A gas circulation and purification system for gas-cell-based low-energy RI-beam production.

A gas circulation and purification system was developed at the RIKEN Radioactive Isotope Beam Factory that can be used for gas-cell-based low-energy RI-beam production. A high-flow-rate gas cell filled with one atmosphere of buffer gas (argon or helium) is used for the deceleration and thermalization of high-energy RI-beams. The exhausted buffer gas is efficiently collected using a compact dry pump and returned to the gas cell with a recovery efficiency of >97%. The buffer gas is efficiently purified using two gas purifiers as well as collision cleaning, which eliminates impurities in the gas. An impurity level of one part per billion is achieved with this method.

GSK-3ß-dependent downregulation of γ-taxilin and αNAC merge to regulate ER stress responses.

The signaling pathway leading to the endoplasmic reticulum (ER) stress responses has not been fully elucidated. Here we showed that glycogen synthase kinase-3ß (GSK-3ß)-dependent downregulation of γ-taxilin and nascent polypeptide-associated complex α-subunit (αNAC) mediates hypoxia-induced unfolded protein responses (UPRs) and the subsequent apoptotic and autophagic pathways. The degradation of γ-taxilin or αNAC was sufficient to initiate UPRs in normoxic cells. However, the ER stress signaling pathways initiated by γ-taxilin or αNAC were distinct, triggering different ER stress sensors and activating different downstream pathways. Hypoxia caused GSK-3ß-dependent tau hyperphosphorylation and cleavage in neuronal cells, but γ-taxilin ablation induced tau hyperphosphorylation alone and αNAC ablation induced neither changes. Notably, downregulation of γ-taxilin and αNAC occurs in the brain of patients with Alzheimer's disease. These results suggest that GSK-3ß-dependent downregulation of γ-taxilin and αNAC, which differently activate the UPRs, merge to regulate hypoxia-induced ER stress responses and provide a new insight into the pathogenesis of neurodegenerative diseases.

Recent advances and developments in the antitumor effect of the HVJ envelope vector on malignant melanoma: from the bench to clinical application.

Inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) are considered to be safe and efficient non-viral vectors used for drug delivery, since they can incorporate DNA, RNA, proteins and drugs. We have recently found that HVJ-E has a novel antitumor immune effect using a colon cancer model. HVJ-E has also been shown to have both direct and immune-mediated indirect actions against malignancy. Intratumoral injection of an inactivated HVJ-E solution significantly reduced the tumor volume and prevented spontaneous lung metastasis, leading to an increased overall survival in C57/BL6 mice transplanted with B16/BL6 mouse melanoma cells, and even in immunodeficient mice transplanted with Mewo human melanoma cells. No severe adverse effects including laboratory data abnormalities or anaphylactic reactions were observed. The comprehensive mechanism(s) underlying the immunological effects of HVJ-E appear to include not only enhanced effector T cell- and/or natural killer (NK) cell-mediated immunity, but also rescue from regulatory T cell (Treg)-mediated immunosuppression, presumably through the interleukin-6 secretion from dendritic cells stimulated by HVJ-E. Since a protocol for a clinical study of HVJ-E in malignant melanoma was approved in 2009 by the ethics committee of Osaka University and of the Medical Center for Translational Research in Osaka University Hospital, a phase I/IIa study for advanced malignant melanoma patients was just started. In this review, we show several favorable results regarding the antitumor effects of HVJ-E and describe the novel mechanism underlying this tumor immune response. Since we are conducting a phase I/IIa clinical trial using HVJ-E in advanced melanoma patients on the basis of preclinical results, detailed clinical information and immune-monitoring data are also introduced. The development of new therapeutic modalities for advanced melanoma patients is urgently needed, and we hope that HVJ-E may provide one such treatment.

First left-right comparative study of topical rapamycin vs. vehicle for facial angiofibromas in patients with tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder causing multiple hamartomas. Treatment of TSC lesions with mammalian target of rapamycin inhibitors is effective. Recently, several reports have shown the efficacy of topical rapamycin (sirolimus) for angiofibromas. However, almost all studies have been case studies and the 0·1% solution caused skin irritation. A comparative study of topical rapamycin and a vehicle has not yet been reported. OBJECTIVES: To compare the efficacy of topical rapamycin formulation with that of vehicle for angiofibromas. METHODS: A left-right comparative study between rapamycin 0·2% topical formulation and vehicle was conducted in 11 patients with TSC. Two formulations, an ointment and a gel, were prepared and in vitro percutaneous absorption of rapamycin was determined. RESULTS: In vitro percutaneous absorption of rapamycin was significantly greater with the gel compared with the ointment. In the clinical study, the rapamycin-treated cheek showed significant improvements relative to the vehicle-treated cheek in all outcome measures after 12 weeks of treatment. The improvement was particularly remarkable in children aged ≤ 10 years. No side-effects were noted, and rapamycin was not detected in the blood of the patients. CONCLUSIONS: Topical rapamycin was significantly effective against angiofibromas. Both formulations used were effective and safe. The 0·2% gel is especially useful because of its better skin penetration and low irritancy. Initiation of topical rapamycin therapy in early childhood would be beneficial for patients with TSC.

Increased frequency of skin-infiltrating FoxP3+ regulatory T cells as a diagnostic indicator of severe atopic dermatitis from cutaneous T cell lymphoma.

Differential diagnosis of cutaneous T cell lymphoma (CTCL) and severe atopic dermatitis (AD) is often difficult because of the similarity in their skin manifestations. However, such differentiation is extremely important because of the differences in remedy and prognosis. The aim of this study was to investigate new, helpful diagnostic aids for distinguishing CTCL from AD. The frequency of forkhead box protein 3(+) (FoxP3(+)) regulatory T cells (T(regs)) in cutaneous lesions was evaluated among the three populations. Serum-soluble interleukin-2 receptor (sIL-2R), immunoglobulin (Ig)E-radioimmunosorbent test, lactate dehydrogenase (LDH) and blood eosinophil count were measured in 11 CTCL patients (including three CTCL patients misdiagnosed previously with intractable AD), 10 adult AD patients and nine psoriasis patients. The frequency of T(regs) was increased significantly in cutaneous lesions of AD compared with those of CTCL. Serum IgE and LDH levels were also elevated significantly in AD compared with CTCL, whereas there were no significant differences in serum sIL-2R levels between CTCL and AD. In the three CTCL patients who were misdiagnosed with intractable AD, IgE and LDH levels were lower than in AD patients, whereas serum sIL-2R levels were as high as in AD patients and higher than in the other eight CTCL patients. The higher frequency of T(regs) in the cutaneous lesions of patients with AD than in those with CTCL and higher serum IgE and LDH levels in patients with AD than in those with CTCL might be helpful reference values for the differential diagnosis of these two diseases.

A case of generalized acanthosis nigricans with positive lupus erythematosus-related autoantibodies and antimicrosomal antibody: autoimmune acanthosis nigricans?

Acanthosis nigricans (AN) is a hyperpigmented keratotic skin lesion known to be associated with malignant disease and endocrinopathy. We report a very rare case of generalized AN with Sjögren's syndrome- and systemic lupus erythematosus-like features but without type B insulin resistance. Neither internal malignancy nor other endocrinological disorders, including glucose intolerance, were detected during a 10-year clinical course with benign diffuse papillomatosis extending from the mucosa of the larynx to the esophagogastric junction. The case was complicated with chronic thyroiditis and interstitial pneumonia, which were not treated with any medication. AN skin lesions and mucosal papillomatosis regressed with oral cyclosporine A, accompanied by the lowering of autoantibody titers. This is the first report of generalized AN involving an area from the mucosa of the larynx to the esophagogastric junction accompanied by autoimmune manifestations which responded to systemic immunosuppressive therapy.

Revised classification/nomenclature of vitiligo and related issues: the Vitiligo Global Issues Consensus Conference.

During the 2011 International Pigment Cell Conference (IPCC), the Vitiligo European Taskforce (VETF) convened a consensus conference on issues of global importance for vitiligo clinical research. As suggested by an international panel of experts, the conference focused on four topics: classification and nomenclature; definition of stable disease; definition of Koebner's phenomenon (KP); and 'autoimmune vitiligo'. These topics were discussed in seven working groups representing different geographical regions. A consensus emerged that segmental vitiligo be classified separately from all other forms of vitiligo and that the term 'vitiligo' be used as an umbrella term for all non-segmental forms of vitiligo, including 'mixed vitiligo' in which segmental and non-segmental vitiligo are combined and which is considered a subgroup of vitiligo. Further, the conference recommends that disease stability be best assessed based on the stability of individual lesions rather than the overall stability of the disease as the latter is difficult to define precisely and reliably. The conference also endorsed the classification of KP for vitiligo as proposed by the VETF (history based, clinical observation based, or experimentally induced). Lastly, the conference agreed that 'autoimmune vitiligo' should not be used as a separate classification as published evidence indicates that the pathophysiology of all forms of vitiligo likely involves autoimmune or inflammatory mechanisms.

The combination of chemotherapy with HVJ-E containing Rad51 siRNA elicited diverse anti-tumor effects and synergistically suppressed melanoma.

Dacarbazine (DTIC) is one of the most popular alkylating agents used for the treatment of malignant melanoma. DTIC induces apoptosis of melanoma cells via double-strand breaks (DSBs). Melanoma cells, however, tend to increase their expression of DNA repair molecules in order to be resistant to DTIC. Here, we show that DTIC increases expression of Rad51, but not Ku70, in a cultured B16-F10 mouse melanoma cell line in dose- and time-dependent manners. On introducing Rad51 short interfering RNA (siRNA) with the hemagglutinating virus of Japan envelope (HVJ-E) to B16-F10 cells, DSBs induced by DTIC treatment were not efficiently repaired and resulted in enhanced apoptotic cell death. Colony formation of B16-F10 cells that received Rad51 siRNA was significantly decreased by DTIC treatment as compared with cells that received scramble siRNA. In melanoma-bearing mice, the combination of three intratumoral injections of HVJ-E containing Rad51 siRNA and five intraperitoneal injections of DTIC at a clinical dose synergistically suppressed the tumors. Moreover, HVJ-E demonstrated anti-tumor immunity by inducing cytotoxic T lymphocytes to B16-F10 cells on administration of DTIC. These results suggest that the combination of chemotherapy with HVJ-E containing therapeutic molecules will provide a promising therapeutic strategy for patients bearing malignant tumors resistant to chemotherapeutic agents.

Erythroderma as a paraneoplastic cutaneous disorder in systemic anaplastic large cell lymphoma.

BACKGROUND: Paraneoplastic cutaneous disorders (PCDs) or dermadromes are skin conditions that have an association with internal malignancies but are not themselves malignant. We report the first two cases of systemic anaplastic large cell lymphoma (s-ALCL) accompanied by erythroderma and multiple leg ulcers as PCDs. CASE 1: A 52-year-old Japanese man presented with disseminated itchy papular erythemas which he had over his entire body for the preceding 5 years that later exacerbated to erythroderma. Multiple punched-out ulcers also developed on his lower legs. Superficial lymph nodes (LNs) were swollen, and a left axillary LN biopsy demonstrated dense CD30(+) atypical large cell (ALC) infiltration. By contrast, lymphocytes infiltrating into the erythroderma and leg ulcers were CD30(-) , and T-cell receptor ß (TCRß) chain gene rearrangement was negative in skin biopsy specimens. Thus, he was diagnosed with s-ALCL. Not only his s-ALCL but also his erythroderma and leg ulcers responded well to chemotherapy. CASE 2: A 71-year-old Japanese woman presented with erythroderma that persisted for approximately 20 years after mastectomy. At her initial hospital visit, she was diagnosed with s-ALCL by biopsy of swollen left inguinal LNs. Similar to Case 1, CD30(+) ALCs were negative in skin samples with normal TCRß chain gene rearrangement. As the erythrodermic skin lesion responded well to chemotherapy for s-ALCL, it was considered a PCD. CONCLUSION: s-ALCL development may be predicted by the precedence and concurrence of intractable paraneoplastic erythrodermic and ulcerative skin lesions, as reported in our two cases.

Diminished regulatory T cells in cutaneous lesions of thymoma-associated multi-organ autoimmunity: a newly described paraneoplastic autoimmune disorder with fatal clinical course.

Thymoma-associated multi-organ autoimmunity is a rare, autoimmune disease that causes colitis, liver dysfunction and cutaneous graft-versus-host (GVH)-like skin damage. This paraneoplastic autoimmune disorder may be due to inadequate T cell selection in the tumour environment of the thymus. Although sporadic case reports have revealed its clinical features, little is known about its pathological mechanism. By comparing the skin-infiltrating T cell subsets with those of GVH disease (GVHD) and other inflammatory skin diseases, we sought to elucidate the pathological mechanism of thymoma-associated multi-organ autoimmunity. Histopathological and immunohistochemical analysis of skin biopsies was performed for three patients with thymoma-associated multi-organ autoimmunity. Histopathological findings of thymoma-associated multi-organ autoimmunity were indistinguishable from those of patients with acute GVHD, although the aetiologies of these diseases are completely different. The frequency of regulatory T cells (T(regs)) is reduced in cutaneous lesions and CD8+ cytotoxic T lymphocytes that massively infiltrate into the epidermis of patients with thymoma-associated multi-organ autoimmunity. Additionally, the ratio of T helper type 17 (Th17) cells to CD4+ cells in patients with thymoma-associated multi-organ autoimmunity and acute GVHD was higher than that in healthy controls, but similar to that in psoriasis vulgaris patients. Similarity of the skin-infiltrating T cell subsets with those of acute GVHD suggested that skin damage in patients with thymoma-associated multi-organ autoimmunity might be induced by self-reactive cytotoxic T lymphocytes under the diminished suppressive capacity of T(regs).

A topical combination of rapamycin and tacrolimus for the treatment of angiofibroma due to tuberous sclerosis complex (TSC): a pilot study of nine Japanese patients with TSC of different disease severity.

BACKGROUND: Dysregulation of mTOR signalling by mutations in tuberin and/or hamartin leads to the formation of tuberous sclerosis complex (TSC). Trials to treat TSC using mTOR inhibitors, including rapamycin, have been performed. Although rapamycin improves many TSC lesions, significant side-effects appear after systemic administration. Topical administration has been recommended. OBJECTIVES: The efficacy of rapamycin-tacrolimus ointment was examined for TSC-related angiofibroma. METHODS: Left-right comparisons of the tacrolimus ointments with/without 0·2% rapamycin was conducted in symmetrical facial angiofibromas in nine patients with definitive TSC. After the 3-month treatment, a cumulative score for redness, flatness and papule size was used to evaluate the efficacy of the treatment. Blood rapamycin levels were analysed by liquid chromatography-electrospray mass spectrometry (LC-ESI/MS). RESULTS: At the end of the treatment, all of the scores significantly improved for rapamycin-tacrolimus treatment compared with tacrolimus alone. No adverse reactions were noted and blood levels of rapamycin were below the detection limit in all cases. CONCLUSIONS: Topical application of rapamycin-tacrolimus ointment is a safe and useful treatment for TSC-related angiofibroma.

Multiple lymphadenopathy as an initial sign of extramammary Paget disease.

Extramammary Paget disease (EMPD) often develops in external genitalia. Paget cells can, however, adopt an invasive phenotype and metastasize to regional lymph nodes and beyond, leading to poor patient outcomes. Based on this clinical observation, multiple lymphadenopathy may represent an initial sign of EMPD. To address the potential significance of multiple lymph node swelling in EMPD, we report two patients with cutaneous primary EMPD who showed multiple lymphadenopathy as an initial sign during the clinical course of the disease as well as tumour metastasis. Significantly, marked lymphatic vessel growth was observed in regional lymph nodes that underwent massive tumour cell invasion. Therefore, nodal lymphangiogenesis may promote tumour cell invasion and metastasis to distant organs, including the lymph nodes, emphasizing the clinical relevance of multiple lymphadenopathy.

Epithelioid sarcoma on the foot masquerading as an intractable wound for > 18 years.

Slow-growing sarcomas may give rise to intractable wounds, which may be attributed to commoner causes. A 57-year-old man with diabetes mellitus presented with a 24-year history of a chronic wound that originated on his left great toe. Because of the long history, the nonspecific histological findings and the complication of ulcerative colitis, we misdiagnosed his ulcer as pyoderma gangrenosum. The wound was eventually diagnosed correctly by histological examination of a skin biopsy and the use of immunohistochemistry to detect cytokeratin, epithelial membrane antigen and vimentin. Specimens obtained 16 years earlier showed the same staining pattern. Radiological examinations revealed no metastasis. The patient received a below-knee amputation without further chemotherapy or radiotherapy. When patients have intractable ulcers, appropriate biopsies and immunohistochemical examinations are sometimes necessary to exclude a malignancy even if the history and symptoms do not suggest a diagnosis of sarcoma.

alphaNAC depletion as an initiator of ER stress-induced apoptosis in hypoxia.

Accumulation of unfolded proteins triggers endoplasmic reticulum (ER) stress and is considered a part of the cellular responses to hypoxia. The nascent polypeptide-associated complex (NAC) participates in the proper maturation of newly synthesized proteins. However, thus far, there have been no comprehensive studies on NAC involvement in hypoxic stress. Here, we show that hypoxia activates glycogen synthase kinase-3beta (GSK-3beta) and that the activated GSK-3beta destabilizes alphaNAC with the subsequent apoptosis of the cell. Hypoxia of various cell types and the mouse ischemic brain was associated with rapid downregulation of alphaNAC and ER stress responses involving PERK, ATF4, gamma-taxilin, elF2alpha, Bip, and CHOP. Depletion of alphaNAC by RNA interference specifically activated ER stress responses and caused mitochondrial dysfunction, which resulted in apoptosis through caspase activation. Interestingly, we found that the hypoxic conditions activated GSK-3beta, and that GSK-3beta inhibition prevented alphaNAC protein downregulation in hypoxic cells and rescued the cells from apoptosis. In addition, alphaNAC overexpression increased the viability of hypoxic cells. Taken together, these results suggest that alphaNAC degradation triggers ER stress responses and initiates apoptotic processes in hypoxic cells, and that GSK-3beta may participate upstream in this mechanism.

Diffusion-weighted MR imaging of ameloblastomas and keratocystic odontogenic tumors: differentiation by apparent diffusion coefficients of cystic lesions.

BACKGROUND AND PURPOSE: Ameloblastomas and keratocystic odontogenic tumors are major aggressive odontogenic tumors in the maxillomandibular regions, but the differentiation between these 2 tumors is frequently ineffective based on only conventional CT and MR imaging findings. Here, we evaluated diffusion-weighted MR imaging for the differentiation of these 2 odontogenic tumors. MATERIALS AND METHODS: We prospectively studied 9 patients with ameloblastoma and 7 patients with keratocystic odontogenic tumor using diffusion-weighted MR imaging. Apparent diffusion coefficients (ADCs) of the nonenhancing and solid lesions in these tumors were determined with use of 2 b factors (500 and 1000). RESULTS: Two types of nonenhancing lesions were identified; one with high signal intensity on fat-suppressed T2-weighted images (type A) and the other with low or intermediate intensity (type B). The type A nonenhancing lesions were observed in all the ameloblastomas, but they were evident in only 2 keratocystic odontogenic tumors. It is interesting to note that the ADCs of the nonenhancing lesions in the ameloblastomas were significantly higher than those of the nonenhancing lesions in the keratocystic odontogenic tumors (2.48 +/- 0.20 x 10(-3) mm(2)/s vs 1.13 +/- 0.56 x 10(-3) mm(2)/s; P < .001). The ADCs of the solid lesions in the ameloblastomas (1.39 +/- 0.15 x 10(-3) mm(2)/s) were significantly lower than those of the nonenhancing lesions in the ameloblastomas and were similar to those of the nonenhancing lesions in the keratocystic odontogenic tumors. CONCLUSION: ADC determination may be used as an adjunct tool for differentiation between ameloblastomas and keratocystic odontogenic tumors.

Akt activation induces epidermal hyperplasia and proliferation of epidermal progenitors.

Various common signaling pathways maintain tissue stem cells, including Notch and Wnt/beta-catenin signals. Phosphoinositide-3 kinase (PI3K)/Akt signaling regulates the 'stemness' of several stem cells in culture, specifically in maintaining embryonic stem and neural stem cells, and in deriving embryonic germ cells from primordial germ cells. We examined the effect of Akt signaling in epidermal cells in transgenic mice expressing an Akt-Mer fusion protein whose kinase activity was conditionally activated by treatment with 4-hydroxytamoxifen (4OHT). The topical application of 4OHT to adult skin of the transgenic mice induced new hair growth in resting phase follicles. In addition, the mice showed hyperplasia in interfollicular epidermis (IFE) and hair follicles, which was presumably caused by the extensive proliferation of keratinocytes in basal layer of IFE and outer root sheath of hair follicles, respectively. The progenitor cell population increased consistently in 4OHT-treated transgenic mice. Our results show that PI3K/Akt signaling induces epidermal hyperplasia and proliferation of epidermal progenitors.

Suppression of tumorigenicity, but not anchorage independence, of human cancer cells by new candidate tumor suppressor gene CapG.

Previously, we isolated a series of cell lines from a human diploid fibroblast lineage as a model for multistep tumorigenesis in humans. After passaging a single LT-transfected fibroblast clone, differently progressed cell lines were obtained, including immortalized, anchorage-independent and tumorigenic cell lines. In the present paper, we analysed the gene expression profiles of these model cell lines, and observed that expression of the CapG protein was lost in the tumorigenic cell line. To examine the possibility that loss of CapG protein expression was required for tumorigenic progression, we transfected CapG cDNA into the tumorigenic cell line and tested for tumor-forming ability in nude mice. Results showed that ectopic expression of CapG suppressed tumorigenicity, but not growth in soft agar or liquid medium. We also found that certain cancer cell lines including stomach cancer, lung cancer and melanoma had also lost CapG expression. One such cancer cell line AZ521 also became non-tumorigenic after the introduction of CapG cDNA. Moreover, we showed that CapG expression was repressed in small-cell lung cancer tissues. Together, our findings indicated that CapG is a new tumor suppressor gene involved in the tumorigenic progression of certain cancers.

Tumour necrosis factor-alpha but not interferon-gamma is the main inducer of inducible protein-10 in skin fibroblasts from patients with atopic dermatitis.

BACKGROUND: Inducible protein (IP)-10 belongs to the CXC chemokine subfamily and acts through the CXCR3 receptors that attract T lymphocytes. Keratinocytes are thought to be the main cell source of this chemokine in the skin, but other sources need to be elucidated. OBJECTIVES: To determine whether skin fibroblasts, besides keratinocytes, are able to produce IP-10 and the possible involvement of these cells in pathogenesis of atopic dermatitis (AD). METHODS: We studied the production pattern of IP-10 in dermal fibroblasts obtained from healthy donors, AD patients and in the HaCaT cell line (normal human keratinocytes) used as control. We stimulated fibroblasts after the sixth and seventh passage with tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-4, and by means of reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay analysis detected the production pattern of IP-10. To determine whether a different pattern of production of IP-10 by fibroblasts corresponds to the level of this chemokine in the plasma of patients with AD, we also checked the plasma IP-10 levels in 33 AD patients and 10 healthy donors. RESULTS: The pattern of chemokine production between dermal fibroblasts and HaCaT cells was different. The main inducer of IP-10 in fibroblasts was TNF-alpha, whereas IFN-gamma was the main inducer of IP-10 in HaCaT cells. We demonstrate that fibroblasts from AD patients have higher IP-10 expression and are more sensitive to TNF-alpha stimulation compared with healthy controls. Consequently, IP-10 levels in plasma of AD patients were higher than in healthy donors. CONCLUSIONS: Skin fibroblasts could be an important source of IP-10. TNF-alpha is the main inducer of IP-10 by skin fibroblasts, but not IFN-gamma or IL-4. The increased level of IP-10 in the plasma of patients with AD could be connected with increased activity of skin fibroblasts.