CHARACTERISTICS OF A172 AND T98G CELL LINES.

During continuous cultivation cell lines can lose a number of innate characteristics or acquire new ones. In this work we compared growth and phenotypic characteristics of human glioblastoma À172 and Ò98G lines from cell culture collection of Research Institute of Influenza of Ministry of Health of Russian Federation (St. Petersburg). The activity of genes encoding intracellular proteins that determine cell lines belonging to mesenchymal type, as well as several growth factor genes and extracellular matrix genes were estimated. Cell lines A172 and T98G varied in morphology and surface markers expression. A172 cells were characterized by higher expression of mesenchymal markers CD90, CD105, fibroblast activation protein, and tenascin C. Both cell lines showed high level of a2 smooth muscle actin expression. The obtained data indicating high activity of genes encoding major inductors of angiogenesis (VEGF, FGF2 (b), TGFb1) and thrombospondin-1 in foregoing cell lines are in agreement with published data. Reduction of fetal serum in culture medium from 10 to 5 % in both cell lines resulted in the increase of proportion of cells with surface antigens CD73 and CD105. Both A172 and T98G cell lines sustain the main features of glioblastomas and therefore can serve as research objects in investigation of this kind of neoplasms.

[PRODUCTION AND CHARACTERIZATION OF THE PANEL OF MONOCLONAL ANTIBODIES AGAINST HUMAN ENDOGLIN].

Endoglin (CD105) is the marker of endothelial and mesenchymal stem cells and the component of TGF-ß, BMP-9 and BMP-10-binding receptor complexes. Its expression is significantly increased on blood vessels endothelium of ischemic tissues and growing tumors. Measurement of concentration of the soluble endoglin in the serum or urine is used as a method for diagnosing cancer and pregnancy disorders. The aim of this work was to create a novel family of monoclonal antibodies recognizing endoglin on the cell surface and in biological fluids. Murine myeloma cells' derived recombinant protein representing the whole extracellular part of endoglin was used as an antigen. F1(SJL/JxBALB/c) mice were the donors of immune splenocytes. Hybridoma screening procedures were performed using E. coli-produced copies of the antigen, endoglin-expressing immortalized human cell lines, and primary cultures of human mesenchymal stromal cells. Ten novel monoclonal antibodies recognizing at least eight distinct epitopes were produced. Eight antibodies bind membrane form of endoglin on the surface of normal and transformed human cells derived from different tissue sources. Two antibodies recognize linear antigenic determinants of the molecule and can be used to detect endoglin by western blot. Sandwich ELISA system was designed in order to measure soluble endoglin in cell culture medium.

[Endoglin (CD105)--a target for visualization and anti-angiogenic therapy for malignant tumors].

Drugs currently used for anti-angiogenic therapy which are based on monoclonal antibodies to VEGF and its receptors are of limited efficiency. Endoglin (CD105) is a protein receptor of TGF-beta superfamily involved in ligand binding and signal transduction regulating VEGF-independent mechanisms of angiogenesis. CD105 is highly expressed on membranes of endothelial cells of vessels in growing tumors. It plays a crucial role in determination the state of activation or quiescence of endotheliocytes. CD105 is present also on membranes of tumor stromal cells (macrophages, fibroblasts, pericytes). High density of CD105-positive microvessels in tumors corresponds with its aggressivness, spreading to regional lymph nodes and poor prognosis. In patients with progressing tumors soluble form of endoglin in peripheral blood may be detected. Monoclonal antibodies to CD105 and their derivatives are regarded as a basis for creation of new generation of anti-angiogenic reagents for visualization of tumor vessels, for direct effect on endothelium or for targeted drugs delivery to growing tumors.

[Intracellular immunoglobulins in Namalwa and U266 cells co-cultivated with mesenchymal stromal cells].

There are contradictory data concerning the influence of mesenchymal stromal cells (MSC) on immunoglobulin (Ig) production. Most of them were obtained using MSC from bone marrow. Properties of MSC from other tissues are elusive. In the present work MSC cultures were derived from umbilical cord, adipose tissue, and bone marrow of healthy donors, as well as from bone marrow of patients with autoimmune diseases. MSC from all these sources had similar surface markers phenotype. The influence of co-cultivation with MSC at exponential or stationary phase on IgM and IgE content in Namalva and U266 cells was evaluated. MSC from bone marrow of healthy donors had no effect on IgM and IgE production. Proliferating MSC obtained from patients with Crohn's disease and multiple sclerosis stimulated Ig production. Exponentially growing MSC derived from umbilical cord and adipose tissue also stimulated Ig synthesis. MSC at stationary cultures amplified IgM production in Namalva cells and suppressed IgE synthesis in U266. Thus, MSC with similar phenotype but derived from different sources differ in their capacity to modulate Ig production in B-lymphoid cells. The effect of MSC depends on their growth stage and may differ for lymphoblastoid and myeloma cells.

Characteristics of mesenchymal stromal cells isolated from patients with breast cancer.

Mesenchymal stromal cells were isolated from the adipose tissue obtained during surgery for breast cancer and cultured under conditions of normal or low oxygen concentrations. In patients that had received a course of radiation and polychemotherapy prior to surgery, the proliferative potential of mesenchymal stromal cells was irreversibly disturbed. In patients receiving no therapy prior to surgery, the morphological, growth, phenotypic, and differentiation characteristics of mesenchymal stromal cells did not differ from the corresponding parameters of mesenchymal stromal cells from healthy donors. Culturing under hypoxic conditions increased adipogenic differentiation potencies of mesenchymal stromal cells from donors and patients.

[Influence of mesenchymal stromal cells on B-cell line growth and immunoglobulin synthesis].

A number of publications contain contradictory data about influence of mesenchymal stromal cells (MSC) on B-lymphocyte growth, differentiation and production of immunoglobulins (Ig). The aim of the study was to investigate the influence of MSC derived from adipose tissue of healthy donors and cancer patients on the proliferation and Ig synthesis of lymphoblastoid cell line Namalva and myeloma cell line U266. Co-cultivation of Namalva cells with MSC stimulated their proliferation, decreased the doubling time and the minimal effective seeding dose and therefore made cloning of these lymphoblastoid cells possible. The presence of MSC supported the survival and proliferation of Namalva cells cultivated in growth factor deficient medium. MSC also stimulated proliferation of U266 myeloma cells. Both MSC derived from adipose tissue from the healthy donors and from patients with breast cancer effectively stimulated B-cell lines proliferation. Presence of MSC in mixed cultures had no influence on the production of IgM or IgE by Namalva or U266 cells respectively. Co-cultivation of Namalva or U266 with MSC resulted in the formation of close intercellular contacts between cells of both types.

[Development and characterization of Helicobacter pylori CagA recombinant fragments].

AIM: The goal of the work was to produce, purify and characterize recombinant fragments of Helicobacter pylori CagA protein. MATERIALS AND METHODS: The methods of molecular cloning, recombinant protein expression in Escherichia coli, affinity chromatography, gel electrophoresis and western-blotting as well as several in silico algorithms of nucleotide and aminoacid sequence analysis were used. RESULTS: Four N-terminal His6-tagged recombinant fragments of CagA protein were produced. Protein rCagAfr.1 (65 kDa) represents the most conserved N-terminal part of the cytotoxin. Fragment rCagAfr.2 (44 kDa) corresponds to the central conserved region of CagA whereas rCagAfr.3 (39 kDa) represents the highly variable C-terminal part of CagA. Finally, the protein rCagAfr.4 (75 kDa) incorporates the sequences of rCagAfr.2 and rCagAfr.3. In silico analysis of fragments' sequences allowed to suppose that rCagAfr.2 and rCagAfr.4 are highly immunogenic proteins. By means of chromatographic purification, high levels of purity (up to 97%) and yield (about 15 mg per L of culture) of recombinant proteins were achieved. CONCLUSION: Use of recombinant proteins technology allowed to solve the problem of producing the CagA pure protein of H. pylori, which open new perspectives for the development of immunodiagnostic assays for detection of CagA protein or antibodies to this cytotoxin.

[Monoclonal antibodies against human secretory component: epitope specificity and utility for immunoanalysis].

AIM: To develop and characterize by immunochemical methods the panel of monoclonal antibodies (MAbs) recognizing different antigenic determinants ofhuman secretory component (SC) molecule. MATERIALS AND METHODS: sIgA and SC were obtained from colostrum by combination of ion-exchange chromatography and gel filtration. Recombinant SC was expressed in Escherichia coli cells transformed by construction that contained fragment of gene coding extracellular domain of receptor for polymeric Ig. MAbs were produced and studied using hybridoma technologies and different methods of immunoenzyme analysis respectively. RESULTS: Panel comprising 10 MAbs against human SC of which 4 types of antibodies recognize cryptic epitopes of free SC and other 6 types recognize epitopes exposed both on SC and sIgA. MAbs panel contains antibodies interacting with conformational and linear epitopes of antigen. Three from obtained MAbs bind to SC epitopes which structure is determined by presence of carbohydrate residues in the molecule of antigen. Immunometric systems were developed which allow to differentially detect free SC and sIgA. CONCLUSION: Developed and characterized MAbs panel recognizing different epitopes of SC molecule opens new opportunities for laboratory and basic research of human secretory immunity.

[Development of panel of monoclonal antibodies for detection of CagA cytotoxin of Helicobacter pylori].

AIM: To obtain and study of immunochemical characteristics of monoclonal antibodies (MAbs) to CagA cytotoxin of Helicobacter pylori employing recombinant fragments of CagA protein. MATERIALS AND METHODS: Standard methods of construction and selection of hybridomas, different variants of immunoenzyme analysis and immunoblotting were used. Molecular genotyping of H. pylori cultures by amplification of cagA gene fragments was performed. RESULTS: Panel of MAbs recognizing 4 different linear epitopes on the CagA molecule, three of which are localized in conservative parts of cytotoxin and one--in variable region of CagA, was developed. On the basis of two obtained antibodies, system of two-center immunoenzyme assay for quantitative detection of CagA protein which is characterized by high sensitivity and specificity, was developed. Obtained MAbs allow to differentiate CagA-positive and CagA-negative strains of H. pylori by immunochemical methods. CONCLUSION: Employing pure recombinant fragments of CagA protein, first panel of MAbs to CagA cytotoxin of H. pylori was developed and characterized. Obtained MAbs open perspectives for study of the H. pylori cytotoxin molecule and construction of immunodiagnostic assays aimed on detection of CagA antigen.

[The antigen of the differentiated basal disc cells of Hydra].

A new antigenic marker of the differentiated basal disk cells of Hydra was characterized. An antigen named 3G11 was revealed by monoclonal antibody in granules of the basal disk gland cells of the ectoderm. The antigen appearance during budding, regeneration and ectopic foot formation evidences for the differentiation of the body column epithelial cells into basal disk gland cells. Antigen 3G11 is species-specific: among six hydra species investigated, the antigen was observed exclusively in polyps of vulgaris group which is a special taxon of the filum Hydra. Cell and tissue localization of the antigen 3G11 was similar to that of the well-established biochemical hydra marker, foot specific peroxidase, reported formerly. However, ELISA data suggest that the molecule bearing antigen 3G11 does not possess any peroxidase activity. Thus the new hydra antigenic marker 3G11 extends the number of previously used markers of differentiation and allows to improve the technique of the basal disk differentiated tissue identification.

[Problem of J-chain of immunoglobulins].

Molecules of secretory immunoglobulins (Ig) of classes A and M (sIgA and sIgM) play the main role in protection of mucosae from pathogenic factors. The apparatus of synthesis of these molecules represents the most powerful part of the immune system. One of the key elements of the sIgA and sIgM is J-chain. It represents an acid polypeptide of molecular mass of about 15 kDa composed of 137 amino acid residues including 8 cysteine residues and one site of N-glycosylation. The primary structure of the J-chain is unique: attempts to ascribe it to any family of known proteins so far have failed. The J-chain is inserted into the sIgA and sIgM molecules to form disulfide bonds with C-terminal sites of alpha- or mu-chains. It is necessary for formation of IgA dimers and IgM pentamers, for reception of these molecules by epithelial cells, binding of secretory component to them, and for transfer of sIgA and slgM molecules onto mucosal surfaces and into secrets of endocrine glands. The J-chain has been revealed in the cytoplasm of the early T- and B-lymphocyte precursors not producing Ig. The J-chain is detected in the human embryonic liver cells earlier than the expression of the mu-chain gene begins. Study of mice with knockout of J-chain B-lymphocytes-producents has shown their block of function of T-helpers providing formation of immunologic memory. Comparison of J-chain genes of mammals, amphibians, reptiles, and cartilaginous fishes has shown the degree of interspecies homology of these proteins to vary from 33% to 70%. The J-chain genes were revealed in representatives of all vertebrate classes except for cyclostomes and bony fishes. In 1996, data were published about the presence of the J-chain genes-homologs in invertebrates, tunicates, and cyclostomes. No papers reproducing or confirming these data have been published. On the contrary, in the literature an opinion appeared that indicate necessity to revise the notion about the presence of J-chain in invertebrates. The main unsolved issues on the J-chain involve the tertiary structure of this protein, its relation to some particular protein family, its functions in cells of the T- and B-lymphocytic differentiation lineages as well as its evolutionary age.

[Cultivation of human plasmacytoma cells]. / Kul'tivirovanie kletok plazmotsitom cheloveka.

Current knowledge of primary culturing and establishment of continuous cell lines from human plasmacytomas are reviewed. The cells derived from chronic monoclonal tumors are capable of proliferating in culture no more than for 3 weeks. The enrichment of the media with nutrient supplements and growth factors does not improve the cell survival. Most of the described stable cell lines were derived from malignancies of the late stages of tumor progression. Cell lines producing complete molecules of immunoglobulins were established with the aid of sophisticated culturing methods. The microenvironment and growth factors in culture system favoured the survival of transformed plasma cells which retained a high level of differentiation.

[Functional morphology of nucleolus organizer regions of chromosomes and nucleoli in human multiple myeloma cell lines. I. Variation of the morphology and silver staining of nucleolus organizer regions of chromosomes in RMPI 8226 and U 266 cell lines with different level of differentiation of during 7 days after cell passage]. / Funktsional'naia morfologiia iadryshkoobrazyiushchikh raionov khromosom i iadryshek v kletkakh linii mnozhestvennoi mielomy cheloveka.I.Izmenenie morfologii i kharactera serebreniia iadryshkoobr azuiushchikh raionov khromosom kletochnykh linii RPMI 8226 i U 266, razlichaiushchikhsia po stepeni differentsirovki, na protiazhenii 7 sut posle pereseva kletok.

The morphology and Ag-staining of nucleoli in human multiple myeloma cell lines RPMI 8226 and U 266, distinguished from each other in the differentiation degree, were quantitatively studied, and the production of immunoglobulins or their fragments by the line cells was evaluated throughout 7 days after cell seeding. The less differentiated cell line RPMI 8226 and the high differentiated cell line U 266 were revealed to differ in both the initial level of immunoglobulin production and dynamics of immunoglobulin accumulation in culture medium. The total number of Ag-stained nucleolar-organizer regions (AgNORs) per nucleus in cells RPMI 8226 was significantly higher than in cells U 266 in all times after seeding of the cells. In both cell lines changes in the quantity and shape of nucleoli and also in the total number of AgNORs per nucleus and pattern of AgNORs distribution within nucleoli correlated with the cell cycle phase. Relationships between morphofunctional changes in nucleoli and the differentiation degree and proliferative activity of the cells, and also between the number of Ag-positive nucleolar-organizing metaphase chromosomes and the functional activity of interphase AgNORs are discussed.

[Functional morphology of nuclear organizer regions of chromosomes and of nucleoli in cells of human multiple myeloma cell lines. II.Relationship between immunoglobulin E production and argentophilia of nuclear organizer regions of chromosomes in U 266 cell line]. / Funktsional'naia morfologiia iadryshkoobrazuiushchikh raionov khromosom i iadryshek v kletkakh linii mnozhestvennoi mielomy cheloveka.II. Vzaimootnoshenie produktsii immunoglobulina E i argentof il'nosti iadryshkoobrazuiushchikh raionov khromosom v kletkakh linii U 266.

The IgE content in both cytoplasm and culture medium of U 266 myeloma cells, was studied by the enzyme-linked immunoassay in correlation with Ag-staining of NORs of chromosomes in their nuclei throughout 9 days after cell seeding. Proliferative activity of the cells was evaluated with 3H-thymidine labeling. The average values of both the cytoplasmic IgE content and its secretion level in U 266 cell population, being in logarithmic growth phase, were higher in S-phase cells as compared with G1-cells. On comparison of results of the present and previous (Turilova et al., 1998) studies it was revealed that U 266 myeloma cell line had a high stability of cell proliferation kinetics and IgE secretion and the dynamics of Ag-NORs-staining, while the number of argentophilic grains in the cell nuclei in the present experiment was higher to correlate with an enhanced IgE production. It is suggested that Ag-NORs-staining reflects the level of specific functional activity of cells in the U 266 line.

[Defining the optimal conditions for immunoperoxidase cytochemistry at the submicroscopic level]. / Opredelenie optimal'nykh uslovii immunoperoksidaznoi tsitokhimii na submikroskopicheskom urovne.

The authors tested a number of experimental protocols and chemicals known to facilitate permeabilization of tissues to immunoperoxidase markers without ultrastructural alterations of the cells to be examined. Monoclonal antibodies producing hybridoma lymphomas served as a primary test object. None of the procedures employed (i.e., quenching of the fixative aldehydes by some reducing agents; cryopermeabilization; treatment by detergents) were shown either to intensify stainability or to increase the penetration of immunoreagents into the tissue depth. The diffusion efficiency depended only on the marker molecular mass and the thickness of the vibratome section incubated. The Elder and coworkers (1983) two-step technique has been found superior in the preservation of both immunoreactivity and fine structure of the cell.

[Characteristics of monoclonal antibodies to RS virus in immunoenzyme and immunofluorescence techniques]. / Kharakteristika monoklonal'nykh antitel k RS-virusu v immunofermentnykh i immunofliuorestsentnykh reaktsiiakh.

According to the competitive ELISA test data, new preparations of monoclonal antibodies (MAb) to respiratory syncytial virus (RSV) differed in their blocking activity and they were directed to 3 different virus epitopes. MAb 9C5 and MAb 131-2A (CDC, Atlanta) competed against each other strongly and they were directed to epitope F1a of RSV F-protein. MAbs 8C5 and 10D8 showed a two-way blocking and were presumably topologically linked. MAb 8B10 did not compete against other MAbs. The fact that MAbs 8B10, 9C5, and 10D8 may be used in indirect ELISA test to detect RSV antigens was shown. The specific activity of MAbs 9C5 was observed, by applying low antibody concentrations to ng/mg. The highest specific activity using the homologous pair MAbs-con 9C5 and 8C5 was observed in direct ELISA employing MAbs for capture and peroxidase conjugate of MAbs for detection. MAbs 8B10 were successfully used for direct (FITC-conjugate of MAbs) and indirect immunofluorescence detection of RSV antigens in the infected cell cultures and clinical materials from patients.

[Development of new approaches to standartization of enzyme immunoassay test systems for detection of antibodies to respiratory syncytial virus using electron microscopy and monoclonal antibodies]. / Razrabotka novykh podkhodov k standartizatsii immunofermentnykh test-sistem dlia detektsii antitel k respiratorno-sintsitial'nomu virusu s ispol'zovaniem élektronnoi mikroskopii i monoklonal'nykh antitel.

Respiratory syncytial virus (RSV), strain Long, was purified through 20-60% sucrose gradient. The virions from different sucrose density zones were tested by ELISA for reactivity with monoclonal antibodies (MAB) to F- (MAB 9C5) and N- (MAB 8B10) proteins of RSV. Comparative study of the same patterns of RSV by electron microscopy after negative staining showed a close relationship between the virion morphology and MAB binding in ELISA. MAB 9C5 were highly reactive with the surface domains of both mature RSV virions and "empty" virion envelopes without formed inner nucleocapsid structures. MAB 8B10 reacted well only with mature virions with completely assembled nucleocapsids. These MAB failed to reorganize the N-protein epitope of immature and destroyed virions, which indicated a conformation dependence of the 8B10 binding site. For practical purposes, MAB tests can be used to determine the RSV patterns, which can be used in ELISA for serologic diagnosis of RSV infection. Testing with these MAB demonstrate the stability of RSV to extreme exposures (lyophilization, storage, heating), which is important for creation of sensitive ELISA test systems and their standardization.