[Safety of retroviral vectors in gene therapy].

Retroviral vectors are widely used in gene therapy and found to be an effective tool for the delivery of genetic constructs into cells. A unique feature of these vectors is the ability to incorporate therapeutic genes into a chromosome that ensures its passage to all progeny cells and enables to cure the diseases requiring genetic correction of dividing cells such as hematopoietic cells or skin cells. Retroviral vectors have been successfully used in gene therapy clinical trials for the treatment of 2 forms of severe combined immunodeficiencies and some other hereditary blood disorders. However, the integration of the vector into the chromosome was accompanied by genotoxicity and caused development of hematologic malignancies in several patients. Later it was shown that genotoxicity is not a general feature of retroviral vectors but it depends on many factors. In the present article we discuss safety issues concerning the use of different retroviral vectors in gene therapy. The description of modern vectors which designed to avoid the genotoxicity and other possible side effects are given.

[Treatment of HIV-infection by means of gene therapy].

Current methods of HIV treatment can contain a progression of the disease; however they do not lead to a cure. Lifelong antiretroviral therapy is therefore necessary, leading to problems of cost and toxicity of chemical drugs. The recent advances in science have allowed a new approach to the HIV-treatment - gene therapy. In the present publication we focus on one strategy of the gene therapy called "intracellular immunization". The strategy is based on the introducing of antiviral genes into the HIV-sensitive cells. We highlight the mechanisms of action of various antiviral genetic agents and discuss some issues concerning target cells and genes delivery. Finally we summarize the results of certain gene therapy clinical trials.

Investigation of Taurine Derivative Magnesium-Bis-(2-Aminoethanesulfonic)-Butadioateon Alleviation of Neurological Defects in Simulated Hemorrhagic Stroke in Rats.

Stroke or ischemia is caused by a blockage in a specific blood vessel that partially or completely reduces the blood flow to the brain. Nutritional factors such as antioxidants and healthy eating patterns are important variables in preventing stroke. Molecular composition properties such as molecular binding and screening can be used to evaluate the specific activity and morphological changes. The present study aimed to evaluate the effectiveness of pharmacological correction of the consequences of a hemorrhagic stroke in rats with a new derivative of taurine magnesium-bis-(2-aminoethanesulfonic)-butadioate. The animals (n=170) were divided into four groups as follows: 1) control group (n=20), 2) group 2 suffered a hemorrhagic stroke without pharmacological correction (n=50), 3) group 3 (n=50) underwent simulation of hemorrhagic stroke received Taurine at the dose of 50 mg/kg, 4) Group 4 underwent simulation of hemorrhagic stroke with correction of hemorrhagic stroke with magnesium-bis-(2-aminoethanesulfonic)-butadioate at the dose of 150 mg/kg (LKHT 3-17) (n=50). Hemorrhagic stroke was induced by transfusing autologous blood into the parietal lobe of the right hemisphere of the brain. Lethality, neurological status, locomotor, and exploratory behavior, as well as the morphological pattern of the brain damage, were assessed on the 1st, 3rd, and 7th days after the pathology simulation. Neurological deficit was determined in animals by the McGrow stroke index scale. The locomotor and exploratory behavior was evaluated using the Acti-track software and hardware complex. Two criteria were considered when assessing morphological changes in the brain: the average thickness of the cerebral cortex (in micrometers) and the number of neurons without degenerative changes. LKHT 3-17 (150 mg/kg) and taurine (50 mg/kg) reduced lethality by 1.7 and 1.36 times, respectively, on the 3rd day after stroke compared to that of the control (p<0.05). In parallel, a neurological deficit was effectively corrected LKHT 3-17 and taurine to 5.3±0.8 and 6.5±0.9, respectively, on the 1st day in contrast to the control of 8.1±0.7 points. The locomotor and exploratory behavior was significantly different on the 7th day and was accompanied by a significant increase in total activity under the influence of LKHT 3-17 to 491 conventional units (CU) compared to the control of 110 conventional units. On the 1st day, the thickness of the cortex was 1943.7±44.08 µm, and 1491.0±38.61 µm in the control and LKHT 3-17 groups, respectively. The number of neurons without neurodegenerative changes prevailed in LKHT 3-17 group (18.7±4.32), and the lowest number was observed in the group without pharmacological correction of the pathology (14.3±3.78). The taurine derivative magnesium-bis-(2-aminoethanesulfonic)-butadioate, which is a combination of the amino acid, magnesium ion, and succinic acid, decreases the neurological deficits, lethality, and enhances the locomotor and exploratory behavior in experimental hemorrhagic stroke in rats. The effect of the studied medication on the dynamics of molecular pathophysiological mechanisms occurring in the cell requires additional research.

Evaluation of the Neuroprotective Effect of Magnesium-Bis-(2-Aminoethanesulfonic)-Butanedioate in Simulated Ischemic Stroke in Rats.

Sudden loss of blood flow to an area of the brain causes ischemic stroke, which leads to the loss of nerve function in the brain. The brain tissue leads to the death of brain cells in less than a few minutes due to the lack of oxygen and nutrients. This study aimed to evaluate the effectiveness of pharmacological correction of the consequences of ischemic stroke with a new derivative of taurine magnesium-bis-(2-aminoethanesulfonic)-butanedioate under laboratory code LKHT 3-17 in rats. The ischemic stroke was simulated by electrocoagulation of the right middle cerebral artery. The assessment of lethality, neurological status, locomotor, exploratory behavior, and morphological pattern of the brain damage was carried out on the 1st, 3rd, and 7th day after the pathology simulation. Neurological deficit was determined by the McGrow stroke index scale. The locomotor and exploratory behavior was evaluated using the Acti-track software and hardware complex. When assessing the morphological changes in the brain, attention was paid to two criteria, including the average thickness of the brain cortex and the number of neurons without degenerative changes. The substances were administered 60 minutes before the start of surgery. The animals were divided into an intact group (n=20); ischemic stroke simulation group without pharmacological correction (n=50); a group with correction of the ischemic stroke with taurine at the dose of 50 mg/kg (n=50); and a group with correction of ischemic stroke with magnesium-bis-(2-aminoethanesulfonic)-butadioate (LKHT 3-17) at the dose of 150 mg/kg (n=50).LHT 3-17 (150 mg/kg) and taurine (50 mg/kg) reduced lethality by 1.55 and 1.47 times, respectively, on the 7th day after stroke, compared to the control group (P<0.05). In parallel, an effective correction of neurological deficit was found for LKHT 3-17 and taurine to 4.0±0.8 and 7.6±0.9, respectively, on the 3rd day in contrast to the control of 8.1±0.8 points. The locomotor and exploratory behavior was most significantly different on the 1st and 7th days and was accompanied by a significant increase in the speed of movement under the influence of LKHT3-17 to 20 and 20 conventional units, compared to the control of 7 and 5 cu. On the 1st day, the thickness of the cortex was 1877.3±43.3 µm in the control group, and 1531.8±39.1 µm in the LKHT 3-17 group. The number of neurons without neurodegenerative changes prevailed in the group administered with LHT 3-17 (19.3±4.3), and the lowest number was observed in the group without pharmacological correction of the pathology (14.3±3.7).LKHT 3-17 at a dose of 150 mg/kg is more effective than taurine 50 mg/kg in protecting nerve activity in experimental ischemic stroke and reducing lethality, minimizing nerve defects, reducing volume, accelerating the process of tissue repair, helping stroke, and activating the regenerative processes.

[Treatment of infection caused by human immunodeficiency virus in the Russian Federation].

AIM: To study efficacy and safety of highly active antiretrovirus treatment (HAAT) used in the Russian Federation for development of recommendations for HIV infection treatment. MATERIAL AND METHODS: A total of 285 patients with chronic HIV infection and 42 patients with acute HIV infection participated in investigation of efficacy and safety of different HAAT schemes in 1997-2008. Efficacy of the treatment was assessed by percentage of the patients who had HIV RNA undetectable by the test system (< 400 copy/ml) after 24-48 treatment weeks, by a mean reduction of HIV RNA in blood plasma and an increase in the number of CD4-lymphocytes in 1 mcl of blood. RESULTS: A 12-year experience in antiretrovirus therapy administration for management of HIV infection is reviewed. Efficacy and safety of Russian antiretrovirus drug Phosphaside in HAAT schemes are shown in patients with both chronic and acute HIV infection. The model of HIV patients consulting before the treatment, psychological support during the treatment, methods of individual and group consulting, conception of the school for patients on HAAT are presented. Recommendations are proposed for administration of antiretroviral therapy and a model of calculation of the number of HIV-infected patients in need of antiretroviral therapy in Russia. Basing on the results of investigations on efficacy and safety of HAAT schemes, the physicians of the National Anti-AIDS Center have developed clinical recommendations, guidelines on the treatment of HIV infection, standards of medical care for HIV-infected patients.

[Molecular and epidemiological characterization of HIV-infection in the Altai Territory].

The genetic analysis of the variants of HIV, type 1, circulating in the Altai Territory was made. The results obtained with the use of the serological analysis and the method of the comparative evaluation of the electrophoretic mobility of heteroduplexes demonstrated that almost all analyzed samples (98.3%) belonged to subtype A. Genetic differences between these viruses did not exceed 9.20%. Moreover, 86.8% of them contained mutation V771 in the protease-coding area. Thus, HIV of subtype A, characteristic of CIS countries and containing mutation V771, may be regarded as the dominating viruses in the Altai Territory and not the viruses of subtypes B, C or A/E, typical of comparatively less remote China.

[Protease and reverse transcriptase genetic polymorphism in HIV type 1 subtype A variants predominating in cis countries].

To define frequencies of drug resistance mutations among HIV-1 variants circulating within the territory of Russia, subtype A HIV-1 nucleotide sequences encoding protease and reverse transcriptase were analyzed. The analysis was carried out in 141 antiretroviral-naive individuals. Low frequency (less than 1%) of primary drug resistance mutations was shown. However, high frequencies of secondary mutations V77I in protease and A62V in RT (67% H 63%, respectively) linked to each other in most cases were observed. The HIV-1 isolates bearing both substitutions (MutV77I/A62V) were also characterized by the presence of several synonymous mutations, suggesting common origin for these viruses. HIV Biochip Hybridization microarray and/or Restriction fragment-length polymorphism analyses were performed to characterize gene pol polymorphism in additional 178 subtype A HIV-1 isolates. Among total 319 samples studied, Mutv77IA62V variant accounted for 56%, and was found to predominate in Russia in terms of both its geographical distribution and number of cases caused. Moreover, these viruses were prevalent in the regions known to have highest incidence of HIV-1 infection (Irkutsk, Samara, and Moscow regions). In addition, three other variants were found: viruses not containing the substitutions V77I or A62V, and variants bearing only one of them. Evolutional relationships between all four HIV-1 variants, as well as potential impact of the gene pol polymorphism on HIV-1 replicative fitness and drug resistance development are discussed.

[Biological properties of HIV-1 variants circulating among drug users in Russia].

Human immunodeficiency virus type 1 variants belonging to subtype A, as well as recombinant gaga/engvB variants, derived from HIV-infected patients living in the Moscow and Perm Regions, were isolated and characterized. Intravenous administration of psychoactive drugs was a major risk factor of the infection for all the patients. All the examined isolates of HIV-1 types A and A/B were shown to be characterized by a low virus-specific activity and to be used as secondary CCR5 and CXCR4 protein receptors. The findings suggest that the domination of subtype A variant in this risk group is unassociated with fundamental differences in biological properties between the isolates of this subtype and recombinant viruses.

[Polymerase and immunologic activity of reverse transcriptase of the HIV-1 virus, isolated from Escherichia coli]. / Polimeraznaia i immunologicheskaia aktivnost' rekombinantnoi obratnoi transkriptazy virusa HIV-1, vydelennoi iz Escherichia coli.

The recombinant reverse transcriptase of HIV-1 virus has been isolated from Escherichia coli cells transformed by the plasmid pRT40 DNA. The 103 Kd protein produced by these cells is shown to be processed to proteins with lower molecular masses by the reverse transcriptases own protease activity as well as Escherichia coli proteases. The resulting 103-66 Kd proteins possess the polymerase activity while 51 Kd and smaller proteins are lacking the activity. The 66 and 51 Kd reverse transcriptase fragments demonstrate the positive immunological reaction with the human blood serum from the people possessing antibodies to HIV-1 virus. The recombinant reverse transcriptase of HIV-1 produced by Escherichia coli cells is shown to be useful in AIDS diagnosis in humans.

[Psychoneurologic aspects of infections caused by the human immunodeficiency virus]. / Psikhonevrologicheskie aspekty infektsii, vyzyvaemoi virusom immunodefitsita cheloveka.

The clinical and follow-up analysis of 213 cases (including 144 males and 69 females) with human immunodeficiency virus-induced infection at different stages and 14 autopsies characterizes the factors which have a negative influence on the CNS. These include opportunistic infections, concomitant infections, chronic alcoholism, drug abuse, premorbid altered background, endogenous diseases, psychosexual disorientation concerning the sex of the object, psychogenic reactions. It is concluded that there are difficulties of distinguishing the symptomatology directly associated with human immunodeficiency virus neurotropism. There are positive and negative trends in the lifestyle of the HIV-infected persons and AIDS patients.

[Hemostatic disorders in patients with HIV infection]. / Narusheniia v sisteme gemostaza u bol'nykh s VICh-infektsiei.

Impaired hemostasis was studied in 50 adult patients with HIV infection. The blood coagulative potential, the number and functional activity of platelets were examined. Platelet aggregation and secretion were shown to change earlier than thrombocytopenia developed and clinical signs of HIV infection appeared. The disturbance in the plasma section of hemostasis are due to concurrent opportunistic diseases and infections.

[Pneumocystis carinii infection in a HIV infection department]. / Pnevmotsistoz v otdelenii VICh-infektsii.

To determine the prevalence of Pneumocystis carinii in the HIV-infection department, a simultaneous survey was made of 33 HIV-infected patients at various stages of the disease, of close relatives that were nursing the patients in the unit (n-7), and of medical staff of the department (n-20). Patients with toxic infections who were on another floor of the same hospital and medical students were examined as a control group. For detection of P. carinii antigen, smears from the deep airways were tested in the immunofluorescence. P. carinii was detected in 87.7% of HIV-infected patients, in 71.4% of their relatives and in 80.0% of the medical staff, in 16.6% of control patients and 27.7% of students. The main type of the infectious process in pneumocystosis is its carriage; 2 patients at a stage of relapses (IIIB) that corresponds to AIDS were recorded as having pneumocystis pneumonia.

[Clinico-experimental study of mental processes in men with HIV infection]. / Kliniko-éksperimental'noe issledovanie psikhicheskikh protsessov u muzhchin, infitsirovannykh VICh.

A total of 55 males aged 19.5 to 62 years who were infected with HIV were examined psychopathologically+ and neuropsychologically by A.R. Luria's methods. Twenty-one of these patients were homosexuals. Syphilis was recorded in 34.7% in this group. Lymphadenopathy was the major clinical sign of HIV infection. Symptoms of organic involvements+ of the central nervous system were revealed by a psychopathological method. Neuropsychological studies detected dysfunction of the right cerebral hemisphere, particularly in a group of homosexuals suffering from syphilis.

[Clinical manifestations and the problems of classification of HIV infection]. / KLinicheskie proiavleniia i voprosy klassifikatsii VICh-infektsii.

The clinical manifestations and some immunological parameters (CD4 lymphocytes, CD4/CD8 ratio, IgM, IgA, IgG levels, skin test) were examined in 226 adult patients (148 males and 78 females) infected with HIV. These included 58 (26%) asymptomatic patients with seropositive test, 109 (48%) with the only clinical manifestation generalized lymphadenopathy; 54 (24%) with AIDS-related infections, 5 (2%) with AIDS. A subsequent follow-up of 3 months to 3 years demonstrated that AIDS developed in 7 patients, 9 died. The period of infection with HIV and death ranged from 1.5 to 9 years. The signs of cell immunodeficiency were found in 70% of the examinees. Recommendations are given on the classification of HIV infection.

[Molecular genetic characteristics of HIV-1 in Russia]. / Molekuliarno-geneticheskaia kharakteristika VICh-1 na territorii Rossii.

The paper presents data on the variants of human immunodefficiency virus type 1 (HIV-1) currently circulating in Russia. The subtype A HIV-1 variant dominating is shown to be most widespread among drug-injected users in the most regions under study. By using the results of an analysis of 1,464 blood samples taken in the past 4 years in 69 subjects of the Russian Federation, the authors have estimated that this HIV-1 variant is responsible for 93% of all HIV-infection cases in the country. The greatest regional genetic diversity was observed in Moscow and its mean (2.35(1.59) was found to be comparable to that (2.41(1.85) in the whole country. Penetration of the subtype A IV-1 variant early detectable among drug-users into other risk groups was noted.

[HIV-infection in Russia: a prediction]. / VICH-infektsiia v Rossii: prognoz.

It is pointed out in the paper that the number of HIV-infected persons is to a great extent understated in Russia. Demographic parameters of sexually-transmitted HIV-infection are briefly forecasted for the country. A variety of scenarios for further epidemic progression are elucidated.

[The factors and cofactors affecting the heterosexual transmission of human immunodeficiency virus (HIV) infection]. / Faktory i kofaktory, vliiaiushchie na geteroseksual'nuiu peredachu infektsii, vyzyvaemoi virusom immunodefitsita cheloveka (VICh)]

HIV-infected women and men as well as their sexual partners were examined with the purpose of detecting the factors and cofactors facilitating the risk of HIV transmission in heterosexual contacts. Among HIV-infected women were predominant those who led promiscuous sex life, practicing sex life during menstruation, those with traumatic sex, with cervical erosion in the anamnesis, and women whose sexual partners showed signs of marked immunodeficiency. Cervical erosion in women with heterosexual contacts is the leading factor increasing the risk of infection transmission from women to man and vice versa.

[The risk factors for the transmission of HIV infection among intravenous narcotic users in Russia]. / Faktory riska peredachi VICh-infektsii sredi upotrebliaiushchikh vnutrivenno narkotiki v Rossii.

Blood sera (316,281 specimens) from registered drug and toxic substance addicts were examined revealing no subjects seropositive to HIV. A special epidemiological questionnaire was developed and used for questioning of 86 drug addicts using drugs prepared domestically by the intravenous route. The group included 56 men and 30 women varying in age from 17 to 33 years. Lack of registered cases of HIV infection among drug addicts in Russia was explained by some peculiar features of using drugs. Intravenous inoculation of drugs with the same syringe and needle without sterilization, and large numbers of sexual contacts without the use of condoms suggest that as soon as HIV gets into this environment it will spread very rapidly.

[Replicative properties of HIV-1 strains, resistant to 3'-azido-2'3'-dideoxythymidine]. / Replikativnye svoistva shtammov VICh-1, rezistentnykh k 3'-azido-2',3'-didezoksitimidinu.

The replicative activity of two strains of human immunodeficiency virus type I, 1974 and 1978, resistant to 3'-azido-2',3'-dideoxythymidine (AZT), was investigated. Assessment of AZT sensitivity by the standard method showed the 50% effective dose (ED50) for strain 1974 to be 0.1 mumole and for strain 1978, 1 mumole. Both strains had the slow/low. 3 phenotype. The level of virus-specific antigens in these strains was 3-7 times lower than in AZT-sensitive strain IIIB. In order to study the effects of AZT on virus replication, the strains were passaged in Jurkat-tat cells in the presence of 3.5 mumole of AZT. Virus activity was assessed from the level of intracellular (S-12) and extracellular (P-100) antigens, cell viability, and syncytium formation. Cell viability increased by 10%, syncytium formation dropped 5 times, and antigen accumulation increased 1.5-2 times in the culture with strain 1974. For strain 1978 these parameters were as follows: 17% decrease of cell viability, increase of syncytium formation by 1.7 times, and no changes in the level of viral antigen. After four serial passages in the presence of AZT ED50 for strain 1974 was 0.5 mumole and at least 1 mumole for strain 1978.

[Detection and quantitative determination of antigen to the human immunodeficiency virus during development of a test-system for the serodiagnosis of AIDS]. / Vyiavlenie i kolichestvennoe opredelenie antigena virusa immunodefitsita cheloveka v protsesse polucheniia test-sistemy dlia serodiagnostiki SPID.

One of the variants of IFA using a conjugate on the basis of anti-HIV-IgG was used for control of the content of human immunodeficiency virus (HIV) antigen at different stages of production of a test system for serodiagnosis of AIDS. This method permits HIV antigen quantitation in virus lysates, its nonpurified concentrates, and in native culture fluid in titres from 2000 to 60,000 and from less than 27 to greater than 729, respectively. In most cases, high titres of antigen in the liquid fraction of the culture corresponded to high values of the antigen-containing cells on the basis of immunofluorescence data and signs of intensive HIV production in electron microscopic culture control. The authors developed a method allowing to determine the antigen titre in the study material using only one dilution (1:9) of it in IFA technique.