Expression of Toll-like receptors 4 and 7 in the embryonic and adult pancreas, liver and adrenal gland of the mouse

The role of Toll protein in development and immunity is very well understood in Drosophila melanogaster (Anderson et al.,1985). Conversely, the contribution of Tolllike receptors (TLRs) in mammalian development is just beginning to be revealed. In this study, we evaluated the expression of TLR4 and TLR7 by immunohistochemistry on paraffin-embedded tissue in the adrenal gland, liver and pancreas of mouse embryos from stages E12, E14 and E16 and in the adult organs. Results show that TLR4 and TLR7 start to be detectable during embryonic development already at the first stage examined (E12). This expression follows the maturation of the organs and is still present in the adult with a different distribution pattern. Before this study no data in the literature were present on TLR4 and 7 expression in mammalian splanchnic organs development and in the adult no localization studies were available for TLR7. A possible interpretation of the results suggests that, besides their immunitary function, TLRs might be involved in a shared mechanism that regulates proliferation and differentiation both in embryonic organs and adult organs (Sato et al., 2009). These results also suggest that the contribution of TLRs in the context of carcinogenesis should be investigated not only in relation to chronic inflammation and tissue damage but also in relation to their contribution to the process of organogenesis

Expression of TLR7 in the murine eye during the embryonic period and in the adult animal

In the present study, we evaluated Toll-like receptor 7 (TLR7) expression at different stages of the murine eye development and in the adult organ. In mammals, TLRs are best known for their immunitary function, however data from the literature are demonstrating that in analogy to their Drosophila homologue Toll, they also participate in developmental mechanisms (Okun, Griffioen and Mattson 2011, Shechter et al. 2008). Immunohistochemistry for TLR7 and double immunofluorescence for TLR7/PCNA were performed on E12, E14 and E16 formalin-fixed paraffin-embedded mouse heads and on eyes enucleated from 3 months adult mice Results of experiments indicate that TLR7 expression is present in different compartments of the mouse eye (cornea, pigmented epithelium, neural retina, and lens) during gestation both in proliferating and differentiating cells and that such expression persists also in the adult organ. These observations indicate that besides being involved in protective mechanisms in the adult eye, TLR7 is also likely involved in the morphogenetic processes of this complex organ to which cells and tissues of different embryological origin contribute

Treatment of primary shoulder stiffness: Results of a survey on surgeon practice patterns in Italy

Objectives Shoulder stiffness is a condition of restricted glenohumeral range of motion (ROM), which can arise spontaneously or as consequence of a known cause. Several treatment options are available and currently no consensus has been obtained on which treatment algorithm represents the best choice for the patient. The aim of this study was to investigate surgeon practice patterns in Italy regarding treatment of primary shoulder stiffness. Methods A literature review was performed to identify randomized controlled trials reporting results of shoulder stiffness treatment. The following controversial or critical points in the treatment of primary shoulder stiffness were identified: modalities of physical therapy; indication for oral corticosteroid; indication and frequency for injective corticosteroid; technique and site of injection; and indication, timing, and technique for surgery. A survey composed by 14 questions was created and administrated to the members of a national association specialized in orthopaedics and sports traumatology (SIGASCOT at the time of survey completion, recently renamed SIAGASCOT after the fusion of the societies SIGASCOT and SIA). Results A total of 204 completed questionnaires were collected. Physical therapy was recommended by 98% of the interviewed. The use of oral corticosteroids was considered by 51%, and injections of corticosteroids by 72%. The posterior injection approach was the one preferred and a number of three was considered the upper limit..

Effect of aging on extracellular matrix and collagen turnover related pathways in human tendons and cultured human tenocytes

The aging process involves different organs, including the musculoskeletal system. Age-related modifications affecting the tendon such as reduced cell proliferation and decreased glycosaminoglycan and proteoglycan content were previously described, but data are incomplete and discordant. Therefore, aim of our study was to characterize the effect of aging on tendons, with particular attention to collagen turnover. For this purpose, tendons and cultured tenocytes were obtained by healthy young (age <65 years) and aging subjects (≥65 years), and analyzed by morphological and molecular methods. Our data show that aging tenocytes have a reduced proliferation rate. Haematoxylin and eosin, Sirius red and Alcian blue staining, respectively, revealed that tendon structure is maintained, and that collagen and proteoglycan content is similar in young and aging tendons. However, decreased lysyl hydroxylase 2b gene expression was observed in aging tenocytes, suggesting that differences in collagen maturation could be responsible for a decreased ability to resist mechanical loading. By fluorescence microscopy, actin cytoskeleton modifications such as fewer and shorter stress fibers were observed in some aging tenocytes, consistent with a decreased ability to form focal adhesions and, therefore, a reduced migration potential. Intermediate filaments and microtubules were not modified by aging. Considered as a whole, our results suggest that the structure of aged tendons is preserved, but the biomechanical properties could be impaired by reduced collagen cross-linking. Moreover, modifications of actin filaments could affect the mechanotransduction system allowing tenocytes to adapt their ability for extracellular matrix remodeling in response to mechanical loading. Therefore, aged tendons could be likely more prone to develop tendinopathies

Analysis of tissue structure and remodeling ion alveolar ridges augmented with human palate or tuberosity mucosa

Previous clinical reports found different clinical outcomes of localized alveolar ridge augmentation with soft tissue harvested either from the palate or from the tuberosity over time, showing that the palatal grafts had a better tissue stability than those from the tuberosity, which tended to a hyperplastic reaction. The mechanisms responsible for a different maturation of the grafted tissue using the two donor sites are still unclear, very likely depending on differences of the structure and extracellular matrix of connective tissue (CT). The current study aimed at comparing the morphology and collagen turnover-related molecular pathways of sites grafted with CT from either the palate (group A = 7 patients) or the tuberosity (group B = 7 patients) one year after surgical procedures for ridge augmentation. Cultured fibroblasts were obtained to analyze by real-time PCR the mRNA levels for collagen type I and III (COL-I, COL-III), matrix metalloproteinases (MMP-1 and 2), long lysyl hydroxylase 2b (LH2b). Collagen protein levels were assessed by slot blot, collagen degradation by SDS-zymography. No significant differences in collagen content were found. COLI and III, MMP-1 and 2 expression was similar in cell culture supernatants from palate and tuberosity fibroblasts, although COL-I and COL-III protein levels resulted up-regulated, respectively, in 57% and 66% of the samples. LH2b/COL-I mRNA ratio 69% was higher in the tuberosity fibroblasts, suggesting that the tuberosity collagen could be cross-linked at a higher extent, and therefore less susceptible to degradation by MMPs, leading to its excessive accumulation. Our data show that in group B the higher LH2b/COL-I mRNA ratio may be responsible for differences in collagen maturation as the major determinants in the hyperplastic response, and that grafting using the maxillary tuberosity could avoid unwanted tissue contraction over time

O LIVRO DIGITAL COMO POSSIBILIDADE DE UM TRABALHO INTERDISCIPLINAR ENTRE LITERATURA E ASTRONOMIA

O presente trabalho tem por objetivo investigar as potencialidades de um livro digital para o ensino de Literatura e Astronomia numa perspectiva interdisciplinar, em uma turma de primeiro ano do Ensino Médio, de uma instituição privada da cidade de Maringá- PR. Esta pesquisa insere-se no campo das investigações qualitativas do tipo descritiva. Como instrumentos de constituição de dados utilizamos mapas mentais, construídos pelos alunos no início e no final da implementação da proposta, e o diário de campo elaborado pelos pesquisadores. Para a análise e interpretação dos dados utilizamos os pressupostos qualitativos propostos pelos autores Novak, Gowin e Buzan. Nossos resultados evidenciam que os alunos estiveram mobilizados para a realização das atividades, caracterizando assim nosso livro digital como um material potencialmente significativo. Os mapas mentais produzidos nos mostraram ainda que houve uma melhora significativa nos elementos abrangência do assunto e hierarquia, caracterizando assim uma diferenciação progressiva, o que a princípio podemos associar a um indício de aprendizagem significativa

Epithelial-to-mesenchymal transition in pancreas adenocarcinoma cells: effect of 2D versus 3D arrangement

It was shown that three-dimensional (3D) cell cultures allow mimicking the functions of living tissues and provide pivotal information encoded in the tissue architecture [1]. Considered the primary role of epithelial-to-mesenchymal transition (EMT) in carcinoma progression [2], we aimed at investigating the effect of the 3D arrangement on the expression of some key markers of EMT in cultured human pancreas adenocarcinoma cells (PDAC). HPAC cells were cultured in both two-dimensional (2D) monolayers and in 3D spheroids, and were analyzed by morphological and molecular approaches. Immunofluorescence analysis for E-cadherin, β-catenin, actin, vimentin and collagen type I was performed on cells grown on 12 mm coverslips or on free-floating spheroids after 4% paraformaldehyde fixation. E-cadherin gene expression was assessed by real time PCR. Fluorescence and confocal microscopy analysis revealed that the E-cadherin/β-catenin complex was similarly expressed at the cell boundaries on the plasma membrane in 2D monolayers as well as in the 3D spheroids. Phalloidin-stained F-actin was mainly arranged into cortical actin filaments while vimentin was undetectable, suggesting an epithelial-like phenotype for HPAC cells in 2D and 3D arrangement. Interestingly, after 3D arrangement decreased E-cadherin mRNA levels and some cells expressing collagen type I were observed in spheroids. Our findings suggest that the 3D arrangement induced the expression of mesenchymal phenotype-related markers in HPAC cells, providing a model to better understand the biology of PDAC

Cigarette Smoke Affects ABCAl Expression via Liver X Receptor Nuclear Translocation in Human Keratinocytes

Cutaneous tissue is the first barrier against outdoor insults. The outer most layer of the skin, the stratum corneum (SC), is formed by corneocytes embedded in a lipid matrix (cholesterol, ceramide and fatty acids). Therefore, the regulation of lipids and, in particular, of cholesterol homeostasis in the skin is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a key role in maintaining cellular cholesterol levels. Among the many factors that have been associated with skin diseases, the environmental stressor cigarette smoke has been recently studied. In the present study, we demonstrate that ABCA1 expression in human cells (HaCaT) was increased (both mRNA and protein levels) after CS exposure. This effect was mediated by the inhibition of NFkB (aldehydes adducts formation) that allows the translocation of liver X receptor (LXR). These findings suggest that passive smoking may play a role in skin cholesterol levels and thus affect cutaneous tissues functions

Dysregulation of MS risk genes and pathways at distinct stages of disease

OBJECTIVE: To perform systematic transcriptomic analysis of multiple sclerosis (MS) risk genes in peripheral blood mononuclear cells (PBMCs) of subjects with distinct MS stages and describe the pathways characterized by dysregulated gene expressions. METHODS: We monitored gene expression levels in PBMCs from 3 independent cohorts for a total of 297 cases (including clinically isolated syndromes (CIS), relapsing-remitting MS, primary and secondary progressive MS) and 96 healthy controls by distinct microarray platforms and quantitative PCR. Differential expression and pathway analyses for distinct MS stages were defined and validated by literature mining. RESULTS: Genes located in the vicinity of MS risk variants displayed altered expression in peripheral blood at distinct stages of MS compared with the healthy population. The frequency of dysregulation was significantly higher than expected in CIS and progressive forms of MS. Pathway analysis for each MS stage–specific gene list showed that dysregulated genes contributed to pathogenic processes with scientific evidence in MS. CONCLUSIONS: Systematic gene expression analysis in PBMCs highlighted selective dysregulation of MS susceptibility genes playing a role in novel and well-known pathogenic pathways

BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers

Background: The K3326X variant in BRCA2 (BRCA2*c.9976A>T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers. Methods: Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided. Results: The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10- 6) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10-3). These associations were stronger for serous ovarian cancer and for estrogen receptor–negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10-5 and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10-5, respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed. Conclusions: Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations