Natural Interferon Research Articles

Immunosorbent Binding Bioassay: A Solid Phase Biological Immunoassay for the Titration of Antisera to Alpha Interferons

An Immuno Sorbent Binding Bio-Assay (ISBBA) for the detection and the titration of antisera to the highly biologically active proteins, alpha interferons, is described. This method is similar to a classical solid phase immunoassay, except for the last step which uses the biological activity of the captured antigen. With specific serum antibodies the bound antigen prevents further virus induced cell lysis. On the contrary, with negative serum or preserum the antigen is washed out and virus induced cell lysis occurs, therefore no labelling is required. ISBBA exhibits three main differences when compared to the reference method i.e. the neutralization assay (NA): i) ISBBA is 10 to 1000 fold more sensitive than NA; ii) in contrast to NA, ISBBA is based on the production of an antiviral effect; iii) ISBBA makes it possible to use unpurified antigen. The applications of ISBBA to subtypes study in natural alpha interferon samples are discussed.

What roles have anti-interferon antibodies in physiology and pathology?

Until recently the generation of antibodies to interferons (IFNs) was considered an unlikely event, while it is now clear that natural interferons (except IFN-beta) are practically nonimmunogenic, although recombinant interferons give rise to antibodies in about 30% of patients with occasional clinical complications. By realizing that normal individuals display spontaneously traces of IFN autoantibodies, in this review it is suggested that (if immunotherapy has to succeed) new generations of recombinant proteins should be the least antigenic as possible.

Natural alpha interferon as maintenance therapy for small cell lung cancer

Natural alpha interferon as maintenance therapy for small cell lung cancer

Induction of interleukin-1 and -6 in human gingival fibroblast cultures stimulated with Bacteroides lipopolysaccharides

Normal human gingival fibroblasts stimulated in vitro by lipopolysaccharides (LPS) from oral Bacteroides species produced cell-free and cell-associated thymocyte-activating factors (TAF). Neutralization assays using antisera to human interleukin-1 alpha (HuIL-1 alpha), HuIL-1 beta, and HuIL-6 revealed that cell-free TAF was attributable mainly to IL-1 beta and that IL-6 augmented the TAF activity of IL-1 beta in the culture supernatant. Another factor(s), however, may also be involved in cell-free TAF. By contrast, the active entity of cell-associated TAF was ascribed to IL-1 alpha alone. Furthermore, IL-6 was detected mainly in the supernatant of fibroblast cultures stimulated with Bacteroides LPS. Fibroblasts pretreated with natural human beta or gamma interferon, but not those pretreated with alpha interferon, synthesized higher levels of cell-associated IL-1 alpha in response to stimulation by Bacteroides LPS; however, no interferons exhibited direct IL-1-inducing activity or synergistic IL-1-inducing activity with LPS. Endogenously induced beta interferon was suggested to be necessary for fibroblasts to produce cell-associated IL-1 alpha in response to Bacteroides LPS.

Treatment of Squamous Cell Carcinoma with Interferon

Human natural leukocytic interferon and recombinant HulFN alpha 2c can be used in the therapy of squamous cell carcinoma. The duration of treatment was 3-6 weeks. A single dose was 400,000-5,000,000 units given weekly for 3-6 weeks. Clinically and histologically 19 of 32 patients were cured and tumor cells were not found in the material taken after interferon treatment for the second biopsy. In ten patients tumor size was reduced 25-90%, and in three patients tumor size was not reduced according to clinical findings. With recombinant HulFN alpha 2c therapy given 5 times per week for 4 weeks. Four of ten patients with similar tumors were cured clinically and histologically clinical findings. In five patients tumor size was reduced 25-90%, while in one patient there was no reduction in tumor size. Both types of interferons are effective in the treatment of squamous cell carcinoma. Side reactions were mild.

EVIDENCE FOR EXTENDED MAINTENANCE OF THE CORPUS LUTEUM BY UTERINE INFUSION OF A RECOMBINANT TROPHOBLAST α-INTERFERON (TROPHOBLASTIN) IN SHEEP

Ovine trophoblastin (oTP) is a natural interferon of the class-II interferon-alpha subfamily. Recombinant ovine trophoblastin (r.oTP), produced by genetic engineering, was purified by anion-exchange HPLC. The product exhibited a high degree of homogeneity (greater than 98%), and similar immunological cross reaction and antiviral activity to natural oTP. Antiluteolytic activity of r.oTP was established by intrauterine injection in two groups of cyclic recipient ewes. Control group A included 10 ewes which received sterile BSA in saline twice daily for 8 days (from day 10-12 of oestrous cycle). Experimental group B included 17 ewes which received 80 micrograms (4 ewes), 170 micrograms (8 ewes) or 340 micrograms (5 ewes) r.oTP daily for 8 days. Maintenance of functional corpora lutea for 1 month or more was observed in 4 out of 5 ewes which received high doses of r.oTP. These results indicate that oTP alone extends luteal secretory activity.

Antitumor Activities of Interferon Alpha, Beta, and Gamma and their Combinations on Human Melanoma Cells In Vitro: Changes of Proliferation, Melanin Synthesis, and Immunophenotype

The antitumor activities of human interferon (IFN) alpha, beta, and gamma alone or in combination were studied on four human melanoma cell lines (StML-11, StML-12, StML-14, and SKMel-28) in various concentrations (1-50,000 IU/ml IFN alpha, 0.1-1000 IU/ml IFN beta, 1-10,000 IU/ml IFN gamma) in vitro. In all experiments IFN beta exhibited the most potent antiproliferative effect of all IFN tested. After 3 d of incubation a 50% growth inhibition was achieved with 20-40 IU/ml for natural IFN beta and with 600-1200 U/ml for recombinant IFN gamma. Substantially higher doses (7,000 to more than 50,000 IU/ml) of recombinant IFN alpha 2a were required to achieve a 50% growth inhibition. A strong synergistic antiproliferative activity resulted from the combination of IFN alpha with IFN gamma and IFN beta with IFN gamma. None of the IFN tested induced terminal differentiation of melanoma cells in vitro. The formation of dendrites was inhibited, and the portion of differentiated cells in vitro was reduced after treatment with IFN in comparison to the untreated controls (untreated controls: 100%; portion of differentiated cells after treatment with IFN alpha: 58%-74%, IFN beta: 48%-96%, IFN gamma: 10%-33%). The melanin synthesis was slightly elevated after treatment with IFN alpha (untreated controls: 100%; after treatment with IFN alpha: 103%-157%, ns.) and decreased significantly after treatment with IFN beta (49%-71%, p less than 0.05) as well as with IFN gamma (80%-88%, ns.). Cell surface markers were modulated varyingly by the IFN: HLA-I antigens were enhanced by all IFN, with IFN beta emerging as the most potent inducer. Only IFN gamma, however, induced a de novo expression of HLA-DR and -DQ antigens and increased the expression of the ICAM-1 molecule and of the melanoma progression marker A.1.43. Possibly, these findings indicate a biologically more aggressive phenotype of melanoma cells.

Low-dose oral administration of human interferon alpha can control the development of Theileria parva infection in cattle

Two natural human interferon alpha preparations, (nHuIFN-alpha [Cantell]) and (nHuIFN-alpha [ISI]), were used for the oral treatment of cattle experimentally infected with Theileria parva parva. In the first experiment, 8 Friesian bulls were inoculated with a 1 in 10 dilution of a sporozoite stabilate of T.p. parva (Marikebuni) stock. Four of the cattle were treated daily with 1 international unit/kg body weight (i.u./kg bwt) of nHuIFN-alpha (Cantell) from day -2 to day 8 p.i. None of the 4 calves given IFN developed clinical theileriosis, but 3 of the 4 control calves died of theileriosis while the fourth had a mild infection. Three of 4 treated calves and the 1 surviving control calf developed a detectable antibody response to T.p. parva schizont antigen but, on challenged with a 10-fold higher dose of stabilate, the surviving control animal and only 1 of the 4 treated calves proved to be immune. In a second experiment, 4 groups of 4 calves were inoculated with the same stabilate dilution. Three treatment groups were given either 1 i.u. nHuIFN-alpha (Cantell), 1 i.u. nHuIFN-alpha (ISI), or 10 i.u. nHuIFN-alpha (ISI)/kg bwt from day -2 to day 8 p.i. once daily and the fourth group were controls. Clinical theileriosis occurred in 2 controls, 2 calves given 10 i.u. nHuINF-alpha (ISI), 1 calf given 1 i.u. nHuIFN-alpha (ISI) and no calves given 1 i.u. nHuIFN-alpha (Cantell)/kg bwt. Of these, 2, 1, 0 and 0 cattle died in the respective groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin A Deficiency Decreases Natural Killer Cell Activity and Interferon Production in Rats

We examined the effect of vitamin A deficiency on natural killer (NK) cell activity and interferon (IFN) production. Rats were weaned at 16 or 21 d of age onto semisynthetic diets containing either 0 or 4 micrograms retinol/g diet. At the time of study, retinol-depleted rats had serum vitamin A concentrations less than 7% of those of pair-fed controls. In two studies, rats exhibited no external signs of retinol deficiency, but with further depletion some symptoms were observed. Splenic NK cell activity against chromium-51-labeled YAC-1 cells was significantly decreased in vitamin A-depleted rats (22-80% of values for control rats, depending on the degree of retinol deficiency), regardless of the ratio of effector to target cells used. When vitamin A-depleted rats were repleted orally with retinol, NK cell activity was consistently normalized. To understand the possible mechanisms involved in decreasing NK cell activity, we investigated IFN production by concanavalin A-stimulated spleen cells from vitamin A-depleted, from repleted and from control animals. IFN titers were significantly decreased (22-33% of values for control rats) in supernatants of spleen cell cultures of the vitamin A-depleted rats. Repletion with vitamin A resulted in IFN activities ranging from 80 to 130% of controls. Adding alpha/beta IFN in vitro to the spleen cells of vitamin A-depleted animals increased their NK cell activity. The number of spleen cells reacting with a monoclonal antibody specific for rat NK cells was slightly lower in retinol-depleted rats, but not enough to account for the differences in NK cell and IFN activities. These data suggest that vitamin A deficiency affect the nonspecific arm of the immune system, possibly by altering the functional capacity of cells to produce lymphokines needed for the generation of an appropriate cytolytic response.

Antiproliferative effects of natural interferon beta alone and in combination with natural interferon gamma on human breast carcinomas in nude mice

Nude mice bearing bilateral xenografts of human breast carcinoma cells (MCF-7 and BT20) were treated with 2 or 45-day cycles of intralesional (i.l.) injections of human natural interferon beta (nIFN-beta) alone or in combination with human natural interferon gamma (nIFN-gamma). The injections were administered to only 1 of the 2 tumors in each animal, thus making it possible to assess at the same time local therapeutic effects in the injected tumors and systemic effects in the contralateral ones. When n-IFN-beta was used as a single agent only mild local antitumor effects and virtually no systemic effects were observed. In contrast, the combined administration of nIFN-beta/nIFN-gamma produced marked antiproliferative effects, presumably as a result of the synergistic action of type I and type II IFNs. These effects ranged from complete regression documented histologically in 2 MCF-7 tumors to varying degrees of growth inhibition with persistence of residual microscopic or grossly detectable tumor. Local effects were more pronounced than systemic effects. The therapeutic efficacy of nIFN-beta proved to be greater than that of recombinant interferon beta (rIFN-beta). In MCF-7 tumors nIFN-beta appeared to be less effective than nIFN-alpha, whereas the opposite was true for BT20 tumors.

Studies in the in vivo expression of the influenza resistance gene Mx by in-situ hybridisation

The inbred laboratory mouse strain A2G carries a functional, interferon type 1 inducible gene, Mx which upon expression confers specific resistance to an otherwise lethal dose of influenza virus. We investigated in vivo Mx gene expression by performing Northern hybridisation and in-situ hybridisation on A2G (Mx+) and (CBA/J x C57)F1 (Mx-) mice that were induced either with human, natural interferon; human, recombinant interferon or double stranded poly(I):(C). All 3 inducers were able to stimulate Mx expression in all organs examined in the A2G strain. However, contrary to previous reports, Mx expression was confined to a small number of cell types; the main contributor was most probably mononuclear cells. Specialised cells such as hepatocyte, nephron, ovarian follicle and seminiferous tubules did not show detectable Mx level. There was also constitutive Mx expression in the epithelia of uterus and duodenum which suggested direct gene activation independent of blood-bourne interferon.

Effect of bovine fibroblast interferon on rhinovirus infection in calves

Summary The prophylactic/therapeutic activity of natural bovine fibroblast interferon (BoF-ifn) against bovine rhinovirus infection in calves was assessed. Six calves were each given 8 intranasal inoculations of partially purified BoF-ifn (3.25 × 105 U at 8 am, 11 am, 5 pm, and 8 pm on day 1 and 8 am, 11 am, 2 pm, and 5pm on day 2), and 6 calves were given placebo. All calves were challenge exposed with 105.1 TCID50) of bovine rhinovirus after the first 2 treatments (6 hours after the first ifn or placebo treatment). Nasal excretion of rhinovirus, ifn concentration in the nasal secretions, and nasal secretion and serum rhinovirus antibodies were measured before and at selected times after calves were inoculated. Interferon-treated calves excreted rhinovirus in their nasal secretions in lesser amounts (mean value, 0.84 log10 TCID50/ml vs 1.58 log10 TCID50/ml on postchallenge exposure days 1 and 2; (P < 0.05) and for a shorter duration (P < 0.05) than did placebo-treated calves. No calves developed clinical signs of respiratory tract illness. Rhinovirus antibody titer was not significantly different between ifn- and placebo-treated calves.

Purification and characterization of natural human interferon omega 1. Two alternative cleavage sites for the signal peptidase.

Human interferon omega 1 (IFN-omega 1 = IFN-alpha II1) is a recently discovered protein structurally related to IFN-alpha and -beta; the biological activities of IFN-omega 1 and its physiological role are not known to date. We have purified IFN-omega 1 from preparations of human leukocyte IFN, derived from peripheral blood leukocytes induced with Sendai virus, by two sequential cycles of monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase high performance liquid chromatography and showed an Mr of 24,500 upon sodium dodecyl sulfate-gel electrophoresis (theoretical Mr, 19,984). Amino acid sequence analysis revealed that only about 40% of the molecules have the NH2 terminus expected on the basis of the sequence similarity to IFN-alpha, whereas the others contain two additional amino acids. This difference probably results from variable cleavage of the pre-protein by the signal peptidase. No evidence for COOH-terminal heterogeneity was found. Essentially all IFN-omega 1 molecules are glycosylated; enzymatic deglycosylation resulted in a reduction of the Mr to 20,500. Experiments using several plant lectins indicated the presence of biantennary complex oligosaccharides containing neuraminic acid. Two major peaks were observed upon chromatofocusing, with isoelectric points of 8.1 and 8.5. The specific antiviral activity of purified IFN-omega 1 assayed on human cells was determined to be 2.7 x 10(8) IU/mg, similar to that of other human class I IFNs; potent antiviral activity was also observed on cells of bovine and ovine but not of equine or murine origin.

Effects of Subchronic Treatment with Natural Human Interferons on Antipyrine Clearance and Liver Function in Patients with Chronic Hepatitis

To determine whether natural human interferon administered under the usual therapeutic dosing scheme would inhibit the hepatic drug metabolism, we performed an antipyrine test in eight patients with chronic B or non-A, non-B hepatitis before and after a subchronic interferon therapy (6 megaunits/day for 17 +/- 4 days, mean +/- SD). Six patients received interferon-beta and 2 received interferon-alpha. To circumvent a possible influence of interferon-induced fever on the hepatic drug metabolism, the antipyrine test during the interferon therapy was performed at least 14 days after the interferon-induced fever disappeared. The kinetic parameters of antipyrine were obtained from seven saliva samples over 32 hours postdose. There were no significant differences in any kinetic parameters of antipyrine observed before and during the interferon therapy. With the sample size of the study, there was only a 20% chance (i.e., beta-power = 0.8 at alpha = 0.05) that we might have missed a 17% reduction in antipyrine clearance by the interferon therapy (type II error). On the other hand, the subchronic interferon therapy lowered serum aminotransferases and DNA polymerase activity significantly (P less than .05) compared with the respective baseline values. Our results suggest that the subchronic therapeutic dosing scheme of interferon as conducted in the present study does not cause the inhibitory effect on the oxidative drug metabolism to a statistically significant or clinically relevant degree in patients with chronic hepatitis, while it improves their liver function. Further studies are required for determining if different types of interferons administered under the different dosing schemes would alter the hepatic drug metabolism and the inhibitory effect would be time-dependent.

Existence and unique N-terminal sequence of alpha II (omega) interferon in natural leukocyte interferon preparation

Human alpha interferon produced in Sendai virus-stimulated leukocytes was purified by immunosorbent affinity chromatography and subjected to subtype analysis. All subtypes in leukocyte culture supernatant were confirmed in purified preparation. One of these subtypes was reactive in Western blotting with antibody specific to alpha II (omega). N-terminal amino acid sequence analysis of this particular subtype showed addition of two amino acids to the N-terminus of alpha II predicted through cDNA studies. The apparent cleavage site for leader sequence of alpha II was different from the site common to all other alpha subtypes. This is the first report on the isolation of natural alpha II and its unique N-terminal amino acid sequence.

Studies on subtype composition in natural leukocyte interferon preparations

Antisera raised against partially purified human leukocyte interferon in goat and horse were exhaustively adsorbed and purified. Affinity chromatography using these antibodies gave 10000-fold purification in one step without losing interferon activity. Then enzyme immunoassay was established with these antibodies and utilized for subtype analysis. Crude and affinity purified leukocyte interferons showed identical profiles of antiviral activity and immunoreactivity when fractionated by means of reversed phase high performance liquid chromatography. These results suggested the possibility of providing mixture of subtypes of leukocyte interferon occurring in natural host response, with comparable purity of recombinant ones.

Effects of human recombinant alpha and gamma and of highly purified natural beta interferons on simian Spumavirinae prototype (simian foamy virus 1) multiplication in human cells

The present study demonstrates the inhibitory effect of human recombinant interferons (r-Hu-IFN) alpha and gamma, and that of highly purified natural human interferon beta on the replication of simian foamy virus type 1 (SFV1) in human AV3-cell cultures. All IFN led to strong inhibition of the SFV1 cytopathic effect. Electron microscopy showed a 70 to 95 % decrease in viral particles. Significant inhibition of virus-associated reverse transcriptase activity was found in supernatant fluids of infected IFN-treated cultures. Metabolic labelling of the virus confirmed the inhibition of extracellular release of SFV1. PAGE analysis of immunoprecipitates indicated a reduction in viral-specific protein bands. Altogether, these results indicate that the mechanism of inhibition of Spumavirinae infection by interferon differs from that described for the other Retroviridae, and particularly for types B, C and D viruses. Our data is of therapeutic interest since Spumavirinae have been linked to pathological processes such as de Quervain thyroiditis.

Biotechnology and the Third World: development strategies in Cuba.

In March 1981, six researchers started production of natural interferon in a modest building adapted as a laboratory. By May the first yields of this protein were achieved. Applications of this interferon were already taking place in 1981 in the first clinical trial with the drug in the country. In June, the top level of the government decided to organize the Biological Group. In January, 1982, a new institution (CIB) began work with about 30 scientists and developed activities in gene manipulation, molecular virology, work with monoclonal antibodies, immunochemistry and tissue cultures. By 1983 interferon genes were cloned and expressed, and the group continued working, increasing the number of personnel. A decision was made in 1984 to build a new center, the CIGB. In July 1986, the center was officially inaugurated. Between 1986-89 important results were achieved by members of the biological group, among which was a vaccine against meningococcal meningitis. Three years later, in July 1989, the CIGB produced 15 new recombinant proteins, drugs, enzymes and antigens for diagnostic purposes. With the application of the most advanced technology, research has been conducted in areas such as gene regulation, bacterial and yeast genetics, protein structure and mammalian development. In addition, 43 types of monoclonal antibodies were produced as were 38 types of restriction enzymes (three of them recombinant), as well as several linkers, markers, and phages. Interferons, monoclonal antibodies, restriction enzymes, and epidermal growth factor, among others, are all being marketed. The CIGB became the leading institution in the country for work in modern biotechnology, generating new work styles in the country and developing a special selection procedure to include young scientists on the staff, simultaneously providing intense training in the center and abroad. This provided the stimulus of participating in the achievement of important results for the country, producing a positive feedback loop to maintain high standards. Interferon has been applied in more than 10,000 cases in the country. The diagnostic kit for AIDS based on recombinant proteins has been used in millions of tests and epidermal grow factor is already registered as a drug after fulfilling all the requirements of the preclinical and clinical testing with hundreds of patients in different clinical trials. A close connection with different institutions working in the field of biotechnology and with different universities has provided a greater strength for the work in modern biotechnology in Cuba.

Natural killer (NK) activity and interferon (IFN) production by a fraction of spleen and blood lymphocytes in swine

After carbonyl iron treatment and gradient isolation, spleen and blood pig lymphocytes exhibited NK activity and produced IFN after viral induction. Removal of plastic-adherent cells, including the majority of B cells, did not change these activities. The plastic-non-adherent cells were further separated into two subsets of roughly similar size by panning using a monoclonal, anti-T, and anti-null cell antibodies (81+cells). NK activity and IFN production were found in the 81 − cell fraction. A significantly higher proportion of null lymphocytes from blood and of splenic Fc-gamma receptor-bearing lymphocytes was also found among the 81 − cell fraction as compared to the 81 + fraction, without any change among other subsets. Similar proportions of helper (PT4+), cytotoxic (PT8+) and total T cells (MSA4+) were found among lymphocytes bound to target K562 cells and among the whole lymphocyte population. In contrast, lymphocytes that bound K562 cells demonstrated a striking increase in the proportion of Fc-gamma receptor-positive cells of high affinity. These results show that NK cells and IFN-producing cells are mainly included in the same blood and spleen fraction, and suggest that among 81 − cells only those expressing an Fc-gamma receptor of high affinity are active.

Mill Hill Strain of Parainfluenza 1 Virus Is a Potent Human Interferon Inducer

The interferon (IFN)-inducing capacities of the widely used Cantell strain of Sendai and the Mill Hill strain of parainfluenza 1 were compared. The Mill Hill strain proved to be a more potent inducer of IFN than the Cantell strain when tested in human peripheral blood leukocytes and amniotic membrane cultures. The Mill Hill strain did not produce defective particles readily, nor did these particles appear necessary for IFN induction. The Mill Hill strain of parainfluenza 1 virus appears superior to the Cantell strain of Sendai virus for the production of natural human IFN.