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.

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.

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.

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.

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.

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.

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.

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.

Profile of Expression of Genes Encoding Matrix Metallopeptidase 9 (MMP9), Matrix Metallopeptidase 28 (MMP28) and TIMP Metallopeptidase Inhibitor 1 (TIMP1) in Colorectal Cancer: Assessment of the Role in Diagnosis and Prognostication.

BACKGROUND Studies on the pathomechanism of colorectal cancer (CRC) expansion indicate a significant role of metalloproteinases and their inhibitors in the extracellular matrix. The results of the analysis of a profile of transcriptional activity of genes encoding metalloproteinases were the basis of the hypothesis indicating changes in the expression of genes encoding MMP9, MMP28, and TIMP1 as an additional diagnostic and prognostic marker of CRC. MATERIAL AND METHODS The material consisted of samples obtained from resected tumors and healthy tissue samples from 15 CRC patients (aged 46-72 years) at clinical stages (CSs) I and II-IV. Gene expression analysis was done using microarrays. Microarray data analysis was done using the GeneSpring 11.5 platform. The results were validated using the qRT-PCR technique. RESULTS We found high levels of expression of MMP9 at each CS, as well as in the tissues at the early stage of CRC. Additionally, we observed high levels of expression of TIMP1 and low levels of MMP28 genes in CS II-IV. No statistically significant differences based on the stage of CRC were observed. CONCLUSIONS MMP9 gene profile may be a complementary diagnostic marker in CRC. The results suggest a crucial role of MMP9 at the early stage of carcinogenesis in the large intestine. The increase in MMP9 and TIMP1 mRNA concentration and the decrease in MMP28 in the large intestinal tissue may be a confirmation of cancer, but it may not indicate the advance of CRC.

Impact of Antibiotics on the Proliferation and Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

Adipose tissue is a promising source of mesenchymal stem cells. Their potential to differentiate and regenerate other types of tissues may be affected by several factors. This may be due to in vitro cell-culture conditions, especially the supplementation with antibiotics. The aim of our study was to evaluate the effects of a penicillin-streptomycin mixture (PS), amphotericin B (AmB), a complex of AmB with copper (II) ions (AmB-Cu2+) and various combinations of these antibiotics on the proliferation and differentiation of adipose-derived stem cells in vitro. Normal human adipose-derived stem cells (ADSC, Lonza) were routinely maintained in a Dulbecco's Modified Eagle Medium (DMEM) that was either supplemented with selected antibiotics or without antibiotics. The ADSC that were used for the experiment were at the second passage. The effect of antibiotics on proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine-B (SRB) tests. Differentiation was evaluated based on Alizarin Red staining, Oil Red O staining and determination of the expression of ADSC, osteoblast and adipocyte markers by real-time RT-qPCR. The obtained results indicate that the influence of antibiotics on adipose-derived stem cells depends on the duration of exposure and on the combination of applied compounds. We show that antibiotics alter the proliferation of cells and also promote natural osteogenesis, and adipogenesis, and that this effect is also noticeable in stimulated osteogenesis.

Differential expression of microRNAs and their predicted targets in renal cells exposed to amphotericin B and its complex with copper (II) ions.

MicroRNAs (miRNAs) have been reported to regulate essential biological processes, and their expression was shown to be affected by pathological processes and drug-induced toxicity. Amphotericin B (AmB) can cause liver and kidney injury, but a recently developed complex of AmB with copper (II) ions (AmB-Cu2+) exhibits a lower toxicity to human renal cells while retaining a high antifungal activity. The aim of our study was to assess AmB-Cu2+-induced changes in the miRNA profile of renal cells and examine which biological processes are significantly affected by AmB-Cu2+. We also aimed to predict whether differentially expressed miRNAs would influence observed changes in the mRNA profile. miRNA and mRNA profiles in normal human renal proximal tubule epithelial cells (RPTEC) treated with AmB-Cu2+ or AmB were appointed with the use of microarray technology. For differentially expressed mRNAs, the PANTHER overrepresentation binomial test was performed. miRNA target interactions (MTIs) were predicted using the miRTar tool. The mRNA profile was much more strongly affected than the miRNA profile, in both AmB-Cu2+- and AmB-treated cells. AmB-Cu2+ influenced both the miRNA and mRNA profiles much more strongly than AmB. The most affected biological processes were intracellular signal transduction (AmB-Cu2+) and signal transduction (AmB). Only a few interactions between differentiating miRNAs and mRNAs were found. Changes in the profiles of genes involved in signal transduction and intracellular signal transduction may not result from interactions with differentially expressed miRNAs. Changes in the miRNA profile suggest the possible influence of tested drugs on the regulation of fibrosis via a miRNA-dependent mechanism.

TNF-α in a molecularly targeted therapy of psoriasis and psoriatic arthritis.

Psoriasis is a chronic immunological skin disease and patients with this disorder typically experience a significant decrease in their quality of life. The disease is traditionally managed with topical and systemic agents (retinoids, ciclosporin A, methotrexate), but these treatment options are often long-term and their effects can be inconsistent and not ideal. The use of biological drugs in dermatological treatment is relatively new and began in the early 2000s. It should be noted that, in most countries, in order for biological treatment to be administered, specific criteria must be met. The current treatment options for psoriasis and psoriatic arthritis include tumour necrosis factor alpha (TNF-α) blockers, interleukin (IL)-12 and IL-23 inhibitors, T cell inhibitors and B cell inhibitors. These classes of biological drugs are characterised by protein structure as well as high molecular weight and their effectiveness is evaluated based on the Psoriasis Area and Severity Index (PASI), Body Surface Area (BSA) and the Dermatology Life Quality Index (DLQI). TNF-α antagonists are one such class of biological drugs which includes infliximad, etanercept and adalimumab. Infliximab is a chimeric protein that is administered via intravenous infusions as a monotherapy in psoriasis vulgaris. Etanercept is indicated for use in both psoriasis vulgaris and psoriatic arthritis and it is the only drug that can be used as a treatment for children under the age of 8 with psoriasis. The drug is administered subcutaneously. Finally, adalimumab is a fully human monoclonal antibody that neutralises both free and membrane-bound TNF-α and is used in the treatment of psoriasis vulgaris and psoriatic arthritis. This article reviews the latest research in the use of TNF-α for the treatment of moderate to severe psoriasis and psoriatic arthritis. The results of research in this field are promising and confirm the effectiveness and safety of biological drugs as dermatological treatments for psoriasis. In particular, adalimumab, etanercept and infliximab are promising therapeutic options for patients with moderate to severe psoriasis and psoriatic arthritis who are unresponsive to conventional treatment strategies and they can significantly improve the quality of lives in patients with this disease.

Anti­cytokine therapy for psoriasis - not only TNF­α blockers. Overview of reports on the effectiveness of therapy with IL­12/IL­23 and T and B lymphocyte inhibitors.

TNF­α inhibitors - infliximab, etanercept and adalimumab - can be used in the treatment of psoriasis vulgaris and psoriatic arthritis, along with other inhibitors of proinflammatory cytokines, such as interleukin­12 (IL­12) and IL­23. This paper presents the results of research on the use of biological drugs other than the tumor necrosis factor blockers (TNF­α), namely inhibitors of IL­12 and IL­23 (ustekinumab), T­cell inhibitors (alefacept and efalizumab), B­cell inhibitors (rituximab), anti­IL­17 agents (secukinumab, ixekizumab, and brodalumab) and IL23p19 inhibitors (guselkumab and tildrakizumab). The paper presents an analysis of the mechanism of action, recommended doses and methods of therapy, taking into account the adverse events associated with the use of anti­cytokine therapy. The use of biological drugs is discussed based on a review of the current literature.

CYCLOSPORIN A AFFECTS THE PROLIFERATION PROCESS IN NORMAL HUMAN DERMAL FIBROBLASTS.

Cyclosporin A is an immunosuppressant drug that is used not only in solid transplant rejection, but also in moderate and severe forms of psoriasis, pyoderma, lupus or arthritis. Serious side effects of the drug such as skin cancer or gingival hyperplasia probably start with the latent proliferation process. Little is known about the influence of cyclosporin A on molecular signaling in epidermal tissue. Thus, the aim of this study was to estimate the influence of cyclosporin A on the process of proliferation in normal human dermal fibroblasts. Fibroblasts were cultured in a liquid growth medium in standard conditions. Cyclosporin A was added to the culture after the confluence state. Survival and proliferation tests on human dermal fibroblast cells were performed. Total RNA was extracted from fibroblasts, based on which cDNA and cRNA were synthesized. The obtained cRNA was hybridized with the expression microarray HGU-133A_2.0. Statistical analysis of 2734 mRNAs was performed by the use of GeneSpring 13.0 software and only results with p < 0.05 were accepted. Analysis of variance with Tukey post hoc test with Benjamini-Hochberg correction for all three (8, 24, 48 h) culture stages (with and without cyclosporin A) was performed to lower the number of statistically significant results from 679 to 66, and less. Between statistically and biologically significant mRNAs down-regulated were EGRJ, BUBIB, MKI67, CDK1, TTK, E2F8, TPX2, however, the INSIG1, FOSL1, HMOX1 were up-regulated. The experiment data revealed that cyclosporin A up-regulated FOSL1 in the first 24 h, afterwards down-regulating its expression. The HMOX1 gene was up-regulated in the first stage of the experiment (CsA 8 h), however, after the next 16 h of culture time its expression was down-regulated (CsA 24 h), to finally increased in the later time period. The results indicate that cyclosporin A had a significant effect on proliferation in normal human dermal fibroblasts through the changes in the expression of genes related to the cell cycle and transcription regulation process.

[Night work and shift work - effects on the health of workers].

Number of shift workers increases in developed as well as in developing countries every year and equals 15- 20% of total amount of working people in Europe, 20% of total count of workers in United States of America, 6-32% in Asian countries and 8.1% workers in Poland. This type of employment is connected with such sectors of economy as medical care, industry, mining, transportation, communication and hospitality. The literature review analyses health effects of shift work and night work in the area of gastroenterology, circulatory system, oncologic diseases, neuropsychiatric and sleep disorders. In summary shift and night work have negative impact on human health. Further investigations analyzing impact of shift and night work are needed.

Expression of insulin-like growth factor family genes in clear cell renal cell carcinoma.

AIM OF THE STUDY: Despite significant progress in the pathology of clear cell renal cell carcinoma (ccRCC), diagnostic and predictive factors of major importance have not been discovered. Some hopes are associated with insulin-like growth factors. The aim of the study was to compare the expression of genes for insulin-like growth factor family in tumours and in tissue of kidneys without cancer. MATERIAL AND METHODS: Fifty-two patients years with clear cell renal cell cancer were qualified to the study group; patients nephrectomised because of hydronephrosis were included in the control group. Expression of genes were evaluated by RT-PCR. RESULTS: Expression of IGFR-1 gene in tumour accounts for about 60% of cases. The incidence is higher than in corresponding adjacent non-cancerous kidney tissues and higher (but with no statistical significance) than in kidney without cancer. Expression of IGFR-2 gene in tumours has not been established. The incidence of the expression in corresponding adjacent non-cancerous kidney tissues is small. Expression of this gene has been present in all specimens from kidneys without cancer. Expression of IGFBP-3 gene ascertained in all (except four) cases of ccRCC and in the majority of clippings from adjacent tissue. It was not found in kidneys from the control group. IGF-1, IGF-2, and IGFR-1 mRNA copy numbers in ccRCC were higher than in the material from the control group.

Screening pigs for xenotransplantation: expression of porcine endogenous retroviruses in transgenic pig skin.

Pigs seem to be the answer to worldwide organ donor shortage. Porcine skin may also be applied as a dressing for severe burns. Genetic modifications of donor animals enable reduction of immune response, which prolongs xenograft survival as temporary biological dressing and allows achieving resistance against xenograft rejection. The risk posed by porcine endogenous retroviruses (PERVs) cannot be eliminated by breeding animals under specific-pathogen-free conditions and so all recipients of porcine graft will be exposed to PERVs. Therefore our study has been focused on the assessment of PERV DNA and mRNA level in skin samples of transgenic pigs generated for xenotransplantation. Porcine skin fragments were obtained from 3- to 6-month-old non-transgenic and transgenic Polish Landrace pigs. Transgenic pigs were produced by pronuclear DNA microinjection and were developed to express the human α-galactosidase and the human α-1,2-fucosyltransferase gene. The copy numbers of PERV DNA and RNA were evaluated using real-time Q-PCR and QRT-PCR. Comparative analysis of all PERV subtypes revealed that PERV-A is the main subtype of PERVs in analyzed skin samples. There was no significantly different copy number of PERV-A, PERV-B and PERV-C between non-transgenic pigs, pigs with the human α-galactosidase and pigs expressing the human α-1,2-fucosyltransferase gene, except of PERV-C DNA. It brings the conclusion, that transgenesis process exerts no influence on PERVs transinfection. That is another step forward in the development of pig skin xenografts as burn wounds dressing.

Expression Level of Genes Coding for Cell Adhesion Molecules of Cadherin Group in Colorectal Cancer Patients.

BACKGROUND: Colorectal Cancer (CRC) is one of the most frequently diagnosed neoplasms and also one of the main death causes. Cell adhesion molecules are taking part in specific junctions, contributing to tissue integrality. Lower expression of the cadherins may be correlated with poorer differentiation of the CRC, and its more aggressive phenotype. The aim of the study is to designate the cadherin genes potentially useful for the diagnostics, prognostics, and the treatment of CRC. MATERIAL AND METHODS: Specimens were collected from 28 persons (14 female and 14 male), who were operated for CRC. The molecular analysis was performed using oligonucleotide microarrays, mRNA used was collected from adenocarcinoma, and macroscopically healthy tissue. The results were validated using qRT-PCR technique. RESULTS: Agglomerative hierarchical clustering of normalized mRNA levels has shown 4 groups with statistically different gene expression. The control group was divided into 2 groups, the one was appropriate control (C1), the second (C2) had the genetic properties of the CRC, without pathological changes histologically and macroscopically. The other 2 groups were: LSC (Low stage cancer) and HSC (High stage cancer). Consolidated results of the fluorescency of all of the differential genes, designated two coding E-cadherin (CDH1) with the lower expression, and P-cadherin (CDH3) with higher expression in CRC tissue. CONCLUSIONS: The levels of genes expression are different for several groups of cadherins, and are related with the stage of CRC, therefore could be potentially the useful marker of the stage of the disease, also applicable in treatment and diagnostics of CRC.

[Expression of melatonin receptors genes and genes associated with regulation of their activity in endometrial cancer]. / Ekspresja genów receptorów melatoninowych i genów zwiazanych z regulacja ich aktywnosci w gruczolakoraku endometrium.

OBJECTIVES: The aim of the study was to evaluate transcription activity of melatonin receptors and genes associated with regulation of their activity in endometrial adenocarcinoma to identify probable diagnostic and prognostic molecular markers. MATERIAL AND METHODS: The material included endometrial adenocarcinoma tissue samples of histopathological grades G1, G2, G3, and normal endometrium. The molecular analysis was performed on 37 patient samples. Total RNA was extracted and used for the microarray HG-U133A analysis. Among 22 283 ID mRNA, only entities of genes associated with regulation of melatonin receptors activity were selected. qRT-PCR was employed for validation, what allowed to compare melatonin receptor genes activation in endometrial cancer tissues to the normal endometrium. RESULTS: The results of the microarray experiments showed that only 18 ID mRNA were differential in endometrial cancer samples as compared to the control at p-value<0.05 and FC(log2)>1.5. These genes were identified as differentially expressed in grade G2-ASMTL, GNA 11, PER2, PTGDS and in grade G3-GNA12, GNA 11. Silencing of RGS4 encoding RGP4, which regulates signal transmission by G protein, was observed in all cancer groups, independently of the histopathological grade. CONCLUSIONS: The profile expression of genes associated with regulation of melatonin receptors activity was different and dependent on the histopathological grade of endometrial cancer and can be an additional diagnostic and prognostic marker Statistically significant was the down-regulation of melatonin biosynthesis genes (ASMTL) and melatonin signal transmitters (GNA 11, GNA 12, RTGS).

Lymphangiogenesis in cervical cancer evaluated by expression of the VEGF-C gene in clinical stage IB-IIIB.

INTRODUCTION: The aim of the present study was to evaluate the profile of VEGF-C gene expression in particular stages of cervical cancer (IB-IIIB) and to estimate the correlation between VEGF-C mRNA quantity profile and clinical stage. MATERIAL AND METHODS: Material for molecular analysis consisted of cervical cancer tissue specimens collected from 38 women (10, 15, 13 cases were classified as IB, IIB and IIIB, respectively). The control group was composed of normal cervical tissues collected from 10 women who underwent hysterectomy for non-oncological reasons. The number of VEGF-C mRNA copies in particular groups was estimated by the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. RESULTS: In the control group the average number of mRNA copies was 134 ± 36 (median: 106), in a group with stage IB it was 16 077 ± 7090 (median: 580), for stage IIB - 35 019 ± 8945 (median: 40 870). The highest number of mRNA VEGF-C copies was derived in a group of patients with cervical cancer of stage IIIB. The average quantity was 56 155 ± 12 470, whereas median 55 981. A statistically significantly higher level of VEGF-C gene expression was disclosed in cervical cancer specimens with stage IIB and IIIB than in the control group. In stage IIIB, the VEGF-C gene expression was significantly higher than in specimens derived from individuals in stage IB. CONCLUSIONS: In squamous cell carcinoma of the uterine cervix of stage IB-IIIB genes involved in lymphangiogenesis, especially VEGF-C, are expressed, which expression increases as the clinical stage of cervical cancer is higher.