Acute infectious mononucleosis is a widespread viral disease, which most often manifests in childhood. The development of acute infectious mononucleosis is accompanied by the change of the CD4+/CD8+ T-lymphocytes ratio and the increase of the virus-specific CD8+ cytotoxic T-lymphocytes number. One of the T-lymphocytes number regulation mechanisms is the modulation of their progenitor cells apoptosis. The death receptor CD95 takes part in the regulation of T-lymphocytes apoptosis, including naïve T-cells. We studied the effect of CD95 receptor activation on apoptosis of naïve CD4+ and naïve cytotoxic CD8+ T-lymphocytes in healthy children and children with acute infectious mononucleosis. In this study children with acute infectious mononucleosis at the age of 9 to 16 years were included. For comparison healthy children of the same age with no clinical and laboratory signs of the disease were used. Naïve CD4+ and naïve cytotoxic CD8+ T-lymphocytes were isolated by negative magnetic immunoseparation. The analysis of naïve T-cells apoptosis and the CD95 receptor surface expression density was performed by using the flow cytometry analysis. The analysis of T-cells was performed in three variants: freshly isolated naïve CD4+ T-lymphocytes and naïve cytotoxic CD8+ T-lymphocytes, and also cells after 24 hours of the cultivation with anti-CD95 monoclonal antibodies or without them. In healthy children both CD95– and CD95+ naïve CD4+ T-lymphocytes underwent apoptosis. In children with acute infectious mononucleosis CD95– naïve CD4+ T-lymphocytes lost their susceptibility to apoptosis induction. In healthy children and children with acute infectious mononucleosis CD95– naïve cytotoxic CD8+ T-lymphocytes were resistant to apoptosis in contrast to CD95+ naïve CD4+ T-lymphocytes. In healthy children CD95 receptor did not induce apoptosis of isolated naïve CD4+ T-lymphocytes and naïve cytotoxic CD8+ T-lymphocytes. In children with acute infectious mononucleosis CD95 receptor was involved in inhibition of apoptosis of naïve cytotoxic CD8+ T-lymphocytes and did not effect on the level of apoptosis of naïve CD4+ T-lymphocytes. We suggest that CD95-dependent suppression of naïve cytotoxic CD8+ T-lymphocytes apoptosis is a protective mechanism for the maintenance of a sufficient number of cytotoxic T-lymphocytes in the blood for the realization of effective antiviral immune response.

Human herpesvirus type 6 (HHV-6) is a lymphotropic virus that is an etiological agent of infectious mononucleosis (IM) in children. HHV-6- mediated infectious mononucleosis (HHV-6M) does not have clearly  defined clinical features. Nowadays immunopathogenetic aspects of  this disease have not been fully understood. The purpose of this  work was to study the characteristics of the quantitative composition  of populations of immunocompetent cells of peripheral  blood in children with HHV-6M. The material for the study was  samples of peripheral blood from children with “infectious  mononucleosis” diagnosis and from virtually healthy children.  Depending on the etiologic cause of the disease, children with IM  were divided into three groups: HHV-6M, IM of other etiology and  mixed infection (combination of HHV-6 and Epstein–Barr virus  and/or Cytomegalovirus). Virtually healthy children formed the  fourth group. In blood samples, the absolute content of the following populations of immunocompetent cells was determined by the  method of flow cytometry: the total population of T-lymphocytes, T- helpers, cytotoxic T-lymphocytes, double positive T-lymphocytes  (CD4+CD8+), NK cells and B-lymphocytes. Discriminant analysis  was carried out: based on the obtained data on the population  composition of blood cells we constructed a model of a child’s  attribution to one of the four groups analyzed in pairs. We used the  method of machine learning — the algorithm of gradient boosting  over decision trees. It was determined whether it is possible to  classify patients on the basis of the studied indicators and which  combination of indicators is optimal for classification. As a result of  the study it was possible to classify the following pairs of groups:  healthy children — children with HHV- 6M, healthy children —  children with IM of other etiology, children with HHV-6M — children  with IM of other etiology. When solving the problem of classifying  children from group with mixed infection and from any other group,  it was not possible to find a model of satisfactory quality. In  comparison with virtually healthy children, children with HHV-6M were characterized by an increased content of the total  population of T-lymphocytes and cytotoxic T-cells, as well as by a  reduced content of doub le-positive T-lymphocytes. Compared with  children with IM of other etiology, children with HHV-6M were  characterized by an increased content of cytotoxic T-lymphocytes, T- helpers, B-lymphocytes and a reduced number of double-positive T  cells. Our results indicate that HHV-6-mediated infectious  mononucleosis causes changes in the quantitative composition of  certain populations of immunocompetent cells of peripheral blood,  different from those of other etiology, in children.

Introduction. The search for specific molecular and genetic markers of the risk of developing infectious disease complications is a current area of research in modern medical and biological science. Materials and methods. In order to solve this issue, we developed a MiDA software that implements an integrated approach allowing for selection of potential markers on the basis of indicators of expression fold change of a number of genes in the comparison groups and the feature importance for classification, i.e. the assignment of samples to the analyzed groups. Results. Using the MiDA software, we searched for molecular and genetic markers of the risk of developing severe dengue fever and chronic brucellosis. As a result of the study, the HSPA6 gene was proposed as a risk marker for the dengue complication. HSPA6 expression was reduced in the peripheral blood samples of severe dengue cases. Markers of chronic brucellosis included a decrease in the expression of miRNA hsa-miR-198 and hsa-miR-501-3p, as well as an increase in the expression of miRNA hsa-miR-618 in CD4+ T-lymphocytes. Conclusion. We demonstrated the possibility of applying the MiDA software to the analysis of big data obtained using modern techniques (sequencing, biochips, etc.). It is possible to expand the scope of the software application in order to analyze the expression of genes, transcripts and proteins in diseases of various origins, to determine molecular mechanisms of the pathological process, to search for diagnostic and prognostic markers of the disease, as well as potential targets for the development of specific therapies.

Despite that human herpes virus type 6 (HHV-6) is extremely spread worldwide, molecular mechanisms of behind HHV-6 infection pathogenesis remain largely unexplored. No molecular markers were found linked to unfavorable course of HHV-6 infection which could allow to ease up selecting proper therapy and preventing development of complications. The aim of the study was to analyze expression of apoptosis and survival-related genes in blood leukocytes from 7–17-year-old children upon various forms of HHV-6 infection. The analysis was carried out by using DNA microarrays developed by us allowing to assess changes in expression level both of individual mRNAs and total gene set (-Σ). It was shown that during the acute phase of HHV-6 infection mRNA level was shifted toward pro-apoptotic factors. In the convalescence phase, most altered mRNA levels returned to normal. We have identified a set of mRNAs and genes whose expression level was significantly changed in acute disease phase. According to available data, these factors play an important role in regulation of studied signaling pathways. In order to search for HHV-6-associated factors, which markedly affect disease pattern of severe herpesvirus mixed infection, we analyzed significant changes of mRNA and genes expression levels in patients with severe HHV-6+EBV+CMV mixed infection and EBV+CMV mixed infection of moderate severity compared with healthy donors. The levels of 5 mRNAs (FAF1-NM_007051, DAPK2-NM_014326, CASP8AP2-NM_001137667, CASP8-NM_033356, BTK-NM_001287345) and 3 genes (FAS-Σ, Puma/BBC3-Σ, ITCH-Σ) were significantly increased in severe mixed infection comorbid with HHV-6 (EBV+CMV+HHV-6) but without HHV-6 (EBV+CMV) compared with healthy donors. Most of detected factors belong to Fas-mediated apoptosis pathway, and may be considered as candidate prognostic development factors of severe herpes virus infection involving HHV-6. This study profoundly extends existing understanding on molecular pathogenesis of HHV-6 infection involving apoptosis and pro-survival signaling pathways. Marked changes of mRNA and gene levels most likely contributed to the pathogenesis of HHV-6 as well as severe HHV-6+EBV+CMV mixed infection.

Acute EBV-associated mononucleosis develops mainly in children and in patients with functionally impaired immune system. Consequently, it may result in developing secondary immunodeficiency, neoplasms as well as diverse alterations in cell-mediated immune reaction. Despite extensively examining molecular mechanisms of EBV infection, it is also necessary seek for new molecular and genetic factors underlying pathogenesis of EBV-mediated mononucleosis and EBV-associated malignant cell transformation is necessary, which might be used in clinical practice to monitor clinical score as well as predictive parameters for EBV-associated complications such as immunocompromised conditions and neoplasms. Here, we proposed to use our splicing sensitive DNA microarrays to perform a comprehensive semi-quantitative mRNA expression analysis for major apoptosis- and survival-related signaling components in peripheral blood leukocytes collected from children with acute EBV infectious mononucleosis as well as during recovery period. Using such DNA microchips allowed to assess both total (denoted by Σ) and separate transcript expression resulting from alternative splicing. It was shown that the balance of mRNA levels in acute phase of EBV-infectious mononucleosis was shifted towards upregulated expression of anti-apoptotic factors and components of of NF-κB-linked pro-survival signaling able to profoundly augment apoptosis resistance. Moreover, some EBV-associated changes (BIM/BCL2L11-Σ, PUMA/ BBC3-NM_001127241, BID-Σ, CASP3-Σ, NFKB1-Σ, RELA-Σ) were in agreement with the data published before. In addition, we also found previously unknown changes in level of EBV-associated coding and noncoding transcripts (DCR1/ TNFRSF10C-NM_003841, DR5/TNFRSF10B-NR_027140, CASP6 beta/CASP6-NM_032992, CASP7-NM_033338). Analyzing their properties allowed to suggest that they play an important role in the pathogenesis of EBV-associated mononucleosis. However, at asymptomatic recovery stage, level of some mRNA expression was kept altered compared to healthy volunteers (DCR2/TNFRSF10D-NM_003840, CASP8-Σ, CASP3-Σ, BIM/BCL2L11-Σ, BCL2-NM_000633, MCL1-Σ, BCL-W/BCL2L2-Σ, BCL-XL/BCL2L1-NM_138578, BIRC2-NM_001166, XIAP-NM_001167, TRAF2-NM_021138, MAP3K14-Σ, NFKB1-Σ), which may point at postponed EBV-associated molecular consequences. On one hand, such changes may be due to long-lasting anti-EBV immune response, whereas, on the other hand, they might be influenced by EBV-associated factors facilitating virus persistence. Overall, we identified the molecular features predisposing to chronic course of EBV-infection. The data obtained further expand our understanding about the molecular pathogenetic mechanisms for EBV infectious mononucleosis.