Higher Infectivity of the Human Immunodeficiency Virus in Sensitive Cells with a Modification of the CCR5 Gene.

As a natural mutation of the human ccr5 gene has been shown to confer resistance to human immunodeficiency virus type 1 (HIV-1) infection, a new avenue has opened in the development of alternative treatment approaches through genome editing. One of the two chemokine co-receptors of the plasma membrane is utilized by HIV-1 to infect CD4+ cells. HIV-1 strains that utilize CCR5 circulate in early infection, and strains that utilize CXCR4 circulate at advanced stages. A complex relationship may exist in the expression regulation of the receptors and may affect virus replication in cells that normally do not express CCR5 on the membrane, such as the MT-4 cell line. MT-4 cells were used to study the effect of ccr5 modification HIV-1 replication in vitro. Genetic modification of ccr5 in MT-4 cells was shown to increase the activities of HIV-1 strains, especially in homozygote. The results indicate that genome editing should be performed with caution in human cells and that the issue needs comprehensive investigation.

[Antiviral and virucidal activity of sodium deoxyribonucleate and its complex with iron against viruses of different kingdoms and families].

INTRODUCTION: The urgent problem of modern medicine is the fight against acute respiratory viral infections (ARVI). To combat ARVI, drugs of wide antiviral potency are needed, as well as immunomodulating drugs. Such antiviral and immunomodulatory effects has sodium deoxyribonucleate (DNA-Na) and its complex with iron (DNA-Na-Fe) developed on the basis of double-stranded DNA of natural origin. AIM OF THE STUDY: To assess antiviral and virucidal activity of DNA-Na and DNA-Na-Fe against viruses of different kingdoms and families. MATERIALS AND METHODS: Antiviral and virucidal activity of DNA-Na and DNA-Na-Fe was assessed in cell cultures infected with viruses. RESULTS AND DISCUSSION: DNA-Na and DNA-Na-Fe had antiviral activity against adenovirus at concentrations of 2501000 mcg/ml. Antiviral effect of both drugs was not detected in case of poliovirus. DNA-Na and DNA-Na-Fe had antiviral activity against coronavirus in all administration schemes. EC50 for DNA-Na ~ 2500 mcg/ml, for DNA-Na-Fe ~ 1000 mcg/ml. In cells treated with DNA-Na-Fe, secretion of following proinflammatory cytokines was detected: Interleukin (IL) 1, IL-2, IL-6, IL-18, interferon- (IFN-), IFN-, as well as anti-inflammatory cytokines: IL-4, IL-10, antagonist of IL-1 receptor. Evidently, DNA-Na and DNA-Na-Fe have antiviral effect, but mechanism of action does not seem to be associated with specific effect on viral replication. Presence of virucidal activity of drugs against representatives of Coronaviridae, Adenoviridae, Picornaviridae, Retroviridae, Herpesviridae in vitro test in range of 1.03.0 lg TCID50 was identified. CONCLUSION: Presence of simultaneous antiviral and virucidal activity of DNA-Na and DNA-Na-Fe against adeno- and coronaviruses shows their prospects for prevention and treatment of ARVI.

[Expression of integrins ß1, α4 and cell adhesion molecule ICAM-1 in the presence of sodium deoxyribonucleate with ferrum complex (DNA-Na-Fe) by MT-4 cells transformed by human T-lymphotropic virus type 1 (Retroviridae: Orthoretrovirinae: Deltaretrovirus: Human T-lymphotropic virus type 1)].

INTRODUCTION: The important role of integrins (IG) in the initiation and development of cancer processes makes these structures convenient targets for the development of immunomodulatory therapeutic drugs that have an effect directly on these molecules. Among the latter, IG ß1, α4 and cell adhesion receptor ICAM-1 (intercellular adhesion molecule 1) are of particular interest. Immunomodulators are capable of changing the IG activity through non-specific mechanisms, which, however, in some cases can cause a decrease in the protective functions of the immune system and health deterioration.The aim of the study was to determine the effect on the levels of cellular expression and the nature of IG metabolism of the drug sodium deoxyribonucleate with ferrum complex, DNA-Na-Fe, which is having been used in the Russian Federation as an immunomodulatory agent, but whose action has not been studied in details so far. MATERIAL AND METHODS: We used 2 variants of the neoplastic CD4+ T-lymphocyte cell line transformed with human T-lymphotropic virus type 1 (HTLV-1) of the Retroviridae family, MT-4 (MT-4/1 and MT-4/2). The indicated variants were characterized by different levels of expression of the protein activation markers CD28 and CD38. After cell culture in the presence of 500 µg/ml DNA-Na-Fe, the expression levels of IG ß1 (CD29), α4 (CD49d), and ICAM-1 (CD54) were studied by flow cytometry. RESULTS: The cells of the both lines contained many membrane proteins CD29+ (90.4% ± 4.5) and CD54+ (97.9% ± 1.4), while small percentage of cells contained protein CD49d+ (1.9% ± 1.0). No changes in the expression of the studied proteins were observed in the presence of the drug. DISCUSSION: The levels of IG ß1, α4 and ICAM-1 expression may serve as one of the phenotypic characteristics of MT-4 cells. The obtained data are of great importance because the peculiarities of CD4+ T-lymphocytes transformation and their metabolism during HTLV-1 infection have not been sufficiently studied so far. CONCLUSION: The results of this work may be helpful in determining the pathogenesis of HTLV-1-induced diseases, some types of malignancies, and in searching for new specific pharmacological agents, including molecularly targeted ones. The results of the study will help to expand the existing knowledge on the markers of MT-4 cell line.

[The study of the innate and acquired cellular immunity chains indicators in the peripheral blood of patients with HIV infection.]

In this study was made an attempt to reveal additional laboratory markers of white blood for preliminary estimation level of HIV-infection development. Essentially such markers these are in progress without complex equipment and expensive reagent. It was studied alterations of basic values cells of innate and acquired immunity of peripheral blood HIV-infected individuals with and without antiretroviral treatment (ART) during infection. It was estimate value leukocytes, neutrophils, monocytes, lymhpocytes, T-lymhpocytes, CD4+, CD8+ T-cells, CD4/CD8 index. It was used the first analysis in the time of registration for regular medical check-up and the intermediate derived during 2017-2018 years. Patients without ART and with ART before and after treatment had rates of leukocytes, lymhpocytes, T-lymhpocytes, monocytes and neutrophils within the normal guideline. Essential changes were observed in basic conventional laboratory parameters evaluation of HIV-infection dynamic (parameters of CD4+, CD8+ T-cells, CD4/CD8 index). Thereby it was impossible to reveal supplementary immunological markers of HIVinfection.

[The effect of ferrovir on to expression of surface markers of activation by cells neoplastic line MT-4.]

The immune-modulating activity of "Ferrovir" medication in system in vitro was analyzed using neoplastic cellular line MT-4 as a model. Ferrovir decreased number of cells containing such markers of activation as CD28+, CD38+, CD62L+, CD69+ and HLA-DR+.Under 24 hours incubation period of cells in presence of 500 mkg per ml of medication, indices of decreasing of number of cells expressing these proteins (IRE), for proteins CD28, CD38, CD62L and HLA-DR made up to 1,9 ± 0,4, 1,3 ± 0,4, 1,2 ± 0,4, 1,1 ± 0,06 correspondingly. At prolonged incubation of cells in presence of Ferovir, the maximal effect was observed after 7 days of incubation and IRE for proteins mentioned above made up to 3,2, 3,4, 6,2, 1,4 и 3,1 correspondingly. Only for protein CD62L was marked a significant decreasing of number of cells bringing this marker and at 11th day of cells cultivation in presence of Ferrovir (IRE 3.89). It is possible that such an action of Ferrovir can decrease the process of spreading of cells containing integrated pathogenic material through organs and tissues of organism and slow down generalization of infectious process. The obtained results indicate that Ferrovir has an immune-modulating activity in vitro since it can decrease activating potential of neoplastic line of cells MT-4. These features can be useful in treatment of various type of cancer, HIV-infection and other human diseases. The decreasing of level of activation of cells of immune system also decreases risk of development of opportunistic infections.

[CYTOKINES DURING THE HUMAN IMMUNODEFICIENCY VIRUS INFECTION TYPE 1(HIV-1)].

In this work the proinflammatory (IL-1ß, IFN-γ, TNF-α, IL-2) and anti-inflammatory (IL-4, IL-10) plasma cytokine levels were evaluated in HIV-infected patients with or without antiretroviral treatment (ART). IFN-γ was detected in 94% samples with and without ART, TNF-α in 88% and IL-2 in 38% samples without ART, as well as in 12% and 30% samples with ART, respectively. Positive correlation was detected between viral RNA and IFN-γ levels (rs = 0.13) and negative correlation (rs = -0.242) in the patients without or with ART. Cosecretion of three cytokines (IFN-γ, TNF-α, IL-2) was detected in 31% samples and two cytokines (IFN-γ, TNF-α) in 35% samples of persons without ART. Cosecretion of three cytokines (IFN-γ, TNF-α, IL-2) was detected in 20% samples with ART; cosecretion of IFN-γ and IL-2 was detected in 10% samples. The higher percentage of the proinflammatory cytokines with cosecretion was detected in plasma HIV-infected patients without ART in the course of 6 and more years, which suggests that their immune system is able to provide disease control.

[The superficial markers of neoplastispecificity of c cell line MT-4 and perspectives of its application as a model for studying activity of immune modulating preparations.]

The article considers expression of markers of activation of neoplastic CD4+ T-lymphocytic transplantable cellular line M T-4, transformed by T-lymphotropic human virus type I. It is demonstrated that in cells are detected such external proteins as CD25+, CD28+, CD38+, CD62L+, CD69+, CD95+ and HLA-DR+. The maximal number of these components was detected in three days after transplantation of cells. These indices reached average level for markers CD25+, CD28+, CD38+, CD69+, CD95+ and HLA-DR+ - more than 90% and for CD62L+ - 48%. The obtained results and cultivation of cells indicate that cells MT-4 can be used as a convenient model for testing of activity of immune modulation preparations.

[Parameters of the CD4-Cell count and viral load in human immunodeficiency virus type 1 (HIV-1) infected patients].

In this work the specific features of parameters of plasma CD4 T-lymphocytes count and level virus RNA in the HIV-infected patients were studied. 22% correlation between reduction of CD4 cell count and an increase in virus RNA level was observed in persons that did not receive antiretroviral treatment during the third HIV-infection phase. During this phase of infection patients exhibited a growth of the median value of virus load in cases of both rise as decline in CD4 cell count during long observation period. In addition, towards the end of the observation period, the percentage of patients with virus load > 3.3 Ig copies/ml considerably expanded. 43% correlation between CD4 cell count and duration of the HIV-infection was detected during the fourth infection phase in persons that did not receive antiretroviral treatment. Most of the patients in the third and the fourth infection phases had essential CD4 cell count growth during antiretroviral treatment. Best values were observed in patients with the initial value of CD4 > 400 cells/µl belonging to the third HIV-infection phase.

[THE IMMUNOMODULATORY ACTIVITY OF PLASMA OF PATIENTS INFECTED WITH HUMAN HIV VIRUS].

The study was carried out to investigate impact of plasma of patients infected with human HIV virus receiving and not receiving highly active antiviral therapy on: expression of phenotypic markers of lymphocytes (CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD3-/CD (16+56)+, CD3+/CD(16+56)+, CD3+/HLA-DR+, CD4+/CD62L+, CD8+/CD38+) in mononuclear cells of blood of donors and secretion of pro-inflammatory (interleukin-1ß, interferon-γ, tumor necrosis factor-α, interleukin-4 and interleukin-10) cytokines. After 24 hours of activation of mononuclear cells with plasmas it was demonstrated that as compared with control groups, in of plasmas of patients with highly active antiviral therapy increasing of number of CD4+ T-cells and decreasing of CD8+ T-cells is observed. The plasmas of patients with highly active antiviral therapy activate in most instances CD4+ T-cells whereas plasmas of patients without treatment--CD8+ T-cells. The results of detection of cytokines in blood indicate that in patients without treatment inflammatory potential is increased as compared with group of highly active antiviral therapy. The data concerning accumulation of interleukin-1ß under cultivation of mononuclear cells with plasmas indicates at its role in preservation of vitality of natural killers. The analysis of immunomodulatory activity of plasma of patients infected with human HIV virus can be recommended as an additional technique of evaluation of functioning of immune system.

[The indicators of immune status of peripheral blood of donors].

The expanded analysis of 57 samples of peripheral blood from conditionally healthy patients was implemented concerning phenotype of main populations of lymphocytes, activated pools of cells and level of cytokines. The samples were received in the department of storage of blood and its components of the research institute of blood transfusion of the hematology research center. It is demonstrated that number of T-lymphocytes, T-helpers and activated TY-cells with phenotype CD3+HLA-R+ and level of detected cytokines by standard indicators had no difference with publications data. In particular cases an increase of number of cytolytic T-lymphocytes, B-lymphocytes and natural killers and decrease or increase of CD4/CD8 index relative to standard were detected. The decrease of number of natural killers was the most frequent aberration. The study demonstrates that among conditionally healthy patients giving blood as donors persons with disorders of immune system were presented.

[CD4+ and CD8+ T-lymphocyte counts in human immunodeficiency virus type 1 subtype A-infected patients carrying mutations V77I in protease and/or A62V in reverse transcriptase].

The peripheral blood counts of CD4+, CD8+, and CD4/CD8 in human immunodeficiency virus type 1 subtype A-infected patients were comparatively analyzed with the data of a genetic study of the pol gene. Thirty peripheral blood samples from antiretroviral-naïve patients with grade 3 (sublinical) HIV infection were analyzed. According to the presence or absence of mutations V77I in protease and/or A62V in reverse transcriptase, the patients were divided into 2 study groups. The genetic analysis of the groups indicated that 19.5% of the study samples had no mutations; 75% contained one or both mutations, of them 36% contained both mutations. Immunological study showed that the median CD4+, CD8+, and CD4/CD8 in the patients infected with virus variants containing mutations V77I and/or A62V were increased by 25, 20, and 16%, respectively. The findings suggest that these mutations may be associated with an immune response in HIV-infected patients.

[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.

[Variants of human immunodeficiency virus type 1, detected in Russia among those infected by the sexual route]. / Varianty virusa immunodefitsita cheloveka tipa 1, obnaruzhivaemye v Rossii sredi infitsirovannykh polovym putem.

We analyzed 52 blood samples obtained independently from among individuals, who had never practiced the intravenous administration of drugs, for the purpose of detecting the subtypes of HIV-1 that circulated during 1999-2002. The study materials were analyzed by the methods of gag/env heteroduplex mobility assay and by env gp120 C2-V3 sequencing. Four viral subtypes (A,B,C and G) and a recombinant gagA/envB were detected in subjects contaminated through heterosexual contacts. Noteworthily, HIV-1 variations of subtype A, which were found in 22 (73.3%) of 30 analyzed samples, were predominant in this risk group. An analysis of nucleotide sequences exposed a high degree of homology between the viruses, detected previously among drug-addicts, and the isolates detected by us in subjects contaminated heterosexually. However, HIV-1, subtype B, detected by us in all 16 studied cases, still continue to circulate among the males infected through homosexual contacts with men.

[Decrease of HIV-1 replicative capacity after serial passage in MT-4 cells under conditions of the combined antiretroviral therapy]. / Snizhenie replikativnoi sposobnosti VICh-1 posle passirovaniia v kletkakh MT-4 v usloviiakh kombinirovannoi antiretrovirusnoi terapii.

The replicative activity of HIV-1/IIIB was determined in MT-4 cells under conditions of combined HIV reverse transcriptase and protease inhibitors in the presence of four different drug cocktails: (1) phosphazide, didanosine, nevirapine; (2) stavudine, didanosine, nevirapine; (3) phosphazide, didanosine, nevirapine, indinavir; (4) stavudine, didanosine, nevirapine, indinavir. The concentration of every inhibitor was 10 times higher than the 50% effective concentration. Alpha interferon was used as a natural antiretroviral agent in addition. The virus was subjected to 5 serial passages in the presence of the drug cocktails and then to 5 serial passages without the agents using cocultivation of infected and uninfected cells at ratio 1:5 to increase virus activity. Virus replication in the presence of all the drug combinations resulted in the appearance of HIV-1 variants with low replicative activity that was insignificantly increased during further passages even without the antiretroviral agents. If extrapolated to the clinical practice, these results indicate that all the drug cocktails were effective inhibitors of HIV-1 replication because the virus variants with high replicative activity did not emerge. Moreover, the results showed that the clinical use of the drug cocktails was promising.

[Effect of home-made narcotics on infectious activity of HIV-1]. / Vliianie narkoticheskikh veshchestv, izgotavlivaemykh kustarnym sposobom, na infektsionnuiu aktivnost' VICh-1.

The stability of human immunodeficiency virus, type 1 (HIV-1), strain IIIB, was studied in liquid preparations of homemade drugs. The "Vint" preparation (containing Methamphetamine and obtained from Ephedrine) as well as "Khanka" (a liquid surrogate opiate made from poppy straw) were analyzed within the case study. HIV-1/IIIB was shown to maintain its infectious activity in "Khanka" at room temperature for least 7 days. The HIV-1 activity in neutralized "Vint" did not essentially change after a 30-minute incubation at pH 7.0. While an incubation in the acid "Vint" solution entailed a more rapidly decreasing activity. However, the virus infection ability preserved during the entire time period, during which the drug was fit for injections, i.e. for 30 minutes at room temperature or for 20 hours at 4 degrees C. Therefore, the infection virus could well preserve in the "Khanka" and "Vint" solutions after its entry, with infected blood, of large volumes of the discussed drugs. The mentioned big volumes of HIV-1 contaminated drugs, shared later into ready-to-use portions, could be the cause for HIV-1 dissemination among those who practice the parenteral administration of these substances. Besides, "Khanka" was shown to have little or no effect on the virus replication to cell culture MT-4. Its presence brought about an insignificant 1.5-fold increase in the viral stock (observed on days 2 and 3 after contamination) only when 2 x 10(5) MT-4 cells per ml and HIV-1/IIIB TCID 50 0.005 were used.

[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.

[Anti-interferon activity of peripheral blood leukocytes in patients infected with HIV-1]. / Antiinterferonovaia aktivnost' leikotsitov perifericheskoi krovi patsientov, infitsirovannykh VICh-1.

The study was aimed at anti-interferon activity of different components of the in vitro system cells MT-4--HIV-1 as well as the culture fluid of peripheral blood leukocytes from patients at different stages of HIV infection and from patients with mixed infection (HIV and chronic hepatitis C) in comparison with patients infected with hepatitis C virus alone and healthy persons. Anti-interferon activity was detected in all groups of patients, its detection rate varying within 33% and 68%. The tendency towards increased detection rate of anti-interferon activity in HIV-infected patients in parallel with decreased number of CD4+ lymphocytes was noted. These data made it possible to suggest that increased detection rate of anti-interferon activity in the culture fluid of peripheral blood leukocytes from HIV-infected patients in parallel with decreased number of CD4+ lymphocytes could result from pathogenetic processes in the body, leading to a decrease in therapy effectiveness of HIV-infected patients with the preparations of alpha-interferon, especially in patients with a low content of CD4+ cells.

[Effect of heroin-containing substances on the infectivity of the human immunodeficiency virus type 1 in vitro]. / Vliianie preparata, soderzhashchego geroin, na infektsionnost' virusa immunodefitsita cheloveka tipa 1 in vitro.

At room temperature, HIV-1 IIIB is shown to remain infectious in a dose of 25 mg/ml of heroin solution for more than 8 days. The large batch of HIV-1-contaminated heroin solution may therefore remain infectious for a long period sufficient for transportation, packing, and sale in any area of Russia. At the same time 41-day incubation under the same conditions caused a complete loss of viral infectivity. Under certain conditions (the concentration of MT-4 cells being less than 2 x 105 cells/ml and the multiplicity of infection, less than 0.01 ID50/cell), heroin was demonstrated to be able to increase HIV-1 replication at the early stages of its life cycle. The findings should be borne in mind in elaborating measures to prevent the spread of HIV-1 among intravenous psychoactive drug users.

[Molecular and epidemiologic characteristics of HIV-infection outbreak in the Irkutsk region]. / Molekuliarno-épidemiologicheskaia kharakteristika vspyshki VICh-infektsii v Irkutskoi oblasti.

The genetic analysis of the variants of human immunodeficiency virus of type 1 (HIV-I) circulating among drug addicts in the Irkutsk region was carried out. The results of serological tests and comparative evaluation of electrophoretic mobility of heteroduplexes (HMA) revealed that all 74 samples under study belonged to subtype A. Genetic differences between these viruses did not exceed 2%. Thus, it was the variant of subtype A prevalent in CIS countries which caused the outbreak of HIV infection among drug addicts in the Irkutsk region, but not viruses of subtypes B, C or A/E typical for this risk group in relatively nearby China.