Epitopes recognized by antibodies to denatured core protein of hepatitis B virus.

Particulate and denatured core protein as well as e-antigen (HBe) of hepatitis B virus (HBV) differ in part immunologically but this has not been studied in sufficient detail. Therefore, in this study the B-cell immune response to native and denatured HBV core protein which both can exhibit HBe-specific epitopes was examined using a panel of mouse MABs and rabbit polyclonal antibodies to native and denatured core protein and polyclonal anti-HBe/anti-HBc antibodies from sera of infected patients. Epitope mapping was performed using a set of partially overlapping synthetic HBc peptides, carboxy-terminally truncated HBc proteins and various HBc fusion proteins. A major immunogenic region between amino acids 134-140 and two less immunogenic regions, one spanning amino acids 2-10 and one with three partially overlapping epitopes between amino acid positions 138 and 154, were defined by mouse MABs. Polyclonal rabbit antibodies to denatured HBc, woodchuck and ground squirrel hepatitis core proteins (WHc and GSHc) recognized similar epitopes but in addition occasionally region 61-85, and the latter was also recognized on particulate HBc. Two antigenic regions (amino acid positions 2-10 and 138-145) were found to be exposed on HBe from human serum, and were recognized by mouse anti-HBe but not by anti-HBc antibodies from sera of infected patients. This study demonstrates a more complex pattern of HBc and HBe epitopes than detected previously and provides tools to study conformational changes which may take place during HBc/HBe processing, transport and core particle assembly.

Expression of hepatitis B virus surface antigen gene in Escherichia coli.

Direct expression of hepatitis B virus (HBV) surface antigen (HBsAg) gene under the control of the Escherichia coli tryptophan operon (trp) promoter has been achieved. Synthesis of HBsAg (both complete and lacking its N-terminal segment) as a part of hybrid proteins with the N-terminal portion coded by genes cat, kan or bla is controlled by the appropriate promoters, as well as by the trp promoter. The highest levels of expression, including those for direct synthesis of HBsAg, provide the accumulation of about 10(5) polypeptide molecules per bacterial cell.

Recombinant core particles of hepatitis B virus exposing foreign antigenic determinants on their surface.

Insertion of foreign oligopeptide sequences (40-50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells. These capsids are morphologically and immunologically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acid-long C terminus of HBcAg.

Relationship between antigenicity and immunogenicity of chimeric hepatitis B virus core particles carrying HIV type 1 epitopes.

We have developed a comparative study of antigenic and immunogenic properties of selected immunodominant HIV-1 epitopes from p24 and gp120 proteins added to C-terminally truncated hepatitis B virus (HBV) core protein and exposed on the surface of chimeric core particles. Inserted p24 (121-210) and gp120/MN (306-328) epitopes induced the appropriate humoral and cellular immune responses against HIV-1. Superficially exposed region 160-192 of p24 also showed maximal B cell immunogenicity whereas buried region 148-162 induced maximal T cell response. Both recombinant proteins were also able to be recognized in vitro by T lymphocytes of HIV-1 asymptomatic carriers.

Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.

Status of the "protein kinase CK2-HMG14" system in age-related amnesia in rats.

The experiments described here demonstrate that disruption of the phosphorylation of transcription factors of the HMG cAMP/Ca-independent protein kinase CK2 class may be the cause of decreased gene expression in age-related cognitive deficits. Amnesia for a conditioned passive avoidance reaction (CPAR) in aged rats (24 months old) was accompanied by decreases in the synthesis of synaptosomal proteins and transcription in nuclei isolated from cortical, hippocampal, and striatal neurons. There was a decrease in chromatin protein kinase CK2 activity and a significant decrease in the phosphorylation of HMG14 by protein kinase CK2. Selective activators of protein kinase CK2 (1-ethyl-4-carbamoyl-5-methylcarbamoylimidazole and 1-ethyl-4,5-dicarbamoylimidazole) increased HMG14 phosphorylation by protein kinase CK2, increased transcription, increased the synthesis of synaptosomal proteins, and decreased amnesia for the CPAR in aged rats. Thus, activation of the "protein kinase CK2-HMG14" system is accompanied by optimization of synaptic plasticity in aged animals. The results provide evidence for the high therapeutic potential of protein kinase CK2 activators.

[Exposure of the major immunodominant epitope of the gp51 envelope protein of bovine leukosis virus on the surface of the hepatitis B core antigen capsid]. / Eksponirovanie mazhornogo immunodominantnogo épitopa obolochechnogo belka gp51 virusa leikoza krupnogo rogatogo skota na poverkhnosti kapsid kor-antigena virusa gepatita B.

Insertion of 48 amino acid long sequence of envelope protein gp51 of bovine leukemia virus (BLV), located from position 56 till 103 of mature protein, into Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids. These capsids preserve morphology of intact HBcAg but expose on their outer surface BLV epitopes which are localised in the inserted gp51 fragment and responsible for the recognition of chimeras by monoclonal anti-gp51 antibodies MAK14. The anti-genicity of gp51 epitopes within chimeric capsids is not disturbed after shortening of C terminal part of inserted gp51 fragment by deletion of amino acids 73-103. The resulting chimeras show the same capsid-forming ability as well as HBcAg and gp51 antigenic properties.

[Bacterial synthesis of immunogenic epitopes of foot-and-mouth disease virus fused either to human necrosis factor or to hepatitis B core antigen]. / Bakterial'nyi sintez immunogennykh épitopov virusa iashchura v sostave gibridov s faktorom nekroza opukholei i kor-antigenom virusa gepatita B.

Using recombinant DNA technology, construction and bacterial expression of genes was carried out which code for hybrid proteins, human tumor necrosis factor and hepatitis B core protein fused to immunogenic epitopes of foot-and-mouth disease virus, strains A22 and O1-194. Hybrids of tumor necrosis factor with foot-and-mouth disease antigenic determinants protected laboratory animals against the experimental challenge with a homologous strain of foot-and-mouth disease virus. Hybrid protein that contained immunogenic regions of two strains, A22 and O1-194, protected animals against infection with both A and O serotypes. Hybrid proteins based on hepatitis B virus core antigen retained the ability to assemble into core-like particles.

[The interrelationship between the inhibitory activity of milk with different types of beta-lactoglobulins and the resistance of cattle to mastitis]. / Izuchenie vzaimosviazi ingibitornoi aktivnosti moloka s razlichnymi tipami beta-laktoglobulinov i rezistentnosti korov k mastitam

In the course of the investigation of piebald (black-white) cattle it is found that 17,62% animals produce the AA type beta-lactoglobulin, 49,52%--the AB type and 32,86%--the BB type. The higher inhibitory activity of milk with the BB type beta-lactoglobulin was found which retained in dilution 1 : 32. The total flora of teat, cyst and parenchyma milk of animals with the BB type beta-lactoglobulin as well as Enterococcus bacteria were much lower than in milk of cows with the AA type homogenous form. The animals with the first type of milk protein had mastitis more rarely as compared to those with the second type. The animals with heterogenous form of beta-lactoglobulin had intermediate values in most of their characteristics.

[The participation of RNA synthesis in the process of the acquisition and retention of conditioned reflexes (a study using antifeins)]. / Uchastie sinteza RNK v protsesse vyrabotki i sokhraneniia uslovnykh refleksov (issledovanie s ispol'zovaniem antifeinov).

RNA-synthesizing activity of neuronal nuclei in the neocortex of rats increases after the termination of conditioning depending on the degree of learning. RNA synthesis shifts induced by propylnorantifein and the demethylated derivatives of ethylnorantifein are correlated only with the influence of the drugs on the retention but not the learning. Participation of RNA synthesis by the neurons of the neocortex in the mechanisms of long-term memory is discussed.

[Effect of ethymisole on long-term memory]. / O vliianii étimizola na dolgovremennuiu pamiat'.

Influence of ethymisol (bismethylamid of 1-ethylimidasol-4,5-dicarbonic acid) on learning and retention of habit was studied in experiments on rats by the method of active conditioned avoidance of electrical stimulation. At the same time optimal conformation form of the drug was determined by the method of proton magnetic resonance and by means of infrared spectra and theoretical conformation analysis. A single administration of ethymisol in the dose of 3 mg/kg reduced the deficit of the avoidance habit, when tested in 30 and 60 days following learning. A stable conformation of the ethymisol molecule with a planar arrangement of atoms of its heterocycle and amide groups was found in which the interaction of this substance with biological macromolecules was most active. The ethymisol effect on the long-term memory apparently results from a stable facilitation of synaptic transmission based on stabilization of spatial structure of biological macromolecules in the given conformation.

[Pathologic anatomy of experimental listeriosis]. / Patologicheskaia anatomiia éksperimental'nogo listerioza

The authors present a survey of the Soviet and foreign literature and findings of their own experimental studies. On the basis of these data the pathologo-anatomic picture of listerosis in laboratory animals (white mice, guinea-pigs and rabbits) is given in dynamics and histogenesis of focal changes in this disease in elucidated. The most typical feature of listerosis in animals is lesions of the kidney where focal changes in the form of granulems could be detected macroscopically at an early period and with great consistency. Several stages in its histogenesis may be discerned: I. focal necroses of hepatic cells associated with their invasion with lister Listeria; 2. appearance of cellular elements around the foci of necroses with subsequent formation of granulemas consisting mainly of leucocytes and lymphoid cells; 3. development of necrobiotic changes in the central areas of granulemas with concomitance of exudative processes; 4. organization of necrotic foci with subsequent scarring.

[Development of diagnostic methods based on a core antigen of hepatitis B, obtained by methods of genetic engineering]. / Razrabotka diagnosticheskikh metodov na osnove korantigena virusa gepatita B, poluchennogo metodami geneticheskoi inzhenerii.

The possibility of using recombinant HBcAg, synthesized in Escherichia coli, for diagnostic purposes has been studied. The antigen can be used in highly sensitive immunological assays for the detection of IgG and IgM antibodies to HBcAg.

[Psychophysiological aspect of the therapeutic use of pyridinolcarbamate]. / Psikhofiziologicheskii aspekt lechebnogo deistviia piridinokarbamata.

The effect of prodectin was examined in 79 patients with coronary disease after a course of treatment, and in 16 normal subjects. A series of electrocardiographic and hemodynamic parameters were investigated at rest and during bicycle ergometric exercise. Resultant psycho-physiologic shifts were reviewed with the aid of the information test recording the time of response, errors and vegetative changes in response to colour and mathematical stimuli. Pyridinol carbamate was found to produce a psychostimulating effect, with information processing and vegetative control improved significantly. Therefore the drug appears to be advisable for patients with coronary disease exposed to increased information inflow which requires greater concentration of their attention in short time.

[Cordaron and nonachlasine in treatment of chronic coronary insufficiency]. / Kordaron i nonakhlazin v lechenii bol'nykh s khronicheskoi koronarnoi nedostatochnost'iu

In a double blind study of the clinical effect of Cordaron conducted in 55 patients with chronic ischaemic heart disease a positive effect was obtained in 80.4% of the cases, an effect of placebo-in 24.3%. Cordaron was especially effective in patients with localized stenoses of the coronary arteries. Nonachlasine (an activator of the cardiac beta-adrenergic receptors) proved effective in 10 of 13 patients with chronic ischaemic heart disease.

[Comparative study of the effectiveness of korinfar, izoptin and obzidan in patients with variant stenocardia combined with effort stenocardia]. / Sravnitel'noe izuchenie éffektivnosti korinfara, izoptina i obzidana u bol'nykh s variantnoi stenokardiei, sochetaiushcheisia so stenokardiei napriazheniia.

Twelve patients with variant angina combined with angina of effort were examined. Repeated exercise tests prior to the administration of antianginal drugs revealed the spontaneous variability of the patients' tolerance to the exercise and of the pulse-pressure index at the exercise peak which may serve as an indirect sign of the angiospastic factor involvement in clinical manifestations of angina of effort. The treatment of such patients with corinfar and isoptin was associated with the elimination and significant reduction of spontaneous attacks of angina attended by a considerable increase in the exercise tolerance and in the pulse-pressure index at the exercise peak. The treatment with obsidan in these cases was less effective: the incidence of spontaneous anginal attacks changed insignificantly, the tolerance to exercise increased with the reduction of the maximum value of the pulse-pressure index.

[The role of ultrasonics in the diagnosis of kidney diseases]. / Mesto ul'trazvuka v diagnostike zabolevanii pochek.

A retrospective comparative assessment of the diagnostic value of excretory urography (EU), scintigraphy (SCG) and ultrasonography (USG) of the kidneys was conducted in 100 patients. The diagnosis in 26 patients was confirmed by CT, angiography, at operation or autopsy. Chronic pyelonephritis was diagnosed in 54% of the patients, renal changes in essential hypertension--9%, hydronephrosis in 8%, abnormal shape and position of the kidneys in 8%, malignant tumors in 7%, chronic glomerulonephritis in 8%, renal cysts in 2%, multicystosis in 1%, kidney contusion in 1%, paranephritis in 1%, and apostematous nephritis in 1%. The sensitivity of EU was 71%, that of SCG 52%, that of USG 93%. The specificity of EU was 66%, SCG--8%, that of USG 90%.

[The state of microcirculation, hemoperfusion and tissue oxygen consumption in patients with ischemic heart disease and angina pectoris]. / Sostoianie mikrotsirkuliatornogo rusla, gemoperfuzii i kislorodnogo rezhima tkani u bol'nykh ishemicheskoi bolezn'iu serdtsa so stenokardiei.

In patients with associated coronary heart disease and angina pectoris functional classes II-IV, microcirculatory disorders are linked with the gravity of the patients' clinico-functional status. Application of the functional tests (inhalation of 100% moistened oxygen, regional occlusion of the vessels of the limb under study) revealed the presence of latent deficiency of oxygen transport to the cellular substrate. In functional classes III and IV of disease, an appreciable reduction in the level of tissue hemoperfusion, of tissue oxygen supply, and an increase in the summary conjunctival index were recorded.

[Progression of coronary arteriosclerosis based on the data from repeat coronary angiography and the clinical course of ischemic heart disease]. / Progressirovanie ateroskleroza koronarnykh arterii serdtsa po dannym povtornoi koronarografii i klinicheskoe techenie ishemicheskoi bolezni serdtsa.

The clinical and instrumental studies data are compared with the repeated coronarography data in 16 patients with coronary heart disease associated with stable angina pectoris. Repeated coronarography was performed within 4 to 8 years (5.2 years on the average). In 14 out of the 16 patients (87.5%), repeated coronarography revealed the development of coronary atherosclerosis which was accompanied clinically by the patients' status deterioration, the decreased exercise tolerance, the occurrence of new cases of myocardial infarction, appearance of the symptoms of heart insufficiency and heart rhythm disorders.