Youichi Omoto

Human Mast Cell Chymase Cleaves Pro-IL-18 and Generates a Novel and Biologically Active IL-18 Fragment

Increased release of IL-18 in the skin causes atopic dermatitis (AD)-like skin lesions, suggesting a role of IL-18 in the pathogenesis of AD. Caspase-1 is a well-known activator of IL-18, but caspase-1 knockout mice still have biologically active IL-18. Normal human keratinocyte constitutively produces pro-IL-18, but it is unable to activate it, suggesting the existence of an alternative pathway for IL-18 in the skin. Dermal accumulation of mast cells is commonly observed in AD patients and in experimental mouse models of AD. Connective tissue mast cells contain high amounts of chymase and tryptase in their cytoplasmic granules. In the present study, we demonstrated that activation of IL-18 is a novel function of human mast cell chymase. Human mast cell chymase rapidly cleaves recombinant pro-IL-18 at 56-phenylalanine and produces a biologically active IL-18 fragment that is smaller than any other reported IL-18-derived species. The human mast cell chymase and the novel IL-18-derived active peptide may be novel therapeutic targets in AD- and IL-18-associated diseases

Granzyme B is a novel interleukin-18 converting enzyme

BACKGROUND Granzyme B (GrB) is recognized to induce apoptosis; however, little is known about its possible role in other biological events. IL-18, a potent inflammatory cytokine, is produced as an inactive precursor (proIL-18). Several cells, including monocytes/macrophage lineage and non-hematopoietic cells such as keratinocytes, produce proIL-18. ProIL-18 requires appropriate processing to become active. Caspase-1 is the authentic IL-18 processing enzyme and is essential for IL-18 release from monocyte/macrophage lineage cells. However, caspase-1 is absent in non-hematopoietic cells, suggesting that there is another candidate to cleave proIL-18 except for caspase-1. OBJECTIVE GrB can invade and be active in cytoplasm of non-hematopoietic cells via perforin, therefore we investigated whether GrB converts proIL-18 into the biologically active form. METHODS Recombinant proIL-18 (rproIL-18) was produced and purified for protease reaction with GrB; this incubate was evaluated by immunoblotting. Biological activity of the proteolytic fragment cleaved by GrB was determined by IFN-gamma assay using KG-1 cells. IFN-gamma induction was also analyzed between extracts from GrB(+)/caspase-1(-) human CD8+ T cells and proIL-18 from normal human keratinocytes (NHK). RESULTS The proteolytic fragment that GrB cleaved proIL-18 had the same sequence and biological activity compared with mature IL-18 cleaved by caspase-1. Culture extracts from CD8+ T cells was able to cleave proIL-18 into authentic mature IL-18. IFN-gamma induction was also detected in NHK treated with CD8+ T cells. CONCLUSION GrB is a potent IL-18 converting enzyme and suggest that GrB secreted by CTLs and/or NK cells may initiate IL-18 release from target cells, leading to the development of inflammation.

IL-4/IL-13 antagonist DNA vaccination successfully suppresses Th2 type chronic dermatitis

Background  Atopic dermatitis (AD) is a chronic disease with a Th2‐type‐cytokine dominant profile. Several cytokines and related peptides have been used for the treatment of AD but they were ineffective because of their limited biological half‐life. We have recently developed a highly efficient mouse dominant negative interleukin (IL)‐4/IL‐13 antagonist (IL‐4DM), which blocks both IL‐4 and IL‐13 signal transductions.

Novel acoustic evaluation system for scratching behavior in itching dermatitis: Rapid and accurate analysis for nocturnal scratching of atopic dermatitis patients

Quantitative analysis of itching in patients with itching dermatitis including atopic dermatitis (AD) is indispensable for the evaluation of disease activity and response to therapy. However, the objective evaluation system for itching is limited. We have developed a new objective and quantitative scratching behavior detection system using a wristwatch‐type sound detector. The scratch sound detected on the wrist is recorded on a personal computer through a filtering, squaring and smoothing process by specific hardware. Subsequently, the data is automatically processed and judged for the scratching movement using specific software based on the periodicity and energy of the signal. Twenty‐four measurements for healthy volunteers and those with AD by this system were evaluated by comparison with a simultaneously recorded video analysis system. The ratio of scratching time in sleeping time evaluated by these two systems was almost identical. The healthy subjects scratched their skin approximately 2 min during 6 h of sleeping time, while the mean scratching time of AD subjects was 24 min in their sleeping time. In contrast to the time‐consuming video analysis system, this system takes only several minutes for evaluation of an overnight record. This scratch sound detection system is expected to serve as a new objective evaluation tool for itching dermatitis, namely, AD, and development of anti‐itch therapies for dermatitis.

Complete remission of advanced extramammary Paget's disease treated with docetaxel: a case report.

reported; however, its effects on metastatic tumours are limited. Interestingly, we found an increase in antitumour cytokine levels in PBMCs, which indicates a similar activation of the immunocytes as seen in the injected lesion. The locally administered BRM successfully augmented the systemic antitumour immune responses. It is likely that the induced cytokines contribute in part to control of local and distant metastasis. Of course, this single case result cannot be generalized, but this patient has survived for 5 years without distant metastasis, with a high quality of life. In conclusion, we found that intratumoral administration of OK-432 had a beneficial effect on SCC metastases, with systemic IFN-c and TNF-a production. Intratumoral injection of OK-432 could be a potent therapeutic candidate for patients with advanced cancer.

Magnetic resonance (MR) imaging‐induced deep second‐degree burns of lower extremities by conducting loop

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CD8+ T cell granzyme B activates keratinocyte endogenous IL-18.

IL-18 is a pro-inflammatory cytokine of the IL-1 family involved in Th1/Th2 polarization. IL-18 is produced and stored as an inactive precursor (proIL-18) in several cells including keratinocytes, and thus appropriate processing is required to release its active form. In a previous study using recombinant protein, we demonstrated that granzyme B (GrB) cleaves proIL-18 into its active forms in a similar fashion as caspase-1 and human mast cell chymase. GrB released from cytotoxic T lymphocyte (CTL) and NK cells has roles in apoptosis and cytotoxic activity. In certain inflammatory skin diseases with epidermal cell death, the epidermal keratinocytes are targets of CTL and NK cells. However, IL-18 activation during the direct interaction of CTL/NK with keratinocytes has not been described so far. We investigated the interaction between CTL and keratinocytes, and IL-18 processing by CTL-derived GrB using cultured CD8+ T cells and keratinocyte cell line HaCaT. GrB(+)/caspase-1(−) CD8+ T cells cultivated from healthy human PBMC were co-cultured with interferon(IFN)-γ-treated HaCaT cells. The expression of GrB and caspase-1 in HaCaT cells was analyzed by flow cytometry and PCR. The IL-18 concentration in the culture supernatant was measured by specific ELISA. The interaction between HaCaT cells and CTL co-culture increased the number of cytoplasmic GrB-positive HaCaT cells with limited endogenous GrB mRNA expression. The concentration of mature IL-18 levels increased in the co-culture supernatant. GrB from CTLs acts double roles to keratinocytes: a IL-18 converting enzyme and pro-apoptotic factor in the skin inflammatory diseases.

Olopatadine, a non‐sedating H1 antihistamine, decreases the nocturnal scratching without affecting sleep quality in atopic dermatitis

We have demonstrated for the first time that a second‐generation antihistamine ameliorates nocturnal scratching behavior in atopic dermatitis patients using a modified wristwatch‐type acoustic scratching counting system that we have recently developed. We also analyzed the sleep quality by simultaneous recording of electroencephalogram, and found that sleep quality was unaffected.

Folliculosebaceous cystic hamartoma differentiates toward the infundibulum, sebaceous duct and sebaceous cells: immunohistochemical study of keratins and filaggrin.

SIR, Folliculosebaceous cystic hamartoma (FSCH) is a rare cutaneous hamartoma with varying proportions of epithelial components and mesenchymal overgrowth. The epithelial components consist of an infundibular cystic structure to which mature sebaceous lobules are attached via sebaceous ducts. The histogenesis of FSCH remains unclear. We report a case of FSCH occurring on the nasolabial fold in an elderly Japanese man. To determine the differentiation of FSCH, we performed an immunohistochemical study of keratins and filaggrin (filament aggregating protein). To our knowledge, this is the first report of FSCH with an immunohistochemical study of keratins and filaggrin. Since this tumour was first described by Kimura et al. in 1991, about 30 cases have been reported. A 78-year-old man presented with a 3-year history of a slow-growing, pink-yellow, elastic hard, pedunculated asymptomatic nodule 17 · 13 mm in size on the right side of his nose. Specimens were fixed in neutral formalin, embedded in paraffin and stained with haematoxylin and eosin. Serial sections were used for the immunohistochemical study. We used 10 antikeratin antibodies: 34bB4 [keratin 1 (K1)], LP5K (K7), LP3K (K8), LHP1 (K10), LL022 (K14), LHK15 (K15), LL025

Intratumoral injection of OK-432 suppresses metastatic squamous cell carcinoma lesion inducing interferon-γ and tumour necrosis factor-α

A 79-year-old man presented with a subcutaneous lesion on his upper arm. He had a history of surgically treated squamous cell carcinoma (SCC) on his arm, and axillary lymph-node metastasis 7 years previously. On physical examination, a large subcutaneous tumour was seen on the upper arm, involving the brachial artery and vein (Fig. 1a), which was diagnosed as an in-transit relapse of the SCC. The first-line choice of treatment was amputation of the patient s arm from the shoulder, but he refused. The patient had renal dysfunction, thus chemotherapy was contraindicated. The patient underwent irradiation to the lesion with limited effect. We then started intratumoral injection of OK-432 [2 KE (Klinische Einheit; clinical units)] every 2 weeks. The tumour size gradually diminished (Fig. 1b), and no further metastases have been seen during a followup of 5 years. OK-432 is a biological response modifier (BRM), which is a penicillin G-inactivated and lyophilized preparation of the low-virulence strain Su of Streptococcus pyogenes. OK-432 is an approved adjuvant therapeutic agent for malignancies in Japan. OK-432 exerts its effect by activating the immune system to secrete antitumour agents; however, little is known about its in vivo immunological mechanism. Therefore, we carried out a study to identify the immunological basis of OK-432. We focused on production of the antitumour cytokines interferon (IFN)-c and tumour necrosis factor (TNF)-a in the peripheral blood. The study was approved by the Mie University institutional review board, and informed consent was obtained from the patient. Blood samples were collected before and 24 h after injection of OK-432. Purified peripheral blood mononuclear cells (PBMC) were stimulated with anti-CD3 and CD28 antibodies in the presence of brefeldin A, and cultured for 12 h at 37 C in 5% CO2. Using flow-cytometry analysis, we found that the percentages of CD4 and CD8 T cells producing IFN-c before treatment were higher in this patient than in healthy volunteers matched for age and gender, and these percentages increased further 1 day after treatment. The percentage of CD14+ monocytes producing TNF-a was also increased (Fig. 2). In vitro or in mice experiments, OK-432 activates neutrophils, macrophages, lymphocytes and natural killer cells, inducing multiple inflammatory cytokines including interleukin-12, TNF-a and IFN-c, and polarizes the T-cell response to a T-helper-1 dominant state. OK-432 also activates dendritic cells, which are potent antigen-presenting cells, through Toll-like receptor (TLR)4 signalling, and enhances antigen-specific cytotoxic T-lymphocyte responses. Some successful results after OK-432 therapy