The detection of hepatitis c virus core antigen using afm chips with immobolized aptamers.

In the present study, the possibility of hepatitis C virus core antigen (HCVcoreAg) detection in buffer solution, using atomic force microscope chip (AFM-chip) with immobilized aptamers, has been demonstrated. The target protein was detected in 1mL of solution at concentrations from 10-10М to 10-13М. The registration of aptamer/antigen complexes on the chip surface was carried out by atomic force microscopy (AFM). The further mass-spectrometric (MS) identification of AFM-registered objects on the chip surface allowed reliable identification of HCVcoreAg target protein in the complexes. Aptamers, which were designed for therapeutic purposes, have been shown to be effective in HCVcoreAg detection as probe molecules.

[Investigation of efficacy of phospholipovit for correction of the hepatic encephalopathy]. / Issledovanie éffektivnosti preparata fosfolipovit dlia korrektsii pechenochnoi éntsefalopatii.

This paper presents the results of clinical studies on the efficacy and safety of the drug Phospholipovit in different groups of patients, in particular with hepatic encephalopathy and with a high risk of its development (chronic alcohol intoxication). Efficacy of treatmernt was evaluated by the LNT test (link numbers test), and standard liver plasma markers (ALT, AST, GGT, AP). The LNT test in patients with encephalopathy showed better improvement after 5 days course of Phos-pholipovit than after standard therapy. In both clinical trials liver enzyme activities, assayed in pa-tients declined more rapidly in the group of patients treated with Phospholipovit, as compared in patients received standard therapy alone. The highest clinical effect of the drug on the liver function tests was observed at a daily dose of 6.4 g of phospholipids (infusional 2 times a day) for 5-10 days. At the end of treatment a two-fold decrease in the activity of AST was observed in patients receiv-ing Phospholipovit compared to the control. This results of clinical results can be regarded as a manifestation of the expressed membrane repairing action of essential phospholipids, reinforced by their introduction into the body in the form of nanoparticles.

Mass spectrometric detection of the amino acid sequence polymorphism of the hepatitis C virus antigen.

A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.

[The concept of infectious diseases pathogenesis].

In this article a concept of infectious disease pathogenesis as consisted with clinical symptoms is provided. The course of disease, immediate and long-term consequences depend on the mode of entry. If the infection comes via oropharynx, airway, gastrointestinal tract or via skin, the immune system provides adequate immune response. This leads to typical symptoms, cyclical clinic progression and usually to the recovery with the formation of full sterile immunity. In case of parenteral way of infection, which includes perinatal way, there is no full mode of entry, the disease takes chronic course involving visceral organs because of different mechanisms of affinity and new tropic organ involving. For the full sanogenesis germ or its mediators should persist in the primary focus of infection. It is suggested, that HIV, HCV, hepatitis B virus, tetanus, rabies and other infectious diseases with inner organs involvement, as well as all slow infections, should be treated as infectious diseases with the parental way of infection, proceeding with affinity changings, which lead to the appearance of new tropic sites in visceral organs. The theory of the mode of entry, affinity, appearance of tropic sites in visceral organs should form the basis of modern infectology.

[Comatose states: etiopathogenesis, experimental studies, treatment of hepatic coma].

The review presents the modern concepts on biochemical mechanisms of processes, that result in comatose states (CS), with emphasis on the search of new therapeutic approaches. CS of various origin causes severe suppression of brain cells functioning and stable unconsciousness. Numerous reasons of various CS are classified into two main groups: primary brain damages (ischemia, tumor, trauma) and secondary damages originating from system injuries in the body (endocrine, toxic e. c.). The most often primary CS is the hypoxic-ischemic one, as result of corresponding encephalopathy. Its mechanism is the brain cells "energy crisis"--because of decreased blood supply or its deficiency by energy substrates or/and by oxygen. Among secondary CS the substantial place takes hepatic coma as a consequence of hepatic encephalopathy in severe liver diseases--cirrhosis, acute liver failure, sharp intoxication. Its main reason is associated with exess of ammonia entering the brain tissue (it accumulates in blood because of lack of its removing by damaged hepatocytes). Ammonia reacts with glutamate in brain astrocytes and the product of this reaction, glutamine, induced osmotic imbalance, that results in change of form and functions of these important brain cells. It induces, in turn, neurons functions damages, changes in neurotransmission and cerebral blood flow and all these may give rise CS. The most of CS studies are carried out in human. Experimental models ofhepatic CS are reproduced mainly in rats, the most often by surgery methods. Other models included administration of thioacetamide or D-galactosamine, sometimes in combination with lipopolysaccharide. In earlier studies ammonia administration together with liver damages by ligation or by CCl4 was used. The main principles of hepatic coma treatment include the care of encephalopathy, detoxification, and liver treatment. Elaboration of new nanodrugs with increased penetration into tissues and cells, in particular, on the base of phospholipid nanoparticles, may increase substantially the therapeuti efficiency. One of such drug is thought to be a new hepatoprotective preparation phosphogliv--nanoparticles of soy phosphatidylcholine with glycyrrhizic acid. It is supposed, that the further development of phospholipid nanoforms, with minimal particle sizes, may reveal the more action in CS treatment.

[Atomic force microscopy detection of serological markers of viral hepatites B and C].

The aim of the study was to demonstrate the possibility of detection of serological markers, containing the hepatitis B surface antigen (HBsAg) and hepatitis C virus core-antigen (HCVcoreAg) in human serum, by use of a new atomic force microscopy (AFM)-based nanotechnological approach. In this study, the immobilization on AFM-chip of antibodies against the hepatitis B virus surface antigen (anti-HBsAg) as well as the antibodies against the hepatitis C virus core antigen (anti-HCVcoreAg) was performed. It was shown that such approach enables to detect: HBsAg, HCVcoreAg and the viral fragments containing these antigens in the serum. Comparative analysis of detection of HBsAg- and HCVcoreAg-containing particles by use of the AFM method vs. traditional methods (ELISA, PCR) has demonstrated the 75% coincidence of results between the AFM and the latter two methods.

[Detection of hepatitis B virus surface antigen with the use of an optical biosensor]. / Opredelenie poverkhnostnogo antigena virusa gepatita B s pomoshch'iu opticheskogo biosensora.

The detection of hepatitis B virus surface antigen (HBsAg) with the use of a model IAsys+ two-channel optical biosensor is based on the registration of interaction between anti-HBs monoclonal antibodies forming the surface layer of the biochip of the biosensor cuvette and blood serum HBsAg. For the first time a two-channel optical biosensor has been used for the detection of HBsAg in blood serum samples. The comparative analysis of the detection of HBsAg by two methods, viz. with the use of an optical biosensor and the enzyme immunoassay, has demonstrated lower sensitivity, but higher specificity of the detection of this antigen by means of a model IAsys+ biosensor with the biochip, prepared in the process of the work. The main advantages of the biosensor detection lie in the registration of interaction in real time without introducing special markers into the molecules under study.

[Treatment and outcome of chronic hepatitis B in children]. / Techenie i iskhody khronicheskogo gepatitia B u detei.

225 children at the age from 6 months to 15 years with chronic hepatitis type B (CHB) were under observation. In addition to clinical biochemical evaluation and morphological recording, there also was the serologic monitoring of the pathologic process condition by means of determination of hepatitis type B and other hepatitis viruses markers. The patients were observed during 1-10 years. It was found that almost all of the patients, with the exception of 3 children (1.3%), had no acute onset of the disease. According to the morphological study data, changes in the liver varied from minimal to apparent activity, up to the formation of hepatocirrhosis in single cases. The clinical presentation of CHB in children included mainly the enlargement and induration of the liver, enlargement of the spleen and anhepatic signs (capillaritis and telangiectasia). After 4 years from the onset of CHB stable and prolonged remission was formed in most of the patients (64.6%); in 35.4% of cases clinical biochemical activity of the disease remained for a longer period of time (5-10 years or longer). The gradual cessation of the disease activity correlates with seroconversion of HBeAg on anti-Hbe. Children with the continuous pathologic process in the liver have HBV DNA in their blood. The main outcome of CHB in children is a prolonged remission with permanent HBs-antigenemia in 89% of cases. The recovery was recorded in 9.68% of cases (disappearance of HbsAg and acquisition of anti-HBs). Hepatocirrhosis was formed in 3 children (1.32%).

[Detection of the hepatitis B virus surface antigen with a biosensor]. / Detektsiia poverkhnostnogo antigena virusa gepatita B s pomoshch'iu opticheskogo biosensora.

A new method for detection of hepatitis B surface antigen (HBsAg) has been developed. It employees IAsys optical biosensor registration kinetics of HBsAg complexes with monoclonal antibodies. Detection of intermolecular interactions is accompanied by changes of the light refraction coefficient in the sensitive layer of the biosensor cuvette. The main advantage of this diagnostic technique consists in rapid registration of these interactions in real time, without any introduction of special labels into analysing molecules. The optical biosensor method was successfully employed for the detection of HBsAg in human blood serum. A comparative study of HBsAg detection by the optical biosensor and by immunoenzyme analysis demonstrated high specificity of HBsAg detection by this new method.

[New domestic phospholipid preparation "Fosfogliv" as an effective treatment for patients with acute viral hepatitis]. / Novyi otochestvennyi fosfolipidnyi preparat "Fosfogliv" kak effektivnoe sredstvo pri lechenii bol'nykh s ostrymi virusnymi gepatitami.

Patients with acute viral hepatitis B, A and mixed hepatitis B + C were treated in two independent clinics with phosphogliv--a new hepatoprotective drug based on polyunsaturated phosphatidylcholine and glycyrrhizic acid salt. Phosphogliv removed some symptoms of intoxication (nausea, weakness, jaundice, etc.) quicker than basic therapy. Among biochemical hepatitis markers, serum bilirubin level was most responsive to phosphogliv. Standard therapy decreases bilirubin by 30% on the average for 5 days, phosphogliv reduces bilirubin for one more week to half those values observed in control patients. At that point low aminotransferase activities were seen in phosphogliv treated patients. No side effects were seen. The new hepatoprotector phosphogliv which repairs biomembranes represents drugs of new generation compared to phospholipid drug essential.

[Hepatoprotective action of phosphogliv in surgery of patients with chronic calculous cholecystitis]. / Gepatoprotektornoe deistvie fosfogliva u bol'nykh, operirovannykh po povodu khronicheskogo kal'kuleznogo kholetsistita.

32 patients with chronic calculous cholecystitis were treated by a new preparation. Phosphogliv (on the base of polyunsaturated phospholipids)--5 days before planned surgery and 5 days after. The increase of alanine and asparagine aminotransferases activities and of bilirubin concentration (2-2.5 fold) was observed in all the patients, cirespective of the drug treatment. However, the essential difference between treated and untreated patients was revealed in the course of post-operative period. In the control group asparagine aminotransferase activity and bilirubin level remained high, and alanine aminotransferases activity even more increased. In contrast, both two aminotransferases activities and bilirubin level fell substantially in Phosphogliv group, that may show more active liver regeneration after surgery. Besides, in this group operation did not result even in short-time changes of plasma protein fractions ratios--opposite to control. It may testify on some protective action of Phosphogliv on liver protein-synthesis system. The observed action of new phospholipid preparation Phosphogliv, together with the absence of side effects, allows to recommend it as an adjuvant in the surgery of patients with chronic calculous cholecystitis. Besides, the above results broaden usage field of polyunsaturated phosphatidylcholine as cell membranes repair agent: it appears to be effective not only for liver diseases treatment, but also for soonest overcome of post-surgery liver changes.

[Detection of TT DNA virus (TTV) in blood donors and patients with hepatitis of unclear etiology]. / Vyiavlenie DNk virusa TT (TTV) u donorov krovi i bol'nykh gepatitom neiasnoi étiologii.

A new human infective agent: TT virus (TTV) has been recently identified. The polymerase chain reaction detected TTV DNA in the sera of 5 (31.3%) out of 16 children with acute hepatitis, 5 (17.2%) out of 29 children and 3 (14.3%) out of 21 adults with liver diseases of unknown etiology, and 18 (13.2%) out of 136 free-of-charge blood donors. These results indicate a high prevalence of TTV infection in Russia and absence of an obvious correlation between this infection and nonA, nonB, nonC hepatitis in examined patients. Phylogenetic analysis of amplified fragments of viral DNA from 3 patients selected at random showed that the isolated strains belong to subtype 1a, most prevalent in the world.

[The vaccination of children with severe somatic pathology]. / Vaktsinatsiia detei s tiazheloi somaticheskoi patologiei.

1,696 children were vaccinated; of these, 1,487 children had different kinds of somatic pathology, including 1,181 children with CNS lesions, 29 children with malignant tumors, 45 children with congenital defects, 82 children with allergic diseases, etc. The group of relatively healthy vaccinees consisted of 209 children. The following vaccines were used for immunization: Tetracoq 05, D.T.Vax, Rudivax, Imovax Polio, Vaxigrip (Pasteur Mèrieux Connaught, France); HBVax, MMRII (Merck Sharp & Dohme, USA); as well as vaccines against hepatitis B produced by Smith Kline Beecham (UK) and Combiotech (Russia). In no case severe vaccine-associated complications were observed. The frequency and manifestation of reactions in children with somatic pathology did nor essentially differ from those in relatively healthy children. The increase of the number of vaccine components did not lead to the increase of the number of side effects of the severity of their manifestation. These investigations demonstrated the safety of vaccination for children with somatic pathology.

[The characteristics of the specific immune response in vaccinated children in an area of high endemicity for viral hepatitis B]. / Kharakteristika spetsificheskogo immunnogo otveta u vaktsinirovannykh detei v regione vysokoi éndemichnosti po virusnomu gepatitu B.

The effectiveness of vaccination against virus hepatitis B (VHB) in the arid region of the Aral, endemic with respect to VHB, was evaluated. The character of specific immune response in 101 immunized children and in 12 adults belonging to a high risk group with respect to VHB. The vaccination of children was shown to be highly effective (96%). The average concentration of anti-HBs antibodies in these children was 856--133 IU/L after completed vaccination and 733--238 IU/L after revaccination. The comparative analysis of antibody levels in children at different periods after complete vaccination indicated that the level of anti-HBs antibodies in recipients decreased with the increase of time elapsed after the moment of vaccination. The effectiveness of the immunization of adults was low, which was probably due to the specific features of immune response to vaccination characteristic of their age and, in one case (a female recipient), to the presence of HBsAg.

[Chronic hepatitis C in children]. / Khronicheskii gepatiti C u detei.

The clinico-laboratory manifestations and outcomes of chronic hepatitis C have been studied in 49 children. The proportion of chronic hepatitis C in the structure of chronic viral hepatitides in children is 20.5%. Among chronic hepatitis C patients, in 18.4% chronic persisting hepatitis, in 20.4% chronic active hepatitis and in 61.4% chronic active hepatitis with transition to cirrhosis of the liver have been diagnosed. In patients with chronic hepatitis C manifested as chronic persisting hepatitis or chronic active hepatitis the course of the disease is characterized by intermittent periods of prolonged exacerbations and short remissions. In cases of chronic active hepatitis with cirrhosis of the liver the signs of the active process can be constantly detected in the course of prolonged observations. In some patients with chronic active hepatitis the lethal outcome is possible as the consequence of progressing liver insufficiency. The data obtained in this investigation indicate that autoimmune chronic hepatitis in children, extensively described in Russian and foreign literature, may be etiologically linked with hepatitis C virus.

[The clinical picture, course and outcome of viral hepatitis A in children with chronic HBV infection]. / Klinika, techenie i iskhody virusnogo gepatita A u detei s khronicheskoi HBV-infektsiei.

The clinical picture, course and outcomes of hepatitis A developing as a superinfection in children with chronic hepatitis B virus (HBV) infection, hospitalized in connection with acute viral hepatitis, were determined on the basis of the dynamic clinico-laboratory investigation and identification of viral hepatitides A, B and delta by the method of enzyme immunoassay. In all children hepatitis B virus surface antigen (HBsAg), i.e. the serological sign of HBV infection, and anti-HAV IgG, the serological sign of hepatitis A virus (HAV) infection, were present in the blood. 11 out of 12 children had never before been examined for diseases of the liver. Only in one child chronic HBsAg carriership had been detected 14 months before this hospitalization. Considering the data of epidemiological history and clinical observations, as well as the results of serological survey, these children were found to have hepatitis A superimposed on chronic HBV infection. As revealed in the present investigation, in cases of HAV superinfection observed simultaneously with chronic hepatitis B viral hepatitis A was manifested by typical clinico-laboratory symptoms.

[Prevalence of hepatitis B in the focus of HIV infection]. / Rasprostranennost' virusnogo gepatita B v ochage infektsii, vyzvannoi virusom immunodefitsita cheloveka.

Highly sensitive up-to-date research methods (enzyme immunoassay) for HbsAg and antibodies against HBsAg and HBcAg were employed to examine 565 adults and children living in the Kalmuck++ ASSR (Elista and adjacent regions), where in 1988-1989 cases of HIV-infection were recorded. Markers of HBV-infection were detected with a high enough frequency in the control group of children (15.2%). The prevalence of the infection in the adult population was at an intermediate level (38.9%). The high infectivity with HBV-infection was recorded among children who had been hospitalized before (for different diseases including chronic ones), among women who had undergone laboratory studies because of the diagnosis of HIV-infection in their children; among adult patients suffering from tuberculosis of morons from a closed boarding school. The highest frequency of HBV-infection marker demonstration was found in HIV-infected children (70%). The data obtained point to the necessity of vaccination against viral hepatitis B in risk groups (children and adults) and in the newborn as well as of raising the efficacy of preventing measures against infections transmitted via blood.