OBJECTIVES:Considering growing evidence of mucosal involvement in RA induction, this study investigated circulating free secretory component (SC) in patients with either recent-onset RA or with ACPA and musculoskeletal pain.METHODS:Two prospective cohorts were studied: TIRA-2 comprising 452 recent-onset RA patients with 3 years of clinical and radiological follow-up, and TIRx patients (n = 104) with ACPA IgG and musculoskeletal pain followed for 290 weeks (median). Blood donors and three different chronic inflammatory diseases served as controls. Free SC was analysed by sandwich ELISA.RESULTS:Serum levels of free SC were significantly higher in TIRA-2 patients compared with TIRx and all control groups (P < 0.01). Among TIRx patients who subsequently developed arthritis, free SC levels were higher compared with all control groups (P < 0.05) except ankylosing spondylitis (P = 0.74). In TIRA-2, patients with ACPA had higher baseline levels of free SC compared with ACPA negative patients (P < 0.001). Free SC status at baseline did not predict radiographic joint damage or disease activity over time. In TIRx, elevated free SC at baseline trendwise associated with arthritis development during follow-up (P = 0.066) but this disappeared when adjusting for confounders (P = 0.72). Cigarette smoking was associated with higher levels of free SC in both cohorts.CONCLUSION:Serum free SC levels are increased in recent-onset RA compared with other inflammatory diseases, and associate with ACPA and smoking. Free SC is elevated before arthritis development among ACPA positive patients with musculoskeletal pain, but does not predict arthritis development. These findings support mucosal engagement in RA development.

During last years monoclonal antibodies (MAB) directed against vascular endothelium markers demonstrated their efficiency for visualization and targeted delivery of therapeutic drugs to tumors. Endoglin (CD105) which serves as a key element that determines endothelial cells quiescence or activation is one of such markers. Endoglin is highly expressed on the vascular endothelium of growing tumors. A first panel of MAB against endoglin in our country was produced at the hybridoma technology laboratory of RRC RST named after A.M. Granov. On the basis of these MAB ELISA was created allowing detection of endoglin in human plasma and other biological fluids. Several MAB had been shown to bind endoglin on the membrane of the cultured endothelial cells and to persist there for several hours. During the first 30 min after binding some of the immune complexes “endoglin-MAB” were internalized into the cytoplasm and were found included in the endosomes. In future these MAB can be used to create the reagents for the addressed delivery of isotope tags both on the membrane and into the cytoplasm of endothelial cells.

The transmembrane protein endoglin (CD105) is a component of the receptor complex for TGF-β family growth factors. It is expressed primarily in endothelial cells, mesenchymal stromal cells, and early hematopoietic precursors. The density of CD105 on the membrane of endothelial cells increases upon their proliferation. A soluble endoglin form (sEng) is produced by a cleavage of an extracellular fragment from CD105, performed by the matrix metalloproteinase MMP-14. sEng blood level serves as an indicator of angiogenesis during the progression of some tumors. The contribution of tumor cells into the sEng pool remains unknown. We investigated the expression of CD105, production of sEng, as well as the mRNA level of two splice-variants of endoglin and mmp-14 genes using a collection of 43 cultures of tumorous and normal cells of different histological origin. Over half of the tumor cell lines and normal stromal cells contained cell populations expressing the membrane-bound endoglin. Cytoplasmic endoglin with unknown function was found in cells of three tumor lines. In all cell cultures, the mRNA expression of L-endoglin splice-variant prevailed over the expression of S-endoglin. We have investigated, for the first time, the formation of sEng in the stromal and tumor cells, and revealed the endothelial cells as the main source of sEng. Normal stromal cells and the majority of CD105+ tumor cells produce only low levels of sEng. Among the tumor cell lines, the highest sEng production was detected in the MeWo melanoma cells, that are characterized by the highest activity of mmp-14 gene.

Detection of total free light chains (FLC) of immunoglobulins and their ratio (kappa/lambda quotient) are used in diagnostics and monitoring of multiple myeloma and other gammapathies, primary amyloidosis and multiple sclerosis. Previously described immunoassays with monoclonal antibodies (Mabs) against cryptic and constantly exposed epitopes of FLC failed to recognize rare variants of lambda Bence-Jones proteins and a significant proportion of lambda chains excreted with urine. Aiming to improve this approach, a novel murine Mab (IgG2b coded as 1C8) was employed, which specifically binds free lambda chains but doesn’t interact with native IgA, IgG, and IgM. The novel Mab recognized an epitope exposed at free lambda chains in peripheral blood of healthy donors and patients with multiple myeloma. It is not destroyed or masked upon renal filtration.The aim of this study was to determine basic features of improved assay system, and to estimate its potential in diagnostics of monoclonal gammapathies. The mixtures of three Bence-Jones proteins of either kappa- or lambda- types purified from the urine of multiple myeloma patients were used as calibrator samples.Improved immunometric assay is able to detect free kappa and lambda chains in serum and urine at a scale of 1 to 100 ng/ml, thus being three orders more sensitive than, e.g., detection levels of Freelite method based on polyclonal antibodies.A novel assay allows to detect free kappa and lambda chains at comparable levels in serum or urine, and to deduce kappa/lambda ratio. The proposed assay is able to detect FLC in 10,000-fold excess of whole IgG molecules. The calibrating plots for both antigens are linear on log-log scales, with very similar slopes. Detection thresholds for kappa or lambda chains proved to be 5 and 3 ng/ml, respectively. Mean concentrations of free kappa chains in sera of healthy donors were 6.7±2.1, in urine, 4.2±3.8 mcg/ml. Mean concentrations of free lambda chains were 4.7±1.96, and 1.6±1.0 mcg/ml, respectively. This method, if applied to serum and urine samples from multiple myeloma patients, revealed free light chains were similar to the paraproteins detected by means of electrophoresis/immunofixation. The values of kappa/lambda ratios corresponded to the types of gammapathies revealed.

1 Ioffe Physical-Technical Institute, Russian Academy of Sciences, Politekhnicheskaya 26, 194021, St. Petersburg 2 ITMO University, Kronverkskii 49, 197101, St. Petersburg 3 Aalto University, Tietotie 3, 02150, Espoo, Finland 4 Nitride Crystals Corp. group, Engel'sa 27, 194156, St. Petersburg 5 Nitride Crystals Inc., 181 E Industry Court, Suite B, Deer Park, NY 11729, USA 6 Russian Research Center for Radiology and Surgical Technologies, Ministry of Health of the Russian Federation, Leningradskaya 70, Pesochnyi stlmnt, 197758, St. Petersburg *) corresponding author shura.lebe@mail.ioffe.ru

Results of developing and testing graphene-based sensors capable of detecting protein molecules are presented. The biosensor operation was checked using an immunochemical system comprising fluorescein dye and monoclonal antifluorescein antibodies. The sensor detects fluorescein concentration on a level of 1–10 ng/mL and bovine serum albumin–fluorescein conjugate on a level of 1–5 ng/mL. The proposed device has good prospects for use for early diagnostics of various diseases.

Endoglin (CD105) is the marker of endothelial and mesenchymal stem cells and the component of TGF-β, 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.

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.

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.

Mesenchymal stromal or stem cells (MSC) represent a population of primitive fibroblastlike cells that are able to differentiate into cellular lineages of connective tissue (osteocytes, chondrocytes, adipocytes), skeletal muscles, and blood vessels. The review deals with contemporary views concerning descent, immunophenotype and immunogenicity of MSC. The studies over last decade revealed an ability of MSC to function as immunomodulatory populations which may suppress innate immunity factors (dendritic cells, natural killers, complement), as well as T-helpers and cytotoxic T-lymphocytes, along with activation of regulatory T lymphocytes (Treg). Special attention is given to analysis of conflicting data about influence of MSC on functions of B-lymphocytes and plasma cells in experimental settings, both in vitro and in vivo conditions. Data presently available are insufficient to explain the controversions revealed. In future investigations, it is necessary to consider attribution of B-cells to B1a, B1b, or B2 subpopulations as well as possible presence of poorly studied regulatory B-cells. It is also important also to take into account potential heterogeneity of initial MSC populations being isolated according to previously approved protocols. (Med. Immunol., 2014, vol. 16, N 2, pp 107-126)

A panel of monoclonal antibodies (Mab) recognizing at least four epitopes of Tamm-Horsfall protein (THP), or Uromodulin, the specific kidney antigen and most abundant urine protein, was developed. All epitopes retain their ability to bind Mab after protein deglycosilation. Three Mab are directed to a cluster of conformation-dependent spatially close epitopes. The interaction with these determinants was increased after reduction of THP by means of dithiothhreitol Among others the first Mab recognizes linear epitope in the stretch including Zona pellucid domain, the second one interacts with epitope located in elastase-sensitive part of molecule, the third is specific to determinant topographically isolated from all other mentioned epitopes. On the basis of developed Mab a multideterminant ELISA system for THP determination in urine was constructed. The mixture of three Mab recognizing different epitopes of THP were used for capture the antigen on the solid phase. As developing reagent the peroxidase-labelled Mab directed to most spatially isolated epitope was used. Calibrating samples were prepared on the basis of THP purified from the urine of healthy individuals and solibilised in the buffer preventing its aggregation. Mab also may be used for affinity purification and for investigation of THP structure.

Abstract. Daily IgA production in human organism comprises 3 to 5 g, thus exceeding total synthesis of other Ig classes. IgA in human body is presented by 9 structural variants. Its molecules belong to two subclasses, IgA1 and IgA2, the latter represented by two allotypes. In human serum, IgA1 monomers predominate, that are produced by the bone marrow cells. Mucosa-associated lymphoid tissues produce dimeric IgA1 and IgA2 molecules containing an accessory polypeptide J-chain. When transported across epithelial layer to the mucosal surface, an extracellular segment of polymeric IgA receptor (pIgAR) is joining the dimeric IgA1, which becomes a ‘secretory’ component being a part cesretory IgA (sIgA) molecule. The main function of sIgA is to bind bacteria and viruses at the mucosal surfaces, thus preventing pathogens to invade the internal spaces of the organism (immune exclusion). If transferred across epithelium, IgA may neutralize the viruses penetrating the cells, like as bind and deliver proteins and other antigens to the mucosal surface. The leukocyte IgA receptor (FcαRI, CD89) is expressed on the neutrophils, eosinophiles, monocytes/macrophages, as well as dendritic and Kupffer cells. The cytoplasmic domain FcαRI is devoid of an activation ITAM motif. To transduce signal, an FcαRI-associated chain of Fcγ receptor is used. Due to this mechanism, IgA binding leads to activation of phagocytosis, endocytosis, antigen presentation, synthesis of proinflammatory mediator and other immune functions. Fcα/μR receptor is a structural homologue of pIgR, and it is able to bind IgA and IgM, being, however, expressed only at the surface of mature B lymphocytes and macrophages. Interaction of IgA with asialoglycoprotein and transferrin (CD71) receptors, like as with some other molecules, that have yet undetermined role in immune defense and development of pathological events.

A sufficient increase of infectious complications rate during early posttransplant period in allogeneic bone marrow (allo-BM) recipients has been traditionally explained by the prominent drop of serum IgG levels in the patients. That was the main reason for therapeutic application of intravenous human immunoglobulins in allo-BM-recipients. In present study, multiple repeated measurements of different IgG subclasses were performed in blood serum of thirty-six patients at early terms after allogenic bone marrow transplantation period, along with analysis of individual clinical data. The study revealed several factors that show significant associations with IgG levels posttransplant. Moderate decrease in concentrations of IgG subclasses was registered after conditioning treatment carried out before the transplantation. Significantly decreased concentrations of IgG subclasses have been revealed during bleedings, diarrhea and glucocorticosteroid therapy. In general, however, no regular prolonged deficiency for total IgG subclasses was found in allogeneic bone marrow recipients during early posttransplant period.

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.

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.