Thyroxine interaction with actinomycin D and possible biological implications.

Actinomycin D and thyroxine interact in solution (pH 8 to 10), as revealed by changes in the absorbance of actinomycin D. Thyroxine can prevent the growth-inhibitory effect of actinomycin D on Bacillus subtilis if it is present in a molar ratio of 3000 (thyroxine to actinomycin D).

Cytokine and natural killing regulation of growth of a hairy cell leukemia-like cell line: the role of interferon-alpha and interleukin-2.

Hairy cell leukemia (HCL) is a lymphoproliferative disorder of B-lymphocytes, with pathological manifestations usually including splenomegaly and pancytopenia. Naturally occurring and recombinant interferons (IFNs), specifically of the alpha subtype, have shown a significant anti-tumor effect in HCL patients, with improvement of hematologic parameters within the first few months of treatment. The mechanisms responsible for the beneficial action of IFN-alpha in HCL patients are unclear, but several hypotheses have been suggested. Recently, a continuous line of cells (Eskol) from a patient diagnosed with hairy cell leukemia was established and shown to have several properties of a leukemic hairy cell. In the present study, we investigated the direct effect of IFN-alpha and interleukin (IL-2) on the Eskol cell line, and lymphokine regulation of natural killing (NK) activity against these cells. It was found that IFN-alpha has a direct antiproliferative effect on Eskol cells. Furthermore, Eskol cells were found to be completely resistant to NK-cell mediated cytotoxicity (CMC) but were somewhat sensitive to either IFN-alpha-primed NK or lymphokine-activated killer (LAK) cells-CMC. The resistance of Eskol cells to NK-CMC is due to a low binding ability to effector cells. Moreover, it was found that like IFN, IL-2 can protect Eskol cells from activated NK-CMC. Both cytokines reduced the ability of Eskol cells to induce NK-cytotoxic factor (NKCF) release from NK cells following conjugate formation between Eskol cells and effector cells. Moreover, cycloheximide treatment abolished the protective effect against NK-CMC induced by IFN-alpha or by IL-2. Therefore, it seems that the protective effect against NK-CMC induced by both cytokines is mediated via the same mechanism.

Type 1 interferon as an antiinflammatory agent: inhibition of lipopolysaccharide-induced interleukin-1 beta and induction of interleukin-1 receptor antagonist.

The effect of two type 1 interferons (r-metIFN-con1 and IFN-alpha 2b) on the induction of specific cytokines and IL-1Ra in whole blood was examined. IFN-gamma was induced at low levels following treatment of diluted whole blood in some but not all subjects. IL-1Ra was induced by both r-metIFN-con1 and IFN-alpha 2b, but with 10- to 100-fold higher induction per ng IFN with r-metIFN-con1 than with IFN-alpha 2b. There was no detectable induction of TNF-alpha, IL-4, or IL-6 by either IFN. The effect of both IFN preparations was measured on LPS-induced inflammatory cytokines. Both IFN preparations inhibited the production of IL-1 beta when added to the diluted blood samples before LPS addition. However, neither IFN had any effect on IL-1 beta synthesis when added at the same time or after LPS induction. When added to total blood cells in the absence of LPS at low concentration (up to 100 pg/ml), IFN slightly stimulated IL-1 beta production, but at 1000 pg/ml or greater there was significant inhibition of IL-1 beta production. These results suggest that type I IFNs play a role in regulating the inflammatory response.

Cell surface binding characteristics correlate with consensus type I interferon enhanced activity.

The binding characteristics of a genetically engineered consensus interferon with unusually high biologic activity were compared to the characteristics of recombinant interferon-alpha 2. Both interferon-alpha 2 and the consensus interferon produced typical biphasic Scatchard plots, indicating multiple independent binding sites. The consensus interferon, which exhibited a biologic potency more than 10-fold greater than all other type I interferons, also exhibited binding site affinities greater than those for IFN-alpha 2b. In addition, a larger number of high, and low-affinity cell surface sites were recognized by the consensus interferon, resulting in equivalent numbers of sites at reduced molar concentrations compared to IFN-a2b. Thus, at any given biologic activity, similar numbers of sites were bound by the consensus interferon and IFN-alpha 2, despite differences in their molar concentrations. No differences in internalization kinetics were identified between the two interferons, indicating that the differences in cell surface binding may be sufficient to produce the differences in biologic activity.

Comparison of the in vitro host range of recombinant met-interferon-con1, interferon-alpha 2b, and interferon-beta [corrected].

The antiviral activity of human r-metIFN-con1 was compared with that of IFN-alpha 2b and IFN-beta on a number of human, other primate, rodent, feline, and canine cell lines. Although the specific activities of r-metIFN-con1 and IFN-alpha 2b differed 10-fold, the host range was very similar. The host range of IFN-beta differed from that of r-metIFN-con1 and IFN-alpha 2b in that Vero cells were 100-fold better protected by IFN-beta and MDBK protected at a 100-fold less efficiency. In general, there were only minor differences between the host ranges of the three interferons, human and primate cells being better protected than those of other species. However, the tissue of origin of the cell appears to be more important than the species of origin in defining host range [corrected].

The biologic activity and molecular characterization of a novel synthetic interferon-alpha species, consensus interferon.

Consensus interferon (Infergen) is a wholly synthetic type I interferon (IFN), developed by scanning several interferon-alpha nonallelic subtypes and assigning the most frequently observed amino acid in each position, resulting in a consensus sequence. The antiviral, antiproliferative, NK cell activation activity, cytokine induction, and interferon-stimulated gene-induction activity of consensus interferon has been compared with naturally occurring type I interferons. In all of these comparisons, consensus interferon had a higher activity when compared, on a mass basis, with IFN-alpha 2a and IFN-alpha 2b, although the activity was the same for all of these parameters on an antiviral unit basis. That a synthetic type I interferon could have higher activities than naturally occurring molecules is surprising and may be a result of the higher affinity for the array of type I interferon receptors demonstrated for consensus interferon when compared with IFN-alpha. In contrast, consensus interferon was shown to be an inferior inducer of IL-1 beta when compared with IFN-alpha. These results may reflect differential binding to multiple accessory proteins interacting with a type I interferon receptor. These unique biologic properties may lead to a favorable clinical benefit for consensus interferon when compared with the naturally occurring recombinant molecules. Ongoing clinical trials will ascertain whether consensus interferon can be used in a wide array of disease situations, such as chronic viral infections and certain malignancies.

Discordance between serum alanine aminotransferase (ALT) and virologic response to IFN-alpha2b in chronic hepatitis C patients with high and low pretreatment serum hepatitis C virus RNA titers.

Although serum alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA concentrations are primary markers used to assess the clinical benefit of interferon (IFN) therapy in patients with chronic HCV infection, discrepancies between these two variables exist. In this study, 103 patients with chronic hepatitis C were treated with 3 MIU IFN-alpha2b three times weekly for 24 weeks, followed by 24 weeks of observation. ALT and virologic responses were compared in patients with high pretreatment HCV RNA titers (defined as pretreatment HCV RNA concentrations at or above the 75th percentile of the distribution or >5,000,000 copies/ml) and low pretreatment HCV RNA titers (defined as pretreatment concentrations below the 75th percentile or < or =5,000,000 copies/ml). Analysis of the virologic response for the high-titer and low-titer groups demonstrated a significantly greater HCV RNA sustained response in the low-titer group (21%) compared with the high-titer group (7%) (p < 0.05). In contrast, the ALT sustained response was not significantly different between the low-titer group (21%) and the high-titer group (18%). Analysis of the correspondence between biochemical and virologic responses showed that only 38% of patients with high pretreatment HCV RNA titers had both a sustained ALT response and a sustained loss of HCV RNA compared with 75% of patients with low pretreatment HCV RNA titers. The level of agreement between the ALT and HCV RNA responses was greater for the low-titer group compared with the high-titer group (kappa = .6848 and kappa = .4966, respectively). Our results indicate that chronic HCV patients with high pretreatment HCV RNA titers showed greater discordance between sustained ALT and HCV RNA responses compared with patients with low pretreatment HCV RNA titers and that measurement of HCV RNA should be included in the assessment of response to IFN therapy in chronic hepatitis C patients.

Treatment of chronic hepatitis C virus infection with recombinant consensus interferon.

To assess the safety and efficacy of consensus interferon (IFN-Con-1), 55 patients with chronic hepatitis C infection were treated with either 3, 6, 9, 12, or 15 microg IFN-Con-1 s.c. three times a week for 24 weeks, followed by 24 weeks of observation. There was a dose-response relationship with respect to the number of patients with normalized ALT concentrations or undetectable HCV RNA. At the end of the 24-week treatment period, the serum ALT had normalized in 18% of patients given the 3 microg dose and 42% of patients given the 12 microg or 15 microg doses of IFN-Con-1. At the end of the posttreatment observation period, the serum ALT was still normal in 10% of patients given the 3 microg, 6 microg, or 9 microg doses and in 50% of patients given the 15 microg dose. Also, at the end of the 24-week treatment period, 27% of patients given the 3 microg dose and 75% given the 15 microg dose had undetectable serum HCV RNA. At the end of the posttreatment observation period, the proportion of patients with undetectable HCV RNA ranged from 9% of those given the 3 microg dose to 50% of those given the 15 microg dose. Our study indicates that treatment with IFN-Con-1 appears to be safe and effective. In addition, use of 15 microg of IFN-Con-1 resulted in significantly more patients with sustained ALT normalization and absence of HCV RNA 6 months after cessation of therapy compared with treatment with lower doses of IFN-Con-1. Additional trials are underway to confirm these findings.

A pilot study of daily subcutaneous interleukin-10 in patients with chronic hepatitis C infection.

The Th1/Th2 cytokine balance is important in persistence of infection and liver injury in chronic hepatitis C. The aim of this study was to administer the anti-inflammatory cytokine, recombinant human interleukin-10 (rHuIL-10), for 28 days in patients with chronic hepatitis C and to assess the safety and measure the effect on alanine aminotransferase (ALT, a marker of hepatic inflammation) levels and serum hepatitis C virus (HCV) RNA values. Three treatment-naive and 13 interferon (IFN) nonresponder patients (total 16 patients) with compensated chronic HCV infection were enrolled in this study. Patients were randomized to receive rHuIL-10 at a dose of 4 or 8 microg/kg/day as a single daily subcutaneous injection for 28 days. ALT values and serum HCV RNA were measured at days 0, 1, 3, 8, 15, 22, and 28 during therapy and at follow-up 2 and 4 weeks after cessation of the 4-week treatment period. ALT values normalized in 9 of 16 patients during therapy and remained normal until the end of treatment in 8 patients. The decreases in ALT values occurred in both the 4 microg and 8 microg dosage groups and were seen in both IFN naive and nonresponder patients. Mean ALT values fell significantly during the study period but usually returned to pretreatment levels by the end of the 4-week follow-up period (p < 0.05). HCV RNA concentrations did not vary significantly during or after therapy. (No patient had either an increase or a decrease in HCV RNA levels of > or =1.5 log during the study.) The drug was well tolerated, with no adverse symptoms noted. Platelet counts fell transiently to 73,000 and 63,000 in 2 patients. No other toxicity was observed, and no patients discontinued therapy. In chronic hepatitis C, short-term therapy with IL-10 was well tolerated and caused transient normalization of ALT values in 50% of patients, which returned to pretreatment levels on cessation of treatment. There were no significant changes observed in serum HCV RNA concentrations during the study. These immunomodulatory effects are similar to those observed with ribavirin monotherapy in chronic hepatitis C. Further study of rHuIL-10 alone or in combination with antiviral agents in chronic hepatitis C is warranted.

An analysis of acute changes in interleukin-6 levels after treatment of hepatitis C with consensus interferon.

Cytokine production has been implicated in the antiviral response to interferon-alpha (IFN-alpha) in hepatitis C and in the development of IFN-alpha-related side effects. We characterized acute changes in serum cytokine levels following administration of a single dose of consensus IFN (IFN-con1) and during continuous treatment of chronic hepatitis C patients. Serum samples were collected at baseline, at multiple times early after IFN administration, and weekly thereafter. Viral RNA titers were assessed by RT-PCR, and viral kinetics were followed. ELISA assays were used to measure IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, and IL-16. Serum cytokine levels were low at baseline. IL-6 was detected in patients with hepatitis C but not in healthy control subjects by either ELISA or RT-PCR, indicating that low levels of circulating IL-6 were associated with hepatitis C infection. None of the cytokines measured increased significantly after IFN administration except for IL-6. IL-6 levels rose rapidly, peaked at 6-15 h in a dose-dependent manner, and returned to baseline by 48 h in both patients receiving a single dose of IFN and those receiving continuous treatment. This was confirmed by RT-PCR. Pretreatment IL-6 levels were directly correlated with area under the curve (AUC) for IL-6 during the 24 h after IFN dosing (r = 0.611, p = 0.007). Viral titers decreased within 24-48 h after a single dose of IFN-con1. Changes in hepatitis C RNA titers were not significantly associated with pretreatment IL-6 levels or with changes in IL-6 levels. In conclusion, (1) baseline serum cytokine levels, except for IL-6, were low or within the normal range in patients with hepatitis C, (2) IL-6 levels were detected in some patients with hepatitis C before treatment but not in healthy controls, (3) IL-6 levels increased acutely after a single dose of IFN-alpha, and IL-6 induction was related to baseline IL-6 level, and (4) changes in IL-6 levels did not correlate with the early virologic response to IFN.

A possible role for interferon-alpha and activated natural killer cells in remission of AIDS-related Kaposi's sarcoma: in vitro studies.

The effect of consensus interferon-alpha on the growth of AIDS-related Kaposi's sarcoma-derived cells was studied. Interferon caused a low but significant in vitro inhibition of cell growth. Whereas Kaposi's sarcoma cells were resistant to the cytotoxic effect of natural killer (NK) cells, treatment of NK cells with either interferon or interleukin-2 activated the cell-mediated cytotoxic response. Pretreatment of the Kaposi's sarcoma cells with interferon reduced their sensitivity to interferon-primed natural killer cell or lymphokine (interleukin-2)-activated killer (LAK) cell cytotoxicity. The resistance of Kaposi's sarcoma cells to NK cell-mediated cytotoxicity did not reside in conjugate formation but was due to an inability of Kaposi's sarcoma cells to induce NK cytotoxic factor. Although interferon reduces the sensitivity of Kaposi's sarcoma cells to interferon-primed NK cell and LAK cell activities, the level of natural killing susceptibility remained significantly higher than the poor sensitivity of Kaposi's sarcoma cells to unprimed NK cell activity. Thus, the potential antitumor effect of interferon against Kaposi's sarcoma cells could be mediated both directly (antiproliferative) and/or indirectly by activation of NK cells.

Gene therapy with an adeno-associated virus carrying an interferon gene results in tumor growth suppression and regression.

Adeno-associated virus (AAV) vectors were constructed containing both a synthetic type I interferon gene, (IFN-con1) and the bacterial neomycin-resistant gene. Recombinant virions were used to infect a number of human tumor cell lines, including 293, Hela, K562, and Eskol (a hairy cell leukemia-like cell), and geneticin-resistant cells were selected. All IFN-con1-transduced cell lines produced low levels of IFN-con1 and grew at the same rate as nontransduced cell lines. Although these cell lines were resistant to IFN in vitro, when injected into nude mice, 293, K562, and Eskol cells failed to form tumors up to 3 months after the initial inoculum, although mice receiving nontransduced cells developed tumors within 7 to 10 days. Transduced Hela cells grew much slower in vivo and formed much smaller tumors than did the parental cells. When equal numbers of transduced and nontransduced cells were injected into nude mice, tumors initially developed slowly and then completely regressed. Treatment of an established Eskol tumor (histologically a malignant immunoblastic lymphoma) with AAV/IFN-con1-transduced 293 cells resulted in tumor regression, whereas treatment of Eskol tumors with IFN-con1 resulted in a small decrease in tumor size. These results indicate that the human IFN-con1 gene in a viral vector can be used successfully in the treatment of tumors both directly and by tumor-targeted gene therapy.

Pharmacokinetics and tolerability of an antiangiogenic ribozyme (ANGIOZYME) in healthy volunteers.

The pharmacokinetics and tolerability of a chemically stabilized synthetic ribozyme (ANGIOZYME) targeting the Flt-1 VEGF receptor mRNA were evaluated in healthy volunteers. In a placebo-controlled, single-dose escalation study, ribozyme was administered as a 4-hour i.v. infusion of 10 or 30 mg/m2 or as a s.c. bolus of 20 mg/m2. Peak ribozyme plasma concentrations of 1.5 and 3.8 micrograms/mL were observed after the 10 and 30 mg/m2 i.v. infusions, respectively. When normalized to dose, AUC values as well as peak concentrations increased proportionally as the dose was increased from 10 to 30 mg/m2. Peak concentrations of 0.9 microgram/mL were observed approximately 3.25 hours after a 20 mg/m2 s.c. bolus of ribozyme. The dose-normalized AUCs obtained after s.c. dosing were compared to the mean dose-normalized AUC after i.v. dosing to estimate an absolute s.c. bioavailability (f) of approximately 69%. An average elimination half-life of 28 to 40 minutes was observed after i.v. administration, which increased to 209 minutes after s.c. administration. Only 4 of 12 reported adverse events were possibly related to administration of ribozyme (headache and somnolence). Thus, ribozyme administration was well tolerated after a single 4-hour i.v. infusion of up to 30 mg/m2 or a single s.c. bolus of 20 mg/m2.

Analysis of the viability of freezer stored serum samples for hepatitis C virus RNA analysis by the SUPERQUANT method: results of a 16 year retrospective study.

Prior to the discovery of the hepatitis C virus (HCV), virological analysis of serum from patients with non-A non-B hepatitis was not possible. Since the finding that HCV is the causative agent of most non-A non-B hepatitis, several reliable methodologies have been developed that allow for quantification of HCV RNA. To determine the viability of stored serum samples for HCV RNA analysis. 256 samples were examined for HCV RNA using a multi-cycle RT-PCR assay. All samples were stored unopened in a -70 degree C freezer until the time of testing. Collection years ranged from 1981 to 1995. To examine the integrity of stored serum samples, the distribution of quantitative HCV RNA values for each year was compared: year-to-year; and, to the distribution of HCV RNA concentrations from 1510 chronic HCV patients determined by the same assay in 1996 and 1997. Pairwise year-to-year analysis revealed that samples collected prior-to-and-including 1991 had significantly lower HCV RNA concentrations as compared to samples collected after 1991 (P < 0.001). Likewise, comparison of the stored samples to the 1510 fresh samples demonstrated that samples collected prior-to-and-including 1991 had significantly lower HCV RNA concentrations as compared to samples collected after 1991 (P < 0.001). The results demonstrate a method for determination of the integrity of stored serum samples from chronic HCV patients. The mechanism of RNA degradation is unknown but it is most likely to be due to poor sample collection procedures in place prior to 1991.

Consensus interferon induces peak mRNA accumulation at lower concentrations than interferon-alpha 2a.

The biological activity of a novel recombinant interferon, r-metIFN-con1, which represents a consensus sequence of the most commonly appearing amino acids at each locus of 14 naturally occurring IFN-alpha s, was assessed and compared to that of IFN-alpha 2a. The increase in cellular mRNA levels for three IFN-inducible genes served as a quantitative measure of the effectiveness of the stimulation by each of the IFNs. Three cell lines were treated with equimolar amounts of two IFNs encompassing a 5 log range and mRNA was extracted at five different times after treatment. In all cases, r-metIFN-con1 produced mRNA increases at lower concentrations than IFN-alpha 2a. HLA-DR alpha mRNA, which is not affected by IFN-alpha in ME180 or Daudi cells, was also not affected by r-metIFN-con1. However, in Eskol cells, both IFNs effected an increase in HLA-DR alpha mRNA to similar levels. The r-metIFN-con1 was effective at approximately 10-fold lower molar concentrations. At effective concentrations (10-fold lower molar dose of r-metIFN-con1), both IFNs produced similar kinetics of accumulation of all three mRNAs tested. r-metIFN-con1 is therefore more effective than IFN-alpha 2a at the level of mRNA regulation as well as the antiviral and antiproliferative activities that have been reported previously.