UV irradiation-induced DNA hypomethylation around WNT1 gene: Implications for solar lentigines.

Wnt/ß-catenin signalling promotes melanogenesis in melanocytes and also induces melanocytogenesis from melanocyte stem cells (McSCs). Previous study reported that WNT1, a ligand which activates Wnt/ß-catenin signalling pathway, was more highly expressed in the epidermis at SLs than in normal skin areas, suggesting that WNT1 causes hyperpigmentation. To elucidate the mechanism by which WNT1 expression is increased in SLs, we examined the methylation of 5-carbon of cytosine (5mC), that is 5-methylcytosine (5mC) level, in a region within the WNT1 promoter; the methylation of the region was known to negatively regulate WNT1 gene expression. We used an immortalized cell line of human interfollicular epidermal stem cells to analyse the effect of UVB irradiation on DNA methylation level of WNT1 promoter and found that UVB irradiation caused demethylation of WNT1 promoter and promoted WNT1 mRNA expression. It was also found that UVB irradiation reduced the expression of DNA methyltransferase 1 (DNMT1), an enzyme responsible for maintaining methylation patterns during cell division. Pathological analysis of SLs and non-SL regions in the human skin revealed that both DNMT1 expression and 5mC level were decreased at SLs compared to non-SL skins. Furthermore, bisulphite sequencing showed that the methylated CpG level in WNT1 promoter was also lower at SLs than in non-SL skins. Thus, in the skin exposed to a high amount of UV rays, excessive expression of WNT1 is thought to be caused by the demethylation of WNT1 promoter, and the upregulated WNT1 promotes melanocytogenesis and melanogenesis, then resulting in SL formation.

Evaluation of the cross-reactivity of antigens in Glupearl 19S and other hydrolysed wheat proteins in cosmetics.

BACKGROUND: In Japan, over 2000 users of a facial soap containing Glupearl 19S (GP19S), a hydrolysed wheat protein (HWP), developed immediate-type systemic wheat allergy (HWP-IWA), and ∼70% of them developed associated contact urticaria. OBJECTIVES: We investigated whether HWP-IWA patients cross-react with other HWPs, and analysed HWP antigenic characteristics. METHODS: We used 10 types of HWP that are commercially available as cosmetic ingredients, and 16 subjects with HWP-IWA. We performed an enzyme-linked immunosorbent assay (ELISA) to evaluate the reactivity to each HWP, and western blotting to evaluate the characteristics of the antigens by using HWP-IWA patients' serum IgE antibodies. We also performed prick tests with the HWPs. RESULTS: The patients reacted to four other HWPs in addition to GP19S, according to ELISA, and this was confirmed by strong reactions in the prick tests to the same four types of HWP. Smears of antigens with molecular weights ranging from the high range to the low range were seen on western blotting with the four HWPs that showed strong reactions in the ELISA and prick tests. CONCLUSIONS: HWP-IWA patients cross-react with other HWPs. The antigens that they cross-reacted to had a molecular weight distribution similar to that of GP19S present in the HWPs.

A varicella outbreak in B-cell lymphoma patients receiving rituximab-containing chemotherapy.

Varicella, characterized by a vesicular rash, occurs primarily in young children. Although older individuals can also be affected or vaccinated, outbreaks among adults are rare. We investigated a small outbreak of varicella in B-cell lymphoma patients for elucidation of risk factor of the disease. We experienced four cases of varicella after an index herpes zoster case. All varicella cases were confirmed varicella zoster virus (VZV) infection by PCR. All varicella cases occurred in diffuse large B-cell lymphoma patients receiving rituximab-containing chemotherapy. On the other hand, only three of the 18 non-varicella patients in the same room were receiving rituximab-containing chemotherapy (P = 0.005). All varicella patients had detectable serum anti-varicella zoster virus IgG antibodies before chemotherapy. Even in the presence of neutralizing antibodies to the virus, lymphoma patients treated with rituximab-containing chemotherapy can possibly become re-infected with varicella. These findings suggest that zoster patients should be strictly isolated in hematology and oncology ward, and prophylactic acyclovir should be considered for such patients when exposed to zoster/varicella.

A new reliable method for detecting specific IgE antibodies in the patients with immediate type wheat allergy due to hydrolyzed wheat protein: correlation of its titer and clinical severity.

BACKGROUND: Immediate-type wheat allergy caused by a specific hydrolyzed wheat protein (HWP-IWA), Glupearl 19S (GP19S), typically develops food-dependent exercise-induced anaphylaxis (FDEIA), but is different from conventional FDEIA, or simple wheat allergy in many aspects. The skin prick test (SPT) is considered to be the most effective method for diagnosis of HWP-IWA. As SPT is a relatively qualitative method, we developed quantitative and high-throughput test method for HWP-IWA. METHODS: An enzyme-linked immunosorbent assay (ELISA)-based GP19S-specific IgE assay was tested using sera from 14 HWP-IWA and five conventional wheat-dependent exercise-induced anaphylaxis (CO-WDEIA) patients, as well as five healthy subjects. Then a validation study at five different institutions was carried out using sera from 10 HWP-IWA and five CO-WDEIA patients, as well as five healthy subjects different from the previous studies. RESULTS: The mean unit values converted from measured absorbance of ELISA were 68.3, 1.3 and 1.1 respectively. Furthermore, the validation study revealed reproducible results across all five institutions, with the standard deviation (SD) being 0.3-0.4 for the healthy group, 0.2-0.6 for the CO-WDEIA group, and 3.8-9.6 for HWP-IWA group except for one case. One case of HWP-IWA was excluded from analysis due to the high SD of 53.3 units, indicating that samples with a unit value > 100.0 will affect inter-laboratory reproducibility. CONCLUSIONS: Our findings suggest that the ELISA-based GP19S-specific IgE assay can be used to test HWP-IWA using venous blood samples, except for those with a unit value > 100.0.

Increase of gremlin 2 with age in human adipose-derived stromal/stem cells and its inhibitory effect on adipogenesis.

INTRODUCTION: Adipose-derived stromal/stem cells (ASCs) have attracted attention as a promising material for regenerative medicine. Previously, we reported an age-related decrease in the adipogenic potential of ASCs from human subjects and found that the individual difference in this potential increased with age, although the mechanisms remain unclear. Recently, other groups demonstrated that a secreted antagonist of bone morphogenetic protein (BMP) signaling, Gremlin 2 (GREM2), inhibits the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts and the adipogenesis of 3T3-L1 cell. Here, we examined the effects of GREM2 on the differentiation of ASCs into adipocytes. METHODS: To examine changes in GREM2 expression levels with age, immunohistochemistry was performed on subcutaneous adipose tissues from subjects 12-97 years of age. Next, GREM2 gene expression levels in ASCs collected from subjects 5-90 years of age were examined by RT-PCR, and the change with age and correlation between the expression level and the adipogenic potential of ASCs were analyzed. In addition, to assess whether GREM2 affects adipogenesis, ASCs (purchased from a vendor) were cultured to induce adipogenesis with recombinant GREM2 protein, and siRNA-induced GREM2 knockdown experiment was also performed using aged ASCs. RESULTS: In adipose tissues, GREM2 expression was observed in cells, including ASCs, but not in mature adipocytes, and the expression level per cell increased with age. GREM2 expression levels in ASCs cultured in vitro also increased with age, and the individual differences in the level increased with age. Of note, partial correlation analysis controlled for age revealed that the adipogenic potential of ASCs and the GREM2 gene expression level were negatively correlated. Furthermore, based on a GREM2 addition experiment, GREM2 has inhibitory effects on the adipogenesis of ASCs through activation of Wnt/ß-catenin signaling. On the other hand, GREM2 knockdown in aged ASCs promoted adipogenesis. CONCLUSIONS: The GREM2 expression level was confirmed to play a role in the age-related decrease in adipogenic potential observed in ASCs isolated from adipose tissues as well as in the enhancement of the individual difference, which increased with age. GREM2 in adipose tissues increased with age, which suggested that GREM2 functions as an inhibitory factor of adipogenesis in ASCs.

CXCL12 regulates differentiation of human immature melanocyte precursors as well as their migration.

Melanocyte stem cells (McSCs) are localized in the bulge region of hair follicles and supply melanocytes, which determine hair color by synthesizing melanin. Ectopic differentiation of McSCs, which are usually undifferentiated in the bulge region, causes depletion of McSCs and results in hair graying. Therefore, to prevent hair graying, it is essential to maintain McSCs in the bulge region, but the mechanism of McSC maintenance remains unclear. To address this issue, we investigated the role of CXCL12, a chemokine which was previously suggested to induce migration of melanocyte lineage cells, as a niche component of McSCs. Immunohistological analysis revealed that CXCL12 was highly expressed in the bulge region of human hair follicles. CXCL12 mRNA expression level was significantly lower in white hairs plucked from human scalps than in black hairs. CXCL12 attracted the migration of early-passage normal human epidermal melanocytes (eNHEMs), an in vitro model of McSCs, which had characteristics of immature melanocyte precursors. We also found that CXCL12 suppressed their differentiation. These results suggest that CXCL12 regulates differentiation of McSCs as well as their proper localization, and maintaining McSCs by regulating CXCL12 expression level in the bulge region may be a key to preventing hair graying.

Extracellular proteoglycan decorin maintains human hair follicle stem cells.

Hair follicle stem cells (HFSC) are localized in the bulge region of the hair follicle and play a role in producing hair. Recently, it has been shown that the number of HFSC decreases with age, which is thought to be a cause of senile alopecia. Therefore, maintaining HFSC may be key for the prevention of age-related hair loss, but the regulatory mechanisms of HFSC and the effects of aging on them are largely unknown. In general, stem cells are known to require regulatory factors in the pericellular microenvironment, termed the stem cell niche, to maintain their cell function. In this study, we focused on the extracellular matrix proteoglycan decorin (DCN) as a candidate factor for maintaining the human HFSC niche. Gene expression analysis showed that DCN was highly expressed in the bulge region. We observed decreases in DCN expression as well as the number of KRT15-positive HFSC with age. In vitro experiments with human plucked hair-derived HFSC revealed that HFSC lost their undifferentiated state with increasing passages, and prior to this change a decrease in DCN expression was observed. Furthermore, knockdown of DCN promoted HFSC differentiation. In contrast, when HFSC were cultured on DCN-coated plates, they showed an even more undifferentiated state. From these results, as a novel mechanism for maintaining HFSC, it was suggested that DCN functions as a stem cell niche component, and that the deficit of HFSC maintenance caused by a reduction in DCN expression could be a cause of age-related hair loss.

Enhancement of individual differences in proliferation and differentiation potentials of aged human adipose-derived stem cells.

BACKGROUND: Adipose-derived stem cells (ASCs) are a robust, multipotent cell source. They are easily obtained and hold promise in many regenerative applications. It is generally considered that the function of somatic stem cells declines with age. Although several studies have examined the effects of donor age on proliferation potential and pluripotency of ASCs, the results of these studies were not consistent. OBJECTIVE: This study tested whether the donor age affects the yield of ASCs from adipose tissue, as well as the proliferation and differentiation potentials of ASCs. METHODS: This study used ASCs obtained from adipose tissues of 260 donors (ages 5-97 years). ASCs were examined for individual differences in proliferation, and adipogenic, osteogenic and chondrogenic differentiation potentials in vitro. Characteristics of ASCs from each donor were evaluated by the principal component analysis (PCA) using their potential parameters. RESULTS: Analyses on ASCs demonstrated that adipogenic potentials declined with age, but proliferation, osteogenic and chondrogenic potentials were not correlated with age. Interestingly, in all ASC potentials, including adipogenesis, individual differences were observed. Principal component analysis (PCA) revealed that individual differences became evident in the elderly, and those variations were more prominent in females than in males. CONCLUSIONS: This study demonstrated age-related changes in the potentials of ASCs and revealed that the individual differences of ASCs become significant in people over 60 years of age (for females over 60, and for males over 80). We believe that it is important to carefully observe ASC potentials in order to achieve effective regenerative medicine treatments using ASCs.

Two Cases of Occupational Contact Urticaria Caused by Percutaneous Sensitization to Parvalbumin.

BACKGROUND: In recent years, it has been proposed that the primary mechanism for the development of food allergies is percutaneous sensitization. Since 2010, in Japan, the number of immediate-type wheat allergy due to hydrolyzed wheat protein has dramatically increased among those who have been using soap containing hydrolyzed wheat. This incidence supports the hypothesis that food allergens arise through percutaneous sensitization. CLINICAL SUMMARY: A 25-year-old man (case 1) and an 18-year-old girl (case 2) with atopic dermatitis visited our Department because of food allergy and hand eczema. After starting their work with fish, severe itchy eczema appeared on their hands. They subsequently started to experience oral allergic symptoms, intraoral itchiness and dyspnea after eating fish. Specific IgE antibodies were detected for many fishes, and skin prick tests showed positive reactions for a variety of fishes in both cases. Furthermore, the fluorescence intensities of specific IgE antibodies against parvalbumin from various types of fish in microarray immunoassay analysis showed positive reactions. We diagnosed them as contact urticaria caused by percutaneous sensitization to parvalbumin through job-related physical contact with fish. CONCLUSION: The patients' histories and findings indicate the possibility of percutaneous sensitization through occupational exposure to parvalbumin, leading to food allergy.

Concurrent reactivation of herpes simplex and varicella zoster viruses confirmed by the loop-mediated isothermal amplification assay.

Concurrent reactivation of herpes simplex and varicella zoster viruses is rare. Here, we describe the case of an elderly patient with herpes labialis and herpes zoster manifesting as a right-side facial eruption with vesicles and crusting. The loop-mediated isothermal amplification (LAMP) assay demonstrated the presence of both herpes simplex virus type 1 and varicella zoster virus in swab samples taken from the face, which was confirmed by real-time PCR, suggesting concurrent reactivation of both viruses. The use of the LAMP assay in the present case indicates its usefulness in the diagnosis of atypical herpes infections.

A Case of Malignant Melanoma with In-Transit Metastasis That Responded to Intravenous Infusion of Interferon-ß.

A 77-year-old man with a history of surgical resection of malignant melanoma involving the fifth toe of his left foot 14 years ago presented at the Kariya Toyota General Hospital with a 3-month history of skin ulcer at the same site and red nodules on the lower left leg. Malignant melanoma was suspected, and the patient was referred to our department. On examination, a skin ulcer measuring 25 × 20 mm was observed at the amputation site on the left foot. In addition, multiple red nodules were observed on the lower left leg. Skin biopsies of the ulcer and nodules revealed recurrent malignant melanoma with in-transit metastasis. Two weeks later, he developed acute myocardial infarction and was hospitalized at the Kariya Toyota General Hospital. One month later, the myocardial infarction ameliorated, and he was transferred to our department. As the myocardial infarction had decreased the patient's tolerance to surgery, interferon-ß was administered by intravenous infusion. The skin ulcer and red nodules on the lower left leg disappeared 26 weeks after infusion had been initiated. The patient's progress has been satisfactory, with no evidence of recurrence or metastasis at 1 year and 9 months after the initiation of intravenous infusion.

Clinical utility of loop-mediated isothermal amplification assay for the diagnosis of common alpha herpesvirus skin infections.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method with a high specificity, efficiency and speed. No reports exist regarding the usefulness of LAMP for clinically suspected skin infections caused by herpes simplex virus (HSV) or varicella zoster virus (VZV). The aim of this study was to evaluate the clinical usefulness of LAMP in the diagnosis of common cutaneous alpha herpesvirus (HSV type 1 and 2, and VZV) infections. LAMP and real-time polymerase chain reaction (PCR) were performed using swab samples collected from 106 patients with clinically suspected alpha herpesvirus skin infections. The results of LAMP performed with DNA extraction did not differ from those performed without DNA extraction. The sensitivity of LAMP tested against real-time PCR was 96% in herpes simplex, 78% in eczema herpeticum, 93% in herpes zoster and 100% in varicella. No viral DNA was detected by LAMP in all negative real-time PCR samples. Viral DNA load was significantly lower in samples with false-negative LAMP results than in the LAMP-positive samples. LAMP enables confirmation of clinically suspected cutaneous HSV and VZV infections. However, the sensitivity of LAMP is lower than real-time PCR. The accuracy of LAMP may increase if sufficient viral DNA is obtained from lesions. LAMP performed without DNA extraction remains sensitive; thus, LAMP represents a quick and economical method for the diagnosis of common alpha herpesvirus skin infections.

Severe hepatitis associated with varicella zoster virus infection in a patient with diffuse large B cell lymphoma treated with rituximab-CHOP chemotherapy.

Severe disseminated varicella zoster virus (VZV) infection rarely occurs in patients who are not recipients of hematopoietic stem cell transplantation. This report concerns severe disseminated VZV infection in a diffuse large B cell lymphoma (DLBCL) patient treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP). The patient was an 82-year-old male with DLBCL who had a history of type II diabetes mellitus. He incurred VZV infection with severe hepatitis and disseminated intravascular coagulopathy after three courses of R-CHOP. When the VZV infection occurred, anti-VZV IgG was not detected and lymphopenia was observed. We initiated treatment with acyclovir, immunoglobulin, and thrombomodulin alpha, and rescued this patient. We suggest that the use of chemotherapy for immune-suppressed elderly lymphoma patients may involve the risk of severe VZV infection.