Jiří Pikula

Detoxification and oxidative stress responses along with microcystins accumulation in Japanese quail exposed to cyanobacterial biomass

The cyanobacterial exposure has been implicated in mass mortalities of wild birds, but information on the actual effects of cyanobacteria on birds in controlled studies is missing. Effects on detoxification and antioxidant parameters as well as bioaccumulation of microcystins (MCs) were studied in birds after sub-lethal exposure to natural cyanobacterial biomass. Four treatment groups of model species Japanese quail (Coturnix coturnix japonica) were exposed to controlled doses of cyanobacterial bloom during acute (10 days) and sub-chronic (30 days) experiment. The daily doses of cyanobacterial biomass corresponded to 0.2-224.6 ng MCs/g body weight. Significant accumulation of MCs was observed in the liver for both test durations and slight accumulation also in the muscles of the highest treatment group from acute test. The greatest accumulation was observed in the liver of the highest treatment group in the acute test reaching average concentration of 43.7 ng MCs/g fresh weight. The parameters of detoxification metabolism and oxidative stress were studied in the liver, heart and brain. The cyanobacterial exposure caused an increase of activity of cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase representing the activation phase of detoxification metabolism. Also the conjugation phase of detoxification, namely the activity of glutathione-S-transferase, was altered. Cyanobacterial exposure also modulated oxidative stress responses including the level of glutathione and activities of glutathione-related enzymes and caused increase in lipid peroxidation. The overall pattern of detoxification parameters and oxidative stress responses clearly separated the control and the lowest exposure group from all the higher exposed groups. This is the first controlled study documenting the induction of oxidative stress along with MCs accumulation in birds exposed to natural cyanobacterial biomass. The data also suggest that increased activities of detoxification enzymes could lead to greater biotransformation and elimination of the MCs at the longer exposure time.

Piezoelectric Biosensor for a Simple Serological Diagnosis of Tularemia in Infected European Brown Hares (Lepus europaeus)

Piezoelectric biosensor was used for diagnosis of infection by Francisella tularensis subsp. holarctica in European brown hares. Two kinds of experiments were performed in this study. First, sera from experimentally infected European brown hares (Lepus europaeus) were assayed by piezoelectric biosensor and the seventh day post infection was found as the first one when statistically significant diagnosis of tularemia was possible; all other sera collected from hares later than on day 7 following the infection were found tularemia positive. Typing to classify the field strain of F. tularensis used for the experimental infection was confirmed by proteome study. Second, sera from 35 European brown hare specimens sampled at hunting grounds and tested as tularemia positive by slow agglutination allowed diagnosis of tularemia by the piezoelectric biosensor. All these sera of naturally infected hares were found as tularemia positive, too. Efficacy of the piezoelectric biosensor for the serological diagnosis of tularemia is discussed.

Macrophage-Assisted Inflammation and Pharmacological Regulation of the Cholinergic Anti-Inflammatory Pathway

Macrophages play an important role in the immune system. They also participate in multiple processes including angiogenesis and triggering of inflammation. The present study summarizes pieces of knowledge on the importance of macrophages in disease, especially the inflammation. Special attention is paid to the cholinergic anti-inflammatory pathway (CAP) associated with the nicotinic acetylcholine receptor (nAChR) and the parasympathetic nervous system. The current pharmacological effectiveness in suppressing the inflammation in general and the septic shock in particular, is limited. CAP was discovered recently and it seems to be a suitable target for the development of new drugs. Moreover, available drugs binding to either nAChR or acetylcholinesterase (AChE) are candidates for either an inhibition or enhancement of CAP. Though the current scientific databases do not include all necessary data on the association of CAP with body functions and the research is quite intensive, the objective of the present review is to introduce the current trends and to critically evaluate CAP and macrophage-associated pathways.

Establishment of Myotis myotis cell lines--model for investigation of host-pathogen interaction in a natural host for emerging viruses.

Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of M. myotis.

Effect of Intramuscular Injection on Oxidative Homeostasis in Laboratory Guinea Pig Model

In animal models, there was observed alteration of various physiological processes caused by microtraumas. Here reported experiment was aimed on the research of link between injection and development of an oxidative imbalance. Laboratory guinea pig was chosen as a suitable model for examining of the oxidative stress. Markers indicating oxidative homeostasis were assayed in the frontal, temporal and occipital brain lobe, cerebellum, liver, kidney, spleen and heart one hour after an intramuscular injection. Common biochemical parameters were measured in plasma samples as well. The most extensive effect was observed in the heart where the thiobarbituric acid reactive substances value was more than twice increased after the injection. The level of carbonylated proteins was significantly elevated in the kidney and ferric reducing antioxidant power value was increased in the brain compartments. The enzyme activities in the organs were not influenced except the activity of superoxide dismutase, which was moderately decreased in the brain. In the plasma samples, there was observed increase of the blood urea nitrogen. The results showed significant the influence of the intramuscular injection on a development of an oxidative insult. The injection can be considered as an adverse effect with quite extensive stress consequences.