Comparative EPR Studies on the Influence of Genistein on Free Radicals in Non-Irradiated and UV-Irradiated MCF7, T47D and MDA-MB-231 Breast Cancer Cells.

The antioxidant activity and the association of genistein with carcinogenesis are widely documented. Few studies directly measure the number of free radicals generated in cells, either during the action of factors stimulating their formation, e.g., ultraviolet (UV), or after exposure to antioxidants. The most suitable method for analysing free radicals is electron paramagnetic resonance (EPR) spectroscopy. The EPR method detects a paramagnetic centre with a single electron. Antioxidants neutralize free radicals, therefore, EPR analysis of antioxidant efficacy is as valuable and important as studying the paramagnetic centres of radicals. The aim of the study was to determine the influence of genistein on free radicals basal level and after UV exposure in breast cancer cell lines MCF7, T47D and MDA-MB-231 cell lines. The impact of genistein on cell viability was investigated at concentrations of 0.37 µM, 3.7 µM, 37 µM and 370 µM. Genistein at a concentration of 370 µM revealed a cytotoxic effect on the cells of all three tested breast cancer lines. Genistein at a concentration of 0.37 µM showed no significant effect on the cell viability of all tested breast cancer lines. Therefore, cell proliferation and antioxidant properties were examined using genistein at a concentration of 0.37 µM and 37 µM. X-band (9.3 GHz) EPR spectra of three different types of breast cancer cells (ER-positive, PR-positive and HER-2 negative: MCF7 and T47D and triple-negative MDA-MB-231) were compared. UV irradiation was used as a factor to generate free radicals in cells. The effect of free radical interactions with the antioxidant genistein was tested for non-UV-irradiated (corresponding to the basal level of free radicals in cells) and UV-irradiated cells. The levels of free radicals in the non-irradiated cells studied increased in the following order in breast cancer cells: T47D < MDA-MB-231 < MCF7 and UV-irradiated breast cancer cells: MDA-MB-231 < MCF7 < T47D. UV-irradiation altered free radical levels in all control and genistein-cultured cells tested. UV irradiation caused a slight decrease in the amount of free radicals in MCF7 cells. A strong decrease in the amount of free radicals was observed in UV-irradiated MDA-MB-231 breast cancer cells. The amount of free radicals in T47D cancer cells increased after UV irradiation. Genistein decreased the amount of free radicals in non-irradiated and UV-irradiated MCF7 cells, and only a weak effect of genistein concentrations was reported. Genistein greatly decreased the amount of free radicals in UV-irradiated T47D cancer cells cultured with genistein at a concentration of 3.7 µM. The effect of genistein was negligible in the other samples. Genistein at a concentration of 3.7 µM decreased the amount of free radicals in non-irradiated MDA-MB-231 cancer cells, but genistein at a concentration of 37 µM did not change the amount of free radicals in these cells. An increase in the amount of free radicals in UV-irradiated MDA-MB-231 cancer cells was observed with increasing genistein concentration. The antioxidant efficacy of genistein as a potential plant-derived agent supporting the treatment of various cancers may be determined by differences in signalling pathways that are characteristic of breast cancer cell line subtypes and differences in activation of oxidative stress response pathways.

10H-1,9-diazaphenothiazine and its 10-derivatives: synthesis, characterisation and biological evaluation as potential anticancer agents.

10H-1,9-diazaphenothiazine was obtained in the sulphurisation reaction of diphenylamine with elemental sulphur and transformed into new 10-substituted derivatives, containing alkyl and dialkylaminoalkyl groups at the thiazine nitrogen atom. The 1,9-diazaphenothiazine ring system was identified with advanced 1H and 13C NMR techniques (COSY, NOESY, HSQC and HMBC) and confirmed by X-ray diffraction analysis of the methyl derivative. The compounds exhibited significant anticancer activities against the human glioblastoma SNB-19, melanoma C-32 and breast cancer MDA-MB-231 cell lines. The most active 1,9-diazaphenothiazines were the derivatives with the propynyl and N, N-diethylaminoethyl groups being more potent than cisplatin. For those two compounds, the expression of H3, TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The proteome profiling study showed the most probable compound action on SNB-19 cells through the intrinsic mitochondrial pathway of apoptosis. The 1,9-diazaphenotiazine system seems to be more potent than known isomeric ones (1,6-diaza-, 1,8-diaza-, 2,7-diaza- and 3,6-diazaphenothiazine).

The influence of genistein on free radicals in normal dermal fibroblasts and keloid fibroblasts examined by EPR spectroscopy.

Normal and keloid fibroblasts were examined using X-band (9.3 GHz) electron paramagnetic resonance spectroscopy. The effect of genistein on the concentration of free radicals in both normal dermal and keloid fibroblasts after ultraviolet irradiation was investigated. The highest concentration of free radicals was seen in keloid fibroblasts, with normal fibroblasts containing a lower concentration. The concentration of free radicals in both normal and keloid fibroblasts was altered in a concentration-dependent manner by the presence of genistein. The change in intra-cellular free radical concentration after the ultraviolet irradiation of both normal and keloid fibroblasts is also discussed. The antioxidant properties of genistein, using its 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity as a model, were tested, and the effect of ultraviolet irradiation on its interaction with free radicals was examined. The electron paramagnetic resonance spectra of DPPH showed quenching by genistein. The interaction of genistein with DPPH free radicals in the absence of ultraviolet irradiation was shown to be slow, but this interaction was much faster under ultraviolet irradiation. Ultraviolet irradiation enhanced the free radical-scavenging activity of genistein.

MOLECULAR EFFECTS OF AMINE DERIVATIVES OF PHENOTHIAZINE ON CANCER CELLS C-32 AND SNB-19 IN VITRO.

Cancer therapy is challenging for scientists because of low effectiveness of so far existing therapies (especially in case of great invasiveness and advanced tumor stage). Such need for new drug development and search for more efficient new findings in therapeutical applications is therefore still valid. There are also conducted studies on modifying so far existing drugs and their new methods of usage in oncology practice. One of them is phenothiazine and its derivatives which are used in psychiatric treatment for years. They also exhibit antiprion, antiviral, antibacterial and antiprotozoal properties. Cytotoxic activity, influence on proliferation, ability to induce apoptosis suggest also a possibility of phenothiazine derivatives usage in cancer cells termination. The aim of our the study was to evaluate the influence of two amine derivatives of phenothiazine on cancer cells in vitro. Amelanotic melanoma C-32 cell line (ATCC) and glioma SNB-19 cells (DSMZ) were used in this study and two derivatives were analyzed. In view of examined substances tumor potential toxicity cells proliferation and viability exposed to phenothiazine derivatives were established. Cell cycle regulatory genes expression (TP53 and CDKN1A), S-phase marker--H3 gene and intracellular apoptosis pathway genes (BAX, BCL-2) were analyzed using RT-QPCR method. The influence of examined derivatives on total cell oxidative status (TOS), total antioxidative status (TAS), malondialdehyde concentration (MDA) and superoxide dismutase activity (SOD) were analyzed. As a result, examined phenothiazine derivatives cytotoxic action on C-32 and SNB-19 and also cells proliferation inhibition were determined. Cell cycle regulatory genes (TP53, CDKN1A) expression and protein products of genes involved in mitochondial apoptosis pathway (BAX, BCL-2) expression are changed by the presence of phenothiazine derivatives during culturing. There were also noted small changes in redox potential in cells exposed to two mentioned phenothiazine derivatives.

Caffeic Acid phenethyl ester and ethanol extract of propolis induce the complementary cytotoxic effect on triple-negative breast cancer cell lines.

Chemotherapy of breast cancer could be improved by bioactive natural substances, which may potentially sensitize the carcinoma cells' susceptibility to drugs. Numerous phytochemicals, including propolis, have been reported to interfere with the viability of carcinoma cells. We evaluated the in vitro cytotoxic activity of ethanol extract of propolis (EEP) and its derivative caffeic acid phenethyl ester (CAPE) towards two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and Hs578T, by implementation of the MTT and lactate dehydrogenase (LDH) assays. The morphological changes of breast carcinoma cells were observed following exposure to EEP and CAPE. The IC50 of EEP was 48.35 µg∙mL-1 for MDA-MB-23 cells and 33.68 µg∙mL-1 for Hs578T cells, whereas the CAPE IC50 was 14.08 µM and 8.01 µM for the MDA-MB-231 and Hs578T cell line, respectively. Here, we report that propolis and CAPE inhibited the growth of the MDA-MB-231 and Hs578T lines in a dose-dependent and exposure time-dependent manner. EEP showed less cytotoxic activity against both types of TNBC cells. EEP and, particularly, CAPE may markedly affect the viability of breast cancer cells, suggesting the potential role of bioactive compounds in chemoprevention/chemotherapy by potentiating the action of standard anti-cancer drugs.

Evaluation of genistein ability to modulate CTGF mRNA/protein expression, genes expression of TGFß isoforms and expression of selected genes regulating cell cycle in keloid fibroblasts in vitro.

Keloids are characterized by overgrowth of connective tissue in the skin that arises as a consequence of abnormal wound healing. Normal wound healing is regulated by a complex set of interactions within a network of profibrotic and antifibrotic cytokines that regulate new extracellular matrix (ECM) synthesis and remodeling. These proteins include transforming growth factor ß (TGFß) isoforms and connective tissue growth factor (CTGF). TGFß1 stimulates fibroblasts to synthesize and contract ECM and acts as a central mediator of profibrotic response. CTGF is induced by TGFß1 and is considered a downstream mediator of TGFß1action in fibroblasts. CTGF plays a crucial role in keloid pathogenesis by promoting prolonged collagen synthesis and deposition and as a consequence sustained fibrotic response. During keloids formation, besides imbalanced ECM synthesis and degradation, fibroblast proliferation and it's resistance to apoptosis is observed. Key genes that may play a role in keloid formation and growth involve: suppressor gene p53.,cyclin-depend- ent kinase inhibitor CDKN1A (p21) and BCL2 family genes: antiapoptotic BCL-2 and proapoptotic BAX. Genistein (4',5,7-trihydroxyisoflavone) exhibits multidirectional biological action. The concentration of genistein is relatively high in soybean. Genistein has been shown as effective antioxidant and chemopreventive agent. Genistein can bind to estrogen receptors (ERs) and modulate estrogen action due to its structure similarity to human estrogens. Genistein also inhibits transcription factors NFκB. Akt and AP-l signaling pathways, that are important for cytokines expression and cell proliferation, differentiation, survival and apoptosis. The aim of the study was to investigate genistein as a potential inhibitor of CTGF and TGFß1, ß2 and ß3 isoforms expression and a potential regulator of p53. CDKN1A(p21), BAX and BCL-2 expression in normal fibroblasts and fibroblasts derived from keloids cultured in vitro. Real time RT-QPCR was used to estimate transcription level of selected genes in normal and keloid fibroblasts treated with genistein. Secreted/cell-associated CTGF protein was evaluated in cell growth's medium by ELISA. Total protein quantification was evaluated by fluorimetric assay in cells llsates (Quant-iT TM Protein Assay Kit). It was found that TGFß1, ß2 and ß3 genes expression are decreased by genistein. Genistein suppresses the expression of CTGF mRNA and CTGF protein in a concentration dependent manner, p53 and p21 genes expression are modulated by genistein in concentration dependent manner. The agent also modulates BAX/BCL-2 ratio in examined cells in vitro.

Hepatic tissue changes in rats due to chronic invasion of Babesia microti.

The etiological agents of babesiosis are intraerythrocytic parasites of the genus Babesia, which are transmitted by ticks. The course of disease is characterized by variable severity. The risk of a complicated course of babesiosis occurs in premature infants, the elderly, splenectomized patients and other immunocompromised patients. Severe cases of this disease can lead to multiple organ dysfunction. The study focuses on the impact assessment of chronic Babesia microti invasion on the morphology and ultrastructure of rat liver. The analyzed material was comprised of liver samples collected from Wistar rats infected with a reference strain of B. microti (ATCC 30221). None of the livers collected from rats with babesiosis was enlarged. The histopathological analyses showed signs of intensive inflammatory processes, especially in the perivascular areas. The hepatic mononuclear phagocyte system was characterized by increased activity. The ultrastructral analyses confirmed disintegration of hepatocytes with vacuolization in the perivascular areas. In addition, the perisinusoidal space (space of Disse) had irregular structure. In some areas, the space of Disse was enlarged or compressed. The morphological and ultrastructural analyses of rat liver with chronic babesiosis caused by B. microti showed significant pathological changes in perivascular areas which may be the cause of hepatic dysfunction.

Influence of genistein on c-Jun, c-Fos and Fos-B of AP-1 subunits expression in skin keratinocytes, fibroblasts and keloid fibroblasts cultured in vitro.

Genistein is a well known flavonoid that exhibits antioxidant, antiproliferative, proapoptotic, antiangiogenic, as well as estrogenic and anti-estrogenic activity. Extensive studies in the field of dermatology and cosmetology in recent years revealed that genistein is a promising anti-aging, anti-photoaging and anti-carcinogenic agent for skin care. Essential role in skin prematuring aging and carcinogenesis play AP-1 transcription factors that are activated, among others, by environmental factors: ultraviolet light and free radicals. Genistein is a potent antioxidant and inhibitor of AP-1 activity. The aim of the study was to investigate genistein as a potential regulator of C-JUN, C-FOS and FOS-B of AP-1 subunits expression in skin keratinocytes, fibroblasts and keloid fibroblasts cultured in vitro. In presented study, genistein modulated C-JUN expression in epidermal keratinocytes and dermal fibroblasts cultured in vitro. The expression of C-JUN was higher in keratinocytes treated with 37 and 370 microM genistein. In dermal fibroblasts genistein regulated C-JUN expression in dose-dependent manner. The expression of C-JUN was lower in fibroblasts treated with 370 microM genistein and higher in fibroblasts treated with 37 microM genistein. Genistein in 370 microM concentration inhibited C-FOS expression in fibroblasts, whereas in 370 and 37 microM concentration genistein inhibited FOS-B expression in keratinocytes. Furthermore, genistein was able to modulate C-JUN and C-FOS genes expression in keloid fibroblasts cultured in vitro. In these cells, transcriptional activity of C-JUN and C-FOS expression depended on employed concentration of genistein. The expression of C-JUN and C-FOS was higher in keloid fibroblasts treated with 370 microM genistein and lower in keloid fibroblasts treated with 37 microM genistein.

The effect of inositol hexaphosphate on the expression of selected metalloproteinases and their tissue inhibitors in IL-1ß-stimulated colon cancer cells.

INTRODUCTION: Matrix metalloproteinases (MMPs) have repeatedly been shown to play a very active role in extracellular matrix degradation associated with tumor invasion and metastasis. Tissue inhibitors of MMPs (TIMPs) are well-known for their ability to inhibit MMP activity thereby inhibiting malignant progression. Inositol hexaphosphate (IP6 phytic acid) has been recognized to have both preventive and therapeutic effects against various cancers including that of colon. In in vitro studies, IP6 has been demonstrated to inhibit cancer cell adhesion and migration. In the present study, the effect of IP6 on the expression of MMP and TIMP genes was evaluated in unstimulated and IL-1ß-stimulated colon cancer cell line Caco-2. MATERIALS AND METHODS: Real-time QRT-PCR was used to validate the transcription level of selected MMP and TIMP genes in Caco-2 cells after treatment with 1 ng/ml of IL-1ß, 2.5 mM of IP6, and both for 6, 12, and 24 h. RESULTS: Stimulation of cells with IL-1ß only resulted in an overexpression of MMP and their TIMP mRNAs. A significant decrease in MMP-13, MMP-3, MMP-2, and TIMP-1 basal expression was achieved by IP6. IP6 was also an efficient downregulator of MMP-1, MMP-9, and TIMP-2 genes transcription stimulated by IL-1ß in 6 h lasting culture. After 12 h, IL-1ß-induced MMP-2 mRNA expression was significantly reduced by IP6. CONCLUSION: Proinflammatory cytokine IL-1ß upregulates MMP and TIMP mRNAs expression in colon cancer epithelial cells Caco-2. IP6 (2.5 mM) influences constitutive expression of both MMP and TIMP genes and downregulates IL-1ß stimulated transcription of some of these genes. IP6 exerts its anti-metastatic activity through modulation of MMP and TIMP genes expression to prevent cancer cell migration and invasion.

Effect of Gly-Gly-His, Gly-His-Lys and their copper complexes on TNF-alpha-dependent IL-6 secretion in normal human dermal fibroblasts.

Cosmeceuticals represent a marriage between cosmetics and pharmaceuticals. There are numerous cosmeceutically active products which can be broadly classified into the following categories: antioxidants, oligopeptides, growth factors and pigment lightning agents. Much attention has been focused on the tripeptides such as Gly-His-Lys (GHK) and Gly-Gly-His (GGH) and their copper complexes, which have a high activity and good skin tolerance. Recent data suggested their physiological role in process of wound healing, tissue repair and skin inflammation. The mechanism of anti-inflammatory properties of these peptides is not clear. The aim of the study was evaluation of influence of two peptides GGH. GHK and their copper complexes and saccharomyces/copper ferment (Oligolides Copper) on secretion of pro-inflammatory IL-6 in normal human dermal fibroblasts NHDF cell line. IL-6 was evaluated using the ELISA kit. GGH, GHK, CuCl2 and their copper complexes decreased TNF-alpha-dependent IL-6 secretion in fibroblasts. IL-6 is crucial for normal wound healing, skin inflammation and UVB-induced erythema. Because of the anti-inflammatory properties, the copper-peptides could be used on the skin surface instead of corticosteroids or non-steroidal anti-inflammatory drugs, which have more side effects. Our observations provide some new information about the role of these tripeptides in skin inflammation.

Evaluation of the expression of metalloproteinases 2 and 9 and their tissue inhibitors in colon cancer cells treated with phytic acid.

Matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) belong to a zinc dependent family of enzymes that degrade components of extracellular matrix. One postulated mechanism by which inositol hexaphosphate (phytic acid, IP6), an ubiquitous plant component, prevents the activation of MMPs may be due to its ability to chelate minerals. The aim of the study was to evaluate the expression profile of MMP-2, MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 at the mRNA level in human colorectal cancer cell line Caco-2 treated with IP6. A kinetic study of MMP-2, MMP-9 and TIMP-1, TIMP-2 mRNAs was performed after cells treatment with 1; 2.5; 5 mM IP6 for 1, 6, 12 and 24 h. Quantification of genes expression was carried out using real time QRT-PCR technique. The gene encoding MMP-9 was neither constitutively expressed nor induced by IP6 in Caco-2 cells. IP6 at the concentration of 1 mM evoked increase in MMP-2 transcript level, however, its higher doses (2.5; 5 mM) caused a decrease in this gene expression at 1 h incubation. In 24 h lasting culture along with increasing IP6 concentration, the cells expressed lower and lower MMP-2 mRNA level. In response to 1 and 2.5 mM at 6 h, the cells demonstrated an increased transcriptional activity of the TIMP-2 gene which was accompanied by a decrease in TIMP-1 gene transcription. Treatment of cells with 2.5 mM IP6 at 12 h resulted in a strong increase in both TIMP-1 and TIMP-2 expression. The results of this study show that IP6 modulates MMP-2, TIMP-1 and TIMP-2 genes expression in colon cancer cells at the transcriptional level in a way dependent on its concentration and time of interaction.

[Characteristic of the endogenous peptides--endothelins and their role in the connective tissue fibrosis]. / Charakterystyka endogennych peptydów--endotelin i ich rola w procesach chorobowych przebiegajacych z wlóknieniem tkanek.

The endothelins (ET) are the family of 21 amino acid endogenous peptides with potent vasoconstriction function. There are 3 isoforms of the endothelin protein (ET-1, ET-2 and ET-3) encoded by separate genes and exhibit distinct tissue distribution and function. Endothelin 1 is the significant isoform in humans. Endothelin 1 is the most abundant, best characterized isoform with truly pluripotent properties. Endothelin 1 is involved in physiological processes of vascular tone and mitogenesis, whereas under pathological conditions fibrosis, vascular hypertension and inflammation are induced. In human body there are 2 separate ET receptors, ET(A)R and ET(B)R belonging to the G-protein family which produce differing, sometimes opposite effects. Both receptors are differentially expressed by different cell types as well as in different disease entities, In fibroblast cell culture in vitro ET-1 through its receptors modulates cell proliferation, differentiation, contraction and migration. Endothelin 1 is implicated in extracellular matrix (ECM) components synthesis. The dual regulatory role of ET-1 consist on stimulation of collagen I and III synthesis and simultaneously on inhibition of MMP-1 expression through inhibition of tissue inhibitors of metalloproteinase: TIMP-1 and TIMP-3. Endothelin 1 promotes the differentiation of fibroblasts into myofibroblast's phenotype via elevated expression of procontractile proteins alpha-SMA, ezrin, paxillin and moesin. The elevated level of endogenous ET-1 expression cause deficient of myofibroblast apoptosis and increased ECM components deposition. Endothelin 1 is a potent vasoconstrictor, a potent mitogen for fibroblast and smooth muscle cells, a strong stimulant of matrix biosynthesis and is a survival factor for myofibroblasts. Endothelin 1 plays a key role in inflammatory disease and in the connective tissue fibrosis. Elevated level of ET-1, TGF-beta and their receptors has been reported in the pathogenesis of systemic sclerosis.

Transforming growth factor beta isoforms (TGF-beta1, TGF-beta2, TGF-beta3) messenger RNA expression in laryngeal cancer.

PURPOSE: Cancerogenesis is a multistage process controlled by many cytokines, including growth factors. The aim of the study was the comparison of transcriptional activity of transforming growth factor beta (TGF-beta) genes in laryngeal squamous cell carcinomas and adjacent nonneoplastic tissues. MATERIALS AND METHODS: Tissues samples were obtained from 32 patients with laryngeal squamous cell carcinoma in histologic grades G1 to G3 who underwent surgical treatment at the ENT Clinics of Medical University of Silesia in Katowice, Poland. Quantification of gene expression was performed by real-time quantitative reverse transcriptase polymerase chain reaction technique. RESULTS: In tumor cells, expression of TGF-beta1 and TGF-beta2 isoforms (P < .001) was higher than in normal tissues. There was a positive correlation between the expression of TGF-beta1 and TGF-beta2 genes in tumors (R = 0.78, P = .0000) and adjacent normal tissues (R = 0.77, P = .0000). CONCLUSIONS: The results suggest that TGF-beta1 and TGF-beta2 messenger RNAs may be useful as molecular markers in distinguishing cancer from nonneoplastic tissues in laryngeal area.

Influence of retinoids on skin fibroblasts metabolism in vitro.

The most dangerous environmental factor for our skin condition is ultraviolet light radiation. Chronic exposition to ultraviolet light can induce epidermal atrophy, keratosis, depigmentation and dysplasia. In the dermis, UV light causes dramatic up-regulation of extracellular matrix-degrading enzymes. Matrix metalloproteinases (MMPs) are engaged in collagen, elastin and other extracellular matrix components degradation. In addition, to increase level of destructive enzymes, UV light has been shown to decrease collagen production. As a consequence of UV impact on skin, it shows signs of aging including loss of tone and elasticity, increased skin fragility, blood vessels weakness and wrinkles. The most dangerous effect of UV on skin is an increased risk of melanoma and other skin cancers. Retinoids are well known antiaging agents. For many years this vitamin has been used for the prevention and treatment of photoaging. Retinoids abolish cellular atypia, increase compacting of the stratum corneum and reduce skin hyperpigmentation caused by sun light. Recent evidence suggests that retinoids also play a role in the prevention of aging, because of its inhibitory effects on metalloproteinases expression. The aim of this study was to examine if all-trans-retinoic acid (ATRA) effects MMP-1, MMP-2, MMP-3 and MMP-14 gene expression in fibroblasts cultured in vitro.

Expression of TGF beta1 genes and their receptor types I, II, and III in low- and high-grade malignancy non-Hodgkin's lymphomas.

BACKGROUND: Transforming growth factor beta (TGF ) is involved in a variety of important cellular functions. The lack of TGF -dependent cell-growth control might be related to oncogenesis, as it has been shown in lung, breast, and colon carcinomas. Current observations have revealed that TGF is rather an inhibiting, not a stimulating, factor as far as malignant tumor development is concerned. Recently, however, there has been a growing number of reports on increased expression of TGF genes in certain tumors. In patients with a diagnosis of non-small-cell lung carcinoma, the tumors expressing high levels of TGF were the ones that had a higher proliferation and metastasis capability, whereas more promising were the cases with lower levels of TGF expression. MATERIAL/METHODS: A pilot study of 14 patients was conducted comprising 8 patients with a low-grade lymphoma and 6 patients with a high-grade lymphoma. The QRT-PCR method was employed to assess the activity of TGF 1 and of its receptor types I, II, and III. RESULTS: The expression values for TGF 1 and its receptors I, II, and III were twice as high in the group of patients with a diagnosis of high-grade lymphomas as in the group of patients diagnosed with low-grade lymphomas. CONCLUSIONS: Results showed a clear difference in TGF 1 expression in patients with NHL depending on the subtype of the lymphoma, suggesting its significant role in the pathomechanism of this group of malignant diseases as well as its potential value as a prognostic factor.

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.

Blood serum glutathione alpha s-transferase (alpha GST) activity during antiviral therapy in patients with chronic hepatitis C.

BACKGROUND: Glutathione transferases (GST) belong to enzymes involved primarily in the processes of detoxification of exo- and endogenous substances. Immunohistochemical studies have demonstrated that alpha-GST present in the liver is localized exclusively in hepatocytes. The activity of alpha-GST is reported to reflect interstitial liver damage better than that of aminotransferases, especially in patients with autoimmune hepatitis, and, according to some authors, also in patients with chronic hepatitis C. The GST system has also been proposed to be indirectly involved in hepatocellular damage due to hepatitis C virus (HCV) infection. THE AIM OF THE STUDY: Was to assess the utility of alpha-GST as an accessory marker in monitoring antiviral therapy in patients with chronic hepatitis C. MATERIAL/METHODS: 21 patients (12 males and 9 females) with chronic HCV infections were evaluated. The diagnosis was based on clinical presentation and detection of anti-HCV, as well as the presence of HCV-RNA in blood serum detected by PCR. Fifteen patients (group I) were treated with interferon alpha-2b (IFN alpha-2b) at 3 MU s.c. doses administered three times a week (tiw) and ribavirin at 0.8 do 1.2 g/day doses, whereas 6 remaining patients (Group II) were treated with IFN alpha-2b alone at 5 MU s.c. tiw doses. The activity of alpha GST was determined with ELISA before and after 6 months of treatment, together with assessment of HCV-RNA viremia determining the decision concerning continuation of the therapy. RESULTS: The results are presented in Table 1 as mean values +/- SD. *p, 0.05. CONCLUSIONS: Combined antiviral treatment with IFN alpha-2b and ribavirin, and IFN alpha-2b monotherapy reduce blood serum alpha-GST activity in patients with chronic hepatitis C. alpha-GST is less useful as a liver damage parameter than ALT in monitoring of antiviral treatment.

Hepatitis C virus RNA level changes in sera during different regimens of treatment in patients with chronic hepatitis C.

BACKGROUND: Interferon alfa-2b (IFN) has been shown to be effective for chronic hepatitis C infection, but treatment efficacy is still limited. Sustained response after interferon alfa monotherapy is achieved in about 15-25%. Great efforts are made to identify more effective forms of therapy. Some of them include modifications of dosage of antiviral drugs, duration of the therapy and more optimal selection of patients for the treatment. Several virus- and host-related predictive factors for response to antiviral treatment have been proposed. One of the key predictive factors of sustained response to therapy are the pretreatment levels of viremia as well as the dynamics of initial changes of HCV-RNA levels assessed during antiviral therapy. AIM: The aim of our study was to compare changes of HCV-RNA viremia in patients with chronic hepatitis C during different regimens of antiviral treatment. MATERIAL/METHODS: 21 patients chronically infected with HCV (anti-HCV positive, HCV-RNA positive by PCR) were enrolled in the study. 11 patients (Group I) were treated with interferon alfa-2b (IFN-alpha 2b) at 3 MU tiw doses administered subcutaneously for 12 months. 10 patients (Group II) received 3 MU of IFN-alpha 2b daily during the first month and later the treatment was continued with the same dosage tiw for the next 11 months. The response to the treatment was assessed according to the generally accepted criteria after 6-month follow-up. The initial decline of HCV-RNA after 4 weeks of therapy was assessed. RESULTS: Results are presented in the Table. HCV-RNA expressed in copy/ml. CONCLUSION: The initial decline of HCV-RNA level during the first 4 weeks of antiviral therapy with interferon correlates with the final response to the treatment. The greater decline of viremia was observed in the group of patients treated with the 'induction' variant of treatment.

Distribution of hepatitis C virus RNA in whole blood of patients with HCV infection and HBV-HCV coinfection.

BACKGROUND: Although HCV and HBV are essentially hepatoptropic, several lines of evidence suggest that these viruses can infect other cells, also PBMC in most patients with chronic HCV. MATERIAL/METHODS: The presence of HBV DNA and HCV-RNA was determined by a polymerase chain reaction (multiplex PCR and RT-PCR - nested PCR) in a group of patients with chronic liver disease. HCV-RNA was investigated in serum, plasma and peripheral blood mononuclear cells (PBMC) while HBV-DNA only in serum or plasma. RESULTS: Among 374 patients tested, HCV-RNA was detected in the venous blood of 208 patients; HCV RNA alone was detected in 154 patients and 54 patients were co-infected by HCV and HBV. HBV-DNA was found in 128 of 374 patients, while infection by HBV only was found in 74 patients. It was also shown that in the presence of HBV the replication ability of HCV is lower (p=0.085, Goodman-Kruskal Gamma = 0.561 and YuleQ = 0.5610). CONCLUSIONS: Since coexistence of HBV and HCV is not a rare case, diagnostics of hepatitis cannot be limited to detection of one type of the virus only. Misinterpretation of the virus type that caused the infection may lead to serious complications, especially in those cases when interferone is used for treatment.