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1.
J Viral Hepat ; 9(2): 134-40, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11876796

RESUMO

Our aims were to measure the kinetics of serum tumour necrosis alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) levels as markers of progression of disease in nontreated chronic hepatitis C virus (HCV)-infected patients with minimal or no fibrosis and minimal histology activity index (HAI) scores. Our study group consisted of 56 patients diagnosed with minimal (1) or no fibrosis (0) and minimal HAI (0-1) on their first biopsy as defined by Knodell and METAVIR scores. We compared their initial (entry of study) cytokine levels with a group of 103 HCV controls with minimal (0-1) to mild fibrosis (0-3) and mild HAI (5.5). Serum TNF-alpha and TGF-beta levels were measured by enzyme-linked-immunosorbent-assay. A significant difference was seen in TNF-alpha levels at baseline in the study group vs. controls. Regardless of their HAI, there was a correlation between TGF-beta and degree of fibrosis. As shown by their biopsies, during the 3 years (from entry to follow up), many of the patients that initially had minimal fibrosis progressed to higher degree of fibrosis. This progression is paralleled by an increase in TGF-beta levels when comparing initial and follow-up levels. In conclusion, serum TNF-alpha reflects the progression of inflammation as seen in liver biopsies and TGF-beta reflects the degree of fibrosis in HCV patients.


Assuntos
Hepatite C Crônica/patologia , Cirrose Hepática/patologia , Fígado/patologia , Fator de Crescimento Transformador beta/sangue , Fator de Necrose Tumoral alfa/análise , Adulto , Progressão da Doença , Feminino , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C Crônica/sangue , Hepatite C Crônica/imunologia , Humanos , Cinética , Cirrose Hepática/sangue , Cirrose Hepática/imunologia , Masculino
2.
Clin Biochem ; 34(3): 173-82, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11408014

RESUMO

OBJECTIVES: (i) To characterize serum cytokine levels of tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL 6), IL 8 and IL 12 in non-cirrhotic patients with chronic hepatitis C, (ii) to correlate the levels of these cytokines with the degree of the disease at the basal level, (iii) to correlate these levels with the response to therapy, (iv) to compare profiles of cytokines in monotherapy (MT) versus combination therapy (CT), and (v) to compare the immunomodulatory effects of MT versus CT. DESIGN AND METHODS: 47 patients were enrolled in the study. The controls were 120 volunteers (recruited from students and staff) that did not present HCV RNA positive and were not known to suffer any other metabolic disease. Thirty patients formed the other group of controls, with alcoholic liver disease (ALD). Serum cytokine levels were assessed using enzyme-linked immunosorbent assay (ELISA). RESULTS: The sustained responders (SRs) have basal values much lower than relapsed responders (RRs) and non-responders (NRs) regardless of the therapy. CONCLUSIONS: Cytokines can be used as non-invasive markers for sustained response and as monitors for the outcome of therapy.


Assuntos
Biomarcadores , Hepatite C Crônica/imunologia , Interferons/uso terapêutico , Interleucinas/sangue , Ribavirina/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Alanina Transaminase/sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Hepacivirus/genética , Hepatite C Crônica/sangue , Hepatite C Crônica/tratamento farmacológico , Humanos , Interferons/administração & dosagem , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , Ribavirina/administração & dosagem , Carga Viral
3.
Clin Biochem ; 34(3): 183-8, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11408015

RESUMO

OBJECTIVE: (i) to characterize the profile of tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), IL 10, Fas-ligand and transforming growth factor beta (TGF beta), chronic hepatitis C (HCV) patients with genotype 1; (ii) to determine the influence of triple therapy (TT) with interferon alpha (IFN alpha) + ribavirin + ursodeoxycholic acid on these cytokines and (iii) to establish the relationship between the pro-inflammatory cytokines and the outcome of treatment. DESIGN AND METHODS: 22 patients infected with HCV-genotype 1 a/b and non responsive to IFN-alpha monotherapy were enrolled in the TT. The controls were 49 HCV naïve patients with genotype 1 a/b. Cytokine levels were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The baseline TNF alpha values (pg/mL) in the sustained responders (SRs) (63+/-3) were significantly lower than non-responders (NRs) (140+/-16) (p < 0.001). Baseline Fas (ng/mL) levels were also lower in SRs (4.3+/-0.2) than NRs (5.4+/-0.4) (p < 0.05). CONCLUSIONS: Fas and TNF alpha may be used as serological markers of inflammation and effectiveness of therapy.


Assuntos
Citocinas/sangue , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Ribavirina/uso terapêutico , Ácido Ursodesoxicólico/uso terapêutico , Adulto , Quimioterapia Combinada , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Hepatite C Crônica/sangue , Humanos , Interferon-alfa/administração & dosagem , Masculino , Pessoa de Meia-Idade , Ribavirina/administração & dosagem , Ácido Ursodesoxicólico/administração & dosagem
4.
Clin Biochem ; 34(3): 211-8, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11408019

RESUMO

OBJECTIVES: To determine the cytotoxicity of valproic acid (VPA) and its metabolite, 4-ene-valproic acid (4-ene-VPA) in human hepatoblastoma cells (Hep G2), and to study the modulatory effect of cytochrome P450 2E1 induction in this model. METHODS: Cells were exposed to VPA or 4-ene-VPA in the presence of either ethanol (EtOH), or EtOH combined with disulphiram (DS). Some cells were exposed to alpha-fluoro-VPA or to alpha-fluoro-4-ene-VPA in the absence of CYP2E1 inducers. Apoptosis and necrosis were measured by analyzing 6000 cells per sample using transmission electron microscopy, while cytokine release and apoptosis were quantitated by ELISA. RESULTS: VPA + EtOH increased VPA cytotoxicity. 4-ene-VPA + EtOH significantly increased toxicity, while DS + EtOH significantly reduced this toxicity. Alpha-fluorinated analogues reduced cytotoxicity compared to the corresponding VPA compounds. Neither VPA nor alpha-fluorinated VPA increased the release of IL-6 or TNF-alpha in media. A significant increase in the release of TNF-alpha was observed in cells exposed to 4-ene-VPA that further increased with EtOH exposure. CONCLUSIONS: Cells exposed to 4-ene-VPA experience greater cytotoxicity than those treated with VPA. Cytochrome P450 2E1 inducers enhance toxicity in VPA-exposed cells, while alpha-fluorination of VPA diminishes cytotoxicity by directly interfering with the beta-oxidation of the 4-ene-VPA metabolite.


Assuntos
Anticonvulsivantes/toxicidade , Citocromo P-450 CYP2E1/metabolismo , Fígado/efeitos dos fármacos , Ácido Valproico/toxicidade , Apoptose , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP2E1/biossíntese , Indução Enzimática , Humanos , Interleucina-6/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Fígado/ultraestrutura , Microscopia Eletrônica , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo
5.
Clin Biochem ; 34(3): 219-27, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11408020

RESUMO

OBJECTIVES: To evaluate whether caspases are involved in ethanol (EtOH)-induced apoptosis and if polyenylphosphatidylcholine (PPC) affects apoptosis, in vitro in Hep G2 cells. METHODS: Cells were treated with 100 mmol/L EtOH for 24 h and with 2 doses of 100 mmol/L EtOH (1/24 h) in the presence of absence of 20 mmol/L of PPC or 50 micromol/L caspase 3 inhibitor (IDN). Cells were analyzed for apoptosis by transmission electron microscopy (TEM) 6000 cells/treatment, DNA fragmentation by ELISA and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (T dt-mediated d-UTP) nick-end-labeling, TUNEL. RESULTS: 100 mmol/L dose of EtOH resulted in 22 +/- 2.5% (p < 0.001) apoptosis (vs. control). Two consecutive doses of 100 mmol/L EtOH for 24 h each caused 36 +/- 3.0% (p < 0.001 vs. control and p < 0.05 vs. one dose). PPC significantly reduced apoptosis (vs. non exposed to PPC): 100 mmol/L -12 +/- 1.5% (p < 0.05) and 2 x 10(-)(0) mmol/L -20 +/- 2.0% (p < 0.001). Pretreatment with 50 micromol caspase inhibitor reduced EtOH-induced apoptosis in a similar proportion. CONCLUSIONS: PPC downregulates EtOH-apoptosis by a mechanism similar to caspase inhibition.


Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Reparo do DNA/efeitos dos fármacos , Etanol/farmacologia , Transdução de Sinais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica
6.
Clin Biochem ; 33(7): 517-24, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11124336

RESUMO

OBJECTIVES: To validate the novel lymphocyte toxicity assay (LTA) based on the mitochondrial succinate dehydrogenase (SDH) activity vs. the LTA by using trypan blue exclusion and to determine the utility of the assay to confirm drug hypersensitivity syndrome (DHS) to sulphonamides (SMX) and aromatic anticonvulsants. METHODS: Incubation of patient lymphocytes, with or without murine hepatic microsomes with anticonvulsants or SMX. The viability of lymphocytes was based on SDH activity that can be measured spectrophotometrically. The percentage of cells displaying cytotoxicity compared to controls (cells treated only with drug) was calculated. Seventy-two immunocompetent and 16 immunocompromised (HIV) patients with DHS to SMX were sampled. The results were validated vs. 26 controls that had not experienced DHS to SMX. Sixty-two patients who had DHS to anticonvulsants were compared with 24 controls that did not have any DHS to the same anticonvulsants. RESULTS: The results showed a very good percentage of sensitivity 98 and specificity 96 with a kappa-score of 0.96. LTA higher than 13.5% was considered positive for the immunocompetent population and LTA higher than 22% was positive for the immunocompromised population. In two of the 26 controls, LTA was positive. CONCLUSION: The high quantitative kappa-value 0.96 emphasizes that the novel LTA is at least as good as the trypan blue assay. The latter is labor intensive, time consuming, and prone to human error. The new assay is objective, faster, and has been reproducible in assessing cytotoxicity.


Assuntos
Química Clínica/métodos , Hipersensibilidade a Drogas , Linfócitos/efeitos dos fármacos , Adolescente , Adulto , Idoso , Animais , Anticonvulsivantes/farmacologia , Carbamazepina/farmacologia , Estudos de Casos e Controles , Sobrevivência Celular , Criança , Estudos de Coortes , Corantes/farmacologia , Saúde da Família , Feminino , Soropositividade para HIV/sangue , Humanos , Lamotrigina , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , Mitocôndrias/enzimologia , Fenobarbital/farmacologia , Fenitoína/farmacologia , Sensibilidade e Especificidade , Succinato Desidrogenase/metabolismo , Sulfadiazina/farmacologia , Sulfametoxazol/farmacologia , Sulfonamidas/farmacologia , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Fatores de Tempo , Triazinas/farmacologia , Trimetoprima/farmacologia , Azul Tripano/farmacologia
7.
Clin Biochem ; 32(7): 519-36, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10614715

RESUMO

OBJECTIVES: To study the effect of cytochrome P450 2E1-inducers on methotrexate (MTX)-induced cytotoxicity in human hepatocytes, and investigate the role of silymarin in preventing this toxicity. DESIGN AND METHODS: Cells were exposed to MTX in the presence of either ethanol (EtOH) or acetaminophen (APAP), or either combined with silymarin (S). Apoptosis and necrosis were measured by analyzing 6000 cells/sample using transmission electron microscopy, while cytokine release and apoptosis were quantitated by ELISA. Cytokine expression was measured by RT-PCR. Gluthatione (GSH) content was determined in cytosolic (c) and mitochondrial (m) fractions. RESULTS: MTX+EtOH and MTX+APAP increased MTX cytotoxicity 2.9-fold and 1.9-fold, respectively. S abolished this toxicity. MTX + EtOH increased the release of IL 6, IL 8 and TNF alpha by 1.0, 1.2, and 1.1 times, respectively. Cytokine expression was upregulated versus control for IL 6 (22%), IL 8 (38%), and TNF alpha (29%). Addition of 0.5 mmol/L S downregulated TNF alpha expression and reduced cytokine release. TNF alpha increased cytotoxicity by 22%, while anti-TNFalpha antibody eradicated it. MTX+EtOH depleted 45% mGSH (0 < 0.001) while S replenished it to 87% (p < 0.001), when both were compared to control levels. CONCLUSIONS: Cytochrome P450 2E1-inducers contribute to increase oxidative stress in MTX-exposed cells by increasing TNF alpha and depleting both cGSH and mGSH. This enhances MTX-cytotoxicity and promotes apoptosis.


Assuntos
Citocromo P-450 CYP2E1/biossíntese , Fígado/efeitos dos fármacos , Metotrexato/toxicidade , Apoptose , Linhagem Celular , Citocinas/metabolismo , Sinergismo Farmacológico , Indução Enzimática , Etanol/toxicidade , Glutationa/metabolismo , Humanos , Fígado/citologia , Fígado/enzimologia , Microscopia Eletrônica , Silimarina/farmacologia
8.
Skin Pharmacol ; 8(6): 279-91, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-8688194

RESUMO

The skin and liver may be targets for cytotoxicity induced by oxidative drug metabolites. We used human epidermoid A431 cells and human hepatoblastoma Hep G2 cells as the experimental model. The aim of the study was to investigate and evaluate the effect of silymarin on acetaminophen (APAP)-induced toxicity under controlled conditions. Silymarin is known to be a potent antioxidant that diminishes toxicity induced by a variety of other hepatotoxins (e.g. Amanita phaloides, algae's toxins, carbon tetrachloride). Glutathione (GSH) depletion was enhanced by adding to the medium buthionine sulfoximine [L-buthionine-(S,R)-sulfoximine, BSO]. Cells were incubated with high-concentration 5-20 mM APAP or alpha-(minimum essential medium for 2-24 h to evaluate the drug's ability to reduce cytoviability. Viability was then quantitated by metabolism of the tetrazolium dyes (MTT) and neutral red (NR). Cytoviability was 100% for controls. For Hep G2 treated for 24 h with 20 mM, APAP viability was 56.0% by MTT and 62.5% by NR. BSO-treated cells showed an enhanced cytotoxicity, determined by both assays. Administration of 0.5 mM silymarin reduced cytotoxicity significantly. In A431 cells, treatment with 20 mM APAP reduced viability by 57% (MTT) and 69% (NR) versus control (100%). BSO further decreased viability. Since incubation with silymarin showed significant protection against APAP toxicity, it can be considered a cytoprotective agent in this in vitro model of drug toxicity. GSH concentrations in both cell lines decrease significantly after exposure to 20 mM APAP, or 0.5 mM versus control (p < 0.05), and increased (p < 0.001) if incubated with APAP and silymarin. The protective effect could be through mitochondrial membrane stabilization and/or an increase in available GSH.


Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Antioxidantes/farmacologia , Fígado/efeitos dos fármacos , Silimarina/farmacologia , Pele/efeitos dos fármacos , Acetaminofen/antagonistas & inibidores , Analgésicos não Narcóticos/antagonistas & inibidores , Linhagem Celular/efeitos dos fármacos , Glutationa/metabolismo , Hepatoblastoma , Humanos , Neoplasias Hepáticas , Células Tumorais Cultivadas/efeitos dos fármacos
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