Antiproliferative and apoptotic effects of anti-human HB-EGF neutralizing polyclonal antibodies in vitro.

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.

Transcriptional regulation of NOX genes express ion in human breast adenocarcinoma MCF-7 cells is modulated by adaptor protein Ruk/CIN 85.

NADPH oxidases are key components of redox-dependent signaling networks involved in the control of cancer cell proliferation, survival and invasion. The data have been accumulated that demonstrate specific expression patterns and levels of NADPH oxidase homologues (NOXs) and accessory genes in human cancer cell lines and primary tumors as well as modulation of these parameters by extracellular cues. Our previous studies revealed that ROS production by human colorectal adenocarcinoma HT-29 cells is positively correlated with adaptor protein Ruk/CIN85 expression while increased levels of Ruk/CIN85 in weakly invasive human breast adenocarcinoma MC F-7 cells contribute to their malignant phenotype through the constitutive activation of Src/Akt pathway. In this study, to investigate whether overexpression of Ruk/CIN85 in MC F-7 cells can influence transcriptional regulation of NOXs genes, the subclones of MCF-7 cells with different levels of Ruk/CIN85 were screened for NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 as well as for regulatory subunit p22Phox mRNA contents by quantitative RT-PCR (qPCR). Systemic multidirectional changes in mRNA levels for NOX1, NOX2, NOX5, DUOX2 and p22Phox were revealed in Ruk/CIN85 overexpressing cells in comparison to control WT cells. Knocking down of Ruk/CIN85 using technology of RNA-interference resulted in the reversion of these changes. Further studies are necessary to elucidate, by which molecular mechanisms Ruk/CIN85 could affect transcriptional regulation of NOXs genes.

Kinetic parameters of polyreactive immunoglobulins interaction with antigens in the presence of protamine.

The discovered earlier phenomenon of the enhancment of polyreactive immunoglobulines (PRIGs) binding to antigens in the presence of protamine and Tween 20 was investigated in more details. The comparative analysis of PRIGs reaction dynamics with immobilized antigen was provided. In addition, the rate constants for the reaction and the affinity constants of PRIGs-antigen binding in the presence or absence of optimal protamine concentration were determined. The rate constant of PRIGs-antigen reaction did not increase in the presence of protamine optimal concentration and was even reduced approximately twice. However, in the presence of protamine the concentration of reactive PRIGs molecules, that were able to interact with antigen, increased approximately 30 times, and this led to strong reaction rate increase. Protamine also influenced the affinity constant of PRIGs-antigen binding, which increased approximately three times. The suggestion was made that such protamine effect was due to its influence on the PRIGs molecules special structure, and, as a result of the conformational change PRIGs became able to bind more effectively to the antigens.

Mycobacterium tuberculosis antigens MPT63 and MPT83 increase phagocytic activity of murine peritoneal macrophages.

Macrophages (MΦ) are the most described and characterized target and host of mycobacteria. Like other cells of innate immunity MΦ have a wide range of receptor molecules which interact with different pathogen associated molecular patterns (PAMPs). Immunodominant proteins MPT63 and MPT83 that are synthesized in abundance by Mycobacterium bovis or Mycobacterium tuberculosis strains could be involved in development of tuberculosis infection. The aim of this study was to search for effects of these mycobacterial antigens on target cells. For this aim full-sized sequences of MPT83 (rMPT83full) and MPT63 antigens were cloned into plasmid pET24a(+). The increase of phagocytic activity of murine peritoneal macrophages was demonstrated, but not of macrophage-like cells from J774 cell line, which were treated by rMPT63 and rMPT83full proteins for 24 h. This effect of such antigens can be considered as a way to facilitate the consumption of mycobacterial cells by macrophages to avoid other effector mechanisms of innate and adaptive immunity.

EFFECT OF DIPHTHERIA TOXIN T-DOMAIN ON ENDOSOMAL pH.

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.

[BIOLOGICAL AND IMMUNOCHEMICAL PROPERTIES OF POLYREACTIVE IMMUNOGLOBULINS].

A previously unknown phenomenon of acquired polyreactivity for serum immunoglobulins, which were subjected either to solutions of KSCN (3.0-5.0 M), low/high pH (pH 2.2-3.0), or heating to 58-60 degrees C, was described by us in 1990 year. Much later, eleven years after that, similar data were published by others, which completely confirmed our results concerning the influence of either chaotropic ions or the drastic shift of pH on immunoglobulins polyreactive properties. Our further investigations of polyreactive serum immunoglobulins (PRIG) properties have shown that the mechanism of non-specific interaction between PRIG and antigens much differs from the mechanism of interaction between specific antibodies and corresponding antigens. Later we have shown that the increasing of PRIG reactivity could be induced in vivo, and PRIG are one of serum components for human or animal sera. Then, it could be suggested that PRIG can perform certain biological functions. Studying of PRIG's effect on the phagocytosis of microbes by peritoneal cells or the tumor growth have shown that PRIG can play a certain role in protecting the body from infections and probably can influence on the development of various pathological processes. Recently we have also found that PRIG IgG contents significantly increases in aged people. These data demonstrate that further investigations of PRIG's immunochemical properties and studying of their biological role in organism protection from various diseases is very intriguing and important.

RECOMBINANT SINGLE CHAIN VARIABLE FRAGMENT ANTIBODIES (scFv) AGAINST Pro144-Leu155 FRAGMENT OF HUMAN PROTEIN C.

The aim of this work was to obtain the recombinant single chain variable fragments of antibodies (scFv) against human protein C, the key component of blood anticoagulation system. For this purpose a peptide that mimics a Pro144-Leu155 sequence of protein C was synthesized and the murine immune scFv library against this peptide was constructed. The protein C specific scFv 9E were selected from the constructed library by the phage-display method. The scFv 9E dissociation constant was found to be 2∙10(-9) M. It was shown that scFv 9E were suitable for protein C detection by ELISA and Western blotting. Selected scFv could be further used for protein C investigation and for the development of quantitative methods for protein C detection in human blood.

FUNDAMENTAL DIFFERENCES BETWEEN NATURAL ANTIBODIES AND POLYREACTIVE IMMUNOGLOBULINS.

A problem of similarity and differences between so-called polyreactive immunoglobulins (PRIGs) and natural antibodies (NAbs), capable of cross-reacting with some structurally dissimilar antigens, has been considered. The analysis of mechanisms of an unspecific interaction between PRIGs or NAbs and antigens evidences for the fact that essential differences exist between these substances. These differences permit classifying the abovementioned substances as different types of immunoglobulin molecules. The major difference between PRIGs and NAbs may include both the mechanisms of the above mentioned immunoglobulin molecules binding to antigens and their interaction affinity, as well as an absolutely different influence of some low-molecular substances on the efficiency of the interaction with antigens. Relying on the obtained data it can be assumed that, since PRIGs and NAbs have fundamental differences, they may perform not only similar but also different functions of the immune system.

[Cell model for the study of receptor and regulatory functions of human proHB-EGF].

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.

Inhibition of fibrin polymerization by synthetic peptides corresponding to Aα195-205 and γ69-77 sites of fibrin molecule.

Using the idea of "proline brackets" we have found four sites in fibrin amino acid sequence, and appropriate peptides were synthesized: γ69NPDESSKPN77, Bß228QPDSSVKPY236, Bß455RPFFPQ460 and Aα195LPSRDRQHLPL205. Turbidity and electron-microscopy analyses have demonstrated that synthetic peptide Aα195-205 specifically inhibited the stage of fibrin protofibril formation and peptide γ69-77 - the stage offibrin protofibril lateral association. The data obtained testify that there are the sites involved in these processes in the appropriate amino acid sequences of fibrin molecule.

Effect of the T-domain on intracellular transport of diphtheria toxin.

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.

[Interaction peculiarities of polyreactive immunoglobulins and various antigens].

The influence of twin 20, lysozyme and protamine on the capability of polyreactive immunoglobulins (PRIG) to attach to various antigens was investigated. Twin 20 can inhibit the binding of PRIG to antigens on immunological plates but lysozyme and protamine can enhance it. As far as the mixture of the optimal concentrations of lysozyme and protamine cannot increase PRIG-antigen interaction in comparison to the optimal dose of protamine, we have concluded that the mechanism of their effect on PRIG binding is similar. Of special interest is the fact that twin 20 at optimal concentration of lysozyme or protamine does not decrease PRIG binding to various antigens but, on the contrary, increases PRIG-antigen interaction.

Age changes of human serum polyreactive immunoglobulins (PRIG) activity.

It has been determined that activity of serum polyreactive immunoglobulins (PRIG) changes with age in practically healthy people at the age from twenty five to seventy. Therewith, the activity of serum IgG PRIG increases most of all (about 3-4 times), while IgM PRIG activity, on the contrary, does not increase, but sometimes even decreases with age. IgA PRIG activity varies significantly more than IgG PRIG activity and, besides, IgA PRIG significantly less depends on age than IgG PRIG. The age changes in the activity of human serum PRIG, belonging to different types of immunoglobulins, may evidence for the important functional role of these immunoglobulins that has to be clarified.

[Recombinant fluorescent models for studying of diphtheria toxin].

Diphtheria toxin is the main pathogenicity factor of causative agent of diphtheria Corynebacterium diphtheriae. Due to the small molecule size, it is of considerable interestfor the development of synthetic protein molecules with transporting function, e.g. immunotoxins. Expression and characterization of nontoxic recombinant fluorescent derivates of diphtheria toxin and its nontoxic mutant CRM 197 were described in this article. Obtained proteins may be applied in studies of receptor-binding and transporting functions of the toxin in cells, for determination of toxin receptor proHB-EGF expression level, immunization and antibody generation against the toxin and in development of diagnostic test-systems, detection of diphtheria toxin and antitoxic antibodies.

[Simultaneous quantification of soluble fibrin and D-dimer in blood plasma for the assessment of the threat of thrombosis].

Soluble fibrin and D-dimer are the most specific markers of blood coagulation cascade and the threat of thrombosis. Two immunoassay test systems were designed using the fibrin-specific and D-dimer-specific monoclonal antibodies. The clinical trials of the test systems were carried out in Ukraine. The high informative value of soluble fibrin quantification as a prognostic indicator of the threat of thrombosis associated with hip replacement and endoprosthetics of the abdominal aorta was shown. Independent D-dimer quantification is less informative. Simultaneous quantification of soluble fibrin and D-dimer before operation and at different time intervals after it is required for the prediction of postoperative thrombotic complications and monitoring the efficiency of antithrombotic therapy. Only in this case it is possible to get information about the state of the balance between blood coagulation and fibrinolytic systems, and determine the degree of the threat of thrombosis.