Luka Traven

Murine cytomegalovirus regulation of NKG2D ligands

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes morbidity risk in immunologically suppressed and immunodeficient patients including congenital infections. Approaches to curb the consequences of HCMV infections are restricted by a lack of complete understanding of viral pathogenesis. The infection of mice with murine cytomegalovirus (MCMV) as a model of HCMV infection has been particularly useful in elucidating the role of innate and adaptive immune response mechanisms. A large number of cytomegalovirus genes modulate the innate and the adaptive host immune response. The products of several MCMV genes are involved in subverting the natural killer (NK) cell response by down-modulating cellular ligands for the NKG2D receptor expressed on NK cells and CD8+ T cells. Mutant viruses lacking these immunoevasion genes are attenuated with respect to virus growth in vivo. Given the importance of the NKG2D receptor in controlling both NK- and T cell-mediated immunity, it is of tremendous importance to understand the molecular mechanisms and consequences of viral regulation of the NKG2D ligands.

GENOTOXICITY OF MARINE SEDIMENTS IN THE FISH HEPATOMA CELL LINE PLHC-1 AS ASSESSED BY THE COMET ASSAY

The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.

CYP1A induction potential and the concentration of priority pollutants in marine sediment samples – In vitro evaluation using the PLHC-1 fish hepatoma cell line

The use of in vitro biotests in combination with chemical determination of priority pollutants is considered a promising approach in environmental risk assessment. The main goal of this study was to evaluate the relationship between the CYP1A induction potential and the concentration of priority pollutants (PAHs, PCBs and heavy metals) in contaminated marine sediments. Six sediment samples characterized by different types of pollution were collected from the Bay of Kvarner, Croatia. CYP1A induction potency was determined in vitro by the measurement of ethoxyresorufin-O-deethylase (EROD) activity in PLHC-1 fish hepatoma cells. The results were compared to the potency of the model CYP1A inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and expressed in 2,3,7,8-TCDD equivalents. All of the tested sediment samples were able to induce CYP1A activity in a dose-dependent manner. On a general scale, there was a good correlation between CYP1A induction and the concentration of priority pollutants in the tested samples. However, some samples, which had relatively low levels of priority pollutants, exhibited a strong CYP1A induction response. Therefore, apart from the confirmed usability and sensitivity of the EROD determination in the PLHC-1 cells as a suitable in vitro model in ecotoxicology, the results of this study indicate that the list of priority pollutants usually determined in the attempt to evaluate the risk of adverse effects to marine wildlife should be reconsidered.

Innate immunity regulates adaptive immune response: lessons learned from studying the interplay between NK and CD8+ T cells during MCMV infection

Natural killer (NK) cells play a crucial role in early immune response against cytomegalovirus infection. A large and mounting body of data indicate that these cells are involved in the regulation of the adaptive immune response as well. By using mouse cytomegalovirus (MCMV) as a model, several groups provided novel insights into the role of NK cells in the development and kinetics of antiviral CD8+ T cell response. Depending on infection conditions, virus strain and the genetic background of mice used, NK cells are either positive or negative regulators of the CD8+ T cell response. At present, there is no unique explanation for the observed differences between various experimental systems used. In this review we discuss the mechanisms involved in the interplay between NK and CD8+ T cells in the early control of MCMV infection.

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.

The responses of the hepatosomatic index (HSI), 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione-S-transferase (GST) activity in sea bass (Dicentrarchus labrax, Linnaeus 1758) caged at a polluted site: implications for their use in environmental risk assessment

The present study investigates the response of three hepatic biomarkers in adult sea bass (Dicentrarchus labrax, Linnaeus 1758) caged at a wastewater outlet of an oil refinery with fish caged at a pristine site used as controls. The biomarkers that were investigated were the hepatosomatic index (HSI), 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione-S-transferase (GST) activity. In addition, we have measured the levels of polycyclic aromatic hydrocarbons (PAHs) and selected heavy metals (lead, cadmium, mercury, copper and zinc) in sediment samples at the polluted site. Although the polluted site had high environmental levels of PAHs and heavy metals, there was no difference in hepatic EROD activity and HSI between fish caged at the polluted site and controls. On the other hand, GST activity was significantly lower in fish caged at the polluted site compared to controls. Our results point out that the studied biomarkers have limited use in environmental risk assessment studies, at least when caged adult sea bass is used as the sentinel species and complex toxicant mixtures are involved.

Sources, trends and ecotoxicological risks of PAH pollution in surface sediments from the northern Adriatic Sea (Croatia)

In this study, the levels, sources and historical trends (2007-2012) of PAH pollution in surface sediments in the northern Adriatic Sea (Croatia) have been assessed. In addition, ecotoxicological risks have been estimated by evaluating the PAH dataset against Sediment Quality Guidelines (SQGs). The results indicate that PAH in surface sediments are of pyrolitic origin with no clear positive or negative trend in PAH concentration over the investigated period. Evaluations against SQG and mSQGq indicate that surface sediments at all sampling sites have a low to medium ecotoxicological risk. In order to improve trend analysis and characterization of PAH pollution in surface sediments in this region an increase in spatial resolution of sampling points is recommended.

The specific NK cell response in concert with perforin prevents CD8+ T cell-mediated immunopathology after mouse cytomegalovirus infection

Abstract Natural killer (NK) and CD8+ T cells play a crucial role in the control of mouse cytomegalovirus (MCMV) infection. These effector cells exert their functions by releasing antiviral cytokines and by cytolytic mechanisms including perforin activation. In addition to their role in virus control, NK cells play an immunoregulatory role since they shape the CD8+ T cell response to MCMV. To investigate the role of perforin-dependent cytolytic mechanism in NK cell modulation of CD8+ T cell response during acute MCMV infection, we have used perforin-deficient C57BL/6 mice (Prf1−/−) and have shown that virus control by CD8+ T cells in Prf1−/− mice is more efficient if NK cells are activated by the engagement of the Ly49H receptor with the m157 MCMV protein. A lack of perforin results in severe liver inflammation after MCMV infection, which is characterized by immunopathological lesions that are more pronounced in Prf1−/− mice infected with virus unable to activate NK cells. This immunopathology is caused by an abundant infiltration of activated CD8+ T cells. The depletion of CD8+ T cells has markedly reduced pathohistological lesions in the liver and improved the survival of Prf1−/− mice in spite of an increased viral load. Altogether, the results of our study suggest that a lack of perforin and absence of the specific activation of NK cells during acute MCMV infection lead to an unleashed CD8+ T cell response that is detrimental for the host.

Historical trends (1998-2012) of nickel (Ni), copper (Cu) and chromium (Cr) concentrations in marine sediments at four locations in the Northern Adriatic Sea.

Historical trends (1998-2012) nickel (Ni), copper (Cu) and chromium (Cr) concentrations in marine sediments were assessed at four locations in the Northern Adriatic Sea (Croatia) in the proximity of an oil refinery. Ecological risks were characterized by benchmarking the dataset against Sediment Quality Guidelines (SQG). A significant number of samples had Ni values above ERL with no exceedance of the ERL values for Cu and Cr. Weak positive historical trends were found for only for Cu. At all sites there were statistically significant correlations between Ni and Cr indicating a common origin of these heavy metals in the investigated marine sediments. There were statistically significant differences between the sites under the direct influence of the oil refinery compared to the control site indicating the possibility that the oil refinery is contributing to the concentration of these heavy metals in the marine sediments.

Management of municipal solid waste in Croatia: Analysis of current practices with performance benchmarking against other European Union member states:

The European Commission has adopted a very ambitious circular economy package and has consequently revised many legislative proposals on waste. The new targets include achieving a recycling rate of 65% by 2030 and imposing a cap on landfilled waste to no more than 10% (as a percentage of weight). Using available European and national databases on municipal solid waste indicators, we have analysed municipal solid waste production trends for Croatia and have benchmarked the indicators against other EU member states and EU averages. Our analysis points out that the production of municipal solid waste has steadily risen over the last two decades. Although Croatia produces a relatively low amount of municipal solid waste compared with other EU countries and EU averages, gross domestic product adjusted waste production reveals that for every euro of gross domestic product Croatia produces substantial amounts of municipal solid waste. As a matter of fact, among all the EU member states, only Bulgaria had a worse performance than Croatia. Regarding recycling rates, Croatia recycles 15% of its municipal solid waste and incinerates approximately 3% (by weight). The rest (82% by weight) is currently being landfilled. Regarding the treatment of waste, Croatia has implemented mechanical–biological treatment technology, but our analysis reveals that the initial number of mechanical–biological treatment plants will likely have to be scaled down taking into consideration the new EU waste management targets.