[The Minor T Allele of the Single Nucleotide Polymorphism rs 13360222 Decreases the Activity of the HAVCR2 Gene Enhancer in a Cell Model of Human Macrophages].

The TIM-3 receptor, encoded by the Hepatitis A Virus Cellular Receptor 2 (HAVCR2) gene, is an immune checkpoint and plays an important role in preventing the development of autoimmune reactions. This receptor is expressed on the surface of various immunocytes and its functions in myeloid cells remain poorly understood, compared to the role of T cell specific TIM-3 that is actively studied in the context of the search for promising therapeutic targets in cancer immunotherapy. During this study, we performed deletion analysis of the promoter region of the HAVCR2 gene, as well as functional characterization of its enhancer, and studied the effect of a number of single nucleotide polymorphisms (SNPs) on the activity of these regulatory elements in the relevant model of human macrophage-like cells-U937 activated monocytes. We have shown that the SNPs rs10515746(A) and rs4704853(A) located in the HAVCR2 gene promoter and associated with the development of a number of pathologies, do not affect the activity of the promoter in activated monocytes. However, a minor T variant of SNP rs13360222 located in the enhancer in the third intron of the gene, significantly reduces the ability of the enhancer to activate the HAVCR2 promoter, presumably due to weakening of the binding of nuclear receptor ESR2 to the respective region.

[Regulation of IL33 Gene Expression by SP1 and Foxa1 in Breast and Lung Cancer Cells].

Interleukin-33 (IL-33) is a member of the IL-1 cytokine family, primarily known as a mediator of the humoral immune response. It provides protection of barrier tissues and participates in the development of a range of diseases. This cytokine promotes carcinogenesis by induction of proliferation and survival of cancer cells, remodeling of the tumor microenvironment, and promoting immunosuppressive conditions. Elevated levels of IL-33 were observed in many types of cancers. This elevation correlates with a poor prognosis, making IL33 a promising target for cancer immunotherapy. The mechanisms of IL-33 expression regulation in human tumor cells are not well understood. Here, we show that that expression of IL-33 in breast and lung cancer cell lines depends, at least in part, on the activity of the SP1 and FOXA1 transcription factors. Increases in the activity of these transcription factors may be responsible for elevated levels of IL-33 and subsequent tumor progression.

[Relative Efficiency of Transcription Factor Binding to Allelic Variants of Regulatory Regions of Human Genes in Immunoprecipitation and Real-Time PCR].

The efficiency at which specific transcription factors interact with DNA may vary in the presence of single nucleotide polymorphisms (SNPs), and the variation provides an important mechanism that regulates expression of human genes and contributes to the individual susceptibility to various diseases. Ample genetic and epigenetic data make it possible to predict both functional polymorphic variants and the transcription factors whose binding they affect. However, predictions of the kind require experimental verification. An original method developed for the purpose includes immunoprecipitation of DNA-protein complexes, followed by quantification of the bound DNA by real-time PCR. The method does not require chemical modification of the DNA probes and yield reproducible results with total nuclear extracts from cultured human cells.

[The Novel Short Isoform of Securin Stimulates the Expression of Cyclin D3 and Angiogenesis Factors VEGFA and FGF2, but Does Not Affect the Expression of MYC Transcription Factor].

Pituitary tumor-transforming gene-1 (PTTG1) encodes securin, a multifunctional protein involved in development of various types of cancer. Securin participates in the regulation of sister chromatids separation and the expression of multiple genes involved in the control of the cell cycle, metabolism, and angiogenesis. In several human cell lines, we have found a novel short isoform of securin mRNA, which does not contain exons 3 and 4. After the translation of this new mRNA, a shortened protein is produced that, like the full-size form, is able to activate the transcription of cyclin D3 gene (CCND3), which controls the G1/S transition and angiogenesis factors VEGFA (vascular endothelial growth factor), and FGF2 (fibroblast growth factor 2) in HEK293 cells. However, unlike the full-size protein, the short isoform of PTTG1 does not affect the MYC gene expression because it lacks the DNA-binding domain, which is needed for its interactions with the MYC promoter. Furthermore, the short form of securin does not influence the expression of MYC transcriptional targets, such as TP53 and IL-8. Thus, we found a novel isoform of securin which is able to activate a more restricted repertoire of genes compared to the full-size protein.

Neonatal Lethality and Inflammatory Phenotype of the New Transgenic Mice with Overexpression of Human Interleukin-6 in Myeloid Cells.

To model human interleukin-6 (hIL-6) associated diseases, unique mice with transgenic overexpression of human IL-6 and reporter fluorescent protein EGFP in cells of macrophage-monocyte lineage were generated using loxP-Cre system. High level of hIL-6 production by macrophages and monocytes, as confirmed in vitro in primary culture of bone marrow-derived macrophages, in vivo resulted in early postnatal death in vivo, presumably, due to the effect of overexpression of hIL-6 on hematopoiesis.

Potential Markers of Autoimmune Diseases, Alleles rs115662534(T) and rs548231435(C), Disrupt the Binding of Transcription Factors STAT1 and EBF1 to the Regulatory Elements of Human CD40 Gene.

CD40 receptor is expressed on B lymphocytes and other professional antigen-presenting cells. The binding of CD40 to its ligand CD154 on the surface of T helper cells plays an important role in the activation of B lymphocytes required for production of antibodies, in particular, against autoantigens. Association of several single nucleotide polymorphisms (SNPs) located in the non-coding areas of human CD40 locus with the elevated risk of autoimmune diseases has been demonstrated. The most studied of these SNPs is rs4810485 located in the first intron of the CD40 gene. Expression of the CD40 gene in B lymphocytes of donors homozygous for the common allelic variant of this polymorphism (G) is higher than in B cells from donors carrying the minor (T) variant. We investigated the enhancer activity of this fragment of the CD40 locus in human B cell lines and showed that it is independent on the rs4810485 alleles. However, the minor allelic variants of the rs4810485-linked SNPs rs548231435 and rs115662534 were associated with a significant decrease in the activity of the CD40 promoter due to the impairments in the binding of EBF1 and STAT1 transcription factors, respectively.

The Minor Variant of the Single-Nucleotide Polymorphism rs3753381 Affects the Activity of a SLAMF1 Enhancer.

The SLAMF1 gene encodes CD150, a transmembrane glycoprotein expressed on the surface of T and B-lymphocytes, NK-cells, dendritic cells, and subpopulations of macrophages and basophils. We investigated the functional regulatory polymorphisms of the SLAMF1 locus associated with autoimmune processes, using bioinformatics and a mutational analysis of the regulatory elements overlapping with polymorphic positions. In the reporter gene assay in MP-1 and Raji B-cell lines, the enhancer activity of the regulatory region of the locus containing the rs3753381 polymorphism demonstrated a twofold increase upon the introduction of the rs3753381 minor variant (G → A) associated with myasthenia gravis. An analysis of the nucleotide context in the vicinity of rs3753381 revealed that the minor version of this polymorphism improves several binding sites for the transcription factors of FOX and NFAT, and RXR nuclear receptors. All mutations that disrupt any of these sites lead to a decrease in the enhancer activity both in MP1 and in Raji cells, and each of the two B-cell lines expresses a specific set of these factors. Thus, the minor variant of the rs3753381 polymorphism may contribute to the development of myasthenia gravis by modulating SLAMF1 expression, presumably in pathogenic B-lymphocytes.

Molecular and Cellular Mechanisms of Inflammation.

Inflammation is one of the most fundamental and pronounced protective reactions of the organism. From ancient times to the present day, complex and diverse patterns of inflammation development and their role in various diseases have attracted attention of investigators. This issue of Biokhimiya/Biochemistry (Moscow) includes experimental studies and reviews dedicated to various aspects of this important and interesting problem.

Mechanisms of Changes in Immune Response during Bacterial Coinfections of the Respiratory Tract.

Acute diseases of the respiratory tract are often caused by viral pathogens and accompanying secondary bacterial infections. It is known that the development of such bacterial complications is caused mainly by a decreased infiltration with immune system cells and by suppressed inflammation in the lungs. There are significant advances in understanding the mechanisms of secondary infections, although many details remain unclear. This review summarizes current knowledge of the molecular and cellular changes in the host organism that can influence the course of bacterial coinfections in the respiratory tract.

Chemokines, cytokines and exosomes help tumors to shape inflammatory microenvironment.

Relationship between inflammation and cancer is now well-established and represents a paradigm that our immune response does not necessarily serves solely to protect us from infections and cancer. Many specific mechanisms that link chronic inflammation to cancer promotion and metastasis have been uncovered in the recent years. Here we are focusing on the effects that tumors may exert on inflammatory cascades, tuning the immune system ability to cause tumor promotion or regression. In particular, we discuss the contributions of chemokines, cytokines and exosomes to the processes such as induction of inflammation and tumorigenesis. Overall, tumor-elicited inflammation is a key driver of tumor progression and an essential component of tumor microenvironment.

Modeling of viral-bacterial coinfections at the molecular level using agonists of innate immunity receptors.

The combined effect of innate immunity receptors in viral-bacterial coinfections was studied in vitro using the primary culture of murine macrophages activated by different combinations of ligands of innate immunity receptors belonging to the family of Toll-like receptors. The activation of macrophages first with a viral ligand and then with a bacterial one significantly decreased the expression of proinflammatory cytokine genes. Such attenuation of immune responses may occur during the development of bacterial complications in viral infections.

[Genetic mechanisms of adaptive immunity emergence in vertebrates].

The adaptive immune system in vertebrates emerged in a multistep process that can be reconstructed on the basis of the data concerning the structure of immune systems of modern cartilaginous and bony fishes, as well as of cyclostomes. The most probable evolutionary scenario is likely to be as follows: the T cell receptor loci emerged on the basis of NK cell-like receptor genes; the antibody loci evolved on the basis of T cell receptor loci; the MHC locus arose on the basis of the locus responsible for innate immunity of early chordates. The ancestral MHC molecules likely participated in the transplantation immunity before they acquired the ability of antigen peptide presentation.

Distinct biological activity of lipopolysaccharides with different lipid A acylation status from mutant strains of Yersinia pestis and some members of genus Psychrobacter.

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 ΔlpxM/ΔlpxP 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.

Upstream open reading frames regulate translation of the long isoform of SLAMF1 mRNA that encodes costimulatory receptor CD150.

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.

[Interleukin-11, an IL-6 like cytokine].

The aim of this review is to consolidate various data about different functions of interleukin-11 (IL-11), a member of the IL-6 family. Numerous in vitro experiments have suggested a long list of IL-11 activities, including support and control of proliferation and differentiation of hematopoietic progenitors, as well as participation in osteoclastogenesis, neurogenesis and development of some other tissues. However, many of the in vitro effects of IL-11 have not been confirmed in experiments using animal models, hampering understanding of the physiological role of this cytokine. We discuss possible reasons for this apparent discrepancy between in vitro and in vivo data as well as perspectives of using conditional gene targeting to assess the role of IL-11 in ontogenesis and immune responses.

[Transcriptional regulation of TNF/LT locus in immune cells].

Tumor necrosis factor (TNF) is one of the most important proinflammatory cytokines. It demonstrates a complex pattern of tissue-specific expression and behaves as a product of immediate early transcriptional response in macrophages. These properties have made the regulation of TNF gene, as well as regulation of tightly linked related lymphotoxin alpha (LTalpha) and beta (LTbeta) genes the object of thorough investigation for more than two decades. Some aspects of TNF/LT locus regulation, such as the role of distal TNF-promoter and of NF-kappaB factors in TNF gene transcription, still remain the object of discussion. Moreover, several recent studies uncovering the molecular mechanisms of immediate early gene activation and directly related to TNF gene regulation have not been reflected in published reviews yet. Here we briefly overview the modern concepts of transcriptional regulation of the TNF/LT locus, with an accent on new data and unanswered questions.

Autoantibodies to tumor-associated antigens as cancer biomarkers.

Malignant tumors induce humoral immune response in cancer patients, although the incidence of such autoantibody responses against individual tumor-associated antigens (TAA) is rather low. To increase predictive value of TAA-recognizing autoantibodies as potential cancer biomarkers, TAAs should be combined into protein arrays. Here we review recent advances in the application of such arrays and summarize data concerning most promising antigens. We also review the methods of cloning TAA-recognizing autoantibodies, generation of human hybridomas and screening of recombinant human immunoglobulin libraries.

[New putative control elements in the promoter OF CXCL13 chemokine gene, a target of alternative NF-kappaB pathway].

We searched the proximal promoter region of CXCL13/BLC chemokine gene for new putative control elements, including potential NF-kappaB binding sites. Using electrophoretic mobility shift assay and reporter gene analysis we identified two new promoter elements. The first element contains Rel/NF-kappaB binding site and seems to participate in inducible gene expression, while another site binds transcription factor Sp1 and is critical for basic transcription. It is the first indication that alternative NF-kappaB pathway target genes are probably cooperatively controlled by factors Rel/NF-kappaB and Sp1. Identification of a functional Sp1 site in the promoter of a target gene of alternative NF-kappaB pathway will be useful for investigation of molecular mechanisms and signal pathwaysinvolved.