Joanna Gola

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.

Detection of porcine endogenous retrovirus in xenotransplantation

Xenotransplantation can provide a virtually limitless supply of cells, tissues and organs for a variety of therapeutic procedures. Cells and tissues for use in human transplantation procedures could be supplied using material taken from pigs. However, there is a potential risk of transmission of porcine infectious agents, including porcine endogenous retroviruses (PERVs), to a novel human host, with as yet unknown consequences. Three subtypes of PERV have been identified, of which both PERV-A and PERV-B have the ability to infect human cells in vitro. The third subtype, PERV-C, does not show this ability. Recombinant PERV-A/C forms have demonstrated infectivity in human cell culture. Monitoring in xenotransplantation should comprise screening of the source pig herd (PERV-A and PERV-B level expression assessment, PERV-C detection) and screening of recipients (differentiation between PERV transmission and chimerism). The detection of PERVs includes analyses of both DNA and RNA (PCR and RT-PCR), quantitative determination of the level of PERV nucleic acids (real-time PCR and real-time RT-PCR), assessment of reverse transcriptase (RT) activity (RT assays) and viral and recipient protein detection (immunological methods). In summary, all available methods should be used in monitoring of PERVs in xenotransplantation, and caution should be exercised at all stages of monitoring. Such monitoring has enormous significance for eliminating the possibility of transmission of PERV infection, thus contributing to higher levels of safety in xenotransplantation.

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.

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.

Structure and properties of slow-resorbing nanofibers obtained by (co-axial) electrospinning as tissue scaffolds in regenerative medicine

With the rapid advancement of regenerative medicine technologies, there is an urgent need for the development of new, cell-friendly techniques for obtaining nanofibers—the raw material for an artificial extracellular matrix production. We investigated the structure and properties of PCL10 nanofibers, PCL5/PCL10 core-shell type nanofibers, as well as PCL5/PCLAg nanofibres prepared by electrospinning. For the production of the fiber variants, a 5–10% solution of polycaprolactone (PCL) (Mw = 70,000–90,000), dissolved in a mixture of formic acid and acetic acid at a ratio of 70:30 m/m was used. In order to obtain fibers containing PCLAg 1% of silver nanoparticles was added. The electrospin was conducted using the above-described solutions at the electrostatic field. The subsequent bio-analysis shows that synthesis of core-shell nanofibers PCL5/PCL10, and the silver-doped variant nanofiber core shell PCL5/PCLAg, by using organic acids as solvents, is a robust technique. Furthermore, the incorporation of silver nanoparticles into PCL5/PCLAg makes such nanofibers toxic to model microbes without compromising its biocompatibility. Nanofibers obtained such way may then be used in regenerative medicine, for the preparation of extracellular scaffolds: (i) for controlled bone regeneration due to the long decay time of the PCL, (ii) as bioscaffolds for generation of other types of artificial tissues, (iii) and as carriers of nanocapsules for local drug delivery. Furthermore, the used solvents are significantly less toxic than the solvents for polycaprolactone currently commonly used in electrospin, like for example chloroform (CHCl3), methanol (CH3OH), dimethylformamide (C3H7NO) or tetrahydrofuran (C4H8O), hence the presented here electrospin technique may allow for the production of multilayer nanofibres more suitable for the use in medical field.

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

BACKGROUND The transforming growth factor β (TGFβ) 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 TGFβ isoforms, their receptors, and TGFβ-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 TGFβ-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 TGFβ2 and TGFβR3 after tacrolimus treatment, and for TGFβR3 after tacrolimus and LPS treatment in RPE cells were decreased. In turn, in the presence of LPS-induced inflammation, TGFβ2 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 TGFβ, such as inflammation, proliferation, epithelial-mesenchymal transition (EMT), and fibrosis.

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.

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.

Changes in expression of genes related to caspases and BCL-2 family in RPTEC treated with amphotericin B and its modified forms

INT RO DUCT ION: The main limitation of the use of amphotericin B (AmB) – effective in the treatment of systemic fungal infections – is its high toxicity to human cells. The mechanism of AmB toxicity is not clear. Caspase-related and BCL-2 proteins participate in the regulation of apoptosis. Thus, they may be involved in drug toxicity. In this study we evaluated the influence of AmB on the transcriptional activity of genes related to caspases and the BCL-2 family. We also tested the influence of modified forms of AmB: AmB-Cu 2+ (the complex with copper(II) ions) and the AmB-ox (oxidized form). MAT ERIAL AND MET HO DS : Human RPTECs (Renal Proximal Tubule Epithelial Cells) were treated with AmB, AmB-Cu 2+ and AmB-ox. Total RNA was extracted using the phenol-chloroform method. The expression profiles of genes related to caspase activity and BCL-2 were determined using oligonucleotide microarrays (HG-U133A 2.0, Affymetrix). Analysis included 67 ID related to caspases and 32 ID associated with BCL-2, according to the Affymetrix database. RES ULTS : The analysis revealed upregulation of the BCL-2 and BCL2L1genes in the cells treated with AmB-Cu 2+ , in comparison to the control. In both the AmB and AmB-Cu 2+ -treated cells, differentiating genes were associated with inflammation and mitophagy activated by intrinsic signals. In the cells treated with AmB-ox, the BCL-2 genes were downregulated. CO NCL US IO NS : The results suggest that AmB and AmB-Cu activate genes involved in the regulation of inflammation and autophagy induced by intrinsic signals, but overexpression of BCL-2 and BCL2L1 may protect AmB-Cu 2+ -treated cells from death. In the cells treated with AmB-ox extrinsic signals prevail, indicating the distinct molecular mechanism of its cytotoxicity. Received: 24.01.2017 Revised: 23.07.2017 Accepted: 24.07.2017 Published online: 26.03.2018 Address for correspondence: Dr n. med. Joanna Gola, Katedra i Zakład Biologii Molekularnej, Wydział Farmaceutyczny z Oddziałem Medycyny Laboratoryjnej w Sosnowcu, Śląski Uniwersytet Medyczny w Katowicach, ul. Jedności 8, 41-200 Sosnowiec, tel. tel. + 48 32 364 10 27, e-mail: jgola@sum.edu.pl Copyright