Transforming growth factor ß-related genes in human retinal pigment epithelial cells after tacrolimus treatment.

BACKGROUND: The transforming growth factor ß (TGFß) family plays an important role in the pathogenesis of many diseases, including fibrotic pathologies of the eyes. The difficulties of surgical procedures contribute to the search for new treatment strategies for proliferative vitreoretinopathy. Therefore, the aim of this study was to investigate the expression profile of TGFß isoforms, their receptors, and TGFß-related genes in human retinal pigment epithelial cells (RPE) after tacrolimus (FK-506) treatment in the presence or absence of lipopolysaccharide (LPS)-induced inflammation. METHODS: The expression profile was analyzed using oligonucleotide microarrays and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) techniques. RESULTS: Analysis using oligonucleotide microarrays revealed 20 statistically significant differentially expressed TGFß-related genes after LPS treatment in relation to control cells, and after tacrolimus and LPS treatment in relation to LPS-treated cells. Moreover, our results showed that mRNA levels for TGFß2 and TGFßR3 after tacrolimus treatment, and for TGFßR3 after tacrolimus and LPS treatment in RPE cells were decreased. In turn, in the presence of LPS-induced inflammation, TGFß2 mRNA level was increased. CONCLUSIONS: These results can be important in regard to the treatment of proliferative vitreoretinopathy, pathogenesis of which is associated with processes regulated by TGFß, such as inflammation, proliferation, epithelial-mesenchymal transition (EMT), and fibrosis.

Effects of intravitreal ranibizumab on the untreated eye and systemic gene expression profile in age-related macular degeneration.

The purpose of this study was to evaluate the systemic effects of intravitreal ranibizumab (Lucentis) treatment in patients with neovascular age-related macular degeneration (AMD). The impact of intravitreal ranibizumab injections on central retinal thickness (CRT) of treated and contralateral untreated eyes, and differences in gene expression patterns in the peripheral blood mononuclear cells were analyzed. The study included 29 patients aged 50 years old and over with diagnosed neovascular AMD. The treatment was defined as 0.5 mg of ranibizumab injected intravitreally in the form of one injection every month during the period of 3 months. CRT was measured by optical coherence tomography. The gene expression profile was assigned using oligonucleotide microarrays of Affymetrix HG-U133A. Studies have shown that there was a change of CRT between treated and untreated eyes, and there were differences in CRT at baseline and after 1, 2, and 3 months of ranibizumab treatment. Three months after intravitreal injection, mean CRT was reduced in the treated eyes from 331.97±123.62 to 254.31±58.75 µm, while mean CRT in the untreated fellow eyes reduced from 251.07±40.29 to 235.45±36.21 µm at the same time. Furthermore, the research has shown that among all transcripts, 3,097 expresses change after the ranibizumab treatment in relation to controls. Among these transcripts, 1,339 were up-regulated, whereas 1,758 were down-regulated. Our results show the potential systemic effects of anti-VEGF therapy for AMD. Moreover, our study indicated different gene expression in peripheral blood mononuclear cells before and after intravitreal ranibizumab treatment.

Microarray analysis of retroviral restriction factor gene expression in response to porcine endogenous retrovirus infection.

Microarray analysis has been used for screening genes involved in specific biological processes. Many studies have shown that restriction factors may play an important role in xenotransplantation safety, but it is still unclear whether porcine endogenous retroviruses (PERVs) may be inhibited by these factors. Therefore, the present study focused on the microarray analysis retroviral restriction factors gene expression in normal human dermal fibroblasts (NHDFs) in response to PERVs. PERV infectivity was analyzed using a co-culture system of NHDFs and porcine kidney epithelial cells (PK15 cell line). Detection of the copy number of PERV A, PERV B DNA and PERV A, PERV B RNA was performed using real-time Q-PCR and QRT-PCR. The expression of retroviral restriction factor genes was compared between PERV-infected and uninfected NHDF cells using oligonucleotide microarray. The up-regulated transcripts were recorded for two differentially expressed genes (TRIM1, TRIM16) with the use of GeneSpring platform and Significance Analysis of Microarrays. In conclusion, our results suggest that the TRIM family may play an important role in innate immunity to PERV infection. These results can allow a better understanding of restriction mechanism of PERV infection and probably design molecularly targeted therapies in the future. Moreover, knowledge of retroviral restriction factor gene expression in human cells may help to uncover strategies for determining their exact function. Microarray analyses seem to be promising in biological and biomedical studies, however, these results should be further confirmed by research conducted at the protein level.

Porcine endogenous retroviruses in xenotransplantation--molecular aspects.

In the context of the shortage of organs and other tissues for use in human transplantation, xenotransplantation procedures with material taken from pigs have come under increased consideration. However, there are unclear consequences of the potential transmission of porcine pathogens to humans. Of particular concern are porcine endogenous retroviruses (PERVs). Three subtypes of PERV have been identified, of which PERV-A and PERV-B have the ability to infect human cells in vitro. The PERV-C subtype does not show this ability but recombinant PERV-A/C forms have demonstrated infectivity in human cells. In view of the risk presented by these observations, the International Xenotransplantation Association recently indicated the existence of four strategies to prevent transmission of PERVs. This article focuses on the molecular aspects of PERV infection in xenotransplantation and reviews the techniques available for the detection of PERV DNA, RNA, reverse transcriptase activity and proteins, and anti-PERV antibodies to enable carrying out these recommendations. These methods could be used to evaluate the risk of PERV transmission in human recipients, enhance the effectiveness and reliability of monitoring procedures, and stimulate discussion on the development of improved, more sensitive methods for the detection of PERVs in the future.

Porcine endogenous retrovirus infection changes the expression of inflammation-related genes in lipopolysaccharide-stimulated human dermal fibroblasts.

BACKGROUND: The present study focuses on explaining the interaction between porcine endogenous retroviruses (PERVs) and human cells in inflammatory conditions. The differences in expression of selected inflammation-related genes in human dermal fibroblasts (NHDF) infected with PERVs with and without lipopolysaccharide stimulation were identified. MATERIAL AND METHODS: The PERV infectivity was analyzed using a co-culture of NHDF and PK15 cells. Quantification of PERV A, B DNA and PERV A, B RNA was performed by real-time QPCR and QRT-PCR. The analysis of the expression profile was performed using HG-U133A 2.0 oligonucleotide microarrays. RESULTS: PERV infection of NHDF cells with LPS stimulation resulted in a statistically significant decrease in the copy number of PERV A DNA, and an increase in the copy number of PERV A RNA compared to fibroblasts without stimulation. There was no statistically significant difference between the copy number of PERV B RNA of LPStreated and untreated NHDF cells. Typing of differentiation genes was performed in a panel of 571 selected transcripts of inflammation-related genes. Among all studied genes, 23 were differentially regulated with a change greater that 1.1-fold and p<0.05 in all studied groups. Of these 23 genes, 3 were found to be regulated by more than 2.0-fold at least in 2 studied groups (IL6, IL8, and IL33). CONCLUSIONS: The interaction between porcine endogenous retroviruses and human cells changes in inflammatory conditions. PERV infection of NHDF cells may alter the expression of inflammation-related genes. Further investigations concerning PERV infection of human cells in different conditions seem to be necessary.

Quantitative analysis of SOD2, ALDH1A1 and MGST1 messenger ribonucleic acid in anterior lens epithelium of patients with pseudoexfoliation syndrome.

PURPOSE: The aim of the study was to investigate the expression of selected genes encoding enzymes involved in the antioxidant defense system (superoxide dismutase 2, SOD2; aldehyde dehydrogenase 1, ALDH1A1; microsomal glutathione S-transferase 1, MGST1) in fragments of anterior lens capsules of patients with pseudoexfoliation syndrome (PEX). The specificity and sensitivity of these molecular markers for PEX development were also assessed. METHODS: The study group consisted of 20 patients (9 women and 11 men) with diagnosed PEX and cataract. The control group included 23 patients (8 women and 15 men) who needed cataract surgery but did not have PEX. Quantification of SOD2, ALDH1A1, and MGST1 messenger ribonucleic acid (mRNA) was performed with quantitative real-time PCR. RESULTS: SOD2, ALDH1A1, and MGST1 mRNAs were detected in all studied samples. The examined genes had statistically significant higher expression in the group of patients with PEX than in the control group (SOD2, p=0.0015; ALDH1A1, p=0.0001; MGST1, p=0.0001, Mann-Whitney U test). The areas under the curve (AUC) of SOD2, MGST1, and ALDH1A1 were 0.766, 0.818, and 0.957, respectively. CONCLUSIONS: Differential expression of SOD2, ALDH1A1, and MGST1 genes in the anterior lens capsules of patients with PEX suggest that diseased tissue appears to respond to the previously reported oxidative stress. A possible role of ALDH1A1 mRNA level as a risk factor or marker for PEX needs further confirmation.

Expression patterns of kinin-dependent genes in endometrial cancer.

OBJECTIVE: The present study has focused on the identification of the differences between expression patterns of kinin-dependent genes in endometrial cancer with the use of real-time quantitative reverse transcription polymerase chain reaction and oligonucleotide microarray. MATERIALS AND METHODS: The study group consisted of 50 endometrium samples collected from women with endometrial cancer. Gene expression of kinin receptors BR1 and BR2 was evaluated with real-time quantitative reverse transcription polymerase chain reaction. The analysis of the expression profile of genes related to the kinin mitogenic signal transduction pathway was performed using HG-U133A oligonucleotide microarrays. RESULTS: The transcriptional activity of the B1 receptor for kinins increased in patients with grade 1 (G1) and grade 2 (G2) endometrial cancer when compared to the control group, whereas it decreased in patients with grade 3 (G3) endometrial cancer. The expression of the B2 receptor showed a growing trend reaching the peak in the G2, whereas G3 was characterized by a decrease in the gene transcriptional activity. Significant differential gene expression was recorded for GNB1, PRKAR1A, KRAS, MAP2K2, GNG5, MAPK1, ADCY9, GNG11, JUN, PRKCA, PRKACB, FOS, PLCB4, ADCY8, and GNG12. CONCLUSION: The expression changes in kinin-dependent genes might cause disturbance in the underlying biological processes, which could be important for the pathogenesis of endometrial cancer. This will eventually help to improve treatment strategies for patients with endometrial cancer in the future.

Degradation effect of diepoxide fixation on porcine endogenous retrovirus DNA in heart valves: molecular aspects.

PURPOSE: Xenotransplantations of porcine cells, tissues, and organs involve a risk of zoonotic viral infections in recipients, including by porcine endogenous retroviruses (PERVs), which are embedded the genome of all pigs. An appropriate preparation of porcine heart valves for transplantation can prevent retroviral infection. Therefore, the present study focuses on the effect of epoxy compounds and glutaraldehyde on the PERV presence in porcine heart valves prepared for clinical use. METHODS: Porcine aortic heart valves were fixed with ethylene glycol diglycidyl ether (EDGE) at 5 °C and 25 °C as well as with glutaraldehyde (GA) for 4 weeks. Salting out was used to isolate genomic DNA from native as well as EDGE- and GA-fixed fragments of valves every week. Quantification of PERV-A, PERV-B, and PERV-C DNA was performed by real-time quantitative polymerase chain reaction (QPCR). RESULTS: All subtypes of PERVs were detected in native porcine aortic heart valves. The reduction of the PERV-A, PERV-B, and PERV-C DNA copy numbers was observed in the heart valves which were EDGE-fixed at both temperatures, and in GA-fixed ones in the following weeks. After 7 and 14 days of EDGE cross-linking, significant differences between the investigated temperatures were found for the number of PERV-A and PERV-B copies. PERV DNA was completely degraded within the first week of EDGE fixation at 25 °C. CONCLUSIONS: EDGE fixation induces complete PERV genetic material degradation in porcine aortic heart valves. This suggests that epoxy compounds may be alternatively used in the preparation of bioprosthetic heart valves in future.

Quantitative relationships between transforming growth factor beta mRNA isoforms in congenital and traumatic cataracts.

PURPOSE: The aim of this study was to determine differences in the expression profiles of transforming growth factor (TGF) ß isoforms in the fragments of anterior lens capsules (ALCs) and peripheral blood mononuclear cells (PBMCs) of pediatric patients with congenital and traumatic cataracts. METHODS: Forty children with congenital cataracts (19 girls and 21 boys) and 22 children with traumatic cataracts (six girls and 16 boys) participated in the study. Fragments of ALCs obtained during cataract surgery and whole blood samples were analyzed. Quantification of TGFß1, TGFß2, and TGFß3 mRNA was performed by real-time quantitative reverse transcription (QRT)-PCR using SYBR Green I chemistry. RESULTS: TGFß1, TGFß2, and TGFß3 mRNA was detected in all the studied samples. Significant differences were found for TGFß1 and TGFß2 expression profiles in PBMCs between the patients with congenital and traumatic cataracts. The expression profiles of TGFß isoforms in ALCs did not differ significantly between the groups. CONCLUSIONS: Overexpression of TGFß1 and TGFß2 in the PBMCs of patients with congenital cataracts might indicate that these cytokines are involved in the development of lens opacity.