The novel peptide LCGM-10 attenuates metabotropic glutamate receptor 5 activity and demonstrates behavioral effects in animal models.

We employed a structural bioinformatics approach to develop novel peptides with predicted affinity to the binding site for negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5). Primary screening in zebrafish (Danio rerio) revealed a stimulatory effect of two peptides, LCGM-10 and LCGM-15. Target validation studies using calcium ion flux imaging and a luciferase reporter assay confirmed mGluR5 as the target. LCGM-10 showed greater potency than LCGM-15; it was comparable to that of the mGluR5 NAM 2-methyl-6-(phenylethynyl) pyridine (MPEP). Rodent behavioral screening in the open field and elevated plus maze revealed increased locomotor activity in both tests after acute LCGM-10 treatment, supported by further analysis of home cage spontaneous locomotor activity (SLA). The stimulating effect of a single LCGM-10 administration on SLA was evident up to 60 min after administration and was not accompanied by hypokinetic rebound observed for caffeine. According to our results, LCGM-10 has therapeutic potential to treat hypo- and dyskinesias of various etiologies. Further investigation of LCGM-10 effects in the delay discounting model of impulsive choice in rats revealed reduced trait impulsivity after single and chronic administrations, suggesting potential implication for attention deficit hyperactivity disorder, obsessive compulsive disorder, and addictions.

The relationship between physical performance and alcohol consumption levels in Russian adults.

Investigating the relationship between alcohol consumption and physical performance, we used data from the 2015-2018 Know Your Heart study on 4215 adults aged 35-69 from Arkhangelsk and Novosibirsk, Russia. We classified participants' drinking status into non-drinking, non-problem drinking, hazardous drinking, and harmful drinking based on their self-reported drinking behaviors. To evaluate physical performance, we developed a Composite Physical Performance Scale (CPPS), which combined the results of three functional tests: grip strength (GS), closed-eyes balance, and chair rises (CR). We applied multivariable linear regression to assess the relationship between alcohol consumption and CPPS score, and ordinal logistic regression to explore the associations between alcohol consumption and the three functional tests separately. The results showed that harmful drinking was associated with lower CPPS scores compared to non-problem drinking. Among harmful drinking men, the decrease in CPPS scores was explained by all three tests equally and exceptionally by GS among women. Non-drinking was also associated with decreased CPPS, linked to lower GS and CR scores in men, and only lower GS scores in women. The study revealed a reduced physical performance in the non-drinking and harmful drinking groups compared to non-problem drinking.

Differentially activated B cells develop regulatory phenotype and show varying immunosuppressive features: a comparative study.

Regulatory B lymphocytes (Bregs) are B cells with well-pronounced immunosuppressive properties, allowing them to suppress the activity of effector cells. A broad repertoire of immunosuppressive mechanisms makes Bregs an attractive tool for adoptive cell therapy for diseases associated with excessive activation of immune reactions. Such therapy implies Breg extraction from the patient's peripheral blood, ex vivo activation and expansion, and further infusion into the patient. At the same time, the utility of Bregs for therapeutic approaches is limited by their small numbers and extremely low survival rate, which is typical for all primary B cell cultures. Therefore, extracting CD19+ cells from the patient's peripheral blood and specifically activating them ex vivo to make B cells acquire a suppressive phenotype seems to be far more productive. It will allow a much larger number of B cells to be obtained initially, which may significantly increase the likelihood of successful immunosuppression after adoptive Breg transfer. This comparative study focuses on finding ways to efficiently manipulate B cells in vitro to differentiate them into Bregs. We used CD40L, CpG, IL4, IL21, PMA, and ionomycin in various combinations to generate immunosuppressive phenotype in B cells and performed functional assays to test their regulatory capacity. This work shows that treatment of primary B cells using CD40L + CpG + IL21 mix was most effective in terms of induction of functionally active regulatory B lymphocytes with high immunosuppressive capacity ex vivo.

rs71327024 Associated with COVID-19 Hospitalization Reduces CXCR6 Promoter Activity in Human CD4+ T Cells via Disruption of c-Myb Binding.

Single-nucleotide polymorphism rs71327024 located in the human 3p21.31 locus has been associated with an elevated risk of hospitalization upon SARS-CoV-2 infection. The 3p21.31 locus contains several genes encoding chemokine receptors potentially relevant to severe COVID-19. In particular, CXCR6, which is prominently expressed in T lymphocytes, NK, and NKT cells, has been shown to be involved in the recruitment of immune cells to non-lymphoid organs in chronic inflammatory and respiratory diseases. In COVID-19, CXCR6 expression is reduced in lung resident memory T cells from patients with severe disease as compared to the control cohort with moderate symptoms. We demonstrate here that rs71327024 is located within an active enhancer that augments the activity of the CXCR6 promoter in human CD4+ T lymphocytes. The common rs71327024(G) variant makes a functional binding site for the c-Myb transcription factor, while the risk rs71327024(T) variant disrupts c-Myb binding and reduces the enhancer activity. Concordantly, c-Myb knockdown in PMA-treated Jurkat cells negates rs71327024's allele-specific effect on CXCR6 promoter activity. We conclude that a disrupted c-Myb binding site may decrease CXCR6 expression in T helper cells of individuals carrying the minor rs71327024(T) allele and thus may promote the progression of severe COVID-19 and other inflammatory pathologies.

Novel Potential Mechanisms of Regulatory B Cell-Mediated Immunosuppression.

B lymphocytes play an important role in the regulation of immune response in both normal and pathological conditions. Traditionally, the main functions of B cells were considered to be antibody production and antigen presentation, but in recent decades there have been discovered several subpopulations of regulatory B lymphocytes (Bregs), which maintain immunological tolerance and prevent overactivation of the immune system. Memory (mBregs, CD19+CD24hiCD27+) and transitional (tBregs, CD19+CD24hiCD38hi) subpopulations of Bregs are usually considered in the context of studying the role of these B cells in various human pathologies. However, the mechanisms by which these Breg subpopulations exert their immunosuppressive activity remain poorly understood. In this work, we used bioinformatic analysis of open-source RNA sequencing data to propose potential mechanisms of B cell-mediated immunosuppression. Analysis of differential gene expression before and after activation of these subpopulations allowed us to identify six candidate molecules that may determine the functionality of mBregs and tBregs. IL4I1-, SIRPA-, and SLAMF7-dependent mechanisms of immunosuppression may be characteristic of both Breg subsets, while NID1-, CST7-, and ADORA2B-dependent mechanisms may be predominantly characteristic of tBregs. In-depth understanding of the molecular mechanisms of anti-inflammatory immune response of B lymphocytes is an important task for both basic science and applied medicine and could facilitate the development of new approaches to the therapy of complex diseases.

Association between Alcohol Consumption and Body Composition in Russian Adults and Patients Treated for Alcohol-Related Disorders: The Know Your Heart Cross-Sectional Study.

There is conflicting evidence about the association between alcohol consumption and body composition (BC). We aimed to investigate this association in Russian adults. The study population included 2357 residents of Arkhangelsk aged 35-69 years, and 272 in-patients treated for alcohol problems (narcological patients) who participated in the Know Your Heart (KYH) cross-sectional study in 2015-2017. The participants were divided into five subgroups based on their alcohol use characteristics: non-drinkers, non-problem drinkers, hazardous drinkers, harmful drinkers, and narcological patients. Considering men, hazardous drinkers had a larger waist circumference (WC), waist-to-hip ratio (WHR), and percentage of body fat mass (%FM) compared to non-problem drinkers. In harmful drinking men, these differences were the opposite: a lower body mass index (BMI), hip circumference (HC), and %FM. Men among narcological patients had the lowest mean BMI, WC, HC, WHR, and %FM compared to other subgroups of men. As for women, non-drinkers had a lower BMI, WC, HC, and %FM compared to non-problem drinkers. Women among narcological patients had the lowest mean BMI and HC but an increased WHR compared to other subgroups of women. In conclusion, alcohol consumption levels had an inverted J-shaped association with adiposity-related BC parameters: they were elevated in hazardous drinkers but were reduced in harmful drinkers, and were even lower in patients with alcohol-related diagnoses.

The Novel Peptide Chm-273s Has Therapeutic Potential for Metabolic Disorders: Evidence from In Vitro Studies and High-Sucrose Diet and High-Fat Diet Rodent Models.

The aim of this study was to develop a novel peptide potentially applicable for the treatment of metabolic conditions, such as obesity and type 2 diabetes (T2D). We identified CHM-273S from the list of peptides from milk hydrolysate obtained by HPLC/MS-MS. In vitro analysis of primary murine fibroblasts indicated the potential of CHM-273S to upregulate IRS2 mRNA expression. CHM-273S showed a prominent anorexigenic effect in mice with the induction of a key mechanism of leptin signaling via STAT3 in the hypothalamus as a possible effector. In the animal model of metabolic disease, CHM-273S alleviated glucose intolerance and insulin resistance, and induced phosphorylation of Akt at Ser473 and Thr308 in the hepatocytes of high-sucrose diet-fed rats. In a murine model of T2D, CHM-273S mitigated high-fat diet-induced hyperglycemia and insulin resistance and improved low-grade inflammation by diminishing serum TNFα. Mice treated with chronic CHM-273S had a significant reduction in body weight, with a lower visceral fat pad weight and narrow adipocytes. The effects of the peptide administration were comparable to those of metformin. We show the potential of CHM-273S to alleviate diet-induced metabolic alterations in rodents, substantiating its further development as a therapeutic for obesity, T2D, and other metabolic conditions.

Peptide LCGA-17 Attenuates Behavioral and Neurochemical Deficits in Rodent Models of PTSD and Depression.

We have previously described the LCGA-17 peptide as a novel anxiolytic and antidepressant candidate that acts through the α2δ VGCC (voltage-gated calcium channel) subunit with putative synergism with GABA-A receptors. The current study tested the potential efficacy of acute and chronic intranasal (i.n.) LCGA-17 (0.05 mg/kg and 0.5 mg/kg) in rats on predator odor-induced conditioned place aversion (POCPA), a model of post-traumatic stress disorder (PTSD), and chronic unpredictable stress (CUS) that produce a range of behavioral and physiological changes that parallel symptoms of depression in humans. CUS and LCGA-17 treatment effects were tested in the sucrose preference (SPT) social interaction (SI), female urine sniffing (FUST), novelty-suppressed feeding (NSFT), and forced swim (FST) tests. Analysis of the catecholamines content in brain structures after CUS was carried out using HPLC. The efficacy of i.n. LCGA-17 was also assessed using the Elevated plus-maze (EPM) and FST. Acute LCGA-17 administration showed anxiolytic and antidepressant effects in EPM and FST, similar to diazepam and ketamine, respectively. In the POCPA study, LCGA-17 significantly reduced place aversion, with efficacy greater than doxazosin. After CUS, chronic LCGA-17 administration reversed stress-induced alterations in numerous behavioral tests (SI, FUST, SPT, and FST), producing significant anxiolytic and antidepressant effects. Finally, LCGA-17 restored the norepinephrine levels in the hippocampus following stress. Together, these results support the further development of the LCGA-17 peptide as a rapid-acting anxiolytic and antidepressant.

In silico Screening and Behavioral Validation of a Novel Peptide, LCGA-17, With Anxiolytic-Like Properties.

The aim of the study was to develop better anxiolytics and antidepressants. We focused on GABA A receptors and the α2δ auxiliary subunit of V-gated Ca2+ channels as putative targets because they are established as mediators of efficacious anxiolytics, antidepressants, and anticonvulsants. We further focused on short peptides as candidate ligands because of their high safety and tolerability profiles. We employed a structural bioinformatics approach to develop novel tetrapeptides with predicted affinity to GABA A receptors and α2δ. In silico docking studies of one of these peptides, LCGA-17, showed a high binding score for both GABA A receptors and α2δ, combined with anxiolytic-like properties in a Danio rerio behavioral screen. LCGA-17 showed anxiolytic-like effects in the novel tank test, the light-dark box, and the social preference test, with efficacy comparable to fluvoxamine and diazepam. In binding assays using rat brain membranes, [3H]-LCGA-17 was competed more effectively by gabapentinoid ligands of α2δ than ligands of GABA A receptors, suggesting that α2δ represents a likely target for LCGA-17. [3H]-LCGA-17 binding to brain lysates was unaffected by competition with ligands for GABAB, glutamate, dopamine, serotonin, and other receptors, suggesting specific interaction with α2δ. Dose-finding studies in mice using acute administration of LCGA-17 (i.p.) demonstrated anxiolytic-like effects in the open field test, elevated plus maze, and marble burying tests, as well as antidepressant-like properties in the forced swim test. The anxiolytic effects were effectively blocked by bicuculline. Therefore, LCGA-17 is a novel candidate anxiolytic and antidepressant that may act through α2δ, with possible synergism by GABA A receptors.

EGR1 and RXRA transcription factors link TGF-ß pathway and CCL2 expression in triple negative breast cancer cells.

Transforming growth factor beta (TGF-ß) is the main cytokine responsible for the induction of the epithelial-mesenchymal transition of breast cancer cells, which is a hallmark of tumor transformation to the metastatic phenotype. Recently, research demonstrated that the chemokine CCL2 gene expression level directly correlates with the TGF-ß activity in breast cancer patients. CCL2 attracts tumor-associated macrophages and is, therefore, considered as an important inductor of breast cancer progression; however, the precise mechanisms underlying its regulation by TGF-ß are unknown. Here, we studied the behavior of the CCL2 gene in MDA-MB-231 and HCC1937 breast cancer cells representing mesenchymal-like phenotype activated by TGF-ß. Using bioinformatics, deletion screening and point mutagenesis, we identified binding sites in the CCL2 promoter and candidate transcription factors responsible for its regulation by TGF-ß. Among these factors, only the knock-down of EGR1 and RXRA made CCL2 promoter activity independent of TGF-ß. These factors also demonstrated binding to the CCL2 promoter in a TGF-ß-dependent manner in a chromatin immunoprecipitation assay, and point mutations in the EGR1 and RXRA binding sites totally abolished the effect of TGF-ß. Our results highlight the key role of EGR1 and RXRA transcription factors in the regulation of CCL2 gene in response to TGF-ß pathway.

Active immunization against serum alcohol dehydrogenase normalizes brain dopamine metabolism disturbed during chronic alcohol consumption.

Chronic ethanol consumption in high doses is associated with constitutively elevated activity of the serum alcohol dehydrogenase I (ADH I) isoform, which demonstrates a high affinity not only for ethanol but also for a number of bioamine metabolites. Such excessive ADH activity is probably associated with disruptions in the metabolism of neurotransmitters (dopamine, serotonin, and norepinephrine) and subsequent long-term changes in the activity of their receptors. Ultimately, a stable depressive-like condition contributes to the development of patients' craving for ethanol intake, frequent disruptions during therapy, and low efficacy of treatment. We applied active immunization against ADH to investigate its efficacy in the reduction of excessive serum ADH activity and regulation of ethanol consumption by chronically ethanol-fed Wistar rats (15% ethanol, 4 months, free-choice method), and we analyzed its ability to influence the levels of bioamines in the brain. Immunization (2 injections, 2-week intervals) was performed using a combination of recombinant horse ADH isozyme as an antigen and 2% aluminum hydroxide-based adjuvant. The efficacy of immunization was demonstrated by the production of high titers of ADH-specific antibodies, which was consistent with the significantly reduced ADH activity in the serum of chronically ethanol-fed rats. On the 26th day after the first vaccine injection, we registered significantly lower levels of alcohol consumption compared to ethanol-fed control animals, and the difference reached 16% on the 49th day of the experiment. These observations were accompanied by data that showed reduced levels of ethanol preference in immunized rats. Chronic alcohol drinking led to a decrease in dopamine and DOPAL (a direct dopamine metabolite and a high-affinity ADH substrate) levels in the striatum,while immunization neutralized this effect. Additionally, we observed that inhibition of serum ADH activity caused a decrease in peak dopamine levels during acute alcohol intake in chronically ethanol-fed rats during ethanol withdrawal that was associated with reduced tyrosine hydroxylase activity in the striatum. The obtained data suggest a significant contribution of ADH to the changes in neurotransmitter systems during chronic alcohol consumption and make available new prospects for developing innovative strategies for treatment of excessive alcohol intake.

Minor C allele of the SNP rs7873784 associated with rheumatoid arthritis and type-2 diabetes mellitus binds PU.1 and enhances TLR4 expression.

Toll-like receptor 4 (TLR4) is an innate immunity receptor predominantly expressed on myeloid cells and involved in the development of various diseases, many of them with complex genetics. Here we present data on functionality of single nucleotide polymorphism rs7873784 located in the 3'-untranslated region (3'-UTR) of TLR4 gene and associated with various pathologies involving chronic inflammation. We demonstrate that TLR4 3'-UTR strongly enhanced the activity of TLR4 promoter in U937 human monocytic cell line while minor rs7873784(C) allele created a binding site for transcription factor PU.1 (encoded by SPI1 gene), a known regulator of TLR4 expression. Increased binding of PU.1 further augmented the TLR4 transcription while PU.1 knockdown or complete disruption of the PU.1 binding site abrogated the effect. We hypothesize that additional functional PU.1 site may increase TLR4 expression in individuals carrying minor C variant of rs7873784 and modulate the development of certain pathologies, such as rheumatoid arthritis and type-2 diabetes mellitus.

Glucocorticoid Receptor Binding Inhibits an Intronic IL33 Enhancer and is Disrupted by rs4742170 (T) Allele Associated with Specific Wheezing Phenotype in Early Childhood.

Interleukin 33 (IL-33) is a cytokine constitutively expressed by various cells of barrier tissues that contribute to the development of inflammatory immune responses. According to its function as an alarmin secreted by lung and airway epithelium, IL-33 plays a significant role in pathogenesis of allergic disorders. IL-33 is strongly involved in the pathogenesis of asthma, anaphylaxis, allergy and dermatitis, and genetic variations in IL33 locus are associated with increased susceptibility to asthma. Genome-wide association studies have identified risk "T" allele of the single-nucleotide polymorphism rs4742170 located in putative IL33 enhancer area as susceptible variant for development of specific wheezing phenotype in early childhood. Here, we demonstrate that risk "T" rs4742170 allele disrupts binding of glucocorticoid receptor (GR) transcription factor to IL33 putative enhancer. The IL33 promoter/enhancer constructs containing either 4742170 (T) allele or point mutations in the GR-binding site, were significantly more active and did not respond to cortisol in a pulmonary epithelial cell line. At the same time, the constructs containing rs4742170 (C) allele with a functional GR-binding site were less active and further inhibitable by cortisol. The latter effect was GR-dependent as it was completely abolished by GR-specific siRNA. This mechanism may explain the negative effect of the rs4742170 (T) risk allele on the development of wheezing phenotype that strongly correlates with allergic sensitization in childhood.

The Risk G Allele of the Single-Nucleotide Polymorphism rs928413 Creates a CREB1-Binding Site That Activates IL33 Promoter in Lung Epithelial Cells.

Cytokine interleukin 33 (IL-33) is constitutively expressed by epithelial barrier cells, and promotes the development of humoral immune responses. Along with other proinflammatory mediators released by the epithelium of airways and lungs, it plays an important role in a number of respiratory pathologies. In particular, IL-33 significantly contributes to pathogenesis of allergy and asthma; genetic variations in the IL33 locus are associated with increased susceptibility to asthma. Large-scale genome-wide association studies have identified minor "G" allele of the single-nucleotide polymorphism rs928413, located in the IL33 promoter area, as a susceptible variant for early childhood and atopic asthma development. Here, we demonstrate that the rs928413(G) allele creates a binding site for the cAMP response element-binding protein 1 (CREB1) transcription factor. In a pulmonary epithelial cell line, activation of CREB1, presumably via the p38 mitogen-activated protein kinases (MAPK) cascade, activates the IL33 promoter containing the rs928413(G) allele specifically and in a CREB1-dependent manner. This mechanism may explain the negative effect of the rs928413 minor "G" allele on asthma development.

Protective C allele of the single-nucleotide polymorphism rs1335532 is associated with strong binding of Ascl2 transcription factor and elevated CD58 expression in B-cells.

CD58 is expressed on the surface of antigen-presenting cells, including B-cells, and provides co-stimulation to regulatory T-cells (Treg) through CD2 receptor binding. Tregs appear to be essential suppressors of tissue-specific autoimmune responses. Thereby, CD58 plays protective role in multiple sclerosis (MS) and CD58 was identified among several loci associated with MS susceptibility. Minor (C) variant of the single-nucleotide polymorphism (SNP) rs1335532 is associated with lower MS risk according to genome-wide association studies (GWAS) and its presence correlates with higher CD58 mRNA levels in MS patients. We found that genomic region containing rs1335532 has enhancer properties and can significantly boost the CD58 promoter activity in lymphoblast cells. Using bioinformatics and pull-down assay we found that the protective (C) rs1335532 allele created functional binding site for ASCL2 transcription factor, a target of the Wnt signaling pathway. Both in B-lymphoblastoid cell lines and in primary B-cells, as well as in a monocytic cell line, activation of Wnt signaling resulted in an increased CD58 promoter activity in the presence of the protective but not the risk allele of rs1335532, whereas ASCL2 knockdown abrogated this effect. In summary, our results suggest that ASCL2 mediates the protective function of rs1335532 minor (C) allele in MS.

Hypoacylated LPS from Foodborne Pathogen Campylobacter jejuni Induces Moderate TLR4-Mediated Inflammatory Response in Murine Macrophages.

Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni, the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.

p63 and p73 repress CXCR5 chemokine receptor gene expression in p53-deficient MCF-7 breast cancer cells during genotoxic stress.

Many types of chemotherapeutic agents induce of DNA-damage that is accompanied by activation of p53 tumor suppressor, a key regulator of tumor development and progression. In our previous study we demonstrated that p53 could repress CXCR5 chemokine receptor gene in MCF-7 breast cancer cells via attenuation of NFkB activity. In this work we aimed to determine individual roles of p53 family members in the regulation of CXCR5 gene expression under genotoxic stress. DNA-alkylating agent methyl methanesulfonate caused a reduction in CXCR5 expression not only in parental MCF-7 cells but also in MCF-7-p53off cells with CRISPR/Cas9-mediated inactivation of the p53 gene. Since p53 knockout was associated with elevated expression of its p63 and p73 homologues, we knocked out p63 using CRISPR/Cas9 system and knocked down p73 using specific siRNA. The CXCR5 promoter activity, CXCR5 expression and CXCL13-directed migration in MCF-7 cells with inactivation of all three p53 family genes were completely insensitive to genotoxic stress, while pairwise p53+p63 or p53+p73 inactivation resulted in partial effects. Using deletion analysis and site-directed mutagenesis, we demonstrated that effects of NFkB on the CXCR5 promoter inversely correlated with p63 and p73 levels. Thus, all three p53 family members mediate the effects of genotoxic stress on the CXCR5 promoter using the same mechanism associated with attenuation of NFkB activity. Understanding of this mechanism could facilitate prognosis of tumor responses to chemotherapy.

Individual roles of brain and serum alcohol dehydrogenase isoforms in regulation of alcohol consumption in SPF Wistar rats.

Alcohol dehydrogenases (ADH) are key enzymes of ethanol metabolism that mediate its oxidation to acetaldehyde. ADHs are also able to oxidize some types of neurotransmitters such as dopamine, serotonin and norepinephrine. Increased level of ADHs activity, induced by chronic alcohol consumption, is presumably associated with disturbed neurotransmitters metabolism that leads to stable alcohol craving. As earlier reported, intraperitoneal administration of 4-methilpirasole (non-specific inhibitor of ADHs) has shown to provide a short-term anti-alcoholic effect, but individual roles of ADH isoforms in this process were still unclear. The aim of this work was to study the roles of brain and serum ADH isoforms in alcohol consumption and neurotransmitter metabolism in the rats. In the study we used specific-pathogen-free (SPF) Wistar rats chronically alcoholized with 15% ethanol. 4-methilpirasole intranasal administration in small doses led to local inhibition of ADH III activity in the brain estimated by spectrophotometric assay. It correlated with dose-dependent reduction of dopamine concentration and increased level of its metabolic products in the brain but did not influence alcohol consumption. These data allowed us to propose an important role of brain ADHs (predominantly ADH III) in metabolism of dopamine in chronically alcoholized rats but not in regulation of alcohol consumption. To evaluate the role of serum ADH isoforms we immunized the rats with recombinant horse ADH that led to production of high levels of cross-reactive anti-ADH antibodies verified by ELISA assay. Immunization led to 30% decrease in alcohol consumption and recovery of general behavioral parameters such as motor activity, anxiety and depression level. At the same time active immunization did not cause any impairments in animal blood composition. We can conclude that immunization against ADHs appeared to be a safe way to decrease alcohol consumption that could be possibly associated with neurotransmitters metabolism correction.

The A Allele of the Single-Nucleotide Polymorphism rs630923 Creates a Binding Site for MEF2C Resulting in Reduced CXCR5 Promoter Activity in B-Cell Lymphoblastic Cell Lines.

Chemokine receptor CXCR5 is highly expressed in B-cells and under normal conditions is involved in their migration to specific areas of secondary lymphoid organs. B-cells are known to play an important role in various autoimmune diseases including multiple sclerosis (MS) where areas of demyelinating lesions attract B-cells by overexpressing CXCL13, the CXCR5 ligand. In this study, we aimed to determine the functional significance of single-nucleotide polymorphism rs630923 (A/C), which is located in cxcr5 gene promoter, and its common allele is associated with increased risk of MS. Using bioinformatics and pull-down assay in B-lymphoblastic cell lines, we showed that protective minor rs630923 "A" allele created functional binding site for MEF2C transcription factor. Elevated MEF2C expression in B-cells correlated with reduced activity of cxcr5 promoter containing rs630923 "A" allele. This effect that was fully neutralized by MEF2C-directed siRNA may mechanistically explain the protective role of the rs630923 minor allele in MS. Using site-directed mutagenesis of the cxcr5 gene promoter, we were unable to find any experimental evidence for the previously proposed role of NFκB transcription factors in rs630923-mediated CXCR5 promoter regulation. Thus, our results identify MEF2C as a possible mediator of protective function of the rs630923 "A" allele in MS.

p53-dependent expression of CXCR5 chemokine receptor in MCF-7 breast cancer cells.

Elevated expression of chemokine receptors in tumors has been reported in many instances and is related to a number of survival advantages for tumor cells including abnormal activation of prosurvival intracellular pathways. In this work we demonstrated an inverse correlation between expression levels of p53 tumor suppressor and CXCR5 chemokine receptor in MCF-7 human breast cancer cell line. Lentiviral transduction of MCF-7 cells with p53 shRNA led to elevated CXCR5 at both mRNA and protein levels. Functional activity of CXCR5 in p53-knockdown MCF-7 cells was also increased as shown by activation of target gene expression and chemotaxis in response to B-lymphocyte chemoattractant CXCL13. Using deletion analysis and site-directed mutagenesis of the cxcr5 gene promoter and enhancer elements, we demonstrated that p53 appears to act upon cxcr5 promoter indirectly, by repressing the activity of NFκB transcription factors. Using chromatin immunoprecipitation and reporter gene analysis, we further demonstrated that p65/RelA was able to bind the cxcr5 promoter in p53-dependent manner and to directly transactivate it when overexpressed. Through the described mechanism, elevated CXCR5 expression may contribute to abnormal cell survival and migration in breast tumors that lack functional p53.