First confirmed sheep scrapie with A136R154Q171 genotype in Slovakia.

The first confirmed evidence of scrapie in Slovakia was demonstrated in one sheep of the autochthonous Merino breed from the southeastern part of the country. The reported scrapie was diagnosed during compulsory transmissible spongiform encephalitis (TSE) screening of sheep over 9 months of age assigned for consumption. The positive ewe was 5-year-old, which did not show any clinical signs of scrapie. The presence of the proteinase-resistant prion protein (PrP) in brain was proved independently by two laboratories using two different immunochemical screening systems, namely the Prionics Check (Western blot analysis) and Enfer TSE enzyme-linked immunosorbent assay (ELISA). In addition, the genotyping analysis of PrP gene demonstrated the presence of PrP genotype from the high risk group R4. The affected sheep was homozygous for the allele PrP(ARQ) (ARQ/ARQ) coding for alanine (A), arginine (R) and glutamine (Q) at three most relevant codons (136, 154 and 171, respectively). The healthy sister of the positive ewe was heterozygous in the PrP locus and carried alleles ARQ/ARR.

The human interferon system: characterization and classification after discovery of novel members.

The human interferon (IFN) system is the best characterized of all animal IFN systems. Until recently it is thought that all IFNs and IFN-related genes and proteins have been discovered. However, in the last three years, the discovery and characterization of IFNs including IFN-epsilon (IFN-epsilon), IFN-kappa (IFN-kappa) and a novel IFN-lambda (IFN-lambda) family, in particular, substantially changed this opinion. In this article, we attempt to review recent developments in the field of interferon discovery and present an overview of current classification of the human IFN system. Characterization of the constituent parts of the human IFN system including ligands, receptors and players involved in the signal transduction pathway are discussed.

Significance of anti-interferon-alpha2 and sICAM-1 activities in the sera of viral hepatitis B and C patients treated with human recombinant interferon-alpha2.

In this study the presence of an IFN-binding activity in the sera of patients with chronic viral hepatitis B or C treated with rIFN-alpha2 was screened by a radioimmune assay (RIA) using radiolabeled rIFN-alpha2. Incidence of an anti-IFN activitywas compared with hepatitis B virus (HBV) or hepatitis C virus (HCV) serum markers as hepatitis B s antigen (HBsAg), hepatitis B e antigen (HBeAg), antibodies to HBsAg (anti-HBsAg), antibodies to HBeAg (anti-HBeAg), seroconversion, HBV DNA, HCV RNA, and serum soluble intracellular adhesion molecule I (sICAM). Injections (intramuscular) of rIFN-alpha2 caused an anti-rIFN activity formation in 8 (27.6%) of 29 patients with chronic active hepatitis B (CAH-B) and in 8 (30.8%) of 26 patients with chronic active hepatitis C (CAH-C). The presence of the anti-rIFN activity in CAH-B patients correlated frequently with the persistence of HBsAg, HBeAg and HBV-DNA, while its absence was often accompanied by the anti-HBeAg and anti-HBsAg seroconversion, respectively, and HBV-DNA negativity. In two CAH-C patients who became HCV RNA-negative no anti-IFN activity was found. Levels of serum sICAM-1 in CAH-B patients responding to the IFN treatment were higher than those in non-responders or in which the anti-IFN activity was present. The anti-IFN activity may negatively influence the effect of the IFN therapy of CAH-B or CAH-C patients at early stages of the therapy. The appearance of the anti-IFN activity at the end of a long-term IFN therapy does not seem to influence the outcome of the therapy. sICAM-1 may be involved in the process of CAH-B reactivation and IFN-triggered cytotoxicity during the IFN therapy.

[Virokines and viroceptors--viral immunomodulators with clinical and therapeutic implications]. / Virokíny a viroceptory--imunomodulátory vírusov s klinickým a terapeutickým dosahom.

During evolution viruses have developed variety of sophisticated strategies for interactions with the immune system of the host. One of the defense strategies that counteract the immune responses of the infected organism exploits viral proteins that directly interfere with the host's cytokine system. Among such immunomodulatory molecules are classed also viral homologs of cytokines (virokines) and viral homologs of cytokine receptors (viroceptors), produced and secreted by the virus-infected cell. Virokines and viroceptors are encoded by large DNA viruses--herpesviruses and poxviruses. The respective genes have been obviously "stolen" by viruses from the host genomes and then have been modified. Detailed characterization of these viral elements, which induce or subvert the host's cytokine responses against viral infection, may contribute to a better understanding of the mechanisms which help the viruses to escape immune surveillance. Such knowledge have potential implications for viral epidemiology, treatment or prevention of viral and inflammatory diseases, and for the development of safer vaccines. Examples of viruses indicate, that "capturing" of the immunomodulatory genes may be a more general strategy used also by other types of pathogenic or parasitic organisms to evade the immune responses of their hosts. (Tab. 2, Fig. 2, Ref. 73.)

The carboxyterminal domains of human IFN-alpha2 and IFN-alpha8 are antigenically homologous.

The antigenic properties of human hybrid IFN-alpha8(60)/alpha1(92)/alpha8 were compared with those of human IFN-alpha1 and IFN-alpha2 using monoclonal antibodies (mAb). Hybrid IFN demonstrated a significantly closer antigenic relationship to the subtype alpha2 than to the subtype alpha1. In particular, high homology was observed between antigenic structures located in the C-terminal domains (93-166) of IFN-alpha8 and IFN-alpha2, whereas the corresponding N-terminal receptor-binding domains (30-53) showed distinct antigenic characteristics. The 100% homology between IFN-alpha8 and IFN-alpha2 in the region 114-131 (helix D) indicated the role of this region in formation of the common antigenic structure. In IFN-alpha8/1/8, this shared antigenic structure was important for antiviral activity and exhibited immunodominant properties, consistent with functional and antigenic properties of the corresponding structure in IFN-alpha2. Based on this antigenic homology, we suggest that IFN-alpha8 and IFN-alpha2 are evolutionarily more closely related to each other than to IFN-alpha1. This study will contribute to a better understanding of evolutionary events in the human IFN-alpha family.

Engineered acid-stabile human interferon gamma.

Loss of anti-viral potency upon pH2-treatment is an inherent feature of interferon (IFN)-gamma. The phenomenon seems to be caused by dissociation of IFN-gamma homodimer into subunits upon acidification and subsequent self-association of monomers into aggregates with reduced activity after neutralization. We demonstrated that acid-stability could be engineered into human IFN-gamma without affecting its specific activity. An artificial intra-monomer disulphide bond E7C/S69C stabilizes the dimeric form of the cytokine, which retained its full bioactivity after exposure to pH2. Acidification did not modify the antigenic structure of IFN-gamma as proved by a panel of mouse anti-human IFNgamma antibodies.

Radioiodination of human interferon-alpha2 interferes with binding of C-terminal specific antibodies.

Radioimmunoassays based on reactivity between a monoclonal antibody (mAb) and human 125I-interferon (IFN)-alpha2 are frequently exploited in interferon research. In general, epitopes of antibodies specific for human IFN-alpha2 are located on the two immunodominant structures formed in the N- and C-terminal domains, respectively. We found that labelling of IFN-alpha2 with Na(125)I by the chloramine-T method did not affect the binding of antibodies recognising the N-terminal region 30-53. In contrast, radioiodination of IFN was associated with a dramatic decrease in IFN reactivity with mAbs specific for the C-terminus (residues approximately 120-145 approximately ). We suggest that steric hindrance araising from the incorporation of 125I into the tyrosine residues at positions 123, 130 and 136 may be responsible for the change in immunoreactivity. The adverse effect of radioiodination of IFN-alpha2 on the binding potency of C-terminal specific mAbs must be taken into consideration in experiments based on the interaction of such antibodies (i.e. NK2) with the labelled antigen.

[Immunogenicity of recombinant interferon alpha-2 in therapy]. / Problém imunogénnosti rekombinantného interferónu alfa 2 v terapii.

Recombinant human (r)IFN-alpha 2 is successfully used in treatment of haematopoietic malignancies, lymphomas and viral hepatitis B and C as well. One of undesired side-effects of rIFN-alpha 2 is its immunogenicity which could decrease therapeutic potential of the drug. Formation of antibodies against rIFN-alpha 2 in man represents a complex process, in which many mutually interacting variables are involved. Factors influencing the humoral response against the recombinant homologue of human IFN-alpha 2 have not been unambiguously determined yet. In general, two categories of these factors--exogenous and endogenous (physiological)--are considered. The exogenous factors determine structural differences of the recombinant protein from the native IFN-alpha 2. Their influence on the formation of therapy-induced antibodies could be limited by the selection of suitable rIFN-alpha 2 subvariant, suitable storage of the preparation and by treatment regimen. On the other side, current knowledge of the endogenous factors, which function at the level of patient's organism, do not allow to propose efficient ways of their elimination. (Tab. 2, Ref. 28.)

Structural and functional heterogeneity of the amino-terminal receptor-binding domain of human interferon-alpha 2.

Structural immunoanalysis of human interferon (IFN)-alpha 2c revealed antigenic and functional heterogeneity in its N-terminal receptor-binding domain (loop AB). Monoclonal antibodies (mAbs) mapped to the region 30-53 of IFN-alpha 2 defined three partially overlapping antigenic sites designated here as 'a', 'b' and 'c'. For the high-affinity binding of IFN-alpha 2c to the cellular receptor, site b located in segment 34-41 and site c (residues 43-53) appeared to be most important. Only the part of site a (amino acids 30-33) seemed to be involved in the interaction with receptor. The segment of residues 30-46 forms a relatively straight structure on the protein surface, according to the three-dimensional model of human IFN-alpha 2.

Immunogenicity of interferon-alpha 2 in therapy: structural and physiological aspects.

Recombinant human interferon (rIFN)-alpha 2 has been approved for therapeutic application in a range of human oncological and viral diseases. However, some patients can develop strictly specific antibody response to rIFN-alpha 2, which may diminish its therapeutic potential. Such humoral response appears to be quite complex and obviously depends on multiple parameters. Our review is aimed primarily to factors associated with structural modifications of rIFN-alpha 2 that we consider crucial for formation of therapy-induced antibodies. These factors are either related to inherent conformational differences between three IFN-alpha 2 subvariants or to immunogenically active contaminating derivatives resulting from production, purification and storage of this recombinant protein. In addition, the role of treatment regimen and physiological variables modulating the immune response to rIFN-alpha 2 in the challenged organism are mentioned.

[Present views on interferons]. / Súcasný pohl'ad na interferón.

The nature of interferons and mechanisms of their action have been studied for more than four decades. The review article brings the current view of interferon, putting emphasis on historical transitions in its definition and changes in the understanding of the physiological role of interferon in organism. The recent classification of interferons together with the more detailed characterization of human interferons are presented. (Tab. 1, Ref. 40.)

Vaccinia virus protein B18R inhibits the activity and cellular binding of the novel type interferon-delta.

The soluble vaccinia virus-encoded protein B18R inhibits the antiviral activity and cellular binding of the type I interferons (IFN)-alpha, -beta and -omega of different mammalian species. Recently, a novel type I IFN was detected in pigs and classified as a member of a distinct IFN family designated IFN-delta. Our study aimed to determine if the structural properties of this shortest (149 residues long) type I IFN allow its interaction with the type I IFN-binding protein B18R. Experiments using bovine (MDBK) cells demonstrated that B18R neutralized the antiviral activity of porcine IFN-delta with high efficiency. Preincubation of B18R with radiolabelled IFN-delta specifically inhibited binding of IFN to bovine cells. These data indicate that the overall conformation of the novel IFN-delta might be similar to that of other type I IFNs.

Therapy-induced antibodies to interferon-alpha 2a recognise its receptor-binding site.

Fifty-eight patients with chronic hepatitis B (HB) or C (HC) were treated with recombinant human interferon (rIFN)-alpha 2 and their sera were assayed for antibodies to rIFN-alpha 2c. Twelve of these patients produced low titres and two high titres of the antibodies. We localized the region which was recognised by the high-titre therapy-induced antibodies on the IFN molecule by testing the antibodies with a set of murine monoclonal antibodies (MoAbs) to IFN-alpha 2 in a competitive radioimmune assay (RIA). Only MoAbs with epitopes located in the amino-terminal portion of IFN-alpha 2 could inhibit the binding of radiolabelled IFN-alpha 2 by patients' sera. Our data indicate that the therapy-induced antibodies were directed to the receptor-binding domain of IFN-alpha 2 formed by amino acids (aa) 30-53. In accordance with this observation, human anti-IFN sera inhibited the binding of rIFN-alpha 2 to human cells.

Structural immuno-analysis of human and porcine interferon gamma: identification of shared antigenic domain.

We examined the antigenic resemblance between human (h) and porcine (p) interferon (IFN)-gamma by binding (ELISA) and neutralization assays. The murine polyclonal antisera and sets of murine monoclonal antibodies (mAbs) raised against either IFN were tested in confrontation with recombinant IFNs of either species, and with site-specific mutants of hIFN-gamma. Several of the mAbs raised against pIFN-gamma cross-reacted in ELISA with hIFN-gamma. In contrast, none of the anti-hIFN-gamma mAbs cross-reacted. By employing site-specific mutants of recombinant hIFN-gamma as antigens in ELISA we succeeded in identifying the C-terminal portion 97-111 as the antigenic site in hIFN-gamma recognized by the cross-reactive anti-pIFn-gamma mAbs. None of the mAbs recognizing the common antigenic structure had neutralizing potency, although His111 was determined by others as the residue important for bioactivity of hIFN-gamma. Mutations in the domain 97-111 had no or little influence on homospecific reactivity of anti-hIFN-gamma mAbs, indicating that this domain, while being mouse-immunodominant in the case of pIFn-gamma was poorly immunogenic in the case of hIFN-gamma. The epitopes of three out of five anti-hIFN-gamma mAbs mapped in the N-terminal region 1-23, indicating immunodominance of this region in hIFN-gamma. Another mAb (D9D10), also directed to the N-terminus of hIFN-gamma, apparently recognized a conformational epitope. This antibody lacked ELISA-reactivity with the wild-type hIFN-gamma but strongly bound mutant protein with an engineered disulfide bridge Cys7-Cys69. Surprisingly, D9D10 showed high reactivity also with the wild type hIFN-gamma produced by baculovirus construct coding for the mature protein with signal sequence or with wild type protein possessing residues Cys-Tyr-Cys from the signal sequence.

Different stabilities of the N- and C- terminal domains of human interferon alpha.

The present results are consistent with the hypothesis predicting two structurally independent polypeptide domains in the regions 1-92 and 111-166 of the type I IFNs. However, we observed differences in molecular unfolding between the N- and C-terminal portions of human IFN-alpha during denaturation in SDS solutions. Monoclonal antibodies (mAbs) detected changes in the N-terminal region (residues 1-85) of denaturated IFN-alpha 1 or IFN-alpha 2. In contrast, SDS-denaturation of antigens did not affect the reactivity of mAbs with epitopes located in the C-terminal portion (residues 105-166) of both IFNs. The N-terminal domain is known to be involved in the high affinity receptor binding of IFN-alpha. Therefore a theory that the active sites may be conformationally more flexible than the rest of the polypeptide might explain the lower conformational stability of this domain.

Analysis of an interaction between the soluble vaccinia virus-coded type I interferon (IFN)-receptor and human IFN-alpha1 and IFN-alpha2.

The soluble B18R protein coded by vaccinia virus exerts properties of a type I interferon (IFN)-receptor with broad species specificity. We analyzed neutralizing and binding activity of the B18R protein against several recombinant human type I IFNs. The B18R protein inhibited the antiviral potency of IFN-alpha1, IFN-alpha2, IFN-alpha8/1/8, and IFN-omega on human cells. The N-terminal domain of human type I IFN is involved in the high affinity binding to its cellular receptor. To localize the binding domain(s) of IFN with the B18R protein, competition experiments between B18R, and mapped monoclonal antibodies to IFN-alpha1 and IFN-alpha2 were performed. Surprisingly, our data indicated that the contact area between the B18R protein and IFN comprised in addition to the N-terminal region of IFN-molecule also its C-terminal portion. We suggest that this different pattern of interaction with a ligand might determine the ability of B18R protein to bind type I IFNs of different species.

Forty years of interferon.

The nature and mode of action of interferon (IFN) have been intensively studied for more than 40 years. In this review, we summarize the current knowledge of IFN putting emphasize on transitions in its definition, understanding of its physiological role and nomenclature, and on brief characterization of individual IFN families. Finally, the evolution of IFN and relations between the IFN families are discussed.

Interferon-omega suppresses hepatitis B surface antigen production in human hepatoma cell line.

Biological activities of human interferon (IFN) omega are less well characterized than those of other type I human IFNs. We compared the ability of recombinant IFN-omega, IFN-alpha 2 and IFN-gamma to inhibit the production of viral hepatitis B surface antigen (HBsAg) in the human hepatoma cell line PLC/PRF/5. The results demonstrated that the capacity of IFN-omega to suppress the HBsAg synthesis was similar to that of IFN-alpha 2. The kinetics of the inhibitory effect of IFN-gamma differed from those of the two other IFNs.

Conformational changes in pH2-treated human interferon-alpha 2 detected with monoclonal antibodies.

Monoclonal antibodies allowed to demonstrate the existence of alternative antigenic forms of the same molecule as of human interferon (IFN)-alpha 2. Exposure of recombinant IFN to pH 2, although not affecting its bioactivity, induced structural modulation of molecular surface. The antigenic structure of IFN-alpha 2 appeared to be built of the acid-stable and acid-labile epitopes. In general, the acid-stable sites determined subtype-specific antigenic properties of the protein, whereas the acid-labile determinants were responsible for antigenic characteristics shared by some other human IFNs. Acidification of IFN-alpha 2 to pH 2 for at least 1-2 h resulted in simultaneous structural rearrangement of all acid-labile sites.