Histaminergic System and Inflammation-Related Genes in Normal Large Intestine and Adenocarcinoma Tissues: Transcriptional Profiles and Relations.

Transcriptional analyses such as microarray data have contributed to the progress in the diagnostics and therapy of colorectal cancer (CRC). The need for such research is still present because of the disease being common in both men and women with a high second position in cancer rankings. Little is known about the relations between the histaminergic system and inflammation in the large intestine and CRC. Therefore, the aim of this study was to evaluate the expression of genes related to the histaminergic system and inflammation in the CRC tissues at three cancer development designs: all tested CRC samples, low (LCS) and high (HCS) clinical stage, and four clinical stages (CSI-CSIV), to the control. The research was carried out at the transcriptomic level, analysing hundreds of mRNAs from microarrays, as well as carrying out RT-PCR analysis of histaminergic receptors. The following histaminergic mRNAs: GNA15, MAOA, WASF2A, and inflammation-related: AEBP1, CXCL1, CXCL2, CXCL3, CXCL8, SPHK1, TNFAIP6, were distinguished. Among all analysed transcripts, AEBP1 can be considered the most promising diagnostic marker in the early stage of CRC. The results showed 59 correlations between differentiating genes of the histaminergic system and inflammation in the control, control and CRC, and CRC. The tests confirmed the presence of all histamine receptor transcripts in both the control and colorectal adenocarcinoma. Significant differences in expression were stated for HRH2 and HRH3 in the advanced stages of CRC adenocarcinoma. The relations between the histaminergic system and inflammation-linked genes in both the control and the CRC have been observed.

Expression pattern of circadian rhythm-related genes and its potential relationship with miRNAs activity in endometrial cancer.

OBJECTIVES: The circadian clock is an autonomous oscillator that controls key aspects of cell physiology, including metabolism, transcriptional state, and cell signaling. Disturbances of circadian rhythms lead to disruption of cell and tissue homeostasis, which promotes carcinogenesis. The aim of the study was to determine the expression of circadian rhythm-related genes in endometrial cancer and to select miRNAs involved in the regulation of their expression. MATERIAL AND METHODS: 50 endometrial tissue samples were collected from patients who underwent hysterectomy: 40 diagnosed with endometrial cancer and 10 without cancer. Expression profile of circadian rhythm-related genes was evaluated using microarrays and validated with RT-qPCR. MicroRNA expression was assessed using microarrays. Then mirTAR tool was used to identify miRNAs involved in the expression regulation of circadian rhythm-related genes. RESULTS: CLOCK expression is disrupted in endometrial cancer, which may be due to miR-15b, miR-331-3p and miR-200a overexpression. Elevated NPAS2 and CSNK1D levels may be associated with miR-432 silencing. In addition, high miR-874 and miR-200a expression may be potentially responsible for the reduction of PER3 level. CONCLUSIONS: Change of CLOCK, CSNK1D, NPAS2 and PER3 expression may suggest that circadian rhythms are disrupted in endometrial cancer. A possible mechanism of the observed changes may be related to miRNAs activity.

Placental expression of Fatty Acid Desaturases 1, 2 and 3 in selected pregnancy pathologies.

Intrauterine development is a key period in human life. The foetal progress largely depends on the function of the placenta, whose responsibility is transportation and biosynthesis of fatty acids. Desaturation enzymes play a key role in placental fatty acid metabolism. Expression of genes coding for desaturases may be associated with pregnancy abnormalities. The objective of this study was to determine the transcriptional activity of the placental genes Fatty Acid Desaturases 1, 2 and 3 (FADS 1, 2 and 3) in women who gave birth to the infants appropriate for gestational age, large for gestational age, small for gestational age, with intrauterine growth restriction and born preterm. 34 pregnant women aged 21-37 years old participated in the study. The placental samples were taken from a site located 2-3 cm away from the umbilical cord attachment. The collected tissue sections were stored in RNAlater according to the manufacturer's protocol, until required for molecular analysis. The expression profiles of FADS1, FADS2 and FADS3 were determined with RT-qPCR. There was no difference in FADS1 and FADS2 expression between the groups. However, the differences in the expression of the FADS3 were found. Analysis of the FADS1, FADS2 and FADS3 transcription showed significant differences between most of the examined groups. Our findings suggest that the transcriptional activity of FADS genes changes with the severity of intrauterine disorders and is associated with foetal lipid disorders linked to a greater accumulation of fat in the foetal tissues.

The transcriptional activity profile of inhibitor apoptosis protein encoding genes in colon cancer patients: A STROBE-compliant study.

ABSTRACT: The inhibitor of apoptosis family proteins (IAPs) plays a crucial role in the process of carcinogenesis by regulating apoptosis and maintaining the tissue balance.In this study, a transcriptomic analysis of IAP-encoding genes in colon cancer was performed using oligonucleotide microarrays.Adenocarcinoma and healthy colon tissue samples were collected from 32 patients (16 females and 16 males) who underwent surgery due to colon cancer. The mRNA was extracted from tissue samples and tested using oligonucleotide microarrays (Affymetrix). The results were validated using the qRT-PCR technique. Hierarchical grouping was used to allocate 37 samples of normalized mRNA concentrations into 4 groups, with statistically significant differences in gene expression between these groups. The group of genes associated with colon cancer, including IAP-encoding gene - BIRC5 (Survivin), was selected for further testing.Our study confirmed an increased expression of BIRC5 in colon cancer tissue when compared to the control group. Increased levels of Neuronal Apoptosis Inhibitory Proteins were detected only in low-stage colon cancer, while the expression of Human X Chromosome-Encoded inhibitor of apoptosis family proteins decreased in colon cancer.The transcriptional activity of IAP-encoding genes varied, depending on the severity of colon cancer. The concentration of mRNA, encoding BIRC5 was elevated in samples obtained from more advanced colon cancer. Hence BIRC5 could be used as a complementary parameter for the diagnosis and prognosis of colon cancer.

Expression Profile of EMT-related Genes and miRNAs Involved in Signal Transduction via the Wnt Pathway and Cadherins in Endometrial Cancer.

BACKGROUND: Epithelial-Mesenchymal Transition (EMT) is a molecular reprogramming that leads to an increased ability to migrate, which can promote invasion and metastasis. EMT can be initiated in response to the activity of signaling pathways such as Wnt as well as miRNAs. OBJECTIVE: The aim of the study was to determine the expression profile of EMT-related genes involved in signal transduction via the Wnt pathway and cadherins and to assess which miRNAs can participate in the regulation of their expression. METHODS: The study material consisted of 50 endometrial samples: 40 with diagnosed endometrial cancer and 10 without neoplastic changes. The expression profile of EMT-related genes was assessed with microarrays and validated by RT-qPCR. MicroRNA expression profiling was performed using microarrays. It was also determined which miRNAs may participate in the expression regulation of EMT-related genes. RESULTS: CDH1 overexpression was observed in all three endometrial cancer grades using both mRNA microarrays and RT-qPCR. The microarray experiment showed a decrease in CDH2 level regardless of the endometrial cancer grade, however, it was only partially validated with RT-qPCR. Low levels of WNT2, WNT4, WNT5A have also been observed. Decreased expression of WNT2 and WNT5A may be caused by miR-331-3p and miR-200b-5p, respectively. CONCLUSION: The Wnt signaling is disrupted in endometrial cancer, which may be due to miR-331- 3p and miR-200b-5p activity. In addition, a change in WNT5A level in endometrial cancer compared to control may indicate that it acts as a suppressor gene and that its low expression is associated with tumor progression.

Osteogenesis of adipose-derived stem cells from patients with glucose metabolism disorders.

BACKGROUND: Adipose derived stem cells (ADSCs) are clinically widely used somatic stem cells obtained from white adipose tissue. They are characterized by ability to differentiate e.g. into osteoblasts and might successfully regenerate bone tissue in fracture repair. However, the main problem of somatic stem cells is a documented influence of various diseases, drugs or age which can inhibit cells activity. Therefore, in the present study, we investigated the influence of insulin resistance (IR) and type 2 diabetes (T2D) on the proliferation and differentiation potential of ADSCs. METHODS: The fat from subcutaneous abdominal adipose tissue was acquired by lipoaspiration from 23 voluntary participants, divided into three groups: with diabetes type 2, with insulin resistance and control healthy donors. The proliferative potential was analyzed by cell cytotoxicity assays and by mRNA expression of genes connected with proliferation. Flow cytometry was done for identifying proteins characteristic for mesenchymal stem cells and an analysis of osteogenic differentiation potential based on the assessment of osteogenic markers by real time RT-qPCR, and the evaluation of calcium deposition were also performed. RESULTS: The results showed that diabetes type 2 lowered the activity of ADSCs in proliferation assays and changed their phenotypical characteristics. Interestingly, we observed differences in the proliferation potential of ADSCs in patients with insulin resistance, which is often the first phase of diabetes, compared to the control. It might suggest that insulin resistance, early-stage T2D, alters the activity of cells. Moreover, expression of osteogenesis markers was higher in cells from T2D patients than in cells from patients with IR and control. CONCLUSION: We conclude that type 2 diabetes changes the activity of stem cells, and insulin resistance influences on the proliferation of ADSCs.

Analysis of the clinical response and changes in the expression of TNF-α and its TNFR1 and TNFR2 receptors in patients with psoriasis vulgaris treated with ustekinumab.

BACKGROUND: Ustekinumab is a monoclonal antibody that shows the ability to bind to subunit p40, common for interleukin 12 (IL-12) and IL-23, which prevents the activation of the JAK STAT signaling pathway. OBJECTIVES: The objective of the study was to evaluate the efficacy of therapy that uses anti-IL-12/23 medicine in patients with psoriasis vulgaris, based on the disease clinical progression indices (Psoriasis Area and Severity Index (PASI), Dermatology Life Quality Index (DLQI) and Body Surface Area (BSA)) and to determine the possibilities of using changes in the expression profiles of tumor necrosis factor α (TNF-α), tumor necrosis factor receptor (TNFR1) and TNFR2 as molecular markers showing the response to ustekinumab therapy. MATERIAL AND METHODS: The group under study was composed of 14 patients (10 men and 4 women, aged 49.3 ±10.2 years) with diagnosed psoriasis vulgaris, treated with ustekinumab. The group was divided into subgroups because of the selected 3 stages of therapy. The control group consisted of 20 healthy volunteers (11 men and 9 women, aged 46 ±10 years). The 120-week long observation involved a clinical assessment of the patients (PASI, BSA and DLQI), based on the following scheme: 0-4-12 weeks of the observation. The analysis of molecular changes in the TNF-α, TNFR1 and TNFR2 expression profiles was performed with the quantitative reverse-transcription polymerase chain reaction (RT-qPCR) method, using the patients' full blood. The statistical analysis was performed with STATISTICA v. 12.0 PL (StatSoft Inc., Tulsa, USA) with the level of statistical significance p < 0.05. RESULTS: Gradually reduced PASI, BSA and DLQI values were observed during anti-IL-12/23 therapy. An increased level of the TNF-α transcription activity was observed in the analyzed group when compared to the control. Correlations between the clinical and molecular parameters were also indicated. CONCLUSIONS: Ustekinumab constitutes an efficient and safe form of pharmacotherapy in psoriasis vulgaris. We did not observe any reduced efficacy of the treatment when reclassifying patients for the therapy. Tumor necrosis factor α, TNFR1 and TNFR2 may serve as supplementary markers of molecular response to the medicine.

Interplay between miRNAs and Genes Associated with Cell Proliferation in Endometrial Cancer.

Endometrial cancer develops as a result of abnormal cell growth associated with uncontrolled cell proliferation, excessive activation of signaling pathways and miRNA activity. The aim of this study was to determine the expression profile of genes associated with cell proliferation and to assess which miRNAs can participate in the regulation of their expression. The study enrolled 40 patients with endometrial cancer and 10 patients without neoplastic changes. The expression profile of genes associated with cell proliferation and the expression profile of miRNAs were assessed using microarrays. RT-qPCR was performed to validate mRNA microarray results. The mirTAR tool was used to identify miRNAs that regulate the activity of genes associated with cell proliferation. Decreased expression of IGF1 and MYLK, as well as SOD2 overexpression, were observed in endometrial cancer using both mRNA microarrays and RT-qPCR. Microarray analysis showed low levels of NES and PRKCA, but this was only partially validated using RT-qPCR. Reduced activity of MYLK may be caused by increased miR-200c, miR-155 and miR-200b expression. Cell proliferation is disturbed in endometrial cancer, which may be associated with an overexpression of miR-200a, miR-200c, and miR-155, making it a potential diagnostic marker.

Pressure Injuries Treated With Anodal and Cathodal High-voltage Electrical Stimulation: the Effect on Blood Serum Concentration of Cytokines and Growth Factors in Patients With Neurological Injuries. A Randomized Clinical Study.

It remains unclear whether electrical currents can affect biological factors that determine chronic wound healing in humans. PURPOSE: The aim of this study was to determine whether anodal and cathodal high-voltage monophasic pulsed currents (HVMPC) provided to the area of a pressure injury (PI) change the blood level of cytokines (interleukin [IL]-1ß, IL-10, and tumor necrosis factor [TNF]-α) and growth factors (insulin-like growth factor [IGF]-1 and transforming growth factor [TGF]-ß1) in patients with neurological injuries and whether the level of circulatory cytokines and growth factors correlates with PI healing progression. METHODS: This study was part of a randomized clinical trial on the effects of HVMPC on PI healing. All patients with neurological injuries (spinal cord injury, ischemic stroke, and blunt trauma to the head) and a stage 2, stage 3, or stage 4 PI of at least 4 weeks' duration hospitalized in one rehabilitation center were eligible to participate if older than 18 years of age and willing to consent to donating blood samples. Exclusion criteria included local contraindications to electrical stimulation (cancer, electronic implants, osteomyelitis, tunneling, necrotic wounds), PIs requiring surgical intervention, patients with poorly controlled diabetes mellitus (HbA1C > 7%), critical wound infection, and/or allergies to standard wound treatment. Participants were randomly assigned to 1 of 3 groups: anodal (AG) or cathodal (CG) HVMPC treatment (154 µs; 100 Hz; 360 µC/sec; 1.08 C/day) or a placebo (PG, sham) applied for 50 minutes a day, 5 days per week, for 8 weeks. TNF-α, IL-1ß, IL-10, TGF-ß1, and IGF-1 levels in blood serum were assessed using the immunoenzyme method (ELISA) and by chemiluminescence, respectively, at baseline and week 4. Wound surface area measurements were obtained at baseline and week 4 and analyzed using a digitizer connected to a personal computer. Statistical analyses were performed using the maximum-likelihood chi-squared test, the analysis of variance Kruskal-Wallis test, the Kruskal-Wallis post-hoc test, and Spearman's rank order correlation; the level of significance was set at P ≤.05. RESULTS: Among the 43 participants, 15 were randomized to AG (mean age 53.87 ± 13.30 years), 13 to CG (mean age 51.08 ± 20.43 years), and 15 to PG treatment (mean age 51.20 ± 14.47 years). Most PIs were located in the sacral region (12, 74.42%) and were stage 3 (11, 67.44%). Wound surface area baseline size ranged from 1.00 cm2 to 58.04 cm2. At baseline, none of the variables were significantly different. After 4 weeks, the concentration of IL-10 decreased in all groups (AG: 9.8%, CG: 38.54%, PG: 27.42%), but the decrease was smaller in the AG than CG group (P = .0046). The ratio of pro-inflammatory IL-10 to anti-inflammatory TNF-α increased 27.29% in the AG and decreased 26.79% in the CG and 18.56% in the PG groups. Differences between AG and CG and AG and PG were significant (AG compared to CG, P = .0009; AG compared to PG, P = .0054). Other percentage changes in cytokine and growth factor concentration were not statistically significant between groups. In the AG, the decrease of TNF-α and IL-1ß concentrations correlated positively with the decrease of PI size (P <.05). CONCLUSION: Anodal HVMPC elevates IL-10/TNF-α in blood serum. The decrease of TNF-α and IL-1ß concentrations in blood serum correlates with a decrease of PI wound area. More research is needed to determine whether the changes induced by anodal HVMPC improve PI healing and to determine whether and how different electrical currents affect the activity of biological agents responsible for specific wound healing phases, both within wounds and in patients' blood. In clinical practice, anodal HVMPC should be used to increase the ratio of anti-inflammatory IL-10 to pro-inflammatory TNF-α , which may promote healing.

Adipose-derived stem cells undergo differentiation after co-culture with porcine limbal epithelial stem cells.

Mesenchymal stem cells (MSCs) are objects of interest in regenerative medicine. They are used for various therapies such as for the regeneration of bone, chondrocytes and other tissues. Adipose derived stem cells (ADSCs) inter alia are particularly easy to access, they are relatively abundant in fat tissue. ADSCs could be differentiated into many types of cells. To date, it has been proven that ADSCs only differentiate into mesodermal cell lineages. In this study, we present the differentiation of ADSCs into the corneal epithelium. Human ADSCs were placed in a co-culture with porcine limbal epithelial stem cells (LESCs). After 14 days of cultivation, total RNA was extracted for the analysis of the molecular markers (expression of genes of interest). The gene expression was assessed by real-time RT-qPCR. The expression of the surface molecular markers of ADSCs is modulated after co-culturing. We have observed the decrease in CD73, CD90 and CD105 mRNA expression, while the expression of mRNA coding for CK3 and CK12 mRNA was increased in ADSCs co-cultured with porcine limbal epithelial stem cells as compared to the control. We conclude that the co-culture of LESCs and ADSCs changed ADSCs' molecular markers gene expression indicating initiation of differentiation towards limbal cells.

Expression profile of human porcine endogenous retrovirus A receptors (HuPAR-1, HuPAR-2) and transcription factor activator protein-2γ (TFAP-2C) genes in infected human fibroblasts-Model in vitro.

BACKGROUND: Xenotransplantation of porcine tissues raises concerns, especially in the context of the potential interspecies transmission of porcine endogenous retroviruses (PERVs). To date, the possibility of PERV infections of various human cells has been confirmed in vitro. PERVs infect cells coupling viral Env protein with adequate functional receptor on the surface of the host cell. So far, two PERV-A receptors have been described in humans: HuPAR-1 and HuPAR-2. TFAP-2C was described as one of the transcription factors engaged in the expression of HuPAR-2. METHODS: Bacterial LPS, well known as a strong inflammation inducer, was used in this study to stimulate changes of the expression profile of inflammation-related genes in human cells in vitro. The aim of the study was to investigate the expression profile of HuPAR-1 and HuPAR-2 and TFAP-2C genes in human NHDF cells treated with LPS and/or infected with PERVs from PK15 cells. PERV infection and expression was confirmed by qPCR and RTqPCR. The expression of HuPAR-1, HuPAR-2, and TFAP-2C genes was studied using HGU 133A 2.0 microarrays and RTqPCR. RESULTS: NHDF cells expressed both HuPAR-1 and HuPAR-2 genes with a higher expression of HuPAR-1. LPS down-regulated the expression of HuPAR-1 and TFAP-2C in NHDF cells, but had no effect on HuPAR-2 expression. These changes induced by LPS were more pronounced in the presence of PERV infection. CONCLUSION: As reported previously, treatment of NHDF cells with LPS decreased PERV-A provirus integration and increased PERV-A mRNA expression. PERV infection alone did not modulate the expression of HuPAR-1, HuPAR-2, and TFAP-2C. This is the first study analyzing the expression profile of HuPAR-1, HuPAR-2, and TFAP-2C in NHDF cells treated by LPS and/or infected by PERVs.

Clinical and molecular evaluation of therapy with the use of cyclosporine A in patients with psoriasis vulgaris.

BACKGROUND: Psoriasis course involves increased secretion of pro-inflammatory cytokines, among others, a beta transforming growth factor (TGFßs) and its receptors. Cyclosporine A (CsA), an immunosuppressive medicine with the molecular mechanism of operation connected with the properties of cell cycle suppression, is often used in the treatment of severe forms of psoriasis. The efficacy of therapy is assessed based on the disease clinical progression indexes - Psoriasis Area and Severity Index (PASI), body surface area (BSA), and Dermatology Life Quality Index (DLQI). The aim of the study was the evaluation of the efficacy of the CsA treatment of patients with psoriasis vulgaris, based on the clinical parameters and an assessment of the expression profiles of TGFßs and TGFßRs, depending on the concurrent diabetes and metabolic syndrome. METHODS: The group under study composed of 32 patients (15 with the metabolic syndrome, seven with diabetes) treated with CsA for 84 days. The molecular analysis included extraction of RNA, assessment of TGßF1-3, TGFßRI-III gene expression with the use of the RTqPCR method. The clinical assessment of the effects of this pharmacotherapy involved evaluation of the parameters: PASI, BSA, DLQI before therapy commencement, on the 42nd and 84th days of therapy. RESULTS: A statistically significant change in the transcription activity of TGFß1 in patients with and without diabetes (P = 0.018) and patients with and without metabolic syndrome (P = 0.023) was shown that on the 84th day of therapy. CONCLUSIONS: TGFb1 may be claimed as the supplementary molecular marker to evaluate the efficacy of CsA therapy. It seems that systemic diseases have an effect on the efficacy of the applied pharmacotherapy and the course of psoriasis.

Variances in the mRNA expression profile of TGF-ß1-3 isoforms and its TGF-ßRI-III receptors during cyclosporin a treatment of psoriatic patients.

INTRODUCTION: Psoriasis is a chronic, immunologic, multi-factor inflammatory skin disease, strongly associated with a higher level of a number of cytokines, such as isoforms of transforming growth factor ß (TGF-ß1-3) and its receptors (TGF-ßRI-III). One of the most popular and important drugs used to treat this disease is cyclosporin A (CsA). AIM: The aim of this study was to investigate the expression of genes encoding the transforming growth factor (TGF)-ß isoforms and receptors of the cytokine TGF-ßRs in psoriatic patients during an 84-day long observation of the effects of cyclosporin A therapy. It made an attempt to determine the usefulness of testing mRNA expression of TGF-ß1-3 and its receptors TGF-ßRI-III as the supplementary molecular markers of lost sensitivity to the medicine. MATERIAL AND METHODS: The study group consisted of 32 patients with psoriasis (20 men and 12 women) treated with cyclosporin A. The changes in expression patterns of TGF-ß1-3 and TGF-ßRI-III were performed by real-time quantitative reverse transcription PCR (RTqPCR). RESULTS: The expression of TGF-ß1-3 and TGF-ßRI-III were detected in the whole period of therapy with CsA. Changes in transcriptional activities of TGF-ß1-3 and TGF-ßRI-III during pharmacotherapy were observed. Differences in the expression of these genes were found before and after 42 and 84 days of using CsA. CONCLUSIONS: The changes in expression profiles of TGF-ß1-3 and TGF-ßRI-III during CsA therapy can be a useful molecular marker of lost sensitivity to the medicine.

Genes involved in the regulation of different types of autophagy and their participation in cancer pathogenesis.

Autophagy is a highly conserved mechanism of self-digestion that removes damaged organelles and proteins from cells. Depending on the way the protein is delivered to the lysosome, four basic types of autophagy can be distinguished: macroautophagy, selective autophagy, chaperone-mediated autophagy and microautophagy. Macroautophagy involves formation of autophagosomes and is controlled by specific autophagy-related genes. The steps in macroautophagy are initiation, phagophore elongation, autophagosome maturation, autophagosome fusion with the lysosome, and proteolytic degradation of the contents. Selective autophagy is macroautophagy of a specific cellular component. This work focuses on mitophagy (selective autophagy of abnormal and damaged mitochondria), in which the main participating protein is PINK1 (phosphatase and tensin homolog-induced putative kinase 1). In chaperone-mediated autophagy, the substrate is bound to a heat shock protein 70 chaperone before it is delivered to the lysosome. The least characterized type of autophagy is microautophagy, which is the degradation of very small molecules without participation of an autophagosome. Autophagy can promote or inhibit tumor development, depending on the severity of the disease, the type of cancer, and the age of the patient. This paper describes the molecular basis of the different types of autophagy and their importance in cancer pathogenesis.

Psoriasis Treatment Changes the Expression Profile of Selected Caspases and their Regulatory MicroRNAs.

BACKGROUND/AIMS: Psoriasis, an autoimmune diseases of the skin, characterized by patches of abnormal/inflammed skin, although not usually life-threatening, it causes severe discomfort, esthetic impairments, and may lead to impaired social functions and social withdrawal. Besides UV-phototherapy, various anti-inflammatory treatments are applied, depending on the severity of symptoms. In 2008, adalimumab (fully humanized human anti-TNF antibody) was launched for the treatment of psoriasis. In the quest to better understand the pathomechanism of adalimumab's therapeutic effects, and the acquired resistance to the drug, we have investigated how its administration affect the regulation of the expression of selected caspases, including those activated by inflammosome. METHODS: The research was initially carried out on normal human dermal fibroblasts (NHDF) treated with adalimumab for 2, 8 and 24 hours in vitro. Then, expression profile of genes encoding caspases and their regulatory micro-RNAs was determined with the use of oligonucleotide microarray. The validation of the microarray results was carried out by qRT-PCR. The in vitro study was followed by ex-vivo investigation of adalimumab's effects on the expression of caspase-6 in blood of the psoriatic patients. The samples were collected before, and 2 hours after adalimumab's administration and the analysis was determined by qRT-PCR. RESULTS: The result of the analysis indicated that introduction of adalimumab to the NHDF culture resulted in the change of the transcription activity of genes encoding caspases and genes encoding miRNAs. The analysis revealed 5 different miRNA molecules regulating the expression of: CASP2, CASP3 and CASP6. There were no statistically significant differences in the expression of gene encoding caspase-6 in the patients' blood before and 2 hours after the anti-TNF drug administration. CONCLUSION: We have found that adalimumab administration affects caspases expression, thus they may be used as molecular markers for monitoring the therapy with the use of an anti-TNF drugs, including adalimumab. It is likely that the mechanisms responsible for changed expression profiles of genes encoding caspase-2,-3, and -6, may be caused by the upregulation of the respective microRNA molecules. Increased expression of genes encoding specific caspases may induce inflammatory processes, as well as trigger apoptosis. Furthermore, the proapoptotic activity of caspases may be enhanced by miRNA molecules, which exhibit proapoptotic function. The overexpression of such miRNAs was observed in our study.

Effect of adalimumab on the expression of genes encoding TNF-α signal paths in skin fibroblasts in vitro.

INTRODUCTION: Tumour necrosis factor (TNF-α) is one of the main cytokines participating in inflammation and immune response. Biological effects of the cytokine action, mediated by two receptors: TNFRSF1A and TNFRSF1B involve activation of many signal paths, thus change the transcription activity of many genes. The mechanism of action of an anti-TNF medicine consists in blocking TNF-α though preventing activation of signal paths. AIM: To single out mRNA and microRNA genes relating to TNF-α signal paths, the expression of which could indicate sensitivity of cells to the medicine in question. MATERIAL AND METHODS: The material used in the research consisted in the cell line of regular human skin fibroblasts NHDF (CC-2511 Lonza, Basel, Switzerland) exposed to adalimumab with a concentration of 8.00 µg/ml of the medium for 2, 8 and 24 h, compared with the control material, i.e. non-stimulated cells. Molecular analysis was performed using the oligonucleotide expressive micro-matrices technology HG-U133A, miRNA 2.0 Array micro-matrices and RTqPCR. RESULTS: mRNA: BIRC5, MAP3K4, ZFAND5, JUN differentiate cells exposed to the anti-TNF medicine, regardless of the time of cell/medicine incubation. TNF-α transcription activity is reduced during exposure of NHDF cells to adalimumab. miRNA regulating transcription activity of the said 4 mRNA and miRNA related to TNF-α and its receptors was also singled out. CONCLUSIONS: It was ascertained that adalimumab has therapeutic potential and affects genes engaged in signal paths activated by TNF-α. The results indicate the TNF-α usefulness as the molecular, supplementary marker in diagnostics and control of treatment effects.

Changes in the Expression Profile of JAK/STAT Signaling Pathway Genes and Mirnas Regulating their Expression Under the Adalimumab Therapy.

OBJECTIVE: The aim of this study was to evaluate the influence of adalimumab on the expression pattern of genes associated with JAK/STAT signaling pathway in Normal Human Dermal Fibroblast (NHDF) cells stimulated with 8 µg/ml of adalimumab and the identification of miRNAs regulating these genes' expression. METHOD: NHDFs were cultured with or without the presence of adalimumab for 2, 8, and 24h. The microarray technology was used to determine expression profile of mRNAs and miRNAs. RESULTS: Out of 22283 ID mRNA, 37 are associated with the JAK/STAT signaling pathway. It can be observed that 18 mRNAs differentiate NHDFs cultures with adalimumab from control. The analysis of miRNAs showed that, among 1105 ID miRNAs, 20 miRNAs are differentiating in cells treated with adalimumab for 2h, 9 miRNAs after 8h, and only 3 miRNAs after 24h. CONCLUSION: It can be observed that miRNAs play an extremely important role in the regulation of the expression of genes associated with JAK/STAT signaling pathway. The results of this study show the possibility of using changes in mRNAs and miRNAs profile expression, as complementary molecular markers of adalimumab treatment effectiveness.

Potential Mechanism of Action of Cyclosporin A in Human Dermal Fibroblasts-Transcriptomic Analysis of CYPs.

Effect of cyclosporin A (CsA) in a therapeutic concentration, on the expression of cytochrome P450 genes (CYPs), was investigated in normal human dermal fibroblast cells. The expression of 57 genes, encoding cytochrome P450 isoforms, was estimated using the microarray method. Amongst 396 normalized fluorescence signals related to cytochrome P450 activity, only 91 were strictly connected to CYPs and were analyzed using two methods: a self-organizing feature map of artificial neural networks and typical statistical analysis with significance level at p ≤ 0.05. Comparing the samples from fibroblasts cultured with CsA and those cultured without, up-regulated changes of CYP19A1, 1B1, 7A1, 7F1, 17A1 and down-regulated 2D6 gene expression were observed. The mRNAs with increased changes were in the same neuron of the self-organizing feature map. All distinguished CYPs encode monooxygenases, which plays an important role in steroids biosynthesis and metabolism. Based on the obtained results, we can conclude that CsA in therapeutic concentration changes the expression profile of CYPs in human dermal fibroblasts, especially affecting genes linked to steroids synthesis and/or metabolism. It shows the potential mechanism of action of CsA in human dermal fibroblast cells.

Porcine Endogenous Retrovirus (PERV) - Molecular Structure and Replication Strategy in the Context of Retroviral Infection Risk of Human Cells.

The xenotransplantation of porcine tissues may help overcome the shortage of human organs for transplantation. However, there are some concerns about recipient safety because the risk of porcine endogenous retrovirus (PERV) transmission to human cells remains unknown. Although, to date, no PERV infections have been noted in vivo, the possibility of such infections has been confirmed in vitro. Better understanding of the structure and replication cycle of PERVs is a prerequisite for determining the risk of infection and planning PERV-detection strategies. This review presents the current state of knowledge about the structure and replication cycle of PERVs in the context of retroviral infection risk.

Influence of Adalimumab on the Expression Profile of Genes Associated with the Histaminergic System in the Skin Fibroblasts In Vitro.

OBJECTIVE: The aim of this study was to evaluate the influence of adalimumab on expression profile of genes associated with the histaminergic system in Normal Human Dermal Fibroblast (NHDF) cells stimulated with 8.00 µg/ml of adalimumab and the identification of miRNAs regulating these genes' expression. METHODS: NHDFs were cultured with or without the presence of adalimumab for 2, 8, and 24 hours. The expression profile of genes and miRNA were determined with the use of microarray technology. RESULTS: Among 22283 ID mRNA, 65 are associated with the histaminergic system. It can be observed that 15 mRNAs differentiate NHDFs cultures with adalimumab form control. The analysis of miRNAs showed that, among 1105 ID miRNA, 20 miRNAs are differentiating in cells treated with adalimumab for 2 hours, 9 miRNA after 8 hours, and only 3 miRNAs after 24 hours. CONCLUSION: It was also determined that miRNAs play certain role in the regulation of the expression of genes associated with the histaminergic system. The results of this study confirmed the possibility of using both genes associated with this system as well as miRNAs regulating their expression, as complementary molecular markers of sensitivity to the adalimumab treatment.