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Previously, we showed that incorporation of methotrexate (MTX) in the form of a lipophilic prodrug (MTXDG) in 100-nm lipid bilayer liposomes of egg phosphatidylcholine can allow one to reduce toxicity and improve the antitumor efficiency of MTX in a mouse model of T-cell leukemic lymphoma. However, in our hemocompatibility tests in vitro, MTX liposomes caused complement (C) activation, obviously due to binding on the liposome surface and fragmentation of the C3 complement factor. In this work, we studied the interactions of MTX liposomes carrying stabilizing molecules phosphatidylinositol (PI), ganglioside GM1, or a lipid conjugate of N-carboxymethylated oligoglycine (CMG) in the bilayer with subpopulations of human blood leukocytes. Liposomes labeled with BODIPY-phosphatidylcholine were incubated with whole blood (30 min and 1 h, 37°C), blood cells were lysed with a hypotonic buffer, and the fluorescence of the liposomes bound but not internalized by the leukocytes was quenched by crystal violet. Cell suspensions were analyzed by flow cytometry. Incorporation of MTXDG dramatically enhanced the phagocytosis of liposomes of any composition by monocytes. Neutrophils consumed much less of the liposomes. Lymphocytes did not accumulate liposomes. The introduction of PI into MTX liposomes practically did not affect the specific consumption of liposomes by monocytes, while CMG was likely to increase the consumption rate regardless of the presence of MTXDG. The GM1 ganglioside presumably shielded MTX liposomes from phagocytosis by one of the monocyte populations and increased the efficiency of monocyte uptake by another population, probably one expressing C3b-binding receptors (C3b was detected on liposomes after incubation with blood plasma). MTX liposomes were shown to have different effects on TNF-α production by activated leukocytes, depending on the structure of the stabilizing molecule.

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We demonstrated similarities and differences in the effects of IFN-α and IFN-ß compared to IFN-γ on the production of factors deposited in the Weibel-Palade bodies in cultures of endothelial cells (intact and infected with herpes simplex virus 1). IFN-α and IFN-ß reduced the content of von Willebrand factor, endothelin-1, and soluble P-selectin and increased IL-8 concentration in the culture medium of human umbilical vein endothelial cells. IFN-γ reduced the content of all studied factors in the endothelial cell culture medium. Possible mechanisms of these effects are discussed.

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BACKGROUND: To study impact of interferon (IFN) alpha, beta and gamma on the Herpes simplex virus type 1 (HSV-1) infected endothelial cells functional activity related with participation in the inflammation development. MATERIALS AND METHODS: In the work endothelial cells isolated from umbilical vein were used. Intact and infected cultures were treated by interferon and in the dynamics of cultivation tested mediators in the cultural medium. RESULTS: All investigated interferons activated the production of IL-6. IFN alpha, beta activated the production of IL-8, while IFN gamma inhibited her. IFN alpha and gamma increased synthesis of nitrogen oxides and reduced the synthesis of endothelin-1, while IFN beta activated the production of endothelin-1. CONCLUSION: Infection of endothelial cells isolated from umbilical vein with HSV-1 does not alter the ability of interferon in modulating of proinflammatory cytokines, nitric oxide and endothelin-1 synthesis. It is obvious in the body modulation manifestations of innate immunity under the influence of exogenous interferon is implemented both intact and infected with HSV-1-vascular endothelium and nature modulation is determined by the type of IFN.

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We studied the effect of type 1 herpes simplex infection on the production of innate immunity mediators in human vascular endothelial cells in culture. It was found that production of anti-inflammatory cytokines IL-1ß, IL.6, and TNF-α in infected cultures depended on the level of their spontaneous production, while IL-8 production was suppressed irrespective of its spontaneous level. Shedding of cell adhesion molecules of early (P-selectin and E-selectin) and late (PECAM-1 and VE-cadherin) phases of leukocyte recruitment depended on individual capacity of human vascular endothelial cell cultures to maintain reproduction of type 1 herpes simples virus. The production of vasodilator NO and vasoconstrictor endothelin-1 by infected cultures also depended on spontaneous synthesis of this transmitter by non-infected cultures.

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Cell adhesion molecules (CAM) expressed in vascular endothelium ensure integrity of the endothelial layer, recruitment and transmigration of leukocytes. Being receptors of many viruses, they play a role in immune control and infectious processes. Monoclonal anti-ICAM-1 antibodies enhance infection of primary human umbilical vein endothelial cell (HUVEC) cultures with HIV-1 due to incorporation into virions. IFN-gamma activates expression of ICAM-1 on HIV-infected HUVEC and thereby promotes binding of this molecule to complementary molecules on a greater number of sensitive cells, virion transfer onto them, and broad dissemination of the virus. Recombinant human IFN-alpha, IFN-beta and IFN-gamma influence (activate, inhibit) CAM shedding from HUVEC both intact and infected wit HSV-1. Activated shedding in the blood stream due to competition between soluble and endothelial CAM slows down recruitment and transmigration of leukocytes, i.e. regulates inflammation. CAM incorporated in microparticles can influence a wide spectrum of pathological processes Endothelial CAM may be a target for the delivery of pharmaceuticals for the treatment of vascular (including infectious) pathology.

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Immunoaffinity chromatography was shown to be the method achieving the most complete elimination of antigenic admixtures from leukocyte interferon preparations without the loss of the preparation activity. An affinity sorbent has been developed on the basis of covalently linked polyvinyl alcohol (PVA). The immobilization of the antigen-specific rabbit globulin in the preparation of the sorbent is achieved by reaction of protein amino groups with the activated matrix. The proposed sorbent achieved the elimination of the antigenic admixtures from interferon preparations as effectively as those prepared on the basis of sepharose 4B, the productivity of the purification process being at least 5 times higher. The proposed sorbent is stable at the limit values of rH, is not destroyed by detergents, is sterilized in the process of preparation. Owing to the strong linkage of the immobilized immunoglobulin with the PVA-carrier, immunoaffinity chromatography on this sorbent does not involve contamination of the preparations with rabbit globulin allergenic for man. The combination of a large pore structure, wetting ability, stiffness, mechanical and chemical stability allows the proposed sorbent to be recommended for use in modern large-scale biotechnological production.

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Transgenic mice carrying a hybrid gene consisting of ovine beta-lactoglobulin gene sequences and human gamma-interferon (hIFN-g) cDNA were produced. hIFN-g expression in the mammary gland of two lactating transgenic founder females was found. The concentration of active hIFN-g in the milk was estimated as being ca. 1800 IU/ml. The hIFN-g ability to express in the mammary gland was found in the progeny of transgenic founder male.

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In response to virus induction a culture of donor leukocytes alongside with interferon (IF-alpha) produced a factor of tumor necrosis (TNF). The kinetics of TNF and IF-alpha biosynthesis did not depend on the kind of IF used for priming, was rapid, with maximum production within 7-8 hours. Antibodies to IF-alpha and IF-alpha had no effect on TNF production, while antibody to TNF did not reduce IF-alpha yields. TNF in detectable titres was present in medical preparations of native IF-alpha but was absent in preparations of recombinant IF-alpha and IF-alpha as well as in an injection preparation of IF purified by chemical methods.

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Production of gamma-interferon was increased considerably by priming with homologous gamma- or alpha-interferons as well as by T-activin or vitamins of group B. The factors under study were effective both in the population of mononuclears and in the total fraction of nucleated cells of the donor blood. The maximal yields of gamma-interferon, up to 10,000 IU/ml, could be obtained by priming with gamma-interferon in a dose of 5 IU/ml or alpha-interferon in a dose of 1000 IU/ml. T-activin and vitamin B12 also enhanced production 8-10-fold. The combined use of priming, T-activin, and vitamin B12 in various combinations gave no further increase in the yield of activity.

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The highest yields of gamma-interferon activity were obtained by using a fraction of mononuclears recovered from freshly collected donor blood in ficoll-verografin density gradient without using hemolysis. Unification of mononuclears from individual donors into a common pool stimulated interferon production. Staphylococcal enterotoxins A and B, concanavalin A, and lentyl-lectin were found to be the most effective inducers. Immobilization of inducers on neutral carriers reduced their effectiveness. Upon induction with lectin the synthesis was complete within 24 hours, and with enterotoxin in 3 days. In the latter instance the synthesis dynamics was of a two-phase nature. Gamma-interferon produced the antiviral condition later (in 10 hours) than alpha-interferon.

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