Methodological basics for differential detection of EBV1/EBV2 and HHV6A/HHV6B

Among a whole variety of EBV- and HHV6-linked diseases only infectious mononucleosis is subject to official statistical reporting in Russia that substantially complicates objective assessment of etiological structure, incidence rate, characteristics of developing epidemic process. Currently, the data on the genetic EBV heterogeneity, even at the level of the main types (EBV1 and EBV2), as well as HHV6A and HHV6B, prevalence and clinical importance are mainly limited to foreign research publications. Few publications assessing this issue are available in Russian scientific papers. At the same time, examining circulation of virus genetic types (variants) and use of such data in implementing epidemiological surveillance after some other infections have been commonly practiced. One of the key issues is the level of developed laboratory support for molecular genetic monitoring. The goal of the study was to improve methodological basics for differential detection of HHV6A/B and the major EBV types. There were used samples of peripheral blood leukocytes collected from children aged 1–15 years with acute (n = 50) and asymptomatic infectious mononucleosis (n = 29). The detection and quantification of EBV and HHV6 DNA was performed by using real-time PCR. For differential determination of EBV1/EBV2 and HHV6A/HHV6B, an optimized one-round PCR with electrophoretic agarose gel detection amplification products was used. The data from our own study showed that frequency of detected EBV and HHV6 DNA in acute infectious mononucleosis patients comprised 74 and 72% compared to control group reaching 35 and 74%, respectively. It was found that among the examined children of Nizhny Novgorod Region, EBV1 and HHV6B prevailed in the viral population that agrees with existing insights about their geographical distribution in the adjacent territories. EBV2 was found in a single sample only in the control group. HHV6A was not detected in any of the groups. The methodological approach optimized in this study allows to separately detect HHV6A/HHV6B and the main EBV types according to a unified laboratory protocol, whereas combining it with additional stage of DNA enrichment increases the diagnostic sensitivity of PCR analysis, minimizes proportion of discordant and false negative results. Such an integrated approach can be applied for diagnostic, epidemiological and research purposes

Сравнительная характеристика бета-герпес-вирусов человека 6А и 6В. Современный взгляд на проблему

This review is devoted to the comparative characteristics of human herpesvirus 6A (HHV6A) and human herpesvirus 6B (HHV6B), taking into account their exogenous and endogenous (inherited chromosomally integrated) forms. The analysis of the literature data on the main interspecies differences and intraspecies features of these viruses in molecular-genetic, biological, epidemiological and clinical aspects has been consistently carried out. Modern views about HHV6A and HHV6B, including their unique inherited chromosomal-integrated form, are the basis for organizing a system of epidemiological surveillance of infections caused by these viruses, as well as developing standardized methodological approaches to differential diagnosis, treatment and specific prevention of a wide range of virus-associated diseases. The development of this direction requires a greater evidence base and intensification of joint efforts of the scientific and medical communities.Представленный обзор посвящен сравнительной характеристике вируса герпеса человека 6А (ВГЧ6А) и вируса герпеса человека 6В (ВГЧ6В) с учетом их экзогенной и эндогенной (наследуемой хромосомно-интегрированной) форм. Последовательно проведен анализ литературных данных об основных межвидовых различиях и внутривидовых особенностях этих вирусов в молекулярно-генетическом, биологическом, эпидемиологическом и клиническом аспектах. Современные представления о ВГЧ6А и ВГЧ6В, в том числе их уникальной наследуемой хромосомно-интегрированной форме, являются основой для организации системы эпидемиологического надзора за инфекциями, вызываемыми данными вирусами, а также разработки стандартизованных методических подходов к дифференциальной диагностике, лечению и специфической профилактике широкого спектра вирус-ассоциированных заболеваний. Развитие данного направления требует большей доказательной базы и интенсификации совместных усилий научного и врачебного сообществ

Thyroid Hormone May Regulate mRNA Abundance in Liver by Acting on MicroRNAs

MicroRNAs (miRNAs) are extensively involved in diverse biological processes. However, very little is known about the role of miRNAs in mediating the action of thyroid hormones (TH). Appropriate TH levels are known to be critically important for development, differentiation and maintenance of metabolic balance in mammals. We induced transient hypothyroidism in juvenile mice by short-term exposure to methimazole and perchlorate from post natal day (PND) 12 to 15. The expression of miRNAs in the liver was analyzed using Taqman Low Density Arrays (containing up to 600 rodent miRNAs). We found the expression of 40 miRNAs was significantly altered in the livers of hypothyroid mice compared to euthyroid controls. Among the miRNAs, miRs-1, 206, 133a and 133b exhibited a massive increase in expression (50- to 500-fold). The regulation of TH on the expression of miRs-1, 206, 133a and 133b was confirmed in various mouse models including: chronic hypothyroid, short-term hyperthyroid and short-term hypothyroid followed by TH supplementation. TH regulation of these miRNAs was also confirmed in mouse hepatocyte AML 12 cells. The expression of precursors of miRs-1, 206, 133a and 133b were examined in AML 12 cells and shown to decrease after TH treatment, only pre-mir-206 and pre-mir-133b reached statistical significance. To identify the targets of these miRNAs, DNA microarrays were used to examine hepatic mRNA levels in the short-term hypothyroid mouse model relative to controls. We found transcripts from 92 known genes were significantly altered in these hypothyroid mice. Web-based target predication software (TargetScan and Microcosm) identified 14 of these transcripts as targets of miRs-1, 206, 133a and 133b. The vast majority of these mRNA targets were significantly down-regulated in hypothyroid mice, corresponding with the up-regulation of miRs-1, 206, 133a and 133b in hypothyroid mouse liver. To further investigate target genes, miR-206 was over-expressed in AML 12 cells. TH treatment of cells over-expressing miR-206 resulted in decreased miR-206 expression, and a significant increase in two predicted target genes, Mup1 and Gpd2. The results suggest that TH regulation of these genes may occur secondarily via miR-206. These studies provide new insight into the role of miRNAs in mediating TH regulation of gene expression

Estrogenic Plant Extracts Reverse Weight Gain and Fat Accumulation without Causing Mammary Gland or Uterine Proliferation

Long-term estrogen deficiency increases the risk of obesity, diabetes and metabolic syndrome in postmenopausal women. Menopausal hormone therapy containing estrogens might prevent these conditions, but its prolonged use increases the risk of breast cancer, as wells as endometrial cancer if used without progestins. Animal studies indicate that beneficial effects of estrogens in adipose tissue and adverse effects on mammary gland and uterus are mediated by estrogen receptor alpha (ERα). One strategy to improve the safety of estrogens to prevent/treat obesity, diabetes and metabolic syndrome is to develop estrogens that act as agonists in adipose tissue, but not in mammary gland and uterus. We considered plant extracts, which have been the source of many pharmaceuticals, as a source of tissue selective estrogens. Extracts from two plants, Glycyrrhiza uralensis (RG) and Pueraria montana var. lobata (RP) bound to ERα, activated ERα responsive reporters, and reversed weight gain and fat accumulation comparable to estradiol in ovariectomized obese mice maintained on a high fat diet. Unlike estradiol, RG and RP did not induce proliferative effects on mammary gland and uterus. Gene expression profiling demonstrated that RG and RP induced estradiol-like regulation of genes in abdominal fat, but not in mammary gland and uterus. The compounds in extracts from RG and RP might constitute a new class of tissue selective estrogens to reverse weight gain, fat accumulation and metabolic syndrome in postmenopausal women

Current and Future Drug Targets in Weight Management

Obesity will continue to be one of the leading causes of chronic disease unless the ongoing rise in the prevalence of this condition is reversed. Accumulating morbidity figures and a shortage of effective drugs have generated substantial research activity with several molecular targets being investigated. However, pharmacological modulation of body weight is extremely complex, since it is essentially a battle against one of the strongest human instincts and highly efficient mechanisms of energy uptake and storage. This review provides an overview of the different molecular strategies intended to lower body weight or adipose tissue mass. Weight-loss drugs in development include molecules intended to reduce the absorption of lipids from the GI tract, various ways to limit food intake, and compounds that increase energy expenditure or reduce adipose tissue size. A number of new preparations, including combinations of the existing drugs topiramate plus phentermine, bupropion plus naltrexone, and the selective 5-HT2C agonist lorcaserin have recently been filed for approval. Behind these leading candidates are several other potentially promising compounds and combinations currently undergoing phase II and III testing. Some interesting targets further on the horizon are also discussed

Клинико-лабораторная характеристика инфекционного мононуклеоза, вызванного вирусом Эпштейна – Барр 1-го типа, у госпитализированных детей

Aim. The aim of the study was to provide clinical and laboratory characteristics of infectious mononucleosis caused by Epstein-Barr virus (EBV) genotype 1 in hospitalized children.  Materials and methods. The material of the study was blood leukocytes and saliva of children aged 1-17 years, hospitalized with a diagnosis of infectious mononucleosis caused by EBV (n=67). For differential detection of EBV-1/ EBV-2, we used an optimized one-round PCR variant with electrophoretic detection of amplification products in agarose gel. Clinical symptoms and laboratory data of patients were analyzed separately and combined into groups of signs. Statistical data processing was carried out using the R programming language and the RStudio environment.  Results. In all children, only one type of virus (EBV-1) was detected in blood leukocytes and saliva. The first data on clinical and laboratory signs of EBV-1 infectious mononucleosis, characterized by a typical symptom complex, have been obtained. The age criterion for dividing patients into groups of younger (1–5 years) and older (6–17 years) age, which were characterized by the most pronounced differences in the manifestations of overt EBV-1 infection, was determined. In young children, the leading was the syndrome of intoxication, in older children, signs of cytolytic syndrome were more pronounced. According to laboratory data, in the age group of 1–5 years, monocytopenia and a decrease in hemoglobin were more often observed, and in children of 6–17 years, lymphocytosis, monocytosis, elevated levels of hepatic transaminases and hemoglobin.  Conclusion. For the first time, EBV typing was performed in children with infectious mononucleosis. The dominant genotype of the virus in infectious mononucleosis in children living in the territory of a large city in the European part of Russia is EBV-1. For the first time, the characteristics of EBV-1 infection are given. The data obtained on the different severity of clinical and laboratory signs of the disease are prerequisites for continuing research aimed at finding the relationship between the characteristics of the clinical course of the infection and the genetic heterogeneity of the EBV population. Цель: клинико-лабораторная характеристика инфекционного мононуклеоза, вызванного генотипом 1 вируса Эпштейна – Барр, у госпитализированных детей. Материалы и методы. Материалом исследования послужили лейкоциты крови и слюна детей в возрасте 1–17 лет, госпитализированных с диагнозом «Инфекционный мононуклеоз», вызванный ВЭБ (n=67). Для дифференциальной детекции ВЭБ-1/ВЭБ-2 в работе применялся оптимизированный однораундовый вариант ПЦР с электрофоретической детекцией продуктов амплификации в агарозном геле. Клинические симптомы и лабораторные данные пациентов анализировали отдельно и объединяя их в группы признаков. Статистическая обработка данных проводилась с использованием языка программирования R и среды RStudio. Результаты. У всех детей в лейкоцитах крови и слюне выявлен только один тип вируса (ВЭБ-1). Получены первые данные о клинико-лабораторных признаках ВЭБ-1-инфекционного мононуклеоза, характеризующегося типичным симптомокомплексом. Определен возрастной критерий разделения пациентов на группы младшего (1–5 лет) и старшего (6–17 лет) возраста, которые характеризовались наиболее выраженными различиями проявлений манифестной ВЭБ-1-инфекции. У детей младшего возраста ведущим являлся синдром интоксикации, у детей старшего возраста более выражены признаки цитолитического синдрома. По лабораторным данным в возрастной группе 1–5 лет чаще наблюдали моноцитопению и снижение гемоглобина, а у детей 6–17 лет – лимфоцитоз, моноцитоз, повышенные уровни печеночных трансаминаз и гемоглобина. Заключение. Впервые проведено типирование вируса Эпштейна – Барр у детей при инфекционном мононуклеозе. Доминирующим генотипом вируса при инфекционном мононуклеозе у детей, проживающих на территории крупного города европейской части России, является ВЭБ-1. Впервые дана характеристика ВЭБ-1- инфекции. Полученные данные о различной выраженности клинических и лабораторных признаков заболевания являются предпосылками для продолжения исследований, направленных на поиск взаимосвязи особенностей клинического течения инфекции и генетической гетерогенности популяции вируса Эпштейна – Барр.

Диагностическое значение количественного определения ДНК вируса Эпштейна – Барр в лейкоцитах крови у детей при инфекционном мононуклеозе

Aim. To determine the threshold value of the Epstein-Barr virus (EBV) viral load (VL) in blood leukocytes to improve the laboratory diagnostics of infectious mononucleosis in children.Materials and methods. EBV DNA quantification in blood leukocytes in children aged 1-17 years (n=163) was determined by real-time polymerase chain reaction. VL were compared in groups of EBV mononucleosis (n=67), non-EBV mononucleosis (n=25) and healthy donors (n=25). Threshold was determined based on VL data from children with active and latent EBV infection. The R program and the RStudio environment were used for satistic analysis.Results. EBV DNA is found in blood leukocytes in infectious mononucleosis not associated with EBV and in healthy virus carriers, however VL in these groups is significantly lower than in patients with EBV mononucleosis (p<0.001). The threshold value was determined – 41 copies/105 cells (or 1.6 lg of EBV DNA/105 cells), which was characterized by acceptable values of specificity and sensitivity (0.90 and 0.85, respectively) of laboratory diagnostics. High EBV VL (equal to or above the set threshold) is associated with an 8.5-fold increased risk of detecting active EBV infection compared to children who have a low VL (below a set threshold) (RR 8.5; 95% CI: 3.7–19.7, p<0.001).Conclusion. In general, the results obtained create prerequisites for more intensive implementation of quantitative studies of EBV DNA in blood leukocytes, both in the context of improving the early diagnosis of infectious mononucleosis and its etiological interpretation, and in terms of detailing the features of the course of EBV infection.Цель: определение порогового значения вирусной нагрузки вируса Эпштейна – Барр в лейкоцитах крови для совершенствования лабораторной диагностики инфекционного мононуклеоза у детей.Материалы и методы: в лейкоцитах крови детей 1–17 лет (n=163) проведено количественное определение ДНК ВЭБ методом полимеразной цепной реакции в реальном времени. Сравнительный анализ вирусной нагрузки выполнен в группах пациентов с инфекционным мононуклеозом ВЭБ-этиологии (n=67), не ВЭБ-этиологии (n=25) и здоровых доноров (n=25). Пороговое значение установлено на основе анализа данных вирусной нагрузки у детей с активной и латентной ВЭБ-инфекцией. Статистическая обработка данных проводилась с использованием языка программирования R и среды RStudio.Результаты. Показано, что ДНК вируса Эпштейна – Барр обнаруживается в лейкоцитах крови при инфекционном мононуклеозе, не связанном с ВЭБ, и у здоровых вирусоносителей, однако вирусная нагрузка в этих группах значительно ниже, чем у пациентов с ВЭБ-мононуклеозом (p<0,001). Установлено пороговое значение вирусной нагрузки на уровне 41 копия/105 клеток (или 1,6 lg ДНК ВЭБ/105 клеток). Применение такого порога позволяет достичь приемлемых значений специфичности и чувствительности (0,90 и 0,85 соответственно) лабораторной диагностики. Высокая вирусная нагрузка (равная или выше установленного порогового значения) ассоциирована с повышенным в 8,5 раз риском выявления активной ВЭБ-инфекции по сравнению с детьми, у кого определяется низкая вирусная нагрузка (ниже установленного порогового значения) (ОР 8,5; 95% ДИ: 3,7–19,7; p<0,001).Заключение. Предложена диагностическая стратегия лабораторного подтверждения активной ВЭБ-инфекции, а также инфекционного мононуклеоза как ее основной клинической формы. В целом, полученные результаты создают предпосылки для более интенсивного внедрения количественных исследований ДНК ВЭБ в лейкоцитах крови как в контексте совершенствования ранней диагностики инфекционного мононуклеоза и его этиологической расшифровки, так и в разрезе детализации особенностей течения ВЭБ-инфекции