Biochemistry (Moscow) (v.79, #12)

Innate immunity underlies symbiotic relationships by E. P. Kisseleva (1273-1285).
Here, the modern data regarding interactions between normal microbiota and barrier tissues in plants, humans and animals are reviewed. The main homeostatic mechanisms responsible for interactions between epithelium and innate immune cells with symbiotic bacteria are described. A key step in this process is recognition of soluble microbial products by ligation to pattern-recognition receptors expressed on the host cells. As a result, epithelial cells secrete mucus, antibacterial peptides and immunoregulatory molecules. The main outcomes from immunological reactions towards symbiotic bacteria involve development of conditions for formation and maintenance of microbial biocenosis as well as providing safety for the host. Also, it is considered important to preserve and transfer beneficial bacteria to progeny.
Keywords: normal microbiota; pattern-recognition receptors; epithelium; innate immunity; symbiotic relationships

Recent insights into the specific type of cell death characteristic of neutrophils, called NETosis, are summarized. NETosis is a process of generation of Neutrophil Extracellular Traps (NETs), whose main components are DNA, granular antimicrobial peptides, and nuclear and cytoplasmic proteins. The structure of NETs determines their bactericidal, fungicidal, antiprotozoal, and antiviral properties. Therefore, NETs production by neutrophils is an essential immune response to infection. In addition to the antimicrobial function, NETosis is involved in many inflammatory and autoimmune disorders and participates in the regulation of noninfectious processes. The molecular mechanisms of NET formation, bactericidal effect, and involvement in some noninfectious, autoimmune, and inflammatory processes are discussed in detail in this review.
Keywords: neutrophils; neutrophil extracellular traps; NETosis; autoimmune diseases; inflammation

Molecular barriers to processes of genetic reprogramming and cell transformation by I. V. Chestkov; E. A. Khomyakova; E. A. Vasilieva; M. A. Lagarkova; S. L. Kiselev (1297-1307).
Genetic reprogramming by ectopic expression of transcription factor genes induces the pluripotent state in somatic cells. This technology provides an opportunity to establish pluripotent stem cells for each person, as well as to get better understanding of epigenetic mechanisms controlling cell state. Interestingly, some of the molecular processes that accompany somatic cell reprogramming in vitro are also characteristic for tumor manifestation. Thus, similar “molecular barriers” that control the stability of epigenetic state exist for both processes of pluripotency induction and malignant transformation. The reprogramming of tumor cells is interesting in two aspects: first, it will determine the contribution of epigenetic changes in carcinogenesis; second, it gives an approach to evaluate tumor stem cells that are supposed to form the entire cell mass of the tumor. This review discusses the key stages of genetic reprogramming, the similarity and difference between the reprogramming process and malignant transformation.
Keywords: genetic reprogramming; transformation; tumor; epigenetics; induced pluripotent stem cells

Modern anti-cytokine therapy of autoimmune diseases by I. V. Astrakhantseva; G. A. Efimov; M. S. Drutskaya; A. A. Kruglov; S. A. Nedospasov (1308-1321).
The emergence of genetically engineered biological agents opened new prospects in the treatment of autoimmune and inflammatory diseases. Cytokines responsible for regulation of a wide range of processes during development of the normal immune response are among the most successful therapeutic targets. Studies carried out in recent decades and accompanied by rapid development of biotechnology have promoted establishing in detail the role and place of cytokines in autoimmune and inflammatory pathologies. Nevertheless, mechanisms that underlie anti-cytokine therapy are still not fully understood. This review examines the role of such cytokines as TNF, IL-1, and IL-6 in the development of inflammatory processes and the action mechanisms of their inhibitors.
Keywords: cytokines; TNF; IL-6; IL-1; autoimmunity; chronic inflammation

Plant cell death caused by fungal, bacterial, and viral elicitors: protective effect of mitochondria-targeted quinones by D. B. Kiselevsky; O. Yu. Frolova; A. G. Solovyev; Yu. L. Dorokhov; S. Yu. Morozov; V. D. Samuilov (1322-1332).
Chitosan (partially deacetylated chitin), a component of fungal cell walls, caused epidermal cell (EC) death in the leaves of pea (Pisum sativum L.) and tobacco Nicotiana tabacum or Nicotiana benthamiana detected by destruction of cell nuclei. The mitochondria-targeted quinone SkQ1 prevented the destruction of EC nuclei induced by chitosan. Chitosan increased and SkQ1 suppressed the activity of protein kinases in N. benthamiana and P. sativum and eliminated the effect of chitosan. Chitosan induced the generation of reactive oxygen species (ROS) in the guard cells (GC) of pea plants. Treatment with chitosan or H2O2 did not cause destruction of GC nuclei; however, it resulted in disruption of the permeability barrier of the plasma membrane detected by propidium iodide fluorescence. Treatment with bacterial lipopolysaccharide but not peptidoglycan caused destruction of pea EC nuclei, which was prevented by SkQ1. Leaves of tobacco plants containing the N gene responsible for resistance to tobacco mosaic virus (TMV) were infiltrated with Agrobacterium tumefaciens cells. These cells contained a genetic construct with the gene of the helicase domain of TMV replicase (p50); its protein product p50 is a target for the N-gene product. As a result, the hypersensitive response (HR) was initiated. The HR manifested itself in the death of leaves and was suppressed by SkQ3. Treatment of tobacco epidermal peels with the A. tumefaciens cells for the p50 gene expression stimulated the destruction of EC nuclei, which was inhibited by SkQ1 or SkQ3. The p50-lacking A. tumefaciens cells did not induce the destruction of EC nuclei. The protective effect of mitochondria-targeted antioxidants SkQ1 and SkQ3 demonstrates the involvement of mitochondria and their ROS in programmed cell death caused by pathogen elicitors.
Keywords: programmed cell death; mitochondria-targeted quinones; chitosan; lipopolysaccharide; peptidoglycan; tobacco mosaic virus; guard cells; epidermal cells

Distinct biological activity of lipopolysaccharides with different lipid a acylation status from mutant strains of Yersinia pestis and some members of genus Psychrobacter by K. V. Korneev; A. N. Kondakova; N. P. Arbatsky; K. A. Novototskaya-Vlasova; E. M. Rivkina; A. P. Anisimov; A. A. Kruglov; D. V. Kuprash; S. A. Nedospasov; Yu. A. Knirel; M. S. Drutskaya (1333-1338).
Correlation between the chemical structure of lipid A from various Gram-negative bacteria and biological activity of their lipopolysaccharide (LPS) as an agonist of the innate immune receptor Toll-like receptor 4 was investigated. Purified LPS species were quantitatively evaluated by their ability to activate the production of tumor necrosis factor (TNF) by murine bone marrow-derived macrophages in vitro. Wild-type LPS from plague-causing bacteria Yersinia pestis was compared to LPS from mutant strains with defects in acyltransferase genes (lpxM, lpxP) responsible for the attachment of secondary fatty acid residues (12:0 and 16:1) to lipid A. Lipid A of Y. pestis double ΔlpxMlpxP mutant was found to have the chemical structure that was predicted based on the known functions of the respective acyltransferases. The structures of lipid A from two members of the ancient psychrotrophic bacteria of the genus Psychrobacter were established for the first time, and biological activity of LPS from these bacteria containing lipid A fatty acids with shorter acyl chains (C10–C12) than those in lipid A from LPS of Y. pestis or E. coli (C12–C16) was determined. The data revealed a correlation between the ability of LPS to activate TNF production by bone marrow-derived macrophages with the number and the length of acyl chains within lipid A.
Keywords: lipopolysaccharide; lipid A; Toll-like receptor 4; tumor necrosis factor; Yersinia pestis ; Psychrobacter spp.

Bacterial lipopolysaccharide activates CD57-negative human NK cells by L. M. Kanevskiy; S. A. Erokhina; M. A. Streltsova; W. G. Telford; A. M. Sapozhnikov; E. I. Kovalenko (1339-1348).
NK cells play an important regulatory role in sepsis by induction and augmentation of proinflammatory reactions in early stages of the septic process and by suppression of immune response in later stages of inflammation. The present work was aimed at the effect of bacterial lipopolysaccharide (LPS), the main pathogenic factor of sepsis development, on human NK cells ex vivo. We show that LPS activates immature CD57-negative NK cells, which typically constitute less than half of the normal NK cell population in human peripheral blood. Under conditions of NK cell stimulation with IL-2, addition of LPS provokes an increase in IFN-γ production. However, LPS both increased and inhibited NK cell cytotoxic activity. It is important to note that the activation of NK cells on LPS addition was observed in the absence of TLR4 on the NK cell surface. These results confirm our previous data arguing for a direct interaction of LPS with NK cells and evidence an atypical mechanism of LPS-induced NK cell activation without the involvement of surface TLR4.
Keywords: NK cells; lipopolysaccharide; IFN-γ production; CD57; cytotoxicity; TLR4

Experimental models of arthritis in which pathogenesis is dependent on TNF expression by M. S. Drutskaya; G. A. Efimov; R. V. Zvartsev; A. A. Chashchina; D. M. Chudakov; S. V. Tillib; A. A. Kruglov; S. A. Nedospasov (1349-1357).
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by joint damage as well as systemic manifestations. The exact cause of RA is not known. Both genetic and environmental factors are believed to contribute to the development of this disease. Increased expression of tumor necrosis factor (TNF) has been implicated in the pathogenesis of RA. Currently, the use of anti-TNF drugs is one of the most effective strategies for the treatment of RA, although therapeutic response is not observed in all patients. Furthermore, due to non-redundant protective functions of TNF, systemic anti-TNF therapy is often associated with unwanted side effects such as increased frequency of infectious diseases. Development of experimental models of arthritis in mice is necessary for studies on the mechanisms of pathogenesis of this disease and can be useful for comparative evaluation of various anti-TNF drugs. Here we provide an overview of the field and present our own data with two experimental models of autoimmune arthritis — collagen-induced arthritis and antibody-induced arthritis in C57Bl/6 and BALB/c mice, as well as in tnf-humanized mice generated on C57Bl/6 back-ground. We show that TNF-deficient mice are resistant to the development of collagen-induced arthritis, and the use of anti-TNF therapy significantly reduces the disease symptoms. We also generated and evaluated a fluorescent detector of TNF overexpression in vivo. Overall, we have developed an experimental platform for studying the mechanisms of action of existing and newly developed anti-TNF drugs for the treatment of rheumatoid arthritis.
Keywords: rheumatoid arthritis; tumor necrosis factor; anti-cytokine therapy; autoimmune diseases

Formation of compact aggregates of B-lymphocytes in lung tissue during mycobacterial infection in mice depends on TNF production by these cells and is not an element of the host’s immunological protection by T. K. Kondratieva; I. A. Linge; E. V. Kondratieva; A. V. Dyatlov; M. S. Drutskaya; R. V. Zvartsev; S. A. Nedospasov; A. S. Apt (1358-1362).
Tumor necrosis factor (TNF) plays a pivotal role in the early control of Mycobacterium tuberculosis and M. avium infections by a host. It was previously shown that both phagocyte-derived and T-cell-derived TNF productions are critical for protective immunity against M. tuberculosis, but the role of TNF produced by B-cells remained unclear. By comparing mice with B-cell-specific TNF deletion to littermate control mice, here we show that TNF production by B-lymphocytes is essential for the formation of infection-specific aggregates of B-cells in the lung. It is likely that these compact foci represent a pathogenic feature of inflammatory response rather than an element of protective immunity, since the capacity to form aggregates has no influence on the severity of M. tuberculosis- and M. avium-triggered diseases.
Keywords: mycobacterial infections; B-lymphocytes; TNF; lung pathology; mouse models

Analysis of results of acute graft-versus-host disease prophylaxis with donor multipotent mesenchymal stromal cells in patients with hemoblastoses after allogeneic bone marrow transplantation by I. N. Shipounova; N. A. Petinati; A. E. Bigildeev; E. A. Zezina; N. I. Drize; L. A. Kuzmina; E. N. Parovichnikova; V. G. Savchenko (1363-1370).
Allogeneic bone marrow transplantation (allo-BMT) is currently the only way to cure many hematoproliferative disorders. However, allo-BMT use is limited by severe complications, the foremost being graft-versus-host disease (GVHD). Due to the lack of efficiency of the existing methods of GVHD prophylaxis, new methods are being actively explored, including the use of donors’ multipotent mesenchymal stromal cells (MMSC). In this work, we analyzed the results of acute GVHD (aGVHD) prophylaxis by means of MMSC injections after allo-BMT in patients with hematological malignancies. The study included 77 patients. They were randomized into two groups — those receiving standard prophylaxis of aGVHD and those who were additionally infused with MMSC derived from the bone marrow of hematopoietic stem cell donors. We found that the infusion of MMSC halves the incidence of aGVHD and increases the overall survival of patients. Four of 39 MMSC samples were ineffective for preventing aGVHD. Analysis of individual donor characteristics (gender, age, body mass index) and the MMSC properties of these donors (growth parameters, level of expression of 30 genes involved in proliferation, differentiation, and immunomodulation) revealed no significant difference between the MMSC that were effective or ineffective for preventing aGVHD. We used multiple logistic regression to establish a combination of features that characterize the most suitable MMSC samples for the prevention of aGVHD. A model predicting MMSC sample success for aGVHD prophylaxis was constructed. Significant model parameters were increased relative expression of the FGFR1 gene in combination with reduced expression levels of the PPARG and IGF1 genes. Depending on the chosen margin for probability of successful application of MMSC, this model correctly predicts the outcome of the use of MMSC in 82–94% of cases. The proposed model of prospective evaluation of the effectiveness of MMSC samples will enable prevention of the development of aGVHD in the maximal number of patients.
Keywords: multipotent mesenchymal stromal cells (MMSC); allogeneic bone marrow transplantation (allo-BMT); acute graft-versus-host disease (aGVHD); relative gene expression level; multiple logistic regression

Cytokine profile in children with inflammatory bowel disease by A. P. Toptygina; E. L. Semikina; G. V. Bobyleva; L. V. Miroshkina; S. V. Petrichuk (1371-1375).
Crohn’s disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) characterized by immune reactivity against microbial and auto-antigens. This work was designed to study the cytokine profile in blood serum and coproextracts of children with CD and UC. The studied patients consisted of 17 children with CD (group I), 17 children with UC (group II), and 18 controls with intestinal dysbiosis (group III). The diagnosis of UC and CD was based on accepted clinical and endoscopic criteria. The levels of 13 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-17A, TNF-α, TGF-β, and IFN-γ) were determined in blood sera and coproextracts of the patients and controls using the BioPlex technology. The level of IL-17A was significantly increased and that of TGF-β was significantly decreased in the blood serum of the patients with IBDs. Changes in the cytokine profile in the coproextracts affected the wider spectrum of cytokines. The levels of proinflammatory cytokines (IL-2, IL-4, IL-6, IL-12p70, TNF-α, and IFN-γ) were increased 6–9-fold, whereas the level of the anti-inflammatory cytokine IL-10 was increased 3-fold. The cytokine balance was shifted to the proinflammatory cytokines. The TGF-β level was increased 9-fold and that of IL-17A was increased 3-fold. Thus, the cytokine profile in the coproextracts was more informative than that of the blood serum. The determination of cytokines in coproextracts is simple and noninvasive.
Keywords: cytokines; interleukin; Crohn’s disease; ulcerative colitis; children

Novel recombinant anti-HER2/neu immunotoxin: Design and antitumor efficiency by E. A. Sokolova; T. A. Zdobnova; O. A. Stremovskiy; I. V. Balalaeva; S. M. Deyev (1376-1381).
The novel HER2/neu-specific recombinant immunotoxin 4D5scFv-PE40 consisting of 4D5scFv antibody (targeting module) and Pseudomonas exotoxin A fragment (effector module) combined in a single polypeptide chain via a flexible linker has been expressed and purified. This immunotoxin conserves specificity and affinity that are characteristics of the parental antibody 4D5scFv and exhibits selective and strong cytotoxic effect against cancer cells overexpressing HER2/neu receptor. The results of the experiments both in vitro (in cell cultures) and in vivo (in tumor-bearing animals) demonstrate high potential of 4D5scFv-PE40 for targeted therapy of tumors overexpressing HER2/neu.
Keywords: recombinant immunotoxin; 4D5scFv; Pseudomonas exotoxin A; receptor HER2/neu; targeted therapy

Molecular analysis of heavy chain-only antibodies of Camelus bactrianus by S. V. Tillib; A. S. Vyatchanin; S. Muyldermans (1382-1390).
In this work, IgG content and structures of antigen-binding domains and hinge regions of different IgG subtypes of Camelus bactrianus were analyzed in detail for the first time. Our data demonstrate that C. bactrianus contains a very large amount of heavy chain-only antibodies that can be used as a source of VHH domain-containing molecules. Despite some minor sequence differences identified in this study, C. bactrianus VHH domains possess principally the same unique features as those of C. dromedarius and the llama. These features are important for developing an efficient phage display-based anti- body selection technology. We conclude that C. bactrianus is a very suitable animal to raise an immune response that serves as a source to identify antigen-specific VHHs selected after phage display.
Keywords: antibody; IgG; variable domain; single-domain antibody

Highly specific hybrid protein DARPin-mCherry for fluorescent visualization of cells overexpressing tumor marker HER2/neu by K. E. Mironova; O. N. Chernykh; A. V. Ryabova; O. A. Stremovskiy; G. M. Proshkina; S. M. Deyev (1391-1396).
Here we propose a simple and reliable approach for detection of the tumor marker HER2/neu using the targeting fluorescent hybrid protein DARPin-mCherry. As a targeting module, we used DARPin9-29, which is a member of a novel class of non-immunoglobulin targeting proteins that can highly selectively recognize the extracellular domain of the epidermal growth factor receptor HER2/neu. The red fluorescent protein mCherry was used as the detecting module. The hybrid protein DARPin-mCherry was prepared with high yield in a bacterial expression system and purified in one step by affinity chromatography. The purified protein is not prone to aggregation. The specificity of DARPin-mCherry binding with the HER2/neu tumor marker was demonstrated using confocal microscopy, flow cytofluorimetry, and surface plasmon resonance. The dissociation constant of the DARPin-mCherry protein complex with the HER2/neu receptor determined by surface plasmon resonance was calculated to be 4.5 nM. These characteristics of the hybrid protein DARPin-mCherry suggest it as a promising agent for immunofluorescent assay and an attractive alternative to antibodies and their fragments labeled with fluorescent dyes that are now used for this purpose.
Keywords: DARPin; mCherry; tumor marker HER2/neu

Epitope mapping of lymphocyte phosphatase-associated phosphoprotein by A. V. Filatov; T. D. Meshkova; D. V. Mazurov (1397-1404).
Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a transmembrane protein with unknown function. The available data on its close association with phosphatase CD45 and its phosphorylation depending on cell activation suggest that LPAP can play a significant role in the antigenic stimulation of lymphocytes. We have localized three antigenic epitopes of the LPAP molecule that can be detected using monoclonal antibodies prepared earlier. Experiments on reactions of antibodies with point mutants and shortened forms of the LPAP protein revealed regions of the amino acid sequence that correspond to the epitopes recognized by the antibodies.
Keywords: LPAP; monoclonal antibodies; site directed mutagenesis; human lymphocytes

Upstream open reading frames regulate translation of the long isoform of SLAMF1 mRNA that encodes costimulatory receptor CD150 by L. V. Putlyaeva; A. M. Schwartz; K. V. Korneev; M. Covic; L. A. Uroshlev; V. Yu. Makeev; S. E. Dmitriev; D. V. Kuprash (1405-1411).
More than 40% of human genes contain upstream open reading frames (uORF) in their 5′-untranslated regions (5′-UTRs) and at the same time express at least one truncated mRNA isoform containing no uORF. We studied translational regulation by four uORFs found in the 5′-UTR of full-length mRNA for SLAMF1, the gene encoding CD150 membrane protein. CD150 is a member of the CD2 superfamily, a costimulatory lymphocyte receptor, a receptor for measles virus, and a microbial sensor on macrophages. The SLAMF1 gene produces at least two mRNA isoforms that differ in their 5′-UTRs. In the long isoform of the SLAMF1 mRNA that harbors four uORFs in the 5′-UTR, the stop codon of uORF4 overlaps with the AUG codon of the main ORF forming a potential termination-reinitiation site UGAUG, while uORF2 and uORF3 start codons flank a sequence identical to Motif 1 from the TURBS regulatory element. TURBS was shown to be required for a coupled termination-reinitiation event during translation of polycistronic RNAs of some viruses. In a model cell system, reporter mRNA based on the 5′-UTR of SLAMF1 short isoform, which lacks any uORF, is translated 5–6 times more efficiently than the mRNA with 5′-UTR from the long isoform. Nucleotide substitutions disrupting start codons in either uORF2-4 result in significant increase in translation efficiency, while substitution of two nucleotides in TURBS Motif 1 leads to a 2-fold decrease in activity. These data suggest that TURBS-like elements can serve for translation control of certain cellular mRNAs containing uORFs.
Keywords: protein biosynthesis; translation control; uORF; mRNA

Mouse lymphomyeloid cells can function with significantly decreased expression levels of cytochrome c by E. S. Shilov; I. V. Kislyakov; E. A. Gorshkova; R. V. Zvartsev; M. S. Drutskaya; I. A. Mufazalov; V. P. Skulachev; S. A. Nedospasov (1412-1422).
Cytochrome c is an indispensable electron carrier in the mitochondrial respiratory chain and also an important mediator of the internal pathway triggering apoptosis. Mice with a complete deficiency of the Cycs gene encoding the somatic cytochrome c die during the embryogenesis. Using the technology of LoxP-cre-dependent tissue-specific recombination, we obtained some mouse strains with significantly reduced expression of cytochrome c in certain cell types (“conditional genetic knockdown”). This knockdown was achieved by abrogation of the normal splicing of the Cycs locus pre-mRNA due to an additional acceptor site inside the stop-cassette neor. Previously, we observed embryonic lethality in homozygous mice with the same knockdown of cytochrome c in all cells of the organism. In the present work we studied two novel mouse strains with conditional knockdown of the Cycs gene in T lymphocytes and macrophages. Somewhat surprisingly, the mice of these two strains under normal conditions were not phenotypically different from the wild-type mice, either on the whole organism level or on the level of activity of individual target cells. Thus, the amount of cytochrome c in lymphomyeloid cells does not affect their development and normal functioning.
Keywords: cytochrome c ; genetic knockdown; T lymphocytes; macrophages; cre recombinase; splicing