Pharmacokinetic study of a human recombinant interferon (Re-IFN-alpha A) in cynomolgus monkeys by 2'-5' oligoadenylate synthetase assay

We evaluated 2'-5'Oligoadenylate (2-5 A) synthetase assay for pharmacokinetic study of human interferon (IFN) in cynomolgus monkeys. The enzyme was induced in primary cultures of cynomolgus monkey kidney (PMK) cells as well as in FL cells in response to human recombinant IFN-alpha A treatment. The enzyme activity increased with IFN dose and, in parallel with the enzyme elevation, developed the antiviral state of the cells. The enzyme activity induced in the peripheral blood lymphocytes peaked at 6 to 12 hr after iv or im administration. The peak level of the enzyme activity depended on the IFN concentration of the blood and the activity rapidly decreased as serum IFN was cleared from the blood. These results indicate that human recombinant IFN-alpha A induces 2-5 A synthetase in monkey cells both in vitro and in vivo, and that the enzyme assay can be used to quantitatively monitor the host response after IFN administration.     

Sensitivity of human germ-cell-tumor cell lines to human interferons

We examined the sensitivity of four human germ-cell-tumor cell lines exhibiting different stages of differentiation to human interferons (IFNs) in vitro. The cell lines were derived from two embryonal carcinomas (NEC 8 and NEC 14), a choriocarcinoma (IMa), and a yolk-sac tumor (HUOT). Treatment with poly I:C induced IFN production in IMa and HUOT cells, but not in NEC-8 and NEC-14 cells. In the two embryonal-carcinoma cell lines, the addition of IFN-alpha, IFN-beta, and IFN-gamma did not prevent infection by vesicular stomatitis virus and encephalomyocarditis virus. Also, in these two lines, 2-5A synthetase was not induced by the addition of IFN-alpha. In contrast, both IMa and HUOT showed sensitivity to the antiviral action of IFN-alpha and IFN-beta against the two viruses, and 2-5A synthetase was induced by IFN-alpha. IFNs added at doses of up to 1000 IU/ml had no antiproliferative effect on NEC 8, NEC 14, and HUOT, whereas colony formation by IMa cells was greatly suppressed by all three forms of IFN. These results indicate that the production of and sensitivity to IFN are developmentally regulated and are related to the level of differentiation of human germ-cell stem cells.     

Expression of antiviral activity and induction of 2',5'-oligoadenylate synthetase by conceptus secretory proteins enriched in bovine trophoblast protein-1

Establishment of pregnancy in cattle has been proposed to depend on production of a conceptus protein, bovine trophoblast protein-1 (bTP-1), which has a high degree of sequence homology with bovine interferon-alpha (bIFN-alpha), especially the alpha II subfamily. A preparation of bovine conceptus secretory proteins enriched for bTP-1 has antiviral and physico-chemical properties similar to other bIFN-alpha. Antiviral activity is initially detectable in uterine flushings on Day 14 of pregnancy, when the conceptus measures 4-5 mm in length, and increases as the conceptus elongates through Day 18. Day 17 conceptuses produce more than 10(6) U antiviral activity during 24 h of culture. All IFNs induce the enzyme 2',5'-oligoadenylate synthetase, which catalyzes production of 2',5'-oligo(A), which in turn is involved in antiviral and growth inhibitory effects of IFNs. This enzyme activity is induced in Madin-Darby bovine kidney cells by the partially purified bTP-1 preparation similarly to IFN-alpha, -beta, and -gamma. Likewise, the partially purified bTP-1 and bIFN-alpha 1 induce 2',5'-oligo(A) synthetase activity in monolayers of endometrial epithelial and stromal cells. Compared to epithelial cells, stromal cells have higher baseline activity of 2'-5'-oligo(A) synthetase activity (p less than 0.01) and show a greater degree of induction in the presence of either the partially purified bTP-1 or bIFN-alpha 1 (p less than 0.01). Also, 2',5'-oligo(A) synthetase of endometrial stromal cells is induced to a greater degree by our enriched bTP-1 preparation than by bIFN-alpha 1 (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo and in vitro induction of 2'-5' oligoadenylate synthetase by interferon-alpha in nodular non-Hodgkin's lymphoma and correlations with the clinical response

We investigated the correlations between the in vivo-in vitro induction of 2'-5' oligoadenylate synthetase (2-5A synthetase) by IFN-alpha in cells isolated from patients with low-grade nodular non-Hodgkin's lymphoma (NHL) and subsequent clinical responses of these patients to IFN-alpha therapy. Eleven patients were treated daily with 9 x 10(6) U of IFN-alpha 2a in a phase II trial. After an eight week treatment, four patients achieved complete remission, one a partial response, one a minor response, and five failed to respond. Basal levels of 2-5A synthetase in lymph node tumor B cells and peripheral blood mononuclear cells (PBMC) isolated before therapy differed from patient to patient and were significantly lower than in PBMC from healthy donors (P less than 0.03). In vivo single injections of 9 x 10(6) U IFN-alpha 2a induced the 2-5A synthetase in PBMC from all patients to various degrees without quantitative relation to the clinical responses. Injection of a tenfold lower dose resulted in effects of similar extent in most cases. In vitro, IFN-alpha 2a induced the 2-5A synthetase in lymph node tumor B cells isolated before therapy, and the degree of induction was significantly higher in patients who proved to respond to therapy than in patients who displayed no or minor responses (P less than 0.013). This indicates that, in nodular NHL, the 2-5A synthetase assay may have some predictive value for responsiveness to IFN-alpha therapy.     

A CpG oligodeoxynucleotide inducing anti-coxsackie B3 virus activity in human peripheral blood mononuclear cells

Coxsackie B3 virus (CVB3) is the most significant pathogen causing myocarditis in humans, and antiviral therapy would be most effective in the early stages of the disease. Here we provide evidence that BW001, a C-type CpG oligodeoxynucleotide, induces anti-CVB3 activity in human peripheral blood mononuclear cells (PBMCs). In parallel, we have demonstrated that BW001 induces human PBMCs to express mRNAs of multiple types of interferon (IFN), including IFN-alpha, IFN-beta, IFN-omega and IFN-gamma, and to express mRNAs of at least 11 subtypes of IFN-alpha. The induced IFNs may contribute to the anti-CVB3 activity. The results suggest that BW001 could be developed into a medication with the potential to treat CVB3 infectious diseases by inducing natural mixed IFNs.     

Enhancement of interferon-induced 2–5 oligoadenylate synthetase activity by retinoic acid in human histiocytic lymphoma U937 cells and WISH cells

The effect of retinoic acid (RA) on the level of interferon (IFN)-induced 2-5 oligoadenylate (2-5A) synthetase activity was examined in human histiocytic lymphoma U937 cells and WISH cells** in order to ascertain the role of this polymerase in interaction between IFNs and RA. Cultures containing both IFNs (1-100 U/ml) and RA (0.1-10 microM) consistently had higher levels of enzyme activity than corresponding cells treated with IFN alone and this was true for all three types of IFNs in both cell lines. The potentiating effect of RA was dose- and time-dependent and under optimal conditions, the induction of the synthetase was synergistic between IFN-beta (10-100 U/ml) and RA (0.1-10 microM). Furthermore, pretreatment (but not posttreatment) with RA followed by subsequent treatment with IFNs preferentially induced higher levels of enzyme activity in U937 cells but not in WISH cells. In addition, our results indicated that the modulating effect of RA on IFNs did not involve interaction at the receptor level and the level of enhancement of 2-5A synthetase activity was not in parallel with either cell-growth arrest or promotion of differentiation. Lastly, the present study raises the possibility that interactions between IFNs and RA, in either a synergistic or antagonistic manner, may be mediated through amplification of the 2-5A system.     

CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep

Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.     

Clonal derivatives of the RD-114 cell line differ in their antiviral and gene-inducing responses to interferons.

The human rhabdomyosarcoma cell line RD-114 is partially responsive to interferons (IFNs). In these cells, alpha interferon (IFN-alpha) or gamma interferon (IFN-gamma) inhibits the replication of some viruses but not of others. Similarly, some of the IFN-inducible mRNAs are induced poorly, whereas others are induced well. Here we report the isolation of clonal derivatives of this line which display different spectra of responses to IFNs. Among the eight extensively characterized clonal lines, one, C10, did not respond to IFN-alpha or IFN-gamma at all. Retrovirus production by each of the seven other lines was inhibited by both IFN-alpha and IFN-gamma. Replication of vesicular stomatitis virus was inhibited strongly by IFN-alpha in clone B1 but not in others, whereas it was not appreciably affected by IFN-gamma in any clone. Replication of encephalomyocarditis virus was inhibited strongly by IFN-gamma in clones A1, A2, A3, B3, and B8 and by IFN-alpha in clone A2. Neither IFN inhibited the multiplication of these clones greatly, although their doubling times were slightly increased. Five mRNAs were induced by IFNs to varying degrees in the seven clones. mRNA 2A was most strongly induced by IFN-gamma in clone A3. mRNA 1-8 was strongly induced by IFN-alpha in clone A1 and by either IFN in clones A2 and A3. The highest concentrations of 2',5'-oligoadenylate synthetase mRNA, mRNA 561, and mRNA 6-16 were in IFN-alpha-treated clones A1 and A2. These results demonstrated the existence of clonal heterogeneity in IFN responses in a cell line and strengthened the view that IFN treatment of cells generates multiple signals leading to a variety of IFN-induced phenotypes.     

In vivo and in vitro induction of (2'-5') oligoadenylate synthetase by human interferons in leukocytes from healthy donors and patients with renal cancer and hairy cell leukemia

The effect of in vivo administered interferon-alpha (IFN-alpha) on 2-5-oligoadenylate (A) synthetase activity of peripheral blood mononuclear cells (PBMC) was compared in patients with hairy cell leukemia and renal cell cancer. Basic levels of this enzyme varied from donor to donor, but mean levels were not significantly different in patients with renal cell cancer or hairy cell leukemia compared to healthy donors. After a single injection of 3 x 10(6) IU IFN, these basic levels rose 2- to 8-fold within 12-24 h post-injection and reverted to pretreatment levels after 48 h. The extent of this in vivo stimulation by IFN-alpha was similar in patients with hairy cell leukemia and renal cell carcinoma, and was correlated with down-regulation of IFN-alpha receptors. The in vitro effects of IFN-alpha, -beta and -gamma were compared after 18 h treatment with 10, 10(2) and 10(3) IU/ml of each IFN. Unlike IFN-alpha and -beta, IFN-gamma did not induce 2-5 A synthetase activity in either normal PBMC or hairy cells; these results were related to the effects of the three IFN on proliferative response of normal PBMC to phytohemagglutinin. Our data support the idea that 2-5 A synthetase activity is a marker of biological response to interferon treatment in human cancers.     

Multiple molecular forms of interferon display different specific activities in the induction of the antiviral state and 2'5' oligoadenylate synthetase

Both Hu IFN-alpha A and Hu IFN-alpha D, produced by two independent recombinant bacterial clones, are mixtures of monomers, dimers and trimers. These forms, when assayed individually in heterologous MDBK cells, induced different degree of antiviral and 2'5' oligoadenylate synthetase (2'5' A synthetase) activities: the antiviral activity of the monomer is greater than that of the dimer and the trimer, whereas the activity of 2'5' A synthetase induction is lower with the monomer than with the dimer or the trimer. Similar differences are also observed on human cells. Compared to the mononeric form, the dimeric and the trimeric forms of Hu IFN-alpha A show higher antiviral inducing activity on heterologous MDBK cells than on homologous WISH cells, whereas the 2'5' A synthetase inducing activity in these two cell lines is about the same. Thus for the same antiviral activity, the trimer or the dimer compared to the monomer are much better inducers of the 2'5' A synthetase on human than on MDBK cells.     

Possible role of genetic factor(s) on age-related increase of peripheral CD4+CD8+ double positive T cells in cynomolgus monkeys.

Mature TCR alpha beta T cells in peripheral blood are generally classified into either CD4 single positive (sp) T cells or CD8sp T cells. Several studies demonstrated that considerable amounts of CD4+CD8+ double positive (DP) T cells exist in peripheral blood of human and several animals. In particular, we previously reported that peripheral DP T cells increase in an age-related manner in cynomolgus monkeys (Macaca fascicularis), but the finding that DP T cells in some aged monkeys were maintained at a low proportion (under 5%), suggests that the increase in peripheral DP T cells might be genetically controlled in cynomolgus monkeys. To test this hypothesis, 24 families were randomly selected and used in a formal genetic analysis of the proportion of DP T cells. Parents and offspring in selected families were classified into DP-High and DP-Low groups based on a 5% cutoff level of DP T cells. The cutoff value was set by analysis of the distribution of the proportion of DP T cells. Nine out of 13 offspring (69.2%) with DP-High x DP-High parents belonged to the DP-High group, whereas three out of nine offspring (33.3%) belonged to DP-High group in the case of DP-High x DP-Low mating pairs. No offspring (0%) of two offspring with DP-Low x DP-Low parents belonged to the DP-High group. In addition, heritability (h2: narrow sense) obtained from the regression coefficient of offspring on mid-parent values was 0.54 +/- 0.19. Both findings suggest that increases in DP T cells in cynomolgus monkeys may be genetically controlled.     

Microinjection of anti-interferon antibodies into cells does not inhibit the induction of an antiviral state by interferon.

Affinity-purified polyclonal antibodies directed against human lymphoblastoid interferon (IFN), Escherichia coli-derived human IFN-alpha 2, or two synthetic fragments of human IFN-alpha 1 all neutralized the antiviral activity of human alpha IFNs when added to the culture medium of MDBK cells together with IFNs. However, when these antibodies were microinjected into the cytoplasm or the nucleus of cells, subsequent treatment of the cells with IFNs induced full protection against vesicular stomatitis virus. This suggests that IFNs themselves need not act in the cytoplasmic compartment or the nucleus to induce an antiviral state.     

Interferon induction of (2′–5′) oligoisoadenylate synthetase in diploid and trisomy 21 human fibroblasts: Relation to dosage of the interferon receptor gene (IRFC)

Trisomy 21 human fibroblasts are more sensitive to human interferon-alpha (IFN-alpha) than are diploid controls, consistent with the location of the gene (IFRC) which codes for the IFN-alpha receptor on chromosome 21. When compared in the antiviral assay, the difference in sensitivity is five- to tenfold, much greater than the 50% difference in IFRC gene dosage. An understanding of the mechanism by which this amplification of gene dosage occurs is relevant to the specific pathology of Down's syndrome and as a model system for studying the pathogenic effects of chromosomal aneuploidy. The enzyme (2'-5') oligoisoadenylate synthetase (2-5A synthetase), which is believed to be central to the interferon-induced antiviral response, is induced 50% more in trisomy 21 fibroblasts than in diploid controls. Thus the amplification in response occurs subsequent to the binding of IFN-alpha to its receptor and the triggering of the first set of intracellular events, the latter exemplified by the induction of 2-5A synthetase. Similar results were obtained with IFN-gamma, consistent with other evidence which indicates that a gene coding for a separate IFN-gamma receptor is also located on chromosome 21.     

Activation of 2'',5''-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low.