Toshifumi Nomura

Clinical Severity Correlates with Impaired Barrier in Filaggrin-Related Eczema

Mutations in the gene-encoding filaggrin (FLG), a key molecule involved in skin barrier function, have been shown to be a major predisposing factor for atopic dermatitis (AD; eczema). To elucidate the pathomechanisms underlying filaggrin-related AD, we investigated stratum corneum (SC) hydration and transepidermal water loss (TEWL) as parameters of barrier function in AD patients harboring FLG mutations compared to AD patients without any FLG mutation. In filaggrin-related AD, SC hydration was both significantly reduced (P<0.01-0.05) and thicker (P<0.01-0.05) than that in healthy controls. TEWL was demonstrably increased in non-filaggrin AD compared to healthy controls (P<0.01-0.05). The objective score of atopic dermatitis (OSCORAD), a disease clinical severity index, significantly correlated with TEWL (r=0.81, P<0.005), SC hydration (r=-0.65, P<0.05), and SC thickness (r=0.59, P<0.05) in filaggrin-related AD. On the contrary, there was no correlation between these parameters and the OSCORAD in non-filaggrin AD. Furthermore, a significant correlation was obtained between the OSCORAD and specific IgE for house dust (r=0.66, P<0.05), mite allergen (r=0.53, P<0.05), and cat dander (r=0.64, P<0.05) in filaggrin-related AD, but not in non-filaggrin AD. All these data suggest that experimentally demonstrable skin barrier defects due to FLG mutations may play a crucial role in the pathogenesis of AD.

Specific Filaggrin Mutations Cause Ichthyosis Vulgaris and Are Significantly Associated with Atopic Dermatitis in Japan

Mutations in the gene encoding filaggrin (FLG) have been identified as the cause of ichthyosis vulgaris (IV) and shown to be major predisposing factors for atopic dermatitis (AD). However, these studies have been mainly carried out in European populations. In early 2007, we identified two Oriental-specific FLG mutations in four Japanese families with IV and reported that filaggrin mutations were also significant predisposing factors for AD in Japan. However, the frequency of FLG mutations observed in our Japanese AD cohort (5.6%), was much lower than that seen in Europeans (up to 48%). Here, we studied a further seven Japanese families with IV and identified two additional nonsense mutations in FLG, S2889X, and S3296X. We found that more than 20% of patients in our Japanese AD case series carry FLG mutations, and there is significant statistical association between the four mutations and AD (chi(2) P=8.4 x 10(-6); heterozygote odds ratio 7.57, 95% CI 2.84-23.03). These data emphasize that skin-barrier impairment due to reduced filaggrin expression plays an important role in the pathogenesis of AD and sheds further light on the genetic architecture of atopy in Japan.

A group of atopic dermatitis without IgE elevation or barrier impairment shows a high Th1 frequency: possible immunological state of the intrinsic type.

BACKGROUND Atopic dermatitis (AD) can be classified into the major extrinsic type with high serum IgE levels and impaired barrier, and the minor intrinsic type with normal IgE levels and unimpaired barrier. OBJECTIVE To characterize the intrinsic type of Japanese AD patients in the T helper cell polarization in relation to the barrier condition. METHODS Enrolled in this study were 21 AD patients with IgE<200kU/L (IgE-low group; 82.5±59.6kU/L) having unimpaired barrier, and 48 AD patients with IgE>500kU/L (IgE-high group; 8.050±10.400kU/L). We investigated filaggrin gene (FLG) mutations evaluated in the eight loci common to Japanese patients, circulating Th1, Th2 and Th17 cells by intracellular cytokine staining and flow cytometry, and blood levels of CCL17/TARC, IL-18, and substance P by ELISA. RESULTS The incidence of FLG mutations was significantly lower in the IgE-low group (10.5%) than the IgE-high group (44.4%) (normal individuals, 3.7%). The percentage of IFN-γ-producing Th1, but not Th2 or Th17, was significantly higher in the IgE-low than IgE-high group. Accordingly, Th2-attracting chemokine CCL17/TARC, was significantly lower in the IgE-low than the IgE-high group. There were no differences between them in serum IL-18 levels, or the plasma substance P levels or its correlation with pruritus. CONCLUSION The IgE-low group differed from the IgE-high group in that it had much less FLG mutations, increased frequency of Th1 cells, and lower levels of CCL17. In the intrinsic type, non-protein antigens capable of penetrating the unimpaired barrier may induce a Th1 eczematous response.

Impaired Epidermal Permeability Barrier in Mice Lacking Elovl1, the Gene Responsible for Very-Long-Chain Fatty Acid Production

ABSTRACT The sphingolipid backbone ceramide (Cer) is a major component of lipid lamellae in the stratum corneum of epidermis and has a pivotal role in epidermal barrier formation. Unlike Cers in other tissues, Cers in epidermis contain extremely long fatty acids (FAs). Decreases in epidermal Cer levels, as well as changes in their FA chain lengths, cause several cutaneous disorders. However, the molecular mechanisms that produce such extremely long Cers and determine their chain lengths are poorly understood. We generated mice deficient in the Elovl1 gene, which encodes the FA elongase responsible for producing C20 to C28 FAs. Elovl1 knockout mice died shortly after birth due to epidermal barrier defects. The lipid lamellae in the stratum corneum were largely diminished in these mice. In the epidermis of the Elovl1-null mice, the levels of Cers with ≥C26 FAs were decreased, while those of Cers with ≤C24 FAs were increased. In contrast, the levels of C24 sphingomyelin were reduced, accompanied by an increase in C20 sphingomyelin levels. Two ceramide synthases, CerS2 and CerS3, expressed in an epidermal layer-specific manner, regulate Elovl1 to produce acyl coenzyme As with different chain lengths. Elovl1 is a key determinant of epidermal Cer chain length and is essential for permeability barrier formation.

An annexin A1–FPR1 interaction contributes to necroptosis of keratinocytes in severe cutaneous adverse drug reactions

Annexin A1 secreted from drug-stimulated monocytes contributes to keratinocyte necroptosis in serious drug-related adverse events in skin. Subduing a Severe Skin Side Effect Certain pain relievers and antiepileptic drugs can cause a very rare, but sometimes fatal, side effect in which skin painfully blisters and peels, caused by the patients’ immune response to the drug. Saito et al. now find that, in susceptible patients, the drug causes secretion of the protein annexin A1 from immune cells, with deadly effect on skin cells. Annexin acts on these cells to cause necroptosis, a programmed form of cell death. The authors confirmed their results in mice, showing that an inhibitor of necroptosis blocked skin blistering. With these findings, Saito et al. lay the groundwork for a countermeasure to this dangerous side effect of otherwise extremely beneficial drugs. Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening, cutaneous adverse drug reactions that are accompanied by keratinocyte cell death. Dead keratinocytes from SJS/TEN lesions exhibited necrosis, by morphological criteria. Supernatant from peripheral blood mononuclear cells (PBMCs) that had been exposed to the causative drug from patients with SJS/TEN induced the death of SJS/TEN keratinocytes, whereas supernatant from PBMCs of patients with ordinary drug skin reactions (ODSRs) exposed to the same drug did not. Keratinocytes from ODSR patients or from healthy controls were unaffected by supernatant from SJS/TEN or ODSR PBMCs. Mass spectrometric analysis identified annexin A1 as a key mediator of keratinocyte death; depletion of annexin A1 by a specific antibody diminished supernatant cytotoxicity. The necroptosis-mediating complex of RIP1 and RIP3 was indispensable for SJS/TEN supernatant–induced keratinocyte death, and SJS/TEN keratinocytes expressed abundant formyl peptide receptor 1 (FPR1), the receptor for annexin A1, whereas control keratinocytes did not. Inhibition of necroptosis completely prevented SJS/TEN-like responses in a mouse model of SJS/TEN. Our results demonstrate that a necroptosis pathway, likely mediated by annexin 1 acting through the FPR1 receptor, contributes to SJS/TEN.

Comprehensive screening for a complete set of Japanese‐population‐specific filaggrin gene mutations

Mutations in FLG coding profilaggrin cause ichthyosis vulgaris and are an important predisposing factor for atopic dermatitis. Until now, most case–control studies and population‐based screenings have been performed only for prevalent mutations. In this study, we established a high‐throughput FLG mutation detection system by real‐time PCR with a set of two double‐dye probes and conducted comprehensive screening for almost all of the Japanese‐population‐specific FLG mutations (ten FLG mutations). The present comprehensive screening for all ten FLG mutations provided a more precise prevalence rate for FLG mutations (11.1%, n = 820), which seemed high compared with data of previous reports based on screening for limited numbers of FLG mutations. Our comprehensive screening suggested that population‐specific FLG mutations may be a significant predisposing factor for hay fever (odds ratio = 2.01 [95% CI: 1.027–3.936, P < 0.05]), although the sample sizes of this study were too small for reliable subphenotype analysis on the association between FLG mutations and hay fever in the eczema patients and the noneczema individuals, and it is not clear whether the association between FLG mutations and hay fever is due to the close association between FLG mutations and hay fever patients with eczema.

Epidermolysis bullosa acquisita associated with psoriasis vulgaris

Summary We report a case of epidermolysis bullosa acquisita (EBA) associated with psoriasis vulgaris. A 71‐year‐old woman with psoriasis vulgaris developed subepidermal blisters on the extremities. Direct immunofluorescence demonstrated linear deposit of IgG at the basement membrane zone, which bound to the dermal side of normal human skin split with 1 mol/L NaCl. Immunoblot analysis using recombinant full‐length type VII collagen detected a 290‐kDa band, confirming the diagnosis of EBA. A literature search for previous reports found a few cases of EBA associated with psoriasis, and all cases, including our own, presented with widespread inflammatory vesicles and bullae, and responded to conventional therapy with corticosteroids and immunosuppressive agents. This study suggests that western blotting using recombinant full‐length type VII collagen could be useful for diagnosis of EBA, and that EBA associated with psoriasis may have a tendency to be the inflammatory type.

Rapid remission of severe pruritus from angiolymphoid hyperplasia with eosinophilia by pulsed dye laser therapy.

A 48‐year‐old Japanese woman with angiolymphoid hyperplasia with eosinophilia (ALHE) was successfully treated with a flashlamp pulsed dye laser (585 nm, 450 µs pulse duration). The lesion was severely pruritic and had been enlarging slowly for 2 years but was resistant to conventional therapies, including topical, intralesional, and systemic corticosteroid, and cryotherapy. The severe pruritus immediately improved after the first treatment using the pulsed dye laser. The erythema and papules gradually improved without scarring and this was followed by further five treatments over approximately a 4‐month interval. No clinical recurrences have been observed 1 year after completion of the treatment. We think that pulsed dye laser therapy is an effective treatment for ALHE in both Japanese as well as Caucasian patients. Pulsed dye laser therapy is also helpful in reducing the pruritus in ALHE patients.

Eccrine porocarcinoma and eccrine poroma arising in a scar

SIR, Malignant tumours occurring at scar sites have long been reported in the literature. Most of them are squamous cell carcinomas and basal cell carcinomas. To our knowledge, eccrine porocarcinoma and eccrine poroma have not yet been described in association with scars. We report a case of eccrine porocarcinoma and eccrine poroma arising from a scar on the right foot. A 74-year-old woman had had paralysis of the lower half of the body for more than 50 years from spinal injury due to tuberculosis. She had scars caused by skin infections due to osteomyelitis and recurrent decubitus on the lower legs and feet. She presented with a partly ulcerated, fresh, red nodule surrounded by a brownish macule, 5 cm in diameter, situated within a scar on the anterior surface of her right foot (Fig. 1a). In addition, there was a slightly elevated, reddish but partly white nodule surrounded by a brown macule, 4 cm in diameter, situated in a scar on the posterior surface of her right foot (Fig. 1b). Histology of the nodule on the anterior surface of her right foot revealed a tumour composed of cords and broad columns of generally small basal-like cells extending into the dermis from the epidermis. The tumour cells were arranged irregularly and showed moderate atypia. The cells had large, hyperchromatic, irregularly shaped nuclei and some of them were multinucleated. Atypical mitotic figures were also seen. Ducts and small cysts were also seen within the tumour nests (Fig. 2a,b). The tumour cells stained positive with periodic acid–Schiff, alcian blue, epithelial membrane antigen, cytokeratin 7 (Fig. 2c) and carcinoembryonic antigen (Fig. 2d). However, diastase digestion, cytokeratin 20, S-100 protein and gross cystic disease fluid protein-15 were negative in the tumour cells. Based on these clinical and histopathological findings, the tumour on the anterior surface of the right foot was diagnosed as an eccrine porocarcinoma arising from a scar. Histopathologically, the nodule on the posterior surface of the right foot showed nests and islands of uniformly small basaloid cells, which were sharply demarcated from the adjacent keratinocytes. Broad, anastomosing cords and solid columns and nests of large cells extended into the dermis to varying levels. Duct-like structures were also found. The tumour was diagnosed as an eccrine poroma that occurred in association with a scar. Carcinomas are well known to arise frequently from a burn scar, and such carcinomas are termed Marjolin’s ulcers. Skin malignancies are thought to occur in association not only with burn scars, but also with scars in chronically inflamed or traumatized skin. Among such malignant tumours, squamous cell carcinomas occur most frequently in association Figure 1. (a) Anterior view of the right leg upon initial examination. Note a partly ulcerated, fresh, red nodule surrounded by a brownish macule (arrow). (b) On the posterior surface of the right foot there was a slightly elevated, reddish nodule surrounded by a brownish macule (arrow).

Pityriasis rubra pilaris type v as an autoinflammatory disease by card14 mutations

Importance We found CARD14 mutations (2 de novo novel mutations and another previously reported mutation) in 3 of 3 patients with pityriasis rubra pilaris (PRP) type V, but not in patients with PRP of other types. Our findings, combined with the published literature, suggest that type V PRP, both familial and sporadic, can be caused by CARD14 mutations. Detailed clinical observation revealed that all 3 patients displayed unique patchy macular brown hyperpigmentation. Objective To further determine how often patients with PRP have pathogenic mutations in CARD14 and to elucidate which clinical subtype of PRP is caused by CARD14 mutations. Design, Setting, and Participants We sequenced the entire coding regions of CARD14 in genomic DNA from patients with 5 clinical subtypes of PRP. The detailed clinical features were analyzed in all the patients. The pathogenicity of each mutation was evaluated by several computational predictions. PRP was classified into 6 subgroups, types I to VI, based on clinical criteria. We categorized all the patients with PRP into the clinical subtypes using the classic PRP classification; 22 cases of PRP with varying subtypes were studied. Main Outcomes and Measures The prevalence of CARD14 mutations in each subtype of PRP was evaluated. Clinical features and characteristics of patients with PRP with CARD14 mutations were analyzed. Results Overall 22 patients with PRP were included in our study (12 men, 10 women; mean [SD] age, 26 [18] years). Among 3 patients with PRP type V, all were found to have CARD14 mutations: 2 de novo novel mutations (p.Cys127Ser and p.Gln136Leu), and another previously reported mutation (p.Gly117Ser). All were close to the reported pathogenic domains. In silico analysis of all 3 mutations suggested that they are functionally relevant to pathogenesis. All 3 patients displayed unique patchy macular brown hyperpigmentation additionally to other typical features of PRP. Patients with PRP type I and type IV, 1 patient each, had the rare variants in CARD14. Conclusions and Relevance Pityriasis rubra pilaris type V is a distinct variant of PRP that is caused by CARD14 mutations. In addition, a rare variant of CARD14 might also be implicated in the pathophysiology of other forms of PRP.