Magdalena C. Kimsa

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.

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.

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.

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.

Adrenergic Response to Maximum Exercise of Trained Road Cyclists

Abstract The aim of this study was to evaluate adrenergic responses in the peripheral blood of trained road cyclists at rest, at maximal intensity of incremental bicycle exercise test, and during 15 minutes of recovery, as well as the relationship between them. Competitive male road cyclists, in the pre-competitive phase of a season, mean age 21.7 ± 6.4 years, and BMI 20.7 ± 0.8 kg·m-2, performed an incremental test on a bicycle ergometer with unloaded cycling for 5 min, then increased the load to 40 W every 3 min, up to maximal exercise intensity. The plasma catecholamine concentrations (epinephrine, norepinephrine) and oxygen uptake were estimated. The expression of 132 genes related to the adrenergic system in leukocytes was measured. A statistically significant increase in plasma epinephrine concentration (p < 0.01) was observed in response to exercise. The mean of maximal oxygen uptake was 65.7 ± 5.5 ml·kg-1·min-1. The RGS2 gene expression was highest regardless of the test phase for all athletes. The effort had a statistically significant influence on ADRB2 and RAB2A expression. In addition, the RAB2A, ADM and HSPB1 expression level increased during recovery. We can conclude that plasma epinephrine concentration and genes related to the adrenergic system such as ADM, ADRB2, CCL3, GPRASP1, HSPB1, RAB2A, RGS2 and ROCK1 seem to have an influence on the response to high-intensity exercise in trained cyclists.

Interleukin 2 as a potential cancer marker in patients after kidney transplantation

INTRODUCTION Transplant recipients have a significantly greater incidence of cancer, compared with the general population, who are referred to immunosuppressive therapy as an additional malignancy risk factor. Therefore, there is a need to search for an easy in clinical practice neoplasm predictor, especially for this group of patients. MATERIALS AND METHODS A group of 74 (43M and 31F; aged 46.8 ± 12 years) kidney transplant recipients was investigated in a three-year follow-up study. During the time of observation, 7 patients were diagnosed with neoplasm (7.4 ± 1.5 years after transplantation). A serum level of IL2 (ELISA test) and mRNA level of IL1beta, IL10 and TNFalfa in peripheral mononuclear blood cells - PBMCs (QRT - PCR method) were measured in every year of observation. Analysis of variances and t-Student test were used in groups mean comparison: N - patients developing malignant neoplasm group (24 probes); M - set of probes from patients with malignancies at the moment of diagnosis (11 probes); P - set of probes from patients before developing malignant neoplasm (10 probes); C - control group of healthy transplant recipients (31 probes). RESULTS Among the analyzed agents, only serum IL2 level differed between the analyzed groups, with higher values in the M compared with the P group (p<0.05) and with C group (p<0.01). There were no differences neither between N and C or P and C groups (p = 0.98), nor any correlation between IL2 and IL1b, IL2 and TNFalfa. CONCLUSIONS The results may indicate that IL2 serum level might be consider as a useful late unspecific cancer marker, although larger studies should yield verification of this finding.

Expression pattern of the transforming growth factor β signaling genes in human peripheral blood mononuclear cells after exercise—inflammatory aspects

The present study has focused on the identification of differences between the expression pattern of TGF‐β signaling pathway genes in athletes after exercise.