Nikolina Mihaylova

Inflammation-induced enhancement of IgG immunoreactivity

Abstract.Natural polyreactive IgG antibodies are found in the sera of all healthy individuals. The in vitro exposure of pooled human IgG to protein-destabilizing chemical or physical factors has been previously shown to result in the exposure of their “hidden” polyspecificity. We hypothesize that such an enhancement of their pre-existing immunoreactivity may occur in vivo in the aggressive microenvironment of inflammation sites. An increase in the antigen binding intensity as well as of the number of recognized antigens was observed in the sera of IgG-infused immunodeficient SCID mice with induced local inflammation. The expansion of the IgG pathogen-binding repertoire may have important biological consequences.

Antibodies use heme as a cofactor to extend their pathogen elimination activity and to acquire new effector functions.

Various pathological processes are accompanied by release of high amounts of free heme into the circulation. We demonstrated by kinetic, thermodynamic, and spectroscopic analyses that antibodies have an intrinsic ability to bind heme. This binding resulted in a decrease in the conformational freedom of the antibody paratopes and in a change in the nature of the noncovalent forces responsible for the antigen binding. The antibodies use the molecular imprint of the heme molecule to interact with an enlarged panel of structurally unrelated epitopes. Upon heme binding, monoclonal as well as pooled immunoglobulin G gained an ability to interact with previously unrecognized bacterial antigens and intact bacteria. IgG-heme complexes had an enhanced ability to trigger complement-mediated bacterial killing. It was also shown that heme, bound to immunoglobulins, acted as a cofactor in redox reactions. The potentiation of the antibacterial activity of IgG after contact with heme may represent a novel and inducible innate-type defense mechanism against invading pathogens.

Selective silencing of DNA-specific B lymphocytes delays lupus activity in MRL/lpr mice

The pathological DNA‐specific B lymphocytes in lupus are logical targets for a selected therapeutic intervention. We have hypothesized that it should be possible to suppress selectively the activity of these B cells in lupus mice by administering to them an artificial molecule that cross‐links their surface immunoglobulins with the inhibitory FcγIIb surface receptors. A hybrid molecule was constructed by coupling the DNA‐mimicking DWEYSVWLSN peptide to a monoclonal anti‐mouse FcγRIIb antibody. This chimeric antibody was added to cultured spleen cells from sick MRL/lpr mice, immunized with diphtheria toxoid, resulting in reduction of the numbers of anti‐DNA but not of anti‐diphtheria IgG antibody‐producing cells. Intravenous infusions with the DNA‐peptide antibody chimera to 7‐wk‐old animals prevented the appearance of IgG anti‐DNA antibodies and of albuminuria in the next 2 months. The administration of the DNA‐peptide chimeric antibody to 18 wk‐old mice with full‐blown disease resulted in the maintenance of a flat level of IgG anti‐DNA antibodies and in delay of the aggravation of the lupus glomerulonephritis. The use of chimeric antibodies targeting inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of autoreactive disease‐associated B cells in autoimmune diseases.

Modulation of the immune response using Rapana thomasiana hemocyanin

We have investigated the non-specific immunostimulatory and specific immunomodulatory effects of hemocyanin from marine gastropod Rapana thomasiana (RtH). The purified RtH, its structural subunits RtH1 and RtH2 and a construct with influenza virus hemagglutinin intersubunit peptide (IP) were used in immunization protocols of Balb/c mice. Antibody formation against RtH, RtH1, RtH2, RtH-IP as well as anti-RtH IgG antibody isotypes were determined by ELISA. The immune homology between both subunits and the whole RtH molecule was investigated by cross-blotting technique. The retaining of the B-cell epitope of IP, coupled to the RtH was recognised by Western blot. The results obtained demonstrate that the immunization with RtH or its subunits in experimental models resulted in strong immune response in vivo. Common epitope of influenza A virus hemagglutinin jointed to RtH results in generation of molecule with increased immunogenicity. Our results are the first demonstration that RtH and/or its subunits could be used in different immunization protocols as an adjuvant or as a protein-carrier.

IgM‐enriched human intravenous immunoglobulin suppresses T lymphocyte functions in vitro and delays the activation of T lymphocytes in hu‐SCID mice

Previous studies of an experimental human immunoglobulin preparation for intravenous use, containing normal pooled IgM (IVIgM), have shown its beneficial therapeutic effect in experimental autoimmune diseases. The mechanisms of its immunomodulatory activity remain however, poorly understood. In the experiments reported here, IVIgM inhibited the proliferation of various autonomously growing human lymphoid cell lines in vitro, as well as of MLR‐ and of PHA‐stimulated human T‐lymphocytes. These effects of IVIgM were observed at non‐apoptotic concentrations and were stronger on a molar basis than those of normal pooled IgG for intravenous use (IVIg). Both preparations, when administered to SCID mice, repopulated with human peripheral blood mononuclear cells, delayed the expression of the early activation marker CD69 on both human CD4+ and CD8+ T‐lymphocytes, activated by the mouse antigenic environment. The data obtained show that normal pooled human IgM exerts a powerful antiproliferative effect on T‐cells that is qualitatively similar but quantitatively superior to that of therapeutic IVIg. Our results suggest that infusions with IVIgM might have a significant beneficial immunomodulating activity in patients with selected autoimmune diseases.

Anti-cancer properties of gastropodan hemocyanins in murine model of colon carcinoma

BackgroundVarious immunotherapeutic approaches have been used for the treatment of cancer. A number of natural compounds are designed to repair, stimulate, or enhance the immune system response. Among them are the hemocyanins (Hcs) - extracellular copper proteins isolated from different arthropod and mollusc species. Hcs are oxygen transporter molecules and normally are freely dissolved in the hemolymph of these animals. Hemocyanins are very promising class of anti-cancer therapeutics due to their immunogenic properties and the absence of toxicity or side effects. KLH (Megathura crenulata hemocyanin) is the most studied molecule of this group setting a standard for natural carrier protein for small molecules and has been used in anti-tumor clinical trials.ResultsThe Hcs isolated from marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix pomatia (HpH) express strong in vivo anti-cancer and anti-proliferative effects in the developed by us murine model of colon carcinoma. The immunization with RtH and HpH prolonged the survival of treated animals, improve humoral anti-cancer response and moderate the manifestation of C-26 carcinoma symptoms as tumor growth, splenomegaly and lung metastasis appearance.ConclusionHemocyanins are used so far for therapy of superficial bladder cancer and murine melanoma models. Our findings demonstrate a potential anti-cancer effect of hemocyanins on a murine model of colon carcinoma suggesting their use for immunotherapy of different types of cancer.

Simultaneous engagement of FcγIIb and CD22 inhibitory receptors silences targeted B cells and suppresses autoimmune disease activity

All B cell targeting therapeutic approaches used at present are unspecific and there is an urgent need for agents that silence selectively pathological autoreactive B lymphocytes only. We hypothesized that this aim could be achieved by chimeric antibodies that cross-link B cell immunoglobulin receptors with inhibitory receptors on the surface of the same targeted disease-associated cell. A hybrid molecule was constructed by coupling copies of the DNA-mimicking DWEYSVWLSN peptide and of the CD22-binding STN epitope with a free terminal sialic acid to a mouse monoclonal IgG antibody backbone. The DNA mimotope peptide binds to the immunoglobulin B cell receptor of pathological DNA-specific B cells of lupus mice, the STN epitope - to CD22 and the IgG by its Fc fragment - to FcgammaIIb on the surface of the same cell. Mass-spectra analysis showed that 4 STN epitopes plus 5 DNA mimotope peptides were coupled to a single light immunoglobulin chain and 4 STN - and 2 DNA mimotopes - to a heavy chain. Both FcgammaIIb and CD22 receptors on spleen cells from lupus MRL/lpr mice were phosphorylated after exposure to the chimeric antibody, indicating the involvement of both inhibitory pathways. The constructed chimera suppressed specifically in vitro as well as in vivo anti-DNA IgM and IgG antibody production and delayed the development of glomerulonephritis in the lupus-prone animals. The use of chimeric antibodies targeting two independent inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of pathological autoreactive B cells in autoimmune diseases.

New fluorogenic dyes for analysis of cellular processes by flow cytometry and confocal microscopy.

Fluorescent microscopy and fluorescent imaging by flow cytometry are two of the fastest growing areas in the medical and biological research. Innovations in fluorescent chemistry and synthesis of new dye probes are closely related to the development of service equipment such as light sources, and detection techniques. Among compounds known as fluorescent labels, the cyanine-based dyes have become widely used since they have high excitation coefficients, narrow emission bands and high fluorescence upon binding to nucleic acids. The key methods for evaluation of apoptosis and cell cycle allow measuring DNA content by several flow cytometric techniques. We have synthesized new monomethine cyanine dyes and have characterized their applicability for staining of live and/or apoptotic cells. Imaging experiments by flow cytometry and confocal laser scanning microscopy (CLSM) have been also performed. Two of the dyes have shown high-affinity binding to the nuclei at high dilutions, up to 10(-9)M. Flow cytometry and CLSM have confirmed that these dyes labeled selectively non-living, e.g. ethanol-fixed cells that makes them appropriate for estimations of cell viability and apoptosis. The novel structures proved to be appropriate also for analysis of the cell cycle.

Marine gastropod hemocyanins as adjuvants of non-conjugated bacterial and viral proteins

Killed viral vaccines and bacterial toxoids are weakly immunogenic. Numerous compounds are under evaluation as immunological adjuvants and peptide-carriers to improve the immune response. The hemocyanins, giant extracellular copper proteins in the blood of many mollusks, are widely used as immune stimulants. In the present study we investigated the adjuvant properties of hemocyanins isolated from marine gastropods Rapana thomasiana and Megathura crenulata. An immunization with Influenza vaccine or tetanus toxoid combined with Rapana thomasiana hemocyanin (RtH) and Keyhole limpet hemocyanin (KLH) in mice induced an anti-influenza cytotoxic response lasting at least 5 months and an antibody response to viral proteins. The IgG antibody response to the tetanus toxoid (TT) combined with RtH or KLH was comparable to the response of the toxoid in complete Freunds adjuvant. The results obtained demonstrate that the both hemocyanins are acceptable as potential bio-adjuvants for subunit vaccines.

Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue.

Objectives To translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5°C for 24 h and then frozen. Materials and Methods Ovarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5°C for 24 h, then frozen after 24 h pre-cooling to 5°C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). Results For groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm3 was 19.0, 20.2, 12.9, and 12.2, respectively (P1-2, 3-4 >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P1-2, 3-4 >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P1, 3-2, 4 <0.05). Conclusions Long time (24 h) cooling of ovarian tissue to 5°C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing.