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.

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.

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.

Differential expression of inflammation-related genes after intense exercise.

The present study focused on the identification of the difference in expression of inflammation-related genes after intense exercise by oligonucleotide microarray methods. This may finally lead to an improved understanding of underlying cellular and molecular mechanism of the immunological alterations in response to exercises. The study group consisted of three healthy road cyclists. Peripheral blood mononuclear cells (PBMCs) were collected preexercise, immediately post-exercise and after 15 min of recovery. The analysis of the expression profile of genes related to the inflammation was performed in PBMCs using HG-U133A oligonucleotide microarrays. 4 genes were found to be regulated by more than 2.0-fold (IL1R2, IL2RB, IL8, IL8RB). Venn diagram indicated that only one of differentially expressed genes (TXLNA) remains the same in each comparison. The balance of both pro- and anti-inflammatory cytokines after exercise seems to be important for athletes. Optimal inflammatory and immune response may help optimize exercise regimes, link physical activity with health and diagnose or prevent athletes from overtraining.

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 porcine endogenous retroviruses in different organs of transgenic pigs generated for xenotransplantation.

The pig appears to be the most promising animal donor of organs for use in human recipients. Among several types of pathogens found in pigs, one of the greatest problems is presented by porcine endogenous retroviruses (PERVs). Screening of the source pig herd for PERVs should include analysis of both PERV DNA and RNA. Therefore, the present study focuses on quantitative analysis of PERVs in different organs such as the skin, heart, muscle, and liver and blood of transgenic pigs generated for xenotransplantation. Transgenic pigs were developed to express the human α-galactosidase, the human α-1,2-fucosyltransferase gene, or both genetic modifications of the genome (Lipinski et al., Medycyna Wet 66:316-322, 2010; Lipinski et al., Ann Anim Sci 12:349-356, 2012; Wieczorek et al., Medycyna Wet 67:462-466, 2011). The copy numbers of PERV DNA and RNA were evaluated using real-time Q-PCR and QRT-PCR, respectively. Comparative analysis of all PERV subtypes revealed the following relationships: PERV A > PERV B > PERV C. PERV A and B were found in all samples, whereas PERV C was detected in 47 % of the tested animals. The lowest level of PERV DNA was shown in the muscles for PERV A and B and in blood samples for PERV C. The lowest level of PERV A RNA was found in the skin, whereas those of PERV B and C RNA were found in liver specimens. Quantitative analysis revealed differences in the copy number of PERV subtypes between various organs of transgenic pigs generated for xenotransplantation. Our data support the idea that careful pig selection for organ donation with low PERV copy number may limit the risk of retrovirus transmission to the human recipients.

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.

Gene expression of IGF1, IGF1R, and IGFBP3 in epiretinal membranes of patients with proliferative diabetic retinopathy: preliminary study.

The molecular mechanism formation of secondary epiretinal membranes (ERMs) after proliferative diabetic retinopathy (PDR) or primary idiopathic ERMs is still poorly understood. Therefore, the present study focused on the assessment of IGF1, IGF1R, and IGFBP3 mRNA levels in ERMs and PBMCs from patients with PDR. The examined group comprised 6 patients with secondary ERMs after PDR and the control group consisted of 11 patients with idiopathic ERMs. Quantification of IGF1, IGF1R, and IGFBP3 mRNAs was performed by real-time QRT-PCR technique. In ERMs, IGF1 and IGF1R mRNA levels were significantly higher in patients with diabetes compared to control subjects. In PBMCs, there were no statistically significant differences of IGF1, IGF1R, and IGFBP3 expression between diabetic and nondiabetic patients. In conclusion, our study indicated IGF1 and IGF1R differential expression in ERMs, but not in PBMCs, of diabetic and nondiabetic patients, suggesting that these factors can be involved in the pathogenesis or progression of proliferative vitreoretinal disorders. This trial is registered with NCT00841334.