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Influence of ZP2 peptide, a synthetic analogue of the GM-GSF active center on the anticytokine activity of bacteria from Enterococcus genus and their ability to produce cytokine-like substances GM-GSF活性中心合成类似物ZP2肽对肠球菌属细菌抗细胞因子活性及其产生细胞因子样物质能力的影响
Pub Date : 2022-10-07 DOI: 10.46235/1028-7221-1162-ioz
O. A. Pashinina, O. L. Kartashova, T. M. Pashkova, V. Gritsenko
Peptide ZP2, a synthetic analogue of the active center of granulocyte-macrophage colony stimulating factor (GM-CSF), exhibits a wide range of immunobiological effects, including antibacterial activity. At the same time, its effect (in sub-inhibitory concentrations) on the biological properties of Enterococcus spp., the causative agents of many infectious and inflammatory diseases remains poorly understood. The aim of our study was to analyze the nature of the effect of synthetic peptide ZP2 on anticytokine activity (ACA) and its ability to produce cytokine-like substances (CLS) in Enterococcus spp. 18 clinical isolates of Enterococcus spp. were used. Over the experiments, the bacterial strains were cultured in Schaedlers broth with ZP2 peptide at 37 C for 24 hours. No specific peptide was added in the control. ACA for IL-8, TNF and IL-17A and the production of the corresponding CLS were determined by the ELISA method. To assess ACA, the proportion of cytokine inactivation in the experiment relative to the control was calculated and expressed in pg/ml; CLS production was evaluated by the level of cytokines in the experiment and control, expressed as pg/ml. The data were subjected to statistical processing. It was revealed that Enterococcus spp. strains are capable of secreting compounds that inactivate cytokines IL-8, IL-17A and TNF, and produce CLS in the culture medium. Intragenital and intraspecific variability was noted in the presence and frequency of occurrence and in the severity of these properties. It was found that the ZP2 peptide in E. faecium increases ACA with respect to all studied cytokines. When tested with E. faecalis, it either did not affect their ACA against TNF and IL-8, or completely inhibited ACA for IL-17A. At the same time, ZP2 blocked the production of CLS, e.g., IL-17A and IL-8 in E. faecium, but increased the production of CLS similar to TNF, and, with E. faecalis, it increased the number of IL-17A-producing isolates twofold, although the average level of production of these CLS was lower than in the control. Enterococcus spp strains are capable of secreting compounds that inactivate cytokines IL-8, TNF and IL- 17A, and may produce substances similar to these cytokines. The synthetic peptide ZP2 has a modifying effect on the manifestation of these properties by Enterococci. Further studies of biological and pathogenetic features of Enterococci and other bacterial species, as well as modifying effects of the ZP2 peptide are required.
肽ZP2是粒细胞-巨噬细胞集落刺激因子(GM-CSF)活性中心的合成类似物,具有广泛的免疫生物学作用,包括抗菌活性。与此同时,其对肠球菌(许多感染性和炎症性疾病的病原体)生物学特性的影响(在亚抑制浓度下)仍知之甚少。本研究的目的是分析合成肽ZP2对肠球菌抗细胞因子活性(ACA)及其产生细胞因子样物质(CLS)能力的影响。在实验中,菌株在含ZP2肽的Schaedlers肉汤中37℃培养24小时。对照组不添加特异性肽。ELISA法测定IL-8、TNF、IL-17A的ACA及相应CLS的产生。计算实验组相对于对照组的细胞因子失活比例,以pg/ml表示;通过实验和对照细胞因子水平来评价CLS的产生,以pg/ml表示。这些数据经过了统计处理。结果表明,肠球菌菌株能够分泌灭活细胞因子IL-8、IL-17A和TNF的化合物,并在培养基中产生CLS。在这些特性的存在和发生频率以及严重程度上,注意到种群内和种内的变异性。结果发现,粪肠中ZP2肽增加了所有研究细胞因子的ACA。当用粪肠球菌进行测试时,它要么不影响它们对TNF和IL-8的ACA,要么完全抑制IL-17A的ACA。同时,ZP2阻断了粪肠杆菌中IL-17A和IL-8等CLS的产生,但增加了与TNF相似的CLS的产生,并且在粪肠杆菌中,它使产生IL-17A的分离株的数量增加了两倍,尽管这些CLS的平均产量低于对照组。肠球菌菌株能够分泌灭活细胞因子IL-8、TNF和IL- 17A的化合物,并可能产生与这些细胞因子相似的物质。合成的肽ZP2对肠球菌的这些特性的表现具有修饰作用。需要进一步研究肠球菌和其他细菌的生物学和致病特性,以及ZP2肽的修饰作用。
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引用次数: 0
Evaluation of specific T cell immune response to SARS-CoV-2 in COVID-19 infection and following Gam-COVID-Vac vaccine prophylaxis COVID-19感染和Gam-COVID-Vac疫苗预防后对SARS-CoV-2特异性T细胞免疫反应的评估
Pub Date : 2022-09-20 DOI: 10.46235/1028-7221-1111-eos
N. Plekhova, T. Sitdikova, A. A. Dubiy, A. O. Mikhailov, E. V. Prosekovа
An aberrant immune response during SARS-CoV-2 infection has been shown to determine the clinical features, disease severity, and progression of COVID-19 infection. This work aimed for comprehensive assessment of the immune response by comparative evaluation of diagnostic significance of the antibodies to RBD domain of the SARS-CoV-2 spike protein, as well as detection of effector CD4+ and CD8+T cells specific to SARS-CoV-2 antigens. The study was performed in unvaccinated persons, healthy individuals vaccinated with Gam-COVID-Vac, and in the patients who have had COVID-19 infection. We have found that IgG antibodies to the RBD domain of the SARS-CoV-2 spike protein are detectable at a frequency of 73% to 92% of cases in vaccinated persons and COVID-19 reconvalescents. The numbers of effector CD4+ and CD8+T lymphocytes responding to stimulation with SARS-CoV-2 antigens by producing the IFN cytokine varied depending on the introduced antigen and tended to be higher in vaccinated individuals. In non-vaccinated healthy persons who contacted with COVID-19 patients, T cell response to the SARS-CoV-2 nucleoproteins was revealed. For adequate assessment of antiviral and post-vaccination immune response to COVID-19, it would be necessary to study not only humoral immune response by the presence of antibodies, but also functionally active specific T lymphocytes directed for SARS-CoV-2 protein antigens.
研究表明,SARS-CoV-2感染期间的异常免疫反应决定了COVID-19感染的临床特征、疾病严重程度和进展。本研究旨在通过比较评估SARS-CoV-2刺突蛋白RBD结构域抗体的诊断意义,以及检测SARS-CoV-2抗原特异性的CD4+和CD8+效应T细胞,对免疫应答进行综合评估。该研究在未接种疫苗的人、接种了Gam-COVID-Vac的健康个体和感染了COVID-19的患者中进行。我们发现,在接种疫苗者和COVID-19康复者中,SARS-CoV-2刺突蛋白RBD结构域的IgG抗体的检测频率为73%至92%。效应CD4+和CD8+T淋巴细胞通过产生IFN细胞因子对SARS-CoV-2抗原刺激作出反应的数量取决于引入的抗原,并且在接种疫苗的个体中往往更高。在与COVID-19患者接触的未接种疫苗的健康人中,揭示了T细胞对SARS-CoV-2核蛋白的反应。为了充分评估抗病毒和疫苗接种后对COVID-19的免疫反应,不仅需要研究抗体存在的体液免疫反应,还需要研究针对SARS-CoV-2蛋白抗原的功能活跃的特异性T淋巴细胞。
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引用次数: 0
Morphological and functional characteristics of LPS-stimulated microglial cells under the action of orexin A 食欲素A作用下lps刺激的小胶质细胞形态学和功能特征
Pub Date : 2022-09-20 DOI: 10.46235/1028-7221-1144-maf
A. P. Synchikova, E. Korneva
Interest to the orexin-containing neurons is caused by their recent discovery and perspectives of their usage for treatment of different diseases. The studies in this area were launched recently and are of special interest since the opportunity of modulating functional activity of the brain immune system is of pivotal significance for therapy of various central nervous system (CNS) disorders providing novel ways of search and promising data on therapeutic effects of orexins in inflammatory, autoimmune diseases as well as malignant tumors. Some data from literature show that orexins may exert therapeutic effects in different disorders caused by altered neuroimmune interactions. Participation of this neuromediator system is shown in pathogenesis of narcolepsia, obesity, multiple sclerosis, Alzheimer disease, intestinal disorders, septic shock and cancer, due to involvement of orexins in functional regulation of various components of immune syste, e.g., microglial cell populations. Despite only scarce data on these effects, some experimental results obtained over last years, add to our understanding of orexin effects upon functional activity of the brain immune system. A number of previous studies allowed to assess the orexin effects on morpho-functional features of microglial cells activated by lipopolysaccharide (LPS), thus presenting a prospective for development of novel approaches to therapy of infectious, inflammatory, neurodegenerative and autoimmune disorders affecting CNS. In the present study, we aimed for detecting the effects of neuromediator orexin A upon functional traits of of microglial cells activated by LPS (M1 phenotype) as evaluated by changes of their size and length of their processes, as well as density of cell distribution. We have studied the changes of microglia cell numbers following intraperitoneal LPS injection. It was shown that, the LPS causes higher activation degree of these cells, i.e., the contents of microglial cells becomes increased in somatosensory area of the brain cortex. A series of these studies allowed us to demonstrate that intracerebroventricular injection of orexin A in animals following LPS injection does not cause detectable changes of the processes initiated by LPS. The comparative analysis did not detect any changes in length of microglial processes localized in somatosensory or motor cortical areas, and corpus striatum. Other parameters of the microglial cell activation will be studied in future.
对含食欲素的神经元的兴趣是由它们最近的发现和它们用于治疗不同疾病的前景引起的。这一领域的研究是最近才开始的,因为调节脑免疫系统功能活动的机会对各种中枢神经系统(CNS)疾病的治疗具有关键意义,为食欲素在炎症、自身免疫性疾病和恶性肿瘤的治疗作用提供了新的研究方法和有希望的数据。一些文献资料显示,食欲素可能对由神经免疫相互作用改变引起的不同疾病发挥治疗作用。由于食欲素参与免疫系统各种成分(如小胶质细胞群)的功能调节,这一神经介质系统在嗜睡症、肥胖、多发性硬化症、阿尔茨海默病、肠道疾病、感染性休克和癌症的发病机制中都有参与。尽管关于这些影响的数据很少,但过去几年获得的一些实验结果,增加了我们对食欲素对大脑免疫系统功能活动的影响的理解。许多先前的研究允许评估食欲素对脂多糖(LPS)激活的小胶质细胞形态功能特征的影响,从而为开发新方法治疗影响中枢神经系统的感染性、炎症性、神经退行性和自身免疫性疾病提供了前景。在本研究中,我们旨在检测神经介质食欲素A对LPS激活的小胶质细胞(M1表型)功能性状的影响,通过其突起的大小和长度以及细胞分布密度的变化来评估。我们研究了腹腔注射LPS后小胶质细胞数量的变化。结果表明,LPS使这些细胞的活化程度提高,即大脑皮层体感觉区小胶质细胞的含量增加。一系列的这些研究使我们能够证明,在LPS注射后在动物脑室内注射食欲素A不会引起由LPS启动的过程的可检测的变化。对比分析未发现位于体感觉或运动皮质区和纹状体的小胶质突起的长度有任何变化。小胶质细胞活化的其他参数将在未来进一步研究。
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引用次数: 0
Mast cells as biomarkers of inflamm-ageing 肥大细胞作为炎症老化的生物标志物
Pub Date : 2022-09-20 DOI: 10.46235/1028-7221-1107-mca
A. Sadek, Y. Khramtsova, B. Yushkov
Most mechanisms of ageing are believed to be more or less associated with inflammation. With age, a unique form of chronic inflammation develops which is termed as inflamm-ageing. The mechanisms of this process are still not fully clear due to the lack of reliable assessment criteria. Immune system is among those involved in accelerating age-related changes in the body. It also directly participates in the process of inflammation. In its pathogenesis, the reaction of mast cells may be of great importance. The role of mast cells in tissue remodeling deserves special attention, since the latter event is among the main features associated with ageing. Hence, the inflamm-ageing is considered a sufficient indicator of ageing, and the mast cells could provide biomarkers of this process. In order to test the proposed hypothesis, the present study was conducted to determine age-related morpho-functional changes in mast cell populations in various organs in rats. Some morpho-functional parameters of mast cells (number, synthetic and functional activity, degree of maturation) in different animal organs were evaluated in male Wistar rats of different ages (4 months and 2 years). We have found the age-dependent changes upon examination of thymus, adrenal glands, and skin, i.e., a decrease in the number of mast cells and their synthetic capacity, along with significantly increased functional activity. In the stomach, small and large intestines, at the constant number of mast cells, we revealed a decrease in their synthetic ability, and increased functional activity. These changes were accompanied by enlargement of blood vessels in the studied organs. Liver is the only organ which did not exhibit any changes in mast cell populations with age. The detected changes in mast cell populations may play an important role in formation of inflamm-ageing events, which accompany the ageing processes, because these cells are an integral component of inflammatory response. The progression of inflamm-ageing leads to accumulation of cytokines and pro-inflammatory mediators in tissues, which, in turn, activate the mast cells. At the same time, increased degranulation of mastocytes may promote the process of inflamm-ageing. The oberved mutual influence of mast cells and inflamm-ageing makes it possible to consider mastocytes as potential candidates for searching the biomarkers in inflamm-ageing.
大多数衰老机制被认为或多或少与炎症有关。随着年龄的增长,一种独特形式的慢性炎症发展,被称为炎症老化。由于缺乏可靠的评估标准,这一进程的机制仍然不完全清楚。免疫系统是参与加速与年龄相关的身体变化的因素之一。它还直接参与炎症的过程。在其发病机制中,肥大细胞的反应可能是非常重要的。肥大细胞在组织重塑中的作用值得特别关注,因为后者是与衰老相关的主要特征之一。因此,炎症老化被认为是衰老的充分指标,肥大细胞可以提供这一过程的生物标志物。为了验证这一假设,本研究确定了大鼠各器官肥大细胞群与年龄相关的形态功能变化。研究了不同年龄(4月龄和2岁)雄性Wistar大鼠在不同动物器官中肥大细胞的形态功能参数(数量、合成和功能活性、成熟程度)。通过对胸腺、肾上腺和皮肤的检查,我们发现了年龄依赖性的变化,即肥大细胞数量及其合成能力的减少,以及功能活性的显著增加。在胃、小肠和大肠中,在肥大细胞数量不变的情况下,我们发现它们的合成能力下降,功能活性增加。这些变化伴随着所研究器官血管的扩张。肝脏是唯一一个肥大细胞群不随年龄变化的器官。肥大细胞群中检测到的变化可能在伴随衰老过程的炎症老化事件的形成中起重要作用,因为这些细胞是炎症反应的一个组成部分。炎症老化的进程导致细胞因子和促炎介质在组织中的积累,进而激活肥大细胞。同时,乳腺细胞脱颗粒的增加可能促进炎症老化的过程。肥大细胞和炎症老化的相互影响使得肥大细胞成为寻找炎症老化生物标志物的潜在候选者成为可能。
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引用次数: 0
Assessment of lymphocyte populations and their subsets in the patients with acute coronary syndrome 急性冠脉综合征患者淋巴细胞群及其亚群的评估
Pub Date : 2022-09-20 DOI: 10.46235/1028-7221-1135-aol
E. Safronova, L. Ryabova
We examined 23 patients aged 40 to 65 years (mean age 54.526.72) with a diagnosis of acute coronary syndrome (ACS) at admission, who underwent emergency or delayed coronary stenting a day later. All patients had arterial hypertension as a concomitant disease. Upon additional examination, blood troponin levels were determined, ECG was performed in the time dynamics. Acute myocardial infarction with ST elevation was diagnosed in 7 patients, infarction without ST elevation, in 6 patients, the unstable angina rest, in the rest of this group (Grace risk from 75 to 150 points, on average, 107.727.16 points). To assess the immune status, especially, lymphocyte populations and subsets we used standardized techniques, i.e., flow cytometric assays with Navios cytofluorimeter (Beckman Coulter, USA). The following subpopulations were determined: CD45+ (panleukocyte marker for gating lymphocytes), CD45+, CD3+ (T cells), CD45+, CD3+, CD4+ (helper inducers), CD45+, CD3+, CD8+ (cytotoxic T cells), CD45+, CD3+CD16+, CD56+ (TNK cells) CD45+, CD3-, CD16+, CD56+ (natural killer cells), CD45+, CD3-, CD19+CD5+ (B cells), CD45+, CD3+, CD4+, CD25+ (activated helpers, early activation phase), CD45+, CD3+, HLA-DR (activated T lymphocytes late activation phase). The data obtained indicate that the relative indices of T helper subpopulations, T cells at early and late activation step, and B lymphocytes were increased in the patients with acute coronary syndrome, compared with control group. At the same time, there is a trend for increasing absolute values of these indexes. The subpopulation of TNK lymphocytes proved to be significantly increased both in relative and absolute values, whereas percentages of CD45+CD3+CD19- (p 0.01) and T cytotoxic lymphocytes (p 0.001) were decreased and showed the same trend in absolute terms. The ratio of CD4/CD8 lymphocytes was almost doubled (p 0.001), due to increased content of T-helpers and decrease in cytotoxic T lymphocytes. In clinical blood analyses of ACS patients a tendency for leukocytosis was shown, (10.155.22), with a shift to the band forms.
我们检查了23例年龄在40至65岁(平均年龄54.526.72岁)的患者,入院时诊断为急性冠脉综合征(ACS),并在一天后接受了急诊或延迟冠脉支架植入术。所有患者均伴有动脉高血压。在附加检查后,测定血肌钙蛋白水平,心电图时间动态。急性心肌梗死伴ST段抬高7例,梗死无ST段抬高6例,不稳定心绞痛休息6例,其余组Grace风险75 ~ 150分,平均107.727.16分。为了评估免疫状态,特别是淋巴细胞群和亚群,我们使用标准化技术,即使用Navios细胞荧光仪(Beckman Coulter,美国)进行流式细胞分析。测定了以下亚群:CD45+(门控淋巴细胞的泛白细胞标记物)、CD45+、CD3+ (T细胞)、CD45+、CD3+、CD8+(细胞毒性T细胞)、CD45+、CD3+、CD16+、CD56+ (TNK细胞)、CD45+、CD3-、CD16+、CD56+(自然杀伤细胞)、CD45+、CD3-、CD19+、CD5+ (B细胞)、CD45+、CD3+、CD4+、CD25+(活化的辅助细胞,早期活化期)、CD45+、CD3+、CD3+、HLA-DR(活化的T淋巴细胞活化后期)。结果表明,与对照组相比,急性冠脉综合征患者辅助T细胞亚群、T细胞激活早、晚阶段、B淋巴细胞等相关指标均有所升高。同时,这些指标的绝对值都有增大的趋势。TNK淋巴细胞亚群的相对和绝对值均显著增加,而CD45+CD3+CD19-的百分比(p 0.01)和T细胞毒性淋巴细胞(p 0.001)的百分比下降,但绝对趋势相同。由于辅助性T细胞含量增加,细胞毒性T细胞减少,CD4/CD8淋巴细胞的比例几乎增加了一倍(p 0.001)。在ACS患者的临床血液分析中显示出白细胞增多的趋势,(10.155.22),并向带型转移。
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引用次数: 0
Cytokine system in the patients with type 2 diabetes mellitus non-infected with SARS-CoV-2 未感染SARS-CoV-2的2型糖尿病患者的细胞因子系统
Pub Date : 2022-09-20 DOI: 10.46235/1028-7221-1129-csi
K. S. Savchuk, A. Simbirtsev
Diabetes mellitus (DM) is a common chronic non-communicable disease, being the most significant comorbidity in SARS-CoV-2 viral infection. The proportion of DM patients among those with COVID-19 is up to 25.0% in the Russian Federation. In presence of DM, clinical course of COVID-19 is characterized by greater severity and persistence of pulmonary damage, an increased need for immunosuppressive, glucocorticoid and combined antiviral therapy in COVID-19 patients, and with prolonged rehabilitation period. Pathogenetic effects of DM on severe course of the SARS-CoV-2 viral infection are being actively studied. The following factors were considered, e.g., negative impact of hyperglycemia on the course of infection; direct cytotoxic and indirect damage to pancreatic -cells with further activation of pro-inflammatory mechanisms; cumulation and progression of generalized inflammation common to DM and COVID-19 including impaired production of cytokines; influence of SARS-CoV-2 virus on the renin-angiotensin-aldosterone system causing inhibition of insulin secretion and increased insulin resistance. Chronic inflammation and impaired immune response may be among the main mechanisms of association between type 2 DM (T2DM) and COVID-19. It is important to identify systemic inflammatory disorders in patients with type 2 diabetes, which may be associated with greater disease severity, being of negative prognostic value. The aim of the present work was to investigate concentrations of some serum cytokines in the patients with type 2 diabetes not infected with SARS-CoV-2. The study included 20 patients with type 2 diabetes; the control group consisted of 11 clinically healthy volunteers. The serum concentration of 13 cytokines was assessed by multiplex analysis on a MAGPIX-100 immunoanalyzer using a Merck multiplex analysis kit (Germany), in accordance with the manufacturers instructions. Increased serum concentrations in T2DM patients were found, as compared with the control group for some key pro-inflammatory cytokines: CX3CL1, TNF, IFN, IL-8, IL-17A, MIP-1, and MIP-1. We have also revealed a decrease in serum concentrations of IL-4. Serum immunoregulatory cytokines in the T2DM were found to be changed in different directions: a decrease in IL-5, along with increase of IL-12p70 and IL- 17, whereas the serum contents of IL-2, IL-13 did not change. A comprehensive analysis of serum cytokine concentrations may increase clinical significance of assessing serum cytokine concentrations as prognostic and diagnostic markers, as well as therapeutic targets in type 2 DM, like as in SARS-CoV-2 infection.
糖尿病(DM)是一种常见的慢性非传染性疾病,是SARS-CoV-2病毒感染中最重要的合并症。在俄罗斯联邦,糖尿病患者在COVID-19患者中所占比例高达25.0%。在存在糖尿病的情况下,COVID-19临床病程的特点是肺损伤更严重和持续存在,COVID-19患者对免疫抑制、糖皮质激素和联合抗病毒治疗的需求增加,康复期延长。目前正在积极研究糖尿病对SARS-CoV-2病毒感染重症病程的致病作用。考虑以下因素,例如,高血糖对感染过程的负面影响;对胰腺细胞的直接细胞毒性和间接损伤,并进一步激活促炎机制;糖尿病和COVID-19常见的全身性炎症的积累和进展,包括细胞因子的产生受损;SARS-CoV-2病毒对肾素-血管紧张素-醛固酮系统的影响,导致胰岛素分泌抑制和胰岛素抵抗增加。慢性炎症和免疫反应受损可能是2型糖尿病(T2DM)与COVID-19相关的主要机制之一。识别2型糖尿病患者的全身性炎症性疾病很重要,这可能与更严重的疾病有关,具有负面预后价值。本研究的目的是研究未感染SARS-CoV-2的2型糖尿病患者血清中某些细胞因子的浓度。该研究包括20名2型糖尿病患者;对照组由11名临床健康志愿者组成。按照制造商的说明,在使用默克多重分析试剂盒(德国)的MAGPIX-100免疫分析仪上,通过多重分析评估13种细胞因子的血清浓度。与对照组相比,T2DM患者血清中一些关键的促炎细胞因子:CX3CL1、TNF、IFN、IL-8、IL-17A、MIP-1和MIP-1的浓度升高。我们还发现血清IL-4浓度降低。T2DM患者血清免疫调节因子呈不同方向变化:IL-5降低,IL-12p70、IL- 17升高,而IL-2、IL-13含量无变化。全面分析血清细胞因子浓度可能会增加评估血清细胞因子浓度作为2型糖尿病(如SARS-CoV-2感染)的预后和诊断指标以及治疗靶点的临床意义。
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引用次数: 0
Effect of mifepristone under acute stress on thymocyte apoptosis in mice 急性应激下米非司酮对小鼠胸腺细胞凋亡的影响
Pub Date : 2022-09-20 DOI: 10.46235/1028-7221-1147-eom
Y. Shilov, S. Shilov, S. Y. Barkov, N. Shilova
In 1936, the Canadian pathologist Hans Selye, in experiments with adrenalectomy, has shown adrenal cortex hormones to be involved in development of thymic acute involution under stress. By 1970s, the idea of lysis of lymphoid cells by glucocorticoids dominated among researchers as the main mechanism of these changes. Later on, increased migration of T lymphocytes from thymus as well as decreased migration of bone marrow precursors to the thymus were considered. Since 1990s, apoptosis induced by glucocorticoids was also studied in this respect. To elucidate the in vivo contribution of glucocorticoids these events, a convenient tool was developed in experimental endocrinology and pharmacology, i.e., treatment of male rats or mice with antagonist of glucocorticoid and progesterone receptors mifepristone, also known as RU-38486 or RU-486. The purpose of this work is to investigate the effect of mifepristone on thymocyte apoptosis in mice under acute stress. Experimental studies were performed in male white noninbred mice. Mifepristone was injected once subcutaneously 30 min before immobilization at a dose of 50 mg/kg body weight prepared in olive oil solution. Control mice and the animals from comparison group were injected once subcutaneously with an equivalent amount of the drug solvent. A classical model of 24-hour immobilization stress in a plastic restrainer (supine position) was used for experimental simulation of acute stress. Thymocyte apoptosis was assessed by flow laser cytometry with BD PE Annexin V Apoptosis Detection Kit I (BD Pharmingen) reagent kit adapted for Guava EasyCyte flow laser cytometer by Millipore Corporation. It was found that acute stress induced by 24-hour immobilization of mice leads to an increase in the total relative number of 7-AAD-positive cells as well as proportion of cells stained with 7-AAD, but not annexin V-PE (nuclear debris, annexin V(-), 7-AAD (+)). Administration of mifepristone alleviated these changes, thus confirming involvement of glucocorticoids in increasing number of necrotic thymocytes. The number of thymocytes in early apoptosis (i.e., annexin V-PE-positive, 7-AAD-negative), as well as total relative number of cells with phosphatidylserine exposed at the surface (annexin V-PE-positive thymocytes) under acute stress and at stress on the background of the introduction of mifepristone did not differ from the control.
1936年,加拿大病理学家汉斯·塞尔耶(Hans Selye)在肾上腺切除术的实验中表明,肾上腺皮质激素参与了应激下胸腺急性退化的发展。到20世纪70年代,糖皮质激素溶解淋巴样细胞的观点作为这些变化的主要机制在研究者中占主导地位。后来,考虑了T淋巴细胞从胸腺迁移增加以及骨髓前体向胸腺迁移减少。20世纪90年代以来,糖皮质激素诱导的细胞凋亡也在这方面进行了研究。为了阐明糖皮质激素在体内对这些事件的贡献,在实验内分泌学和药理学中开发了一种方便的工具,即用糖皮质激素和孕激素受体拮抗剂米非司酮(也称为RU-38486或RU-486)治疗雄性大鼠或小鼠。本研究旨在探讨米非司酮对急性应激小鼠胸腺细胞凋亡的影响。实验研究是在雄性非近亲繁殖的白色小鼠中进行的。固定前30分钟皮下注射米非司酮1次,剂量为50 mg/kg体重,用橄榄油溶液配制。对照小鼠和对照组动物皮下注射等量药物溶剂1次。采用经典的塑料约束器(仰卧位)24小时固定应力模型进行急性应力的实验模拟。采用Millipore公司的Guava EasyCyte流式激光细胞仪适配的BD PE Annexin V凋亡检测试剂盒(BD Pharmingen)试剂盒,流式激光细胞术检测胸腺细胞凋亡。结果发现,24小时固定小鼠急性应激导致7-AAD阳性细胞的相对总数和7-AAD染色细胞的比例增加,但膜联蛋白V- pe(核碎片、膜联蛋白V(-)、7-AAD(+))不增加。给药米非司酮减轻了这些变化,从而证实糖皮质激素参与了坏死胸腺细胞数量的增加。早期凋亡的胸腺细胞数量(即膜联蛋白v - pe阳性,7- aad阴性),以及急性应激和引入米非司酮应激背景下暴露在表面的磷脂酰丝氨酸细胞(膜联蛋白v - pe阳性胸腺细胞)的总相对数量与对照组没有差异。
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引用次数: 0
Relationships between the indices of innate immunity in sepsis depend on clinical outcomes 脓毒症先天免疫指标之间的关系取决于临床结果
Pub Date : 2022-09-20 DOI: 10.46235/1028-7221-1123-rbt
A. Savochkina, A. Poltorak, K. Nikushkina, M. Zotova
At the present time, sepsis is primarily considered a disorder of the immune system caused by dominant infection, manifesting with individual response reaction of the host organism. Hence, sepsis should not be classified as a distinct pro- or anti-inflammatory syndrome, but rather as a variable continuum of overlapping immune mechanisms. The role of innate immunity in sepsis is probably the leading one, since it exerts an early and nonspecific response to any foreign agent. The nature of appropriate relationships may determine current state of the immune system. To perform a deeper study of the anti-infectious protective mechanisms, we have evaluated the relationships between the immunological parameters of peripheral blood in patients with sepsis, depending on the outcome of the disease. Total number of leukocytes, and neutrophils was determined in whole peripheral blood of the patients with sepsis, along with calculation of leukocyte counts and numbers of neutrophil extracellular traps. Serum levels of procalcitonin, peptidyl-arginine deiminase 4, and cytokines were studied by ELISA assay. To study the relationship between the indices of innate immunity, we used correlation analysis, which was carried out by the Spearman rank criterion. When conducting the statistical analysis, strong correlations between the immunological parameters of peripheral blood in patients with sepsis were not revealed, regardless of the outcome of the disease. In cases of favorable outcome, 10 relationships were identified, with an unfavorable outcome, 7 relationships were registered. All the relationships were of medium strength. Regardless of clinical outcome of sepsis, the significant relationships were established between the number of leukocytes and the level of neutrophil extracellular traps; positive relationships were also found between pro-inflammatory cytokines IL-1 IL-6, TNF IL-18. However, these relationships were significant in cases with favorable outcome, while becoming weaker and losing their significance for the group with lethal outcomes. Among the revealed relationships, the most interesting finding was an association between favorable outcome of sepsis with IL-10 contents, which is an anti-inflammatory cytokine coordinating the innate immune response. In lethal outcomes, such relationship with IL-10 was not revealed. IL-10, a cytokine with anti-inflammatory properties may limit the immune response to pathogens and, thus, potentially prevent damage to the host. Therefore, the relationship between IL-10 and other immunological parameters of peripheral blood in sepsis may affect the outcome of this condition.
目前,脓毒症主要被认为是一种由显性感染引起的免疫系统紊乱,表现为宿主机体的个体应答反应。因此,脓毒症不应被归类为一种明显的促炎或抗炎综合征,而应被归类为重叠免疫机制的可变连续体。先天免疫在败血症中的作用可能是主要的,因为它对任何外来物都能产生早期的非特异性反应。适当关系的性质可能决定免疫系统的当前状态。为了对抗感染保护机制进行更深入的研究,我们评估了脓毒症患者外周血免疫参数之间的关系,这取决于疾病的结果。测定脓毒症患者全外周血白细胞和中性粒细胞总数,计算白细胞计数和中性粒细胞胞外陷阱数量。ELISA法检测血清降钙素原、肽精氨酸脱亚胺酶4和细胞因子水平。为了研究先天免疫各指标之间的关系,我们采用Spearman秩标准进行相关分析。在进行统计分析时,没有发现脓毒症患者外周血免疫参数之间存在强相关性,无论疾病的结局如何。在有利结果的情况下,确定了10种关系,在不利结果的情况下,登记了7种关系。所有的关系都是中等强度。无论脓毒症的临床结果如何,白细胞数量与中性粒细胞胞外陷阱水平之间存在显著关系;促炎细胞因子IL-1、IL-6、TNF - IL-18之间也存在正相关。然而,这些关系在结果有利的情况下是显著的,而在结果致命的情况下变得更弱并失去意义。在揭示的关系中,最有趣的发现是败血症的有利结果与IL-10含量之间的关联,IL-10是一种协调先天免疫反应的抗炎细胞因子。在致死性结果中,IL-10与这种关系未被揭示。IL-10是一种具有抗炎特性的细胞因子,可能会限制对病原体的免疫反应,从而潜在地防止对宿主的损害。因此,IL-10与脓毒症患者外周血其他免疫参数的关系可能影响脓毒症的预后。
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引用次数: 0
Association of gene polymorphism and cytokine content in the blood serum in children with allergic bronchial asthma 变应性支气管哮喘患儿血清基因多态性与细胞因子含量的关系
Pub Date : 2022-09-20 DOI: 10.46235/1028-7221-1115-aog
E. Prosekova, M. S. Dolgopolov, A. I. Turyanskaya, V. A. Sabynych
The study of genes controlling cytokine activities of is among important tasks when assessing predisposition and revealing pathogenetic links of initiation and course of clinical disorders. Aberrant production of cytokines and dysregulation of immune response may be considered genetic predictors associated with differentiation and functioning of T helpers, being of decisive importance in pathogenesis of pediatric allergic bronchial asthma. Our objective was to evaluation of associations between polymorphic genotypes and serum levels of cytokines of various T helper profiles in the children with allergic bronchial asthma. We have observed 175 children aged 3 to 11 years. Of them, we have examined 75 patients diagnosed with allergic bronchial asthma (ABA) as well as 100 healthy children matched for age and gender. All children underwent general clinical and allergological examination. The contents of cytokines attributed to Th1, Th2 and Th17 profiles were determined in blood serum by means of ELISA technique. DNA samples isolated from peripheral venous blood were used for molecular genetic analysis. Using allele-specific PCR technique, the following mutation points were investigated: IFN (T-874 A), IL-4 (C-589 T), IL-6 (C-174 G), IL-17A (G- 197 A), TNF (G-308 A). The analysis of distribution and occurrence of the cytokine gene polymorphisms was carried out, and the odds ratio of the disease risk were calculated. Statistical data processing was carried out using the program Statistica 10 by methods of descriptive, parametric and non-parametric statistics, comparison of unrelated groups was performed by qualitative characteristics of HardyWeinberg equilibrium, and with Chi-square test ( 2). These studies have revealed differences in patterns and occurrence of polymorphic genotypes associated with aberrant production of cytokines typical for various Th profiles among the children with allergic bronchial asthma. A comparative analysis of the mutant allele frequencies and cytokine genotypes of various Th profiles, along with determination of the cytokine contents in blood serum of children with allergic bronchial asthma revealed a predominance of homozygous IFN 874A, IL-4 589T, IL-6 174G, IL-17A 197A, and TNF 308A genotypes. Studies of gene polymorphisms, features of production and content of the cytokines specific for T helpers 1, T helpers 2, T helpers 17 profiles in bronchial asthma in the children revealed differences in distribution and occurrence of mutant alleles associated with aberrant cytokine production, variable risk of developing allergic pathology and development of the distinct disease phenotype.
研究控制细胞因子活性的基因是评估易感性和揭示临床疾病发生和发展的病理联系的重要任务之一。细胞因子的异常产生和免疫反应的失调可能被认为是与辅助性T细胞分化和功能相关的遗传预测因子,在儿童过敏性支气管哮喘的发病机制中具有决定性的重要性。我们的目的是评估过敏性支气管哮喘儿童中多态基因型和各种辅助性T细胞因子血清水平之间的关系。我们观察了175名3至11岁的儿童。其中,我们检查了75名诊断为过敏性支气管哮喘(ABA)的患者以及100名年龄和性别匹配的健康儿童。所有儿童均接受一般临床和过敏检查。采用ELISA技术测定血清中Th1、Th2和Th17基因型相关细胞因子的含量。从外周静脉血中分离DNA样本进行分子遗传学分析。采用等位基因特异性PCR技术,研究IFN (T-874 A)、IL-4 (C-589 T)、IL-6 (C-174 G)、IL-17A (G- 197 A)、TNF (G-308 A)等突变点,分析细胞因子基因多态性分布及发生情况,计算患病风险优势比。统计数据处理采用Statistica 10软件,采用描述统计、参数统计和非参数统计的方法,不相关组间比较采用HardyWeinberg平衡的定性特征;和卡方检验(2)。这些研究揭示了过敏性支气管哮喘儿童中与各种Th谱典型的细胞因子异常产生相关的多态性基因型的模式和发生的差异。通过对不同Th谱的突变等位基因频率和细胞因子基因型的比较分析,以及对变应性支气管哮喘患儿血清中细胞因子含量的测定,发现纯合子IFN 874A、IL-4 589T、IL-6 174G、IL-17A 197A和TNF 308A基因型的优势。对儿童支气管哮喘中辅助性T 1、辅助性T 2、辅助性T 17特异性细胞因子的基因多态性、产生和含量特征的研究揭示了与异常细胞因子产生相关的突变等位基因的分布和发生差异、发生过敏病理的可变风险以及不同疾病表型的发展。
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引用次数: 1
Comparative characteristics of cytokine content in neutrophil supernatants after incubation with daily cultures of S. aureus with or without spA gene 带spA基因和不带spA基因的金黄色葡萄球菌每日培养后中性粒细胞上清细胞因子含量的比较
Pub Date : 2022-09-20 DOI: 10.46235/1028-7221-1140-cco
L. Fomina, V. Gritsenko, A. I. Fayzullina
The study was aimed for assessment of cytokine profile in the whole blood neutrophil supernatants stimulated by Staphylococcus aureus, with and without the spА gene. 20 clinical isolates of S. aureus were tested in vitro. Ten of them carried spА gene and other 10 were without the spА gene. We selected the strains with very high ability to secrete cytokine-like substances (CLS), both in terms of the level and spectrum of the cytokines. The S. aureus strains were divided into two groups depending on the spА status according to the data from the spА genotyping databank (http://spa.ridom.de/). The neutrophils of donors were isolated in a double gradient of Ficoll-Verografin, according to the standard method, at a concentration of 5 106. The bacteria were taken at a ratio of 1:20 to neutrophils (at a concentration of 108 bacteria/mL). The effect of staphylococci on cytokine secretion by neutrophils was determined with a Magpix-100 device (USA), using immunofluorescence multiplex kits from BioRad (USA) for the detection of 17 cytokines (G-CSF, GM-CSF, IFN, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, TNF, MCP-1, MIP-1) after 1 hour incubation with the neutrophils. Results: It has been shown that staphylococci with a high degree of CLS activity, regardless of presence or absence of the spА gene, affected the ability of neutrophils to secrete cytokines. At the same time, S. aureus carrying the spА gene caused increased secretion of IL-1, TNF, IL-6 (pro-inflammatory cytokines), but, at the same time, it reduced secretion of IL-8, MIP-1 (chemokines). S. aureus strains devoid of spА genes have a more diverse spectrum and more pronounced effect on cytokine secretion of proinflammatory cytokines by neutrophils, e.g., IL-10, IL-12p70, IL-17A, IL-1, IL-2, TNF and the G-CSF growth factor. Both variants of staphylococci caused reduction of IL-6 secretion.
该研究旨在评估金黄色葡萄球菌刺激的全血中性粒细胞上清中细胞因子的谱,有和没有spА基因。对20株临床分离的金黄色葡萄球菌进行了体外检测。其中10只携带spА基因,10只不携带spА基因。我们选择了分泌细胞因子样物质(CLS)能力非常高的菌株,无论是在细胞因子的水平还是谱上。根据spА基因分型数据库(http://spa.ridom.de/)的数据,金黄色葡萄球菌菌株根据spА状态分为两组。供者中性粒细胞按标准方法,用双梯度的Ficoll-Verografin分离,浓度为5 106。以中性粒细胞与细菌1:20的比例(浓度为108个细菌/mL)取菌。使用Magpix-100(美国)装置检测葡萄球菌对中性粒细胞分泌细胞因子的影响,使用BioRad(美国)的免疫荧光复合试剂盒检测17种细胞因子(G-CSF、GM-CSF、IFN、IL-1、IL-2、IL-4、IL-5、IL-6、IL-7、IL-8、IL-10、IL-12p70、IL-13、IL-17A、TNF、MCP-1、MIP-1)与中性粒细胞孵育1小时后。结果:研究表明,具有高CLS活性的葡萄球菌,无论是否存在spА基因,都会影响中性粒细胞分泌细胞因子的能力。同时,携带spА基因的金黄色葡萄球菌引起IL-1、TNF、IL-6(促炎因子)分泌增加,但同时使IL-8、MIP-1(趋化因子)分泌减少。缺乏spА基因的金黄色葡萄球菌菌株谱更多样化,对中性粒细胞分泌促炎细胞因子如IL-10、IL-12p70、IL-17A、IL-1、IL-2、TNF和G-CSF生长因子的影响更显著。这两种葡萄球菌变异均导致IL-6分泌减少。
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Russian Journal of Immunology
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