Dermatophyte antigen kit: A useful diagnostic tool for onychomycosis.

A dermatophyte antigen kit (DQT) was released in Japan as an in vitro diagnostic tool to identify tinea unguium in June 2022. From July 2022 to February 2023, we examined 75 potassium hydroxide (KOH)-negative patients (male, n = 23; female, n = 52; mean ± SD age, 63.6 ± 13.9 years) and determined the accuracy in confirming the fungal element with ZoomBlue™ staining at 400× magnification. The DQT results were classified into three categories. DQT-positive onychomycosis was detected in 27 patients with tinea unguium and two with non-dermatophyte onychomycosis. Fungal cultures were positive in 14 (51.8%) patients (Trichophyton rubrum [n = 11], T. interdigitale [n = 1], Fusarium solani [n = 1], and Talaromyces muroii [n = 1]). DQT-negative onychomycosis included ten patients with cured tinea unguium and 3 with Candida onychomycosis. Twenty-three patients had DQT-negative mimics for onychomycosis (onychauxis [n = 11], traumatic onycholysis [n = 8], yellow nail syndrome [n = 5], pincer nail deformity [n = 3], brittle nail syndrome [n = 2], contact dermatitis [n = 2], lichen planus [n = 1] and psoriasis [n = 1]). Because sparse, atrophic and/or fragmented mycelia are invisible in direct microscopy with potassium hydroxide (KOH) at 100× magnification, DQT was beneficial for diagnosing onychomycosis.

Hyperfunctioning Papillary Thyroid Carcinoma with a BRAF Mutation: The First Case Report and a Literature Review.

INTRODUCTION: Hyperfunctioning papillary thyroid carcinoma (PTC) is rare and consequently, little information on its molecular etiology is available. Although BRAF V600E (BRAF c.1799T>A, p.V600E) is a prominent oncogene in PTC, its mutation has not yet been reported in hyperfunctioning PTC. CASE PRESENTATION: Ultrasonography detected a 26-mm nodule in the right lobe of the thyroid gland of a 48-year-old man. Thyroid function tests indicated that he was hyperthyroid with a TSH level of 0.01 mIU/L (reference range: 0.05-5.00) and a free thyroxine level of 23.2 pmol/L (reference range: 11.6-21.9). TSHR autoantibodies were <0.8 IU/L (reference value: <2.0 IU/L). The 99mTc thyroid scintigram revealed a round, right-sided focus of tracer uptake by the nodule with a decreased uptake in the remainder of the gland. The patient underwent total thyroidectomy because fine-needle aspiration cytology revealed a malignancy. The histopathological diagnosis was conventional PTC. Subsequent mutational analysis of BRAF (exon 15), TSHR (exons 1-10), GNAS (exons 7-10), EZH1 (exon 16), KRAS, NRAS, HRAS (codons 12, 13, and 61), and TERT promoter (C250T and C228T) identified a heterozygous point mutation in BRAF V600E in a tumor tissue sample. In addition, we identified a TSHR D727E polymorphism (TSHR c.2181C>G, p.D727E) in both the tumor and the surrounding normal thyroid tissue. DISCUSSION AND CONCLUSIONS: We report a case of hyperfunctioning PTC with a BRAF V600E mutation for the first time. Our literature search yielded 16 cases of hyperfunctioning thyroid carcinoma in which a mutational analysis was conducted. We identified TSHR mutations in 13 of these cases. One case revealed a combination of TSHR and KRAS mutations; the other case revealed a TSHR mutation with a PAX8/PPARG rearrangement. These findings suggest that the concomitant activation of oncogenes (in addition to constitutive activation of the TSHR-cyclic AMP cascade) are associated with the malignant phenotype in hyperfunctioning thyroid nodules.

Laser treatment of 26 Japanese patients with Mongolian spots.

BACKGROUND: Mongolian spots are congenital hyperpigmented areas of varying size and shape and are usually confluent grayish-blue in color. They are found most frequently in the sacral region and typically disappear during childhood. Occasionally, they persist to adulthood. OBJECTIVE: We used Q-switched alexandrite laser treatment for Mongolian spots and examined therapeutic outcomes of 26 Japanese patients who consulted our department. MATERIALS & METHODS: We retrospectively compared 26 Japanese patients before and after treatment. RESULTS: A good therapeutic outcome was achieved overall, but some adult female patients subsequently developed severe postinflammatory hyperpigmentation. Sacral Mongolian spots were more laser-resistant than extrasacral Mongolian spots. CONCLUSION: The outcome correlated with the age of patients at the initiation of treatment; therefore, sacral and extrasacral Mongolian spots should be treated before 20 years of age. To avoid severe postinflammatory hyperpigmentation, the optimal interval between laser treatments and the use of other treatment modalities including Q-switched ruby laser, Q-switched neodymium-doped yttrium aluminium garnet laser, or bleaching creams should be considered. Our results will be of some help in considering the treatment course of patients with Mongolian spots.

Involvement of the constitutive complex formation of c-Ski/SnoN with Smads in the impaired negative feedback regulation of transforming growth factor beta signaling in scleroderma fibroblasts.

OBJECTIVE: The principal effect of transforming growth factor beta1 (TGFbeta1) on mesenchymal cells is its stimulation of extracellular matrix synthesis. Previous reports indicated the significance of the autocrine TGFbeta loop in the pathogenesis of scleroderma. The aim of this study was to examine c-Ski and SnoN, principal molecules in the negative regulation of TGFbeta signaling, to further understand the autocrine TGFbeta loop in scleroderma. METHODS: Levels of expression of c-Ski/SnoN on cultured normal and scleroderma fibroblasts were determined by Western blotting, Northern blotting, and immunohistochemical staining. To determine the protein-protein interaction between c-Ski/SnoN, Smads, and p300, immunoprecipitation was performed. A transient transfection assay was performed to measure promoter activity of the alpha2(I) collagen gene and the 3TP-Lux plasmid construct. RESULTS: Scleroderma fibroblasts exhibited increased c-Ski/SnoN levels compared with normal fibroblasts, both in vivo and in vitro. Although c-Ski/SnoN constitutively formed a complex with Smads by immunoprecipitation, the inhibitory effect of c-Ski/SnoN on the promoter activity of human alpha2(I) collagen and 3TP-Lux was impaired in scleroderma fibroblasts. Immunoprecipitation analyses also revealed that overexpressed c-Ski/SnoN could not compete with p300 in these cells. CONCLUSION: These results indicate that impaired competition with p300 is the possible cause of dysfunction of c-Ski/SnoN in scleroderma fibroblasts and that this might contribute to maintenance of the autocrine TGFbeta loop in this disease.

Treatment of 153 Japanese patients with Q-switched alexandrite laser.

We have recently used Q-switched alexandrite laser for the treatment of various kinds of pigmented skin lesions. We retrospectively compared therapeutic outcomes of 153 Japanese patients who consulted our department. This approach was not very efficient for nevus spilus/café-au-lait spots, which seemed laser-resistant, especially when the pigmentation had appeared after 1 year of age, was treated after 5 years of age, was located on the face, was oval with a smooth border, and the patient was male. This approach was equally effective for senile lentigo, nevus of Ota, and Mongolian spots, but less effective for acquired bilateral nevus of Ota-like macules. Some patients with sacral Mongolian spots or those with light-colored, senile lentigo developed severe post-inflammatory hyperpigmentation after treatment. As a whole, good therapeutic outcome was achieved after multiple treatment sessions. However, the use of other lasers or other treatment modalities should be considered to treat nevus spilus/café-au-lait spots.

Clinical features of scleroderma patients with contracture of phalanges.

Contracture of phalanges (CP) is a frequent complication of patients with systemic sclerosis (SSc). The objective of this study was to examine the prevalance of CP in patients with SSc and investigate the clinical and laboratory features of SSc patients with CP. Three hundred and fifty patients with SSc were examined. CP was found in 164 of the 350 patients (47%) with SSc. The prevalence of oesophageal involvement, pulmonary fibrosis or heart involvement was significantly greater in the patients with CP than that in those without CP. Our study suggested that the presence of CP may be a marker of oesophageal involvement, pulmonary fibrosis and heart involvement.

Anti-p53 antibodies in patients with dermatomyositis/polymyositis.

Dermatomyositis/polymyositis (DM/PM), which is often accompanied by various immunological abnormalities, was reported to be associated with an increased incidence of malignancies. In this study, we analyzed serum levels of anti-p53 antibody (anti-p53 Ab) in DM/PM patients and in normal controls. Serum levels of anti-p53 Abs were significantly higher in DM/PM patients than those in healthy controls. However, there was no significant difference between serum levels in patients with malignancies and those in patients without malignancies. Anti-p53 Abs were positive in 13% (4 out of 31) of the DM/PM patients. Of these four patients, only one had an internal malignancy. Immunoglobulin G levels were significantly higher in patients positive for anti-p53 Ab than those who were not. These results seemed to suggest that the presence of anti-p53 Abs in DM/PM patients is due to immunological abnormalities in this disease.

Involvement of alphavbeta5 integrin in the establishment of autocrine TGF-beta signaling in dermal fibroblasts derived from localized scleroderma.

Localized scleroderma (LSc) is a connective tissue disorder limited to skin and subcutaneous tissue, which may share pathogenic processes with systemic sclerosis (SSc). We previously demonstrated that upregulated expression of integrin alphavbeta5 might contribute to autocrine TGF-beta signaling in SSc fibroblasts. Based on these data, we presently focused on alphavbeta5 and assessed its involvement in pathogenesis of LSc. We initially demonstrated that LSc fibroblasts might be activated by the stimulation of autocrine TGF-beta. Consistent with SSc fibroblasts, expression levels of alphavbeta5 were elevated in LSc fibroblasts in vitro and in vivo. Anti-alphavbeta5 antibody partially reversed expression levels of type I procollagen and MMP-1 and constitutive DNA-Smad3 binding in LSc fibroblasts. In LSc fibroblasts pretreated with antisense TGF-beta1, exogenous latent TGF-beta1 stimulation increased expression of type I procollagen in an alphavbeta5-dependent manner. The luciferase activities of TMLC cells, Mv1Lu cells stably expressing a portion of the plasminogen activator inhibitor 1 promoter, co-cultured with LSc fibroblasts were significantly elevated compared with those co-cultured with normal fibroblasts and were significantly reduced in the presence of anti-alphavbeta5 antibody. Anti-alphavbeta5 antibody reversed the myofibroblastic features of LSc fibroblasts. These results indicate that upregulated expression of alphavbeta5 contributes to autocrine TGF-beta signaling in LSc fibroblasts.

Potential regulatory elements of the constitutive up-regulated alpha2(I) collagen gene in scleroderma dermal fibroblasts.

The promoter activity of the full-length alpha2(I) collagen gene is higher in scleroderma fibroblasts, when compared to normal fibroblasts. In this study, to investigate the molecular mechanisms up-regulating the expression of the alpha2(I) collagen gene in scleroderma dermal fibroblasts more clearly, we compared promoter activities of serial 5'-deletion mutants and the substitution mutants of the alpha2(I) collagen promoter constructs between normal and scleroderma fibroblasts. The transient transfection assays using a series of 5'-deletions of the promoter revealed that the up-regulated fold-increase in scleroderma fibroblasts relative to that in normal fibroblasts was significantly decreased by the removal of bp -353 to -264 fragment or bp -264 to -186 fragment. The substitution mutations introduced into binding sites of Sp1 (bp -303 and -271), Ets1 (bp -285 and -282), as well as Smad (bp -263 and -258) also abrogated the fold-increase in promoter activity in scleroderma fibroblasts synergistically. A DNA affinity precipitation assay showed that the binding activity of Ets1 as well as Smad3 to their binding sites was increased in scleroderma fibroblasts compared with normal cells. Taken together, our promoter analysis emphasized that Ets1 form a transcriptionally active complex with Smad and Sp1 by autocrine transforming growth factor (TGF)-beta signaling, leading to the intrinsic up-regulation of alpha2(I) collagen promoter activity in scleroderma fibroblasts. The blockade of autocrine TGF-beta signaling is thought to be one of the most reliable approaches in the treatment of scleroderma, and further study targeting Ets1, Smad or Sp1 may contribute to this blockade.

Rheumatoid factor isotypes in mixed connective tissue disease.

Mixed connective tissue disease (MCTD) is a connective tissue disorder that is often accompanied by various immunological abnormalities. In this study, we analyzed serum levels of rheumatoid factor (RF) isotypes in patients with MCTD and in normal controls to determine if any of these isotypes reflects the severity of the disease. IgM-RF, IgG-RF, and IgA-RF were positive in 48, 38, and 33% of the patients, respectively. The frequency of positive anti-SS-A antibody and decrease in white blood cell counts were significantly greater in patients with elevated IgA-RF levels than that in those with normal levels. These results suggest that the presence of RF isotypes can be regarded as one of the various immunological abnormalities of MCTD.

Epidermal growth factor affects the synthesis and degradation of type I collagen in cultured human dermal fibroblasts.

EGF and type I collagen are known to play important roles in wound healing. In the present study, we demonstrated that EGF down-regulates the expression of type I procollagen protein as well as alpha2(I) collagen mRNA in cultured human dermal fibroblasts. EGF induced the degradation of type I procollagen protein in conditioned medium through the up-regulation of MMP-1 expression. EGF down-regulated alpha2(I) mRNA expression partially at the post-transcriptional level by reducing the mRNA stability. In contrast, EGF up-regulated MMP-1 mRNA expression mostly at the transcriptional level, in that it had a stimulatory effect on MMP-1 promoter activity, but no effect on MMP-1 mRNA stability. The MEK/ERK signaling pathway was shown to be involved in EGF-mediated type I collagen and MMP-1 expression.

Increased expression of integrin alpha(v)beta3 contributes to the establishment of autocrine TGF-beta signaling in scleroderma fibroblasts.

The constitutive secretion of latent TGF-beta by many cell types in culture suggests that extracellular mechanisms to control the activity of this potent cytokine are important in the pathogenesis of the diseases in which this cytokine may be involved, including fibrotic disorders. In this study, we focused on the alpha(v)beta3 integrin, which is recently demonstrated to function as an active receptor for latent TGF-beta1 through its interaction with latency-associated peptide-beta1, and investigated the involvement of this integrin in the pathogenesis of scleroderma. Scleroderma fibroblasts exhibited increased alpha(v)beta3 expression compared with normal fibroblasts in vivo and in vitro. In scleroderma fibroblasts, ERK pathway was constitutively activated and such abnormality induced the up-regulation of alpha(v)beta3. Transient overexpression of alpha(v)beta3 in normal fibroblasts induced the increase in the promoter activity of human alpha2(I) collagen gene and the decrease in that of human MMP-1 gene. These effects of alpha(v)beta3 were almost completely abolished by the treatment with anti-TGF-beta Ab or TGF-beta1 antisense oligonucleotide. Furthermore, the addition of anti-alpha(v)beta3) Ab reversed the expression of type I procollagen protein and MMP-1 protein, the promoter activity of human alpha2(I) collagen gene, and the myofibroblastic phenotype in scleroderma fibroblasts. These results suggest that the up-regulated expression of alpha(v)beta3 contributes to the establishment of autocrine TGF-beta loop in scleroderma fibroblasts, and this integrin is a potent target for the treatment of scleroderma.

Involvement of alphavbeta5 integrin-mediated activation of latent transforming growth factor beta1 in autocrine transforming growth factor beta signaling in systemic sclerosis fibroblasts.

OBJECTIVE: To confirm the involvement of alphavbeta5 in the self-activation system in systemic sclerosis (SSc) fibroblasts. METHODS: Levels of alphavbeta5 expression were analyzed by immunoprecipitation. The promoter activity of the human alpha2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor beta (TGFbeta) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis. RESULTS: Levels of alphavbeta5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti-alphavbeta5 antibody or beta5 antisense oligonucleotide significantly reduced human alpha2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFbeta1 antisense oligonucleotide, the exogenous latent TGFbeta1 stimulation significantly increased human alpha2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti-alphavbeta5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with beta5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti-alphavbeta5 antibody. Anti-alphavbeta5 antibody reversed the myofibroblastic features of SSc fibroblasts. CONCLUSION: Up-regulated expression of alphavbeta5 contributes to the establishment of autocrine TGFbeta signaling in SSc fibroblasts through activation of endogenous latent TGFbeta1.

Matrix metalloproteinase-1 up-regulation by hepatocyte growth factor in human dermal fibroblasts via ERK signaling pathway involves Ets1 and Fli1.

In this study, we clarified the molecular mechanism(s) underlying the regulation of matrix metalloproteinase (MMP)-1 gene by hepatocyte growth factor (HGF) in cultured human dermal fibroblasts. HGF induced MMP-1 protein as well as mRNA at a transcriptional level via extracellular signal-regulated kinase (ERK) signaling pathway. The region in the MMP-1 promoter mediating the inducible responsiveness to HGF, defined by the transient transfection analysis of the serial 5' deletion constructs, contained an Ets binding site. Mutation of this Ets binding site abrogated the HGF-inducible promoter activity. Ets1 up-regulated the expression of MMP-1 promoter activity, whereas Fli1 had antagonistic effects on them. After HGF treatment, the protein level and the binding activity of Ets1 was increased and those of Fli1 was decreased, which were canceled by PD98059. These results suggest that HGF up-regulates MMP-1 expression via ERK signaling pathway through the balance of Ets1 and Fli1, which may be a novel mechanism of regulating MMP-1 gene expression.

Differential effects of the immunosuppressant FK-506 on human alpha2(I) collagen gene expression and transforming growth factor beta signaling in normal and scleroderma fibroblasts.

OBJECTIVE: To investigate the effects of FK-506 on the expression of the human alpha2(I) collagen gene and transforming growth factor beta (TGFbeta) signaling in normal and scleroderma fibroblasts. METHODS: The expression levels of type I procollagen protein and alpha2(I) collagen messenger RNA (mRNA) were analyzed by immunoblotting and Northern blotting, respectively. The promoter activities of alpha2(I) collagen gene and 3TP-Lux were determined by transient transfection assay. Interaction between TGFbeta receptor type I and FK-506 binding protein 12 (FKBP12) was evaluated by immunoprecipitation. RESULTS: FK-506 did not affect the basal expression of type I procollagen protein or alpha2(I) collagen mRNA, but it significantly reduced the TGFbeta1-induced expression of type I procollagen protein and alpha2(I) collagen mRNA in normal fibroblasts. The effect of FK-506 was regulated posttranscriptionally, but not transcriptionally. In scleroderma fibroblasts, FK-506 significantly reduced the expression of type I procollagen protein and alpha2(I) collagen mRNA through posttranscriptional regulation, but not transcriptional regulation. FK-506 increased the basal activity of the 3TP-Lux promoter, but it did not affect the TGFbeta1-induced promoter activity in normal fibroblasts. In contrast, FK-506 did not affect the basal or the TGFbeta1-induced 3TP-Lux promoter activity in scleroderma fibroblasts. Furthermore, FKBP12, which protects TGFbeta receptor type I from ligand-independent activation by TGFbeta receptor type II, constitutively dissociated from TGFbeta receptor type I in scleroderma fibroblasts. CONCLUSION: FK-506 inhibits alpha2(I) collagen gene expression by reducing the stability of mRNA without exhibiting its activation effect on TGFbeta signaling in scleroderma fibroblasts.

Constitutive thrombospondin-1 overexpression contributes to autocrine transforming growth factor-beta signaling in cultured scleroderma fibroblasts.

The extracellular matrix (ECM) glycoprotein thrombospondin-1 (TSP-1) has been reported to activate the latent complex of transforming growth factor-beta (TGF-beta), the major effects of which in mesenchymal cells is stimulation of the synthesis of ECM. Previous reports suggested the involvement of an autocrine TGF-beta loop in the pathogenesis of scleroderma. In this study, we examined whether TSP-1 plays a role in maintaining the autocrine TGF-beta loop in scleroderma. TSP-1 expression was increased in scleroderma patients compared with in healthy controls in vivo and in vitro. TGF-beta blocking antibody or TGF-beta1 antisense oligonucleotide markedly reduced the up-regulated TSP-1 expression in scleroderma fibroblasts but had little effect on normal fibroblasts. The expression of TSP-1 is up-regulated in scleroderma fibroblasts, possibly at the post-transcriptional level just like in normal fibroblasts stimulated with exogenous TGF-beta1. TSP-1 blocking peptide or antisense oligonucleotide had an inhibitory effect on the up-regulated alpha2I collagen and phosopho-Smad3 levels in scleroderma fibroblasts but had little effects on normal fibroblasts. The transient overexpression of TSP-1 up-regulated alpha2I collagen and phospho-Smad3 levels in normal fibroblasts but had no major effect on scleroderma fibroblasts. Furthermore, these effects of transiently overexpressed TSP-1, which possibly occurred via the activation of latent TGF-beta1, were abolished by the TGF-beta1 antisense oligonucleotide. These results indicate that the constitutive overexpression of TSP-1 may play an important role in autocrine TGF-beta signaling and accumulation of ECM in scleroderma fibroblasts.

Constitutive phosphorylation of focal adhesion kinase is involved in the myofibroblast differentiation of scleroderma fibroblasts.

Most of the cultured scleroderma fibroblasts have been reported to be myofibroblasts that have the ability to express alpha smooth muscle actin (alphaSMA). It is reported that, in human lung fibroblasts, alphaSMA is induced by transforming growth factor-beta (TGF-beta), which requires focal adhesion kinase (FAK) phosphorylation on its Tyr-397 site. In this study, we investigated how alphaSMA expression is upregulated in cultured scleroderma fibroblasts. 4-amino-5-(4-chlorophenyl)-7-(butyl)pyrazolo[3,4-d]pyrimidine, which is a pharmacologic inhibitor of FAK/Src, markedly diminished upregulated alphaSMA expression in scleroderma fibroblasts as well as in normal fibroblasts stimulated with TGF-beta. Likewise, alphaSMA expression was significantly reduced in sclerderma fibroblasts transfected with kinase-deficient FAK mutant. FAK phosphorylation levels on Tyr-397 in scleroderma fibroblasts were significantly higher than those in normal fibroblasts. Both alphaSMA expression and FAK phosphorylation levels in scleroderma fibroblasts were markedly diminished by the treatment with TGF-beta antisense oligonucleotide. These results indicate that the constitutive phosphorylation of FAK, which is possibly because of the autocrine TGF-beta signaling, may play an important role in alphaSMA expression in scleroderma fibroblasts.

Alpha2(I) collagen gene regulation by protein kinase C signaling in human dermal fibroblasts.

We investigated the mechanisms by which protein kinase C (PKC) regulates the expression of the alpha2(I) collagen gene in normal dermal fibroblasts. Reduction of PKC-alpha activity by treatment with Gö697-6 or by overexpression of a dominant negative (DN) mutant form decreased alpha2(I) collagen gene expression. This decrease required a sequence element in the collagen promoter that contains Sp1/Sp3 binding sites. Reduction of PKC-delta activity by rottlerin or overexpression of DN PKC-delta also decreased alpha2(I) collagen gene expression. This effect required a separate sequence element containing Sp1/Sp3-binding sites and an Ets-binding site. In both cases, point mutations within the response elements abrogated the response to PKC inhibition. Forced overexpression of Sp1 rescued the PKC inhibitor-mediated reduction in collagen protein expression. A DNA affinity precipitation assay revealed that inhibition of PKC-delta by rottlerin increased the binding activity of endogenous Fli1 and decreased that of Ets1. On the other hand, TGF-beta1, which increased the expression of PKC-delta, had the opposite effect, increasing the binding activity of Ets1 and decreasing that of Fli1. Our results suggest that PKC-delta is involved in the regulation of the alpha2(I) collagen gene in the presence or absence of TGF-beta. Alteration of the balance of Ets1 and Fli1 may be a novel mechanism regulating alpha2(I) collagen expression.