Variances in the Expression Profile of DUSP1-7 and miRNAs Regulating their Expression in the HaCat Line under LPS and Cyclosporine A.

INTRODUCTION: Cyclosporin A (CsA) treats moderate to severe psoriasis vulgaris. Psoriasis is a chronic inflammatory disease in which hyperproliferation of keratinocytes occurs. One of the most relevant signaling cascades in the development of psoriasis is the mitogen-activated protein kinase (MAPK) signaling pathway. It has been observed that dual-specificity phosphatases (DUSPs) dephosphorylate signaling molecules, such as MAPKs. AIMS: This study aims to determine changes in the expression pattern of Dual Activity Protein Phosphatase (DUSP1-7) and micro RNAs (miRNAs), potentially regulating their expression in the human adult, low-calcium, high-temperature keratinocytes cell line (HaCaT) cultures exposed to lipopolysaccharide A (LPS)-induced inflammation, followed by CsA. METHODS: HaCaT cell line was exposed for 8 hours to 1 µg/mL LPS and then to 100 ng/mL CsA for 2, 8, and 24 hours compared to cultures not exposed to LPS and the drug. The molecular analysis included determining the DUSP1-7 expression and the miRNAs potentially regulating it using an expression microarray technique. An enzyme-linked immunosorbent assay (ELISA) was also performed to assess the concentration of DUSP1-7 in the culture medium. Statistical evaluation was performed assuming a statistical significance threshold (p) of < 0.05. RESULTS: Statistically significant differences were found in the expression of DUSP1-7 mRNAs and the miRNAs that regulate their expression. The most significant changes in expression were observed for DUSP1 and DUSP5, with the differences being most pronounced during the eighthour incubation period of the cells, with the drug predictive analysis showing that miR-34 potentially regulates the expression of DUSP1-4,7, miR-1275: DUSP2, mir-3188: DUSP4, miR-382: DUSP4, miR-27a and miR-27b: DUSP5,6 and miR-16: DUSP7. No expression of DUSP1-7 was demonstrated at the protein level in CsA-exposed cultures. CONCLUSION: Our evaluation of the efficacy of CsA therapy on an in vitro model of HaCaT indicates that treatment with this drug is effective, resulting in changes in the expression of DUSP1-7 and, potentially, the miRNAs that regulate their expression. We also confirmed that the different expression pattern of mRNA and protein encoded by a given transcript is not only due to the regulatory role of miRNAs but also the lack of synchronization between transcription and translation processes.

Detection and Genotyping of Human Papillomavirus (HPV16/18), Epstein-Barr Virus (EBV), and Human Cytomegalovirus (HCMV) in Endometrial Endometroid and Ovarian Cancers.

The purpose of this study was to evaluate the relationship between human papillomavirus (HPV16/18), Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV) infections and the occurrence of ovarian cancer in 48 women, of whom 36 underwent surgery and chemotherapy (group A), 12 in whom surgery was sufficient (group B), and 60 with endometroid endometrial cancer stage G1-G3 (group C), compared to patients in whom the uterus and its appendages were removed for nononcological reasons (control group). The detection of HPV, EBV, and HCMV in tumor tissue and normal tissue was performed using the real-time polymerase chain reaction (RT-PCR) technique. A statistically significantly higher risk of endometrial cancer was noted in patients infected only with HCMV (OR > 1; p < 0.05). In contrast, a significantly higher risk of ovarian cancer in group A was associated with HPV16, HPV18, and EBV (OR > 1; p < 0.05); a significantly higher risk of ovarian cancer in group B was associated with HPV18 and HMCV (OR > 1; p < 0.05). The obtained results suggest that HCMV infection is associated with the development of a stage of ovarian cancer when treatment can be completed with surgery alone. Meanwhile, EBV appears to be responsible for the development of ovarian cancer in more advanced stages.

Effects of Cyclosporine A and Adalimumab on the expression profiles histaminergic system-associated genes and microRNAs regulating these genes in HaCaT cells.

Previous studies have not completely elucidated the role of the histaminergic system in the pathogenesis of psoriasis. This study aimed to evaluate the effects of adalimumab and cyclosporine A on the expression of histaminergic system-related genes and miRNAs regulating these genes in bacterial lipopolysaccharide A (LPS)-stimulated human keratinocyte (HaCaT) cells. HaCaT cells were treated with 1 µg/mL LPS for 8 h, followed by treatment with 8 µg/mL adalimumab or 100 ng/mL cyclosporine A for 2, 8, or 24 h. Untreated cells served as controls. The cells were subjected to ribonucleic acid (RNA) extraction and microarray, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay analyses. Statistical analysis was performed using the Statistica 13.0 PL (StatSoft, Cracow, Poland) and the Transcriptome Analysis Console programs (Affymetrix, Santa Clara, CA, USA) (p < 0.05). The differential expression of the following two miRNAs was not affected in LPS-stimulated cells upon treatment with cyclosporine A or adalimumab: hsa-miR-583 (downregulated expression), involved in the regulation of histamine receptor 1 - HRH1 (overexpression); has-miR-1275 (downregulated expression), involved in the regulation of histamine receptor 1 - HRH3 (overexpression) and Solute carrier family 22 member 3 - SLC23A2 (downregulated expression)). Adalimumab and cyclosporine A modulated the histaminergic system in HaCaT cells in vitro. However, further studies are needed to elucidate the underlying mechanisms.Abbreviations: (-) - downregulated in comparison to the control, (+) - overexpression in comparison to the control, ACTB - ß-actine, ADA - Adenosine deaminase, ADCYAP1 - Adenylate Cyclase Activating Polypeptide 1, BMP - bone morphogenetic protein, bp - base pair, cAMP - adenosine 3' 5'-cyclic monophosphate, CBX7 - Chromobox protein homolog 7, cDNA - double-stranded complementary DNA, CSA - cyclosporine A DAG - diacylglycerol, DIAPH - Diaphanous related formin 1, DNMT - DNA methyltransferases, DRD2 - Dopamine receptor D2, EDN1 - Endothelin 1, EDNRA - Endothelin receptor type A, ELISA - Enzyme-linked immunosorbent assay, EZH2 - Enhancer of zeste homolog 2, FC - fold change, GABRB1 - Gamma-aminobutyric acid (GABA) A receptor, alpha 1, GABRB2 - Gamma-aminobutyric acid (GABA) A receptor, alpha 2, GABRB3 - Gamma-aminobutyric acid (GABA) A receptor, alpha 3, HaCaT - Human adult, low-calcium, high-temperature keratinocytes, HIS - Human Histamine, HLAs - human leukocyte antigens, HNMT - Histamine N-methyltransferase, HNMT - Histamine N-Methyltransferase, HRH1 - histamine receptor 1, HRH2 - histamine receptor 2, HRH3 - histamine receptor 3, HRH4 - histamine receptor 4, HTR6 - 5-Hydroxytryptamine Receptor 6, IGF1 - Insulin-like growth factor 1, IL10 -interleukin 10, IL12 -interleukin 12, IL6 - interleukin 6, IP3 - inositol 1,4,5-triphosphate, LPS - bacterial lipopolysaccharide A, LYN - LYN Proto-Oncogene, Src Family Tyrosine Kinase, MAPKs -mitogen-activated protein kinases, miRNA - micro RNA, MMP2 - matrix metalloproteinase-2, NHDF - Normal Human Dermal Fibroblasts, NHEK - Normal Human Epidermal Keratinocytes, OCT3 - organic cation transporter 3, PANTHER - Protein ANalysis THrough Evolutionary Relationships Classification, PBS - phosphate-buffered saline, PI3K-AKT - phosphatidylinositol 3-kinase-protein kinase B, PIP2 - phosphatidylinositol 4,5 bisphosphate, PMSF - phenylmethylsulfonyl fluoride, PSORS1- psoriasis susceptibility gene 1, qRT-PCR - quantitative Reverse Transcription Polymerase Chain Reaction, RNA - ribonucleic acid, RNAi - RNA interference, RTqPCR - Real-Time Quantitative Reverse Transcription Reaction, SLC223A2 - Solute carrier family 22 member 3, SNX -Sorting nexin, SOX9 - SRY-Box Transcription Factor 9, TGF-α - transforming growth factor α, TGF-ß - transforming growth factor beta, TNF-α - tumor necrosis factor alpha, TP53 - tumor protein 5 z, VAMP2 - Vesicle associated membrane protein 2.

Changes in the Expression Pattern of DUSP1-7 and miRNA Regulating their Expression in the Keratinocytes Treated with LPS and Adalimumab.

BACKGROUND: Increased levels of phosphorylated ERK and p38 MAPK proteins have been observed in psoriatic skin biopsies compared to controls, which may be associated with an impaired expression pattern of dual activity protein phosphatase (DUSP). OBJECTIVE: The purpose of this study was to assessment changes in expression profile of mRNA DUSP 1-7 and miRNA regulating their expression in human keratinocyte cells (HaCaT) had exposed to the liposaccharide A (LPS). METHODS: HaCaT was exposed to 1 µg/ml LPS and next adalimumab by 2,8,24h compared to untreated cells. The microarray method was used to analyze expression pattern of mRNAs, miRNAs, and ELISA to evaluate changes in the level of the proteins. RTqPCR was used to validate the microarray data. Transcriptome Analysis Console and Statistica Software 13 PL were used in statistical analysis (p<0.05). RESULTS: The highest changes in expression was observed for DUSP2 (FC +11.12) and DUSP5 (FC +5.53) in HaCaT culture after 2 hours exposition on adalimumab. It was observed that miR- 1275 (FC -2.39) and miR-34a (FC +6.52) might regulate level of DUSP2, and miR-27a (FC +3.55), miR-27b (FC +2.87) are involved in DUSP5 expression. CONCLUSION: The results obtained suggest that DUSP2 and DUSP5 may be considered as complementary molecular markers in the diagnosis and monitoring of the effectiveness of psoriasis therapy. It was confirmed that hsa-miR-34a, hsa-miR-1275, hsa-miR-3188, hsa-miR-382, hsa-miR- 27a, hsa-miR-27b, hsa-miR-16 have the highest influence on the expression pattern of DUSP1-7.

Evaluation of the Influence of Adalimumab on the Expression Profile of Leptin-Related Genes and Proteins in Keratinocytes Treated with Lipopolysaccharide A.

Psoriasis is a disease with a proinflammatory base, in which an increased expression of leptin, tumor necrosis factor alpha (TNF-α), interleukin (IL) IL-12/23, IL-6, is observed. A drug used in the treatment of psoriasis of moderate and acute strength is the monoclonal antibody anti-TNF-adalimumab. The goal of this study was to evaluate the influence of adalimumab on changes in the expression profile of leptin-related genes in human keratinocyte cells exposed to lipopolysaccharide A and analyze if adalimumab acts via leptin pathways. The evaluation of changes of the pattern of genes connected with leptin and proteins coded by them was marked in a culture of human keratinocytes (HaCaT) exposed to 1 µg/mL lipopolysaccharide A (LPS) for 8 h in order to induce the inflammatory process, then to 8 µg/mL of adalimumab for 2.8 and 24 h in comparison with the control (cells not treated with the substances). The techniques used were mRNA microarray, Real-Time Quantitative Reverse Transcription Reaction (RTqPCR), Enzyme-Linked Immunosorbent Assay (ELISA), as well as transfections of HaCaT culture with leptin small interfering RNA (siRNA) in order to see whether adalimumab works through pathways dependent on leptin. A statistically lower expression of leptin and its receptors was observed under the influence of the drug, independent of the exposition time of keratinocytes to adalimumab. In the cells transfected with leptin siRNA, a lower concentration of JAK2 and STAT3 proteins was observed, which confirms that adalimumab works through pathways dependent on leptin. Adalimumab has a modulatory effect on the gene expression pattern and the proteins coded by them connected with leptin in keratinocytes treated with LPS in vitro.

Geochemical modelling for predicting the long-term performance of zeolite-PRB to treat lead contaminated groundwater.

The feasibility of using geochemical modelling to predict the performance of a zeolite-permeable reactive barrier (PRB) for treating lead (Pb(2+)) contaminated water was investigated in this study. A short-term laboratory column experiment was first performed with the zeolite (clinoptilolite) until the elution of 50 PV (1 PV=ca. 283 mL). Geochemical simulations of the one-dimensional transport of the Pb(2+), considering removal processes including: ion-exchange, adsorption and complexation; the concomitant release of exchangeable cations (Ca(2+), Mg(2+), Na(+), and K(+)) and the changes in pH were subsequently performed using the geochemical model PHREEQC. The results showed a reasonable agreement between the experimental results and the numerical simulations, with the exception of Ca(2+) for which a great discrepancy was observed. The model also indicated the formation of secondary mineral precipitates such as goethite and hematite throughout the experiment, of which the effect on the hydraulic conductivity was found to be negligible. The results were further used to extrapolate the long-term performance of the zeolite. We found the capacity would be completely exhausted at PV=250 (ca. 3 days). The study, thus, generally demonstrates the applicability of PHREEQC to predict the short and long-term performance of zeolite-PRBs. Therefore, it can be used to assist in the design and for management purposes of such barriers.

Combining steam injection with hydraulic fracturing for the in situ remediation of the unsaturated zone of a fractured soil polluted by jet fuel.

A steam injection pilot-scale experiment was performed on the unsaturated zone of a strongly heterogeneous fractured soil contaminated by jet fuel. Before the treatment, the soil was stimulated by creating sub-horizontal sand-filled hydraulic fractures at three depths. The steam was injected through one hydraulic fracture and gas/water/non-aqueous phase liquid (NAPL) was extracted from the remaining fractures by applying a vacuum to extraction wells. The injection strategy was designed to maximize the heat delivery over the entire cell (10 m × 10 m × 5 m). The soil temperature profile, the recovered NAPL, the extracted water, and the concentrations of volatile organic compounds (VOCs) in the gas phase were monitored during the field test. GC-MS chemical analyses of pre- and post-treatment soil samples allowed for the quantitative assessment of the remediation efficiency. The growth of the heat front followed the configuration of hydraulic fractures. The average concentration of total hydrocarbons (g/kg of soil) was reduced by ∼ 43% in the upper target zone (depth = 1.5-3.9 m) and by ∼ 72% over the entire zone (depth = 1.5-5.5 m). The total NAPL mass removal based on gas and liquid stream measurements and the free-NAPL product were almost 30% and 2%, respectively, of those estimated from chemical analyses of pre- and post-treatment soil samples. The dominant mechanisms of soil remediation was the vaporization of jet fuel compounds at temperatures lower than their normal boiling points (steam distillation) enhanced by the ventilation of porous matrix due to the forced convective flow of air. In addition, the significant reduction of the NAPL mass in the less-heated deeper zone may be attributed to the counter-current imbibition of condensed water from natural fractures into the porous matrix and the gravity drainage associated with seasonal fluctuations of the water table.