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HB-EGF is one of the most potent ligands of EGFR and ErbB4 receptors. This growth factor plays a pivotal role in many cellular processes, but its effect differs from one cell type to another and remains not fully understood. The aim of this work was to investigate the dependence between the rate of HB-EGF mediated cell proliferation and activation of EGFR and ErbB4 receptors. Therefore, the effects of human recombinant sHB-EGF (rsHB-EGF) on the proliferation of cell lines with different EGFR and ErbB4 quantity and ratio, as well as activation of the MARK-cascade p38 and ERK1/2 (p42/44) kinases, were analyzed. For comparison, a similar study of the effect of native sHB-EGF secreted by human histiocytic lymphoma cells U937 during co-cultivation with different cell lines was performed. It was proved that cell proliferation in response to sHB-EGF depends not only on the quantity but also on the ratio of EGFR and ErbB4. It was shown that signaling through ErbB4 is associated with activation of p38 kinase and signaling through EGFR associated with activation of ERK1/2 (p42/44) kinase. We assume the existence of two different mechanisms for sHB-EGF-mediated stimulation of cell proliferation, and the simultaneous launch of these mechanisms provides a maximal proliferative response. The results of this study support the feasibility of creating anti-proliferative drugs that target ErbB4.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathophysiological functions. Also, HB-EGF plays a pivotal role in progression of different tumors. So, HB-EGF seems to be a target molecule for the treatment of some cancer types. Aim To obtain HB-EGF neutralizing polyclonal antibodies and test their anti-proliferative properties in vitro. Materials and methods Lab rabbits and mice were used for immunization with recombinant HB-EGF. The effect of generated polyclonal antibodies on viability and apoptosis of human epidermoid carcinoma derived A431 cell line was assessed using MTT and Annexin V-propidium iodide assays. Results Rabbit polyclonal anti-HB-EGF serum could block binding of soluble HB-EGF to epidermal growth factor receptor/human epidermal growth factor receptor. Also, anti-HB-EGF antibodies could bind to surface of A431 cells which express abnormally high levels of membrane bound proHB-EGF and its receptor. It has been shown that immune serum with polyclonal antibodies against HB-EGF was able to block the mitogenic activation of the cells with HB-EGF and cause apoptotic cell death. Conclusion Inhibition of HB-EGF activity with neutralizing polyclonal antibodies can effectively inhibit mitogenic activation and cause apoptosis of cancer cells with significant epidermal growth factor receptor overexpression.

The aim of this work was to characterize the experimental test-system based on MPT83-MPT63 fusion antigen with reference commercial diagnostics and investigate the basic characteristics of enzyme-linked immunosorbent assay (ELISA) tests such as specificity and sensitivity. In addition, we investigated the correlation of biochemical and immunological parameters of blood samples of patients with tuberculosis, which was correctly diagnosed by those test-systems. It was shown that the developed test-system match the existing ones regarding the criteria of reliability and the basic requirements of ELISA kits. Recommendations for the testing of serum samples and indications for the use of the proposed serological diagnostic methods has been suggested.

Several mammalian species are resistant to diphtheria toxin (DT). The DT receptor, proHB-EGF in resistant and sensitive species, has a different primary structure due to the amino acid substitutions; however, there is no definite opinion regarding how the difference in primary receptor structure alters the process of DT internalization by resistant cells compared to sensitive. The aim of the present study was to evaluate the role of DT internalization in the development of DT resistance of mammalian cells. It was shown that L929 cells resistant to toxin and derived from the C3H mouse strain absorb the recombinant fluorescent subunit B of DT with the value of binding constant Kb that was close to that of African green monkey Vero cells highly sensitive to toxin (0.269 and 0.372 μM, respectively). Endocytosis dynamics analysis by confocal microscopy indicated that the amount of subunit B internalized by Vero varied from nearly equal (at early stages of the process) to approximately 2‒5 times bigger (after 30 min) compared to L929 cells. The obtained results suggest that, at the initial stages, DT is internalized by resistant cells as rapidly as by sensitive cells. Nevertheless, at further stages of uptake, toxin amount in cells sufficiently varies depending on the receptor expression level and physiological features of the cell culture. It was concluded that the internalization and, therefore, the resistance of cells to the DT depends insufficiently on receptor structure in resistant and sensitive species but may be dependent on subsequent endosomal transport and accumulation of DT molecules in cells at late stages of internalization.

The level of proHB-EGF expression on J774, Raji, KG-1 cells derived from different types of human and mouse immune system cells under the standard in vitro culture conditions and during functional activation of these cells was investigated. Changes in the proHB-EGF expression on the cell surface were found to depend on the density of cell population, the content of fetal bovine serum in the culture medium, the effect of mitogenic factors – bacterial lipopolysaccharide, an inactive full-size form of diphtheria toxin (CRM197) and recombinant soluble HB-EGF – rsHB-EGF. The results obtained are important for the understanding of the functional role of proHB-EGF receptor on the surface of macrophage-like cells and B lymphocytes and indicate the involvement of this receptor in immune response regulation in an organism.

Subunit B of diphtheria toxin (DT) and its R-domain differ by the presence of T-domain. The aim of the present work was to analyze the interaction of these toxin fragments with mammalian cells in order to evaluate the T-domain’s influence on endocytosis in resistant cells. Internalization of recombinant fluorescent subunit B and R-domain was characterized in toxin-resistant L929 cells derived from mouse connective tissue and toxin-sensitive Vero cells from African green monkey kidney. It was found that during incubation of cells in the presence of both subunit B and R-domain in the culture medium, Vero cells internalize more molecules of subunit B than of R-domain. Under the same conditions, L929 cells internalize more molecules of R-domain than of subunit B. Colocalization of fluorescent subunit B and R-domain in L929 was rapid and proceeded almost completely at the early period of incubation compared to Vero cells in which it was slow and occurred gradually. The obtained data suggest that T-domain affects internalization and endosomal transport of DT in cells indirectly correlated with their toxin sensitivity. It was concluded that T-domain participates in intracellular endosomal transport and sorting of DT only in toxin-sensitive cells by enhancing the internalization of toxin molecules.

The aim of the research was to compare the characteristics of nanoparticles with different chemical structure and size (colloidal gold Gold 1 and Gold 2, calcium phosphate CaP and poly(lactideco-glykolid) PLGA 1 and 2) to find the most efficient carriers of antigen — recombinant diphtheria toxoid for per os immunization. According to the MTT test, all studied particles show no significant cytotoxic impact on the studied cells in vitro, with the exception of CaP nanoparticles, which in high concentrations have cytotoxic effect on the U937 cells, and Gold nanoparticles 1 and 2, that are able to inhibit growth of the L929 cells. The most effective phagocytosis by macrophage-like cells J774 is observed for PLGA nanoparticles 1 and 2 with the immobilized antigen, while Gold nanoparticles 1 and 2 with antigen can interact with the surface of these cells without being phagocytated by them. In BALB/c mice immunized per os with antigen immobilized on PLGA 1 and 2 as well as Gold 2 carriers, the concentration of specific IgA antibodies in blood significantly increases after the second immunization, compared with controls. In the group of mice treated with PLGA 2 conjugated antigen, the concentration of specific IgG in blood after the third immunization also increases. These results show the promise of nanoparticles PLGA 1 and 2 as adjuvant for immunization per os.

A century of science after I. Metchnikoff’s death demonstrated the truth in many of his views and judgments in the field of immunology, pathology, bacteriology, zoology and comparative embryology. Today Metchnikoff is deservedly called the father of the theory of cellular immunity, and also a harbinger of the theo­ry of natural immunity. His work in the field of lactic acid bacteria formed the basis for an entire industry of probiotics. Metchnikoff’s doctrine of the possibility of extending human life is still relevant. This publication is one more attempt to tie the biography of the scientist with his creative legacy and his contribution in the world treasury of biological science.

Diphtheria toxin is a major factor of the pathogenicity of the causative agent of diphtheria Corynebacterium. Due to a small size, it is of considerable interest as the basis for the development of synthetic protein molecules with a transport function, e.g., immunotoxins. In this work we describe the expression and characterized nontoxic recombinant fluorescent derivatives of the diphtheria toxin and its nontoxic CRM197 mutant. The proteins obtained can be used for studying receptor-binding and transport functions of the toxin in cells, evaluation of the expression level of the toxin proHB-EGF receptor membranes, immunization and generation of specific antibodies against the toxin, as well as for the development of diagnostic test-systems for the diphtheria toxin and antitoxic antibodies.

Diphtheria toxin (DT) is a unique bacterial protein which consists of three domains with various biological functions. Using genetic engineering for the creation of various recombinant constructions of DT with definite features, it is possible to create unique tools for cellular biology and toxins with efficient and selective action on certain populations of cells. The review highlights the structural and functional aspects of the DT molecule, its fragments and domains, as well as the major areas of application of its recombinant derivatives. In particular, the perspectives for practical use of recombinant DT derivatives are discussed for creating immunobiological preparations, cytotoxins, blockers of the heparin-binding epidermal growth factor-like growth factor (HB-EGF), protein constructions for direct delivery of substances into the cell, and also the possibility to use DT recombinant derivatives for therapy and prevention of a number of diseases.

Developing of new models and approaches, particularly with fluorescent techniques, for investigation of intracellular transport of proHB-EGF and its ligand-receptor complexes is strongly required. In order to create a model for studying proHB-EGF functions the genetic construction pEGFP-N1-proHB-EGF, encoding proHB-EGF-EGFP which is fluorescent-labeled form of proHB-EGF with enhanced green fluorescent protein EGFP in the cytoplasmic terminus of the molecule, was obtained. Eukaryotic cells expressing fusion protein proHB-EGF-EGFP on the cell surface were obtained by transfection with pEGFP-N1-proHB-EGF. Expressed in the Vero cells proHB-EGF-EGFP could bind fluorescent derivative of nontoxic receptor-binding subunit B of diphtheria toxin mCherry-SubB. After stimulation oftransfected cells with TPA (12-O-Tet-radecanoylphorbol-13-acetate), proHB-EGF-EGFP formed a fluorescentl-labeled C-terminal fragment of the molecule - CTF-EGFP. Thus, the obtained genetic construction pEGFP-N1-proHB-EGF could be helpful in visualization of molecules proHB-EGF and CTF in cells, may open new possibilities for the studying of their functions, such as receptor function of proHB-EGF for diphtheria toxin, intracellular translocation of CTF and provide possibilities for natural proHB-EGF ligands search.

Development of complications during diphtheria depends to a large extent on toxin-neutralizing antibodies level in the patient's blood. Active immunization of people with diphtheria anatoxin is widely used for diphtheria prevention and passive immunization with hyperimmune antitoxic horse serum is used for diphtheria treatment. A traditional component of anti-diphtheria vaccines--diphtheria anatoxin has a number of serious disadvantages, which are mainly associated with complicated procedure of its production. Thus, the search for new antigen substances, which can effectively stimulate protective humoral response to diphtheria toxin, is an urgent task in anti-diphtheria vaccine development. Furthermore, one of the most important objects is the development of new in vitro methods for estimation of diphtheria toxin-neutralizing polyclonal and monoclonal antibodies, which allow to avoid using active diphtheria toxin and toxin-sensitive laboratory animals. Comparative studies of toxin-neutralizing antibodies induction after immunization of laboratory animals with recombinant subunits A and B of diphtheria toxin were carried out. The new method for detection of protective antibodies in serum was proposed. This method is based on the ToBI test (Toxin Binding Inhibition test); namely on the property of anti-diphtheria antibodies to inhibit the biding of toxin subunit B fused with enhanced green fluorescent protein (EGFP) to the sensitive to diphtheria toxin Vero cells. The ability of subunit B to induce toxin-neutralizing antibodies in laboratory animals (rabbits and guinea pigs) was confirmed by the intradermal test, which is traditionally used to detect protective antitoxic antibodies in the serum, and by flow cytometry method, developed for this purpose. The results suggest that diphtheria toxin recombinant subunit B may be used for the induction of the protective immune response. The new developed approach for estimation diphtheria toxin-neutralizing antibodies is more ethical and safe and can substitute successfully the traditional methods.

The effect of nicotine on both the expression of nicotinic acetylcholine receptors (nAChRs) and proliferation of hybridoma cells and normal mouse lymphocytes has been investigated. By means of immunoenzyme assay, nicotine was shown to regulate the number of nAChRs in both hybridoma cells and normal rat splenocytes. According to the data of triazolyl blue inclusion and ELISpot assay, nicotine stimulated proliferation of both hybridoma cells and normal plasma cells generated in the course of immune response in vivo. The cell sensitivity to nicotine depended on the number of nAChRs expressed on the membrane, as well as on their functional activity affected, in particular, by adhesive contacts. The use of the open channel blocker benzohexonium revealed that proliferative signal through nAChR in hybridoma cells was mediated by ion channel opening. The data obtained demonstrate the proproliferative role of nicotine for B lymphocytes, and may account for the development of lymphoproliferative disorders in tobacco smokers.

A key step in the mode of cytotoxic action of diphtheria toxin (DT) is the transfer of its catalytic domain (Cd) from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td), but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT. The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT, which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demons­trated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.

Subunit B of diphtheria toxin (DT), which consists of two domains: R (receptor-binding) and T (transmembrane), plays an important role in toxin-receptor binding on the cell-targets and in transportation of catalytic subunit A to the cell cytosol. Recombinant analogues of the subunit B are promising representatives in the unique class of transporting proteins, able to deliver different types of biologically active molecules to cell cytosol. In the development of these protein constructs understanding of the role of each of the DT fragments in determination of transporting pathways of endocytosed complex toxin-receptor is urgently required. We have studied in this work the T-domain effect on intracellular transport of recombinant fragments of DT. We have compared intracellular transport of the R-domain and the subunit B, the last one consisted of both R-domain and T-domain. Recombinant fragments of DT used in this work were labeled with fluorescent proteins, which allowed applying colocalization technique for our study. Application of confocal microscopy technique revealed differences in transportation of recombinant derivates of DT in Vero cells: R domain moved faster than subunit B to tubular compartments. Analysis of R-domain and subunit B transportation confirmed almost linear increase of their colocalization with the time regarding to Pearsons correlation coefficient (PCC). However, amount of colocalized with R-domain subunit B were not linearly increased with time according to Manders coefficient (M1), this could indicate the ability of subunit B to transport to such compartments that R-domain do not reach. Possible role of the T-domain in intracellular transportation and compartmentalization of the toxin may be associated with the ability of the T-domain to form a proton channels and its ability to interact with COPI complex.