TRAF6 signaling pathway in T cells regulates anti-tumor immunity through the activation of tumor specific Th9 cells and CTLs.

CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T (Th) cells play a critical role in protective immune responses to tumor cells. Particularly, Th9 cells exert anti-tumor activity by producing IL-9. TNF receptor (TNFR)-associated factor 6 (TRAF6) is an adaptor protein that mediates the signals from both the TNFR superfamily and Toll-like receptors (TLRs). We have previously reported that T cell-specific TRAF6-deficent (TRAF6ΔT) mice spontaneously developed systemic inflammatory diseases. However, the physiological role of TRAF6 in T cells in controlling anti-tumor immune responses remains largely unclear. Here, we found that tumor formation of syngeneic colon cancer cells inoculated in TRAF6ΔT mice was accelerated compared to that in control mice. Although TRAF6-deficient naïve T cells showed enhanced differentiation of Th9 cells in vitro, these T cells produced lower amounts of IL-9 in response to a specific antigen. Moreover, CD4+ tumor-infiltrating lymphocytes (TILs) in tumor-bearing TRAF6ΔT mice expressed lower levels of IL-9 than those in WT mice. Importantly, administration of recombinant IL-9 (rIL-9) strongly suppressed tumor progression in TRAF6ΔT mice. Furthermore, expression levels of the T-box transcription factor Eomesodermin (Eomes) and its target molecules IFN-γ, granzyme B and perforin, as well as cytotoxic activity, were reduced in TRAF6-deficient CD8+ T cells in vitro. TRAF6-deficient T cells were found to express significantly increased levels of immune checkpoint molecules, CTLA-4 and PD-1 on the cell surface. These results demonstrate that the TRAF6 signaling pathway in T cells regulates anti-tumor immunity through the activation of tumor specific Th9 cells and CTLs in a tumor microenvironment.

Movements of Ancient Human Endogenous Retroviruses Detected in SOX2-Expressing Cells.

Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.

Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing.

Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nuclease AsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of the RET SNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding gene COL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.

Development of a portable reverse transcription loop-mediated isothermal amplification system to detect the E1 region of Chikungunya virus in a cost-effective manner.

Chikungunya fever is a mosquito-borne disease cause of persistent arthralgia. The current diagnosis of Chikungunya virus (CHIKV) relies on a conventional reverse transcription polymerase chain reaction assay. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and simple tool used for DNA-based diagnosis of a variety of infectious diseases. In this study, we established an RT-LAMP system to recognize CHIKV by targeting the envelope protein 1 (E1) gene that could also detect CHIKV at a concentration of 8 PFU without incorrectly detecting other mosquito-borne viruses. The system also amplified the E1 genome in the serum of CHIKV-infected mice with high sensitivity and specificity. Moreover, we established a dry RT-LAMP system that can be transported without a cold chain, which detected the virus genome in CHIKV-infected patient samples with high accuracy. Thus, the dry RT-LAMP system has great potential to be applied as a novel CHIKV screening kit in endemic areas.

Cutaneous histopathology of the tick-bite region in severe fever with thrombocytopenia syndrome.

A case of severe fever with thrombocytopenia syndrome (SFTS) in which a skin biopsy from the tick-bite region was analyzed is reported. The patient was a 72-year-old woman who developed fever and thrombocytopenia after a tick bite. SFTS was diagnosed from polymerase chain reaction (PCR) analysis of a blood sample. Histopathological analysis of a skin biopsy specimen from the tick-bite region showed CD20-positive perivascular and interstitial immunoblastic cells, which were positive to anti-SFTS virus (SFTSV) nucleoprotein antibody. In addition, SFTSV RNA was detected by real-time PCR from this biopsy specimen. Moreover, hemophagocytosis was also found in the tick-bite region. To the best of our knowledge, this is the first report to analyze the details of the tick-bite region of skin in SFTS, and the first to detect virus-infected cells in the skin. The present findings may help elucidate the mechanisms of entry of SFTSV.

Selective Inhibition of ß-Catenin/Co-Activator Cyclic AMP Response Element-Binding Protein-Dependent Signaling Prevents the Emergence of Hapten-Induced Atopic Dermatitis-Like Dermatitis.

BACKGROUND: The canonical Wnt/ß-catenin signaling pathway is a fundamental regulatory system involved in various biological events. ICG-001 selectively blocks the interaction of ß-catenin with its transcriptional co-activator cyclic AMP response element-binding protein (CBP). Recent studies have provided convincing evidence of the inhibitory effects of ICG-001 on Wnt-driven disease models, such as organ fibrosis, cancer, acute lymphoblastic leukemia, and asthma. However, the effects of ICG-001 in atopic dermatitis (AD) have not been investigated. OBJECTIVE: To investigate whether ß-catenin/CBP-dependent signaling was contributed in the pathogenesis of AD and ICG-001 could be a therapeutic agent for AD. METHODS: We examined the effects of ICG-001 in an AD-like murine model generated by repeated topical application of the hapten, oxazolone (Ox). ICG-001 or vehicle alone was injected intraperitoneally every day during the development of AD-like dermatitis arising from once-daily Ox treatment. RESULTS: Ox-induced AD-like dermatitis characterized by increases in transepidermal water loss, epidermal thickness, dermal thickness accompanied by increased myofibroblast and mast cell counts, and serum levels of thymic stromal lymphopoietin and thymus and activation-regulated chemokine, and decreases in stratum corneum hydration, were virtually normalized by the treatment with ICG-001. Elevated serum levels of periostin tended to be downregulated, without statistical significance. CONCLUSION: These results suggest that ß-catenin/CBP-dependent signaling might be involved in the pathogenesis of AD and could be a therapeutic target.

Mesenchymal stem cells derived from human iPS cells via mesoderm and neuroepithelium have different features and therapeutic potentials.

Mesenchymal stem cells (MSCs) isolated from adult human tissues are capable of proliferating in vitro and maintaining their multipotency, making them attractive cell sources for regenerative medicine. However, the availability and capability of self-renewal under current preparation regimes are limited. Induced pluripotent stem cells (iPSCs) now offer an alternative, similar cell source to MSCs. Herein, we established new methods for differentiating hiPSCs into MSCs via mesoderm-like and neuroepithelium-like cells. Both derived MSC populations exhibited self-renewal and multipotency, as well as therapeutic potential in mouse models of skin wounds, pressure ulcers, and osteoarthritis. Interestingly, the therapeutic effects differ between the two types of MSCs in the disease models, suggesting that the therapeutic effect depends on the cell origin. Our results provide valuable basic insights for the clinical application of such cells.

Establishment and gene expression analysis of disease-derived induced pluripotent stem cells of scleroderma.

BACKGROUND: We recently generated induced pluripotent stem cells (iPSCs) from cultured dermal fibroblasts of systemic sclerosis (SSc-iPSC) to study the disease mechanisms. OBJECTIVE: In the present study, we have performed gene expression analysis using cultured SSc dermal fibroblasts, SSc-iPSC, and fibroblasts re-differentiated from SSc-iPSC (SSc-iPSC-FB). METHODS: mRNA and protein levels of collagen and integrins were analyzed using PCR array, PCR, immunoblotting, and immunofluorescence. RESULTS: We compared expression pattern of TGF-ß-related genes between normal iPSC (NS-iPSC) and SSc-iPSC by PCR array, and found constitutive and significant down-regulation of S100A8, Smad6, and TGF-ß2 in SSc-iPSC. The expression of these genes was not altered in cultured SSc fibroblasts or SSc-iPSC-FB compared to NS fibroblasts or NS-iPSC-FB, respectively. On the other hand, the expression of collagen, integrin α and ß was up-regulated in SSc fibroblasts, while SSc-iPSC-FB showed normalized levels of collagen and integrin ß. CONCLUSIONS: So far, there have been no reports investigating disease-derived iPSCs of SSc. Our results suggest that S100A8, Smad6, and TGF-ß2 may be the key molecules of this disease. On the other hand, the normalization of collagen and integrins by iPSC reprogramming suggests that epigenetic modifications of genes may play a role in the mechanism of collagen accumulation seen in SSc fibroblasts, and that gene reprogramming may become novel therapeutic approach. As the limitation of this study, we established only one iPSC line from each patient, which may not be enough to discuss disease-specific phenotypes. Larger studies including increased number of iPSC lines are needed in the future.

Epiplakin Is a Paraneoplastic Pemphigus Autoantigen and Related to Bronchiolitis Obliterans in Japanese Patients.

All plakin family proteins are known to be autoantigens in paraneoplastic pemphigus (PNP). In this study, we first examined whether PNP sera also react with epiplakin, another plakin protein, by various immunological methods using 48 Japanese PNP sera. Immunofluorescence confirmed that cultured keratinocytes expressed epiplakin. Epiplakin was detected by 72.9% of PNP sera by immunoprecipitation-immunoblotting with KU-8 cell extract, but not by immunoblotting of either normal human epidermal extract or KU-8 cell extract. Epiplakin was essentially not detected by 95 disease and normal control sera. Statistical analyses of various clinical and immunological findings revealed a significant correlation of the presence of anti-epiplakin antibodies with both bronchiolitis obliterans and mortality. No epiplakin-negative PNP case developed bronchiolitis obliterans. However, although 29.4% of European patients with PNP had bronchiolitis obliterans, significant correlation with anti-epiplakin autoantibodies was not observed. In further studies for lung, immunofluorescence showed the presence of epiplakin in normal human lung, particularly respiratory bronchiole, immunoprecipitation-immunoblotting showed that PNP sera reacted with epiplakin in cultured lung cells, and mice injected with polyclonal antibody specific to epiplakin histopathologically showed abnormal changes in small airway epithelia. These results indicated that epiplakin is one of the major PNP autoantigens and is related to PNP-related bronchiolitis obliterans.

Results of second-stage screening for skin cancers in Oita Prefecture, Japan.

We performed skin cancer screenings for 2 or 3 days annually from 2006 through 2013 in Oita Prefecture, Japan. Screening of approximately 3000 people in total allowed us to identify and treat several skin cancers, including five cases of malignant melanoma, four of squamous cell carcinoma, 16 of basal cell carcinoma, 11 of Bowen's disease, 17 of actinic keratosis, one of extramammary Paget's disease and one of metastatic breast carcinoma. The sensitivity and specificity for the category defined by an identified lesion associated with risk of cancer and requiring further examination (category C) were 92.7% and 95%, respectively. We cannot estimate the outcome of our skin cancer screenings in terms of cancer mortality because of the small number of subjects examined and the brief follow-up period. However, we did estimate the effectiveness of these screenings in terms of stages or sizes of cancerous lesions. The relative numbers of subjects with malignant melanoma at various clinical stages, identified during skin cancer screenings and during a routine visit to our hospital, were significantly different. We also compared, statistically, the sizes of lesions in Bowen's disease that were found during cancer screenings and during a direct visit to our hospital. The former lesions were smaller than the latter. Our data suggest the benefits of our skin cancer screenings and the importance of campaigns and education to encourage people to visit dermatologists for the detection of skin cancers at an early stage.

Cutaneous horn malignant melanoma.

A 73-year-old Japanese woman presented with cutaneous horn on the right cheek. The resected tumor was 9 mm in diameter, with 14 mm protrusion, and showed exophytic growth with marked papillomatosis. Histopathology showed proliferation of atypical melanocytes with melanin pigments in the epidermis and dermis under the cutaneous horn. These cells were confined to the base of the cutaneous horn, and did not spread to the surrounding epidermis. The final diagnosis was cutaneous horn malignant melanoma. This pathological entity is considered a specific form of verrucous melanoma, and might be added to the list of cutaneous horn-forming lesions.

Analysis of 256 cases of basal cell carcinoma after either one-step or two-step surgery in a Japanese institution.

Basal cell carcinoma (BCC) is a common skin cancer that arises from the cells of the basal layer of the epithelium or from the external root sheath of the hair follicle. In the present report, 256 cases treated surgically between 1999 and 2008 in our department were retrospectively analyzed. The most frequent BCC locations included the face (77.8%), especially the nose (26.9%) and eyelids (21.5%). Incomplete excisions occurred in 21 cases. Two patients experienced local recurrence; one of these patients exhibited a bone metastasis while the other had a metastasis of the parotid gland without the local recurrence. The rate of local BCC recurrence was 0.78%, which is lower than that described in previous reports. We categorized BCC into four histological types: superficial, solid, adenoid and infiltrative. The solid type was the most frequent histological type (62.1%). For preventive recurrence, we treated BCC patients with two-step surgery when the tumor was large or histologically invasive. At the first step, we excised the tumor with a sufficient safety margin, and at the second step, we performed reconstruction after the histological confirmation that no remnant malignant cells were in the tumor margins. In the present report, no local recurrence occurred in patients following the two-step surgery. Therefore, two-step surgery is recommended for tumors at locations and with histological types related to frequent recurrence.