Kazuki Tatsuno

Kallikrein-related peptidase 5 functions in proteolytic processing of profilaggrin in cultured human keratinocytes

Background: Filaggrin is a skin barrier function-related factor processed from profilaggrin. The identity of human profilaggrin-processing enzymes remains unclear. Results: The protease kallikrein 5 (KLK5) specifically processed a recombinant human filaggrin fragment fused to a linker. Conclusion: KLK5 is potentially a key molecule in human profilaggrin maturation. Significance: KLK5 may function in formation of the skin barrier. Filaggrin protein is synthesized in the stratum granulosum of the skin and contributes to the formation of the human skin barrier. Profilaggrin is cleaved by proteolytic enzymes and converted to functional filaggrin, but its processing mechanism remains not fully elucidated. Kallikrein-related peptidase 5 (KLK5) is a major serine protease found in the skin, which is secreted from lamellar granules following its expression in the stratum granulosum and activated in the extracellular space of the stratum corneum. Here, we searched for profilaggrin-processing protease(s) by partial purification of epidermal extracts and found KLK5 as a possible candidate. We used high performance liquid chromatography coupled with electrospray tandem mass spectrometry to show that KLK5 cleaves profilaggrin. Furthermore, based on a proximity ligation assay, immunohistochemistry, and immunoelectron microscopy analysis, we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis. KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage.

TSLP Directly Interacts with Skin-Homing Th2 Cells Highly Expressing its Receptor to Enhance IL-4 Production in Atopic Dermatitis

Thymic stromal lymphopoietin (TSLP) is overtly expressed on skin lesions of atopic dermatitis (AD), and the initiative role of TSLP-activated DCs in AD has gained much attention in the past few years, while its actions on other immune cells such as T cells have been given less notice. We aimed to clarify whether TSLP receptor (TSLPR) is expressed on certain populations of T cells and whether TSLP possesses the capability to directly interact with T cells from AD patients. Peripheral lymphocytes from 51 AD patients are analyzed by flow cytometry, and ex vivo experiments using peripheral blood and lesional skin-derived T cells were conducted. TSLPR expression was defined to CD4+ T cells, and CD4+CCR4+CXCR3-CCR7-CCR10+CLA+ T cells in AD patients exhibited enhanced TSLPR expression. The frequency of TSLPR+CD4+ T cells correlated with disease activity. CD4+ T cells from AD patients directly interacted with TSLP to produce a higher amount of IL-4 than those from normal subjects, and this action was attenuated with anti-TSLPR antibody. The importance of IL-4 in the induction of TSLPR expression was found in AD T cells. Our findings indicate that T cells from AD patients possess strong potential to directly interact with TSLP to promote Th2 response.

Biochemical, cytological, and immunological mechanisms of rhododendrol-induced leukoderma

Recently, an unexpected outbreak of patients with leukoderma occurred in Japan with the use of brightening/lightening cosmetics containing rhododendrol (RD). Patients developed leukoderma mostly on the skin sites repeatedly applied with RD, but some patients also had vitiligo-like lesions on the non-applied sites. RD is a tyrosinase-competitive inhibiting substance, thereby serving as an inhibitor of melanin synthesis. Upon inhibition of tyrosinase, RD is converted to new products such as tyrosinase-catalyzed hydroxyl-metabolite, which damage melanocytes. The melanocyte cell lysates seem to induce T-cell response. The frequencies of CD8+ T cells in both lesional skin and peripheral blood are significantly higher in the RD leukoderma as well as non-segmental vitiligo patients than in normal controls. In HLA-A*02:01 positive cases, circulating Melan-A-specific cytotoxic T cells can be detected at a high frequency. It is thus suggested that RD-induced leukoderma is induced by not only cytolysis of melanocytes but also subsequent immune reactions toward melanocytes.

Serum interleukin‐22 and vascular endothelial growth factor serve as sensitive biomarkers but not as predictors of therapeutic response to biologics in patients with psoriasis

The T‐helper (Th)17 cell plays a crucial role in the pathogenesis of psoriasis, and several biological therapies have shown to be highly efficient in the treatment. However, some patients respond poorly to these therapies and may even develop paradoxical adverse effects. To evaluate the significance of serum immunological factors or circulating competent cells for biomarkers or predictors to biological therapies, we retrospectively analyzed 28 patients with psoriasis (19 psoriasis vulgaris, three pustular psoriasis and six psoriasis arthropathica). The numbers of patients treated with each agents were 16 for ustekinumab, six for adalimumab and six for infliximab. Patients were classified into three types according to the responsiveness: 13 patients were high‐responders showing a 75% or more reduction of Psoriasis Area and Severity Index (PASI); 10 patients were moderate‐responders showing PASI reduction of less than 75%; and five patients were non‐responders showing PASI elevation. During the treatments, serum levels of interleukin (IL)‐22 and vascular endothelial growth factor (VEGF) were monitored. At baseline, serum IL‐22 levels were significantly higher in the psoriatic patients than the normal controls. Both serum IL‐22 and VEGF levels significantly correlated with PASI. After the treatment, the high‐responders showed significant decreases in serum IL‐22 and VEGF. On the other hand, serum IL‐22 levels in the non‐responders were elevated. However, the baseline levels of serum IL‐22 and VEGF were not significantly different between the three groups. These results suggest that serum IL‐22 and VEGF levels serve as sensitive biomarkers but not as predictors of therapeutic response to biologics in patients with psoriasis.

Proteome analysis of stratum corneum from atopic dermatitis patients by hybrid quadrupole-orbitrap mass spectrometer

may also be anti-inflammatory by reducing nuclear factor kappa B gene expression. Similarly, in addition to their inflammatory role, Langerhans cells are thought to have immunoregulatory functions. Indeed, we see that langerin cells are largely reduced in lesional than in nonlesional AD skin at baseline, and significantly increase on clinical reversal after 12 weeks of CsA treatment (Fig 1, E, and Table E3). In addition to the RDGP, other possible mechanisms for disease recurrence in the same areas need to be considered, including (1) regional differences (increased humidity/friction, transepidermal water loss, pH, and lipids) that allow increased antigen penetration, (2) epigenetic modifications, and (3) microbiome differences. In summary, we have demonstrated that although the CsA RDGP is much smaller than the NB-UVB RDGP, important structural defects and residual inflammation remain and the overall size of the RDGP does not predict relapse kinetics. Given that NB-UVB and CsA have different courses of disease maintenance on discontinuing therapy, some elements in the RDGP of each treatment might explain relevant treatmentand disease-specific mechanisms. Mariya Rozenblit, BA Mayte Suarez-Farinas, PhD Avner Shemer, MD Saakshi Khattri, MD Patricia Gilleaudeau, NP Mary Sullivan-Whalen, NP Xiuzhong Zheng, MSc Hui Xu, MSc Irma Cardinale, MSc James G. Krueger, MD, PhD Emma Guttman-Yassky, MD, PhD

Eruptive milium-like syringoma showing eccrine duct origin of milia.

Dear Editor, Syringoma is a benign eccrine gland tumor, representing multiple small papules frequently appearing on the lower eyelids of young women. On the other hand, milium is a small white superficial keratinous cyst, also commonly seen on the eyelids and cheeks. Thus, these two conditions share similar clinical features with each other. Rare variants of syringoma are known, such as lichen planus type, unilateral linear nevoidal type, alopecia type and eruptive type. Milium-like syringoma is one such exceptional variant and there are 31 documented cases to date. Skin lesions are localized to one area of the body in most cases, and the generalized eruptive form of milium-like syringoma is extremely rare. Here, we report the second case of the generalized form in the English-language published work, with the finding that the milium structure is of eccrine origin. A healthy 18-year-old Japanese woman was referred to our hospital with non-pruritic papules, which had gradually increased in number over the past 4 years. On examination, firm whitish papules, measuring 1–4 mm in diameter, were observed over her face, trunk and extremities. Papules on the upper and lower palpebrae and cheeks strikingly resembled milia because of their white hue (Fig. 1a), and the lesions on her body and limbs were suggestive of eruptive syringoma (Fig. 1b). A skin biopsy specimen from a papule on the abdomen revealed comma-shaped epithelial strands with occasional ductal formation in the dermis (Fig. 2a). Serial sectioning unveiled the presence of a cystic structure filled with keratin, which closely resembled a milium (Fig. 2b). The epithelial strand and the milium-like cyst were connected or located just in the vicinity. Immunohistochemically, the epithelial strands were positive for carcinoembryonic antigen (CEA), and more interestingly, the elongated epithelial cells at the lower part of keratinous cyst also bore CEA (Fig. 2c). Melanin was absent in the wall of the cyst, as demonstrated by Fontana–Masson stain. Both syringoma and milium are frequently encountered in clinical practice, but the combination of two apparently discrete entities had never been realized until Friedman and Butler reported the first case of milium-like syringoma in 1987. Due to the paucity of documentations, conclusive details regarding this type remain unclear, except for the female dominancy and the prevalence among Asian ethnic backgrounds. Milium-like syringoma occasionally appears on aberrant sites such as the vulva and perianal region. Its association with Down syndrome or genetic background has been reported. A rare case presenting with skin lesions distributed over the whole body was described by Weiss et al., and this is the sole case of eruptive milium-like syringoma before the present document. The key histological features observed in our case of milium-like syringoma are the connection of a milium-like cyst to the underlying syringoma, the positive stain of the keratinous cyst for CEA and the absence of staining with Fontana–Masson, all of which consolidate the concept that the cyst is derived from the underlying eccrine duct tumor. Wang et al. investigated the histological features of their case of milium-like syringoma, and recognized positive CEA staining only on the lower portion of the cystic component, a common feature also seen in eccrine duct milia. They suggested that the loss of CEA reactivity in the upper half of the milium-like cyst results from fusion of the milia with the epidermis, as proposed for the incomplete-type sweat duct milia. On the other hand, Tsuji et al. found that 75.4% of secondary milia arise from eccrine ducts. These observations denote the intimate relationship that lies between milium and eccrine duct. Although milium and syringoma are two distinct skin diseases, the correlation between the two entities may not be as bewildering as one imagines. Interestingly, Wenyuan studied 76 patients with syringoma and found milium-like lesions in 21 patients (27.63%). Therapeutically, patients with milium-like syringoma successfully treated with CO2 laser have been documented. (a)

Epstein-Barr virus-associated T-cell lymphoproliferative disorder affecting skin and lung in an elderly patient.

A 70‐year‐old man presented with papular skin lesions and was diagnosed with Epstein–Barr virus (EBV)‐associated T‐cell lymphoproliferative disorder (T‐LPD). The patient showed infiltration of a large number of EBV‐encoded RNA‐positive T cells in the skin and lung, presence of EBV load in the peripheral blood, and expansion of clonal EBV‐infected γδ T cells and CD8+ T cells in the blood and skin, as assessed by EBV‐terminal repeat Southern blot, T‐cell receptor polymerase chain reaction and flow cytometric analyses. In the Japanese or East Asian fatal cases of EBV‐associated T/natural killer (NK)‐LPD, there are two peaks in age at death, approximately 20 years and 60 years. The former age group is associated with chronic active EBV infection (CAEBV), and the latter group typically suffers from extranodal NK/T‐cell lymphoma. Our case is characterized not only by the unique skin and lung manifestations but also the late onset age of the disease, indicating that the skin manifestation of CAEBV can be seen even in elderly patients.

Clearance Efficacy of Autoantibodies in Double Filtration Plasmapheresis for Pemphigus Foliaceus

© 2013 The Authors. doi: 10.2340/00015555-1444 Journal Compilation

Distinctive downmodulation of plasmacytoid dendritic cell functions by vitamin D3 analogue calcipotriol

BACKGROUND In relation to Th17 cell actions, interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs) are involved in the pathogenesis of psoriasis. Vitamin D3 analogues are widely used in the treatment of psoriasis, however, their actions on pDCs are not well understood. OBJECTIVE To investigate the effects of Vitamin D3 analogue calcipotriol (CAL) on pDCs, focusing on the cytokine production and chemotactic activity. METHODS We compared in mice the effects of CAL, cyclosporine A (CyA), and triamcinolone acetonide (TA) on the cytokine production by pDCs (IFN-α), conventional DCs (TNF-α), and γd T cells (IL-17A). pDCs isolated from mouse spleen cells were stimulated with CpG-ODN in the presence or absence of each drug for 48h. Purified splenic conventional DCs (cDCs) and lymph node γδ T cells were stimulated with CpG-ODN or with anti-CD3/CD28 antibody, respectively. IFN-α, TNF-α and IL-17A in the 48-h culture supernatants were quantified by ELISA. We also studied the ability of CAL to inhibit the chemotaxis of freshly isolated pDCs toward chemerin and VEGF-A, representative chemoattractants of pDCs, by a real-time monitoring method, EZ-Taxiscan. To assess the effect of CAL on pDC accumulation in vivo, we painted CAL ointment to the mouse skin inflamed by topical application of imiquimod cream (IMQ) for 4 consecutive days. In the skin samples, we enumerated 440c+ pDCs by immunohistochemistry and evaluated the mRNA expression of cytokines by real-time PCR. RESULTS CAL significantly inhibited CpG-enhanced pDC IFN-α production at a comparable level to T cell IL-17A production, whereas its effect on cDC TNF-α production was minimal. Accordingly, CAL suppressed the CpG-augmented expression of TLR9 and MyD88. On the contrary, CyA strongly suppressed the production of TNF-α and IL-17A, but not IFN-α. TA inhibited the production of all the cytokines tested. The effect of CAL on the chemotactic activity of pDCs was also evaluated, demonstrating a significant downmodulation by exposure to the reagent. CAL depressed chemerin receptor CMKLR1 expression in pDCs. The in vivo mouse study showed that simultaneous application of CAL to the imiquimod-applied skin reduce both the recruitment of pDCs and the expression of IFN-α2 in the skin. CONCLUSIONS Our findings suggest that CAL uniquely downmodulates the cytokine production and chemotactic activity of pDCs. The CAL suppression of the in vivo pDC accumulation to the skin suggests that these actions are therapeutically relevant.

Emergence of Photosensitivity with Decreased Treg Cells in a Patient with Mycosis Fungoides Treated with Anti-CC Chemokine Receptor 4 Antibody Mogamulizumab.

Mogamulizumab is a therapeutic monoclonal antibody that targets the CC chemokine receptor 4 (CCR4). The treatment exhibits strong cytotoxicity for adult T-cell leukaemia/lymphoma (ATLL) cells via antibody-dependent cellular cytotoxicity (ADCC), although it carries the risk of serious adverse reactions to the skin, such as StevensJohnson syndrome (1, 2). We report here a patient with mycosis fungoides (MF) at tumour stage treated with mogamulizumab, who developed a photosensitivity reaction during the course of treatment. At the onset of photosensitivity, a reduction in both circulating and skin infiltrating regulatory T cells (Tregs) was observed.