Barbara De Servi

Occludin gene expression as an early in vitro sign for mild eye irritation assessment

PURPOSE To test a new multiple endpoint analysis (MEA) including occludin gene expression for screening the ocular irritation potential of tear substitutes on human corneal epithelium (HCE), an in vitro model proposed to limit the use of animal testing in pre-clinical studies. METHODS Four chemically-preserved and two non chemically-preserved tear substitutes were tested after acute (24h, 24h+24h post incubation) and repeated applications (for 72h) and compared to the positive control, benzalkonium chloride (BAK) at 0.1% and 0.01%, by assessing complementary parameters. Cellular viability was evaluated using MTT, histomorphologic analysis was performed on H&E stained vertical sections, IL-8 release was measured by ELISA, and occludin gene expression was quantified using qRT-PCR. RESULTS Cellular viability was moderately reduced by Perborate and Polyquad-preserved tear substitutes and dramatically reduced by BAK and by Thiomersal and Oxyd preserved tear substitutes. Thiomersal also increased IL-8 release. Occludin expression profiles were modified by the four chemically-preserved tear substitutes and by the mechanically-preserved Comod, but not by the mechanically-preserved Abak. The behavior of BAK and tear substitutes led us to propose a prediction model for the classification of different levels of irritants, mainly based on the occludin transcriptional study. CONCLUSION The versatility and sensitivity of the HCE model allowed the modeling of cumulative effects that may approach conditions obtained after long term application of tear substitutes. Thus, the modified MEA proposed in this study represents a valuable tool for in vitro eye irritation assessment with the power to detect mild irritants and subclinical eye irritant potential.

Corneal epithelial toxicity of antiglaucoma formulations: in vitro study of repeated applications.

Background By using a biologically relevant and sensitive three-dimensional model of human corneal epithelium and multiple endpoint analysis, assessment of the potential for eye irritation and long-term compatibility of four registered ophthalmological preparations, ie, Timolabak®, Timoptol®, Nyogel®, and Timogel®, was performed. This approach enables classification of the potential for irritation, discriminating between mildly irritant and non-irritant ocular substances. Methods The exposure protocol included two time periods, ie, 24 hours (acute application) and 72 hours (repeated applications twice daily). This approach allows assessment of not only the acute reaction but also possible recovery, as well as mimicking the potential cumulative effects associated with long-term application. Using benzalkonium chloride (BAK) 0.01% as a positive control, the following parameters were quantified: cellular viability by MTT test, histological analysis by hematoxylin and eosin staining, passive release of interleukin-1α by enzyme-linked immunosorbent assay, and OCLN gene expression by quantitative real-time polymerase chain reaction. Results Cell viability was reduced to under the 50% cutoff value after acute exposure (24 hours) to BAK 0.01%, and after repeated application (72 hours) of Timoptol and Nyogel. Histological analysis after acute exposure showed signs of superficial damage with all formulations, and severe changes after repeated applications of Timoptol, BAK 0.01%, and Nyogel. Timolabak and Timogel did not significantly alter the morphology of the human corneal epithelial cells after the different exposure times. Interleukin-1α release was greater than that for the negative control (>20 pg/mL) and the positive control (BAK 0.01%), Nyogel, and Timoptol treatments and not different after treatment with Timolabak and Timogel. Expression of OCLN, a sign of epithelial barrier impairment, was only significantly upregulated at 24 hours by BAK 0.01%, suggesting a toxic reaction at the ocular surface. OCLN was also overexpressed after repeated application of Nyogel and Timogel. Conclusion Overall, the multiple endpoint analysis approach allows classification of these products according to decreasing order of irritation potential as follows: BAK 0.01%, Timoptol, Nyogel, Timogel, and Timolabak.

Safety and efficacy evaluation of an isotonic manganese-enriched seawater solution on human nasal epithelium reconstituted in vitro

Inflammatory upper airway diseases, particularly chronic rhinosinusitis (CRS) and allergic rhinitis (AR), have a high worldwide prevalence. CRS and AR involve sustained and exaggerated inflammation that is associated with marked changes in gene and protein expression under tight regulation. miRNAs represent one of the fundamental epigenetic regulatory mechanisms used by cells that can mediate posttranscriptional gene silencing of target genes. As fine-tuning regulators of gene expression, miRNAs are involved in diverse biological processes, including cell proliferation, apoptosis, and differentiation, organ development, metabolism, stress responses, and signal transduction. Emerging evidence implicates an involvement of miRNAs in shaping the inflammation pattern in upper airways. Studies regarding the roles of miRNAs in allergic diseases have multiplied during the last 4 years, and the functions of miRNAs in the regulation and pathogenesis of these diseases are better and better characterized. Recently, miRNAs have been shown to be detectable in cell-free body fluids such as serum and plasma samples. The circulating miRNAs are protected from blood RNAs either by existing in cell membrane-derived vesicles such as exosomes or by forming a complex with lipid-protein carriers such as high-density lipoprotein. So it becomes possible to use such kind of molecules for a therapeutic purpose, and this is achieved by the Bio Immun(G)en Medicine – BI(G)MED – by introducing high diluted microRNAs in nanocompounds looking for a fine regulation in different upper airways diseases with an allergic aetiology.

Evidences of a cytotoxic activity of S-adenosylmethionine on OCI-AML3 cells

Background The acute myeloid leukemia (AML) cell line OCI-AML3, carrying both NPM1 mutation A and the heterozygous DNMT3A R882C mutation, represents the model for in vitro studies on AML with mutated NPM11. AML with mutated NPM1 harbours a hypo-methylated profile distinct from those of the other AML subtypes2. This characteristic is probably related to the inhibitory effect of the mutant DNMT3A on the wild type protein3. S-adenosylmethionine (SAM) is a universal methyl donor acting as a coenzyme of DNMT3A. There are growing evidences of the antineoplastic effect of SAM in vitro and in murine models of gastric cancer, colon cancer and hepatocellular carcinoma, where SAM induces the downregulation of several oncogenes4-10. Moreover SAM upregulates the expression of DNMT enzymes in lung cancer cells11. In our knowledge there are no published data exploring the effect of SAM on the growth of OCI-AML3 cells and its ability to modulate DNMT3A activity in this cell line. Study design and methods The present data have been generated between August 2013 and April 2014 at the VITROSCREEN facilities in Milan–ITALY. We used a 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyl tetrazolium bromide (MTT) assay to assess the cytotoxic effect of SAM iodide (Sigma-Aldrich) on OCI-AML3 cells (DSMZ Leibniz Institut). We analyzed then the ability of SAM to induce apoptosis by Tali Image-Based Cytometer (green Annexin V – Alexa Fluor 488 for apoptotic cells, red propidium and green Annexin V-Alexa Fluor 488 for necrotic cells). Results The MTT assays were performed after having treated the OCI-AML3 cells with various concentrations of the indicated drug for 24 hours. We observed no significant effects on cells viability using 0.5μM, 10 μM and 100 μM of SAM (data not shown). In contrast, a dose dependent cytotoxic effect of SAM on OCI-AML3 cells was evident for concentrations equal or superior to 500 μM, with an IC50 of 500 μM (Figure 1). Since a Cmax of 211(SD 94)μM after single intravenous infusion of SAM was previously reported in healthy voluntarees12, we decided to investigate the cytotoxic effect of SAM for concentrations close to 211 μM using the MTT test. A significant dose dependent reduction of the cells viability was observed with SAM 200μM (62,74% viable cells) and SAM 300μM (53.32% viable cells), (Figure 2). The Apoptosis assay after 24 hours of treatment with SAM showed no differences in the percentages of apoptotic cells between the OCI-AML3 cells treated with SAM 300-500-2500 μM and the untreated cells (data not shown). After 72 hours, only a minimal effect on the amount of apoptotic cells was obtained, while a clear dose dependent increase in the proportion of dead cells was noted (Figure 3), confirming the results of the aforementioned MTT tests. Conclusions SAM showed remarkable in vitro cytotoxic activity on OCI-AML3 cells at concentrations similar to those achievable in humans after intravenous administration. SAM is not able to induce apoptosis of OCI-AML3 cells in vitro after 72 hours of treatment. However, the increase in the amount of dead cells after SAM treatment may be due to mechanisms other than apoptosis. In order to verify if the observed cytotoxicity was mediated by the enzymatic activity of DNMT3A, we planned to repeat the cytotoxicity assays after DNMT3A silencing. The in vivo antineoplastic effect of SAM could be assessed in NOD/SCID mice engrafted with OCI-AML3 cells. Authors contribution KAvH wrote the study rationale, designed the study, interpreted the data and wrote the article; IP revised the article, MM and BDS planned and interpreted the experiments and BDS performed the experiments.