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.

[Antiviral activity of extracts of basidiomycetes and humic compounds substances against Human Immunodeficiency Virus (Retroviridae: Orthoretrovirinae: Lentivirus: Human immunodeficiency virus 1) and Herpes Simplex Virus (Herpesviridae: Simplexvirus: Human alphaherpesvirus 1)].

INTRODUCTION: One of the most urgent problem of modern medicine is the fight against the disease caused by the Human Immunodeficiency Virus (HIV) - HIV infection. The chemical compounds have improved the situation for infected people, but they are toxic, disrupt the metabolism and cannot eliminate the integrated virus from the body. The emergence of resistant HIV strains makes these treatments ineffective. Often, the death of HIV-infected people occurs as a result of the development of opportunistic infections caused by viruses of the Herpesviridae family. Therefore, the search for new therapeutic and preventive drugs that are less toxic and active against several viruses at the same time is relevant. Basidiomycetes, higher fungi, are a source of medicinal compounds that have antimicrobial properties, as well as antiviral ones. Humic compounds (HS) of various nature also have antiviral activity.The aim of the study was to obtain nontoxic compounds from the basidiomycete Inonotus obliquus and humic compounds from brown coals and to test their activity against viruses that are pathogenic to humans: HIV and Herpes Simplex Virus (HSV). MATERIAL AND METHODS: The antiviral activity of melanin extracts obtained from the culture of the chaga fungus Inonotus obliquus and HS from the brown coal of the Kansko-Achinsk Deposit was studied using a model of MT-4 lymphoblastoid cells infected with HIV type 1 (HIV-1) strains and a monolayer culture of Vero cells infected with HSV type 1 (HSV-1) using virological and statistical research methods. RESULTS AND DISCUSSION: It was found that all the studied compounds did not have a cytotoxic effect on cells at a concentration of 100 mcg/ml. It was shown that extracts of basidiomycetes and HS have antiviral activity against HIV-1 and HSV-1. EC 50 (50%-effective concentration) for HIV-1 was 3.7-5.0 mcg/ml, selectivity index 28-35. Antiherpetic activity was detected at a dose of 50-100 mcg/ml. The antiviral effectiveness of melanin compounds was established both in the «preventive¼ (2 hours before cell infection) and in the «therapeutic¼ regimen of drug administration, both for HIV-1 and HSV-1. The presence of antiviral activity of melanin and HS in relation to the RNA-containing HIV-1 virus and DNA-containing HSV-1 virus in our study coincides with the results of a number of authors in relation to influenza viruses, herpes virus, HIV, hepatitis B virus, Coxsackievirus, smallpox vaccine virus, which suggests that the type of nucleic acid in the virus does not play a fundamental role in the antiviral action of these drugs. It is also clear that HS is effective against both enveloped and non-enveloped viruses. CONCLUSION: In general, it can be concluded that melanin and humic compounds are characterized by low toxicity in the presence of both virucidal and antiviral activity. This allows us to consider the studied compounds as the basis for creating safe medicines that are effective against pathogens of various viral infections.

CYTOTOXIC PROPERTIES OF TRITERPENE SAPONIN TAUROSID SX1 AND ITS EFFECT ON HUMAN IMMUNODEFICIENCY VIRUS AND INFLUENZA VIRUS INFECTION IN MICE.

Triterpene saponin Taurosid Sx1 purified from leaves of the plant Crimean Ivy Hedera taurica Carr. (Araliaceae) was evaluated for its cytotoxic activity against lymphoblastoid cell lines MT-4, Jurkat-tat, U937, and human peripheral blood monocytes. The ability of saponin to influence HIV-1 replication was studied as well. In addition, the ability of Taurosid Sx1 to increase survival of mice infected with influenza virus А/WSN/1/33(H1N1) and its capacity for strengthening the immune responses in mice immunized with the influenza vaccine Grippol® have been studied. Taurosid Sx1 has been shown to inhibit MT-4 cell line at 25 µg ml-1 concentration, IC50 33,3 µmol l-1 (MTT assay). The saponin concentration of 5 µg ml-1 was non-toxic for all the cell lines studied and demonstrated a moderate inhibitory effect on HIV p24 production in Jurkat-tat cells. In the lower concentrations Taurosid Sx1 did not stimulate HIV p24 production. It was shown that oral administration of 200 µg Taurosid Sx1 to the influenza virus infected mice caused 1.5-fold increase in their survival. Taurosid Sx1 given orally amplified immunopotentiating ability of an intramusculary administered subunit influenza vaccine. Antibody production was significantly higher in animals fed Taurosid Sx1 after primary or secondary immunizatuion. In mice given 2 doses of vaccine, from 1 to 3 weeks apart, feeding 200 µg saponin resulted in 2 to 10-fold enhancement in production of anti-H1, anti-H3, and anti-inluenza type B hemagglutinin antibodies. Thus, Taurosid Sx1 can be considered as low-toxic promising immunopotentiating agent uncapable of enhancing HIV-1 replication.

[Application of MT-4 neoplasm cell line for the immunomodulating activity study of patients plasma with HIV-infection.]

It was studied in vitro the immunomodulatory effect of plasma HIV-infected individuals on expression of activation markers when used as a model neoplastic cell line MT-4. Carrying out researches indicated the variation in expression of the activation markers CD28+, CD38+, HLA-DR+ и CD69+. Change dynamics of these indices showed that these proteins can to consider as markers for level evaluation of patients immune system during used of plasma HIV-infected individuals with and without antiretroviral treatment (ART). Analysis revealed reduction of cells activation potential in plasma of patients with ART presence and rise without treatment. Examinations of the expression proteins CD28, CD38, HLA-DR и CD69 MT-4 cells and plasma of patients with HIV-infection application can have prognostic value for infection monitoring and efficacy of different therapeutic approaches.

INFLUENCE OF IMMUNOMODULATORY DRUG STIMFORTE ON THE EXPERIMENTAL HERPES VIRUS INFECTION IN COMBINATION WITH ACYCLOVIR AND ON HIV-INFECTION IN COMBINATION WITH RETROVIR.

The combined action of the immunostimulatory drug Stimforte and the basic etiotropic drug acyclovir commonly used to treat herpes infections was studied using the model of lethal experimental infection of mice BALB/c with herpes simplex virus type 1. It was found that the interaction of these drugs is additive. In addition, Stimforte inhibits infection caused by a strain of virus, which is highly resistant to acyclovir. When administered 24 hours prior to HIV-1 infection of human lymphoblastoid cells MT-4, Stimforte exhibited reliable antiretroviral activity best expressed during the early period of infection (the 3rd day). On the 6th day of observation the effect was almost completely lost. Combined use of Stimforte at a dose of 50-100 µg/ml with a subthreshold dose of retrovir (0.03 µg/ml) had a synergistic antiviral effect. Thus, Stimforte, which exhibits, on the one hand, antiviral activity against viruses of different families and, on the other hand, the immunomodulatory properties, could be promising as an etiopathogenic tool in helping to normalize both nonspecific and specific immunity. It may be used simultaneously with etiotropic antiviral chemotherapy in treatment of generalized herpes infection in patients with immunodeficiency. Furthermore, Stimforte can be used in the case of development of drug resistance in HSV, in particular, in HIV-infected patients.

[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.

A comparative analysis of virucidal efficiency of biocide agents.

The main groups of biocide agents used for inactivation of bacteria and viruses were studied for their virucidal activity against enveloped (HIV, viral hepatitis C, influenza virus A) and non-enveloped viruses (poliovirus, adenovirus). Their efficiency was analyzed. Quarterly ammonium compounds (QAC) themselves are not able to properly inactivate non-enveloped viruses. However, they can be successfully applied in combination with other biocides (guanidines, aldehydes). Effective composition of QAC with amines and guanidines provided inactivation of viruses (4.0 lgTCID50) in concentrations of 0.166-0.280% for non-enveloped viruses and 0.080-00.185% for enveloped viruses. The combination of QAC with aldehydes is especially effective (0.04-0.64% for non-enveloped viruses). The virucidal efficiency does not directly depend on the QAC concentration in the chemical disinfectants.

[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.

[Antiviral activity of aqueous extracts of the birch fungus Inonotus obliquus on the human immunodeficiency virus].

Fractions of aqueous and water-alcohol extracts of the birch fungus Inonotus obliquus have antiviral effect against the human immunodeficiency virus type 1 (HIV-1). Antiviral properties of low toxic extracts were manifested in the concentration of 5.0 µg/ml upon simultaneous application with the virus in the lymphoblastoid cells culture MT-4. The extract of the birch fungus can be used for development of new antiviral drugs, inhibitors of HIV-replication when used both in the form of individual drugs and as a part of complex therapy.

[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.

[Activity of the inositol-containing phospholipid dimer analogues against human immunodeficiency virus].

For the purpose of finding effective inhibitors of virus adsorption the series of inositol-containing phospholipid dimer analogues were previously synthesized. In the present work, the antiretroviral activity of these compounds against HIV-1 was demonstrated on the model of cells infected with the virus. The highest effect was found in the case of dimer poliol 5, EC50 (50%-effective concentration) was 3.9 microg/ml. The development of new polyanionic compounds, which can interfere with early steps of the virus life cycle, is a promising addition to the antiretroviral therapy based on the virus enzyme inhibitors.

[Synthesis, properties and anti-HIV activity of novel lipophilic 3'-azido-3'-deoxythymidine conjugates containing functional phosphoric linkages].

One of the approaches to enhance bioavailability of nucleoside reverse transcriptase HIV inhibitors consists in design of their prodrugs based on 1,3-diacylglycerols, which may simulate nature lipids metabolic pathways promoting the improvement of drug delivery to the target cells. Glycerolipidic AZT conjugates with different functional phosphoric centers were synthesized by H-phosphonate technique in the present work. Study of prepared prodrugs sensibility to the chemical and enzymatic hydrolysis (in buffer solution and under the influence of pancreatic lipase) and also study of their anti-HIV activity on the T-lymphoid human MT-4 cells in regarding to virus HIV-1(899A) strain were carried out.

[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.

[The antiretroviral agent Fullevir].

The antiretroviral properties of Fullevir (sodium salt of fullerenepolyhydropolyaminocaproic acid) manufactured by IntelFarm Co.) were studied in the human cell culture infected with human immunodeficiency virus (HIV). The agent was ascertained to be able to protect the cell from the cytopathic action of HIV. The 90% effective concentration (EF90) was 5 microg/ml. The 50% average toxic concentration was 400 microg/ml. Testing of different (preventive and therapeutic) Fullevir dosage regimens has shown that the drug is effective when used both an hour before and an hour after infection and when administered simultaneously with cell infection. The longer contact time for the agent with the cells increased the degree of antiviral defense. Co-administration of Fullevir and the HIV reverse transcriptase inhibitor Retrovir (azidothymidine) showed a synergistic antiretroviral effect. Thus, Fullevir may be regarded as a new promising antiretroviral drug for the treatment of HIV infection.

[Effect of novel natural hypolipidemic compounds on human immunodeficiency virus]. / Vliianie novykh prirodnykh gipolipidemicheskikh soedinenii na virus immunodefitsita cheloveka.

Target screening among microbial products resulted in isolation of hypolipidemic compounds tested for activity against HIV in culture of transferable lymphoblastoid cells MT-4. The majority of the compounds showed antiviral activity. The highest antiviral effect was observed when before exposure to the virus the cells were preincubated for 1 hour in the presence of the isolated compounds. The compounds showed no effect when added to the cell culture preliminarily infected by HIV.

[Activity of "Ferrovir" preparation towards RNA and DNA viruses]. / Aktivnost' preparata "Ferrovir" v otnoshenii RNK- i DNK- soderzhashchikh virusov.

Ferrovir (trivalent iron in complex with native sturgeon milt DNA) is nontoxic, its 50% inhibiting concentration (IC50) is at least 4000 micrograms/ml, 90% effective concentration (EC90) towards HIV-1 is 800 micrograms/ml. These effects do not depend on the cell culture or individual biological characteristics and subtypes of 7 strains of HIV-1 used in our study. The chemotherapeutic index of the drug is more than 20. Combined therapy with ferrovir and retrovir had an additive antiviral effect. Ferrovir reduced the titer of human CMV in fibroblast culture by 1-2 Ig TCD50. Ferrovir protected mice after intracerebral inoculation with lethal herpes simplex virus (type 1) (survival 33.7%, protection 27.1%, which is close to the reference group treated with zovirax). These facts evidence antiviral activity of ferrovir towards RNA and DNA viruses and prompt further study of this drug with the aim of its clinical application.