230 antibodies is not related with disease activity in some BP cases. Most significantly, by combining the results of BP230 ELISA and BP180 ELISA, 232 (97.1%) of 239 BP sera were positive. CONCLUSION: The combination of BP230 ELISA and BP180 ELISA is the highly sensitive method for the diagnosis of BP.
Autoantigens↗
Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37.
The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 microg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak 2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.
Antimicrobial Cationic Peptides↗
Effects of mandibular advancement on brain activation during inspiratory loading in healthy subjects: a functional magnetic resonance imaging study.
Oral appliances have been a popular treatment option for subjects with obstructive sleep apnea. However, little information is available on how brain activation induced by respiratory challenge is modulated by mandibular advancement with these appliances. We hypothesized that the brain activation caused by respiratory stress may be alleviated by mandibular advancement. Respiratory stress was induced in 12 healthy subjects by resistive inspiratory loading. The effects of mandibular advancement during resistive inspiratory loading were assessed subjectively by using a visual analog scale. These effects were also evaluated objectively by using blood oxygenation level-dependent functional magnetic resonance imaging. The score for the visual analog scale significantly decreased with mandibular advancement. Cortical deactivation, in association with mandibular advancement, was localized to several specific regions, including the left cingulate gyrus and the bilateral prefrontal cortexes. These regions are known to be involved in respiratory control. Our results suggest that mandibular advancement with an oral appliance appears to be useful for reducing respiratory stress, based on both subjective and neuronal criteria.
Adult↗
dsRNA-mediated innate immunity of epidermal keratinocytes.
MIP-1alpha, a CC chemokine, recruits monocytes, natural killer cells, lymphocytes, and neutrophils, and plays a critical role in viral infection. Since, the lesional epidermis of herpes zoster expressed MIP-1alpha, we hypothesized that keratinocytes produce MIP-1alpha in response to virus-associated dsRNA via TLR3. To investigate this, we examined cultured human keratinocytes for MIP-1alpha production induced by poly(I:C), a TLR3 ligand. Poly(I:C) treatment induced MIP-1alpha production, interestingly, poly(I:C)-induced IFN-alpha and -beta production preceded MIP-1alpha production. A neutralizing antibody for IFN-beta significantly inhibited the poly(I:C)-induced MIP-1alpha production indicating that MIP-1alpha production is via IFN-beta. IFN-alpha priming enhanced TLR3 expression and MIP-1alpha production in poly(I:C)-treated keratinocytes. This suggests that IFN-alpha enhanced the TLR3 expression and reinforced the response of keratinocytes to poly(I:C), which resulted in an increase in MIP-1alpha production. In conclusion, normal human keratinocytes produce MIP-1alpha in response to dsRNA via TLR3, and this production is regulated by IFN-alpha/beta.
Cells, Cultured↗
Microbubble-enhanced ultrasound for gene transfer into living skin equivalents.
BACKGROUND: Gene transfer to skin is an attractive therapeutic approach because of the accessibility of the skin and the high rate of cure for many cutaneous diseases. However, safety concerns over viral vectors and the low efficiency of most non-viral gene transfer techniques have encumbered their clinical application for gene transfer. By contrast, efficient gene transfers into various cell types using microbubble-enhanced ultrasound has been reported. OBJECTIVES: The purpose of this study was to investigate whether ultrasound with microbubble enhancement allowed effective transfer of foreign genes into living skin equivalents (LSEs). METHODS: Microbubbles and plasmid DNA encoding green fluorescent protein (GFP) were added to the dermal-epidermal junctions of LSEs, which were then exposed to ultrasound. The LSEs were harvested at different time points to investigate transgene expression using confocal laser microscopy. Transfected LSEs were also transplanted onto nude mice, and the in vivo transgene expression was observed. RESULTS: From days 2 to 7 after transfection, most GFP-positive cells continued to migrate upward from the basal layer, while other GFP-positive cells lagged behind or remained in the basal layer on days 5 and 7. Transfection resulted in 20-30% GFP-positive cells. Multiple transfections further increased the percentage of transfected cells and resulted in multi-layer transgene expression. Grafts from the transfected LSEs survived on nude mice and continued to express GFP up to 2 weeks post-transplantation. CONCLUSION: Gene transfer into LSE using ultrasound with microbubble enhancement is an effective alternative to viral and non-viral methods.
Animals↗
Suppression of Stat3 promotes neurogenesis in cultured neural stem cells.
To investigate the effects of signal transducer and activator of transcription 3 (Stat3) on neural stem cell fate, stem cells were inoculated with an adenovirus vector expressing dominant negative form of Stat3 (Stat3F). One day later, a promoter assay revealed significant reduction of the transcriptional level in the transfected cells. Three days later, Western blot analysis and immunocytochemical analysis revealed that the protein level of microtubule-associated protein (MAP)2 and the number of MAP2-positive cells were increased significantly in the transfected cells whereas the protein level of glial fibrillary acidic protein (GFAP) and the number of GFAP-positive cells were decreased significantly. In addition, mRNA levels of Notch family members (Notch1, 2, and 3) and of inhibitory basic helix-loop-helix (bHLH) factors (Hes5, Id2, and Id3) were significantly downregulated at 3 days after viral inoculation with Stat3F; however, mRNA levels of bHLH determination factors (Math1 and Neurogenin3) and bHLH differentiation factors (NeuroD1 and NeuroD2) were significantly upregulated. These data indicated that suppression of Stat3 directly induced neurogenesis and inhibited astrogliogenesis in neural stem cells.
Animals↗
Urokinase-induced activation of the gp130/Tyk2/Stat3 pathway mediates a pro-inflammatory effect in human mesangial cells via expression of the anaphylatoxin C5a receptor.
Glomerular mesangial cells (MCs) are central to the pathogenesis of progressive glomeruli-associated renal diseases. However, molecular mechanisms underlying changes in MC functions still remain poorly understood. Here, we show that in MCs, the urokinase-type plasminogen activator (uPA) induces, via its specific receptor (uPAR, CD87), upregulated expression of the complement anaphylatoxin C5a receptor (C5aR, CD88), and modulates C5a-dependent functional responses. This effect is mediated via the interaction of the uPA-specific receptor (uPAR, CD87) and gp130, a signal transducing subunit of the receptor complexes for the IL-6 cytokine family. The Janus kinase Tyk2 and the transcription factor Stat3 serve as downstream components in the signaling cascade resulting in upregulation of C5aR expression. In vivo, expression of C5aR and uPAR was increased in the mesangium of wild-type mice in a lipopolysaccharide (LPS)-induced model of inflammation, whereas in uPAR(-/-) animals C5aR expression remained unchanged. This is the first demonstration in vitro and in vivo that uPA acts in MCs as a modulator of immune responses via control of immune-competent receptors. The data suggest a novel role for uPA/uPAR in glomeruli-associated renal failure via a signaling cross-talk between the fibrinolytic and immune systems.
Animals↗
Acute interstitial nephritis predisposed a six-year-old girl to minimal change nephrotic syndrome.
A six-year-old girl was admitted to our hospital with acute renal failure. We made a clinical diagnosis of acute interstitial nephritis and oral corticosteroid therapy was started. Her renal failure soon recovered, and renal biopsy showed acute interstitial nephritis by light microscopy with glomerular foot process effacement by electron microscopy. Although her proteinuria was not heavy at the time of biopsy, her proteinuria subsequently increased to show nephrotic syndrome. We continued to give corticosteroids and her nephrotic syndrome went into remission 13 days after biopsy. Serological and bacteriological examination showed no evidence of known pathogen or drug hypersensitivity. The time changes in proteinuria were monitored by fractional total protein excretion (FETP) and fractional beta2 microglobulin excretion (FEbeta2MG) in order to evaluate the severity of proteinuria under different glomerular filtration rates and different proximal tubular functions. The results revealed that nephrotic syndrome had occurred during recovery from acute interstitial nephritis. This is the first case report to show the sequential occurrence of acute interstitial nephritis and nephrotic syndrome based on evidence from fractional protein excretion.
Acute Disease↗
Heparin-binding EGF-like growth factor accelerates keratinocyte migration and skin wound healing.
Members of the epidermal growth factor (EGF) family are the most important growth factors involved in epithelialization during cutaneous wound healing. Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is thought to play an important role in skin wound healing. To investigate the in vivo function of HB-EGF in skin wound healing, we generated keratinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter. Studies of wound healing revealed that wound closure was markedly impaired in keratinocyte-specific HB-EGF-deficient mice. HB-EGF mRNA was upregulated at the migrating epidermal edge, although cell growth was not altered. Of the members of the EGF family, HB-EGF mRNA expression was induced the most rapidly and dramatically as a result of scraping in vitro. Combined, these findings clearly demonstrate, for the first time, that HB-EGF is the predominant growth factor involved in epithelialization in skin wound healing in vivo and that it functions by accelerating keratinocyte migration, rather than proliferation.
Animals↗
Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial cells.
OBJECTIVES: Antimicrobial peptides are one of the factors involved in innate immunity. The susceptibility of periodontopathogenic and cariogenic bacteria to the major antimicrobial peptides produced by epithelia was investigated. METHODS: Synthetic antimicrobial peptides of human beta-defensin-1 (hBD1), hBD2, hBD3 and LL37 (CAP18) were evaluated for their antimicrobial activity against oral bacteria. They included Actinobacillus actinomycetemcomitans (20 strains), Porphyromonas gingivalis (6), Prevotella intermedia (7), Fusobacterium nucleatum (7), Streptococcus mutans (5), Streptococcus sobrinus (5), Streptococcus salivarius (5), Streptococcus sanguis (4), Streptococcus mitis (2) and Lactobacillus casei (1). RESULTS: Although the four peptides had bactericidal activity against all bacteria tested, the degree of antibacterial activity was variable against the different strains and species. The antibacterial activity of hBD1 was lower than that of the other peptides. Among the bacteria tested in this study, F. nucleatum was highly susceptible to hBD3 and LL37, and S. mutans was highly susceptible to hBD3. We measured the Zeta-potential, representing the net charge of whole bacteria, to study the relationship between susceptibility to cationic peptide and the net charge of the bacteria. Although we found some correlation in A. actinomycetemcomitans strains, we did not find a definite correlation with all the bacterial species. CONCLUSIONS: These results indicate that beta-defensins and LL37 have versatile antibacterial activity against oral bacteria.
Amino Acid Sequence↗
The impact of preoperative serum C-reactive protein on the prognosis of patients with hepatocellular carcinoma.
BACKGROUND: The authors evaluated the significance of the preoperative serum C-reactive protein (CRP) level as a prognostic indicator in patients with hepatocellular carcinoma (HCC). METHODS: One hundred forty-one patients who underwent curative resection for HCC were reviewed retrospectively. Clinicopathologic variables were compared between patients with serum CRP levels >/= 1.0 mg/dL (n = 22 patients; the CRP-positive group) and patients with serum CRP levels < 1.0 mg/dL (n = 119 patients; the CRP-negative group). Univariate and multivariate analyses were conducted to identify factors that affected survival and disease recurrence. RESULTS: There was a significant correlation between the preoperative serum CRP level and tumor size. Invasion to the portal vein in the CRP-positive group was significantly more frequent than that in the CRP-negative group. Even after they underwent curative resection, 75.3% of patients in the CRP-positive group experienced recurrence within 1 year. The overall survival and recurrence-free survival rates in the CRP-positive group were significantly lower compared with the rates in the CRP-negative group. On multivariate analysis, the preoperative serum CRP level was selected as one of the unfavorable indicators regarding survival and recurrence. When CRP levels, albumin levels, and platelet counts that were available before surgery were scored as a combined index, the total score demonstrated a good stratification value for survival after hepatic resection. CONCLUSIONS: The current results showed that the preoperative serum CRP level is an independent and significant indicator predictive of poor prognosis and early recurrence in patients with HCC. The new CRP-based scoring system offers reliable information for predicting survival.
Adult↗
Reversible O-O bond cleavage and formation of a peroxo moiety of a peroxocarbonate ligand mediated by an iron(III) complex.
A mononuclear iron(III) complex containing a peroxocarbonate ligand, [Fe(qn)2(O2C(O)O)]- (qn = quinaldinate), underwent the reversible O-O bond cleavage and reformation of the peroxo group via the formation of FeIV=O or FeV=O species, which was confirmed by the resonance Raman and ESI-TOF/MS measurements.
Journal Article↗
Construction of an electrochemical DNA chip for simultaneous genotyping of single nucleotide polymorphisms.
An electrochemical DNA chip was constructed for simultaneous genotyping of single nucleotide polymorphisms (SNPs) using genomic DNA extracted from blood samples. This chip consisted of electrodes located on a single piece of substrate and allele-specific oligonucleotide probes on the electrodes. As a first application, the 4 SNPs (MxA[-88], MxA[-123], MBL[X/Y], and MBL[A/B]), which have association with the efficacy of interferon therapy for HCV patient, were genotyped on the new DNA chip. Following hybridization of PCR products containing the 4 types of fragments, washing, bisbenzimide H33258 (Hoechst 33258) reaction and electrochemical analyses, 59 blood samples were genotyped by the chip method simultaneously. All procedures were completed within 2 h and the results were 100% concordant with those by the direct sequence method. The electrochemical DNA chip is expected to be a practical tool for SNPs genotyping.
Bisbenzimidazole↗
SOCS3/CIS3 negative regulation of STAT3 in HGF-induced keratinocyte migration.
Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes. Because HGF has strong effects on the motility of keratinocytes and is produced by fibroblasts, HGF is thought to regulate keratinocyte migration during wound healing. However, the intracellular signaling mechanism of HGF-induced keratinocyte migration is poorly understood. In this report, we clarify the roles of STAT3 and SOCS/CIS family in HGF-induced keratinocyte migration. HGF activated STAT3 and strongly induced keratinocyte migration. Transfection with the dominant-negative mutant of STAT3 almost completely abolished HGF-induced keratinocyte migration and STAT3 phosphorylation. Next, we studied the mechanisms that regulate STAT3 phosphorylation. HGF enhanced the expression of SOCS3/CIS3 by sixfold within 1h, but had minimum effect on SOCS1/JAB expression. Transfection with SOCS3/CIS3 almost completely abolished HGF-induced STAT3 phosphorylation and keratinocyte migration, indicating that SOCS3/CIS3 acts as a negative regulator of HGF-induced keratinocyte migration. In conclusion, SOCS3/CIS3 regulates HGF-induced keratinocyte migration by inhibiting STAT3 phosphorylation.
Cell Movement↗
New mechanisms of skin innate immunity: ASK1-mediated keratinocyte differentiation regulates the expression of beta-defensins, LL37, and TLR2.
Epidermal keratinocytes differentiate and form a multilayered epidermis, which is the primary barrier between the body and the outer environment. As the epidermis is constantly exposed to a variety of microbial pathogens, its function of resisting microbial pathogens is vital. This characteristic feature is formed during differentiation. Immunohistochemical analysis revealed that the upper epidermis of normal human skin expresses beta-defensins 1-3 and LL37. We hypothesized that epidermal keratinocytes develop an innate immune barrier based on human beta-defensins (hBD) and LL37 during differentiation. To prove this, we introduced an active form of the apoptosis signal-regulating kinase-1 (ASK1), an intracellular regulator of keratinocyte differentiation, into cultured normal human keratinocytes. Transfection of this active form, ASK1-DeltaN, significantly enhanced the expression of hBD1-3 and LL37. In addition, a p38 inhibitor abolished this induction, indicating that the ASK1-p38 cascade regulates the expression of hBD1-3 and LL37. Furthermore, the ASK1-p38 pathway also regulated the expression of Toll-like receptor (TLR)2 in keratinocytes. Contact between S. aureus and keratinocytes resulted in the phosphorylation of p38 and induced the expression of hBD2 and hBD3. Moreover, the p38 inhibitor reduced this induction. In conclusion, the ASK1-p38 cascade regulates the innate immunity of the skin by forming an immune barrier consisting of hBD, LL37, and TLR2 during epidermal differentiation.
Antimicrobial Cationic Peptides↗
Efficient rescue of measles virus from cloned cDNA using SLAM-expressing Chinese hamster ovary cells.
We here report a highly efficient reverse genetics system for measles virus (MeV), using Chinese hamster ovary cells constitutively expressing a MeV receptor human signaling lymphocyte activation molecule (CHO/hSLAM cells). The recombinant vaccinia virus vTF7-3 that encodes the T7 RNA polymerase under the control of the early/late promoter was used in the system. Replication of vTF7-3 was highly restricted in CHO/hSLAM cells, but the virus could still drive the T7 promoter, allowing us to recover MeV from the transfected cDNA efficiently. With this system the number of infectious centers, in which MeV replication cycles are initiated from transfected cDNAs, was approximately 100 times higher than that with the previous system (. J. Virol. 74, 6643-6647), and the recovery rate was 100%. The wild-type MeV that encodes the lac-Z gene of approximately 3.2kb in length, was easily generated with this CHO/hSLAM system, while such virus could not be recovered with the previous system. Since SLAM acts as a cellular receptor for both MeV vaccine and wild-type strains, the Edmonston vaccine strain was also recovered with this system more efficiently than with any other systems reported previously. Thus, the CHO/hSLAM-based system would expand applications of the MeV reverse genetics by allowing productions of mutant MeVs that have been difficult to generate with less efficient systems.
Animals↗
In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris.
Patients with pemphigus vulgaris (PV) have circulating anti-desmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic strength of anti-Dsg3 IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantly by Dsg3, we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting, and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PV sera (n=10, 77.4+/-21.4) than controls (n=11, 16.0+/-9.6; p=0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n=6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies.
Adult↗