Increased effects of topically applied interferon on herpes simplex virus-induced lesions by caffeine.

Caffeine (Cf, 0.15-0.6 mg/ml) and human leukocyte interferon (IFN, > 5 x 10(2) IU/ml) partially inhibited the replication of herpes simplex virus type 1 (HSV-1) in human diploid LEP cells and HaCaT cells. When the two drugs were applied simultaneously the inhibitory effects exceeded those achieved with either substance alone. In the subsequent clinical trial, which was conducted as a one-center, randomised, and placebo-controlled study, the same substances included in either ointment or solution were used for topical treatment of recurrent herpetic lesions. Of 115 patients taken into the study 20 were treated with Cf only (50 mg/g of ointment or solution), 25 with IFN only (5 x 10(4) IU/g), 25 with mixture of Cf and IFN (same concentrations) and 45 with placebo. Both the healing time (HT, period between the prodromes and reepithelization) and the spreading time (ST, period between the prodromes and the appearance of the last new lesion) were recorded in each patient. While the placebo effects were negligible, the other treatments tended to abort the lesions. HT was shortened by at least 4 days in 75% of patients treated with Cf alone, in 88% of those treated with IFN alone, and in all patients treated with their mixture. The effects on ST were less marked; in the case of Cf alone they were negligible. A shortening of ST by at least 2 days was recorded in 40% and 60% of patients treated with IFN alone and the drug mixture, respectively. Statistical analysis confirmed that both in terms of HT and ST the disease was more significantly alleviated by the mixture of Cf and IFN than by either drug alone.

Porcine leukocyte interferon exhibits close antigenic relatedness to human interferon alpha 2, but not to human interferon alpha 1.

As reported by others using polyclonal antisera, natural human and porcine interferons (IFN)-alpha are antigenically related. Using monoclonal antibodies (mAb) in neutralization and ELISA experiments, we found differences in the subtype/antigenic composition between virus-induced porcine and human leukocyte preparations. Human leukocyte IFN-alpha contains two major antigenically distinct subtypes, IFN-alpha 1 and IFN-alpha 2. However, swine leukocytes produced only a single predominant species of IFN-alpha with high antigenic homology to human IFN-alpha 2. Moreover, we were unable to detect close antigenic relatedness between recombinant porcine and human IFN-alpha 1 subtypes.

Cytokines: the fourth homeostatic system.

The cytokines represent a network of small polypeptides produced by a variety of immune, blood and somatic cells. They can be considered the fourth homeostatic system serving the integrity of the organism. The cytokines cooperate with, substitute or, regulate the activity of cells and products of hormonal, neural and immune systems and function as physiological and/or emergency regulators. The attempts to exploit them in the therapy of viral and/or neoplastic diseases brought, so far, only partial success. However, they remain the goal of a new, more physiological therapy.

Peptide-mapping of three neutralizing epitopes into predicted biologically active sites of human interferon-alpha 2.

Immunologically less reactive but functionally relevant structures were identified on human interferon (IFN)-alpha 2 by three neutralizing monoclonal antibodies (mAb). The binding sites of these mAbs were mapped using a set of synthetic peptides that covered the amino acid sequence of two predicted biologically active segments in the regions 31-53 and 63-85 of IFN-alpha 2. We measured the capacity of fragments to inhibit the IFN-neutralizing activity of mAbs and located three linear epitopes around residues 42-53, 63-76 and 77-85 of the IFN-alpha 2 molecule.

In vitro antiproliferative effect of interferon alpha in solid tumors: a potential predictive test.

An in vitro test for the antiproliferative effect of human leukocyte interferon (IFN-alpha) was performed in primary cultures of tumor cells obtained from 32 patients with either malignant melanoma (13), renal carcinoma (4) or bladder carcinoma (15). Our results demonstrated activity of IFN in all three groups of solid tumors. However, appreciable differences in sensitivity to antiproliferative effect of IFN between individual tumors of the same type were found. The potential of this antiproliferative test for prediction of treatment response in IFN-therapy is discussed.

Immunodominant structures in the aminoterminal portion of human interferon alpha 1.

The analysis of the antigenic structure of human interferon (IFN)-alpha 1 with a panel of monoclonal antibodies revealed four immunodominant regions. Three of them, recognized by 12 of 14 antibodies were mapped into the aminoterminal portion of IFN-alpha 1 around residues 31-38, 43-53 and 63-85. The region 31-85 proved important also for the antiviral and antiproliferative activity of the IFN-alpha 1 molecule. The antibody recognizing the sequence around residues (54)63-67 also inhibited the cellular binding of IFN-alpha 1 to the high-affinity receptors.

Cytokines in health and disease.

Cytokines are cellular regulators of non-immunoglobulin character. The studies of interferon, a representative cytokine, support the view that cytokines are information molecules forming a network in the animal organism. Their main task is to protect the homeostasis of the organism. This may be disturbed both by external and internal causes. The results of the studies of interferon appearing in patients with systems lupus erythematosus do not support the assumption that interferons of this type may play a role in aetiology of autoimmune diseases.

Distinct effect of pH 2 on a common antigenic structure found in human interferons-alpha 1 and -alpha 2 in the region 30-35.

The antigenic similarity between molecules of recombinant human interferon-alpha 1 (IFN-alpha 1) and recombinant human IFN-alpha 2 was demonstrated with neutralizing monoclonal antibody (mAb) 1-46. The common epitope for the mAb 1-46 was localized into amino-terminal region of IFN-alpha molecule around residues 30-35. Following pH 2 treatment, the biological activity of both IFN-alpha 1 and IFN-alpha 2 was retained but the antigenic relatedness between corresponding sequences 30-35 was diminished. The common structure on the IFN-alpha 1 molecule proved acid stable and the mAb 1-46 retained the ability to neutralize the pH 2 treated IFN-alpha 1. However, the neutralization of pH 2-treated IFN-alpha 2 by specific antibody was completely suppressed. These results complemented our earlier finding of the dramatic effect of acidic pH on the antigenic structure of region 132-137 of the IFN-alpha 2 molecule. We conclude that pH 2 may induce a conformational rearrangement of the IFN-alpha 2 molecule, resulting in an altered tertiary structure with deviating antigenic characteristics.

Mapping of two immunodominant structures on human interferon alpha 2c and their role in binding to cells.

Structure-function studies of human recombinant interferon (IFN) alpha 2c were performed using a panel of specific monoclonal antibodies in the binding and neutralizing assays. Two immunodominant structures, designated sites I and II, were detected and localized within two conserved hydrophilic regions of IFN-alpha molecule. Using the NK2 antibody as a marker, site I was mapped into a carboxy-terminal domain around residues 112-148. This site was shown to be, most probably, responsible for inducing the antiviral and antiproliferative activities of the receptor-bound IFN-alpha 2c in the cell. Site II that mapped into the amino-terminal domain of IFN-alpha 2c was, at least partially, formed by the amino acid residues 36-41. This region was shown to be most probably involved in the binding of IFN to its cellular receptor. These findings fit with Sternberg and Cohen's model (Int. J. Biol. Macromol. 4, 137-144, 1982) for the tertiary structure of human IFN-alpha.

Enhancement of neutralizing efficacy by combining three monoclonal antibodies to human interferon-alpha.

Three murine monoclonal antibodies (mAb) directed to distinct epitopes on recombinant human interferon (IFN)-alpha 1, and three mAb recognizing distinct epitopes on recombinant human interferon (IFN) alpha 1, and three mAb recognizing distinct epitopes on recombinant human IFN-alpha sc, were studied by IFN-neutralizing assays. The efficacy of neutralization of the anti-viral and the anti-proliferative activities of IFN-alpha 1, or IFN-alpha 2c, by the specific antibodies used, individually or in combination, were evaluated. In comparison with single mAb, the mixtures of three mAb against IFN-alpha 1 or three mAb against IFN-alpha 2c were capable of neutralizing more than 10-times larger amounts of IFN-alpha 1 and alpha 2c, respectively. The strong potentiation of the neutralization efficacy resulting from mixing different mAb was demonstrated by neutralization of the anti-viral as well as the anti-proliferative activities of both recombinant IFN. The neutralization experiments support the interpretation that the observed potentiation results from simultaneous interaction of anti-IFN mAb with different epitope specificity.

Are the acid-labile interferon alpha and interferon omega-1 identical?

The interferon (IFN) activity found in human leukocyte IFN alpha preparations, autoimmune and AIDS sera, and others was reported to have distinct antigenic and deviating biological properties. This led to its vague designation as acid-labile and thermolabile IFN alpha. However, using specific monoclonal antibodies, the acid-labile component of IFN alpha (not exposed to pH 2) and recombinant IFN omega 1 showed significant relatedness. Monoclonal antibody T19, generated with virus-induced leukocyte IFN alpha that had not been exposed to pH 2, neutralized both the antiviral and antiproliferative activities of IFN omega-1, and vice versa; monoclonal antibody OMG 5, specific for recombinant IFN omega-1, cross-neutralized the antiviral and antiproliferative effects of the acid-labile component of leukocyte IFN alpha. When these two IFN preparations were incubated at pH 2 for 72 hr, their biological activity significantly decreased.

Antigenic link between human interferons-alpha and -beta: the common epitope 1.

An until now unobserved consistent antigenic structure, tentatively named "common epitope 1," was detected on molecules of human recombinant (rHu) IFN-alpha 2 and natural HuIFN-beta by testing with monoclonal and polyclonal antibodies. The monoclonal antibody B6, obtained after immunization of BALB/c mice with human fibroblast IFN-beta, was capable of binding and neutralizing both IFN-alpha 2 and natural IFN-beta. The neutralizing activity of monoclonal antibody B6 was completely inhibited by a synthetic hexapeptide which corresponded to the amino acid sequence of IFN-alpha 2 in positions 132-137. Although a corresponding sequence of amino acids in the IFN-beta molecule was localized to the region 134-139 and shows only a 66% homology with the assumed IFN-alpha 2 binding site, lysine at position 132 in IFN-alpha 2 and at position 134 in IFN-beta seems to be crucial for establishment of the common epitope. Its existence was supported by experiments using polyclonal antibodies. Antiserum to IFN-alpha 2 showed cross-neutralization with IFN-beta, and vice versa, antiserum to IFN-beta cross-reacted with IFN-alpha 2. The ability for cross-neutralization by both polyclonal antisera was abolished in the presence of IFN-alpha 2 hexapeptide SH 132-137. No cross-reacting epitope could be detected on the IFN-alpha 1 molecule. These findings are the first evidence of a homology between human IFNs of alpha and beta types at the antigenic level. They indicate that the antigenic distinction between IFNs of alpha and beta types is not absolute.

Modification of the antigenic structure of human interferon alpha-2 by PH 2 treatment: a further support for the antigenic relationship between alpha and beta interferons.

We have recently reported that a unique antigenic structure, designated as Common epitope 1, was found to be shared by human recombinant IFN alpha-2 and the human fibroblast IFN beta. The Common epitope 1 was identified with the aid of a synthetic IFN alpha-2 fragment SH 132-137. Based on this observation, we proposed the hypothesis that an antigenic relationship should exist also between natural human leukocyte IFN alpha and natural human fibroblast IFN beta. However, we were not able to detect any Common epitope 1 in preparations of conventional human leukocyte IFN alpha. In the present study, we were looking for a possible explanation of absence of the Common epitope 1 in conventional leukocyte IFN alpha. First, we demonstrated its acid labile nature in the recombinant IFN alpha-2 molecule and second, we proposed that the pH 2 lability of this unique epitope might be responsible for the lack of antigenicity also in pH 2-treated (conventional) leukocyte IFN alpha preparations. Actually, when pH 2 non-treated leukocyte IFN alpha was examined, we succeeded in demonstration of the Common epitope 1 in IFN-preparation. Moreover, anti-serum against pH 2 non-treated IFN alpha was capable of neutralizing both the conventional i.e. pH 2-treated leukocyte IFN alpha and fibroblast IFN beta. It is concluded that the nomenclature distinguishing two classes (i.e. alpha and beta as class I and gamma as class II) of IFNs is more appropriate than the current official nomenclature distinguishing three antigenic classes of IFNs.

Murine monoclonal antibodies against human fibroblast (beta) interferon. Correlation of the neutralization of antiviral and antiproliferative activities.

Eight mouse hybridoma lines secreting monoclonal antibodies (MoAb) to human fibroblast interferon (HuIFN-beta) were established. All MoAbs were capable of neutralizing two different biological activities of HuIFN-beta: the antiviral activity and the antiproliferative activity. Positive correlation was demonstrated between the ability of hybridoma culture supernatants to neutralize the antiviral and antiproliferative effects of human fibroblast IFN. The neutralizing capacity of individual hybridoma culture supernatants depended on the concentration of MoAb in the sample. Only one of IFN-neutralizing MoAbs has shown binding capacity to human fibroblast IFN when used in enzyme immunoassay as solid-phase antigen. This MoAb was purified to homogeneity and its specific neutralizing activity against HuIFN-beta was calculated (3.5 X 10(4) I.U. per mg).

Human acid- and thermolabile alpha-interferon-like substance: selective reactivity with a monoclonal antibody.

An acid- and thermolabile alpha-interferon-like substance, designated AL-IFN-alpha, has been found in non-processed normal human leukocyte IFN preparations as well as sera from patients with autoimmune or other chronic diseases. Little is known about origin, production and biological activity of these IFN activities. Monoclonal antibodies were obtained which proved highly selective in neutralizing AL-IFN-alpha in both anti-proliferative and antiviral tests. While the monoclonal antibodies were strict specific, polyclonal antibodies against various interferons showed less specificity in these tests. The results suggest that AL-IFN-alpha represents an antigenically distinct IFN-alpha subtype or, alternatively, a new lymphokine with antiproliferative and antiviral activity.

Abnormal macrophages and NK cell cytotoxicity in human systemic lupus erythematosus and the role of interferon and serum factors.

Macrophage (MO) and natural killer (NK) cell mediated cytotoxicity to K562 target cells were strikingly decreased in patients with systemic lupus erythematosus (SLE). SLE NK cells failed to release soluble factor(s) for lysing the targets. IFN-induced enhancement of both types of cytotoxicity was impaired. NK cells from healthy subjects kept their activity in culture with or without IFN for more than six days whereas SLE NK cell activity declined to zero at day 3. So, the increased IFN level of many SLE patients and a possible prior IFN priming effect seemed unrelated to the insensitivity to exogenous IFN in vitro. Inhibition factor(s) of SLE serum suppressed NK cytotoxicity in the presence of IFN whereas IFN sensitivity of MO remained unaffected indicating the complex regulation by serum components of immune reactions.

Interferon after 30 years.

IFN is a product of a growing family of IFN-genes in the vertebrate cell. It is a polypeptide which fits the definition both of lymphokines and/or "local" hormones, and, cannot be anymore considered an "autonomous antiviral factor", as postulated originally. Rather, IFN increasingly seems to play in the organism the role of a "master-lymphokine" that mediates, potentiates or regulates the effects of various lymphokines in differentiation, growth and antigen expression of cells. The antiviral, antiproliferative, cell- and antigen-activating activities of IFN formed the basis for expectations that IFN will be utilized in therapy. These expectations were only partially fulfilled. In some diseases, however, the results of clinical tests are convincing. In such tests, we learned that the successful application of IFN in disease requires other strategies than with cytostatics etc. IFN may be deleterious for the organism when present for a long time in circulation and/or applied in high doses. This follows both from observed side effects of IFN and from the mitigating effect of anti-IFN sera in some diseases.

Monoclonal antibodies neutralizing human leukocyte acid- and thermolabile interferon alpha.

Using a high titred human leukocyte IFN alpha preparation which contained both acid- and thermolabile (AL-IFN alpha) and acid- and thermostable IFN alpha species in 9:1 proportion for immunization of BALB/c mice, five hybridomas secreting monoclonal antibodies that reacted with AL-IFN alpha were obtained. In antiviral and antiproliferative tests on HL-60 cells, their products showed high degree of specificity for AL-IFN alpha. The results suggest that both the "normal" leukocyte AL-IFN alpha and the IFN alpha found in sera of autoimmune and other chronic patients might belong to the same subtype of IFN alpha.

Monoclonal antibodies neutralizing the antiviral and the antiproliferative activities of human interferon gamma.

Spleen cells from BALB/c mice immunized with human leukocyte interferon gamma (HuIFN-gamma) were fused with mouse NSO myeloma cells. Nine hybridoma lines were found secreting monoclonal antibodies (MoAb) which were able to neutralize the antiviral activity of HuIFN-gamma. All nine MoAb inhibited also the antiproliferative activity of HuIFN-gamma. The ability of tested MoAb to inhibit both the antiviral and antiproliferative activities of HuIFN-gamma supports the suggestion that a common domain on HuIFN-gamma molecule might be responsible for both biological activities. However, ELISA and/or RIA proved unsuitable for detection of these specific MoAb.