INTRACELLULAR ANTIOXIDANT ACTIVITY OF A STREPTOMYCES SP. 8812 SECONDARY METABOLITE, 6,7-DIHYDROXY-3,4-DIHYDROISOQINO- LINE-3-CARBOXYLIC ACID, AND ITS SYNTHETIC DERIVATIVES.

The aim of this study was to determine the antioxidant properties of 6,7-dihydroxy-3,4-dihydroiso- quinoline-3-carboxylic acid (1) and its derivatives in living cells against reactive forms of oxygen and nitrogen, i.e., hydrogen peroxide and nitric oxide. Four of tested compounds scavenged the reactive form of nitrogen more efficiently or similarly to Trolox (EC50 = 55.80 µM). Two compounds exhibited antioxidant activity against reactive oxygen species better than Trolox (EC50 = 51.88 µM). The most active derivative of 1 was the compound containing an iodine atom at position 8 (6,7-dihydroxy-8-iodo-3,4-dihydroisoquinoline-3-carboxylic acid). Our studies showed that some of the derivatives had the ability to cross the cell membrane and scavenge free radicals inside living cells. Thus, they are able to protect DNA and other cellular structures from the dam- aging effects of reactive oxygen and nitrogen species. In addition, some molecular descriptors of the tested compounds were determined with the use of ICM Pro (Molsoft L.L.C.).

Problem of rubella in Poland after compensatory outbreak in 2013 / Problem rózyczki w Polsce po epidemii wyrównawczej w 2013 roku

The attempt to estimate the real number of rubella cases in the years 2015-2016 in Poland was presented in this paper. The relations between number of reported cases of measles in 2006-2015y., the number of laboratory-confirmed cases of rubella among cases suspected of measles and the proportion of rubella among patients suspected of measles in the last 10 years as well as the results of serological examination in samples from 74 patients suspected of rubella collected in the first half of 2016 year were analysed. The sera from patients suspected of rubella were collected in cooperation with the State Sanitary Inspection. The analysis of data collected during the study-cases of suspected measles (2006-2016) and rubella infections (in 2016y.) indicated high over-registration of rubella in recent years in Poland, which is associated with a very low rate of laboratory confirmed cases.

[Measles virus stimulation effect on the expression of Toll-like receptors in PBMC]. / Wplyw stymulacji wirusem odry na ekspresje receptorów Toll-like w komórkach PBMC.

INTRODUCTION: Toll-like receptors (TLRs) are an important component of a innate immune system. Stimulation of TLRs, through action with helper T cells, could change Th1/Th2 balance and thus affect adaptive immune response. Receptors TLR2 and TLR4 play important role in immune response to measles virus. The aim of this work was stimulation of the peripheral blood mononuclear cells (PBMC) and determination of Toll-like gene expression in these cells. METHODS: PBMCs from 20 healthy donors were stimulated with measles viruses and ligands for TLR2 and TLR4. For examinations the real time RT-PCR (QuantiFast® Assay, Qiagen) was used. The expression of Toll-like receptors was determined on muRNA level, using real-time one step RT-PCR (QuantiFast® Assay, Qiagen) with simultaneous detection of TLR2 and TLR4 genes and housekeeping gene (GAPDH). RESULTS: Virus-specific influence of wild measles virus strains activity on PBMC derived from vaccinated seronegative individuals manifested in higher level of expression of TLR2 and TLR4 genes in compare to the expression of these genes in PBMC of seropositive individuals. CONCLUSIONS: Toll-like receptors participate in the development of immune response to measles virus.

The significance for epidemiological studies anti-measles antibody detection examined by enzyme immunoassay (EIA) and plaque reduction neutralization test (PRNT).

The paper discusses the role of anti-measles antibodies for protection and significance for epidemiological studies determination of antibodies by different serological methods. The comparison of anti-measles virus antibodies levels measured by enzyme immunoassay (EIA) and Plaque Reduction Neutralization Test (PRNT) was described. It was found that the 200 mIU/ml of anti-measles activity measured by PRNT (level protection against symp- tomatic disease) is equivalent of 636 mIU/ml measured by EIA (Enzygnost®Anti-Measles Virus/IgG, Simens).

[The prevalence of active infection with viruses from the family Herpesviridae among members of families with children]. / Czestosc wystepowania aktywnych zakazen wirusami z rodziny Herpesviridae wsród czlonków rodzin wychowujacych dzieci.

INTRODUCTION: Cytomegalovirus infection (CMV) is one of the most common viral infections during pregnancy and one of the most common causes of birth defects in newborns. CMV infection occurs mostly through close contact with small children who can secrete the virus in saliva and urine. Children, especially in preschool and early school can also be a source of infection with other herpesviruses. The aim of the study was to determine the prevalence of active infections caused by viruses from the family Herpesviridae (CMV, EBV, VZV) among members of families with children. MATERIAL AND METHODS: The study included 24 families raising children aged from 2 to 18 years. From all members of the families (46 adults and 39 children) saliva samples were collected from which DNA was extracted. The isolated DNA samples were tested for the presence of CMV, EBV, VZV genetic material by nested PCR. In addition, each family carried out a survey. RESULTS AND CONCLUSIONS: The presence of CMV DNA in saliva samples were detected in members of 7 families and the presence of EBV DNA were detected in members of 11 families. Total DNA of CMV was detected in 8/85 samples of saliva (9.41%), of which 1/46 adults (2.17%) and 7/39 children (17.95%) and EBV DNA was detected in 18/85 tested saliva samples (21,18%) - 13/46 samples from adults (28,26%) and 5/39 samples from children (12,82%). VZV DNA was not detected in any of the tested saliva samples. The obtained results indicate that the active, asymptomatic infections with lymphotropic herpesviruses are common and affect more than 10% (CMV) and 20% (EBV) subjects.

Response of viral specific CD4 T cells to in vitro stimulation with vaccine and wild measles virus strains in vaccinated and naturally infected subjects.

With the implementation of the WHO strategic plan for the elimination of measles, the number of measles cases in European Region has decreased. However, outbreaks are still observed. Although most measles cases affect unvaccinated individuals, cases with vaccinated persons are also reported. Furthermore, it was described that a high percentage of young people in Poland exhibit no presence of anti-MeV IgG despite the high level of vaccination covering no less than 97% of the Polish population. Strong evidence exists that immunity to measles is complex and depends on both the humoral and cellular response and although antibodies have been used as correlates of immunity, it is increasingly being considered that antibody-based definitions of vaccine success or failure may be incomplete. Here, we investigated immunity to measles as the reactivity of CD4 T cells to stimulation with vaccine as well as wild strains of measles virus (MeV) isolated in Poland, in young vaccinated persons and subjects infected naturally. Evidence for the presence of MeV-specific memory cells years after infection or vaccination was found, however the cells ofvaccinees and naturally infected subjects reacted differently in contact with wild and vaccine MeV strains. Furthermore, the presence of a significant proportion of non-responder vaccinees was observed. In conclusion, our results may have implications for studies on the monitoring of the complexity of post-vaccine immune response.

[The use of real-time RT-PCR method for the determination of Toll-like genes expression at mRNA level]. / Zastosowanie metody real-time RT-PCR do oznaczania ekspresji genów receptorów Toll-like na poziomie mRNA.

INTRODUCTION: Toll-like receptors (TLRs) are an important component of a innate immune system. Stimulation of TLRs, through action with helper T cells, could change Th1/Th2 balance and thus affect adaptive immune response. The aim of this work was to optimize the stimulation of the peripheral blood mononuclear cells (PBMC) and the validation of real-time RT-PCR method for determination of Toll-like gene expression in these cells. METHODS: PBMCs from healthy donors were stimulated with measles viruses and ligands for TLR2 and TLR4. For examinations the real time RT-PCR (QuantiFast Assay, Qiagen) was used. Fold change of TLRs expression was normalized to GAPDH and estimated by 2(-deltadeltaCt) method. Validation of real-time RT-PCR method was performed for repeatability and efficiency. RESULTS: The level of gene expression varies between individuals and was dose and time of incubation dependent. The efficiency ofreal-time RT-PCR was 90.4% +/- 10.2 for GAPDH, 87.0% +/- 8.2 for TLR2 and 44.5% +/- 9.2 for TLR4. Repeatability, expressed as relative standard deviation (RSD) for Ct values was less than 0.70% for GAPDH, < 3.2% for TLR2 and < 2.84% for TLR4. CONCLUSIONS: Based on obtained results, the optimal conditions for stimulation were: 10 microg/ml/24h for LPS, 1 microg/ml/6h for Pam3CSK4 and 1250 MeV infectious particles/24h.

RSV respiratory infection in children under 5 y.o.--dynamics of the immune response Th1/Th2 and IgE.

INTRODUCTION: The imbalance of Th1/Th2 cytokine concentrations and increased level of IgE might be useful tool for prediction of severity of RSV infection among young children and possibility of sequels. The ratio of cytokines Th1/Th2 varied during the disease. THE AIM of our studies was the assessment of immunological response by dynamics of Th1 and Th2 cytokines and IgE in RSV infections. MATERIAL AND METHODS: Study was done on sera collected from 36 young children hospitalized because of RSV infection and from 16 children with other respiratory tract infection (HMPV, EV, HPIV1-3). Assaying of the serum levels of cytokine Th1 (IL-2, IFN-g, TNF), Th2 (IL-4, IL-6, IL-10) and concentration of IgE has been done. Paired sera (48 patients) were collected in the interval 4-14 days. Reference group consist of 18 children (< 6 months of life) hospitalised because other than respiratory diseases with negative results for viruses tested by PCR. RESULTS: Among children with respiratory infection the Th1/Th2 ratio was shifted towards Th2, level of IgE increased in comparison to the reference group. Changes in concentration of IL-6, IFN-g, IL-10 were related to RSV infection, mainly bronchitis and bronchiolitis, while the dynamic of TNF concentration was independent on aetiological agent. It was found that the risk factors (prematurity, artificial nutrition) correlated with RSV bronchitis and the levels of cytokines and IgE. Increased level of IL-6 and IL-10 were shown in prematures, and increased concentration of IgE--among artificial nourished children. The time of serum collection affected the level of cytokines and IgE and the effect was depended on the aetiological agent. In RSV infections was observed significant decrease with time of IL-6, IL-10 and IFN-gamma but not IgE (still significantly higher than in the reference group). While the significant decrease of IgE was determined only in other than RSV infections. CONCLUSIONS: Determined level of cytokines and IgE varied depending on the time of serum collection. Observed dynamics in paired sera and IgE might have prognostic value in disease and sequels of RSV infections. Prevention RSV infection in premature infants should be done in any possible way. Breastfeeding is one of the critical elements of prevention of RSV infection.

The influence of toll-like receptor stimulation on expression of EBV lytic genes.

Epstein-Barr virus (EBV) establishes latency in the resting memory B-cell compartment. It has been recently suggested that maintenance of chronic infection is dependent on periodic reactivation. Although the stimuli for EBV reactivation in vivo during natural infections are largely unknown, there is evidence indicating that heterologous infections could trigger herpesviruses reactivation. The purpose of this work was to identify the influence of Toll-like receptors stimulation on EBV replication in EBV latently infected Burkitt lymphoma cells (P3HR-1, Raji and Namalwa). The cells were stimulated with Pam3CSK4 (synthetic triacylated lipoprotein), PolyI:C (synthetic analog of dsRNA), LPS (lipopolysaccharide from E.coli), measles virus (MeV) and PMA (phorbol myristate acetate). Non-stimulated cells (NS) served as control. EBV expression was investigated at mRNA level for three viral lytic genes: BZLF1 (immediate early, ZEBRA), BALF2 (early, EA) and BcLF1 (late, VCA). Additionally, the effect of stimulation on NF-kBp65 and inflammatory cytokines (IL-lb, IL-6, IL-8, IL-10, IL-12p70, and TNF) was investigated. Stimulation of TLRs led to limited changes in EBV expression manifesting as increase of ZEBRA at mRNA level in cells treated with PolyI:C and Pam3CSK4. Stimulation with PolyI:C, Pam3CSK4 and LPS also lead to considerable increase of NF-kBp65, while increased levels of inflammatory cytokines were observed for IL-8, TNF and IL-6 in cells treated with PMA and MeV. In conclusion, the results of our experiments support the suggestion that TLRs stimulation with microbial ligands influences EBV virus replication.

[Presence of IgG class antibodies anti-measles virus in sera of subjects in different age]. / Wystepowanie przeciwcial klasy IgG dla wirusa odry w surowicach os6b w róznym wieku.

INTRODUCTION: The increase of measles incidence in Poland was recently observed. Furthermore, the analysis of routine serological tests performed in the department of virology, NIPH-NIH revealed, that nearly half of young people (20-30 years old) have no antibodies against measles virus. The paper presents results of IgG specific for measles virus prevalence in the sera of vaccinated and unvaccinated subjects, which aimed to make the selection of groups for immunological memory research. METHODS: Total of 100 persons were examined based on results of determination of the presence of IgG anti-MeV: 26 people born before and 74 born after 1972 year. From this group, 55 participants were selected for further study and divided into 3 groups (1) subjects born before 1972, unvaccinated against measles and seropositive as a result of natural infection, (2) subjects born after 1972, vaccinated against measles but seronegative or with traces ofanti-MeV IgG presence, (3) subjects born after 1972 seropositive due to vaccination. Selected persons are subject to further examinations include determination the number of leukocytes and lymphocytes profile. RESULTS: The level of anty-MeV IgG antibodies in subjects after natural infection was significantly higher compared to levels obtained by vaccinations. No significant differences in the immunological parameters which could influence on immune response were observed. CONCLUSIONS: Obtained results lead to search other factors that may affect the weak postvaccinal humoral response.

[Effect of stimulation with the measles virus on expression of early activation markers on CD4+ T lymphocytes]. / Wplyw stymulacji wirusem odry ekspresji wczesnych markerów aktywacji limfocytów T CD4+.

Elimination of measles is one of the priority plans of WHO. The success of this plan depends on the development of long lasting, postvaccinal immune response. The aim of this study was to present the effect of stimulation with different strains of measles virus on the expression of T-helper cell (CD4+ T) early activation markers in people with different history of measles infection and to determine the correlation between the activation and dose of virus used for stimulation. The study was conducted using material derived from two patients: one seropositive due to natural infection and one vaccinated, with traces of anti-MeV IgG antibodies. In the CD4 T helper cells, the expression of CD69 receptor and the ability of the cells to produce INF after stimulation with the vaccine-derived or wild-type strain of measles virus was determined. For antigen-specific stimulation the virus suspension containing about 100 infectious particle, its tenfold and hundredfold dilutions was used. We found that the expression of T-helper cells early activation markers depended on the strain of the measles virus used for the stimulation, type of the immune response (postvaccinal, natural infection), and in the case of CD69 expression also on the dose of the virus used for the stimulation.

[Toll-like receptors expression on Burkitt lymphoma cells]. / Ekspresja receptorów Toll-like na komórkach chloniaka Burkitta.

Toll like receptors (TLRs) are an important component of the immune response. They are link between innate and adaptative response. Lymphocytes B express most of the toll-like receptors and they may respond to a broad spectrum of PAMP. Lymphocytes B are one of the major lymphocyte populations in secondary lymphoid tissues, where they represent up to 50% of cells population. These cells are an important element of the defense, largely by using the mechanisms associated with innate response. On the other hand, lymphocytes B are the site of EBV latency, so Burkitt lymphoma cells can may be a convenient model to study the mechanisms associated with EBV infection. The aim of study was to determine the expression of TLRs at the m-RNA level of in Burkitt lymphoma cells treated with ligands for selected TLRs. P3HR, Raji and Namalwa cells were stimulated with Pam3 (10 microg/ml), PolyI:C (25 microg/ml), LPS (10 microg/ml) and measles virus (MeV, moi 0.02). Unstimulated cells and cells treated with PMA (0.5 microg/ml) served as negative and positive controls. After incubation, from stimulated and unstimulated cells mRNA was extracted, RT-PCR reaction was performed and electrophoretic separation was made. The intensity (INT) of bands were determined using the tools for quantitative analysis. In order to analyze the expression of TLR genes, INT values for TLR2, TLR3 and TLR4 in tested cell lines are expressed as %, assuming an average level of GAPDH expression as 100%. The 25% of INT for negative control was accepted as a change in expression level. It was found that the expression of Toll-like receptors in Burkitt lymphoma cells is diverse both in terms of cell type and the type of stimulation.

[Raji, P3HR-1 and Namalwa cells as a model for the study of Epstein-Barr virus (EBV) reactivation]. / Komórki Raji, P3HR-1 i Namalwa jako model badania reaktywacji zakaienia wirusem Epsteina-Barr (EBV).

The aim of the study was to characterize Raji, P3HR-1 and Namalwa cell lines in the aspect of their usefulness for the research on virus Epstein-Barr (EBV) reactivation, with the participation of Toll-like receptors (TLR). During a 12-day experiment, optimal conditions of cultivation (RPMI with 10% FCS at 37 degrees C in 5% CO2) were determined. In these conditions cells showed logarithmic growth. The presence of the DNA EBV was confirmed by the PCR method, showing that 12-day long maintenance of cells does not cause the loss of the virus. The presence of genes encoding TLR2, TLR3 and TLR4 was also confirmed by PCR. The TLRs expression at the mRNA level in cells subjected to 24h stimulation with TLR2, TLR3 and TLR4 agonist (Pam3CSK4, Poly(I:C) and LPS, respectively) was determined by the RT PCR method. The presence ofTLR4 mRNA was confirmed in the case of Namalwa cells stimulated by Pam3CSK and LPS, and P3HR cells stimulated by Pam3CSK4. In the case of Raji cells the expression of none of the receptors was confirmed at the mRNA level in cells with and without stimulation.