[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.

[Mesenchymal stem cells influence on leukocytes allergen-specific reactions in case of atopic hypersencitivity].

Buffy coat samples containing lymphocytes, monocytes and granulocytes, were obtained from the peripheral blood of 16 donors who had clinical manifestations of atopic hypersensitivity in their medical background. After ex vivo incubation with donor-specific allergens, the percentage of B- and T-lymphocytes and natural killers (NK) remained unchanged. Buffy coat incubation with allergens induced production of IgE and IL-4 in all studied samples. In 13 out of 16 cases the reaction to contact with an allergen was also evident in the increasing of T-activated lymphocytes (CD3+, HLA-DR+) subpopulation. Co-cultivation with MSC from bone marrow, adipose tissue and umbilical cord resulted in blocking of allergen-induced IgE and IL4 secretion and HLA-DR+ T-lymphocytes subpopulation increase. There were no significant differences in the effect of MSCs, isolated from three different sources, on allergen-specific responses of leukocytes. Co-culturing of leukocytes with MSCs from all three sources led to an increase in the content of regulatory T-lymphocytes by an average of 30%. Thus, the immunomodulatory activity of MSCs in vitro results in blocking of the effector part of allergic reactions.

[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.

IgM and its receptors: structural and functional aspects.

This review combines the data obtained before the beginning of the 1990s with results published during the last two decades. The predominant form of the IgM molecule is a closed ring composed of five 7S subunits and a J chain. The new model of spatial structure of the pentamer postulates nonplanar mushroom-shaped form of the molecule with the plane formed by a radially-directed Fab regions and central protruding portion consisting of Cµ4 domains. Up to the year 2000 the only known Fc-receptor for IgM was pIgR. Interaction of IgM with pIgR results in secretory IgM formation, whose functions are poorly studied. The receptor designated as Fcα/µR is able to bind IgM and IgA. It is expressed on lymphocytes, follicular dendritic cells, and macrophages. A receptor binding IgM only named FcµR has also been described. It is expressed on T- and B-lymphocytes. The discovery of new Fc-receptors for IgM requires revision of notions that interactions between humoral reactions involving IgM and the cells of the immune system are mediated exclusively by complement receptors. In the whole organism, apart from IgM induced by immunization, natural antibodies (NA) are present and comprise in adults a considerable part of the circulating IgM. NA are polyreactive, germ-line-encoded, and emerge during embryogenesis without apparent antigenic stimuli. They demonstrate a broad spectrum of antibacterial activity and serve as first line of defense against microbial and viral infections. NA may be regarded as a transitional molecular form from invariable receptors of innate immunity to highly diverse receptors of adaptive immunity. By means of interaction with autoantigens, NA participate in maintenance of immunological tolerance and in clearance of dying cells. At the same time, NA may act as a pathogenic factor in atherosclerotic lesion formation and in development of tissue damage due to ischemia/reperfusion.

[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.

[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.

[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.

Human immunoglobulin light chains lambda form amyloid fibrils and granular aggregates in solution.

Myeloma nephropathy is a disorder characterized by deposition of monoclonal immunoglobulin light chains in the kidneys. The chains deposited form either amyloid fibrils or granular (amorphous) aggregates. Distinct molecular mechanisms leading to the formation of different aggregate types in kidney of patients with multiple myeloma are poorly understood. Here we describe the self-association kinetics of human monoclonal immunoglobulin light chains lambda (GRY) isolated from urine of a patient with multiple myeloma. Under physiological conditions, the isolated light chain exists predominantly in a form of covalent dimer with apparent molecular mass of 50.1 kD. Spectral probe binding, analytical gel filtration, Western blot analysis, and electron microscopy indicate that GRY dimer aggregation occurs via two different pathways producing either amyloid fibrils or amorphous aggregates depending on microenvironment. Incubation of GRY (25 microM) for 4-14 days at 37 degrees C in phosphate buffered saline (PBS), pH 7.0, or in PBS containing urea (0.8 M), pH 6.5, leads to amyloid fibril formation. Under electron microscopy, the fibrils show unbranched thread-like structures, approximately 60-80 x 1000 A in size, which can bind thioflavin T and Congo Red. GRY maintained in acetate buffer, pH 3.5, forms granular aggregates. The structure of GRY oligomers formed during the early stage of amyloid fibril formation (1-4 days) has been examined by means of protein cross-linking with homobifunctional reagents. These oligomers are predominantly trimers and tetramers.

Thermodynamic stability and functional activity of tumor-associated antibodies.

Tumor-associated antibodies of human IgG1 subclass were eluted from cell-surface antigens of human carcinoma cells and studied by differential scanning calorimetry and binding to local conformational probes, protein A from Staphylococcus aureus and a monoclonal antibody targeted to the CH2 domain of the Fc fragment. At pH 2.0-7.0, we observed virtually identical enthalpies of thermal unfolding for IgG1 from normal human sera and tumor-associated IgG1. The exact values of calorimetric enthalpy (Delta h) at pH 7.0 were 6.1 and 6.2-6.3 cal/g for IgG1 from normal serum and IgG1 from carcinoma cells, respectively. The affinity constants of protein A binding to the CH2--CH3 domain interface demonstrated differences between serum IgG1 and tumor associated IgG1 that did not exceed 3-8-fold. The binding affinity toward the anti-CH2 monoclonal antibody determined for serum IgG1 and IgG1 from carcinoma cells differed not more than 2.5-fold. The thermodynamic parameters of IgG1 from carcinoma cells strongly suggest that protein conformational stability was essentially unaltered and that the Fc fragment of the tumor-derived IgG1 preserved its structural integrity.

[IgM, IgG and IgG subclass antibodies to herpes simplex virus in persons of different ages]. / antitela k virusu prostogo gerpesa, otnosiashchiesia k IgM, IgG i podklassam IgG, u lits raznogo vozrasta.

IgM, IgG antibodies to herpes simplex virus and their subclases were investigated in 565 subjects of different age tested at virological laboratories of St. Petersburg in 1996-1997. The majority of these subjects had a history of herpes infection and 21.5% had IgM antibodies to herpes simplex virus (HSV), marker of acute herpetic infection. Besides IgM, IgG1 antibodies can be referred to early antibodies appearing during the acute stage of herpetic infection. The predominant subclass was HSV IgG3 antibodies. As for IgG4, they were completely absent in infants aged under 1 year, were detected in 6.2% children aged under 14 years, and were present in 12.2-12.5% adults.

[Free light-chain immunoglobulins in the biological fluids of patients with multiple sclerosis]. / Svobodnye legkie tsepi immunoglobulinov v biologicheskih zhidkostiakh bol'nykh rasseiannym sklerozom.

15 patients with clinically significant multiple sclerosis (MS) were examined in terms of content of free light chains of immunoglobulins of kappa- and lambda-types in blood serum, cerebrospinal liquor, lacrimal fluid, saliva, urine, indices were compared with corresponding data of 12 patients with other neurological diseases and of 10 healthy individuals (control group). Significantly increased content of kappa-chains in cerebrospinal, lacrimal fluids and urine was revealed in patients with MS as compared with other two groups and in saliva of patients as compared with controls. The frequency of alterations was high in all biological fluids of the patients with MS. Elevated content of kappa-chains was most often observed in cerebrospinal fluid of the patients (13 individuals). The changes observed reflected systemic activation of humoral immune response in MS. Examination of cerebrospinal fluid was most informative for differential diagnosis.

[The use of monoclonal antibodies and a biotin-streptavidin system of amplification for the quantitative determination of human IgE]. / Ispol'zovanie monoklonal'nykh antitel i biotin-streptavidinovoi sistemy usileniia dlia kolichestvennogo opredeleniia IgE cheloveka.

A "sandwich"-variant of the solid phase immunoenzyme method of quantitative determination of human IgE in biological liquids has been developed on the basis of two monoclonal antibodies to various epitopes of Fc-fragment of E-chain of human IgE and biotin-streptavidin system of the signal amplification. The total analysis time is 3 h: the range of determined concentrations of IgE is 1-200 kE/l; sensitivity-1 kE/l; the type of calibrating curve is linear. To make an analysis twice 10 microliters of the tested sample are required. Reproducibility of the suggested method is not below 5%.