Treatment with afobazole at delayed time points following ischemic stroke improves long-term functional and histological outcomes.

There is currently a significant lack of therapeutic options for acute ischemic stroke, and no drug has been approved for treating patients at delayed time points (≥6h post-stroke). Afobazole, an anxiolytic currently used clinically in Russia, has been shown to reduce neuronal and glial cell injury in vitro following ischemia. Experiments using the permanent middle cerebral artery occlusion (MCAO) rat model were carried out to determine if afobazole can reduce ischemic stroke damage in vivo and expand the therapeutic window for stroke treatment. Post-stroke (24h) application of afobazole (0.3-3mg/kg) significantly decreased infarct volume at 96h post-surgery, as determined by Fluoro-Jade and NeuN staining of brain sections. Moreover, afobazole helped preserve both the levels and normal histological distribution of myelin basic protein, indicating a reduction in white matter injury. A time-dependence study showed that either pre-treatment or treatment started 6 to 48h post-stroke with the drug yields improved outcomes at 96h. The decrease in infarct volume produced by afobazole was blocked by the application of either a σ-1 (BD 1063, 30mg/kg) or a σ-2 (SM-21, 1mg/kg) antagonist, indicating that both receptor subtypes are involved in the effects of afobazole. Treatment with afobazole starting at 24h post-stroke resulted in enhanced survival one month following surgery. Behavioral testing of animals 28-32days post-surgery using the elevated body swing and forelimb grip-strength tests revealed that treatment with afobazole starting 24h post-stroke significantly reduces behavioral deficits caused by ischemic stroke. The increase in survival and improved functional outcomes are accompanied by a reduction in infarct volume, as determined by thionin staining of brain sections. Taken together, our data support the use of afobazole as a post-stroke pharmacological agent to expand the current therapeutic window.

[Recombinant granulocyte-colony stimulating factor (filgrastim): optimization of conjugation with polyethylene glycol].

In order to create an active pharmaceutical substance of the drug with prolonged action the modification of recombinant human granulocyte colony-stimulating factor GCSF (filgrastim) with polyethylene glycol (PEG, M 21.5 kDa) was conducted. A method for preparation of PEG-filgrastim designed for the development and scaling-up of the technological process of production was described. Modification of proteins with PEG was performed by selective covalent attachment of the molecule alpha-methyl-PEG-propionaldehyde to the alpha-amino group of the N-terminal methionine amino acid residue of the recombinant GCSF. The conditions of the reaction, which provide the desired product yield at least 85% of the total protein, also high protein concentration in the reaction mixture (more than 9 mg/mL) and reduce consumption of PEG in terms of terminal alpha-amino group of the protein was chosen. The data of RP HPLC and MALDI-mass spectrometry showed that the produced drug modified by the N-terminal residue and contains no more than 10% of products with a high degree of modification.

[Interferon inductor cupping of postvaccinal complications after variolation].

Efficacy of arbidol and ridostin in cupping postvaccinal complications due to variolation was studied by the clinico-virological, hematological and biochemical indices and it was shown that arbidol was efficient in cupping development of dermal complications, lowered the severity of the postvaccinal reaction and stimulated the cellular and humoral immune response. Ridostin, a high molecular interferon inductor, was highly efficient in cupping all the forms of the postvaccinal complications, including the neurological and cutaneous ones.

DNA hydrolyzing autoantibodies.

A DNA-nicking activity was detected in the sera of patients with various autoimmune pathologies and was shown to be a property of autoantibodies. The DNA hydrolyzing activity, which was purified by affinity and high-performance liquid chromatography, corresponded in size to immunoglobulin M (IgM) and IgG and had a positive response to antibodies to human IgG. The DNA hydrolyzing autoantibodies were stable to acid shock and yielded a DNA degradation pattern that was different from that of deoxyribonuclease (DNase) I and blood DNase.

[Effectiveness of new diagnostic drug Diaskintest in children for tuberculosis diagnostic].

DST was ascertained to have a high sensitivity in virtually all patients with tuberculosis and a positive reaction was first noted in the infected. With stabilization and regression, the response to DST was much less pronounced than that in clinical and primary infection (that to the Mantoux test being more evident). DST showed its use as a marker of active tuberculosis not only in its local forms, but also in latent tuberculous infection. This makes it possible to apply DST when preventive treatment is performed. The agent may be used to monitor the progress of treatment. DST has a high specificity--healthy individuals had a negative response to DST while the Mantoux test was positive in many cases. The high specificity of DST was suggested by the fact that the persons vaccinated with (this caused BCG ostitis) had a negative reaction to DST while the Mantoux test was positive in all cases BCG-vaccinated BCG. The findings warrant the use of DST for the differential diagnosis of tuberculosis and BCG-associated complications and the possibility of differentiating postvaccinal and infection allergy in children and adolescents.

[Clinical trials of the new skin test Diaskintest for the diagnosis of tuberculosis].

A new reagent for a skin test given the name Diaskintest has been designed for the screening diagnosis of tuberculosis and preclinical and clinical trials conducted. Preclinical trials were carried out on 315 laboratory animals (guinea-pigs, albino mice). The reagent Diaskintest was ascertained to be nontoxic, to have no sensitizing properties, to be safe and specific, and to induce no positive reactions in BCG-vaccinated animals and healthy guinea-pigs. Its specific activity was comparable with that of the national reference--purified tuberculin PPD-L-2. With progression of tuberculous lesions, the guinea-pigs showed higher responses to Diaskintest dilution and the BCG-vaccinated animals lacked responses to Diaskintest with increased delayed type hypersensitivity. The clinical trial was permitted by the Federal Service for Surveillance in Health Care and Social Development of the Russian Federation. Clinical trials were conducted in 150 persons. The safety, specificity, sensitivity of Diaskintest were first examined in the clinical studies and its action was compared with the results of tuberculin skin test (Mantoux test) with 2 TE of PPD L-2. Diaskintest was ascertained to be highly sensitive when given in a dose of 0.2 microg in 0.1 ml. In patients with active tuberculosis and new cases of Mycobacterium tuberculosis infection, the agent induced a positive skin reaction (a papule of more than 10 mm) in 98-100% of cases (p < 0.05). The agent caused no reaction associated with BCG vaccination. The specificity of the test was 93-100% with 95% significance. The rate of overexuberant reactions (vesicular necrotic changes, lymphangitis, and lymphadenitis) was 4-14% with 95% significance. Tuberculosis patients with significant immunopathological disorders might have no skin sensitivity to Diaskintest, as to PPD L-2 (a negative test). The findings substantiate the use of Diaskintest for mass epidemiological surveys for the differential diagnosis of tuberculosis and BCG vaccination-associated complications. The agent may be also used to evaluate the activity of the process in patients with tuberculosis and the efficiency of treatment in combination with other methods and to make a differential diagnosis of tuberculosis.

[Antiviral activity of arbidol and its derivatives against the pathogen of severe acute respiratory syndrome in the cell cultures].

Experimental studies of arbidol and arbidol mesylate versus ribavirin suggest that insertion of these agents into the nutrient medium of the cultured cells GMK-AH-1 (D) after infection at concentrations of 50, 25, and 100 microg/ml, respectively, is effective in suppressing the reproduction of severe acute respiratory syndrome (SARS) virus. Arbidol and arbidol mesylate were shown to have a direct antiviral effect in early viral replication in the cultured cells. The promising antiviral agent is arbidol mesylate that is nearly 5 times as effective as arbidol in reducing the reproduction of SARS virus in the cultured cells. Insertion of arbidol, arbidol mesylate, and ribavirin into the nutrient medium 2 hours after infection of porcine embryonic renal cells caused a reduction in the accumulation of the pathogen by 2.5, 2.1, and 2.6 Ig, respectively.

[Rimantadine and arbidol sensitivity of influenza viruses that caused epidemic morbidity rise in Russia in the 2004-2005 season].

The study of the activity of arbidol against epidemic influenza A and B virus strains (2002-2005) in the cultured MDCK cells showed the higher sensitivity of enzyme immunoassay than that of hemagglutination test. The influenza A virus strains tested, including those resistant to rimantadine (5 microg/ml), were sensitive to arbidol (10 microg/ml). The population of influenza B virus strains was heterogeneous in this indicator, 43% of the strains being less sensitive to arbidol. There was an increase in the number of rimantadine-resistant influenza A(H3N2) virus strains (10-18%) in our country during 3 epidemic seasons. The sequencing analysis of protein M2-endoding gene revealed the amino acid replacement of serine by asparagine in position 31, which is characteristic of rimantadine-resistant strains. Arbidol in combination with rimantadine potentiated the effect of viral reproduction in the cultured cells, as compared with the effect produced by the same concentrations of the drugs used alone.

[Antiviral etiotropic chemicals: efficacy against influenza A viruses A subtype H5N1].

The paper analyzes data of an experimental study of the efficacy of antiviral agents (amantadine, remantadine, ozeltamivir, zanamivir, arbidol, ribavirin) in the cultured cells and on a model of murine influenza pneumonia against influenza A viruses subtype H5N1. It also gives data on their use in the treatment of human beings during avian influenza outbreak. The mechanism of action of the agents, pharmacokinetics, adverse reactions, and their potential resistance are considered.

[Antiviral effectiveness of the combined use of amixine and virasole in experimental hemorrhagic fever with renal syndrome in sucking albino mice].

The antiviral effectiveness of the combined and single use of superlow-dose amixine and virasole on the course of experimental hemorrhagic fever with renal syndrome was studied in sucking albino mice parenterally infected with their virus Hantaan. The co-administration of virasole and amixine was shown to protect 52% of the infected animals from death, which is superior to the effect of their monotherapy. The combined use of the drugs substantially prolongs the survival of albino mice after their infection and the level of brain viral reproduction suppression ( delta = 3.21 g) in the experimental group as compared to the controls and to the mice given only one of the drugs.

[Sensitivity of influenza A/H5 viruses isolated from wild birds on the territory of Russia to arbidol in the cultured MDCK cells].

The effect of the antiviral drug arbidol on the reproduction of avian influenza A/H5 viruses was studied in in vitro experiments. The strains were isolated from the wild birds of Eastern Siberia and they were closely related to the 1997-2000 viruses from South-Eastern Asia. Arbidol was shown to exert a selective inhibiting effect on the reproduction of these viruses in the MDCH cell cultures.

DNA-specific antiidiotypic antibodies in the sera of patients with autoimmune diseases.

Blood sera of patients with autoimmune diseases scleroderma (Scl), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) have been shown to yield a specific immune response to topoisomerase I, the product of expression of a cDNA fragment cloned into lambda gt11 and monoclonal antibodies (MAB) to the enzyme. The 'topoisomerase test' is not absolutely specific for Scl. The stable positive response of autoimmune sera to anti-topoisomerase monoclonal antibodies has a specific character and is associated with the interaction of the Fab fragment of MAB with the IgG fraction of autoimmune serum. The response observed indicates the induction of anti-idiotypic antibodies against topoisomerase. The anti-idiotype, isolated by HPLC and affinity chromatography demonstrated the following functional activities: (i) the immunological reaction against DNA; (ii) high-affinity DNA-binding with topoisomerase-specific consensus; (iii) ability to compete with the native enzyme for binding with DNA and MAB to topoisomerase; (iv) immunological reaction against MAB to topoisomerase.

Cystathionase: high-performance liquid chromatography. Molecular cloning in lambda gt11. Nonradioactive immunodetection of fusion protein.

A method of purification of rat liver cystathionase by high-performance liquid chromatography (HPLC) utilizing non-ideal gel filtration method is proposed. Resolution factors-flow rate, pH values, ionic strength of the mobile phase-were optimized. Antibodies to the enzyme were purified using an immunosorbent synthesized on the basis of epoxylated Toyopearl-65. Radioimmunoassay and immunoblotting demonstrated antibody monospecificity towards cystathionase. These monospecific antibodies were utilized for detecting enzyme amounts (up to 30 pg) using the avidin-biotin system. Rat cDNA expression library in phage lambda gt11 was screened. The cystathionase cDNA clone was isolated, and the structure of the insert was determined.

Avidin-peroxidase: synthesis and HPLC isolation of highly sensitive oligomers.

Optimal conditions for the preparation of avidin-peroxidase conjugates by the periodate method were studied. A method based on hydrophobic interaction chromatography was developed for the isolation of active oligomers. The probable structure of oligomers with highest sensitivity is discussed.

[Gamma-cystathionase: the non-ideal gel filtration high performance liquid chromatography. Physico-chemical and immunochemical characteristics of the enzyme]. / Gamma-tsistationaza: vysokoéffektivnaia zhidkostnaia khromatografiia v rezhime neideal'noi gel'-fil'tratsii. Fiziko-khimicheskaia i immunokhimicheskaia kharakteristika fermenta.

The rapid method for gamma-cystathionase purification was developed. It is based on the non-ideal gel filtration HPLC. The isolated homogeneous enzyme was used for immunization and immunosorbent preparation. A monospecific polyclonal antibody was prepared. The substrate specificity of the isolated enzyme was studied.

[Anti-idiotypic and natural catalytically active antibodies]. / Antiidiotipicheskie i prirodnye kataliticheski aktivnye antitela.

The principal existence of natural catalytic antibodies in the autoimmune sera is discussed. In the course of the autoimmune process, the induction of antiidiotypic antibodies against topoisomerase I has been shown in the sera of patients with scleroderma, systemic lupus erythematosus, and rheumatoid arthritis. The above antibodies were obtained in preparative amounts. Proceeding from the concept of the idiotypic network, the antibodies were suggested to be natural enzymes and their properties were studied. They appeared to be anti-DNA antibodies, competing with the native topoisomerase I for binding to anti-topoisomerase monoclonal antibodies and possessing highly specific DNA-binding activity (Kd is about 0.1 nM). The antiidiotypic antibodies specifically inhibit the topoisomerase-catalysed relaxation reaction and affect the formation of covalent DNA-protein complex. Possible involvement of antiidiotypic antibodies against topoisomerase in the catalysis of reactions of DNA transformation is analysed. Catalytic antibodies that are natural enzymes possessing DNA-nicking activity have been isolated from the blood sera of patients with different autoimmune pathologies.

[Topoisomerase I from human placenta. Functional activity of products of expression of cloned cDNA fragments]. / Topoizomeraza I iz platsenty cheloveka. Funktsional'naia aktivnost' produktov ékspressii klinirovannykh fragmentov kDNK.

Immunoscreening of the human placenta cDNA-library in the expression vector lambda gt11 using non-isotope detection based on the avidin-biotin system allowed to identify a number of clones encoding human topoisomerase I. The fusion protein from an extract of Escherichia coli cells infected with the recombinant phage lambda gt11 interacts with the monoclonal antibody raised against topoisomerase I from calf thymus; the dissociation constant being 5.7.10(-8) M. The restricted DNA fragments coding for the topoisomerase polypeptide in the composition of the fusion protein were recloned, and expression in the pEX vector was obtained. The functional analysis of the expression products has enabled localization of the epitope of binding the monoclonal antibody. It was demonstrated that the identified fusion protein can be applied for diagnosis of autoimmune diseases.

[Efficacy of amixine in experimental hantavirus infection]. / Effektivnost' amiksin pri éksperimental'noi khantavirusnoi infektsii.

The experimental studies conducted on 2-week suckling mice infected with Hantaan virus, Strain 76-118) treated with oral and subcutaneous amoxine showed its prophylactic, therapeutical-and-prophylactic, and therapeutical efficiencies. Oral amoxine exhibited the highest efficiency when used in a dose of 10 mg/kg-1 96 hours before infection and throughout the incubation period. The protective efficiency was 61%. Subcutaneously, the agent was effective when three schemes for injection in a dose of 1 mg/kg-1. Its maximum effect was observed when amoxine was given by the therapeutical-and-prophylactic scheme. The death protection rate was 65%. The agent is effective in suppressing the reproduction of Hantaan virus in the brain tissue.

[Antiviral activity of an interferon inducer amixin in experimental West Nile Fever]. / Protivovirusnaia aktivnost' induktora interferona amiksina pri éksperimental'noi forme likhoradki Zapadnogo Nila.

Experimental research was undertaken to investigate the use of amixin in prevention, emergency prevention schemes and treatment of mice infected with West Nile fever (WNF) agent, strain Eg-101; the results are indicative of the drug efficiency both in its peroral and subcutaneous administrations. Amixin was shown to be most effective in the former case when administered, 10 mg/kg, in 96 hours before mice were infected as well as during the entire incubation period: lethality protection--46%. In the latter case, the drug was effective, when 3 administration schemes were in use, 10 mg/kg. The maximum degree of protection efficiency was registered with amixin administration according to the emergency prevention scheme: lethality protection--33%. The drug suppresses effectively the WNF virus reproduction in cerebral tissues.

[Laccase from Coriolus hirsutus--a new marker enzyme for immunoenzyme analysis]. / Lakkaza Coriolus hirsutus--novyi ferment-marker dlia immunofermentnogo analiza.

A new immunochemical reagent is proposed which contains laccase, isolated from the culture liquid of the basidial fungus Coriolus hirsutus, as a marker enzyme. The feasibility of immunolaccase conjugates for different variants of immunoassay, i.e. "sandwich", competitive and indirect, is demonstrated. The comparison of immunolaccase and immunoperoxidase conjugates showed that the absolute sensitivity of laccase-antibody conjugates was 3 times higher than that of antibody-peroxidase conjugates (7.7 x 10(-11) M and 2.3 x 10(-10) M, respectively). The assay based on antibody-laccase conjugates is simpler than that employing antibody-peroxidase conjugates, since in the former case air oxygen in used as the second substrate of the enzymatic reaction.