RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Influence of Lactic Acid on Endogenous and Viral RNA-Induced Immune Mediator Production by Vaginal Epithelial Cells

OBJECTIVE: To evaluate the influence of lactic acid on immune mediator release from vaginal epithelial cells. METHODS: The human vaginal epithelial cell line, VK2/E6E7, was cultured in the presence or absence of physiological concentrations of lactic acid, and in the presence or absence of the viral Toll-like receptor 3 agonist, poly (inosinic acid: cytidylic acid). Supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-1 beta, IL-6, IL-8, IL-23, transforming growth factor (TGF)-beta and secretory leukocyte protease inhibitor. RESULTS: Vaginal epithelial cells spontaneously released IL-1 beta (25.9 pg/mL), IL-8 (1.0 ng/mL), TGF-beta (175 pg/mL), and secretory leukocyte protease inhibitor (33.8 ng/mL). Only TGF-beta production was marginally enhanced (49%) by addition of lactic acid alone. Poly (inosinic acid: cytidylic acid) by itself stimulated the release of IL-6 (305 pg/mL) and enhanced IL-8 production (2.8 ng/mL). The combination of poly (inosinic acid: cytidylic acid) and lactic acid markedly increased IL-8 production (5.0 ng/mL) and induced the release of IL-1 beta (96.2 pg/mL). The poly (inosinic acid: cytidylic acid)-mediated lactic acid effect on IL-1 beta and IL-8 release was abrogated when the lactic acid was neutralized or if acetic acid was substituted for lactic acid. CONCLUSION: Lactic acid enhances the release of selective mediators from vaginal epithelial cells and stimulates antiviral immune responses. (Obstet Gynecol 2011;118:840-6) DOI: 10.1097/AOG.0b013e31822da9e9

Evidence for a network transcriptional control of promiscuous gene expression in medullary thymic epithelial cells

The expression of peripheral tissue antigens (PTAs) in the thymus by medullary thymic epithelial cells (mTECs) is essential for the central self-tolerance in the generation of the T cell repertoire. Due to heterogeneity of autoantigen representation, this phenomenon has been termed promiscuous gene expression (PGE), in which the autoimmune regulator (Aire) gene plays a key role as a transcription factor in part of these genes. Here we used a microarray strategy to access PGE in cultured murine CD80(+) 3.10 mTEC line. Hierarchical clustering of the data allowed observation that PTA genes were differentially expressed being possible to found their respective induced or repressed mRNAs. To further investigate the control of PGE, we tested the hypothesis that genes involved in this phenomenon might also be modulated by transcriptional network. We then reconstructed such network based on the microarray expression data, featuring the guanylate cyclase 2d (Gucy2d) gene as a main node. In such condition, we established 167 positive and negative interactions with downstream PTA genes. Silencing Aire by RNA interference, Gucy2d while down regulated established a larger number (355) of interactions with PTA genes. T- and G-boxes corresponding to AIRE protein binding sites located upstream to ATG codon of Gucy2d supports this effect. These findings provide evidence that Aire plays a role in association with Gucy2d, which is connected to Several PTA genes and establishes a cascade-like transcriptional control of promiscuous gene expression in mTEC cells. (C) 2009 Elsevier Ltd. All rights reserved.

Henneguya pseudoplatystoma n. sp causing reduction in epithelial area of gills in the farmed pintado, a South American catfish: Histopathology and ultrastructure

The present study is part of an ongoing investigation into the characteristics of Myxozoan parasites of Brazilian freshwater fish and was carried out using morphology, histopathology and electron microscopy analysis. A new Myxosporea species (Henneguya pseudoplatystoma) is described causing an important reduction in gill function in the farmed pintado (a hybrid fish from a cross between Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum), which is a commercially important South American catfish. From a total of 98 pintado juveniles from fish farms in the states of Sao Paulo and Mato Grosso do Sul (Brazil), 36 samples (36.7%) exhibited infection of the gill filaments. infection was intense, with several plasmodia occurring on a same gill filament. The plasmodia were white and measured up to 0.5 mm in length; mature spores were ellipsoidal in the frontal view, measuring 33.2 +/- 1.9 mu m in total length, 10.4 +/- 0.6 mu m in body length, 3.4 +/- 0.4 mu m in width and 22.7 +/- 1.7 mu m in the caudal process. The polar capsules were elongated, measuring 3.3 +/- 0.4 mu m in length and 1.0 +/- 0.1 mu m in width and the polar filaments had six to seven turns. Histopathological analysis revealed the parasite in the connective tissue of the gill filaments and lamella. No inflammatory process was observed, but the development of the plasmodia reduced the area of functional epithelium. Ultrastructural analyses revealed a single plasmodial wall, which was in direct contact with the host cells and had numerous projections in direction of the host cells as well as extensive pinocytotic canals. A thick layer (2-6 mu m) of fibrous material and numerous mitochondria were found in the ectoplasm. Generative cells and the earliest stage of sporogenesis were seen more internally. Advanced spore developmental stages and mature spores were found in the central portion of the plasmodia. (C) 2009 Elsevier B.V. All rights reserved.

The geodiamolide H, derived from Brazilian sponge Geodia corticostylifera, regulates actin cytoskeleton, migration and invasion of breast cancer cells cultured in three-dimensional environment

We are investigating effects of the depsipeptide geodiamolide H, isolated from the Brazilian sponge Geodia corticostylifera, on cancer cell lines grown in 3D environment. As shown previously geodiamolide H disrupts actin cytoskeleton in both sea urchin eggs and breast cancer cell monolayers. We used a normal mammary epithelial cell line MCF 10A that in 3D assay results formation of polarized spheroids. We also used cell lines derived from breast tumors with different degrees of differentiation: MCF7 positive for estrogen receptor and the Hs578T, negative for hormone receptors. Cells were placed on top of Matrigel. Spheroids obtained from these cultures were treated with geodiamolide H. Control and treated samples were analyzed by light and confocal microscopy. Geodiamolide H dramatically affected the poorly differentiated and aggressive Hs578T cell line. The peptide reverted HsS78T malignant phenotype to polarized spheroid-like structures. MCF7 cells treated by geodiamolide H exhibited polarization compared to controls. Geodiamolide H induced striking phenotypic modifications in Hs578T cell line and disruption of actin cytoskeleton. We investigated effects of geodiamolide H on migration and invasion of Hs578T cells. Time-lapse microscopy showed that the peptide inhibited migration of these cells in a dose-dependent manner. Furthermore invasion assays revealed that geodiamolide H induced a 30% decrease on invasive behavior of Hs578T cells. Our results suggest that geodiamolide H inhibits migration and invasion of Hs578T cells probably through modifications in actin cytoskeleton. The fact that normal cell lines were not affected by treatment with geodiamolide H stimulates new studies towards therapeutic use for this peptide.

Vimentin expression and the influence of Matrigel in cell lines of head and neck squamous cell carcinoma

Vimentin is a cytoeskeletal intermediate filament protein commonly observed in mesenchymal cells; however, it can also be found in malignant epithelial cells. It is demonstrated in several carcinomas, such as those of the cervix, breast and bladder, in which it is widely used as a marker of the epithelial to mesenchymal transition that takes place during embryogenesis and metastasis. Vimentin is associated with tumors that show a high degree of invasiveness, being detected in invasion front cells. Its expression seems to be influenced by the tumor microenvironment. The aim of this study was to evaluate vimentin expression in head and neck squamous cell carcinoma (HNSCC) cell lines, and to investigate the contribution of the microenvironment to its expression. HNSCC cell lines (HN6, HN30 and HN31) and an immortalized nontumorigenic cell line (HaCaT) were submitted to a three-dimensional assay with Matrigel. Cytoplasmatic staining of the HN6 cell line cultured without Matrigel and of the HN30 and HN31 cell lines cultured with Matrigel was demonstrated through immunohistochemistry. Western Blotting revealed a significant decrease in vimentin expression for the HN6 cell line and a significant increase for the HN30 and HN31 cell lines cultured with Matrigel. The results suggest that vimentin can be expressed in HNSCC cells and its presence is influenced by the microenvironment of a tumor.

Lineage relationship of prostate cancer cell types based on gene expression

Background: Prostate tumor heterogeneity is a major factor in disease management. Heterogeneity could be due to multiple cancer cell types with distinct gene expression. Of clinical importance is the so-called cancer stem cell type. Cell type-specific transcriptomes are used to examine lineage relationship among cancer cell types and their expression similarity to normal cell types including stem/progenitor cells. Methods: Transcriptomes were determined by Affymetrix DNA array analysis for the following cell types. Putative prostate progenitor cell populations were characterized and isolated by expression of the membrane transporter ABCG2. Stem cells were represented by embryonic stem and embryonal carcinoma cells. The cancer cell types were Gleason pattern 3 (glandular histomorphology) and pattern 4 (aglandular) sorted from primary tumors, cultured prostate cancer cell lines originally established from metastatic lesions, xenografts LuCaP 35 (adenocarcinoma phenotype) and LuCaP 49 (neuroendocrine/small cell carcinoma) grown in mice. No detectable gene expression differences were detected among serial passages of the LuCaP xenografts. Results: Based on transcriptomes, the different cancer cell types could be clustered into a luminal-like grouping and a non-luminal-like (also not basal-like) grouping. The non-luminal-like types showed expression more similar to that of stem/progenitor cells than the luminal-like types. However, none showed expression of stem cell genes known to maintain stemness. Conclusions: Non-luminal-like types are all representatives of aggressive disease, and this could be attributed to the similarity in overall gene expression to stem and progenitor cell types.

Gene expression relationship between prostate cancer cells of Gleason 3, 4 and normal epithelial cells as revealed by cell type-specific transcriptomes

Background: Prostate cancer cells in primary tumors have been typed CD10(-)/CD13(-)/CD24(hi)/CD26(+)/CD38(lo)/CD44(-)/CD104(-). This CD phenotype suggests a lineage relationship between cancer cells and luminal cells. The Gleason grade of tumors is a descriptive of tumor glandular differentiation. Higher Gleason scores are associated with treatment failure. Methods: CD26(+) cancer cells were isolated from Gleason 3+3 (G3) and Gleason 4+4 (G4) tumors by cell sorting, and their gene expression or transcriptome was determined by Affymetrix DNA array analysis. Dataset analysis was used to determine gene expression similarities and differences between G3 and G4 as well as to prostate cancer cell lines and histologically normal prostate luminal cells. Results: The G3 and G4 transcriptomes were compared to those of prostatic cell types of non-cancer, which included luminal, basal, stromal fibromuscular, and endothelial. A principal components analysis of the various transcriptome datasets indicated a closer relationship between luminal and G3 than luminal and G4. Dataset comparison also showed that the cancer transcriptomes differed substantially from those of prostate cancer cell lines. Conclusions: Genes differentially expressed in cancer are potential biomarkers for cancer detection, and those differentially expressed between G3 and G4 are potential biomarkers for disease stratification given that G4 cancer is associated with poor outcomes. Differentially expressed genes likely contribute to the prostate cancer phenotype and constitute the signatures of these particular cancer cell types.

The Effects of Commercially Available Preservative-Free FDA-Approved Triamcinolone (Triesence (R)) on Retinal Cells in Culture

Purpose: To evaluate the effects of Triesence (R) (TRI), a new preservative-free triamcinolone approved by the U. S. Food and Drug Administration (FDA) for intraocular use, on human retina pigment epithelial (ARPE-19) and rat neurosensory (R28) cells in culture. Methods: ARPE-19 and R28 cell cultures were treated 24 h with 1,000, 500, 200, or 100 mu g/mL of crystalline (cTRI) or 1,000, 500, or 200 mu g/mL of solubilized (sTRI). TRI was solubilized by centrifuging the drug, discarding the supernatant containing the vehicle and then resuspending the drug pellet in an equivalent amount of Dimethyl sulfoxide to achieve the same concentration as the commercial preparation. Percentage of cell viability (CV) was evaluated by a trypan blue dye-exclusion assay. The mitochondrial membrane potential (Delta Psi m) was analyzed with the JC-1 assay. The caspase-3/7 activity was measured by a fluorochrome assay. Results: In the ARPE-19 cultures, the cTRI caused a decrease in CV at 1,000 mg/mL (13.03 +/- 6.51; P < 0.001), 500 mu g/mL (28.87 +/- 9.3; P < 0.001), 200 mu g/mL (54.93 +/- 5.61; P < 0.001), and 100 mu g/mL (82.53 +/- 0.65; P < 0.005) compared with the untreated controls (96.98 +/- 0.16). In R28 cultures, the cTRI treatment also reduced CV values significantly (P < 0.001) for the 1,000 mu g/mL (22.73 +/- 2.44), 500 mu g/mL (34.63 +/- 1.91), 200 mu g/mL (58.70 +/- 1.39), and 100 mu g/m (75.33 +/- 2.47) compared with the untreated controls (86.08 +/- 3.54). Once the TRI was solubilized (sTRI), the CV and Delta Psi m remained similar to the untreated controls for both ARPE-19 and R28 cells. The sTRI treatment with 1,000, 500, and 200 mu g/mL increased in caspase-3/7 activity in ARPE-19 cells (P < 0.01) and in R28 cells (P < 0.05) compared with dimethyl sulfoxide equivalent controls. Conclusion: The crystalline form of TRI (cTRI) can cause a significant decrease in CV to cultured retinal cells. Once the TRI is solubilized (sTRI), at the same concentrations, the cells remain viable with no decrease in CV or Delta Psi m. The sTRI can, however, increase caspase-3/7 activity, thus suggesting some degree of apoptosis.

Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background: In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro. Methods: Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 mu M for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples >= 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors. Results: Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction = 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed. Conclusion: Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.

Viability of fibroblasts cultured under nutritional stress irradiated with red laser, infrared laser, and red light-emitting diode

Phototherapy is noninvasive, painless and has no known side effect. However, for its incorporation into clinical practice, more well-designed studies are necessary to define optimal parameters for its application. The viability of fibroblasts cultured under nutritional stress irradiated with either a red laser, an infrared laser, or a red light-emitting diode (LED) was analyzed. Irradiation parameters were: red laser (660 nm, 40 mW, 1 W/cm(2)), infrared laser (780 nm, 40 mW, 1 W/cm(2)), and red LED (637 +/- 15 nm, 40 mW, 1 W/cm(2)). All applications were punctual and performed with a spot with 0.4 mm(2) of diameter for 4 or 8 s. The Kruskal-Wallis test and analysis of variance of the general linear model (p <= 0.05) were used for statistical analysis. After 72 h, phototherapy with low-intensity laser and LED showed no toxicity at the cellular level. It even stimulated methylthiazol tetrazolium assay (MTT) conversion and neutral red uptake of fibroblasts cultured under nutritional stress, especially in the group irradiated with infrared laser (p = 0.004 for MTT conversion and p < 0.001 for neutral red uptake). Considering the parameters and protocol of phototherapy used, it can be concluded that phototherapy stimulated the viability of fibroblasts cultured under nutritional deficit resembling those found in traumatized tissue in which cell viability is reduced. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3602850]

Can established cultured papilloma cells harbor bovine papillomavirus?

Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

Protein aggregation containing beta-amyloid, alpha-synuclein and hyperphosphorylated tau in cultured cells of hippocampus, substantia nigra and locus coeruleus after rotenone exposure

Background: Protein aggregates containing alpha-synuclein, beta-amyloid and hyperphosphorylated tau are commonly found during neurodegenerative processes which is often accompanied by the impairment of mitochondrial complex I respiratory chain and dysfunction of cellular systems of protein degradation. In view of this, we aimed to develop an in vitro model to study protein aggregation associated to neurodegenerative diseases using cultured cells from hippocampus, locus coeruleus and substantia nigra of newborn Lewis rats exposed to 0.5, 1, 10 and 25 nM of rotenone, which is an agricultural pesticide, for 48 hours. Results: We demonstrated that the proportion of cells in culture is approximately the same as found in the brain nuclei they were extracted from. Rotenone at 0.5 nM was able to induce alpha-synuclein and beta amyloid aggregation, as well as increased hyperphosphorylation of tau, although high concentrations of this pesticide (over 1 nM) lead cells to death before protein aggregation. We also demonstrated that the 14kDa isoform of alpha-synuclein is not present in newborn Lewis rats. Conclusion: Rotenone exposure may lead to constitutive protein aggregation in vitro, which may be of relevance to study the mechanisms involved in idiopathic neurodegeneration.

The Fate of Chrysotile-Induced Multipolar Mitosis and Aneuploid Population in Cultured Lung Cancer Cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged alpha-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.

Massive infestation by Perulernaea gamitanae (Crustacea: Cyclopoida: Lernaidae) in juvenile gamitana, cultured in the Peruvian Amazon

The gamitana is a species of socio-economic importance in the Peruvian Amazon, often intensively produced locally for human consumption. Because of this, more studies concerning parasite populations affecting this species culture are necessary. In this study, a heavy copepod infestation of Perulernaea gamitanae is reported in a managed culture of gamitana. The prevalence of infection was 100% and mortality of the fish population was complete. The average intensity and abundance of the parasite was 268.8 parasites per individual.