Selective human cells in vitro transduction with porcine endogenous retrovirus (PERV).

Porcine xenograft transplantation raises concerns in humans about the risk of infection with porcine endogenous retroviruses (PERV) as they are an integral part of the pig genome and are therefore very difficult to exclude. In this study, for the first time, a relationship between the provirus genes sequences and released virions from pig cell line and the embedded sequence of this retrovirus in infected human cells was analyzed. PERV infection of human cells HEK-293 and HeLa and detection of PERV in pig PK-15 cells and supernatant were assessed by QPCR or RT-QPCR using primers specific for envA, envB, gag, pol genes and LTR region. Sequence analysis was performed at the DNA level and changes in the amino acid sequence were deduced in silico. Fifty nucleotide substitutions (45 in pol, 3 in gag and one each in envA and envB) were detected and most of these were heterozygous (42), which were present mainly in PK-15 cells. Our results show that sequence of the pol gene and the Pol protein is less conserved compared to the other PERV genes and PERV with some polymorphisms were not released from pig cells or/and do not infect human cells. PERV virions with a homozygous allele system were released from PK-15 cells, although their sequence replicated on the basis of the heterozygous PERV provirus sequence in PK-15. The newly discovered selective transduction of human cells with PERV will be helpful in studying the characteristics and genetic variability of the retrovirus genes to ensure safe xenotransplantation. Keywords: PERV; porcine endogenous retroviruses; infection; genetic polymorphism; xenotransplantation.

Cytotoxicity of etoposide in cancer cell lines in vitro after BCL-2 and C-RAF gene silencing with antisense oligonucleotides.

BCL-2 and C-RAF genes are overexpressed in most types of cancers. Although these genes are mediators in different molecular pathways their main characteristic is the antiapoptotic activity, thus cells that overexpress either BCL-2 or C-RAF lose their ability to undergo apoptotic death being resistant to chemotherapeutic agents and/or physiologic mediators of cell death (e.g., TNF-alpha). Both anti-C-RAF, and anti-BCL-2 oligonucleotides were tested as chemosensitizers in cancer therapy. The aim of the study was to investigate the effects of the combined use of antisense oligonucleotides (ASOs) targeting BCL-2 and C-RAF transcripts on the in vitro cancer cell cultures exposed to etoposide. Cells were transfected with phosphorothioate BCL-2 and C-RAF ASOs. To sustain high intracellular level of ASOs, 3-day transfection was used, and it was followed by a single treatment with 20 microM etoposide for 5 h. The following cancer cell lines were tested: A549, HeLa, and T24. Sequence-specific decrease in BCL-2, and C-RAF mRNA levels were confirmed by real-time RT-PCR: after 1-day treatment mRNA levels decreased by 9-42% of the normal expression in cells treated with 50-1200 nM ASOs. Also, the induction of cell death in all transfected cultures in a concentration-dependent manner was confirmed by MTT assay,microscopic analysis of cell morphology, and the measurement of histone H3 expression. Results also showed that both ASOs effectively potentiated etoposide-induced cytotoxicity; the strongest effects were obtained in A549 (lung cancer). This observation suggests that lower concentrations of both antisense oligonucleotides may be used, at least for this type of cancer, to obtain high efficiency of etoposide-induced cell death enhancement. Simultaneous use of two ASOs in 3-day treatment allows us to lower concentrations needed to obtain significant treatment results thus enabling to diminish sequence-unspecific toxicity.

Impact of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum bones after sternotomy.

Median sternotomy is the surgical method of choice for many procedures where one of the main problems is the long post-operative wound healing process leading to sternal dehiscence and the development of infection. This leads to prolonged hospital stay and increased mortality due to post-operative complications. A promising solution seems to be the use of allogeneic chondrocytes for wound treatment, whose properties in the field of cartilage reconstruction are widely used in medicine, mainly in orthopedics. In the present study, we investigated the effect of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum after sternotomy. We optimized the culture conditions for the isolated chondrocytes, which were then applied to the sternal incision wound. Chondrocytes in the culture were assessed on the basis of the presence of chondrocyte-specific genes: Sox9, Aggrecan and Collagen II. In turn, the histopathological and immunohistochemical evaluation was used to assess the safety of implantation. In our work, we demonstrated the possibility of obtaining a viable culture of chondrocytes, which were successfully introduced into the sternal wound after sternotomy. Importantly, implantation of allogeneic chondrocytes showed no significant side effects. The obtained results open new possibilities for research on the use of allogeneic chondrocytes in the process of accelerating wound healing after median sternotomy.

Repetitive extragenic palindromic PCR (REP-PCR) as a method used for bulking process detection in activated sludge.

Bulking of activated sludge is a world-widely prevalent problem and can lead to loss of bio-oxidation, further deterioration of effluent quality, and even to a complete breakdown of the entire treatment process. Most common reasons of bulking are bacterial community changes, especially excessive growth of filamentous bacteria or excess of biopolymers on surface of non-filamentous microbes. Because of complex nature of the bulking phenomenon, the successful bulking control strategy finding is still a very important need awaiting new options and advices. The repetitive extragenic palindromic PCR (REP-PCR) fingerprinting method has been applied to distinguish bacterial community in non-bulking and bulking activated sludge. The characteristic REP-PCR fingerprinting patterns, using the Ward's clustering method, have been analyzed to determine homology/similarity relation between particular non-bulking and bulking sludge sampling. The received clustering results were in high concordance with activated sludge typing done based on physicochemical sludge analysis. The choice and application of molecular typing method in sludge analysis will depend upon the needs, skill level, and resources of the laboratory. The proposed REP-PCR method and statistical analysis of fingerprinting patterns seems to be simple, rapid, and effective methods to show differences between population in non-bulking and bulking activated sludge. It is easy to implement, and it may be useful for routinely activated sludge monitoring as well as may be helpful in early detection of bulking process.

Repetitive extragenic palindromic PCR (REP-PCR) as an alternative method for detection of bulking in activated sludge.

Bulking of activated sludge is a world-wide problem which negatively affects wastewater treatment efficiency. The most common reasons of bulking are bacterial community changes, especially excessive growth of filamentous bacteria (filamentous bulking) or excess of biopolymers on the surface of non-filamentous microbes (non-filamentous or Zoogleal bulking). Because of the complex nature of the bulking phenomenon finding a successful bulking control strategy remains a very important issue that awaits new options and advices. The REP-PCR fingerprinting method has been applied to distinguish a bacterial community in non-bulking and bulking activated sludge. The characteristic REP-PCR fingerprinting patterns were compared with each other in terms of the presence or absence of bands and in terms of measured integrated optical density (IOD) of the bands. The obtained fingerprinting patterns, using Ward's clustering method, have been analyzed to determine homology/similarity relations between specific non-bulking and bulking sludge sampling. The received clustering results were in high concordance with activated sludge typing which generally is done based on physicochemical sludge analysis. The proposed REP-PCR method and statistical analysis of fingerprinting patterns seems to be a simple, rapid and effective method revealing differences between populations in non-bulking and bulking activated sludge. It may be useful for routine activated sludge monitoring and may be helpful in the early detection of the bulking process.

Influence of New Synthetic Xanthones on the Proliferation and Migration Potential of Cancer Cell Lines In Vitro.

BACKGROUND: Natural plant metabolites and their semisynthetic derivatives have been used for years in cancer therapy. Xanthones are oxygenated heterocyclic compounds produced as secondary metabolites by higher plants, fungi or lichens. Xanthone core may serve as a template in the synthesis of many derivatives that have broad biological activities. OBJECTIVE: This study synthesized a series of 17 new xanthones, and their anticancer potential was also evaluated. METHODS: The anticancer potential was evaluated in vitro using a highly invasive T24 cancer cell line. Direct cytotoxic effects of the xanthones were established by IC50 estimation based on XTT assay. RESULTS: 5 compounds of the total 17 showed significant cytotoxicity toward the studied cancer cultures and were submitted to further detailed analysis, including studies examining their influence on gelatinase A and B expression, as well as on the cancer cells migration and adhesion to an extracellular matrix. These analyses were carried out on five human tumor cell lines: A2780 (ovarian cancer), A549 (lung cancer), HeLa (cervical cancer), Hep G2 (liver cancer), and T24 (urinary bladder cancer). All the compounds, especially 4, showed promising anticancer activity: they exhibited significant cytotoxicity towards all the evaluated cell lines, including MCF-7 breast cancer, and hindered migration-motility activity of cancer cells demonstrating more potent activity than α-mangostin which served as a reference xanthone. CONCLUSION: These results suggest that our xanthone derivatives may be further analyzed in order to include them in cancer treatment protocols.

Influence of interferon-alpha and ribavirin on the transcription of interferon-gamma and IFN-gamma receptor genes in Jurkat cells.

Interferon-alpha and ribavirin are currently the only drugs registered in the chronic hepatitis C therapy. Their actions are based on both direct antiviral activities, and their influence on genes expressions. In the presented study, using Jurkat cell line as an in vitro model for interferon-gamma synthesis, influence of interferon-alpha and ribavirin on the expressions of IFN-gamma and its receptor subunits (IFNgR1 and IFNgR2) were studied. Expressions of the studied genes were measured at the transcriptional level using Real-Time RT-PCR method. Results indicate that both drugs, IFN-alpha and ribavirin, induced changes in IFN-gamma and its receptor expressions. While IFN-alpha stimulated the expressions of the studied genes (IFN- gamma, IFNgR1, and IFNgR2), ribavirin showed the contradictory influence. The inhibitory effect of ribavirin dominated IFN-alpha action and was responsible for the decrease in the mRNA levels of IFN-gamma and the receptor of IFN-gamma. This phenomenon observed at in vitro model may be responsible for the IFN-gamma decrease during hepatitis C therapy with IFN-alpha and ribavirin which was suggested by some authors.

[Changes in etoposide-induced apoptosis of HeLa tumor cells transfected with antisense oligonucleotide for BCL-2 mRNA]. / Zmiany w indukcji apoptozy komórek linii nowotworowej HeLa za pomoca etopozydu w obecnosci oligonukleotydu antysensownego dla mRNA genu BCL-2.

UNLABELLED: Overexpression of genes involved in proliferation, including genes of BCL family, is often found in cells of most malignant tumors. Currently developed strategies of cancer treatment include trials combining classical chemotherapy with silencing of genes expression using the gene therapy. The aim of the study was to induce silencing of BCL-2 gene expression in HeLa tumor cell line using antisense oligonucleotides (ASOs) technique. MATERIAL AND METHODS: Studies were carried out on in vitro HeLa cell cultures treated with etoposide. Cells were transfected by lipofection with ASO targeting BCL-2 mRNA. Effects of BCL-2 silencing were determined by Real-Time RT-PCR, proliferation/cytotoxicity MTT [3-(4,5-dimethylthiazol-2-yl)-2-5-dipheryl tetrazolium bromide] test, and by microscopic detection of apoptotic cells. RESULTS: We showed a decrease in BCL-2 mRNA level in cells transfected with anti-BCL-2 ASO at concentration ranging from 50 to 1200 nM. Apoptotic cells were detected more frequently in transfected cultures compared with untreated controls. However, MTT tests did not display significant decrease of cell proliferation in the transfected cultures as compared with cultures treated with etoposide alone. CONCLUSIONS: 1. These results indicate that the studied ASO sequence-specifically decreases BCL-2 expression in HeLa cells in vitro, although its action is limited mostly by transfection efficiency. 2. The use of the anti-BCL-2 oligonucleotide in combination with etoposide results in significant deacrease of proliferation in cell cultures and this phenomenon is a result of synergy between used chemotherapy and gene therapy.

Analysis of swine leukocyte antigen class I gene profiles and porcine endogenous retrovirus viremia level in a transgenic porcine herd inbred for xenotransplantation research.

Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.

Synthesis and in vitro Evaluation of the Anticancer Potential of New Aminoalkanol Derivatives of Xanthone.

A series of 15 derivatives of xanthone were synthesized and evaluated for the anticancer activity. The structure of the tested compounds was diversified to establish structureactivity relationships. The following evaluations were carried out: cytotoxicity-proliferation tests, apoptosis detection, expression of apoptosis and proliferation-related genes, expression and activity of gelatinases A and B, wound migration assays, and cell adhesion to MatrigelTMcoated plates. Four compounds (7, 12, 13 and 15) displayed direct cytotoxicity at micromolar concentrations toward the studied cell lines. They also significantly affected the expression of proliferationapoptosis markers, and 13 demonstrated as strong influence as α-mangostin, that served as a natural standard in our study. These four compounds also decreased the expression and activity of gelatinases, and inhibited the migration-motility potential of cancer cells. The influence of compounds 7 and 12 on MMPs mRNA levels even exceeded the activity of α-mangostin and shRNA-mediated silencing; zymography revealed that 7, 13 and 15 were as equally active as α-mangostin, despite their higher IC50 values. The highest activity to inhibit motility and migration of cancer cells was demonstrated by 7, 12, 15, and by α-mangostin; and this was almost equal to shRNA-mediated silencing. Structural features predetermining compound activity were: substitution at position C4 instead of C2, and presence of a chlorine atom and allyl moiety. These results indicate that synthesis of aminoalkanol derivatives of xanthone may lead to successful establishment of new potential anticancer chemicals.

Modulation of porcine endogenous retroviruses (PERVs) expression in vitro by shRNA in the presence of cyclosporine A and dexamethasone.

BACKGROUND: Despite the fact that the risk of Porcine Endogenous Retroviruses (PERV) infection and propagation in human recipients is extremely low, such an event cannot be completely ruled out, especially in immunosuppressed patients. Therefore, the aim of this study was to analyze the expression of PERVs in vitro in the presence of immunosuppression agents: cyclosporine A (CsA), and dexamethasone (DEX). We investigated the possible interactions between immunosuppression drugs, CsA and DEX, and the efficiency of anti-PERV RNAi. MATERIAL/METHODS: Plasmid-based vectors expressing shRNAs against all PERV genes were constructed and analyzed. PERVs expression in cultures transfected with anti-PERV RNAi constructions and treated with CsA or DEX was analyzed by Real-Time RT-PCR, Western blot, and by the measurement of RT activity. RESULTS: Both CsA and DEX inhibited PERVs expression in cell cultures in vitro. RNAi constructions efficiently knocked down PERV expression in Circe, and de novo PERV-infected HeLa and HEK-293 cell cultures. Pretreatment of Circe cultures with CsA or DEX increased PERVs knockdown by RNAi, but no specific interaction between the drugs and transfection efficiency was observed. CONCLUSIONS: Our results demonstrate that cyclosporine A and dexamethasone decrease expression of PERVs in vitro. We also proved that these drugs did not synergize or antagonize RNAi-mediated knockdown of PERVs. These observations may be beneficial in immunosuppressed xenograft recipients; however, due to the controversial literature data concerning influence of immune suppression on graft recipients, our results should be further analyzed.