Lipophilic Prodrug of Methotrexate in the Membrane of Liposomes Promotes Their Uptake by Human Blood Phagocytes.

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.

[Expression patterns of germ line specific genes in mouse and human pluripotent stem cells are associated with regulation of ground and primed state of pluripotency].

One of the main criteria of pluripotency is ability of cell lines to differentiate into the germ line. Pluripotent stem cell lines in ground state of pluripotency differ from the lines in primed state by their ability to give rise to the mature gametes. To understand molecular mechanisms involved in regulation of different states of pluripotency we investigated the expression patterns of germ line specific genes in different type pluripotent stem cells and mouse and human embryonic teratocarcinoma cells. We found that pluripotent stem cells in vitro, in blastocyst and gonocytes at stage E13.5 had similar expression patterns in contrast to the epiblast cells at stage E6.5. Quantitative real time PCR analysis showed that Vasa/Ddx4 expression in mouse and human embryonic stem cells was significantly lower than in blastocyst and gonocytes. Moreover, Vasa/Ddx4 and E_ras expression was significantly higher in mouse embryonic stem cells than in human embryonic stem cells. Our analysis of germ line specific gene expression in differentiating mouse embryonic stem and embryonic germ as well as in mouse embryonic teratocarcinoma cells maintained under conditions promoting cell reprogramming from primed to ground state of pluripotency (2i + LIF) revealed that only pluripotent stem cells are able to regulate the expression level of Oct4 and Vasa/Ddx4 and restore initial ground state, while in embryonic teratocarcinoma cells the expression level of these genes remained unchanged. We suggest that expression patterns of germ lines specific genes, in particular of Vasa/Ddx4, can underlie the regulation of ground and primed states of pluripotency. [corrected].

[The influence of carbohydrate ligands on the cytotoxicity of liposomes bearing a methotrexate-diglyceride conjugate in human acute leukemia cell cultures].

The efficiency of the chemotherapeutic agent methotrexate (MTX) in tumor cells is limited by the frequent development of the drug resistance of tumor cells. We had previously shown in vitro using human acute leukemia cells with various sensitivity to MTX (T-lymphoblastic CCRF-CEM line and resistant CEM/MTX subline) that MTX incorporation into liposomes as a lipophilic prodrug, diglyceride conjugate (MTX-DG), allows for the overcoming of cell resistance due to the impaired active transmembrane transport. In this work, we have studied the profile of binding with carbohydrates of the cell lines mentioned using carbohydrate fluorescent probes (poly(acryl amide) conjugates). Lipophilic conjugates of tetrasaccharide SiaLe(x), 6'-HSO3LacNAc, and also inactive pentaol for incorporation into liposomes, have been synthesized. The cytotoxicity of MTX-DG liposomes equipped with the SiaLe(x) ligand toward the sensitive CCRF-CEM cell culture was demonstrated to be 3.5 times higher than that of MTX-DG liposomes bearing the control inactive pentaol. The activity of MTX liposomes bearing 6'-HSO3LacNAc toward resistant CEM/MTX was 1.6-fold increased. The use of carbohydrate ligands as molecular addresses for drug-carrying liposomes as a potential method of treating heterogeneous tumor tissue is discussed.

Individual characterization of stably expanded T cell clones in ankylosing spondylitis patients.

Ankylosing spondylitis (AS) is commonly characterized by clonal expansions of T cells. However, these clonal populations are poorly studied and their role in disease initiation and progression remains unclear. Here, we performed mass sequencing of TCR V beta libraries to search for the expanded T cell clones for two AS patients. A number of clones comprising more than 5% of the corresponding TCR V beta family were identified in both patients. For the first time, expanded clones were shown to be stably abundant in blood samples of AS patients for the prolonged period (1.5 and 2.5 years for two patients, correspondingly). These clones were individually characterized in respect to their differentiation status using fluorescent cell sorting with CD27, CD28, and CD45RA markers followed by quantitative identification of each clone within corresponding fraction using real time PCR analysis. Stable clones differed in phenotype and several were shown to belong to the proinflammatory CD27 - /CD28 - population. Their potentially cytotoxic status was confirmed by staining with perforin-specific antibodies. Search for the TCR V beta CRD3 sequences homologous to the identified clones revealed close matches with the previously reported T cell clones from AS and reactive arthritis patients, thus supporting their role in the disease and proposing consensus TCR V beta CDR3 motifs for AS. Interestingly, these motifs were also found to have homology with earlier reported virus-specific CDR3 variants, indicating that viral infections could play role in development of AS.

Immunocorrecting properties of GB-115 dipeptide.

We studied the effects of a new dipeptide with anxiolytic activity (GB-115, N-phenylhexanoyl-glycyl-L-tryptophan amide) on parameters of the immune in intact mice and in animals with secondary immunodeficiency caused by cyclophosphamide. GB-115 in doses of 0.1-10 mg/kg stimulated phagocytic activity of peritoneal macrophages and humoral immune response in intact mice. GB-115 exhibited immunocorrecting activity in animals with secondary immunodeficiency.

[Fluorescently labeled differentiating myelopeptide-4: specific binding to and penetration into target cells].

Myelopeptide-4 (MP-4) (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro), inducing the terminal differentiation of HL-60 leukemia cells, was labeled with fluorescein isothiocyanate. The specific binding of this modified peptide to the surface of HL-60 cells and its ability to penetrate into the cells were studied. It was shown by cytometry and confocal microscopy to be bound on the HL-60 cell surface, to penetrate into their cytoplasm, and finally to concentrate around the cell nucleus. These phenomena are probably necessary for the exhibition of MP-4 differentiating activity.

Calcium homeostasis change in CD4+ T lymphocytes from human peripheral blood during differentiation in vivo.

Resting naïve CD4+(CD45R0-)CD45RA+ T cells are sensitive to ionomycin. In contrast, resting CD4+(CD45RA-)CD45R0+ memory T cells show resistance to this Ca2+ ionophore. In the present study, the ability of activated T lymphocytes to respond to ionomycin during the transition from naïve precursors into memory T cells has been analyzed. Activated CD4+(CD45RA+)CD45R0+ T cells are always present both in human peripheral blood (HPB) and in the ionomycin-resistant (IR) fraction. Therefore, some activated T cells are resistant toward the Ca2+ ionophore. CD69 molecules are markers of the very early stage of T cell activation. However, CD4+CD69+ T cells have never been found in the IR fraction. Thus, the majority of CD4+ T lymphocytes at the early stage of activation are ionomycin-sensitive cells. The proportion of CD4+CD25+ T cells did not differ significantly in HPB and in the IR fraction. The presence of CD4+CD25+ T lymphocytes in the IR fraction reflects changes in the Ca2+-signaling pathway at this differentiation step of activated cells. Depending on the expression level of CD25 molecules, the population of CD4+CD25+ cells is divided in T-regulatory (CD25high) and proliferating (CD25low) subpopulations. The action of ionomycin results in a decrease in the portion of the CD4+CD25low T-cells, but it leads to an increase in the proportion of the CD4+CD25high T lymphocytes. Consequently, greater portion of CD4+CD25high T lymphocytes and smaller portion of CD4+CD25low T cells are IR cells. Expression of HLA-DR molecules can be used as the marker for the late activation step. The IR fraction is significantly rich in CD4+HLA-DR+ T lymphocytes in comparison to the blood of the same donor. The link between different differentiation steps of CD4+ T-lymphocytes and alterations in calcium ion homeostasis is discussed.

Induction of differentiation in leukemic cell strains with myelopeptide-4.

We studied the capacity of myelopeptide-4 (regulatory peptide of the bone marrow origin) to induce terminal differentiation of HL-60 and K-562 leukemic cells. Myelopeptide-4 increased the expression of CD14 and CD38 differentiation antigens on the surface of HL-60 cells and of CD44 antigen on K-562 cells, induced the appearance of mature monocyte/macrophages in HL-60 culture and hemoglobin-producing cells in K-562 cell culture, and stimulated phagocytic activity of THP-1 leukemic cells. Myelopeptide-4 is an endogenous factor of cell differentiation, a prospective agent for antileukemic therapy.

[Modification of the target cell surface with lipophilic glycoconjugates and interaction of the modified cells with natural killer cells]. / Modifikatsiia poverkhnosti kletok s pomoshch'iu lipofil'nykh glikokon''iugatov i vzaimodeistvie modifitsirovannykh kletok s natural'nymi killerami.

An experimental model system involving the modification of carbohydrate composition of the target cell surface with neoglycolipids was developed for studying the role of surface carbohydrates of target cells in the NK-cell-mediated cytotoxicity. The polymeric glycoconjugates of the Glyc-PAA-PEA and Glyc-PAA(Flu)-PEA types (where Glyc was an oligosaccharide residue, PAA poly(acrylamide) polymer, and PEA the phosphatidylethanolamine residue, and Flu fluorescein residue) capable of incorporation into the cell membrane were synthesized. The optimum structures of neoglycoconjugates and conditions for their incorporation into K562 and Raji cell lines, which differ in their sensitivity to the NK-cell-mediated lysis were selected. The mechanism of association of glycoconjugates with the plasma cell membrane and the kinetics of their elimination from the cell surface were investigated using the fluorescent-labeled Glyc-PAA(Flu)-PEA derivatives. The spatial accessibility of the carbohydrate ligands for the interaction with human NK cells was demonstrated. The target cells modified with the Le(x) trisaccharide were shown to be more sensitive to the cytotoxic effect of human NK cells than the intact cells. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Expression of type IV collagen by Lewis pulmonary carcinoma cells.

Study of the expression of type IV collagen by Lewis pulmonary carcinoma cells (PLMG-T, PLMG-M1, PLMG-M2) showed that these cells produce type IV collagen. The expression of type IV collagen in PLMG-T and PLMG-M2 cells approximately 2-fold surpassed that in PLMG-M1 cells. The number of active cells (synthesizing type IV collagen) in PLMG-M1 culture was 1.6 and 2.2 times lower than in PLMG-M2 and PLMG-T cultures, respectively. It was found that cell culture medium modulated expression of type IV collagen and this effect was mediated via cell receptors.

[Phenotype characteristic of the ionomycin-resistant CD4+ subset of the peripheral blood T-lymphocytes]. / Ionomitsin-rezistentnaia subpopuliatsiia CD4+ T-limfotsitov perifericheskoi krovi cheloveka. Fenotipicheskaia kharakteristika.

The differential sensitivity of peripheral blood (PB) CD4+ T lymphocytes to the calcium ionophore ionomycin was investigated. Effect of ionomycin exerted on T cells was time- and dose-dependent. We have shown that resistant cells belonged to some distinct T cell subsets. The resting naive CD4+CD45RA+ T cells showed a little, if any, resistance to ionomycin treatment. The primed CD4+CD45R0+ memory T cells behaved similarly as did ionomycin-resistant (IR) cells. Although IR CD4+ T cells had a typical "memory" phenotype, some quantitative differences were found in expression of CD11a, CD28, CD29, CD62L and CD243 markers between PB CD4+CD45R0+ T cells and corresponding IR cells.

Phenotypic characteristics of lewis lung carcinoma cells.

We studied adhesive properties and physiological activity in vivo of cells from Lewis lung carcinoma and its metastases. These cells differed in tumorogenic activity and metastatic potential in the syngeneic system. In vivo non-metastasizing cells are characterized by a lower content of surface lectins to tetrasaccharides SiaLex [Neu5Aca2-3Galb1-4(Fuca1-3) GlcNAcb] and SiaLea [Neu5Aca2-3Galb1-3(Fuca1-4)GlcNAcb] and trisaccharide HSO3Lex [HSO32-3Galb1-4(Fuca1-3)GlcNAcb] compared to cells forming metastases in the syngeneic system. Metastatic cells with low tumorogenic activity weakly expressed lectins to disaccharide ligands 6-SiaLac [Neu5Aca2-6Galb1-4Glc], 6-HSO3LacNAc, and A-di [GalNAca 1-3Galb] and trisaccharides H-type 1 [Fuca1-2Galb1-3GlcNAc and Lex [Fuca1-3(Galb 1-4)GlcNAc] compared to cells that initiated tumor formation in the syngeneic system (similarly to transplanted tumors). We hypothesized that cell receptors to these carbohydrate determinates are involved in the development and growth of primary tumors, while lectins to SiaLex, SiaLea, and HSO3Lex play a role in the progress of tumor process and metastasizing.

Anti-DNA autoantibodies reveal toxicity to tumor cell lines.

Cytotoxicity of anti-DNA autoantibodies from sera of SLE and CLL patients was assayed on permanent cell lines L929, HL-60, Raji, and K562. L929 cells appeared to be the most sensitive to antibody treatment. DNA-hydrolyzing properties of the same autoantibody preparations were analyzed in parallel. The data obtained outlined the correlation between cytotoxicity and DNA-hydrolyzing properties of these autoantibodies. It was shown that treatment of the cells with cytotoxic anti-DNA autoantibodies induced internucleosomal DNA fragmentation and Annexin V binding to the cell surface characteristic of apoptotic pathway of cell death. A time-dependent profile of antibody-mediated toxicity to L929 cells suggested recruitment of at least two distinct mechanisms of cell death. The first peak of cell death observed in 3 h of incubation was completely inhibited by preincubation of cells with caspase inhibitor YVAD-CHO, while the second increase in cell mortality (18-30 h) persisted. Possible mechanisms for anti-DNA autoantibody cytotoxicity are discussed.

Intranasal BCG vaccination protects BALB/c mice against virulent Mycobacterium bovis and accelerates production of IFN-gamma in their lungs.

Local immune reactivity in the lungs of BALB/c mice was studied following (i) intranasal (i.n.) vaccination with Mycobacterium bovis BCG, (ii) intravenous (i.v.) challenge with a virulent M. bovis field isolate and (iii) i.n. vaccination with M. bovis BCG followed by i.v. challenge with an M. bovis field isolate. The results demonstrated that i.n. vaccination with BCG induced a high degree of protection against systemic M. bovis challenge, and that this protection correlated with a rapid production of IFN-gamma after M. bovis challenge by lung T cells from vaccinated mice.

Immunological memory in CBA and CBA/N mice after vaccination with Mycobacterium bovis (BCG).

In inbred CBA and CBA/N mice immunological memory was induced by subcutaneous injection of Mycobacterium bovis (BCG). Experiments with adoptive transfer of spleen T cells and ionomycin-resistant T cells (memory cells) between CBA and CBA/N mice in various combinations showed that immunological memory was not formed in CBA/N mice, but can be induced by adoptive transfer of cells from CBA mice.

[Endogenous Tumor necrosis factor alpha unmasks the binding sites for N-acetylglucosaminyl-(beta1-4)-N-acetylmuramyl-alanyl-D-isoglutamine on the surface of melanoma cells]. / Endogennyi faktor nekroza opukholei demaskiruet na poverkhnosti kletok melanomy tsentry sviazyvaniia N-atsetilgliukozaminil-(beta1-4)-N-atsetilmuramoil-alanil-D-izoglutamina.

The surface of the melanoma BRO cells was shown to contain binding sites for N-acetylglucosaminyl-(beta 1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Their number (1500 +/- 200 per cell) and affinity (Kd = 10 +/- +/- 1.2 nM) were determined. The occurrence of these sites was found to correlate with the ability of the melanoma cells to react in vitro with GMDP by increasing the expression of melanoma-associated antigens (MAA). An increased number of the GMDP binding sites (5200 +/- 500 per cell) was observed upon treating the melanoma BRO cells with tumor necrosis factor alpha (TNF-alpha). The mechanism of the TNF-alpha action most likely involves the unmasking of GMDP binding sites, initially expressed on the cell surface, by activating the endogenous protease that hydrolyzes surface proteins, in particular, highly glycosylated LAMP-2 protein exposed on the melanoma cell surface.

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor.

It is shown that neokyotorphin (the alpha-globin fragment 137-141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.

Cellular localization of the galactose-binding lectin from human serum.

An SL2 lectin was isolated from human serum and characterized previously; cellular localization of the lectin was studied using polyclonal rabbit antibodies. According to cytofluorimetry, anti-SL2 antibodies bound only to lymphocytes and monocytes but not to other blood cells. Antibodies bound to Jurkat T cell lymphoma but did not interact with IM-9 cells of B cell origin. Moreover, the Jurkat cells bound oligosaccharides having the highest affinity to SL2 (GalNAcalpha_and Fucalpha1-2Gal), and this interaction was inhibited by anti-SL2 antibodies. Lysis of the Jurkat cells with subsequent electrophoresis and Western blotting indicates that anti-SL2 antibodies recognized a 14-kD protein.

Comparative analysis of T lymphocytes recovered from the lungs of mice genetically susceptible, resistant, and hyperresistant to Mycobacterium tuberculosis-triggered disease.

Genetic control of susceptibility to tuberculosis (TB) is being intensively studied, and immune responses to mycobacteria are considerably well characterized. However, it remains largely unknown which parameters of response distinguish resistant and susceptible TB phenotypes. Mice of I/St and A/Sn inbred strains and (A/Sn x I/St)F(1) hybrids were previously categorized as, respectively, susceptible, resistant, and hyperresistant to Mycobacterium tuberculosis-triggered disease. In the present work we compared parameters of lung T cell activation and response following M. tuberculosis challenge. In all mice, the disease progression was accompanied by a marked accumulation in the lungs of activated CD4(+) (CD44(high)/CD45RB(low)) and CD8(+) (CD44(high)/CD45RB(+)) T cells capable of secreting IFN-gamma and of activating macrophages for NO production and mycobacterial growth inhibition. However, significantly more CD8(+) T cells were accumulated in the lungs of resistant A/Sn and F(1) compared with I/St mice. About 80% A/Sn and F(1) CD8(+) cells expressed CD44(high)/CD45RB(+) phenotype, while about 40% I/St CD8(+) cells did not express CD45RB marker at week 5 of infection. In contrast, in susceptible I/St mice lung CD4(+) cells proliferated much more strongly in response to mycobacterial sonicate, and a higher proportion of these cells expressed CD95 and underwent apoptosis compared with A/Sn cells. Unseparated lung cells and T cells of I/St origin produced more IL-5 and IL-10, respectively, whereas their A/Sn and F1 counterparts produced more IFN-gamma following infection. F(1) cells overall expressed an intermediate phenotype between the two parental strains. Such a more balanced type of immune reactivity could be linked to a better TB defense.