Tumor necrosis factor alpha gene expression and the response to interferon in chronic hepatitis

Abstract

Tumor necrosis factor alpha (TNF-alpha) is a cytokine with pleiotropic properties that is induced in a variety of pathological situations including viral infections. In this work, we analyzed the expression of TNF-alpha gene in patients with chronic hepatitis C. Serum TNF-alpha levels were found to be elevated in all chronic hepatitis C patients including those cases presenting sustained biochemical remission of the disease after interferon therapy. Untreated patients with chronic hepatitis C showed increased TNF-alpha messenger RNA (mRNA) levels in the liver and mononuclear cells as compared with healthy controls. After completion of treatment with interferon, patients experiencing sustained complete response showed values of TNF-alpha mRNA, both in the liver and in peripheral mononuclear cells, within the normal range, significantly lower than patients who did not respond to interferon and than those with complete response who relapsed after interferon withdrawal. Pretreatment values of TNF-alpha mRNA were lower in long-term responders to interferon than in cases who failed to respond to the treatment. Values of TNF-alpha mRNA in the liver or in mononuclear cells were higher in specimens with positive hepatitis C virus (HCV) RNA than in those samples where the virus was undetectable. Neither the intensity of the liver damage nor the amount of HCV RNA in serum or in cells showed correlation with the levels of TNF-alpha transcripts in peripheral mononuclear cells but it was found that high TNF-alpha values were associated with genotype 1b. In conclusion, there is an enhanced expression of TNF-alpha in HCV infection. High levels of this cytokine may play a role in the resistance to interferon therapy.

Superoxide Dismutase in Patients With Chronic Hepatitis C Virus Infection

“…7 To determine Mn-SOD mRNA levels we used a similar procedure. Either PBMC or liver total RNA (1.25 or 0.75 g, respectively) was reverse-transcribed (60 min at 37°C) with 200 U of M-MuLV reverse transcriptase (BRL, Gaithersburg, MD) in 25 l volume of 5ϫ RT buffer (0.25 mol/l Tris-HCl pH 8.3, 0.375 mol/l KCl, 15 mmol/l MgCl 2 ), supplemented with 5 mmol/l DTT, 0.4 mmol/l deoxynucleoside triphosphate, ribonuclease inhibitor (30 U), and random hexamers (250 ng) (Boehringer-Mannheim, Mannheim, Germany).…”

“…7 Mn-SOD cDNA fragments were amplified by 24 or 23 cycles (PBMC or liver, respectively) (94, 60, and 72°C, 1 min each step), and ␤-actin was amplified by 20 cycles (94, 55, and 72°C, 1 min each step), a protocol that avoided interference of the plateau effect. Oligonucleotides (5Ј-3Ј) d(TGCACCACAG-CAAGCACCAC) and d(AAACCAAGCCAAC-CCCAACC) were the upstream and downstream primers, respectively, used for amplification of 310 bp fragment from human Mn-SOD cDNA.…”

“…Mn-SOD mRNA values were normalized to those of ␤-actin mRNA because we had previously demonstrated that ␤-actin mRNA was constantly expressed in patients with chronic hepatitis C with or without IFN treatment. 7 Validation experiments of PCR assays using known quantities of total RNA (from 0 to 1 g) were carried out. As shown in Fig.…”

“…7 Two sets of primers specific for the 5Јuntranslated region of HCV genome were used (5Ј-CCTGTGAGGAACTACTGTCT-3Ј and 5Ј-CTATCAGGCAGTACCACAAG-3Ј for outer primers; 5Ј-ACTGTCTTCACGCAGAAAGC-3Ј and 5Ј-GACCCAACACTACTCGGCTA-3Ј for inner primers). HCV-RNA was quantitated using a competitive PCR technique as previously reported.…”

“…Several lines of evidence support this contention, including the existence of an activated glutathione turnover, the presence of increased levels of lipid peroxidation products and augmented iron stores in the liver, [2][3][4] and the finding of diminished reduced glutathione values in peripheral blood mononuclear cells (PBMC) and erythrocytes. 5,6 Moreover, it has been shown that patients with chronic hepatitis C exhibit an increased production of tumor necrosis factor-␣ (TNF␣), 7 a cytokine that can produce oxidative stress by stimulating the generation of reactive oxygen species (ROS) such as superoxide ion (O 2 ⅐Ϫ ) and hydrogen peroxide (H 2 O 2 ). 8 Reactive oxygen metabolites are mainly produced in mitochondria during electron transport 9,10 but also, to a lesser extent, in endoplasmic reticulum, peroxisomes, and nuclear and plasma membrane.…”