Differential Expression of Claudin 1 and 4 in Basal Cell Carcinoma of the Skin.

Basal cell carcinoma (BCC) is the most common human malignancy. The biological behavior of this entity is remarkably indolent. Claudin plays an important role in tight junctions, regulating paracellular passage of variable substance including growth factors and maintaining the polarity of epithelia. Up- or downregulated claudin expression has been reported in many cancers. Nevertheless, claudin expression in BCC of the skin remains unclear. We therefore examined the status of claudin 1 and 4 expressions in BCC and adjacent normal skin by immunohistochemistry (IHC). Our IHC results demonstrated high claudin 1 expression and low claudin 4 expression in 33 of 34 lower-grade BCCs. In lower-grade BCC, claudin 1 was increased and claudin 4 was decreased compared with the normal skin. Claudin 1 was inclined to be highly expressed in the membrane and cytoplasm of tumour cells in the periphery of tumour nest. Conversely, almost all lower-grade BCCs (33/34) and one of two higher-grade BCC lacked or showed focal positivity for claudin 4. These results imply that the expression pattern is characteristics of lower-risk BCC. Interestingly, one of the two higher-grade BCCs demonstrated the converse expression patterns of claudins, with decreased claudin 1 and increased claudin 4. The combination of immunohistochemical claudin 1 and 4 expression may offer a useful ancillary tool for the pathological diagnosis of BCC. Furthermore, membranous and intracellular claudins may present future therapeutic targets for uncontrollable BCC.

Telmisartan Exerts Cytotoxicity in Scirrhous Gastric Cancer Cells by Inducing G0/G1 Cell Cycle Arrest.

BACKGROUND/AIM: This study aimed to assess the effects of telmisartan (TEL), a potential antitumor agent, and its mechanism of action in the regulation of apoptosis, autophagy, and cell cycle in scirrhous gastric cancer (SGC). MATERIALS AND METHODS: The effect of TEL on the viability and chromatin condensation of OCUM-2M and OCUM-12 cells was assessed. Protein expression and the cell cycle were analysed using western blotting and flow cytometry, respectively. RESULTS: TEL inhibited cell proliferation in a dose-dependent manner and increased chromatin condensation and autophagy marker LC3-II levels in OCUM-12 cells. TEL also increased the proportion of cells in the G0/G1 phase transition. CONCLUSION: Apoptosis and autophagy are partially involved in the inhibitory effect of TEL on cell proliferation. Additionally, TEL caused G0/G1 cell cycle arrest. Therefore, TEL could be a promising treatment for SGC.

Reirradiation of salivary gland tumors with carbon ion radiotherapy at CNAO.

AIMS: To report oncologic and functional outcomes in terms of tumor control and toxicity of carbon ion radiotherapy (CIRT) in reirradiation setting for recurrent salivary gland tumors at CNAO. METHODS: From November 2013 to September 2016, 51 consecutive patients with inoperable recurrent salivary gland tumors were retreated with CIRT in the frame of the phase II protocol CNAO S14/2012C for recurrent head and neck tumors. RESULTS: Majority of pts (74.5%) had adenoid cystic carcinoma, mainly rcT4a (51%) and rcT4b (37%). Median dose of prior photon based radiotherapy was 60 Gy. Median dose of CIRT was 60 Gy [RBE] at a mean of 3 Gy [RBE] per fraction. During reirradiation, 19 patients (37.3%) experienced grade G1 toxicity, 19 pts (37.3%) had G2 and 2 pts (3.9%) had G3. Median follow up time was 19 months. Twenty one (41.2%) patients had stable disease and 30 (58.8%) tumor progression at the time of last follow up. Furthermore, 9 (18%) patients had G1 late toxicity, 19 (37%) had G2 and 9 (17. 5%) had G3. Using the Kaplan Meier method, progression free survival (actuarial) at one and two years were 71.7% and 52.2% respectively. Estimated overall survival (actuarial) at one and two years were 90.2% and 64%, respectively. CONCLUSIONS: CIRT is a good option for retreatment of inoperable recurrent salivary gland tumors with acceptable rates of acute and late toxicity. Longer follow up time is needed to assess the effectiveness of CIRT in reirradiation setting of salivary gland tumors.

Emergency Responses and Health Consequences after the Fukushima Accident; Evacuation and Relocation.

The Fukushima accident was a compounding disaster following the strong earthquake and huge tsunami. The direct health effects of radiation were relatively well controlled considering the severity of the accident, not only among emergency workers but also residents. Other serious health issues include deaths during evacuation, collapse of the radiation emergency medical system, increased mortality among displaced elderly people and public healthcare issues in Fukushima residents. The Fukushima mental health and lifestyle survey disclosed that the Fukushima accident caused severe psychological distress in the residents from evacuation zones. In addition to psychiatric and mental health problems, there are lifestyle-related problems such as an increase proportion of those overweight, an increased prevalence of hypertension, diabetes mellitus and dyslipidaemia and changes in health-related behaviours among evacuees; all of which may lead to an increased cardiovascular disease risk in the future. The effects of a major nuclear accident on societies are diverse and enduring. The countermeasures should include disaster management, long-term general public health services, mental and psychological care, behavioural and societal support, in addition to efforts to mitigate the health effects attributable to radiation.

Characteristics of neuropathic pain and its relationship with quality of life in 72 patients with spinal cord injury.

STUDY DESIGN: A cross-sectional study. OBJECTIVES: Neuropathic pain (NP) after spinal cord injury (SCI) tends to be hard to treat, and its heterogeneous properties make it difficult to identify and characterize. This study was conducted to assess the characteristics of SCI-related NP in detail. SETTING: A single hospital for SCI rehabilitation. METHODS: This study included 72 patients who were seen at our hospital in 2012 and 2013 and who had sustained SCI at least 3 months before enrollment. The patients completed the Neuropathic Pain Symptom Inventory (NPSI) and the Short Form (SF)-36 Health Inventory. The NPSI score was analyzed for correlations with clinical presentations of SCI and SF-36 subitems. RESULTS: Paresthesia/dysesthesia was the most common subtype of NP after SCI. With regard to location, below-level superficial NP was significantly more intense than at-level pain. Patients who underwent surgery showed significantly less evoked pain compared with patients with non-surgery. Patients reported significantly more severe pain if >1 year had elapsed after the SCI. Patients with an American Spinal Injury Association Impairment Scale grade of B for completeness of injury reported more intense NP than those with other grades. Among the SF-36 subitems, NP correlated significantly with bodily pain, general health and mental health. CONCLUSION: NP in SCI patients was significantly associated with the location of pain, the time period since the injury, surgery and quality-of-life factors. A more detailed understanding of the characteristics of NP may contribute to better strategies for relieving the pain associated with SCI.

A current life table and causes of death for insured dogs in Japan.

The life expectancies and causes of death were evaluated in 299,555 dogs insured in Japan between 1 April 2010 and 31 March 2011, of which 4169 dogs died during this period. The overall life expectancy of dogs was 13.7 years. The probability of death was high in the first year of life, lowest in the second and third years, and increased exponentially after 3 years of age. The life expectancy was 13.8 years in the <5 kg body weight group, 14.2 years in the 5-10 kg body weight group, 13.6 years in the 10-20 kg body weight group, 12.5 years in the 20-40 kg body weight group and 10.6 years in the ≥40 kg body weight group. As body weight increases, life expectancy tended to decrease except in the <5 kg body weight group. The probability of death increased as dogs got older for most potential causes of death. Neoplasia resulted in the highest probability of death, especially in the large and giant breed groups. Cardiovascular system disorders were the second major cause of death and the toy group had a probability of death significantly higher than the other breed groups at age 12+.

Breed, gender and age pattern of diagnosis for veterinary care in insured dogs in Japan during fiscal year 2010.

We calculated the annual prevalence of diseases of 18 diagnostic categories in the insured dog population in Japan, using data from 299,555 dogs insured between April 2010 and March 2011. The prevalence was highest for dermatological disorders (22.6% for females and 23.3% for males), followed by otic diseases (16.4% for females and 17.2% for males) and digestive system disorders (15.7% for females and 16.4% for males). The prevalence of cardiovascular, urinary, neoplasia and endocrine disorders, increased with age; infectious diseases and injuries showed a high prevalence at young ages, and the prevalence of musculoskeletal and respiratory disorders showed a bimodal peak at young and old ages. A large variation in prevalence was observed between breeds for dermatological, otic, digestive, ophthalmological and cardiovascular disorders.

Dysregulated FOXO transcription factors in articular cartilage in aging and osteoarthritis.

OBJECTIVE: Aging is a major risk factor for osteoarthritis (OA). Forkhead-box class O (FoxO) transcription factors regulate mechanisms of cellular aging, including protein quality control, autophagy and defenses against oxidative stress. The objective of this study was to analyze FoxO transcription factors in normal, aging and OA cartilage. DESIGN: Knee joints from humans ages 23-90 and from mice at the age of 4-24 months and following surgically induced OA were analyzed for expression of FoxO proteins. Regulation of FoxO protein expression and activation was analyzed in cultured chondrocytes. RESULTS: Human cartilage expressed FOXO1 and FOXO3 but not FOXO4 proteins. FOXO1 and FOXO3 were more strongly expressed the superficial and mid zone as compared to the deep zone and were mainly localized in nuclei. During human joint aging, expression of FOXO1 and FOXO3 was markedly reduced in the superficial zone of cartilage regions exposed to maximal weight bearing. In OA cartilage, chondrocyte clusters showed strong FOXO phosphorylation and cytoplasmic localization. Similar patterns of FOXO expression in normal joints and changes in aging and OA were observed in mouse models. In cultured chondrocytes, IL-1ß and TNF-α suppressed FOXO1, while TGF-ß and PDGF increased FOXO1 and FOXO3 expression. FOXO1 and FOXO3 phosphorylation was increased by IL-1ß, PDGF, bFGF, IGF-1, and the oxidant t-BHP. CONCLUSIONS: Normal articular cartilage has a tissue specific signature of FoxO expression and activation and this is profoundly altered in aging and OA in humans and mice. Changes in FoxO expression and activation may be involved in cartilage aging and OA.

Simultaneous treatment with azelnidipine and olmesartan inhibits apoptosis of Hl-1 cardiac myocytes expressing E334k cMyBPC.

BACKGROUND: Apoptosis appears to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM). We have previously reported 3 HCM patients carrying the E334K MYBPC3, and that heterologous expression of E334K cMyBPC in cultured cells induced apoptosis. The purpose of this study was to identify pharmacological agents that would inhibit apoptosis in HL-1 cardiomyocytes expressing E334K cMyBPC. METHODS AND RESULTS: E334K cMyBPC expression in cells increased levels of pro-apoptosis (p53, Bax and cytochrome c) and decreased levels of anti-apoptosis (Bcl-2 and Bcl-XL). While the beta blocker carvedilol (1 µM) normalized the level of p53 and Bcl-2 and the calcium channel blocker (CCB) bepridil (0.5 µM) normalized that of Bcl-2, both the CCB azelnidipine (1 µM) and the angiotensin receptor blocker (ARB) olmesartan (10 µM) normalized those of p53, Bax, cytochrome c, and Bcl-XL. Among those proteins, cytochrome c was the one which showed the highest degree of change. Both azelnidipine (0.1 µM) and olmesartan (1 µM) reduced the level of cytochrome c by 40.2 ± 4.3% and 31.3 ± 5.1%, respectively. The CCB amlodipine and the ARB valsartan reduced it only by 19.1 ± 2.1% and 20.1 ± 5.2%, respectively. Flow cytometric analysis and annexin V staining showed that treatment of cells with azelnidipine (0.1 µM) plus olmesartan (0.3 µM) or that with amlodipine (0.1 µM) plus valsartan (0.3 µM) reduced the number of apoptotic cells by 35.8 ± 10.5% and 18.4 ± 3.2%, respectively. CONCLUSION: Azelnidipine plus olmesartan or amlodipine plus valsartan inhibited apoptosis of HL-1 cells expressing E334K cMyBPC, and the former combination was more effective than the latter.

Integration of cap analysis of gene expression and chromatin immunoprecipitation analysis on array reveals genome-wide androgen receptor signaling in prostate cancer cells.

The androgen receptor (AR) is a critical transcriptional factor that contributes to the development and the progression of prostate cancer (PCa) by regulating the transcription of various target genes. Genome-wide screening of androgen target genes provides useful information to understand a global view of AR-mediated gene network in PCa. In this study, we performed 5'-cap analysis of gene expression (CAGE) to determine androgen-regulated transcription start sites (TSSs) and chromatin immunoprecipitation (ChIP) on array (ChIP-chip) analysis to identify AR binding sites (ARBSs) and histone H3 acetylated (AcH3) sites in the human genome. CAGE determined 13 110 distinct, androgen-regulated TSSs (P<0.01), and ChIP-chip analysis identified 2872 androgen-dependent ARBSs (P<1e-5) and 25 945 AcH3 sites (P<1e-4). Both androgen-regulated coding genes and noncoding RNAs, including microRNAs (miRNAs) were determined as androgen target genes. Besides prototypic androgen-regulated TSSs in annotated gene promoter regions, there are many androgen-dependent TSSs that are widely distributed throughout the genome, including those in antisense (AS) direction of RefSeq genes. Several pairs of sense/antisense promoters were newly identified within single RefSeq gene regions. The integration of CAGE and ChIP-chip analyses successfully identified a cluster of androgen-inducible miRNAs, as exemplified by the miR-125b-2 cluster on chromosome 21. Notably, the number of androgen-upregulated genes was larger in LNCaP cells treated with R1881 for 24 h than for 6 h, and the percentage of androgen-upregulated genes accompanied with adjacent ARBSs was also much higher in cells treated with R1881 for 24 h than 6 h. On the basis of the Oncomine database, the majority of androgen-upregulated genes containing adjacent ARBSs and CAGE tag clusters in our study were previously confirmed as androgen target genes in PCa. The integrated high-throughput genome analyses of CAGE and ChIP-chip provide useful information for elucidating the AR-mediated transcriptional network that contributes to the development and progression of PCa.

miR-148a is an androgen-responsive microRNA that promotes LNCaP prostate cell growth by repressing its target CAND1 expression.

Recent advances in cancer biology reveal that microRNAs (miRNAs) are involved in the regulation of cancer-related genes, or they function as tumor suppressors or oncogenes. In prostate cancer, evidence has accumulated for the contribution of the androgen-dependent gene network to tumor growth, although the precise functions of miRNAs in prostate cancer remain to be investigated. Here, we identified androgen-responsive miRNAs by the short RNA sequencing analysis in LNCaP prostate cancer cells. Among 10 miRNAs with known sequences, we have determined that miR-148a reduces the expression of cullin-associated and neddylation-dissociated 1 (CAND1), a negative regulator of SKP1-Cullin1-F-box (SCF) ubiquitin ligases, by binding to the 3'-untranslated region of CAND1 mRNA. CAND1 knockdown by small interfering RNA promoted the proliferation of LNCaP cells. Our study indicates the potential contribution of miR-148a to the growth of human prostate cancer.

Pathologic and immunologic profiles of a limited form of neuromyelitis optica with myelitis.

BACKGROUND: Neuromyelitis optica (NMO) is a demyelinating syndrome characterized by myelitis and optic neuritis. Detection of anti-NMO immunoglobulin G antibody that binds to aquaporin-4 (AQP4) water channels allows the diagnosis of a limited form of NMO in the early stage with myelitis, but not optic neuritis. However, the detailed clinicopathologic features and long-term course of this limited form remain elusive. METHODS: We investigated 8 patients with the limited form of NMO with myelitis in comparison with 9 patients with the definite form. RESULT: All patients with limited and definite form showed uniform relapsing-remitting courses, with no secondary progressive courses. Pathologic findings of biopsy specimens from the limited form were identical to those of autopsy from the definite form, demonstrating extremely active demyelination of plaques, extensive loss of AQP4 immunoreactivity in plaques, and diffuse infiltration by macrophages containing myelin basic proteins with thickened hyalinized blood vessels. Moreover, the definite form at the nadir of relapses displayed significantly higher amounts of the inflammatory cytokines interleukin (IL)-1beta and IL-6 in CSF than the limited form and multiple sclerosis. CONCLUSION: This consistency of pathologic findings and uniformity of courses indicates that aquaporin 4-specific autoantibodies as the initiator of the neuromyelitis optica (NMO) lesion consistently play an important common role in the pathogenicity through the entire course, consisting of both limited and definite forms, and NMO continuously displays homogeneity of pathogenic effector immune mechanisms through terminal stages, whereas multiple sclerosis should be recognized as the heterogeneous 2-stage disease that could switch from inflammatory to degenerative phase. This report is a significant description comparing the pathologic and immunologic data of limited NMO with those of definite NMO.

Gene expression profile during ovarian folliculogenesis.

Assisted reproductive technologies have progressed significantly and have provided successful treatment for many infertile couples. However, more advanced technologies are required for severe infertility such as premature ovarian failure and ovarian impairment due to adjuvant therapy for cancer. Ovarian tissue cryopreservation followed by in vitro growth of isolated follicles is a feasible proposition for such patients. Close coordination of communication among follicle cells including oocytes, granulosa and theca cells is required for follicle growth. Crucial factors may regulate the gonadotropin-independent and -dependent follicle growth stages. To facilitate development of a culture system for early growing follicles, DNA microarray analysis of mouse ovaries recovered at 7, 10, 13, 16 and 19 days of age was performed to identify factors required for the growth of early-stage follicles. These studies showed strong intensity of zona pellucida glycoproteins, bone morphogenic protein-15 (BMP-15) and growth differentiation factor (GDF-9) in 7 days old mice, which gradually declined in 19 days old mice. KIT, KIT ligand, anti-müllerian hormone (AMH) and platelet-derived growth factor (PDGF), known as granulosa cell secreted factors, also showed relatively high expression. These studies will facilitate our understanding of the regulatory factors involved in folliculogenesis and thereby enable establishment of in vitro culture system for ovarian follicles.

CD52 expression is induced in the mouse uterus at the time of embryo implantation.

CD52 is a GPI anchor protein present in lymphocytes, the epithelial cells of the epididymis and sperm. It has been reported that male reproductive tract CD52 induces antibodies interfering with sperm function and causes infertility. CD52 is also expressed in ovulated cumulus cells in female reproductive tissues. In the present study, we examined the distribution and the mechanism of regulation of CD52 in the uterus. CD52 expression was evaluated in uterine tissue recovered at 0.5, 4.5, 8.5 and 12.5dpc (days post-coitum). Immunohistochemistry, RT-PCR, Western blotting and gel shift analysis were performed to determine localization and transcriptional regulation of CD52. Cd52 mRNA and CD52 protein were found to increase simultaneously from 0.5 to 4.5dpc. Gel shift analysis revealed that NKX2.2, a transcriptional factor, binds to the promoter region of the Cd52 gene. CD52 and NKX2.2 were co-localized in the endometrium of the uterus. Pathway analysis using Ingenuity pathway analysis predicted that Cd52 is associated with genes involved in the formation of uterosomes which are necessary for embryo attachment. These findings suggest that CD52 synthesis is regulated by NKX2.2 at a transcriptional level, and that Cd52 may be a member of the network of genes regulating uterine receptivity for embryo implantation.

Neutrophil elastase inhibitor (sivelestat) reduces the levels of inflammatory mediators by inhibiting NF-kB.

OBJECTIVE: Sivelestat sodium hydrate (sivelestat) is a specific synthetic inhibitor of neutrophil elastase (NE). Various studies suggest that sivelestat treatment reduces inflammation. In this study, we tested the hypothesis that sivelestat acts as an inhibitor of inflammatory mediators and prevents nuclear factor-kB (NF-kB) activation. METHODS: In the presence and absence of sivelestat, the mouse macrophage cell line RAW 264.7 was stimulated with lipopolysaccharide (LPS) and the levels of inflammatory mediators (TNF-alpha, IL-6 and high mobility group box 1 (HMGB1)) and nitrite in the cell supernatant were measured, along with inducible nitric oxide synthase (iNOS) expression. RESULTS: While LPS administration increased the secretion of inflammatory mediators and nitric oxide (NO), sivelestat decreased the secretion of these mediators. Cell signaling studies demonstrated that sivelestat decreased NF-kB activation by inhibiting IkB phosphorylation. CONCLUSION: Sivelestat may inhibit the various inflammatory mediators through NF-kB inhibition.

NUCEL (Cell and Molecular Therapy Center): a multidisciplinary center for translational research in Brazil.

Social and economical development is closely associated with technological innovation and a well-developed biotechnological industry. In the last few years, Brazil's scientific production has been steadily increasing; however, the number of patents is lagging behind, with technological and translational research requiring governmental incentive and reinforcement. The Cell and Molecular Therapy Center (NUCEL) was created to develop activities in the translational research field, addressing concrete problems found in biomedical and veterinary areas and actively searching for solutions by employing a genetic engineering approach to generate cell lines over-expressing recombinant proteins to be transferred to local biotech companies, aiming at furthering the development of a national competence for local production of biopharmaceuticals of widespread use and of life-saving importance. To this end, mammalian cell engineering technologies were used to generate cell lines over-expressing several different recombinant proteins of biomedical and biotechnological interest, namely, recombinant human Amylin/IAPP for diabetes treatment, human FVIII and FIX clotting factors for hemophilia, human and bovine FSH for fertility and reproduction, and human bone repair proteins (BMPs). Expression of some of these proteins is also being sought with the baculovirus/insect cell system (BEVS) which, in many cases, is able to deliver high-yield production of recombinant proteins with biological activity comparable to that of mammalian systems, but in a much more cost-effective manner. Transfer of some of these recombinant products to local Biotech companies has been pursued by taking advantage of the São Paulo State Foundation (FAPESP) and Federal Government (FINEP, CNPq) incentives for joint Research Development and Innovation partnership projects.

Biochemical property and immunogenicity of mouse male reproductive tract CD52 (mrt-CD52).

Male reproductive tract CD52 (mrt-CD52) is known to be a pathogenic antigen for immunological infertility. Although human CD52 has been extensively investigated, the properties of mouse CD52 are not well elucidated. This study was conducted, therefore, to examine the tissue distribution, molecular composition and immunogenicity of mouse mrt-CD52. Immunohistological studies with an antibody to a synthetic peptide showed that mouse CD52 was localized mainly in the cauda epididymis and vas deferens, but not in the testis, liver, kidney or spleen. The molecule was composed of Asn (N)-linked and The/Ser (O)-linked carbohydrates as well as a glycosylphosphatidyl (GPI) anchor portion. Purified mrt-CD52 preparations produced antibodies by subcutaneous and intranasal immunization in both male and female mice. These antisera showed sperm-immobilizing activities with complement to mouse sperm. The research indicated mouse CD52 had similar biochemical and immunological properties to human CD52. This animal experiment is a good model for investigating human mrt-CD52 antibody detected in infertile patients.