Biocompatibility of ZrO2 vs. Y-TZP Alloys: Influence of Their Composition and Surface Topography.

The osseointegration of implants is defined as the direct anatomical and functional connection between neoformed living bone and the surface of a supporting implant. The biological compatibility of implants depends on various parameters, such as the nature of the material, chemical composition, surface topography, chemistry and loading, surface treatment, and physical and mechanical properties. In this context, the objective of this study is to evaluate the biocompatibility of rough (Ra = 1 µm) and smooth (Ra = 0 µm) surface conditions of yttria-zirconia (Y-TZP) discs compared to pure zirconia (ZrO2) discs by combining a classical toxicological test, morphological observations by SEM, and a transcriptomic analysis on an in vitro model of human Saos-2 bone cells. Similar cell proliferation rates were observed between ZrO2 and Y-TZP discs and control cells, regardless of the surface topography, at up to 96 h of exposure. Dense cell matting was similarly observed on the surfaces of both materials. Interestingly, only 110 transcripts were differentially expressed across the human transcriptome, consistent with the excellent biocompatibility of Y-TZP reported in the literature. These deregulated transcripts are mainly involved in two pathways, the first being related to "mineral uptake" and the second being the "immune response". These observations suggest that Y-TZP is an interesting candidate for application in implantology.

Elaboration of Sterically Stabilized Liposomes for S-Nitrosoglutathione Targeting to Macrophages.

S-nitrosoglutathione (GSNO) is a potential therapeutic for infectious disease treatment because of its pivotal role in macrophage-mediated inflammatory responses and host defense in addition to direct antibacterial activities. In this study, sterically stabilized cationic liposomes (SSCL) and sterically stabilized anionic liposomes (SSAL) were developed as nanocarriers for macrophage targeting. Elaborated liposomes were characterized in terms of size, zeta potential, morphology, encapsulation efficiency, in vitro drug release behavior and cytotoxicity. Their versatility in targeting monocytes/macrophages was determined by confocal laser scanning microscopy and transmission electron microscopy. Flow cytometry revealed that cellular uptake of both SSCL and SSAL was governed by several endocytic clathrin- and caveolae-dependent mechanisms. Quantitative assessments of intracellular nitric oxide demonstrated highly efficient uptake of GSNO-loaded SSCL that was twenty-fold higher than that of GSNO-free molecules. GSNO-loaded SSCL displayed strong bacteriostatic effects on Staphylococcus aureus and Pseudomonas aeruginosa, which can be involved in pulmonary infectious diseases. These results reveal the potential of liposomal GSNO as an anti-infective therapeutic due to its macrophage targeting capacity and direct antibacterial effects.

Drug delivery by polymeric nanoparticles induces autophagy in macrophages.

Drug delivery nanosystems are currently used in human therapy. In preliminary studies we have observed that Eudragit RS nanoparticles, prepared by nanoprecipitation or double emulsion techniques, are cytotoxic for NR8383 rat macrophages. In this study, we expand our previous analysis and suggest that unloaded Eudragit RS nanoparticles prepared by nanoprecipitation (NP/ERS) may induce important morphological and biochemical cellular modifications leading to cellular death. In NR8383 rat macrophages cell line exposed to doses varying from 15 to 100 µg/mL, NP/ERS nanoparticles are internalized inside the cells, reach the mitochondria and alter the structure of these organelles. In addition, the exposure to nanoparticles induces cellular autophagy as demonstrated by electron microscopy analysis, microchip array, qRT-PCR and Western blot assays. Although toxicity of nanoparticles has already been evidenced, it is the first time that results show clearly that the toxicity of polymeric nanovectors may be related to an activation of autophagy.

Cytotoxicity assessment of heparin nanoparticles in NR8383 macrophages.

The bioavailability of low molecular weight heparin (LMWH) has been increased by encapsulation in nanoparticles. As a complement to these results, the cytotoxicity and apoptosis induced by LMWH nanoparticles prepared by two methods [nanoprecipitation (NP) and double emulsion (DE)] using Eudragit RS (RS) and poly-epsilon-caprolactone (PCL) have been analysed. Particle sizes varied from 54 to 400nm with zeta potential values between -65 and +63mV. Our results showed that the method of nanoparticle preparation affects their properties, especially in terms of drug incorporation and cell tolerance. Cell viability ranged from 6% to 100% depending on the preparation method and physicochemical properties of the particles and the type of toxicity assay. Particle diameter and zeta potential seemed to be the most valuable cytotoxicity markers when cell viability was measured by Trypan blue exclusion and MTT respectively. Nanoparticles prepared by DE were better tolerated than those of NP. LMWH encapsulation into the cationic nanoparticles reduces remarkably their toxicity. Apoptosis evaluation showed activated caspases in exposed cells. However, no nuclear fragmentation was detected in NR8383 cells whatever the tested nanoparticles. DE nanoparticles of RS and PCL can be proposed as a good LMWH delivery system due to their low toxicity (IC(50) approximately 2.33 and 0.96mg/mL, respectively).

APOE genotyping: comparison of three methods.

Apolipoprotein E (apo E) polymorphism is associated with increased risk of cardiovascular and Alzheimer diseases, making its genotyping of potentially predictive value. We developed a rapid, reliable and specific method for determining APOE genotypes by fluorescent resonance energy transfer (FRET) over a high number of samples in a single run using a LightTyper device and dedicated probes. The method, validated with 75 blood samples, was designed to simultaneously detect three common APOE polymorphisms, epsilon(2,) epsilon(3) and epsilon(4), and to identify in a single reaction any of the six following genotypes: epsilon(2)/epsilon(2), epsilon(3)/epsilon(3), epsilon(4)/epsilon(4), epsilon(3)/epsilon(4), epsilon(4)/epsilon(2), epsilon(3)/epsilon(2). The assay involved three phases: (1) DNA extraction, (2) amplification, and (3) melting curve analysis using FRET technique. Briefly, genomic DNA of patients was extracted from total blood. Fragment of APOE was amplified by a first PCR run. Fluorescent labeled probes were added in a second PCR run. FRET genotyping showed following distribution: (1) 1.3% for epsilon(2)/epsilon(2) and epsilon(4)/epsilon(4) homozygotes, (2) 4.0, 6.6 and 14.7% for epsilon(2)/epsilon(4), epsilon(2)/epsilon(3) and epsilon(3)/epsilon(4) heterozygotes, respectively, and (3) 72.0% for epsilon(3)/epsilon(3) homozygotes. Moreover, a careful analysis of the FRET melting curves allowed us to determine the presence of a new polymorphism on the third position of the codon 158 (-AAGCGT-), namely, two nucleotides downstream from the known polymorphism. When the FRET analysis was compared to those obtained by RFLP and sequencing, the presence of this new polymorphism was confirmed only by sequencing thus indicating that RFLP analysis is not always reliable for genotyping.

Internalization, cytotoxicity, apoptosis, and tumor necrosis factor-alpha expression in rat alveolar macrophages exposed to various dusts occurring in the ceramics industry.

In 1997 The International Agency for Research on Cancer classified some exposures to crystalline silica as carcinogenic to humans. Such exposures were acknowledged to be very variable, and even in the same monograph it was admitted that coal dust, containing as much as 20% quartz, could not be classified. Clearly there is a need to develop methods for assessing any risks posed by various silica containing dusts in different workplaces. A European collective research project, SILICERAM, was launched with the aim of assessing the toxicity of various dusts in the ceramics industry and improving worker protection. This study examined the effect of particles, namely, DQ12 quartz, China clay, feldspar, and a sample resembling a typical mixture used in the ceramic industry (a "contrived sample" or CS), on NR8383, a rat alveolar macrophage (AM) cell line. Titanium dioxide and aluminum oxide were also used as negative controls. Confocal microscopy observations showed internalization of DQ12 and CS in NR8383. Cell viability decreased dramatically after a 2-h incubation exposure period with DQ12 (-71%). CS was less toxic than DQ12 at 2 h. China clay and feldspar were slightly cytotoxic to NR8383 cells. DQ12 induced apoptosis, with a smaller effect of CS and China clay. TNFalpha gene expression was analyzed by RT-PCR. DQ12, at a noncytotoxic dose of 10 microg/cm(2), induced a significant expression of TNFalpha (+2 times increase). In contrast, similar doses of CS and China clay did not produce a significant increase, while TiO2 and Al2O3 displayed no effect. Co-treatment with 10 microM aluminum lactate significantly reduced the effects of silica-containing particles on cytotoxicity, apoptosis, and TNFalpha expression.

Molecular characterization of African orthobunyaviruses.

The genus Orthobunyavirus is composed of segmented, negative-sense RNA viruses that are responsible for mild to severe human diseases. To date, no molecular studies of bunyaviruses in the genus Orthobunyavirus from central Africa have been reported, and their classification relies on serological testing. Four new primer pairs for RT-PCR amplification and sequencing of the complete genomic small (S) RNA segments of 10 orthobunyaviruses isolated from the Central African Republic and pertaining to five different serogroups have been designed and evaluated. Phylogenetic analysis showed that these 10 viruses belong to the Bunyamwera serogroup. The S segment sequences differ from those of the Bunyamwera virus reference strain by 5-15 % at the nucleotide level, and both overlapping reading frames, encoding the nucleocapsid (N) and non-structural (NS) proteins, were evident in sequenced genomes. This study should improve diagnosis and surveillance of African bunyaviruses.

Immune Signature of Malignant Pleural Mesothelioma as Assessed by Transcriptome Analysis.

Malignant pleural mesothelioma (MPM) is a highly malignant tumor arising in patients previously exposed to asbestos fibers. Its increasing incidence and its social, financial and human impact have become a frequent problem in many industrialized countries. The unresponsiveness of malignant mesothelioma to conventional therapies has led clinicians to develop new treatments. As immunotherapy has been shown to offer promising and targeted treatment of MPM patients, the knowledge of the immunoresistance level of MPM may be a valuable tool for "à la carte" therapy. In a previous work, we profiled the gene expression of two MPM tissues compared to healthy mesothelial cells using a 10K cDNA microarray. Subsequent clustering analysis identified several clusters of differentially-expressed genes among those that are functionally-related to the immune system. In this report, we focus on genes with expression changes that may facilitate tumor escape from immune-mediated rejection. We also analyzed the immune reaction by staining the immunocompetent cells surrounding the tumor. Interestingly, the tumor with the strongest escape response, as shown by the expression of numerous immunoresistance-associated genes, displayed the strongest T cell infiltrate. The main genes conferring immunoresistance are CD74, HLADOA, HLADMB, PTGS1, IGFBP7 and TGFB3, by favoring immune tolerance, and CFLAR, DFFA, TNFRSF6, BNIP3L by impairing apoptosis. These observations have fundamental consequences in the understanding of immunological properties of MPM, and offer a new insight into the mechanisms whereby MPM may circumvent host-mediated immune activities and promotes its own development. For an immunomodulation strategy to cure mesothelioma, it is crucial to characterize the MPM "immune signature" to design adapted immunotherapies.

Man-made mineral fiber hazardous properties assessment using transgenic rodents: example of glass fiber testing.

Transgenic BigBlue rats were exposed to CM 44 glass fibers (6.3 mg/m3) by nose only, 6 h/day for 5 days. Two endpoints were examined 1, 3, 14, 28, and 90 days following exposure: fiber biopersistence and mutations in lung DNA. The half-time of the fibers >20 microm was 12.8 days, and mutant frequencies of control and exposed rats were similar across all time points. The mutation spectra of both series were similar after 28 days of fixation time. These results showed that a glass fiber with a high clearance in the lung seems to not present any significant effect on mutagenesis on lung DNA and are in marked contrast to results for asbestos, which caused a twofold mutant frequency increase as described in a previous study.

Magnesium content in seminal fluid as an indicator of chronic prostatitis.

Magnesium and zinc are both involved in a high number of enzymic activities vital for mammals. They are found in prostate in remarkably high concentrations and released into seminal fluid. Furthermore, drastic reduction of Zn and Mg concentrations in the semen fluid may lead to disorders in male fertility. We aimed to analyse the differences in Mg and Zn levels in the seminal plasma of 213 males including 48 normozoospermic, 30 azoospermic, 28 oligoasthenozoospermic, 22 asthenozoospermic and 85 chronic prostatitis. Mg and Zn concentrations were measured using an atomic absorption spectrophotometer. While zinc levels did not show correlation either with the volume of the sperm or the percentage of pathological forms, magnesium concentrations in seminal plasma were significantly decreased in chronic prostatitis patients as compared to other groups or normozoospermic patients (p<0.001). We propose therefore magnesium as a marker of prostatitis.

The human type 2 iodothyronine deiodinase is a selenoprotein highly expressed in a mesothelioma cell line.

Types 1 and 3 iodothyronine deiodinases are known to be selenocysteine-containing enzymes. Although a putative human type 2 iodothyronine deiodinase (D2) gene (hDio2) encoding a similar selenoprotein has been identified, basal D2 activity is not selenium (Se)-dependent nor has D2 been labeled with (75)Se. A human mesothelioma cell line (MSTO-211H) has recently been shown to have approximately 40-fold higher levels of hDio2 mRNA than mesothelial cells. Mesothelioma cell lysates activate thyroxine (T(4)) to 3,5,3'-triiodothyronine with typical characteristics of D2 such as low K(m) (T(4)), 1.3 nm, resistance to propylthiouracil, and a short half-life ( approximately 30 min). D2 activity is approximately 30-fold higher in Se-supplemented than in Se-depleted medium. An antiserum prepared against a peptide deduced from the Dio2 mRNA sequence precipitates a (75)Se protein of the predicted 31-kDa size from (75)Se-labeled mesothelioma cells. Bromoadenosine 3'5' cyclic monophosphate increases D2 activity and (75)Se-p31 approximately 2.5-fold whereas substrate (T(4)) reduces both D2 activity and (75)Se-p31 approximately 2-3-fold. MG132 or lactacystin (10 microm), inhibitors of the proteasome pathway by which D2 is degraded, increase both D2 activity and (75)Se-p31 3-4-fold and prevent the loss of D2 activity during cycloheximide or substrate (T(4)) exposure. Immunocytochemical studies with affinity-purified anti-hD2 antibody show a Se-dependent increase in immunofluorescence. Thus, human D2 is encoded by hDio2 and is a member of the selenodeiodinase family accounting for its highly catalytic efficiency in T(4) activation.

Differential gene expression in mesothelioma.

To investigate the molecular events controlling malignant transformation of human pleural cells, we compared constitutive gene expression of mesothelioma cells to that of pleural cells. Using cDNA microarray and high-density filter array, we assessed expression levels of > 6500 genes. Most of the highly expressed transcripts were common to both cell lines and included genes associated with stress response and DNA repair, outcomes consistent with the radio- and chemo-resistance of mesothelioma. Interestingly, of the fewer than 300 genes that differed between cell lines, most functioned in (i) macromolecule stability, (ii) cell adhesion and recognition, (iii) cell migration (invasiveness), and (iv) extended cell division. Expression levels of several of these genes were confirmed by RT-PCR and could be useful as diagnostic markers of human mesothelioma.

Genotoxicity of 3-methylcholanthrene in liver of transgenic big Blue mice.

Transgenic mice provide a unique tool for studying the tissue specificity and mutagenic potential of chemicals. Because 3-methylcholanthrene (3MC) was found mutagenic in bacteria, clastogenic in bone marrow, and induces DNA adducts in animals, we were interested to determinine whether this xenobiotic provokes (1) cell proliferation, (2) transcriptional activity changes, (3) DNA adducts, and (4) hepatic mutations in transgenic Big Blue mice carrying the lambdaLIZ phage shuttle vector. Big Blue C57/Bl male mice were treated with a single intraperitoneal dose of 80 mg/kg 3MC for 1, 3, 6, 14, or 30 days. Cell proliferation was checked by 5-bromo-2-deoxyuridine labeling and immunohistochemical detection. The maximal increase of the mitotic index was evidenced after 3 days (2.9 times the control value; P < 0.01). The relative nucleus area, reflecting the transcriptional activity, was also the highest in the treated group after 3 days: 1.86 times the control value, on average (P < 0.01). Four major DNA adducts, determined according to the [(32)P]-postlabeling method, were evidenced in liver DNA of treated mice, 6 days after the treatment: the spot intensities increased in a time-dependent manner. The mutant frequency of liver DNA was the highest after 14 days: 20.3 +/- 2.9 x 10(-5) in the treated vs. 7.6 +/- 2.7 x 10(-5) in the control mice (P < 0.01). Sequencing of the lambda lacI mutant plaques showed mainly G:C --> T:A and C:G --> A:T transversions. In conclusion, 3MC at first induced nuclear enlargement and a slight increase of cell proliferation in liver, followed by parallel formation of DNA adducts and mutations. This study shows how transgenic models allow in vivo evaluation of mechanistically simultaneous endpoints.

Alpha-lipoic acid in liver metabolism and disease.

R-alpha-Lipoic acid is found naturally occurring as a prosthetic group in alpha-keto acid dehydrogenase complexes of the mitochondria, and as such plays a fundamental role in metabolism. Although this has been known for decades, only recently has free supplemented alpha-lipoic acid been found to affect cellular metabolic processes in vitro, as it has the ability to alter the redox status of cells and interact with thiols and other antioxidants. Therefore, it appears that this compound has important therapeutic potential in conditions where oxidative stress is involved. Early case studies with alpha-lipoic acid were performed with little knowledge of the action of alpha-lipoic acid at a cellular level, but with the rationale that because the naturally occurring protein bound form of alpha-lipoic acid has a pivotal role in metabolism, that supplementation may have some beneficial effect. Such studies sought to evaluate the effect of supplemented alpha-lipoic acid, using low doses, on lipid or carbohydrate metabolism, but little or no effect was observed. A common response in these trials was an increase in glucose uptake, but increased plasma levels of pyruvate and lactate were also observed, suggesting that an inhibitory effect on the pyruvate dehydrogenase complex was occurring. During the same period, alpha-lipoic acid was also used as a therapeutic agent in a number of conditions relating to liver disease, including alcohol-induced damage, mushroom poisoning, metal intoxification, and CCl4 poisoning. Alpha-Lipoic acid supplementation was successful in the treatment for these conditions in many cases. Experimental studies and clinical trials in the last 5 years using high doses of alpha-lipoic acid (600 mg in humans) have provided new and consistent evidence for the therapeutic role of antioxidant alpha-lipoic acid in the treatment of insulin resistance and diabetic polyneuropathy. This new insight should encourage clinicians to use alpha-lipoic acid in diseases affecting liver in which oxidative stress is involved.