Search

Until now, the problem of effective therapy of HIV-infection is not resolved due to integrative type of interaction of HIV virus with target cell - T-lymphocyte. The study was aimed on search of method of deletion of HIV DNA-provirus from cell's genome.Non-pathogenic for humans Mycoplasma arginini was used for coinfection of HIV-infected cells in model systems in vitro.Complex of mechanisms was documented leading to: blocking up to 50 - 60% of extracellular virus (according to titration results), cancel of apoptosis in infected cells stained on Hoechst, formation of defective vif(-) virions, which together with arginine-desaminase of M. arginini arrange permissive conditions for activation of cellular APOBEC3G with subsequent disruption of DNA- provirus and blocking of viral infection. As studies of ultrastructure showed, listed events resulted from direct interaction of HIV with mycoplasma.The elimination of HIV DNA-provirus is possible by co-infection of T-lymphocytes with M. arginini.

Target-aimed synthesis of a new class of water soluble amino acid and dipeptide derivatives of fullurene (C60 - X) for inhibition of specific virus enzymes, i.e. protease and reverse transcriptase of HIV (P HIV and RT HIV) in cell culture lytic and chronic infections was performed. Out of 13 tested substances, 8 showed inhibitory activity and 5 were effective in pharmacological doses (ID50 varied within 0.46 to 1.0 mcm/ml with respect to the lytic infection and 5.0 to 12.5 mcm/ml with respect to the chronic infection). The activity of (1), (2), (6), (7) and (8) was comparable to that of azidothymidine, a nucleozide inhibitor of RT HIV in the cell culture lytic infection. The substances also showed marked virucidal action. The cytotoxicity (survival, antiproliferative effect) varied from low to very low with respect to the rapidly dividing cells MT4 and HTHIV27 (CD50200-800) and was somewhat higher with respect to PBL (CD50100). The selectivity index (SI = CD50/ID50) was equal to 165-2000 for various samples. The prototype derivatives (1) and (2) had a selective (competitive) inhibitory action on the recombinant protease of HIV with IC50 = 1.25-2.76 mcM, while derivatives (1), (la) and (2) had a noncompetitive inhibitory action on the recombinant reverse transcriptase of HIV (Ki = 7.9-12.1 mM). The pharmacokinetic study of the prototype derivative (1) on laboratory animals revealed no acute or chronic toxicity up to the terminal high concentrations. As for (1), its high interspecies (mice--rabbits) relative bioavailability equal to 110% was shown.

For the first time the detailed description of continuous cell line HTHIV27, remaining stable for more than 10 years, has been made. The stability of all biological characteristics and high productivity of the strain has made it possible to use it as a HIV producing strain for the construction of a diagnostic test system for the detection of antibodies to HIV. The lysate obtained on the basis of HIV producing cells HTHIV27 has been shown to possess a number of advantages in comparison with the analogous system based on lytically infected cells. On the basis of strain HTHIV27 an in vitro cell system for the analysis of the specific activity of chemotherapeutic preparations intended for the inhibition of HIV has been developed. The use of this newly obtained continuous cell line HTHIV27 has been shown to permit the evaluation of the antiviral activity of compounds, characterized by different molecular mechanisms for the suppression of viral activity.

New nucleoside-phospholipid conjugates were synthesized based on 1,2-disubstituted glycerides and nucleosides. These contain rac-1-hexadecyl-2-palmitoyl(or 2-methylcarbamoyl)-sn-glycero-3-phosphate as the phospholipid component and 2',3'-didehydro-3'-deoxythymidine, 1-(Z-5-hydroxypentene-2-yl)thymine, or 2',3'-isopropylideneuridine as a nucleoside component. The conjugates were synthesized by three different ways: from rac-1-hexadecyl-2-acyl-sn-glycero-3-phospodichlorides, -3-phosphatidic acids, or -3-H-phosphonates. When subjected to mild alkaline hydrolysis, conjugates containing a 2-palmitoyl group formed conjugates with the lysophospholipid component that had not yet been described. All the conjugates obtained were amorphous compounds stable at room temperature. Their hemolytic and anti-HIV activities were determined. Some conjugates were found to completely inhibit in vitro HIV-1 reproduction in lower doses than the corresponding nucleosides.

When Krebs-2 ascitic carcinoma is transplanted to mice, administration of interferon may produce either a stimulating or an inhibiting effect on replication of carcinoma cells in the peritoneal cavity depending on the responsiveness of mice (intact or vaccinated with Newcastle disease virus) and the state of the transplanted cells ("common" or "tolerant"). Parallelism was observed in changes of the intensity of cell multiplications and indices of their enzymatic activity under the influence of exogenic interferon.

A comparative study of different methods for concentration of porcine leukocyte interferon active in human cells was carried out. Ultrafiltration in an apparatus TCF-10 was found to be the most effective method of concentration by which preparations of porcine interferon showing antiviral activity up to 1 X 10(6) IU/ml were obtained. The total antiviral activity of concentrated interferon was twice as high as that of the native preparation. This increase of the total initial activity is due to the removal in the process of concentration of an inhibitor of antiviral activity which was isolated and concentrated. The concentrated preparation of porcine leukocyte interferon is now undergoing trials in veterinary and medicine.

In the presence of serum from newborn mice the cells of newborn mice produce almost no interferon induced by Newcastle disease virus. When cultivated in a medium containing adult mouse serum, interferon production by the cells increases markedly reaching the level of interferon production by the cells of adult animals. It is suggested that there are factors present in mouse serum which are capable, depending on the age of the animals, either of inhibiting or stimulating the activation of lysosomal apparatus and interferon production.

Comparisons of native preparation of mouse interferons, "macrophage" and "Krebs" revealed some differences, Thus, the minimal time necessary for the development of resistance to vesicular stomatitis virus (VSV) in L cells treated with "macrophage" and "Krebs" interferon was 5 and 2 hours, respectively. The activity of the lysosomal enzyme of acid phosphatase was considerably higher in the cells treated with "Krebs" interferon and infected with VSV than with "macrophage" interferon. Differences in the antiviral and antioncogenic properties of fractions No. 1 and No. 5 of these interferons obtained in fractionation of native interferon preparations by the cryomethod were demonstrated. In particular, fraction No. 5 of "Krebs" interferon, in contrast to that of "macrophage" interferon, had no antioncogenic properties but did have antiviral properties. It is suggested that these differences are due to the presence in native preparations of different substances capable of exerting an effect on interferon.

Tests of various interferon preparations (alpha, alpha2, alpha-beta, beta, and swine leukocyte) in human diploid fibroblast culture for the presence or absence of cytopathic effect of herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) demonstrated antiviral effect of all interferons with the exception of lymphoblastoid one. Preparations of alpha and swine leukocyte interferons were the most effective for inhibition of HSV-1 reproduction. Recombinant interferon was 10-fold and fibroblast interferon 100-fold less active for HSV-1 than alpha- and swine leukocyte interferons. All the interferons under study (alpha-beta) were similarly active in their antiviral effect on HSV-2 reproduction.

The role of chromosomes 2, 5, 16, and 21 in production and effect of human interferon was checked in human diploid cells, human heteroploid cells J-96 and clone J-41 thereof. The J-41 cells were found to have a lower number of chromosomes 2 as compared to the other cells under study; J-41 cells produce less interferon than the other cells. Most J-41 cells lack chromosome 21. Unlike the other two cell cultures, these cells do not produce antivirus state after treatment with interferon. The number of chromosomes 16 is larger in J-96 cells than in diploid ones, and they are less sensitive to interferon than diploid cells. The experimental results confirm the importance of chromosome 2 for coding for interferon production, other chromosomes taking part in the regulation of this process. The gene of sensitivity to interferon is localized in chromosome 21, the regulator gene coding the production of repressor of sensitivity to interferon is in chromosome 16.

Decreased resistance to Krebs-2 ascitic carcinoma was demonstrated in mice which were in the state of hyporesponsiveness (tolerance) to virus interferon inducer (NDV) and increased resistance of the control mice to transplantation of tolerant carcinoma cells. Manifestations of tolerance in peritoneal and carcinoma cells were shown to be identical: interferon production and reproduction of vesicular stomatitis virus were inhibited, the number of viable cells was reduced, masked infection with NDV was detected, and the activity of cell enzymes was similarly changed. It is assumed that cellular manifestations of tolerance are the cause of reduced resistance of tolerant mice to ascitic carcinoma.

The antiviral effect of swine leukocyte interferon was studied in cell cultures of different origins. Swine leukocyte interferon was demonstrated to protect human diploid fibroblasts. The cultures of primarily trypsinized mouse embryo fibroblasts and continuous bovine embryo tracheal cells are as sensitive to swine leukocyte interferon as human diploid cells. Studies of the age sensitivity of mouse fibroblast tissues to swine interferon showed the tissue sensitivity to interferon to decline markedly with age.

In vitro production of interferon by blood leukocytes from patients with lymphosarcoma, lymphogranulomatosis, leukemia, cancer tumours, pneumonia, as well as by leukocytes of mice with Rauscher leukemia, and mice in the condition of hyporeactivity to interferon inducer was studied. Alongside with quantitative differences in interferon production, biological differences in the properties of interferons produced of normal and sick humans and animals were revealed. The biological differences consist in that the interferon produced by leukocytes from cancer and leukemia patients interacting with homologous cell culture is conducive to more rapid formation of resistance to the indicator virus than the interferon produced by normal leukocytes. Thus, resistance of the homologous cell culture to the infection with the indicator vesicular stomatitis virus developed within 1--2 hours after contact with leukocyte interferon from patients and only within 5--6 hours after contact with that of normal subjects. This finding is not specific for cancer and leukemia, as the same was observed with specimens from patients with pneumonia and from mice hyporeactive to interferon inducer. It is suggested that patients with cancer and leukemia have a state of interferon hyporeactivity.

A simple method for increasing the activity of human leukocyte interferon is described. It is based on the removal from the native preparation of a presumed inhibitor of antiviral effect followed by condensation of the resulting fractions by thin-canal cells TCF-10 of an Amicon apparatus. With this method of concentration there was no loss of the general initial antiviral activity. Depending on the titer of the initial interferon and the condensation degree, the native interferon was concentrated 32--350-fold. The resulting concentrated interferon preparation is nontoxic, areactogenic, safe in animal trials and may be recommended for trials as a therapeutic-prophylactic means in various viral diseases.

The effect of human leukocyte interferon on reproduction of poliomyelitis virus in MIO cells resistant to this virus (MIOr) and sensitive MIO cells was studied. Interferon was shown to exert a short-time protective effect in the sensitive cells and to induce virus reproduction in the resistant cells. It is suggested that poliomyelitis virus reproduction in the resistant cells is due to activation of lysosomal enzyme, cathepsin D, in this system.

The fact of non-simultaneous thawing of protein and water was used for concentration of interferon from pre-frozen preparations. As thawing proceeded, 5 sequential fractions equal in a volume were removed from preparations of human leucocyte, mouse macrophage and tissue chick interferons; the titers of their antiveral activity in the first fraction were 4--8-fold higher than the initial titer, and this activity decreased gradually to a titer which was 2-fold lower than the initial in the fifth fraction. The total amount of interferon units in all the fractions was 1.8--3.8-fold higher than in the initial preparation taken in a volume corresponding to the total volume of all the fractions. As addition of the Vth fraction to the Ist fraction reduced the titer of interferon in it to the initial level, it is suggested that in interferon preparations there is an inhibitor of its effect, "anti-interferon".

The results of the study of the anticellular effect of alpha-interferons of various origins (human, porcine, and bovine) on the growth of human lymphoblastoid cells (Namalva and J-96). Both homologous (human) and heterologous (porcine and bovine) leukocyte interferons produced a similar anticellular effect on the cells under study. All 3 types of interferon inhibited proliferation of lymphoblastoid cells. No such effect of these interferons on the control (diploid) cells was observed.

Swine blood leukocytes have first been found capable of producing interferon the antiviral activity in human cells of which is comparable to that of native human leukocyte interferon. The influence of various factors on production of porcine leukocyte interferon active in human cells were studied. Thus, when serum was substituted with MAF in the medium used for leukocyte cultivation the titer of the produced interferon increased 4--16-fold and the content of protein impurities in the preparation decreased 10-fold or more. Marked individual interferon-producing capacity of blood leukocytes from different swine and seasonal variations in the level of this capacity were established. The optimal conditions for porcine leukocyte interferon production are: the use of leukocytes in the first few hours after bleeding of the animals; leukocyte concentration in the medium 10 million cells per 1 ml; medium 199 with 10% MAF; New-castle disease virus as interferon inducer in a dose of 10--100 CPD50 per cell. Priming of leukocytes with interferon enhanced their interferon-synthesizing capacity 2--8-fold. This new source of interferon production is readily available, and interferon is useful for public health and veterinary needs.

The activity (free and total) of cathepsin D and acid phosphatase was studied in cells of peritoneal exudate of mice of different ages in the process of interferon production in the presence of sera from newborn and adult animals. Cathepsin D release in newborn mice upon interferon induction is actively stimulated by serum factors of newborn animals altering lysosome permeability selectively for this enzyme alone. Another lysosomal enzyme, acid phosphatase, was more strongly bound to the structures and showed no such features. With an increased age of the donor the amount of stimulating factors in the serum decreases and that of inhibiting factors increases. Inhibition of cathepsin release from lysosomes by the serum factor of adult animals protects the synthesized interferon molecules from degradation and facilitates intensive interferon production in adult mice. A higher susceptibility of children to virus infections and inefficiency of the earliest defence system, interferon, may be due to degradation of newly synthesized interferon molecules by lysosomal cathepsin D.