Neuroprotective and antiamnestic effects of mutant molecules of erythropoietin on model of photochemical thrombosis of rat brain prefrontal cortex.

Mutant EPO molecules, deprived of erythropoietic activity, but possessing cytoprotective action, were created by the method of genetic engineering. The assessment of the therapeutic effectiveness of the received mutant proteins was carried out by the retention of the conditioned reflex of passive avoidance (PA), developed before the ischemic injury of rat brain prefrontal cortex, and by the MRI-analysis of ischemic damage volume. Antiamnestic and neuroprotective action of mutant molecules - MERO-Fc and MEPO-TR is investigated on model of photothrombosis of rat brain prefrontal cortex at single intranasal introduction in 1 h after cortex ischemic damage. The neuroprotective (MRI) and antiamnestic (PA) effects of mutant molecules of erythropoietin derivatives are shown.

Neuroprotective and antiamnesic effect of erythropoietin derivatives after experimental ischemic injury of cerebral cortex.

Using the model of bilateral photothrombosis of the blood vessels in the prefrontal cortex we have shown that new hybrid proteins derived from recombinant human erythropoietin, carbamylated EPO-Fc and EPO-TR fusion proteins, injected intraperitoneally 1 h after ischemic injury contribute to restoration of passive avoidance response formed before photothrombotic injury and reduction in the volume of the ischemic focus. These data attest to nootropic and neuroprotective activities of these hybrid proteins. Carbamylated glycopeptide derivative ЕPO-TR exhibited prolonged neuroprotective properties.

[Comparison of neuroprotective effects of derivatives of eritropoetine by different way of bringing in drugs with model of bilateral photochemically induced thrombosis of the prefrontal cortex of rat brain].

It was stated with model of bilateral photochemically induced thrombosis of the prefrontal cortex by injected intranasally or intraperitoneally in 1h after operation new derivatives of eritropoetine: Epo, Epo-Fc, Epo-Tr provoked neuroprotective and antiamnestic action. Epo-Fc demonstrated more effective action by intranasal injection.

[Clinical and economic assessment of treatment efficacy of uncomplicated duodenal ulcer with different antisecretory agents in hospital].

A study of 197 patients with uncomplicated duodenal ulcer revealed clinical and pH-metric particular features of the action of pariet (rabeprazole) and six secretolytics: cimetidine, ranitidine, ranisan, quamatel, omez. It was established that the main clinical criteria of treatment efficacy is duration of ulcer cicatrisation and percentage of cicatrized ulcers during 21 days, and additional criteria are duration of disappearance of clinical signs and stage of acid production suppression. Possibilities of applying methods of pharmacoeconomic study were examined: analysis of the treatment cost, cost minimization, and cost-efficacy with the definition of the increase of expense efficacy for drug selection. It was shown that the cost of the antisecretory therapy makes up from 0.45 to 9.14% of inpatient treatment. A cost-efficacy analysis showed that it is most expedient to use pariet.

[Use of synthetic peptides as additional antigens for detecting antibodies to hepatitis C virus]. / Ispol'zovanie sinteticheskikh peptidov kak dopolnitel'nykh antigenov dlia vyiavleniia antitel k virusu gepatita C.

Peptide fragments of hepatitis C virus (HCV) nonstructural protein NS4 capable of reacting with anti-HCV in enzyme immunoassay are synthesized. Addition of synthetic peptides to recombinant nucleocapsid HCV antigen absorbed on solid phase notably improves the efficacy of detection of antibodies to HCV in the sera of patients with hepatitis C.

Specificity of humoral and cellular immune response against recombinant particles of nucleocapsid protein of human hepatitis B virus in rabbits.

Nucleocapsid (core) protein of hepatitis B virus (HBcAg) induces potent cellular and humoral responses that have a clear protective potential. Rabbits were immunized by particles formed by recombinant molecules of HBcAg carrying N-terminally inserted heterologous sequences. Specificity of humoral and cellular immune response against HBcAg and selection of HBcAg epitopes was surveyed. Immunological properties of the recombinant particles were similar to those of the original HBcAg. Recombinant particles were not toxic to the peripheral blood mononuclear cells (PBMC) of non-immune or HBcAg-immunized animals ex vivo. Proliferative response of PBMC (T-lymphocytes) to HBcAg in immunized animals increased in a concentration-dependent manner in the broad interval of HBcAg concentrations (10-104 ng/ml). On the contrary, a narrow bell-shaped HBcAg dose-dependence curve was earlier observed for T-lymphocytes of donors immune to HBV after natural infection that was probably due to the cytotoxic effect of HBcAg on the expressing cells. Specificity of humoral and cellular immune response against HBcAg particles in the immunized animals and in natural infection with hepatitis B virus (HBV) was compared. Immunization with recombinant HBcAg particles induced potent anti-HBcAg antibody responses: high (up to 2.107) titers of anti-HBcAg antibodies were reached. Appearance of anti-HBcAg antibodies was in every case preceded by an increasing T-cell response to the whole protein and HBcAg-derived peptides, thus mimicking immune responses during acute HBV infection in humans. A predominant universal (haplotype-independent) T-helper cell epitope (amino acid residues (aa) 61-85 of HBcAg (p61-85)) was recognized by T-cells of all animals. Transient antibody response against p61-85 was recorded during the early stages of immunization in spite of the fact that a major B-cell epitope localized in this region is supposed to be purely conformational. A sequence representing another cluster of immunodominant T-cell epitopes of mice and HBV infected humans, aa 121-140 (p121-140), was not immunogenic on the T-cell level. However, it appeared to be a potent B-cell immunogen, despite a common assumption that HBcAg and p121-140 are not cross-reactive at the B-cell level. A possibility that anti-p121-140 antibodies were induced by an exposed region of the native particulate HBcAg and not by the denatured protein molecules, was confirmed by recognition of the particulate HBcAg by antibodies specific to synthetic peptides representing aa 120-140 of HBcAg. The data point to the exposition of aa 121-140 on the surface of the particles.

Detection of hepatitis C virus core protein circulating within different virus particle populations.

Progress in studying pathogenesis and increasing the reliability of hepatitis C diagnosis can be achieved by analysis of different forms of virus particles circulating in blood of both patients and infected persons. Detection of hepatitis C virus (HCV) proteins faces two basic difficulties: low concentration of HCV proteins, and their blocking by antibodies. The aim of this work was to develop a method for the detection of nucleocapsid (core) protein in the plasma of HCV-infected persons using monoclonal antibodies (MABs). Twenty-seven anti-HCV-positive donor plasmas were studied of which 21 contained HCV RNA and 6 were negative. The plasmas were centrifuged for 3 hr at 143,000 g and the antigenic activity of core-protein was studied in the pellets by EIA using four MABs able to recognize four nonoverlapping determinants, two at N-terminus and two at C-terminus of recombinant core (1-150 aa). The determinants detected were present in the natural core protein of at least two genotypes (1b and 3a). Maximal efficiency of recombinant protein detection was achieved with 2 MABs, whereas a combination of 4 MABs was necessary for optimal detection of natural core protein. This is indicative of different conformational structures of natural protein and its gene-engineered analog. The sensitivity of core detection by monoclonal sandwich assay was 1 ng/ml in the pellet or 5 pg/ml after normalization to the initial plasma volume. To dissociate immune complexes, the pellet was treated with 2.5 M KBr after first treating the pellet with the nonionic detergent Tween 80 to remove the virus lipid envelope. Using this treatment protocol, core protein was found in 19 of 21 RNA positive plasmas.

[Preparation and purification of a polypeptide containing antigenic determinants of hepatitis C core protein]. / Poluchenie i ochistka rekombinantnogo polipeptida, soderzhashchego antigennye determinanty serdtsevinnogo belka virusa gepatita C.

A method has been developed for preparing and purifying recombinant polypeptide--hepatitis C virus core protein (HCcoreAg) expressed in E. coli from pFC105-302 plasmid coding for 150 N-terminal amino acids of HCcoreAg in the hybrid from the C-terminal with beta-galactosidase. HCcoreAg was purified by ion-exchange chromatography on P-11 phosphocellulose. The bulk of protein carrying HCcoreAg antigenic determinants was found in two polypeptides: with molecular weights 26 and 136 kD. Antigenic and immunogenic properties of the resultant polypeptide were studied. The 26 kD protein can be used in enzyme immunoassay and immunoblotting as antigen for detecting antibodies to the HCV core protein. The results of immunization of rabbits indicate a high immunogenic activity of the protein. High diagnostic value of HCcoreAg preparation has been demonstrated, for the rapid variant of indirect solid-phase enzyme immunoassay among other tests.

[Immunochemical properties and specificity of monoclonal antibodies against N- and C-terminal sites of the recombinant protein of the nucleocapsid of hepatitis C virus (HCcAg)]. / Immunokhimicheskie svoistva i spetsifichnost' monoklonal'nykh antitel v otnoshenii N- i C-kontsevykh uchastkov rekombinantnogo belka nukleokapsida virusa gepatita C (HCcAg).

Highly affine murine monoclonal antibodies (MAB) to recombinant nucleocapsid (core) protein of hepatitis C virus (rHCcAg) expressed in E. coli were obtained. The MABs were analyzed by solid-phase enzyme immunoassay (EIA), immunodot, immunoblotting, and competitive immunochemical analysis. For estimating the epitope specificity of MAB, several immunoreactive fragments of different length were cloned from the HCcAg region overlapping 160 N-terminal amino acid (a. a.) residues. Use of these fragments and the competitive EIA demonstrated that MAB recognize 4 non-overlapping epitopes, 2 of which are localized in the 1-80 a. a. and 2 other in the 80-150 a. a. regions. A protocol of EIA for detecting HCcAg using MABs to two nonoverlapping HCcAg epitopes has been designed. The sensitivity of double-site sandwich is 1 ng/ml for the recombinant protein.

[Expression of HIV-1 epitopes included in particles formed by human hepatitis B virus nucleocapsid protein]. / Ekspressiia épitopov VICh-1 v sostave chastits, obrazuemykh belkom nukleokapsida virusa gepatita B cheloveka.

Hybrids of the core protein of hepatitis B virus (HBcAg) have been designed which carry N-terminal insertions of B- and T-cell epitopes of HIV-1 an immunodominant B-epitope from gp41, a T-cell epitope from p34 pol, and a cluster of B- and T-cell epitopes from p17 gag. The hybrids have been synthesized using two expression systems-one based on the thermoinducible PR promoter of bacteriophage lambda and the other one based on phi 10 promoter of bacteriophage T7 with 3-5% and 7-14% yields, respectively. The hybrids have dual HBV and HIV-1 immunospecificity and are assembled into particles similar to those formed by the protein carrier HBcAg. Sandwich ELISA and immune electron microscopy revealed that HIV-1 epitopes are exposed on the surface of the particles.

[Introduction of heterologous epitopes at the N-terminal part of the hepatitis B core protein]. / Vvedenie geterogologichnykh épitopov v N-kontsevuiu chast' core-belka virusa gepatita B.

For investigation of proteins possessing assigned immunological properties, plasmids pPS31-42, pPS1-5, pPS2-17, and pPS1P-30 were constructed encoding the hepatitis B core protein (HBcAg) with N-terminally inserted immunodominant epitopes of preS regions (amino acids 31-36 or 94-105 of preS1, or 133-143 of preS2). Analysis of the hybrid proteins with the use of ELISA and immunoelectron microscopy showed that the insertions did not prevent specific aggregation of the protein molecules, the inserted sequences being exposed on the surface of the particles obtained, and both HBcAg and the corresponding preS determinants were antigenically active.

[Heterologous epitopes in the central part of the hepatitis B virus core protein]. / Geterologichnye épitopy v tsentral'noi chasti core-belka virusa gepatita B.

A set of plasmids was constructed determining synthesis of hybrid proteins with insertions of antigenic determinants of preS1 and preS2 regions of HBV in the middle part of HBcAg. The polypeptides containing the 31-36 or monomeric 94-105 preS1 epitopes were water-soluble and formed particles similar to the 27-nm ones of native HBcAg, possessing antigenic properties of both HBcAg and the inserted epitope, while the hybrids containing 133-143 of preS2 or a trimeric form of the 94-105 preS1 epitope became membrane-bound. Another hybrid encoded by plasmid pPS2M31 contained two regions of HBcAg modification: insertion of amino acids 133-143 a.a. (preS2 region) in the N-terminal part and 31-36 (preS1) in the middle part of the carrier. The immunogenicity of the epitope inserted into the middle part of the HBcAg molecule was an order of magnitude higher than that of the same epitope in the N-part of the protein; the fact might be important for constructing artificial immunogens.

[Localization of the immunodominant site in the hepatitis B virus core antigen using synthetic peptides]. / Lokalizatsiia immunodominantnogo saita v sostave korovogo anitgena virusa gepatita B s pomoshch'iu sinteticheskikh peptidov.

Based on theoretical analysis of secondary structure and hydrophilicity data, the region (residues 73-89) of the HBV core-antigen presumably containing the antigenic determinant has been revealed. The epitope mapping of this region with the use of synthetic peptides, obtained by the pin technology and solid phase method, was carried out. Peptides were synthesized in two variants with different amino acids residues in the position 80 (Ala, Ile). Free peptides and their conjugates with bovine serum albumin were tested for antigenicity in ELISA. The results indicate that the fragment 78-83 is the shortest region of the core-antigen which reacts with antibodies from hepatitis B patients sera.

A monoclonal inhibition enzyme immunoassay for detection of antibodies against hepatitis B core antigen: confirmation of an immunodominant epitope.

Monoclonal antibodies (mAbs) were raised against hepatitis B virus core produced by a recombinant clone of Escherichia coli (rHBc). The three mAbs recognized rHBc by Western blot, suggesting that they reacted with non-conformational epitopes. Competition experiments between mAbs and human anti-HBc sera confirmed the existence of an immunodominant HBc epitope within the viral antigen. A monoclonal competition enzyme immunoassay using an IgM mAb conjugated to biotin and streptavidin-peroxidase as the detection system yielded 99% sensitivity and 100% specificity, when compared to other commercial assays.

Linear epitopes of HIV-1, presented as hybrids with Escherichia coli beta-galactosidase or synthetic peptides.

HIV-1 B cell epitopes from gp41, the T cell epitope of p34pol, and a cluster of B and T epitopes from p17gag were selected. The epitopes were presented as synthetic peptides and as either N- or C-terminal insertions into beta-galactosidase. Hybrids were efficiently expressed in E. coli and easily purified when epitopes were inserted at the beta-galactosidase C terminus. Sera from HIV-1-infected individuals reacted in peptide- and hybrid protein-based enzyme-linked immunosorbent assays (ELISAs) mostly with the immunodominant site of gp41. The second site of gp41 and also sites from p17 and p34 appeared to be immunorecessive. A few of the HIV-1-positive sera exhibited several immunorecessive reactivities. HIV-1-positive sera from the former Soviet Union and Cuba had reactivities similar to those of American, African, and west European sera. Some sera could not be evaluated as specifically HIV-1 seropositive because of their broad reactivities with a multitude of peptides and proteins, unrelated to HIV-1. Extensive tests were performed to define unspecific reactivities by absorption, blocking, and sandwich ELISAs. The application of the hybrid protein assay substantially improved the specificity of the ELISA tests. Thus, hybrid protein-based ELISAs appeared to be more suitable than peptide-based ELISAs, especially for the evaluation of immunorecessive reactivities.

[Synthesis of peptides of the preS1-region of the viral hepatitis B envelope protein and localization of antigenic determinants]. / Sintez peptidov preS1-oblasti belka obolochki virusa gepatita B i lokalizatsiia antigennoi determinanty.

We synthesized the 24-41, 30-36, 31-36, 24-30 fragments of the preS1-region of the hepatitis B (subtype ayw) envelope. The peptides were prepared by the solid phase synthesis on perfluorpolyethylene polymer grafted with polystyrene. The peptide chains were elongated from C-terminus using activated esters and symmetrical anhydrides of Boc-amino acids, cleaved off the solid phase by HBr or TFMSA in TFA, purified by gel filtration, and, after conjugation with protein carriers, inoculated into test animals. The resultant antibodies were shown to react with peptides. The blood sera from patients with acute hepatitis B reacted with the conjugates of peptides 24-41, 30-36, 31-36 in the immunoenzymic solid phase assay. The monoclonal antibodies for the preS1-polypeptide were shown to react with peptides 24-41, 30-36, 31-36 and with their conjugates. The results obtained were proved by the data of the epitope-mapping with overlapping hexapeptides.

The effect of the structure of the terminal regions of the hepatitis B virus gene C polypeptide on the formation of core antigen (HBcAg) particles.

A series of plasmids encoding native and modified sequences of the hepatitis B virus core antigen (HBcAg) was created. Analysis of the products generated by expression of the plasmid genomes in Escherichia coli showed that a polypeptide with primary structure identical to that deduced for native HBcAg forms particles in the bacterial cells which are indistinguishable from the native nucleocapsids in morphological and antigenic properties. Removal of the thirty-nine C-terminal amino acids which form a protamine-like domain caused insignificant impairment of the particle-forming process. Modification of the N-terminal region of the polypeptide showed that at least part of the structural determinant governing particle formation is localised between amino acid residues 3 and 11. When the plasmid genes were expressed in an E. coli cell-free transcription - translation system, polypeptides devoid of ten to twenty N-terminal amino acids were formed in addition to the full-length products. From the results obtained it is proposed that a protease digestion site situated within the region containing amino acid residues 10 - 20 plays a role in the formation of the HBe antigen.

[Construction and properties of a hybrid operon coding the HBcAG and beta-galactosidase of the hepatitis B virus in Escherichia coli]. / Konstruirovanie i svoistva gibridnogo operona, kodiruiushchego HBcAg virusa gepatita v i veta-galaktozidazy Escherichia coli.

A set of plasmids was constructed, that carries the hybrid operons with an artificial region for translation initiation of the second cistron. The SD-sequence situated close to the termination signal of the previous cistron facilitates the reinitiation of translation. Both HBcAg and beta-galactosidase coding cistrons are functionally active. The analysis of expression efficacy shows: 1) The second cistron possesses its own initiation region; ii) the opportunity of translation reinitiation increases of the protein synthesis level. The correlation between the translation initiation efficacy and the structure of the initiator codon was investigated. AUG and UUG provide comparable protein synthesis levels, AUG being 1.5-3 times more effective. Probably, there exists a different efficacy of recognition of initiator codons by ribosomes for the systems with independent and connected initiation of translation. The influence of mRNA secondary structure in the translation initiation region on expression is discussed.

[Synthesis and immunological properties of the peptide fragment of the hepatitis B virus envelope preS-protein]. / Sintez i immunologicheskie svoistva peptidnogo fragmenta preS-oblasti belka obolochki virusa gepatita B.

We have synthesized the 24-41 fragment of the preS region of the hepatitis B (subtype ayw) envelope. The peptide was prepared by the solid phase synthesis on perfluoropoly-ethylene polymer grafted with polystyrene. The peptide chain was elongated from C-terminus using pentafluorophenyl- and p-nitrophenyl esters of Boc-amino acids. The peptide was cleaved off the solid phase by HBr in CF3COOH, purified by gel filtration, and, after conjugation with serum albumin (BSA), inoculated into mice. The resultant antibodies were shown to react with the peptide. The blood sera from patients with acute hepatitis B reacted with the peptide-BSA conjugates in the immunoenzymic solid phase assay.

[Study of the effect of rations containing dietary fiber on the level of total lipids and cholesterol in the blood and liver of the rat during a 6.5-month experiment]. / Izuchenie vliianiia ratsionov, soderzhashchikh pishchevye volokna, na uroven' obshchikh lipidov i kholesterina krovi i pecheni krys v usloviiakh 6.5-mesiachnogo éksperimenta.

The influence of the following food fibers: wheat bran, citrus pectin, microcrystallized cellulose, methyl cellulose, was studied in experiments on male Wistar rats. Basing on the data obtained it has been suggested that the diets with pectin are conducive to secretion of general lipids and sterols from the liver. Comparison of the action of natural food fibers (wheat bran, pectin) with artificial fibers (microcrystallized cellulose, methyl cellulose) has not revealed any changes in the blood cholesterol level. At the same time a lower content of total lipids and cholesterol was recorded in the liver of rats that had received pectin and bran.