Detection of human papillomavirus gene sequences in cell lines derived from laryngeal tumors

The role of Human Papillomaviruses (HPV) in laryngeal carcinomas has been studied with conflicting results. To evaluate the etiologic relationship between HPV infection and epithelial malignancy of the larynx we studied five laryngeal carcinoma cell lines obtained from patients undergoing surgery for laryngeal tumors. The paraffin embedded biopsy samples of the original tumor and different passages of the new established cell lines were investigated by PCR with consensus primers specific for HPV DNA. The findings indicate that HPV infection is associated with some larynx carcinomas. The positive association has been enhanced when a method of enrichment of epithelial cells from fresh tumor samples was used. All tumor cells enriched smears were positive for HPV DNA not only by PCR but also by in situ hybridization (ISH). Investigated by PCR, different passages of larynx tumor cell lines maintained expression of HPV DNA. At subsequent passages ISH gives constantly no signals suggesting a minimal amount of viral harbored sequences. In one cell line propagated more than 60 population doublings, the chromosomal frequency distribution shifted from modal number 46 at the 5^th passage to 63 at the 60^th passage. The mechanisms by which persistent HPV infection maintains continuous cell proliferation were discussed.     

Ex vivo differentiation of umbilical cord blood progenitor cells in the presence of placental conditioned medium

Hematopoetic stem cells (HSC) are the progenitors for the lympho-hematopoietic system, with long lifespan and high proliferation potential. Transplantation of HSC from bone marrow or peripheral blood represents a standard therapy in severe hematological conditions. A possible alternative source of HSC is the umbilical cord blood, prepared by various separation procedures followed by expansion in cultures supplemented with hematopoietic growth factors. In order to check the effects of placental conditioned medium (PCM) from placental cells culture upon viability of HSC, we added plasma, PCM, dimetil sulfoxyde or hemin in HSC cultures. Flow cytometry or direct scoring of solid cultures using CD45+, CD34+, CD71+ and CD14+ fluorescent-labeled monoclonal antibodies evaluated the effects upon cell proliferation and colony forming ability of HSC cultures, versus controls. PCM produced the highest proliferation, followed by plasma, DMSO and hemin. PCM improved the survival time and maintained a higher proportion of immature cells. PCM stimulates the differentiation towards myeloid lineage progenitor cells (>90% being CD45+), increasing the percentage of CD14+, granulocites /monocytes precursors. It is highly suggestive that PCM contains growth factors or cytokines, which regulate the development of HSC. Characterization of these factors is in progress.     

In vitro assessment of the antimicrobial activity of new N-acyl-thiourea derivatives

The qualitative screening of the susceptibility spectra of different microbial strains to the newly synthesized substances complexes was performed by adapted disk diffusion techniques, while the quantitative assay of the minimal inhibitory concentration (M.I.C., microg/cm3) value was based on liquid medium serial microdilutions. The compounds were solubilized in dimethylsulfoxide (DMSO). The in vitro biological screening effects were tested against a microbial inoculum of approximately 1.5 x 10(8) UFC/cm3, corresponding to 0.5 McFarland standard density, obtained from Gram positive (Staphylococcus aureus, Bacillus subtilis), Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and fungal strains (Candida albicans). In order to investigate the influence of the subinhibitory concentration of the tested substances on the expression of different virulence features, the strains were incubated overnight in the presence of the newly synthesized thiourea derivatives (vol:vol) and different virulence features were investigated, i.e: adherence capacity to the cellular substrate represented by HeLa cells and to inert substrata quantified by slime test and soluble enzymatic virulence factors (haemolysins and other pore-forming toxins, proteases activity, DN-ase and siderophores production). The cytotoxicity was assessed microscopically, by observing the effect of the tested compounds on the cellular substratum integrity.     

Cytokine profiles of HeLa and human diploid cells induced by different fractions of Vibrio parahaemolyticus cultures exposed to stress conditions

Vibrio (V.) parahaemolyticus is an aquatic halophilic bacteria which produces gastroenteritis and in rare cases septicaemia after the consumption of raw or under-cooked contaminated seafood.The severity of diarrheal illness caused by this bacterium is closely related to the presence of two types of hemolysins (the thermostable direct hemolysin-TDH and TDH related hemolysin-TRH) and also of type III secretion system (TTSS) proteins. The TTSS type 1 induces a wide array of effects on infected HeLa cells such as autophagy, oncosis, cell rounding and lysis. Previous studies have shown that heat shock proteins have the ability to stimulate the production of interleukins in different cellular cultures. In our studies we have stimulated two cellular lines (HeLa and human diploid cells) with different V. parahaemolyticus culture fractions in order to observe the effect on cytokines production. Thus, the purpose of this study was to analyze the expression of IL-1, IL-2, IL-4, IL-6, IL-10 and TNF-alpha induced by the cell treatment with total cellular lysate, periplasmic fractions and culture supernatants extracted from V. parahaemolyticus exposed to normal and also to stress conditions. The ELISA assay of the cytokine profile of the HeLa and HDC cell lines stimulated with different bacterial fractions revealed that in the V. parahemolyticus cultures submitted to osmotic and heat shock stress are accumulating factors (probably heat shock proteins) which are exhibiting immunomodulatory activity, responsible for the induction of a pro-inflammatory response associated with increased levels of IL-6 and TNF-alpha expression, however balanced by the stimulation of the anti-inflammatory cytokine IL-4 synthesis.     

MicroRNA-24 regulates the processing of latent TGFβ1 during cyclic mechanical stress in human trabecular meshwork cells through direct targeting of FURIN

Cyclic mechanical stress (CMS) leads to alterations of cellular functions in the trabecular meshwork (TM), including the up-regulation of transforming growth factor beta 1 (TGFβ1), that can potentially contribute to the pathogenesis of glaucoma. Although microRNAs (miRNAs) are known to play important roles in many biological functions, little is known about their potential involvement in the cellular responses elicited by mechanical stress. Here we analyzed changes in miRNA expression induced by CMS, and examined the possible role of miR-24 in the response of human TM cells to CMS. CMS induced the expression of miR-24 that led to the down regulation of the subtilisin-like proprotein convertase FURIN, which is known to play a major role in the processing of TGFβ1. FURIN was confirmed as a novel target of miR-24 by 3' UTR luciferase assay and western blot. Overexpression of miR-24 resulted in a significant decrease in activated TGFβ1. This effect was mimicked by down regulation of FURIN by siRNA. Conversely, inhibition of miR-24 expression with a specific antagomir led to a small but significant increase in TGFβ1. Furthermore, the increase in active TGFβ1 induced by CMS in HTM cells was prevented by miR-24. Altogether, our results suggest that miRNAs might contribute to the regulation of responses to CMS in TM cells. Specifically, miR-24 might play an important role in modulating the induction of TGFβ1 mediated by CMS through direct targeting of FURIN.     

Essential role of intracellular glutathione in controlling ascorbic acid transporter expression and function in rat hepatocytes and hepatoma cells

Although there is in vivo evidence suggesting a role for glutathione in the metabolism and tissue distribution of vitamin C, no connection with the vitamin C transport systems has been reported. We show here that disruption of glutathione metabolism with buthionine-(S,R)-sulfoximine (BSO) produced a sustained blockade of ascorbic acid transport in rat hepatocytes and rat hepatoma cells. Rat hepatocytes expressed the Na(+)-coupled ascorbic acid transporter-1 (SVCT1), while hepatoma cells expressed the transporters SVCT1 and SVCT2. BSO-treated rat hepatoma cells showed a two order of magnitude decrease in SVCT1 and SVCT2 mRNA levels, undetectable SVCT1 and SVCT2 protein expression, and lacked the capacity to transport ascorbic acid, effects that were fully reversible on glutathione repletion. Interestingly, although SVCT1 mRNA levels remained unchanged in rat hepatocytes made glutathione deficient by in vivo BSO treatment, SVCT1 protein was absent from the plasma membrane and the cells lacked the capacity to transport ascorbic acid. The specificity of the BSO treatment was indicated by the finding that transport of oxidized vitamin C (dehydroascorbic acid) and glucose transporter expression were unaffected by BSO treatment. Moreover, glutathione depletion failed to affect ascorbic acid transport, and SVCT1 and SVCT2 expression in human hepatoma cells. Therefore, our data indicate an essential role for glutathione in controlling vitamin C metabolism in rat hepatocytes and rat hepatoma cells, two cell types capable of synthesizing ascorbic acid, by regulating the expression and subcellular localization of the transporters involved in the acquisition of ascorbic acid from extracellular sources, an effect not observed in human cells incapable of synthesizing ascorbic acid.     

Scavenger Receptors Mediate the Role of SUMO and Ftz-f1 in Drosophila Steroidogenesis

SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval–pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi–mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.     

Interaction of bacteria isolated from clinical biofilms with cardiovascular prosthetic devices and eukaryotic cells

Purpose

To identify the relationships between some infectious agents implicated in cardiovascular diseases with the cellular substrate and prosthetic devices in the presence of antibiotics.

Specific objectives

Strains isolation and identification, comparative study of antibiotic resistance of planktonic (disk diffusion, E-test, automatic systems) and sessile (using original experimental models for in vitro development of monospecific biofilms) bacterial cells, virulence assays (adherence and invasion of HeLa cells, slime test, soluble virulence factors expression), dynamic study of biofilm development on inert substrata, under the influence of antibiotics, the influence of cellular and soluble bacterial fractions on HeLa cells (by flow cytometry and real-time PCR).

Results

The identified strains were isolated from different sources, the etiology being dominated by Gram-negative non-fermentative bacilli, Gram-positive cocci and yeasts, harboring invasion enzymes responsible for development of systemic infections. The isolated strains exhibited a high level of antibiotic resistance to beta-lactams, aminoglycosides and quinolones, and an evident tendency of colonizing the cellular and inert substrate, the degree of colonization depending on the physico-chemical nature of the substrate. By comparison with planktonic ones, the sessile bacterial strains expressed a changed profile of antibiotic resistance, this aspect being very important for the readjustment of the treatment and prevention of infections associated with prosthetic devices. In vitro experiments suggested that different fractions of S. aureus cultures could trigger the release of proinflammatory (TNF-α, IL-1b, IL-6) and anti-inflammatory (IL-8) cytokines and induced apoptosis in HeLa cells.     

Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.