Abstract: Interferon-γ (IFN-γ) previously has been shown to inhibit the replication of Chlamydia psittaci in epithelial cells by inducing indoleamine 2,3-dioxygenase, the enzyme that decyclizes tryptophan to N-formylkynurenine. The role of indoleamine 2,3-dioxygenase in IFN-mediated inhibition of C. psittaci in human macrophages has now been examined. Peripheral blood monocytes from normal donors were isolated and cultivated 10–14 days to allow differentiation to macrophages. Cells were then treated with either IFN-γ or IFN-β for 48 h before infection with sufficient C. psittaci to infect approximately 30% of the cells. Infected cells were incubated 24 h, at which time coverslips were fixed, stained with Giemsa, and examined for development of C. psittaci inclusions by light microscopy. Complete inhibition of inclusion development was observed with IFN-γ. In the absence of lipopolysaccharide, inhibition of C. psittaci by IFN-γ was variable; however, in the presence of lipopolysaccharide, IFN-γ also complet...

Abstract: Ovine trophoblast protein-1 (oTP-1), the major product secreted by the trophectoderm of the sheep conceptus between days 13 and 21 of pregnancy, is considered to mediate maternal recognition of pregnancy by maintaining the function of the corpus luteum. Its amino acid sequence has 40–55% identity with various mammalian interferons-α (IFN-α), and it has been shown to have antiviral activity. The present results confirm that oTP-1, which at days 15–17 of pregnancy is produced by a single embryo at more than 100 μg (>1 million antiviral units) per day, is a functional IFN. A preparation of purified oTP-1 was made. Its amino-terminal sequence suggested that it consisted of a single homogeneous protein, so that its antiviral activity probably was not due to a contaminant. In a cytopathic effect inhibition assay with GBK-2 bovine cells challenged with vesicular stomatitis, its specific activity was 1.3 × 107 end point units/mg protein. It also protected GBK-2 cells against four other viruses, and A549 ...

Abstract: Ovine embryos produce an interferon (IFN)-αII in significant quantities during early pregnancy. This IFN, previously termed ovine trophoblast protein-1 (oTP-1), is a 172-amino-acid polypeptide which has been suggested to be the causal agent in maternal recognition of pregnancy in the ewe. Here we report the binding of oTP-1 and a recombinant bovine IFN-αI1 (rBoIFN-αI1; 165–166 amino acids long) to membrane preparations from ovine uterine endometrium. Both oTP-1 and rBoIFN-αI1 competed with each other for receptor binding. Based on Scatchard analysis, [125I]oTP-1 binding was determined to be complex and resolvable into a high-affinity (Kd = 3.8 × 10−11 M, 30 fmoles/mg protein) and a low affinity (Kd = 1.7 × 10−10 M; 96 fmoles/mg protein) component. Conversely [125I]rBoIFN-αI1 bound to only a single high-affinity receptor (Kd = 6.1 × 10−11 M; 174 fmoles/mg protein). Cross-linking experiments using disuccinimidyl suberate revealed that [125I]oTP-1 associated with membrane polypeptides of two molecul...

Abstract: Earlier studies showed that minor differences in primary structure among the interferon-α (IFN-α) protein family are reflected in their potency in selected biological assays. These studies have been extended and results from assays of antiviral, growth inhibitory and 2′,5′-oligoadenylate (2-5A) synthetase activities indicate that the various novel hybrid and analog species are differentially biologically active. Overall these observations suggest a correlation between predicted secondary structure characteristics, receptor binding affinity, and 2-5A synthetase, antiviral and growth inhibitory activities. Studies with a consensus IFN-α analog particularly implicated the region around residues 78 and 79 as influencing antiviral activity. Neutralization experiments with a monoclonal antibody directed against a conserved region from residues 113 to 149 indicated that although this region of the IFN-α molecule may be important for antiviral activity, altering residues at sites removed from this region...

Abstract: Patients receiving recombinant interferon-beta ser (rIFN-beta ser) for cancer and viral diseases were evaluated for formation of antibodies with IFN-neutralizing activity. The assay for serum neutralizing activity measures the ability of the sample to neutralize the antiviral activity of rIFN-beta ser. Neutralizing antibody incidence was route dependent. Of 335 patients treated intravenously, 3 were positive, representing an incidence of 0.9%. The remaining 13 antibody-positive patients had been treated subcutaneously, representing an incidence of 9.6% (13/136). In general, peak titers for neutralizing activity were low to moderate; 15 of the 16 patients (94%) had titers between 100 and 2,000 neutralizing units/ml. The incidence of neutralizing antibodies was higher in patients who had been under study for longer periods of time. Mean time of onset of neutralizing activity for the 16 positive patients was about 5 months. Because length of time on study appeared to be a possible predisposing factor for development of neutralizing antibodies, we recalculated the incidence of this activity for all patients on study for 5 months or longer (90 i.v., 71 s.c.). All of the 16 neutralizing-positive patients were on study for more than 150 days, whereas the remaining 87 in the i.v. group and 58 in the s.c. group were negative. Thus, a longer time on study appears to increase incidence, but in only a portion of subjects treated. No adverse clinical effects could be directly associated with neutralizing antibodies.

Abstract: Treatment of primary chicken kidney (CK) cultures with supernatants from concanavalin A-treated Eimeria tenella-immune avian splenic T cells significantly inhibited the invasion of the cel...

Abstract: In clinical trials with a range of doses of recombinant interferon-alpha 2a, we observed a 25% overall incidence of neutralizing antibody development. Careful data analysis did not reveal a relationship between antibody development and therapeutic response. Of 752 patients evaluable for antibody development and clinical response, 31% of the antibody-positive patients and 28% of the antibody-negative patients had therapeutic responses. Although the formation of anti-IFN antibodies seems to be associated with most IFN preparations, incidence and clinical significance have been difficult to determine for a number of reasons. Different assays with differing sensitivities for the detection of IFN antibodies have been utilized in different studies. Additionally, important clinical variables that affect antibody development have often not been carefully controlled or analyzed. The relationship between antibody development and multiple related factors, such as route of administration, dose regimen, cumulative dose, duration of treatment, and the underlying disease, requires clarification. Initial analyses suggest that the median time to antibody development may vary with the dose regime and cumulative dose, as well as the route of administration. Carefully designed prospective studies controlling for influential clinical variables and utilizing standardized assay techniques are required for a meaningful analysis of the antigenic potential of all therapeutic protein products.

Abstract: The antiviral potential of a novel cross-species active, recombinant human interferon-alpha B/D hybrid (rHuIFN-alpha B/D), was evaluated for its efficiacy in cultured human monocytes and in several murine models of viral disease. When examined in 14-day-old human monocyte cultures, rHuIFN-alpha B/D was highly effective in preventing viral replication and cell destruction caused by herpes simplex virus type 1 (HSV-1/VR3). The effect observed with 100 units of this hybrid IFN was as good or higher than that observed with equivalent amounts of rHuIFN-alpha A or IFN-gamma. In addition, a single dose (5 X 10(7) U/kg) of rHuIFN-alpha B/D administered several hours after intranasal infection with HSV-1/VR3 suppressed pulmonary virus replication and prevented death due to interstitial pneumonia. Similarly, mice infected with a more aggressive strain of HSV-1 (McIntyre) were protected when this IFN preparation was administered at the time of virus infection and 1 day later. The anti-retroviral activity of rHuIFN-alpha B/D was examined in two murine leukemia retroviral models, Rauscher (RMLV) and Friend (FMLV), and a murine model of acquired immunodeficiency (LP-BM5). Treatment of RMLV or FMLV infected mice significantly prolonged mean survival times and the number of long-term FMLV survivors. These therapeutic effects were demonstrated when IFN was administered on the day of virus infection or as late as 3 days following infection. Transient reversal of the immunosuppressive effects induced by LP-BM5 infection was observed when rHuIFN-alpha B/D treatment was initiated at the time of virus infection. Moreover, when rHuIFN-alpha B/D was used together with azidothymidine (AZT), the effect of the combination was better than either drug alone.

Abstract: Eight patients with advanced, previously treated non-small cell lung cancer inhaled natural leukocyte interferon-α (IFN-α) from a dosimeter-equipped jet nebulizer. Single doses of IFN ranged from 1 × 106 to 120 × 106 IU. Serum IFN was undetectable after single doses of 1 × 106 to 18 × 106 IU, but 60 × 106 IU resulted in measurable levels of circulating IFN in 3 out of 6 patients. All 5 patients who inhaled 120 × 106 IU had between 11 and 35 IU of IFN per milliliter of serum for at least 12 h. No systemic or local side effects were observed after 1 × 106 to 18 × 106 IU, but doses of 60 × 106 to 120 × 106 IU resulted in temperature rise, headache, and malaise. All symptoms started within 3–6 h, reached their peak by 8–10 h, and lasted until 12–24 h after inhalation. A decrease (>20%) in peak expiratory flow following inhalation was temporarily found in 2 patients. We conclude that IFN, given by inhalation, penetrates into the blood stream, thus causing systemic side effects similar to those describ...

Abstract: Analogs of human interferon-α1 (IFN-α1) were created in vitro by site-directed mutagenesis to investigate the structural requirements at amino acid position 123 for binding to the IFN rece...

Abstract: Sera obtained from 612 patients enrolled in 40 different clinical trials with an interferon-alpha-n1 (IFN-alpha-n1) preparation have been tested for neutralizing activity against this IFN in a cytopathic effect inhibition assay. Positive samples were assayed in triplicate. Of 391 patients treated for human papillomavirus infections, 13 developed neutralizing antibodies (3.3%); two suffering from condylomata acuminata and 11 from juvenile laryngeal papillomatosis (JLP). None has positive titers prior to receiving IFN-alpha-n1 and none appeared to lose the benefits of IFN treatment. Serial specimens from four JLP patients demonstrated that titers rose during IFN treatment and declined postherapy. None of the 221 cancer patients from whom serum samples were available had positive titers. The overall incidence of IFN neutralizing sera from these IFN-alpha-n1 recipients was 2.1%.

Abstract: Three dosages (0.1, 0.5, and 2.5 mg/animal, subcutaneously), of recombinant bovine interferon-gamma (rBoIFN-gamma) were evaluated for their in vivo influence on neutrophil function and lymphocyte blastogenesis in cattle. The optimal of the three dosages tested (0.5 mg/animal or 1.1 x 10(6) U/animal) was then evaluated for its influence on neutrophils and lymphocytes in both normal and dexamethasone-treated cattle. One animal, which received 2.5 mg of rBoIFN-gamma, died by 24 h after administration due to acute diffuse interstitial pneumonia with interlobular edema and emphysema. The two highest dosages used caused fever at 24 h and the highest dosage caused a decrease in lymphocyte blastogenesis at 24 h after administration. The influence of rBoIFN-gamma on neutrophil function was dose dependent and depended on the baseline values for neutrophil function. Random migration by neutrophils was consistently inhibited in animals that received 0.5 mg or more of rBoIFN-gamma. Staphylococcus aureus ingestion and antibody-dependent cell-mediated cytotoxicity by neutrophils was enhanced by rBoIFN-gamma treatment in both dexamethasone-treated cattle and in nondexamethasone-treated cattle, which had relatively low values for these parameters before treatment. Iodination by neutrophils was also enhanced by rBoIFN-gamma when either a suboptimal concentration of neutrophil stimulant was used or when the cattle were treated with dexamethasone. In summary, the rBoIFN-gamma had greater immunomodulator activity in immunosuppressed than in normal cattle. The in vivo influence of rBoIFN-gamma therefore depends on the physiologic status of the animal.

Abstract: Intranasal recombinant interferon-alpha 2b (rIFN-alpha 2b) protects against natural colds due to rhinoviruses, but apparently not against those caused by viruses. Because rIFN-beta serine17 (rIFN-beta ser) appears less active than rIFN-alpha 2b in preventing natural rhinovirus colds, we compared the two IFNs in two in vitro assays against selected respiratory viruses. In a yield reduction assay, both IFNs had comparable activity against rhinovirus types 39 and 1A and coronavirus 229E, which were inhibited by 90% or more at IFN concentrations of 10(-11) to 10(-10) gram of protein/ml (approximately 2-20 IU/ml). Similar activities were observed with rIFN-beta ser against rhinoviruses isolated from clinical specimens. At concentrations of 10(-9) gram protein/ml, both IFNs inhibited the growth of influenza A and parainfluenza viruses, but not of adenovirus or respiratory syncytial virus in the cell culture systems tested. Thus, the different clinical protection conferred by rIFN-alpha 2b and rIFN-beta ser in studies of natural rhinovirus colds are not accounted for by differences in their in vitro activity against these viruses, and other explanations must be found.

Abstract: Recently, two proteins, ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1), that are major products of the preimplantation ovine and bovine embryo, respectively, have been shown to be interferons (IFNs). Both proteins belong to an IFN-α subfamily whose polypeptide chains are 172 amino acids long

Abstract: Previous studies have reported a low (less than 3%) incidence of anti-interferon (IFN) serum neutralizing antibodies following treatment with IFN-alpha 2b. Since this result contrasts with a higher incidence reported with IFN-alpha 2a, the question has been raised whether differences in assay techniques and patient comparability rather than inherent differences in the molecules might account for the reported differences in antibody incidence. In this report two patient groups, 151 hairy cell leukemia (HCL) patients and 101 patients with other malignancies, who have received long-term dosing with IFN-alpha 2b, are reported. The sera of both groups were studied before, during and after treatment by various assay methodologies. Utilizing three assay techniques, a less than 3% overall incidence of serum antibody formation was confirmed in these retested samples. With over 575 samples tested in multiple assays, the radioimmunoassay, as utilized in prior reports, demonstrated 99% agreement with a bioassay. Therefore, prior speculation that the assay technique for IFN-alpha 2b might produce a high false-negative rate was disproven. Additionally, the clinical outcome of these patients also failed to demonstrate a pattern of clinical relapse. In summary, these new analyses confirm the low (less than 3%) incidence of neutralizing antibody development following treatment with IFN-alpha 2b and confirm that no high rates of clinical relapse have developed in patients treated with chronic long-term dosing. Assay methodology does not appear to be a likely explanation for the low incidence of antibody formation reported with IFN-alpha 2. Rather, the unique molecular structure and pharmaceutical formulation of IFN-alpha 2b remains the most likely explanation of its minimal antigenicity.

Abstract: Interferon (IFN) was shown to inhibit herpes simplex virus type 1 (HSV-1) replication at the transcription of the immediate early (α) genes. This apparent inhibition could be due to a dire...

Abstract: A murine cosmid clone harboring the single-copy interferon-β1 (IFN-β1) gene and extended flanking sequences was isolated. The functional IFN-β1 promoter is contained within a 170-bp DNA fragment located 5′ of the coding sequence. This was shown by fusion of this fragment to a heterologous reporter gene and transient as well as stable expression in mouse L and monkey CV-1 cells. With the help of these functional assays, it could be demonstrated that the 5′-flanking sequences are the target for the typical regulatory action of common type I IFN activators. DNA sequencing reveals a considerable homology to the human IFN-β1 promoter within the 280 upstream base pairs. The homology is particularly pronounced within the DNA region containing the virus responsive element (VRE). This phenomenon may explain the similarity of both genes in the mode of regulation. The mouse promoter fragment compared with the human equivalent was shown to be several times more efficient in transcriptional activation in muri...

Abstract: When 23 ponies were infected with equid herpesvirus-1 or -4 (EHV-1 or EHV-4), nasal shedding of interferon (IFN) correlated closely with the duration of viral excretion. Equine interferon (EqIFN) was detected in the serum only from animals infected with the EHV-1 virus, and here high levels correlated with clinical symptoms of locomoter disorder and indicated a poor prognosis. Low levels of IFN were detected in explanted mononuclear cells from ponies infected with either virus.

Abstract: Poly(I):poly(C) (rIn.rCn), mismatched poly(I):poly(C) [rIn.r(C12U)n], and poly(I):poly(C) poly-lysine carboxymethylcellulose [poly(ICLC)] were studied for their capacity to induce interferon-alpha (IFN-alpha) in human peripheral blood mononuclear cell (MNC) cultures and in their subpopulations. In MNC, poly(I):poly(C) and mismatched poly(I):poly(C) induced IFN-alpha in a dose-dependent manner, whereas poly(ICLC) was unable to do so in concentrations that ranged from 1 to 160 micrograms/ml. In contrast, all three molecules were incapable of inducing IFN-alpha when added into either purified monocyte or lymphocyte cultures. The capacity of poly(I):poly(C) to induce IFN-alpha was reestablished only in monocyte cultures when, prior to the stimulation, the cells were exposed for at least 2 h to supernatants from poly(I):poly(C)-stimulated lymphocyte cultures. IFN-beta and IFN-gamma were found in those supernatants and the ability of each dsRNA to induce IFN-alpha correlated with its ability to induce IFN-gamma. Further studies using recombinant human IFN-gamma and IFN-beta and their specific antibodies corroborated an important role of these molecules for the induction of IFN-alpha with dsRNA. Because each IFN type is produced by different cells, our studies show that complex cell-to-cell interactions via other IFNs have to take place in peripheral blood mononuclear cells (MNC) cultures before IFN-alpha can be induced by dsRNA.

Abstract: Systemically administered interferon (IFN) is not readily detected in the central nervous system (CNS) due to the presence of the blood-brain barrier (BBB). A method of osmotic BBB alterat...

Abstract: Combinations of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) demonstrate synergistic antiproliferative activity in vitro. Therefore, we initiated a clinical study of recombinant TNF-alpha (rTNF-alpha) and rIFN-gamma combination therapy in humans. Twenty-five patients with metastatic cancer received both rTNF-alpha and rIFN-gamma by intramuscular injection for 5 consecutive days every 2 weeks for a total of 4 weeks. The dose levels were 5/5, 10/5, 10/10, 25/10, 25/25, 50/25, 50/50, 75/50, and 75/75 micrograms/m2 per day of rTNF-alpha/rIFN-gamma. A minimum of 2 patients were entered sequentially at each dose level. If the first 2-week cycle of therapy was well tolerated, the dose was increased to the next highest dose level during the second 2-week cycle. Fever, chills, and fatigue were observed at all doses. Severity of symptoms corresponded to increasing dose levels. Although TNF is identical to a molecule known as "cachectin," the vast majority of our patients did not lose weight while on study. However, alterations in lipid metabolism occurred and were manifested by a median change in triglyceride values of +40 mg/dl (range, -130 to +318 mg/dl), and in cholesterol values of -30 mg/dl (range, -103 to +2 mg/dl). The maximum tolerated dose was 75 micrograms/m2 of rTNF-alpha combined with 50 micrograms/m2 of rIFN-gamma, with dose-limiting side effects being mainly constitutional symptoms. A dose-related suppression in granulocyte and platelet counts was observed. Hematologic parameters returned to baseline within 72 h after therapy was discontinued, and neither infection nor bleeding occurred. Ten of 22 evaluable patients had stable disease for a median of 8 weeks (range, 4-21 weeks); 12 patients showed progressive disease. This study will form the framework for phase II trials of rTNF-alpha and rIFN-gamma combination therapy.

Abstract: In porcine Leydig cell culture, incubation with natural or recombinant human interferon-α (IFN-α), or recombinant porcine IFN-γ preparations were effective in inhibiting basal and human ch...

Abstract: We have isolated and characterized two types of cDNA clones corresponding to interferon (IFN)-induced 1.6- and 1.8-kb mRNAs, as encoding two different forms of the 2′,5′-oligoadenylate (2′...

Abstract: Commonly viral infections induce expression of type I interferon (IFN) genes. The induction is primarily due to transcriptional activation of the genes. Soon after the isolation of the genes encoding IFN-alpha and IFN-beta, the IFN system became a subject of intensive study in the context of switching on or off the otherwise silent genes by extracellular stimulation signals. Hence, the IFN system serves as a typical model in studying the genetic events occurring in many cytokine systems. The IFN genes contain within their 5'-flanking region positive elements that function efficiently in cells induced by viruses and other stimuli such as double-stranded RNA. At least three transcription factors have been identified and molecularly cloned. Of these, two factors, designated interferon regulatory factors IRF-1 and IRF-2, are unique in their function; they interact with identical DNA sequence elements of IFN and IFN-inducible genes. Results of the functional studies on these factors suggest that transcription of the IFN and IFN-inducible genes is regulated by two similar trans-acting factors that apparently compete for the same cis-acting recognition sequences, but mediate opposite effects.

Abstract: Circulating autoantibodies constitute a prominent feature of many autoimmune diseases. Recently, antibodies to interferon (IFN) have been recognized as an additional variant occurring spontaneously in about 10% of patients with autoimmune disorders. The reactivity of these antibodies appears to be restricted to IFN-alpha, with antibodies of individual patients recognizing unique epitope patterns. IFN antibodies are preferentially of the IgG type, and they likely constitute part of the normal B-cell repertoire. Whereas antibody titers differ largely among patients, the affinity of IFN binding has not been determined. Likewise, the mechanisms involved in IFN antibody synthesis as well as the clinical impact of these antibodies in autoimmune disorders remain elusive. Based on recent reports and our own observations, this article reviews current knowledge regarding occurrence and properties of IFN antibodies associated with autoimmune conditions. From clinical and experimental evidence, mechanisms possibly involved in IFN antibody production are derived and the significance of antibody-mediated sequelae is evaluated.

Abstract: Twenty-seven patients in the chronic phase of a Philadelphia chromosome-positive chronic myelogenic leukemia (CML) underwent a monotherapy with recombinant interferon-alpha 2b (rIFN-alpha 2b), receiving a mean dose of 3 x 5 Mio. IU per week. Eight of the 27 patients developed both rIFN-alpha 2b-binding and -neutralizing antibodies during therapy. Sera of these patients abrogated both antiviral and antiproliferative activities of rIFN-alpha 2b, but not of nIFN-beta or rIFN gamma. Only one serum neutralized nIFN-alpha. The IFN binding and neutralizing titers rose during therapy, while a decrease occurred over months after cessation of therapy. The IgG fraction obtained by DEAE chromatography contained more than 90% of the IFN-neutralizing capacity of the purified sera. This result strongly suggested that IgG antibodies are responsible for the IFN-alpha-neutralizing activity.

Abstract: An interferon (IFN) inducer and immunomodulator, CL246,738 [3,6-bis(2-piperidinoethoxy)acridine trihydrochloride], protected mice from lethal infection with Semliki Forest (SFV) and Banzi ...

Abstract: Patients receiving large amounts of interferon (IFN), particularly cloned IFN-alpha, have been reported to develop antibodies to IFN-alpha. We used a neutralization technique bioassay to monitor 175 patients undergoing recombinant (r) IFN-alpha 2b therapy in an attempt to correlate antibody production to disease progression. Only 1 patient being treated for chronic myelogenic leukemia produced antibodies. Our findings indicate that rIFN-alpha 2b has minimal antigenicity, even when administered for prolonged periods of time.

Abstract: The object of this study was to investigate the effects of different single doses of recombinant interferon-γ (rIFN-γ) on white blood cell counts, differential blood counts, and the relative composition of T-cell subsets in the peripheral blood of cancer patients. Sixteen patients suffering from metastasizing renal cell carcinoma received 10, 100, or 500 μg of rIFN-γ three times at weekly intervals. After a therapy-free interval of 2 weeks, the next dose level was applied. The order of dose levels was assigned randomly to each patient. White blood cells, differential blood counts, and the number of Leu1, Leu3, Leu2a, Leu7, and HLA DR+ cells were measured immediately before and at 4, 24, 48, 72, 96, and 168 h after the administration of single doses of rIFN-γ. Results indicated that white blood cells were transiently removed from the blood circulation after administration of IFN-γ. Monocytes, HLA DR+ cells, and Leu7+ cells were reduced to below 50% of pretreatment values 4 h after application of t...