Oligoadenylate Synthetase Activity Research Articles

Pilot study of recombinant human granulocyte-macrophage colony-stimulating factor in the treatment of chronic hepatitis B

Recombinant human granulocyte-macrophage colony-stimulating factor is being used to improve the immunological function of patients with various diseases and to ameliorate hematological disorders. We investigated the tolerance and possible antiviral effect of the administration of daily doses of recombinant human granulocyte-macrophage colony-stimulating factor (3, 1 or 0.5 micrograms/kg body wt) to nine patients with chronic hepatitis B, alone or in combination with 5 MU interferon-alpha 2b. Recombinant human GM-CSF reduced significantly (p < 0.02) hepatitis B virus DNA levels. The three doses used were equally effective. Of the eight patients who completed the study, four became negative for HBV DNA and HBeAg; two of them seroconverted to HBe antibody. These four patients showed improvement in the histological activity of their liver disease. Ultimately, two patients regained normal ALT values. 2',5'-Oligoadenylate synthetase activity increased significantly (p < 0.01) in cell lysates of mononuclear cells cultured in vitro, coinciding with the reductions in hepatitis B virus DNA levels. Recombinant human granulocyte-macrophage colony-stimulating factor was well tolerated but produced a dose-dependent increase in white blood cell counts. It became intolerable at doses of 3 micrograms (and was reduced to 1.5 microgram); this effect was reversible after cessation of recombinant human granulocyte-macrophage colony-stimulating factor treatment. No remarkable variations occurred in other parameters. In conclusion, recombinant human granulocyte-macrophage colony-stimulating factor administration is safe and tolerable at doses of 0.5 to 1 microgram/kg body wt and may exert an antiviral effect in chronic hepatitis B.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of interferon-gamma-induced major histocompatibility complex class II gene transcription by interferon-beta and type beta 1 transforming growth factor in human astrocytoma cells. Definition of cis-element.

To address mechanisms by which cytokines inhibit interferon-gamma (IFN gamma)-induced gene expression in astrocytic cells, we have been studying effects of type beta 1 transforming growth factor (TGF beta 1) and interferon-beta (IFN beta) on IFN gamma-induced expression of the well-characterized human major histocompatibility complex (MHC) class II gene DRA. This investigation was motivated by the observations that IFN gamma-induced expression of MHC class II antigen expression on astrocytic cells can be blocked in a tissue-specific fashion by several cytokines and neurotransmitters in tissue culture and that astrocyte expression of MHC class II is severely restricted in vivo. We previously showed that IFN beta inhibited IFN gamma-induced DRA expression at the transcriptional level. This inhibition was not global, since IFN gamma-induction of intercellular adhesion molecule-1 was not affected. Here, TGF beta 1-mediated inhibition of DRA is shown to exhibit similar characteristics. To address the mechanism of this inhibition, sequence requirements for IFN beta and TGF beta 1 to suppress IFN gamma-induced transcription of DRA were determined. A 135-base pair DRA sequence element containing the IFN gamma-responsive region and transcriptional start site was sufficient to direct IFN beta- or TGF beta 1-mediated suppression of a reporter gene. These experiments suggested that either IFN beta or TGF beta 1 could repress IFN gamma-induced DRA transcription directly, without requiring a cis-element extrinsic to the IFN gamma-inducible DRA sequences. Consistently similar effects of IFN beta and TGF beta 1 observed in these experiments prompted comparison of other gene regulatory effects of the two cytokines. TGF beta 1, unlike IFN beta, induced gene expression directed by upstream elements of the gene encoding plasminogen activator inhibitor type-1. IFN beta, unlike TGF beta 1, enhanced levels of 2'-5'-oligoadenylate synthetase activity. Additionally, direct assay of TGF beta 1 and monoclonal anti-TGF beta antibody blocking experiments were used to determine that cells treated with IFN beta did not produce increased amounts of active or latent TGF beta. These data argued that IFN beta and TGF beta 1 utilized distinct pathways to mediate the inhibition of IFN gamma-induced DRA transcription.

Infection with Moloney Murine Sarcoma Virus Inhibits Myogenesis and Alters the Myogenic-Associated (2′-5′)Oligoadenylate Synthetase Expression and Activity

Infection of rat skeletal muscle cultures on the first or second day in vitro with Moloney murine sarcoma virus (MSV) led to the arrest of myotube formation and to inhibition of both the synthesis of the muscle-specific proteins acetylcholine recaptors and creatine kinase and the expression of the myosin light chain-2. Mos-specific RNA transcripts were readily detected at 1 day after infection indicating that viral genes were expressed in infected cells. In parallel, the expression of the cell growth-associated gene- c-myc-in uninfected muscle cultures was drastically reduced with time, while in MSV-infected myoblasts, the amount of c-myc-specific RNA transcripts gradually increased with time after infection. Under these conditions we could demonstrate that the interferon-induced gene (2′-5′)oligoadenylate synthetase (2-5A synthetase) was transiently activated in uninfected muscle culture reaching a peak activity on the third day. Infection of myoblasts with murine leukemia virus did not alter the pattern of 2-5 synthetase activity observed in uninfected cells. However, infection with MSV on the second day led to a slight reduction in activity followed by a significant increase on the sixth and seventh day. Similarly, 2-5A synthetase gene expression was down-regulated with time in culture in uninfected myoblasts while re-expressed between the fourth and seventh days in MSV-infected cultures.

Serum levels of 2',5 oligoadenylate synthetase during interferon therapy in patients with B-cell chronic lymphocytic leukemia.

We assayed 2',5 oligoadenylate synthetase (2'5-AS) activity in the serum of eight patients with B-cell chronic lymphocytic leukemia (B-CLL) who were undergoing therapy with alpha interferon (IFN). Mean pretreatment levels of 2'5-AS were normal, but upon treatment the levels rose to higher than normal values. Moreover, the degree of enzyme induction in serum was significantly higher in CLL responders (P < 0.003) whereas no difference was found in non-responders. The lack of serum 2'5-AS induction in non-responder patients might be related to unresponsiveness to treatment with IFN. These results seem to show that the measurement of serum levels of 2'5-AS activity may be a useful tool in monitoring IFN treatment.

Pharmacodynamics of Biological Response In Vivo After Single and Multiple Doses of Interferon?????

Interferons (IFNs) induce gene regulation in vivo that may be used to identify effective doses, schedules, and potential correlates of therapeutic response. To critically examine minimum effective dose, duration of response, and cumulative effects of repetitive doses, a range of subcutaneous doses of IFN beta ser was studied in 32 healthy human volunteers. IFN-induced products of gene regulation assessed were beta 2-microglobulin, neopterin, and tryptophan in serum and 2',5'-oligoadenylate (2-5A) synthetase activity in peripheral blood mononuclear cells. Eight subjects per group received 0.09, 0.9, 9, or 45 MU of IFN beta. Responses were measured at 24, 48, and 72 h after single and multiple doses. The lowest biologically effective dose was 0.9 MU; significant (p < 0.02) increases were observed at 24 h in beta 2-microglobulin and cellular 2-5A synthetase activity. At the two higher doses, 9 and 45 MU, changes were observed at 24 h in all products (p < 0.01). A dose response (p < 0.01) over the range of 0.09-45 MU was observed for all these serum and intracellular gene products. Changes in neopterin, beta 2-microglobulin, and cellular 2-5A synthetase correlated significantly with each other. The response to a single dose of IFN beta was as great in magnitude as the response to multiple doses, suggesting an alternate-day schedule would maintain biological response.

Mode of Action of the Anti-AIDS Compound Poly(I)·Poly(C12U) (Ampligen): Activator of 2′,5′-Oligoadenylate Synthetase and Double-Stranded RNA-Dependent Kinase

The mismatched double-stranded RNA (dsRNA), poly(I).poly(C12U), also termed Ampligen, exhibits a strong antiviral and cytoprotective effect on cells (human T-lymphoblastoid CEM cells and human T-cell line H9) infected with the human immunodeficiency virus type 1 (HIV-1). Untreated H9 cells infected with HIV-1 start to release the virus 3 days post-infection, while in the presence of 40 micrograms/ml (80 micrograms/ml) of poly(I).poly(C12U) the onset of virus production and release is retarded and does not occur before day 5 (day 6). We demonstrate that poly(I).poly(C12U) markedly extends the duration of the transient increase of 2',5'-oligoadenylate (2-5A) synthetase mRNA level and activity preceding virus production after infection of cells with HIV-1. Treatment of HeLa cells with poly(I).poly(C12U) was found to cause a significant increase in total (activated plus latent) 2-5A synthetase activity; no evidence was obtained that the level of latent (nonactivated) 2-5A synthetase is changed in cells treated with dsRNA plus interferon (IFN). Poly(I).poly(C12U) is able to bind and to activate 2-5A synthetase(s) from HeLa cell extracts. Addition of poly(I).poly(C12U) to HeLa cell extracts results in production of longer 2-5A oligomers (> or = 3 adenylate residues), which are better activators of RNase L. Both free and immobilized poly(I).poly(C12U) also bind to the dsRNA-dependent protein kinase (p68 kinase), resulting in autophosphorylation of the enzyme. Activation of the kinase by the free RNA occurs within a limited concentration range (10(-7) to 10(-6) grams/ml). Addition of HIV-1 Tat protein does not affect binding and activation of p68 kinase to poly(I).poly(C12U)-cellulose but strongly reduces the binding of the kinase to immobilized TAR RNA of HIV-1. We conclude that poly(I).poly(C12U) may antagonize Tat-mediated down-regulation of dsRNA-dependent enzymes.

Biologic effects after a single dose of poly(I):poly(C12U) in healthy volunteers

Poly(I):poly(C12U) (mismatched double-stranded RNA; atvogen), an interferon inducer, is active against human immunodeficiency virus in vitro. To determine the extent and duration of the biologic effects of poly(I):poly(C12U), we administered a single dose of the drug to healthy volunteers in a randomized, double-blind, placebo-controlled 2-week crossover study. We analyzed blood for alpha and gamma interferons, neopterin, 2',5'-oligoadenylate synthetase, lymphocyte surface markers, lymphocyte proliferation after exposure to soluble antigens and mitogens, and natural killer cell activity. Minimal biologic effects were observed after administration of a single 200-mg dose to four volunteers; therefore, the dose was increased to 600 mg in 10 subjects. Only neopterin levels and symptoms were greater after administration of 600 mg of poly(I):poly(C12U) than after administration of placebo (Wilcoxon signed rank sum test, P = 0.06). A definite response in 2',5'-oligoadenylate synthetase activity, however, was seen in a few subjects. Neither alpha nor gamma interferon was detectable in serum after poly(I):poly(C12U) dosing. The neopterin changes after administration of poly(I):poly(C12U) were similar at both poly(I):poly(C12U) dose levels, with an early decrease at 6 h, a peak at 1 day, and a gradual decrease toward the baseline over the following 3 days. A mild flu-like syndrome occurred in one-half of the subjects following administration of poly(I):poly(C12U) and in only one subject following administration of placebo. This syndrome resolved within 16 h after poly(I):poly(C12U) dosing. We conclude that poly(I):poly(C12U) does not induce measurable levels of interferon and causes only minimal biologic or toxic effects among those parameters measured after administration of a single dose in the 200- to 600-mg dose range in health volunteers.

2′,5′-Oligoadenylate Synthetase Activity as a Responsive Marker During Interferon Therapy for Chronic Hepatitis C

The 2',5'-oligoadenylate (2-5A) synthetase is an intracellular enzyme induced by interferon (IFN). We evaluated the serum level of this enzyme in 25 patients affected by chronic hepatitis C and treated with recombinant IFN-alpha 2b. At the end of treatment, 14 patients were classified as responders and 11 as nonresponders. Before therapy initiation no significant differences in 2-5A synthetase levels among the patients were detected, while during therapy responders showed higher mean levels of 2-5A synthetase than nonresponders. An increase in the enzyme activity was observed after 1 month of therapy, and this trend was maintained in the following 2 months. The peak of 2-5A synthetase activity was found at the end of therapy. 2-5A synthetase levels were negatively correlated with serum alanine aminotransferase (ALT). This study suggests that 2-5A synthetase may be a useful marker to monitor IFN efficacy during treatment and to predict the clinical response.

High Levels of 2′,5′-01igoadenylate Synthetase and 2′,5′-Oligoadenylate-Dependent Endonuclease in Human Trophoblast

Human placenta contains a high level of 2',5'-oligoadenylate (2-5A) synthetase activity of the 100-kD form of the enzyme. About 20% of the placental 2-5A synthetase activity was found to be cytosolic, whereas the remaining 80% was released by 0.5 M KCl in the presence of detergent. Most of the enzyme activity was localized in trophoblast cells, which also contain a high level of 2-5A-dependent RNase L activity. The purified trophoblast 100-kD 2-5A synthetase was shown to be activated by human immunodeficiency virus type 1 (HIV-1) 5' RNA 1-311 and 1-707, which both contain the TAR and primer binding site (PBS) structured regions. These two HIV-1 RNAs activated human trophoblast 2-5A synthetase at the same level as poly(I).poly (C), a standard highly efficient activator of the enzyme, and at the same optimal concentration. On the contrary, HIV-1 RNA 311-618, a poorly structured region missing TAR and PBS, was shown to be a poor activator of the enzyme. The specific cellular location of the 2-5A synthetase and its efficient activation by HIV 5' RNA favors the idea that the trophoblast 2-5A system negatively controls HIV replication in trophoblasts.

Pharmacokinetic properties of recombinant feline interferon and its stimulatory effect on 2',5'-oligoadenylate synthetase activity in the cat.

The pharmacokinetic behavior of recombinant feline interferon produced in silkworm infected with recombinant baculovirus harboring cDNA coding for feline interferon was studied in vivo in cats. The decreasing profile of the serum interferon level after intravenous administration was fitted to a two-compartment model. The half-times of the first phase (distribution phase) and second phase (metabolic phase) were 5.0 +/- 0.5 min and 31 +/- 5 min, respectively. In the whole body autoradiogram, at 15 min after the administration, the highest radioactivity was observed in urine in the bladder, and predominant radioactivity in the kidneys, liver, thyroid gland and spleen. Almost no radioactivity was detected in the brain or fat. Three hr after administration, the highest radioactivity was recorded in the thyroid gland, urine in the bladder, intestinal contents, and gastric mucous membrane. The data obtained in this study suggest that recombinant feline interferon has similar pharmacokinetic properties to human interferons and that it is distributed primarily in the liver and kidneys, is catabolized rapidly mainly in the kidneys, and is excreted in the urine without residual accumulation in the body. It was confirmed that 2',5'-oligoadenylate synthetase activity was increased by the interferon in vivo for 3 days after an intravenous bolus injection in cats.

Hormonal and immunological regulation of 2′,5′-oligoadenylate synthetase activity in human peripheral blood mononuclear cells

A newly developed method for assaying 2′,5′-oligoadenylate (2,5A) synthetase activity by polyacrylamide gel electrophoresis was applied to peripheral blood mononuclear cells (PBMC) from normal subjects, HIV-positive subjects, and renal cell carcinoma (RCC) patients. Sex differences were observed in 2,5A synthetase activity of PBMC from normal young adults, males having eightfold higher activities of this enzyme than females. Moreover, compared to values for postmenopausal (PM) females receiving estrogen replacement, untreated PM females had higher activities. Collectively, these results suggest that estrogen downregulates 2,5A synthetase activity. Activities of 2,5A synthetase were investigated in two disease states associated with altered immune function. In one patient with AIDS-related Kaposi's sarcoma, interferon-α (IFN-α) therapy increased 2,5A synthetase activity twofold. In addition, combined therapy with interleukin-2 (IL-2) and IFN-α increased 2,5A synthetase activities in eight of nine patients with RCC. Therefore, in patients receiving immunotherapy with IL-2 and IFN-α, our new assay could contribute to evaluation of immune stimulation. In general, studies in vitro confirmed these observations; however, exposure of PBMC from RCC patients revealed that in vitro IL-2 failed to induce this enzyme activity as it did in PBMC from normal volunteers.

Type I interferons inhibit hepatitis B virus replication and induce hepatocellular gene expression in cultured liver cells.

A hepatoblastoma cell line transfected with hepatitis B virus (HBV) DNA (Hep G2.2.15) was used to investigate the effects of interferons (IFNs) on HBV replication and hepatocellular gene expression. IFN-alpha 2b or -beta inhibited HBV replication transiently. In parallel, there was a decrease in the amount of HBV mRNA. Hepatitis B surface antigen and early antigen secretion were not influenced; however, their intracellular levels diminished during treatment. The cellular 2',5'-oligoadenylate synthetase activity was increased 9- to 18-fold during treatment of cells with IFN-gamma, -alpha, or -beta. The number of IFN-alpha and -beta receptors was down-regulated, while the number of IFN-gamma receptors remained constant. The expression of major histocompatibility complex class I antigens was stimulated by addition of IFN-alpha or -beta. These data show that both IFN-alpha and -beta can effectively inhibit HBV replication and induce a cellular IFN response in Hep G2.2.15 cells similar to that seen in humans.

Spectrophotometric pyrophosphate assay of 2′,5′-ollgoadenylate synthetase

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2′,5′-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Gle pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2′,5′-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.

Opposite sensitivity to the antiproliferative action of interferon-alpha and granulocyte-macrophage colony-stimulating factor in monoblastic U937 cells.

Three variants of the human monoblastic cell line U937 with different degrees of sensitivity to the antiproliferative action of interferon-alpha (IFN-alpha were examined for phenotypic differences. The highly IFN-sensitive variant U937-V expressed twice as many IFN-alpha binding sites as both its IFN-alpha-resistant derivative U937-VR and the cell line U937 exhibiting a 20-fold reduction in IFN-alpha sensitivity as compared to U937-V cells. All three variants were IFN-reactive with regard to induction of 2',5'-oligoadenylate (2-5A) synthetase activity and were similarly sensitive to the growth-inhibiting action of IFN-gamma and tumor necrosis factor. Responsiveness to the antiproliferative effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), however, was confined to cell lines U937 and U937-VR. Although expressing a comparable number of GM-CSF receptors, the highly IFN-sensitive variant U937-V was refractory to GM-CSF. Flow cytometry revealed a marked difference in the expression of the antigen CD11b which was detectable on 85% of cells of the U937-V line but only on approximately 25% of cells derived from the U937 and U937-VR lines. Results thus demonstrate opposite sensitivity of U937 cells to the growth-inhibiting action of IFN-alpha and GM-CSF, apparently dependent on the state of U937 differentiation as determined by expression of the CD11b antigen.

The effects of poly(I).poly(C12U) and interferon on the multiplication of a mammalian type C retrovirus in human cells.

Poly(I).poly(C12U) or interferon treatment inhibited multiplication of the xenotropic baboon type C endogenous retrovirus M7 in chronically infected human AV3-M7 cells, as determined by a reverse transcriptase (RT) assay and electron microscopy. Furthermore, this polynucleotide induced 2'5' oligoadenylate (2'5'A) synthetase activity. In contrast to interferon (IFN), poly(I).poly(C12U) did not give rise to the appearance of a trapping phenomenon observable by electron microscopy. When AV3-M7 cells were treated simultaneously with poly(I).poly(C12U) and anti-IFN-beta/alpha antibodies, the induction of 2'5'A synthetase was abolished without any alteration of the inhibitory effect of RT activity. Taken together, these results suggest that different mechanisms are used by poly(I).poly(C12U) and IFN in blocking type C retrovirus multiplication.

Pentoxifylline Inhibits Certain Constitutive and Tumor Necrosis Factor-α-Induced Activities of Human Normal Dermal Fibroblasts

Pentoxifylline (PFN), analog of theobromine, which phenotypically and functionally alters various cell types including dermal fibroblasts, has been reported to inhibit tumor necrosis factor-alpha (TNF alpha) activation of neutrophils. We investigated the ability of PFN to alter constitutive and TNF alpha-induced biosynthetic activities of human normal dermal fibroblasts. The sixteenfold increase over constitutive intracellular 2'-5' oligo-adenylate synthetase (2'-5' A synthetase) activity induced by TNF alpha (400 U/ml) failed to occur when PFN (1 mg/ml) was added prior to cytokine treatment. This loss of biologic activity paralleled a reduction in 2'-5' A synthetase proteins and 2'-5' A synthetase-specific m-RNA. PFN failed to inhibit constitutive or TNF alpha-induced IL-6 hybridoma proliferative activity, IL-6 protein, or IL-6-specific m-RNA levels. The presence of PFN (1 mg/ml) in fibroblast cultures reduced constitutive synthesis of collagen and glycosaminoglycan (GAG) by 87% and 45%, respectively, and blocked induction of their synthesis by TNF alpha (10(4) U/ml). Total non-collagenous protein synthesis was not inhibited following PFN treatment (1 mg/ml). PFN did not inhibit TNF alpha induction of only those biosynthetic activities also susceptible to PFN in the constitutive state, with PFN failing to reduce constitutive collagenolytic activity but reducing TNF alpha-induced enhanced collagenolytic activity by 26% and collagenase m-RNA by 51%. Furthermore, PFN did inhibit, by 98%, TNF alpha-dependent murine and human fibroblast cytotoxicity. The selective nature of PFN inhibition of certain TNF alpha activities, the failure of PFN (1 mg/ml) to alter constitutive and TNF alpha-induced levels of type 1 and 2 TNF alpha receptor m-RNA, and the finding that PFN-treated fibroblasts express a similar number of receptors, of similar molecular weight and high affinity for TNF alpha as control, untreated cells, suggest that inhibitory activities of PFN are mediated at a locus other than receptors for TNF alpha.

Cell cycle regulation (G1) by autocrine interferon and dissociation between autocrine interferon and 2',5'-oligoadenylate synthetase expression.

In tertiary MEF undergoing cell cycle progression, autocrine interferon (IFN) is released and constitutive levels of 2',5'-oligoadenylate (2-5A) synthetase activity, low through the cell cycle, surge into a peak within S phase. Treatment of MEF with the autocrine IFN they produce elicits a 2-5A synthetase response from cells positioned in G0 but not from cells in G1 and from cells in S phase. Neutralization of the autocrine IFN by antibody shortens the length of G1 leaving unaltered the kinetics of progression through S and G2 and has no effect on the S phase-linked expression of 2-5A synthetase activity. The growth controlling effect of the autocrine IFN has been mapped to the second part of G1.

Clinical and biologic effects of combination therapy with gamma-interferon and tumor necrosis factor

Tumor necrosis factor (TNF) and gamma-interferon (gamma-IFN) are cytokines with synergistic biologic and antiproliferative effects in vitro and in mouse models. The biologic effects of the combination of TNF and gamma-IFN, however, have not been studied well in humans. A Phase I trial was conducted of TNF and gamma-IFN therapy in 24 patients with advanced malignancies to determine the tolerability of the combination and the biologic effects of TNF and gamma-IFN in vivo. Both TNF and gamma-IFN were administered as 30-minute intravenous infusions three times per week. Doses of TNF ranged from 25 to 100 micrograms/m2; all patients received 100 micrograms/m2 of gamma-IFN. Dose-limiting toxicity consisted primarily of orthostatic hypotension and constitutional symptoms. The maximum tolerated dose level (MTDL) of 50 micrograms/m2 of TNF and 100 micrograms/m2 of IFN-gamma was less than the maximum tolerated dose (MTD) observed in previous Phase I trials of gamma-IFN and TNF alone. Biologic responses were studied in seven patients treated at the MTDL. Serum interleukin-2 receptor levels and neopterin secretion were enhanced significantly 24 hours after therapy (P = 0.002); enhancement of monocyte Fc receptor levels had borderline statistical significance (P = 0.07). With the exception of the mean fluorescent intensity on monocytes positive for histocompatibility antigen HLA-DR (P = 0.03), HLA Class I and II cell surface protein expression was not increased. The combination significantly enhanced indoleamine dioxygenase activity and serum beta 2-microglobulin expression (P less than 0.04) but not 2',5'-oligoadenylate synthetase activity, bactericidal function, or chemiluminescence. These results were compared retrospectively with those observed in previous Phase I trials of gamma-IFN and TNF alone. The combination of TNF and gamma-IFN significantly increased urinary kynurenine levels more than either TNF alone or gamma-IFN alone. Given the limitations inherent in any retrospective analysis, however, the enhancement in the other biologic parameters measured at the MTDL during this trial did not differ significantly from the changes observed at the MTD of either TNF or gamma-IFN alone. It was concluded that the combination of TNF and gamma-IFN, when administered at the MTDL of the combination, does not offer any enhancement in biologic responses over either agent alone.

Ethanol induces 2',5'-oligoadenylate synthetase and antiviral activities through interferon-beta production.

We demonstrate here that ethanol, in contrast to heat shock (Chousterman, S., Chelbi-Alix, M.K., and Thang, M.N. (1987) J. Biol. Chem. 262, 4806-4811), induces interferon (IFN) synthesis and its related activities in Madin-Darby bovine kidney (MDBK) cells. The induced IFN is secreted maximally at 6 h, whereas the induction of 2',5'-oligoadenylate synthetase mRNA peaks between 9 and 12 h and its activity at 15 h. The appearance of both 2',5'-oligoadenylate synthetase activity and the antiviral state upon ethanol treatment is prevented by anti-bovine recombinant IFN-beta antibodies. Bovine diarrhea virus infection-free MDBK cells cultured in medium supplemented with serum substitute also gave similar results, thus indicating that IFN synthesis induced by ethanol is not mediated by the activation of bovine diarrhea virus. Together, these results show that: 1) ethanol induces the 2',5'-oligoadenylate synthetase and antiviral activities through IFN-beta production; and 2) the IFN produced does not act directly from inside the cells, but has to be first secreted to bind to its receptor. In MDBK cells, ethanol induces the synthesis of the 70-kDa protein, which precedes the expression of 2',5'-oligoadenylate synthetase; moreover, the transient nature of the synthesis of the hsp 70 in these cells is similar after both heat shock and ethanol treatment.

Gene dosage and down-regulation of the alpha-interferon receptor.

The gene coding for the alpha,beta-interferon (alpha,beta-IFN) receptor is localized to chromosome 21. Cells from patients with Down's syndrome (trisomy 21) contain an extra chromosome 21, which results in a 1.5 times increase of dosage for the genes localized to this chromosome. Trisomy 21 cells express more cell-surface alpha-IFN receptors, consistent with the increased gene dosage. Down-regulation of the alpha-IFN receptors in trisomy 21 and normal cells was studied by incubating the cells with alpha-IFN. The alpha-IFN-induced effects showed 1.6 times more internalized cell-surface alpha-IFN receptors in trisomy 21 cells compared with normal cells, but no statistically significant change in the dissociation constants. A close relationship was found between the alpha-IFN receptor number and the biological response expressed as 2',5'-oligoadenylate synthetase activity.