Institute of Immunological Engineering

Lyubuchany, Russia

Institute of Immunological Engineering

Lyubuchany, Russia

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Kozyr A.V.,State Research Center for Applied Microbiology and Biotechnology | Kolesnikov A.V.,Institute of Immunological Engineering | Khlyntseva A.E.,State Research Center for Applied Microbiology and Biotechnology | Bogun A.G.,State Research Center for Applied Microbiology and Biotechnology | And 3 more authors.
Autoimmune Diseases | Year: 2012

Anti-DNA autoantibodies are responsible for tissue injury in lupus. A subset of DNA-specific antibodies capable of DNA cleavage can be even more harmful after entering the living cells by destroying nuclear DNA. Origins of anti-DNA autoantibodies are not fully understood, and the mechanism of induction of DNA-cleaving activity remains speculative. The autoantibody BV04-01 derived from lupus-prone mouse is the only DNA-hydrolyzing immunoglobulin with known 3D structure. Identification and analysis of antibodies homologous to BV04-01 may help to understand molecular bases and origins of DNA-cleaving activity of autoantibodies. BLAST search identified murine anti-DNA autoantibody MRL-4 with sequences of variable region genes highly homologous to those of autoantibody BV04-01. Despite significant homology to BV04-01, not only MRL-4 had no DNA-cleaving activity, but also reversion of its unusual P23 mutation to the germline alanine resulted in a dramatic loss of affinity to DNA. Contrary to this effect, transfer of the P23 mutation to the BV04-01 has resulted in a significant drop in DNA binding and almost complete loss of catalytic activity. In the present paper we analyzed the properties of two homologous autoantibodies and mutants thereof and discussed the implications of unusual somatic mutations for the development of autoantibodies with DNA-binding and DNA-hydrolyzing activity. © 2012 A. V. Kozyr et al.


Ryazantsev S.,University of California at Los Angeles | Tischenko V.,Russian Academy of Sciences | Nguyen C.,University of California at Los Angeles | Abramov V.,Institute of Immunological Engineering | Zav'yalov V.,University of Turku
PLoS ONE | Year: 2013

Human immunoglobulin G, subclass 2 (hIgG2), plays an important role in immunity to bacterial pathogens and in numerous pathological conditions. However, there is a lack of information regarding the three-dimensional (3D) structure of the hIgG2 molecule. We used electron microscopy (EM), differential scanning microcalorimetry (DSC) and fluorescence for structural analysis of the hIgG2. DSC and fluorescence indicated two types of interaction between CH1 domain of Fab (antigen-binding fragment/subunit) and CH2 domain of Fc (complement fixation fragment/subunit) simultaneously present in the sample: close interaction, which increases the thermostability of both, CH1 and CH2 domains, and weak (or no) interaction, which is typical for most IgGs but not hIgG2. Thermodynamics could not determine if both types of interactions are present within a single molecule. To address this question, EM was used. We employed a single-particle reconstruction and negative staining approach to reveal the three-dimensional structure of the hIgG2. A three-dimensional model of hIgG2 was created at 1.78 nm resolution. The hIgG2 is asymmetrical: one Fab subunit is in close proximity to the upper portion of the Fc subunit (CH2 domain) and the other Fab is distant from Fc. The plane of Fab subunits is nearly perpendicular to Fc. EM structure of the hIgG2 is in good agreement with thermodynamic data: a Fab distant from Fc should exhibit a lower melting temperature while a Fab interacting with Fc should exhibit a higher melting temperature. Both types of Fab subunits exist within one molecule resembling an A/B hIgG2 isoform introduced earlier on physicochemical level by Dillon et al. (2008). In such an arrangement, the access to the upper portion of Fc subunit is partially blocked by a Fab subunit. That might explain for instance why hIgG2 mildly activates complement and binds poorly to Fc receptors. Understanding of the three-dimensional structure of the hIgG2 should lead to better design of antibody-based therapeutics. © 2013 Ryazantsev et al.


Abramov V.,Institute of Immunological Engineering | Khlebnikov V.,Institute of Immunological Engineering | Kosarev I.,Institute of Immunological Engineering | Bairamova G.,Research Center for Obstetrics | And 10 more authors.
Probiotics and Antimicrobial Proteins | Year: 2014

Lactobacillus crispatus 2029 isolated upon investigation of vaginal lactobacilli of healthy women of reproductive age was selected as a probiotic candidate. The aim of the present study was elucidation of the role of L. crispatus 2029 in resistance of the female reproductive tract to genitourinary pathogens using cervicovaginal epithelial model. Lactobacilluscrispatus 2029 has surface layers (S-layers), which completely surround cells as the outermost component of their envelope. S-layers are responsible for the adhesion of lactobacilli on the surface of cervicovaginal epithelial cells. Study of interactions between L. crispatus 2029 and a type IV collagen, a major molecular component of epithelial cell extracellular matrix, showed that 125I-labeled type IV collagen binds to lactobacilli with high affinity (Kd = (8.0 ± 0.7) × 10−10 M). Lactobacilluscrispatus 2029 consistently colonized epithelial cells. There were no toxicity, epithelial damage and apoptosis after 24 h of colonization. Electronic microscope images demonstrated intimate association between L. crispatus 2029 and epithelial cells. Upon binding to epithelial cells, lactobacilli were recognized by toll-like 2/6 receptors. Lactobacilluscrispatus induced NF-κB activation in epithelial cells and did not induce expression of innate immunity mediators IL-8, IL-1β, IL-1α and TNF-α. Lactobacilluscrispatus 2029 inhibited IL-8 production in epithelial cells induced by MALP-2 and increased production of anti-inflammatory cytokine IL-6, maintaining the homeostasis of female reproductive tract. Lactobacilluscrispatus 2029 produced H2O2 and provided wide spectrum of antagonistic activity increasing colonization resistance to urinary tract infections by bacterial vaginosis and vulvovaginal candidiasis associated agents. © 2014, Springer Science+Business Media New York.


Zubkova E.,Institute of Experimental Cardiology | Semenkova L.,Institute of Immunological Engineering | Dudich E.,Institute of Immunological Engineering | Dudich I.,Institute of Immunological Engineering | And 2 more authors.
Molecular and Cellular Biochemistry | Year: 2013

Alpha-fetoprotein (AFP) for long was known as immunomodulator and tumor marker having multifaceted actions on the activity of normal and transformed cells. In present study, we have investigated the involvement of AFP in regulation of THP-1 cell line invasion and underlying mechanisms. Treatment with human recombinant AFP causes up-regulation of MMP9 expression, chemotaxis and calcium mobilization, and increases invasion through Matrigel, with no significant impact on THP-1 cell growth or viability. Using small molecule inhibitors, we have shown that the rhAFP-induced MMP9 expression depends on the activation of ERK1,2, JNK and Akt kinases, with the involvement of NFκB and likely, AP-1 transcription factors. In contrast, inhibition of p38 kinase, but not of JNK, had dramatic suppressive effect on the rhAFP-triggered chemotaxis. In addition, rhAFP-induced MMP9 expression and calcium response were completely blocked by pertussis toxin, indicating that Gi-protein-coupled receptor(s) has a mediatory role in these processes. CCR5 chemokine receptor is the only known Gi-protein binding to AFP. The action of CCR5 inhibitor Maraviroc results in partial suppression of MMP9 up-regulation and calcium response suggesting that CCR5 might be involved in these effects. © 2013 Springer Science+Business Media New York.


Ushakova N.A.,RAS Severtsov Institute of Ecology | Abramov V.M.,Institute of Immunological Engineering | Khlebnikov V.S.,Institute of Immunological Engineering | Semenov A.M.,Moscow State University | And 8 more authors.
Probiotics and Antimicrobial Proteins | Year: 2012

The biofilm formation took place in 48 h within the solid substrate cultivation of Lactobacillus plantarum 8-RA-3 strain on the wheat bran saturated with the MRS medium. The drying of the bran fermented by lactobacilli resulted in a decrease in the number of colony-forming units (CFU) from 23. 0 × 10 8 to 6. 9 × 10 5 CFU/g in daily samples and to less than 10 4 CFU/g in 2- and 3-day samples. However, according to the fluorescence-based live/dead assay data, more than 40 % of the non-cultured bacteria were viable. As a result of mice kept on a diet with the introduction of bran fermented by Lact. plantarum 8-RA-3 for 72 h into the fodder, a recovery of normal level of intestinal lactobacilli, inhibited by administration of antibiotic was noted. The strain genetically identical to the Lact. plantarum 8-RA-3 was isolated from the feces of these mice. The results indicate that solid substrate cultivated Lact. plantarum 8-RA-3 strain formed a biofilm. Once dried and transferred into a non-cultured state, biofilm cells retained its viability and biological activity. © 2012 Springer Science + Business Media, LLC.


Ushakova N.A.,RAS Severtsov Institute of Ecology | Abramov V.M.,Institute of Immunological Engineering | Khlebnikov V.S.,Institute of Immunological Engineering | Semenov A.M.,Moscow State University | And 8 more authors.
Biology Bulletin | Year: 2012

This paper shows that in the solid-state cultivation of Lactobacillus plantarum 8R-A3 on wheat bran, a biofilm was formed on the surface of the carrier within 48 h. Prolongation of fermentation caused a drop in the number of CFU from 96% of the initial total number of cells to 8.8% by 72 h. When the temperature was raised from 37 to 45°C, which led to drying of the fermentation mass, the CFU index decreased to <104. According to fluorescence microscopy data, up to 40% of bacteria with signs of life survived in the dry specimens. After keeping the mice on a diet with the introduction of 0.05% of fermented bran dried for 72 hours, a strain genetically identical to the L. plantarum 8R-A3 was extracted from the colon. In animals with amikacin-inhibited intestinal lactobacilli, their normal level recovered. It is suggested that L. plantarum 8R-A3 generates uncultivable forms, which are reanimated by passage through the animal organism and exhibit probiotic activity. The biofilm formed in the solid-state cultivation contributes to the survival of lactobacilli cells in drying of the fermentation mass. © 2012 Pleiades Publishing, Ltd.


Sergienko O.V.,Russian Academy of Agricultural Sciences | Aksenova E.I.,Gamaleya Institute of Epidemiology and Microbiology | Lyashchuk A.M.,Gamaleya Institute of Epidemiology and Microbiology | Galushkina Z.M.,Gamaleya Institute of Epidemiology and Microbiology | And 13 more authors.
Molecular Genetics, Microbiology and Virology | Year: 2012

The genes of ESAT-6, CFP-10 and Ag85A secreted antigens of Mycobacterium tuberculosis were cloned in Escherichia coli. Fusion proteins Esat6-CBD, Ag85A-CBD and CFP10-CBD with thermostable cellulose binding domain (CBD) of Anaerocellum thermophilum providing high affinity to cellulose were constructed. Content of the fused proteins in overproducing strains was 20% of total protein for CFP10-CBD and 15% for both Esat6-CBD and Ag85A-CBD. Immobilization of the fusion proteins on cellulose is a one-stage process that provides simultaneous antigen purification and absorption on cellulose. The proteins retain their antigenic properties and can be used as the components for subunit tuberculosis vaccine production or in diagnostic kits. © 2012 Allerton Press, Inc.


Arshad N.M.,University of Malaya | In L.L.A.,University of Kuala Lumpur | Soh T.L.,University of Kuala Lumpur | Azmi M.N.,University of Malaya | And 5 more authors.
Oncotarget | Year: 2015

Purpose: Previous in vitro and in vivo studies have reported that 1'-S-1'-acetoxychavicol acetate (ACA) isolated from rhizomes of the Malaysian ethnomedicinal plant Alpinia conchigera Griff(Zingiberaceae) induces apoptosis-mediated cell death in tumour cells via dysregulation of the NF-κB pathway. However there were some clinical development drawbacks such as poor in vivo solubility, depreciation of biological activity upon exposure to an aqueous environment and non-specific targeting of tumour cells. In the present study, all the problems above were addressed using the novel drug complex formulation involving recombinant human alpha fetoprotein (rhAFP) and ACA. Experimental Design: To study the synergistic effect of both agents on human cancer xenografts, athymic nude (Nu/Nu) mice were used and treated with various combination regimes intraperitoneally. Serum levels of tumour markers for carcinoembryonic antigen (CEA) and prostate specific antigen (PSA) were assessed using sandwich ELISA. IHC and Western blotting were also conducted on in vivo tumour biopsies to investigate the involvement of NF-κB regulated genes and inflammatory biomarkers. Quantification and correlation between drug efficacies and AFP-receptors were done using IF-IC and Pearson's correlation analysis. Results: Mice exposed to combined treatments displayed higher reductions in tumour volume compared to stand alone agents, consistent with in vitro cytotoxicity assays. Milder signs of systemic toxicity, such as loss in body weight and inflammation of vital organs were also demonstrated compared to stand alone treatments. Tumour marker levels were consistent within all rhAFP/ACA treatment groups where levels of CEA and PSA were initially elevated upon commencement of treatment, and consecutively reduced corresponding to a decrease in tumour bulk volume. Both IHC and Western blotting results indicated that the combined action of rhAFP/ACA was not only able to down-regulate NF-κB activation, but also reduce the expression of NF-κB regulated genes and inflammatory biomarkers. The efficacy of rhAFP/ACA complex was also found to be weakly negatively correlated to the level of surface AFP-receptors between tumour types. Conclusions: This drug complex formulation shows great therapeutic potential against AFP-receptor positive tumours, and serves as a basis to overcome insoluble and nonspecific anti-neoplastic molecules.


PubMed | Institute of Immunological Engineering
Type: Journal Article | Journal: Probiotics and antimicrobial proteins | Year: 2014

Lactobacillus crispatus 2029 isolated upon investigation of vaginal lactobacilli of healthy women of reproductive age was selected as a probiotic candidate. The aim of the present study was elucidation of the role of L. crispatus 2029 in resistance of the female reproductive tract to genitourinary pathogens using cervicovaginal epithelial model. Lactobacillus crispatus 2029 has surface layers (S-layers), which completely surround cells as the outermost component of their envelope. S-layers are responsible for the adhesion of lactobacilli on the surface of cervicovaginal epithelial cells. Study of interactions between L. crispatus 2029 and a type IV collagen, a major molecular component of epithelial cell extracellular matrix, showed that 125I-labeled type IV collagen binds to lactobacilli with high affinity (Kd = (8.0 0.7) 10(-10) M). Lactobacillus crispatus 2029 consistently colonized epithelial cells. There were no toxicity, epithelial damage and apoptosis after 24 h of colonization. Electronic microscope images demonstrated intimate association between L. crispatus 2029 and epithelial cells. Upon binding to epithelial cells, lactobacilli were recognized by toll-like 2/6 receptors. Lactobacillus crispatus induced NF-B activation in epithelial cells and did not induce expression of innate immunity mediators IL-8, IL-1, IL-1 and TNF-. Lactobacillus crispatus 2029 inhibited IL-8 production in epithelial cells induced by MALP-2 and increased production of anti-inflammatory cytokine IL-6, maintaining the homeostasis of female reproductive tract. Lactobacillus crispatus 2029 produced H2O2 and provided wide spectrum of antagonistic activity increasing colonization resistance to urinary tract infections by bacterial vaginosis and vulvovaginal candidiasis associated agents.


Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved in these two types of biological functional activities attributed to AFP.

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