Biomedical subjects

P Kontsek

Publications and source records attributed to P Kontsek.

At least 19 recordsLinked to original sources

PubMed1995

Priming with interferon-alpha 1 or interferon-alpha 2 enhances the production of both subtypes simultaneously.

A short period of incubation with interferon-alpha ("priming") increases the amounts of IFN-alpha formed by human peripheral blood leukocytes when subsequently induced with a virus. We investigated specifically the effect of priming on the production of two individual subtypes, IFN-alpha 1 and IFN-alpha 2. The rate of interferon synthesis and the amounts formed were equally potentiated in leukocytes primed with either IFN-alpha 1 or IFN-alpha 2. Whichever of these was used for priming had no selective effect on the relative increased production of IFN-alpha 1 or IFN-alpha 2.

Antibodies, Monoclonal↗

PubMed1994

Increased sensitivity of an enzyme immunoassay for human interferon alpha 1 using a mixture of three monoclonal antibodies.

A sensitive sandwich ELISA for the quantification of human interferon (IFN)-alpha 1 was designed. The assay employed IFN-alpha 1- specific polyclonal antibody for coating and monoclonal antibodies (mAbs) to IFN-alpha 1 as a second antibody. A major increase in sensitivity of the assay could be achieved, when polyclonal antibody was combined with a mixture of three mAbs binding to different regions of IFN-alpha 1, compared to the combination of a polyclonal antibody with a single mAb. The sensitivity of the established ELISA was close to that of an antiviral IFN-bioassay. The ELISA did not cross-react with IFN-alpha 2, beta, tau or omega. The immunoassay allowed to estimate the content of IFN-alpha 1 in leukocyte IFN-alpha to about 25-50% or to 2-6% in Namalwa IFN-alpha, respectively.

Antibodies, Monoclonal↗

PubMed1994

Cross-species antiviral and antiproliferative activity of human interferon-omega.

The antiviral and antiproliferative activities of human interferon-omega (IFN-omega) on two human cell lines and on VERO (monkey), MDBK (calf), SPEV (pig), L929 (mouse), BHK-21 (hamster), and MDCK (dog) cell lines were compared with those of human IFN-alpha 1 and IFN-alpha 2. The results are tabulated. Compared with its antiviral titer on human A549 cells, INF-omega was more active on mouse cells and even more active on the pig cells, but had little activity on the hamster cells and virtually none on the dog cells. IFN-omega also inhibited the growth of all these cells to a greater or lesser extent, and there was in general an apparent correlation between its antiviral and antiproliferative activities on the different cells, except that the dog cells were relatively much more sensitive to the antiproliferative effect.

Animals↗

PubMed1994

Human type I interferons: structure and function.

Human type I interferons (IFNs) comprise a family of 13 IFN-alpha subtypes and single species of IFN-beta and IFN-omega. Their 20% overall sequence homology determines identical secondary and tertiary folding of polypeptides. Three-dimensional models suggest that the globular structure of type I IFNs consists of a bundle of 5 alpha-helices, which might form two polypeptide domains. Disulfide bond Cys 29-Cys 139 stabilizes both domains in a bioactive configuration. The IFN molecule exerts its functional entity only as an organic polypeptide complex and therefore molecular fragments apparently lack biological activity. IFN-beta, IFN-omega and some IFN-alpha subtypes are glycoproteins, but the sugar moiety was found to be neither structurally nor functionally relevant. Type I IFNs share a common cellular receptor, a fact that implies a high structural conservativity of their receptor-binding areas. Two conservative hydrophilic regions associated with the amino acids (aa) 30-41 and 120-145 appear to constitute the basic framework of receptor recognition site in type I IFNs. However, the individual IFN-(sub)types induce different spectra of biological effects which reflect some specificity in modelling of binding sites. Besides a subtle sequential heterogeneity in the segments aa 30-41 and 120-145, also the variable hydrophilic aa regions 23-26, 68-85 and 112-121 are responsible for structural and functional individuality among human type I IFNs. The interaction between IFN and its receptor seems to be a complex event which triggers simultaneously antiviral, antiproliferative and immunomodulating actions, although different parts of IFN molecule are not involved equally in eliciting of respective basal activities.

Amino Acid Sequence↗

PubMed1994

Interferon alpha2b is the predominant subvariant detected in human genomic DNAs.

Chromosomal DNAs isolated from eight individuals from the Slovak population and from lymphoblastoid Namalwa cells were analyzed for the presence of genes coding for three subvariants of human interferon-alpha 2 (IFN-alpha 2), namely a, b, and c. The respective genes are regarded allelic, because they differ in the coding nucleotide sequence only at the position 137 (a:A, b/c:G) and/or at the position 171 (a/b:A, c:G). IFN-alpha 2 sequences in genomes were selectively amplified using polymerase chain reaction (PCR). Resulting "consensus" PCR-product (the total mixture of PCR-derived clones) was sequenced and the subvariant-specific nucleotides at position 137 and 171 were determined. In one placental genomic DNA and in a mixture of genomic DNAs from leukocytes of seven donors only nucleotides specific for subvariant IFN-alpha 2b could be detected. This suggests that the placental DNA contained only genes coding for IFN-alpha 2b and these alleles were at least prevailing in donor's genomes. On the other hand, the majority of genomic alpha 2-sequences in Namalwa cells (from which IFN-alpha 2c was originally derived), seems to be corresponding to subvariant IFN-alpha 2c.

Alleles↗

PubMed1993

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.

Animals↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

Comparison of antigenic properties of three interferon (IFN)-alpha 2 subvariants and establishement of a quantitative IFN-alpha 2 ELISA.

Three subvariants of human IFN-alpha 2 (2a, 2b, 2c) were found antigenically highly homologous, using a panel of specific monoclonal antibodies (MoAbs) directed to seven epitopes. Only in the region 30-41 IFN-alpha 2c showed some difference from the corresponding structures of subvariants 2a and 2b. An universal sandwich ELISA for the quantification of all subvariants of human IFN-alpha 2 was designed. A major increase in sensitivity of the immunoassay could be achieved, when polyclonal antibody to IFN-alpha 2 was combined with a mixture of three MoAbs to distinct sites of IFN-alpha 2, compared to the combination of a polyclonal antibody with a single MoAb. The sensitivity of the established ELISA ranged between 1-10 units/ml of IFN-alpha 2 and no cross-reactivity with IFN-alpha 1, -beta, or -omega 1 could be observed. We estimated the content of IFN-alpha 2 to about 56% in leukocyte IFN-alpha or to about 72% in Namalwa IFN-alpha.

Animals↗

PubMed1993

Interferon alpha 2b but not interferon alpha 2a detected in human genomic DNA.

The presence of the genes for human interferon (IFN)-alpha 2a and IFN-alpha 2b subvariants in human genomic DNA was studied using polymerase chain reaction (PCR). The respective genes differ in the coding sequence only at the position 137, adenine (2A) being substituted by guanine (2B). IFN-alpha 2-sequences were selectively amplified from the placental genomic DNA using specific primers. When sequencing the mixture of PCR-derived clones, at the position 137 guanine, specific for subvariant 2b could be detected. This indicates that, at least in the genome analyzed, only sequence coding for IFN-alpha 2b was found. Our finding supports the view, that genes for IFN-alpha 2a and IFN-alpha 2b seem to be rather allelic than occurring at distinct loci.

Base Sequence↗

PubMed1993

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.

Adult↗

PubMed1992

Quadroma-secreted bi(interferon alpha 2--peroxidase) specific antibody suitable for one-step immunoassay.

A mouse hybridoma (quadroma) was prepared by fusing hybridomas producing monoclonal antibody of G1-isotype to human interferon-alpha 2 with hybridomas producing monoclonal antibody of G2a-isotype against horseradish peroxidase. The established quadroma line secreted immunoglobulins of both G1/G2a-isotypes which manifested parental and bispecific binding characteristics. Culture supernatant containing the bifunctional antibody cross-linking interferon and peroxidase was used for a one-step immunoassay. The developed sandwich ELISA was able to detect the human interferon-alpha 2 at a concentration of 10 units/ml (0, 1 ng/ml) within 2-3 hours.

Animals↗

PubMed1992

Conformation-dependent accessibility of the linear epitopes located on the rabies virus glycoprotein.

Seven monoclonal antibodies (MAbs) were derived from mice immunized with the rabies virus glycoprotein of the Pitman-Moore (PM) strain. These antibodies recognized at least five partially overlapping sites located in one immunodominant region. A panel of MAbs was then used to characterize antigenic relationship between PM strain and SAD-Vnukovo strain of these rabies viruses. In immunoblot, all tested antibodies bound to the glycoprotein of both rabies strains, indicating shared antigenic determinants located on the corresponding immunodominant regions. The pattern of reactivity in immunoblot suggested the specificity of antibodies against linear epitopes. However, the supposed close antigenic relation between PM and SAD-Vnukovo strains (evidenced by immunoblot) was not fully confirmed by immunoenzymatic assay. Data provided by ELISA demonstrated two distinct patterns of MAbs reactivity with both antigens. Four antibodies showed specificity for PM strain glycoprotein only, while three MAbs bound with both PM and SAD-Vnukovo strain antigens. We supposed the strain-specific conformation of the native glycoprotein to be responsible for selective access of single MAbs to the respective common linear epitopes.

Animals↗

PubMed1992

Common and different antigenic properties of the rabies virus glycoprotein of strains SAD-Vnukovo and Pitman-Moore.

Two fixed rabies virus strains, SAD-Vnukovo and Pitman-Moore (PM) were used as combined immunogens for the generation of hybridomas secreting specific monoclonal antibodies (MoAbs). The obtained hybridomas were primarily screened by an ELISA for production of MoAbs to antigen of SAD-Vnukovo strain. Six positive clones were established. A panel of MoAbs has been characterized according to reactivity in immunofluorescence, immunoblot, ELISA and neutralization tests. All MoAbs were positive in immunofluorescence when cells infected with the SAD-Vnukovo strain were used. By immunoblot, four MoAbs showed specificity for the viral glycoprotein of both SAD-Vnukovo and PM rabies strains. This pattern of reactivity indicated the existence of shared conformation-independent epitopes located on the related antigens. However, in ELISA, the tested MoAbs did not recognize viral glycoproteins of the PM strain. This indicates, that the different strain-specific conformations of the native glycoprotein determine the accessibility of the common linear determinants for respective antibodies. Only one antibody, with conformation-dependent glycoprotein specificity, was capable to neutralize the CVS strain of rabies virus.

Animals↗

PubMed1992

[Bispecific antibodies: present status and possibile applications].

The review article information on the current status in the research regarding bispecific antibodies. The theoretical and methodical aspects of preparation of heterohybridomas secreting bifunctional monoclonal antibodies are discussed in details. The question of purification of the antibody fraction with dual specificity is also addressed. The possible application of bispecific antibodies in human medicine for immunodiagnostics and aimed immunotherapy is described. (Fig. 7, Ref. 22.).

Antibody Specificity↗

PubMed1991

One-step method for establishing 8-azaguanine-resistant hybridomas suitable for the preparation of triomas.

A simple and rapid one-step method for establishing azaguanine resistant (Agr) hybridomas, which can be used as a fusion partner for the construction of triomas (hybridoma x splenocyte), has been developed. The method relies on cloning the hybridoma cells in soft agar supplemented with 20 micrograms/ml 8-azaguanine. The drug-resistant subclones were isolated after 3-5 days, in comparison with 4-5 weeks reported for the conventional adaptation method. The high frequency (about 10(-3) of Agr-mutants achieved by the cloning method was demonstrated with five different hybridoma clones. One of the derived Agr-hybridomas was fused with mouse immune spleen cells in order to demonstrate its suitability for the generation of triomas secreting bispecific monoclonal antibodies.

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

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.

Acids↗