Novel Xanthone Derivatives Impair Growth and Invasiveness of Colon Cancer Cells In Vitro.

Natural xanthones are a large group of compounds from which promising anticancer properties could be further developed by chemical modifications. This study aimed to investigate the influence of four novel xanthone derivatives based on a naturally occurring xanthone skeleton on the invasiveness of colon cancer cells in vitro. First, the concentrations required to inhibit growth of three colorectal cancer cell lines to 50% (GI50) of all the studied compounds, as well as the natural xanthones used as a reference (gambogic acid and α-mangostin), have been established (MTS reduction test). Next, the assays determining several aspects of the GI25 xanthones influence on colorectal cancer cells, including cytotoxicity, migration and invasion potential, interaction with extracellular matrix and endothelial cells, as well as expression of selected invasiveness related genes have been performed. Our results demonstrate that these novel xanthone derivatives impair colorectal cancer proliferation, motility, adhesion to extracellular matrix and to endothelial cells, and also induce apoptosis and cell death. Moreover, their activity is comparable to cisplatin and 5-fluorouracil, used as reference compounds. Conducted research indicates our compounds for further research and development as novel drugs in colorectal cancer treatment.

Morin exerts anti-metastatic, anti-proliferative and anti-adhesive effect in ovarian cancer cells: an in vitro studies.

The influence of morin hydrate on changes of proliferative, metastatic, and adhesive potential of human ovarian cancer cells concerning the influence of decitabine, and decitabine with trichostatin A, and in comparison to untreated cells, were analyzed. The effect of morin hydrate, decitabine, and trichostatin A were examined in A2780 and SKOV-3 ovarian cancer cell lines using MTS assay, clonogenic assay, adhesion to endothelial HMEC-1 cells, transwell migration assay and cell cycle analysis. The expression level of epithelial to mesenchymal transition (EMT) markers was quantified using PCR Array in relation to the level of global methylation determined with Methylated DNA Quantification Kit. We observed statistically significant inhibition of adhesive and migratory potential of both cell lines and the accumulation of G0/G1 phase A2780 cells after treatment with morin hydrate. Our studies confirmed the influence of morin hydrate on down-regulation of genes considered as up-regulated during EMT, and up-regulation of some genes considered as down-regulated during EMT in A2780 and SKOV-3 cells. Phenotypic changes were associated with molecular changes in cells, eg. decrease of the expression level of genes associated with adhesion, and an increase of genes down-regulated during EMT, after morin hydrate treatment in comparison to untreated control cells in both cell lines, were observed.

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.

MiR-424-3p suppresses galectin-3 expression and sensitizes ovarian cancer cells to cisplatin.

PURPOSE: Assessment of miR-424-3p mimic capability to sensitize SK-OV-3 and TOV-21G ovarian cancer cells to cisplatin by decreasing the expression of galectin-3, which is an anti-apoptotic protein overexpressed in ovarian cancer and associated with resistance to chemotherapy. METHODS: We performed a reverse transfection of miR-424-3p mimic into SK-OV-3 and TOV-21G ovarian cancer cells, followed by Real Time™ RT-PCR analysis of the expression of miR-424-3p and galectin-3 mRNA as well as ELISA assay for galectin-3 protein level. Next, we studied the viability (XTT assay), proliferation (EdU incorporation assay), and apoptosis (ELISA assay) of the both cell lines transfected with the mimic and treated with cisplatin. RESULTS: We demonstrated that miR-424-3p mimic effectively transfects into SK-OV-3 and TOV-21G ovarian cancer cells in which it significantly suppresses the expression of galectin-3 at the protein level, but not at the mRNA level. Reverse transfection of both cell lines with the mimic, followed by treatment with cisplatin, resulted in a reduction in cell viability and proliferation as well as an increase in the induction of apoptosis. CONCLUSIONS: MiR-424-3p mimic sensitizes SK-OV-3 and TOV-21G ovarian cancer cells to cisplatin by decreasing the expression of galectin-3.

Morin decreases galectin-3 expression and sensitizes ovarian cancer cells to cisplatin.

PURPOSE: This study aimed at evaluating whether morin (a natural flavonoid and a known inhibitor of NF-κB) can sensitize ovarian cancer cells to cisplatin by decreasing the expression of galectin-3, which is an anti-apoptotic protein regulated by NF-κB transcription factor. METHODS: To assess the possibility of augmentation the activity of cisplatin by morin, we studied the separate and the combined effect of morin and cisplatin on viability, proliferation, and apoptosis of TOV-21G (cisplatin-sensitive) and SK-OV-3 (cisplatin-resistant) ovarian cancer cells. We also analysed the effect of morin and cisplatin on galectin-3 expression at the mRNA and protein levels. RESULTS: We demonstrated that morin possess antitumor activity against TOV-21G and SK-OV-3 ovarian cancer cells by reducing cell viability and proliferation as well as increasing the induction of apoptosis. Co-treatment of the cells with selected concentrations of morin and cisplatin, accordingly to specific treatment approaches, reveals a synergism, which leads to sensitization of the cells to cisplatin. During this sensitization, morin significantly reduces the expression of galectin-3 at the mRNA and protein level, regardless of the presence of cisplatin. CONCLUSIONS: Morin sensitizes TOV-21G and SK-OV-3 ovarian cancer cells to cisplatin, what is associated with a decrease of the expression of galectin-3.

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.

Contribution of reactive oxygen species to the anticancer activity of aminoalkanol derivatives of xanthone.

Reactive oxygen species (ROS) are critically involved in the action of anticancer agents. In this study, we investigated the role of ROS in the anticancer mechanism of new aminoalkanol derivatives of xanthone. Most xanthones used in the study displayed significant pro-oxidant effects similar to those of gambogic acid, one of the most active anticancer xanthones. The pro-oxidant activity of our xanthones was shown both directly (by determination of ROS induction, effects on the levels of intracellular antioxidants, and expression of antioxidant enzymes) and indirectly by demonstrating that the overexpression of manganese superoxide dismutase decreases ROS-mediated cell senescence. We also observed that mitochondrial dysfunction and cellular apoptosis enhancement correlated with xanthone-induced oxidative stress. Finally, we showed that the use of the antioxidant N-acetyl-L-cysteine partly reversed these effects of aminoalkanol xanthones. Our results demonstrated that novel aminoalkanol xanthones mediated their anticancer activity primarily through ROS elevation and enhanced oxidative stress, which led to mitochondrial cell death stimulation; this mechanism was similar to the activity of gambogic acid.

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.

MMP-9 directed shRNAs as relevant inhibitors of matrix metalloproteinase 9 activity and signaling.

INTRODUCTION: The main function of matrix metalloproteinases is the degradation of extracellular matrix components, which is related to changes in the proliferation of cells, their differentiation, motility, and death. MMPs play an important role in physiological processes such as embryogenesis, angiogenesis and tissue remodeling. The increase of MMPs activity is also observed in pathological conditions including tumorigenesis where MMP-2 (gelatinase A) and MMP-9 (gelatinase B) show the ability to degrade the basement membrane of vessels and they are involved in metastasis. The aim of our study was to verify the changes of MMP-9 enzymatic activity and the mobility of cells after inhibition of MMP-9 gene expression. MATERIAL AND METHODS: The oligonucleotide shRNA insert had been designed to silence MMP-9 gene expression and was cloned into the pSUPER.neo expression vector. The construct was introduced into the HeLa (CCL-2) cervical cancer cells by lipotransfection. Simultaneously in control cells MMP-9 were inhibited by doxycycline. Changes in activity of MMP-9 were analyzed by gelatin zymography and wound-healing assay. RESULTS/CONCLUSIONS: Gelatin zymography allowed us to confirm that activity of MMP-9 in cells transfected by shRNA-MMP-9 and treated by doxycycline were similar and significantly lower in comparison with control cells. Phenotypic tests of migration in vitro confirm statistically significant (P<0.05) changes in cell migration - control cells healed 3 to 5 times faster in comparison with transfected or doxycycline treated cells. Our studies show the significant role of MMP-9 in mobility and invasiveness of tumor cells, thus indicating a potential target point of interest for gene therapy.

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.

Gene expression and activity of antioxidant enzymes in the blood cells of workers who were occupationally exposed to lead.

In this study, we sought to understand the influence of occupational lead-exposure on the gene expression (Sod1) and activity (SOD) of superoxide dismutase, catalase and glutathione peroxidase (GPx, Gpx1) in leukocytes and erythrocytes. The study group consisted of 45 healthy male employees of a lead-zinc works and was divided into two subgroups: those with low exposure to lead (LE) and those with high exposure to lead (HE). In addition, 17 healthy male administrative workers participated in the study as the control group. The gene expression levels of both Sod1 and Gpx1 were significantly increased in the LE group as compared to the control group. By contrast, we noted only an insignificant tendency for increased gene expression of both Sod1 and Gpx1 in the HE group. The expression and activity of catalase were unchanged. Nevertheless, SOD and GPx activities in erythrocytes was significantly elevated in both examined subgroups, whereas SOD activity in leukocytes was raised only in the LE group. The results of this study led us to conclude that lead has a significant influence not only on the activities of antioxidant enzymes but also on the dose-dependent expression in their genes.

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.

The efficiency of silencing expression of the gene coding STAT3 transcriptional factor and susceptibility of bladder cancer cells to apoptosis.

AIM OF THE STUDY: Abnormalities in signaling as well as altered gene expression have been identified in numerous diseases, including cancer. The biological functions of signal transducer and activator of transcription 3 (STAT3) are very broad. It is thought that STAT3 can also contribute to oncogenesis. RNA interference (RNAi) is one of the most efficient tools for silencing gene expression within cells. The main goal of the study was to verify the effectiveness of STAT3 gene silencing and its influence on cell proliferation and activation of apoptosis in bladder cancer cells. MATERIAL AND METHODS: The study was conducted on cellular material, which was the stable human bladder cancer cell line T24. The synthesis of shRNA (short hairpin RNA) interfering with the STAT3 gene was based on pSUPER. neo expression vector. The gene expression at the mRNA level was determined by the real-time PCR method. The influence of STAT3 gene silencing on apoptosis induced in cells with modulated STAT3 expression was evaluated using parallel quantification of mono- and oligonucleosomal DNA degradation of genomic DNA. RESULTS: In transfected T24 cells, the STAT3 mRNA expression decreased to the level of 68.3% compared to the scrambled (SCR) control. Silencing the STAT3 gene induced changes in the phenotype of T24 cells. Statistically significant differences in cell proliferation (p = 0.0318) and apoptosis induction (p = 0.0376) were observed. CONCLUSIONS: Application of the designed shRNA for the STAT3 gene contributed to a decrease of expression of the examined gene. It also decreased the proliferation and increased the susceptibility to apoptosis in T24 bladder cancer cells.

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.

[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.

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.

Distribution of porcine endogenous retroviruses (PERVs) DNA in organs of a domestic pig.

OBJECTIVES: Domestic pig may serve as the most appropriate organ source for human xenotransplantation in the future. However, there is a serious threat of xenogeneic pathogens transmission, especially porcine endogenous retroviruses (PERVs) which are present in genomes of all pigs. The aim of this study was to monitor the prevalence and distribution of PERV DNA in organs of a domestic pig. METHODS: We used a primer set for a highly conserved fragment of PERV gag sequence to monitor a total PERV DNA copy number and genotype-specific primer sets to study PERV subtypes distribution using Real-Time QPCR (SYBR Green I). RESULTS: Our results showed that PERV DNA was present in all studied pigs, however, most PERV DNA molecules carried numerous mutations thus indicating inability to express functional retroviral particles. The level of PERV DNA in kidney was much higher than in heart (p = 0.007) and in the liver (p = 0.009). CONCLUSIONS: It indicates that kidney is potentially the biggest PERV reservoir which makes it the organ of particular concern in xenotransplantation. We also conclude it is possible to monitor pig herds for individuals with the lowest PERV DNA prevalence, especially lacking PERV-C, and perhaps with only defective PERV proviruses that are unable to express functional RNA.

Changes in TNF-alpha mRNA levels in the peripheral blood of patients with chronic hepatitis C virus (HCV) infection during alpha-interferon and ribavirin therapy.

HCV virus infections have become a serious epidemiological problem throughout the world. Hepatitis C therapy includes the administration of IFN-alpha and ribavirin, but results in the complete eradication of HCV viremia in only 30% of patients. TNF-alpha is one of the factors involved in hepatitis C pathogenesis and the results of therapy. In this study, we present the results of applying real-time RT-PCR for assessing the TNF-alpha mRNA level in the peripheral blood of patients treated with IFN-alpha and ribavirin. We found the TNF-alpha mRNA level to be higher in HCV-infected patients compared with healthy controls when analyzed after 4 weeks (p = 0.001) and 3 months (p = 0.003) of IFN-alpha/RIBA therapy. The pretreatment level and the level after six months of therapy were not significantly different from the level of healthy controls. There were no significant differences in TNF-alpha mRNA levels between patients who responded to anti-HCV therapy, resulting in a decrease in HCV viremia below detection limit over 6 months and patients whose HCV RNA was not eliminated (p = 0.881). These results indicate that there is a transient increase of TNF-alpha gene expression during anti-HCV therapy. This fact may be connected with the host organism's response to IFN-alpha/RIBA therapy.