ABSTRACT
Interferon (IFN)-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1) in peripheral blood mononuclear cells (PBMC) is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/á-actin-mRNA ratio (mean ñ SD). Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 ñ 0.15; N = 5; P < 0.05) compared to non-responders (0.35 ñ 0.17; N = 12) and controls (0.30 ñ 0.16; N = 9). Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.
Subject(s)
Humans , Male , Female , Antiviral Agents , Hepatitis C, Chronic , Interferon-alpha/therapeutic use , Leukocytes, Mononuclear , Alanine Transaminase , Aspartate Aminotransferases , Gene Expression , Liver , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger , RNA, ViralABSTRACT
Interferon (IFN)-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1) in peripheral blood mononuclear cells (PBMC) is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha 2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/beta-actin-mRNA ratio (mean +/- SD). Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 +/- 0.15; N = 5; P < 0.05) compared to non-responders (0.35 +/- 0.17; N = 12) and controls (0.30 +/- 0.16; N = 9). Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis C, Chronic/drug therapy , Interferon-alpha/blood , Interferon-alpha/therapeutic use , Leukocytes, Mononuclear/chemistry , Receptors, Interferon/blood , Alanine Transaminase/blood , Aspartate Aminotransferases/blood , Female , Gene Expression , Hepatitis C, Chronic/blood , Humans , Interferon alpha-2 , Interferon-alpha/metabolism , Liver/chemistry , Liver/metabolism , Male , Middle Aged , RNA, Messenger/analysis , RNA, Messenger/blood , RNA, Viral/blood , Receptor, Interferon alpha-beta , Receptors, Interferon/metabolism , Recombinant Proteins , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Abnormal production of interferon alpha (IFN-alpha) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-alpha. IFN-alpha can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-alpha inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-alpha receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-alphaB on Daudi cells, but did not block IFN-alphaB activity at higher concentrations (>6. 25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-alphaB on Hep 2/c cells challenged with encephalomyocarditis virus.
Subject(s)
Antiviral Agents/antagonists & inhibitors , Escherichia coli/chemistry , Interferon Type I/antagonists & inhibitors , Interferon-alpha/antagonists & inhibitors , Peptide Fragments/pharmacology , Receptors, Interferon/chemistry , Viral Proteins/chemistry , Animals , Cattle , Humans , Interferon-alpha/metabolism , Mice , Peptide Fragments/metabolism , Receptors, Interferon/genetics , Recombinant Proteins , Statistics, NonparametricABSTRACT
Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.