Expression analysis of apoptotic and survival genes in blood leukocytes of children with various forms of HHV-6 infection

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

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

Enrichment of laterite ore from Trstenik locality in Kosovo

W artykule omówiono możliwości ekstrakcji niklu z laterytów, wydobywanych z miejscowości Trstenik w Kosowie. Region ten jest jednym z największych w Europie pod względem zasobów niklu. Dlatego konieczne jest prowadzenie badań nad optymalizacją procesów wzbogacania. Do wzbogacania laterytów postanowiono zastosować ługowanie chemiczne stosując następujące rodzaje odczynników chemicznych: kwas solny, kwas azotowy V, kwas siarkowy VI, kwas octowy, kwas szczawiowy, wodorotleneku amonu, wodorotlenek sodu, woda destylowana oraz gorąca woda. Woda królewska była stosowana w postaci mieszaniny kwasu HNO3 i HCl w stosunku 1 do 3. Uzyskane wyniki sugerują, że przy zastosowaniu wody królewskiej możliwe jest uzyskanie roztworu o zawartości 72% Ni. Stosowanie kwasów nieorganicznych, wiąże się z uzyskaniem gorszych wyników ługowania.The paper deals with possible extraction of nickel from laterites coming from the locality of Trstenik in Kosovo. This locality is one of the largest in Europe as for Ni content and thus, it is necessary to test certain benefication processes. With regard to the fact that it is the case of laterites, chemical leaching was applied testing the following types of chemical agents: hydrochloric acid, nitric acid, sulphuric acid, acetic acid, oxalic acid, ammonium hydroxide, sodium hydroxide, distilled water, warm up water. Aqua regia was applied in the form of acid mixture of HNO3 and HCl in 1:3 proportion. The acquired results imply that applying aqua regia it is possible to retrieve 72% of Ni. Mineral acids provided partial results

Bakterijsko izluživanje olovnog metalurškog otpada

The aim of this paper is verification of application of bacterial leaching and calcination to recover heavy metals from metallurgical wastes - matte from metallurgical plant Kovohute Pribram. For bacterial leaching a pure bacterial culture of Acidithiobacillus ferrooxidans was used. For a verification test an original sample of matte and matte from 2004 year were used. This paper further shows changes in the samples after bacterial leaching and after calcination. The paper results imply that the samples in question may be used as pigments.Cilj ovog rada je potvrđivanje primjene bakterijskog izluživanja i kalcinacije za oporabu teških metala iz metalurških otpada – sulfida teških metala i željeza (eng. matte) iz metalurškog postrojenje Kovohute Pribram. Za izluživanje je potrebna bakterijska kultura Acidithiobacillus ferrooxidans. Za ispitivanje je korišten orginalnu uzorak sulfida teških metala i željeza i uzorak sulfida teških metala i željeza iz 2004. godine. U ovom radu pokazane su promjene na uzorku nakon bakterijskog izluživanja i kalcinacije. Iz rezultata slijedi da se uzorci mogu koristiti kao pigmenti

ROLE OF CD95 AND DR3 RECEPTORS IN NA VE T-LYMPHOCYTES APOPTOSIS IN CHILDREN WITH INFECTIOUS MONONUCLEOSIS DURING CONVALESCENCE

Infectious mononucleosis is a widespread disease caused by certain members of Herpesviridae family. Acute infectious mononucleosis develops predominantly in children and is accompanied by an increase of the number of circulating naive CD4+ and naive CD8+ T-lymphocytes in the peripheral blood. The normalization of immunological parameters is achieved within 4–6 months after recovery and that is an indicator of a proper functioning of the immune system. CD95 and DR3 death receptors are involved in the initiation of apoptosis of naive T-lymphocytes in healthy people and in patients with infectious mononucleosis. The aim of the study was to evaluate the ability of CD95 and DR3 receptors to initiate apoptosis of naive CD4+ and naive CD8+ T-lymphocytes in children with infectious mononucleosis during convalescence. The material for the study was the samples of the peripheral blood of children who previously had infectious mononucleosis. The blood sampling was carried out again after 4–6 months after the disease. At the time of the study, children did not display clinical and laboratory signs of infectious mononucleosis. Same children who were examined earlier in the period of the development of acute infectious mononucleosis, as well as relatively healthy children were used as the comparison groups. Isolation of naive CD4+ and naive CD8+ T-lymphocytes was performed by negative magnetic immunoseparation. For specific stimulation of CD95 and DR3 receptors monoclonal antibodies were used. The level of apoptosis and expression of death receptors were evaluated by flow cytometry. Freshly isolated cells were analyzed, as well as cells cultured with the addition of appropriate monoclonal antibodies. It was shown that the recovery period was accompanied by increased apoptosis of freshly isolated naive CD4+ and naive CD8+ T-lymphocytes compared with the acute phase of infectious mononucleosis. Thus in both populations of naive T-cells showed an increase of CD95+ cells’ susceptibility to apoptosis. CD95 stimulation in the cell culture did not lead to the change in the level of apoptosis of naive CD4+ and naive CD8+ T-lymphocytes. The freshly isolated naive CD4+ and naive CD8+ T-lymphocytes DR3+ cells were resistant to apoptosis, and in the process of cultivating their sensitivity varied depending on the subpopulation belonging. Thus in the culture of naive CD4+ T-lymphocytes DR3 was not involved in the transfer of pro-apoptotic signal. In the culture of naive CD8+ T-lymphocytes DR3+ cells were possible to increase the apoptosis of DR3-negative cells. At the same time the DR3 activation by monoclonal antibodies in the culture caused the death of DR3+ naive CD8+ T-lymphocytes that naturally associated with decreased proapoptotic activity of these cells and resulted in inhibition of apoptosis of total pool of naive CD8+ T-lymphocytes. Thus, the functional ability of CD95 and DR3 receptors to trigger an apoptosis of naive T-lymphocytes in children during convalescence of infectious mononucleosis varied and depended on their belonging to naive CD4+ or naive CD8+ T-lymphocytes

DETERMINATION OF SOME IMMUNOLOGICAL FEATURES OF HHV-6-MEDIATED INFECTIOUS MONONUCLEOSIS IN CHILDREN BY THE METHOD OF DISCRIMINATORY ANALYSIS

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

ANTI-APOPTOTIC EFFECT OF CD95 RECEPTOR IN NA VE CD8+ T-LYMPHOCYTES IN CHILDREN WITH ACUTE INFECTIOUS MONONUCLEOSIS

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