Background. Tuberculous inflammation is mediated by a complex molecular signaling pathway, the analysis of which makes it possible to identify promising biomarkers and targets for the development of new diagnostic, prognostic and pharmacological approaches in order to improve the effectiveness of anti-tuberculosis chemotherapy. Determining the relationship between key inflammatory cytokines, the multidrug-resistant protein P-gp and the activity of specific inflammation in the surgical material of patients with pulmonary tuberculosis may prove to be a novel tool in the development of pathogenetic therapy and personalized medicine. Aims — to characterize molecular and genetic profiles of tuberculomas and identify genes that correlate with the expression of the ABCB1 gene of the P-gp protein in the surgical material of patients with pulmonary tuberculosis. Research objectives: 1) to obtain molecular and genetic characteristics of tuberculosis by real-time PCR and compare it with the activity of tuberculous inflammation; 2) to carry out a correlation analysis between the expression of the ABCB1 gene and key cytokines of the tuberculosis process: IL-6, IL-10, IFN-γ, TGF-β, TNF-α, IL-1β. Methods. A prospective cohort study was conducted on the basis of the FSBI CTRI. The object of the study was the surgical material of 35 patients diagnosed with multiple pulmonary tuberculomas. Histological examination methods were used for the morphological assessment of the surgical material. A real-time quantitative PCR method was used to analyze gene expression. Statistical processing was performed using the GraphPad Prism Version 7.04 software package (GraphPad Software, USA). The data is presented as a median with an interquartile range. The nonparametric Mann–Whitney U-test was used to compare the two groups. All p-values were two-sided and p 0.05 was considered statistically significant. The correlation between the variables was estimated using the Spearman correlation coefficient. The correlation analysis was carried out in the Microsoft Office Excel 2010 Software. Results. The study revealed that the highest level of expression of ABCB1 gene of the P-gp protein is observed in tuberculomas with high activity of tuberculous inflammation, and its expression is correlated with the expression of the IL6 gene (p 0.001) and the expression of the IL10 gene (p 0.01). Tuberculomas of this group are also characterized by higher expression of the TGFB1, TNF and IL1B genes, compared with the group of moderate activity of specific inflammation. Conclusions. The data obtained indicate that in addition to pro-/anti-inflammatory cytokines, the P-gp protein plays an important role in the pathogenesis of tuberculous inflammation, especially with its high activity. Further clarification of the P-gp role in tuberculous inflammation may be an important step for the development of new approaches to treat tuberculosis using methods of HDT and personalized medicine.

Accurate normalization of qRT-PCR data in pulmonary tuberculosis (TB) research requires reference genes whose expression is invariant across clinically relevant matrices, yet no studies have addressed this in lesion tissue and blood concurrently. We assessed the expression stability of eight popular housekeeping genes-ACTB, B2M, GAPDH, HPRT1, PPIA, RPL13A, UBC and YWHAZ-in lung tuberculomas and peripheral blood mononuclear cells (PBMCs) from TB patients. Standardized extraction and amplification yielded Cq values that were ranked by geNorm, NormFinder, BestKeeper and comparative Delta CT, with consensus scores generated in RefFinder; and correlation analysis was conducted in order to select the most suitable genes to work collectively for future normalization. The consensus analysis placed PPIA, YWHAZ and HPRT1 at the top, while GAPDH and UBC were the least stable. Our findings endorse a three-gene panel (PPIA, YWHAZ, HPRT1) for robust normalization of host gene-expression studies in both lesion tissue and PBMCs in pulmonary TB and highlight the necessity of context-specific reference-gene validation.

P-glycoprotein (encoded by the ABCB1 gene) has a dual role in regulating inflammation and reducing chemotherapy efficacy in various diseases, but there are few studies focused on pulmonary TB patients. In this study, our objective was to identify a list of genes that correlate with high and low levels of ABCB1 gene expression in the lungs of pulmonary TB patients with different activity of chronic granulomatous inflammation. We compared gene expression in two groups of samples (with moderate and high activity of tuberculomas) to identify their characteristic gene signatures. Gene expression levels were determined using quantitative PCR in samples of perifocal area of granulomas, which were obtained from 65 patients after surgical intervention. Subsequently, two distinct gene signatures associated with high inflammation activity were identified. The first signature demonstrated increased expression of HIF1a, TGM2, IL6, SOCS3, and STAT3, which correlated with high ABCB1 expression. The second signature was characterized by high expression of TNFa and CD163 and low expression of ABCB1. These results provide insight into various inflammatory mechanisms and association with P-gp gene expression in lung tissue of pulmonary TB patients and will be useful in the development of a host-directed therapy approach to improving the effectiveness of anti-TB treatment.

The problem of reduced effectiveness of antituberculosis drugs is currently associated not only with the development of drug resistance in the pathogen but also with the functional activity of the P-gp (P-glycoprotein) multidrug resistance protein of macroorganism cells. One of the main antituberculosis drugs, rifampicin (RIF), is a substrate for P-gp, which reduces its effectiveness. The article presents the data on the P-gp activity in proinflammatory human macrophages and assesses the effect of a new form of RIF encapsulated in lactic acid polymer nanoparticles, approved for medical use, on its activity. It was shown that THP-1 macrophages, in contrast to the THP-1 monocytes, are characterized by the functional activity increase of P-gp during differentiation as well as the preservation of the viability in the presence of RIF, which is associated with the P-gp activation. The new form of RIF is nontoxic to THP-1 monocytes and macrophages in comparison with the traditional form. When using the encapsulated form of RIF, the process of endocytosis/phagocytosis in macrophages is activated and the functional activity of P-gp decreases. The data obtained demonstrate that the development of encapsulated antituberculosis drugs, phagocytosis activators, which targetedly reduce the P-gp activity and thus affect the functional characteristics of human macrophages, seems to be a promising and relevant direction for increasing the effectiveness of antituberculosis drugs.

Background. The effect on the activity of the multidrug resistance protein P-glycoprotein (P-gp, MDR1 gene) in pro-inflammatory (M1) human macrophages is considered one of the promising strategies for increasing the effectiveness of the treatment in patients with pulmonary tuberculosis: P-gp activity is considered a factor that reduces intracellular accumulation of rifampicin (RIF), a substrate for P-gp. The aim of this work was to reveal the effect of the therapeutic concentration of RIF on the activity of P-gp in M1 human macrophages. The objectives were as follows: to determine the expression levels of the MDR1 gene, P-gp protein, as well as its functional activity at different periods of cell differentiation and under the influence of RIF.Material and methods. The following cell lines were used in the work: suspension cells of promonocytic leukemia THP-1 and THP-1 macrophages induced by phorbol ether according to the pro-inflammatory phenotype. Suspension cells of myeloid leukemia K562/IS-9 transfected with the MDR1 gene were used as a comparison group. An important factor is the choice of the experimental concentration of RIF: the average concentration of the drug in patients with pulmonary tuberculosis was 10 µg/ml. The methods of RT-PCR, immunocytochemistry, and flow cytometry were used in the work.Results and discussion. The induction of MDR1 gene expression in M1 macrophages under short-term exposure to a therapeutic concentration of RIF was revealed. This effect is typical only for THP-1 macrophages, in which a significant functional activity of P-gp is registered. This induction does not occur in the cells with no detectable P-gp activity (THP-1 suspension cells). This indicates the presence of different mechanisms of RIF influence on MDR1, which can be used to develop a strategy for P-gp inhibition in inflammatory macrophages.Conclusion. Given the key role of macrophages in tuberculosis, further evaluation of MDR1/P-gp in the surgical material of patients with pulmonary tuberculosis is necessary, which makes it possible to draw a conclusion that it is necessary to develop and apply drug strategies aimed at blocking the functional activity of P-gp and choosing more effective anti-tuberculosis therapy regimens.

Early (preclinical) diagnosis of Parkinsons disease (PD) is a major challenge in modern neuroscience. The objective of this study was to experimentally evaluate a diagnostic challenge test with monoiodotyrosine (MIT), an endogenous inhibitor of tyrosine hydroxylase. Striatal dopamine was shown to decrease by 34% 2 h after subcutaneous injection of 100 mg/kg MIT to intact mice, with the effect not being amplified by a further increase in the MIT dose. The selected MIT dose caused motor impairment in a neurotoxic mouse model of preclinical PD, but not in the controls. This was because MIT reduced striatal dopamine to the threshold of motor symptoms manifestation only in PD mice. Therefore, using the experimental mouse model of preclinical PD, we have shown that a MIT challenge test may be used to detect latent nigrostriatal dysfunction.

Herein, we describe the design, synthesis, and biological evaluation of novel betulin and N-acetyl-d-galactosamine (GalNAc) glycoconjugates and suggest them as targeted agents against hepatocellular carcinoma. We prepared six conjugates derived via the C-3 and C-28 positions of betulin with one or two saccharide ligands. These molecules demonstrate high affinity to the asialoglycoprotein receptor (ASGPR) of hepatocytes assessed by in silico modeling and surface plasmon resonance tests. Cytotoxicity studies in vitro revealed a bivalent conjugate with moderate activity, selectivity of action, and cytostatic properties against hepatocellular carcinoma cells HepG2. An additional investigation confirmed the specific engagement with HepG2 cells by the enhanced generation of reactive oxygen species. Stability tests demonstrated its lability to acidic media and to intracellular enzymes. Therefore, the selected bivalent conjugate represents a new potential agent targeted against hepatocellular carcinoma. Further extensive studies of the cellular uptake in vitro and the real-time microdistribution in the murine liver in vivo for fluorescent dye-labeled analogue showed its selective internalization into hepatocytes due to the presence of GalNAc ligand in comparison with reference compounds. The betulin and GalNAc glycoconjugates can therefore be considered as a new strategy for developing therapeutic agents based on natural triterpenoids.

Objectives. To identify changes in the biochemical composition of the plasma in a group of patients at risk of developing Parkinson’s disease (PD) at the prodromal stage in comparison with age-matched controls. Materials and methods. Subjects in the risk group were selected on the basis of the having impairments to sleep, olfaction, and peristalsis. The risk group consisted of 12 people and the control group of eight people. Results. The results showed that of seven catecholamines and their metabolites, the only blood change was in the L-dihydroxyphenylalanine (L-DOPA) level, which decreased in the risk group from the level in controls. A decreased L-DOPA concentration is regarded as a marker for selective degeneration of central and peripheral catecholaminergic neurons in PD. In contrast to L-DOPA, the blood concentrations of seven of 12 sphingomyelins increased. Given that changes in sphingomyelin metabolism are linked with apoptosis, autophagy, and synucleinopathies, increases in their concentrations in the risk group are regarded as indicators of systemic degeneration of central and peripheral neurons. Furthermore, the risk group showed a tendency to decreased urate concentrations, which are endogenous neuroprotectors. Conclusions. The results obtained here suggest that changes in blood L-DOPA, sphingomyelin, and urate levels can serve as diagnostic markers for the development of PD at the prodromal stage.

The paper summarizes the literature and author's data on the development of early (preclinical) diagnosis of Parkinson's disease (PD). Implementation of this diagnosis will promote the use of preventive therapy and change investments in diagnosis and treatment of patients. The paper declares that at present the only approach to early diagnosis of PD is positron-emission tomography of the nigrostriatal dopaminergic system, but it cannot be used for preventive examination due to its high cost. The authors consider that a less specific, but more promising approach to the development of early diagnosis of PD is the search for markers in body fluids, mainly in the blood, in patients at the prodromal stage of PD. Indeed, a number of markers as changes in the level of metabolites of monoamines, sphingolipids, urates, and indicators of oxidative stress were found in patients selected for the risk group of the prodromal stage of PD, according to characteristic premotor symptoms. In addition, it is assumed that the search for blood markers at an earlier - pre-prodromal stage is possible only in animal models of PD at the early preclinical stage. This approach can also be used to verify blood markers identified in patients at the clinical stage of PD. It is also evident that the complex socio-economic factors influencing the incidence of PD is different in developed versus developing countries. The societal and medical costs of Parkinson's are huge and efforts to improve early preclinical diagnosis of PD will lead to considerable economical and societal benefits. For instance this will allow efficient selection of patients for preclinical diagnostic tests. To assess the effectiveness of this strategy considering the uncertainty of socio-economic issues, a modification of the «cost-utility» analysis is proposed. For the first time, a Markov model of PD including preclinical diagnostic tests and possible neuroprotective therapy was developed and studied. Analytical outcomes of this process suggest that the idea of developing a new multimodal strategy is promising from a socio-economic point of view.

Актуальность: YB-1 - мультифункциональный белок, фактор транскрипции, участвующий в регуляции многочисленных клеточных процессов. YB-1 является важным участником молекулярных каскадов, через которые регулируются реакции организма на присутствие возбудителя, активность воспаления, эффективность заживления и излечения. Эти функции YB-1 являются актуальными и при таком социально-значимом заболевании, как туберкулёз лёгких, при котором выявление транскрипционной активности YBX1 и роли YB1 в патогенезе ранее не проводилось. В связи с этим, была поставлена цель исследования: определить у больных туберкулёзом лёгких (туберкулёмами) наиболее значимые корреляции между экспрессией гена белка YB-1, генами ключевых цитокинов, участвующих в регуляции воспаления при ТБ (IL-6, IL-10, INF-γ, TGF-β, TNF-α, IL-1β), фактором гипоксии HIF1a и геном белка P-gp ABCB1. Методы: уровни экспрессии генов определяли с помощью количественной ПЦР в образцах перифокальной области гранулем, полученных от 35 пациентов в ходе плановой операции. На основе результатов ПЦР был проведен корреляционный и иерархический кластерный анализ. Статистическую обработку проводили с помощью статистического пакета GraphPad Prism Version 7.04. Корреляцию между переменными оценивали с помощью коэффициента корреляции Спирмена. Иерархический кластерный анализ и тепловые карты выполняли с использованием инструмента ClustVis 2.0. Результаты: обнаружена положительная корреляция между экспрессией генов YBX1, TGFB1 и ABCB1. Корреляция между экспрессией генов YBX1 и ABCB1 умеренная, при этом экспрессия гена ABCB1 имеет сильную положительную корреляцию с генами HIF1A и IL6. Наиболее сильная корреляция выявлена между YBX1 и TGFB1 (r=0,62). Корреляция YBX1 с генами других цитокинов отсутствует. При этом TGFB1 имеет умеренную корреляцию с TNF (r=0,56). Взаимосвязь YBX1 с TGFB1 также подтвердил иерархический кластерный анализ, который демонстрирует формирование единого кластера генов YBX1, TGFB1, TNF. Обсуждение и заключение: мы предполагаем, что выявленный в нашей работе кластер генов YBX1, TGFB1, TNF образует регуляторную систему, играющую важную роль в развитии туберкулезного воспаления. Наша работа расширяет данные о молекулярно-генетической характеристике одной из форм туберкулёзом лёгких – туберкулёмах. Мы предполагаем, что белок YB-1 потенциально может выполнять при туберкулёзом лёгких разные функции: выступать в качестве одного из участников туберкулёзного воспаления через влияние на экспрессию генов ключевых цитокинов, а также модулировать активность белка-транспортёра P-gp и изменять фармакокинетику противотуберкулёзных препаратов, что требует дальнейшего изучения.

Nigrostriatal dopaminergic neurons (DNs), involved in the regulation of motor function, are characterized by a high plasticity. Indeed, at the death of up to 50% of DNs in Parkinson’s disease, the survived neurons provide normal regulation. This study was aimed to determine whether the vesicle cycle proteins, syntaxin Ia (Syn Ia), synaptotagmin I (Syt I), Rab5a, and complexins I and II (Cmpx I and II) are involved in the mechanisms of neuroplasticity in the substantia nigra, which mainly contains cell bodies and processes of the DNs. In the neurotoxic models of Parkinson’s disease in mice, it was shown that, at the degeneration of up to 50% of DNs, the content of Syt I, Syn Ia, and Cmpх I and II, involved in vesicle exocytosis, does not change in the substantia nigra as a whole but is compensatorily increased in individual survived DNs. Thus, the data obtained in this study suggest that the impairment of motor behavior, which occurs at the death of half of the nigrostriatal DNs, is not caused by the impairment of the production of vesicle cycle proteins in the survived DNs.

Нигростриатные дофаминергические нейроны (ДН), участвующие в регуляции двигательной функции, обладают высокой пластичностью. При гибели до 50% этих нейронов при болезни Паркинсона выжившие нейроны обеспечивают нормальную регуляцию двигательной функции. В данной работе была поставлена задача определить, вовлечены ли белки везикулярного цикла - синтаксин Iа (Syn Ia), синаптотагмин I (Syt I), Rab5a и комплексины I и II (Cmpх I и II) - в механизмы нейропластичности в чёрной субстанции, содержащей преимущественно тела и отростки ДН. На нейротоксических моделях болезни Паркинсона у мышей показано, что при дегенерации до 50% ДН содержание Syt I, Syn Ia, Cmpх I и II, участвующих в экзоцитозе везикул, в чёрной субстанции в целом не изменяется, а в отдельных выживших ДН компенсаторно увеличивается. Таким образом, данные, полученные в этой работе, позволяют предположить, что нарушение моторного поведения, возникающее при гибели половины нигростриатных ДН, не является следствием нарушения продукции белков везикулярного цикла в выживших ДН.