Analysis of arsenic-modulated expression of hypothalamic estrogen receptor, thyroid receptor, and peroxisome proliferator-activated receptor gamma mRNA and simultaneous mitochondrial morphology and respiration rates in the mouse.

Arsenic has been identified as an environmental toxicant acting through various mechanisms, including the disruption of endocrine pathways. The present study assessed the ability of a single intraperitoneal injection of arsenic, to modify the mRNA expression levels of estrogen- and thyroid hormone receptors (ERα,ß; TRα,ß) and peroxisome proliferator-activated receptor gamma (PPARγ) in hypothalamic tissue homogenates of prepubertal mice in vivo. Mitochondrial respiration (MRR) was also measured, and the corresponding mitochondrial ultrastructure was analyzed. Results show that ERα,ß, and TRα expression was significantly increased by arsenic, in all concentrations examined. In contrast, TRß and PPARγ remained unaffected after arsenic injection. Arsenic-induced dose-dependent changes in state 4 mitochondrial respiration (St4). Mitochondrial morphology was affected by arsenic in that the 5 mg dose increased the size but decreased the number of mitochondria in agouti-related protein- (AgRP), while increasing the size without affecting the number of mitochondria in pro-opiomelanocortin (POMC) neurons. Arsenic also increased the size of the mitochondrial matrix per host mitochondrion. Complex analysis of dose-dependent response patterns between receptor mRNA, mitochondrial morphology, and mitochondrial respiration in the neuroendocrine hypothalamus suggests that instant arsenic effects on receptor mRNAs may not be directly reflected in St3-4 values, however, mitochondrial dynamics is affected, which predicts more pronounced effects in hypothalamus-regulated homeostatic processes after long-term arsenic exposure.

Antiviral Drug Candidate Repositioning for Streptococcus suis Infection in Non-Tumorigenic Cell Models.

The increasing prevalence of antimicrobial resistance against zoonotic bacteria, including Streptococcus (S.) suis, highlights the need for new therapeutical strategies, including the repositioning of drugs. In this study, susceptibilities of bacterial isolates were tested toward ten different 3-amidinophenyalanine (Phe(3-Am)) derivatives via determination of minimum inhibitory concentration (MIC) values. Some of these protease inhibitors, like compounds MI-432, MI-471, and MI-476, showed excellent antibacterial effects against S. suis. Their drug interaction potential was investigated using human liver microsomal cytochrome P450 (CYP450) measurements. In our work, non-tumorigenic IPEC-J2 cells and primary porcine hepatocytes were infected with S. suis, and the putative beneficial impact of these inhibitors was investigated on cell viability (Neutral red assay), on interleukin (IL)-6 levels (ELISA technique), and on redox balance (Amplex red method). The antibacterial inhibitors prevented S. suis-induced cell death (except MI-432) and decreased proinflammatory IL-6 levels. It was also found that MI-432 and MI-476 had antioxidant effects in an intestinal cell model upon S. suis infection. Concentration-dependent suppression of CYP3A4 function was found via application of all three inhibitors. In conclusion, our study suggests that the potential antiviral Phe(3-Am) derivatives with 2',4' dichloro-biphenyl moieties can be considered as effective drug candidates against S. suis infection due to their antibacterial effects.

Immune response to zinc oxide inhalation in metal fume fever, and the possible role of IL-17f.

Metal fume fever (MFF) is a work-related disease caused by the inhalation of metal particles, including zinc oxide. Chronic asthma may develop as a long-term consequence of exposure, particularly for welders and metal workers who are most at risk. In this study, we investigated the effects of ZnO fume inhalation on multiple inflammation-related cytokine- and cytokine receptor genes in mice from lung and lymph node samples, to explore the role of these in the pathogenesis of MFF. In our experiments, the animals were treated with a sub-toxic amount of ZnO fume for 4 h a day for 3 consecutive days. Sampling occurred 3 and 12 h post-treatment. We are the first to demonstrate that ZnO inhalation causes extremely increased levels of IL-17f gene expression at both sampling time points, in addition to increased gene expression rates of several other interleukins and cytokines, such as IL-4, IL-13, CXCL5, CSF-3, and IFN-γ. Our animal experiment provides new insights into the immunological processes of early metal fume fever development. IL-17f plays a crucial role in connecting immunological and oxidative stress events. The increased levels of IL-4 and IL-13 cytokines may explain the development of long-term allergic asthma after exposure to ZnO nanoparticles, which is well-known among welders, smelters, and metal workers.

Metal-oxide inhalation induced fever - Immuntoxicological aspects of welding fumes.

Metal fume fever is a well-known occupational disease that arises from prolonged exposure to subtoxic levels of zinc oxide-containing fumes or dust. This review article aims to identify and examine the possible immunotoxicological effects of inhaled zinc oxide nanoparticles. The current most widely accepted pathomechanism for the development of the disease involves the formation of reactive oxygen species following the entry of zinc oxide particles into the alveolus resulting the release of pro-inflammatory cytokines by activation of the Nuclear Factor Kappa B transcriptional signal, thus evoking the symptoms. The role of metallothionein in inducing tolerance is believed to be a key factor in mitigating the development of metal fume fever. The other, poorly proven hypothetical route is that zinc-oxide particles bind to an undefined protein in the body as haptens to form an antigen and act as an allergen. After activation of the immune system, primary antibodies and immune complexes are developed and type 1. hypersensitivity reaction occurs, that can cause asthmatic dyspnoea, urticaria and angioedema. The development of tolerance is explained by the formation of secondary antibodies against primary antibodies. Oxidative stress and immunological processes cannot be completely separated from each other, as they can induce each other.

Preliminary study to investigate the distribution and effects of certain metals after inhalation of welding fumes in mice.

The most important welding processes used are the gas metal arc (GMA) welding, the tungsten inert gas (TIG) welding, and the manual metal arc (MMA) welding processes. The goal of our investigation was to monitor the distribution of iron (Fe), manganese (Mn), calcium (Ca), and magnesium (Mg) in the lung, spleen, liver, and kidney of mice after inhalation exposure of different welding methods using different steel base materials. The treatment groups were the following: MMA-mild steel, MMA-molybdenum-manganese (MoMn) alloy, TIG-mild steel, and TIG-stainless steel. The samples were taken 24 and 96 h after the treatments. Most importantly, it was found that the Mn concentration in the lung' samples of the MMA-mild steel and the MMA-MoMn groups was increased extremely at both sampling times and in the spleen' samples also. In the TIG groups, the rise of the Mn concentration was only considerable in the lungs and spleens at 24 h, and emerged concentration was found in the liver in 96 h samples. Histopathology demonstrated emerged siderin content in the spleens of the treated animals and in siderin filled macrophages in the lungs mostly in all treated groups. Traces of high-level glycogen retention was found in the MMA groups at both sampling times. Similar glycogen retention in TIG-Ms and TIG stainless group's liver samples and emerged number of vacuoles, especially in the hepatocytes of the TIG-stainless steel 96 h group were also found. The mentioned results raise the consequence that there is a considerable difference in the kinetics of the Mn distribution between the MMA- and the TIG-fume-treated groups. Hence, the result suggests that manganese has a particle-size-dependent toxico-kinetics property. The anomaly of the glycogen metabolism indicates the systemic effect of the welding fumes. Also, the numerous vacuoles mentioned above show a possible liver-specific adverse effect of some components of the TIG-stainless steel welding fumes.

Effects of Acute Heat Stress on a Newly Established Chicken Hepatocyte-Nonparenchymal Cell Co-Culture Model.

Heat stress is one of the most important issues in broiler flocks impairing animal health and productivity. On a cellular level, excess heat exposure can trigger heat shock response acting for the restoration of cell homeostasis by several mechanisms, such as affecting heat shock protein synthesis, redox homeostasis and pro-inflammatory cytokine production. The major aim of this study was to establish a novel avian hepatocyte-nonparenchymal cell co-culture as a model for investigating the cellular effects of heat stress and its interaction with inflammation in chicken liver. Cell fractions were isolated by differential centrifugation from a freshly perfused chicken liver, and hepatocyte mono-cultures as well as hepatocyte-nonparenchymal cell co-cultures (with cell ratio 6:1, hepatocytes to nonparenchymal cells, mimicking a milder hepatic inflammation) were prepared. Isolated and cultured cells were characterized by flow cytometry and immunocytochemistry applying hepatocyte- and macrophage-specific antibodies. Confluent cell cultures were exposed to 43 °C temperature for 1 or 2 h, while controls were cultured at 38.5 °C. The metabolic activity, LDH enzyme activity, reactive oxygen species (H2O2) production, extracellular concentration of heat shock protein 70 (HSP70), and that of the pro-inflammatory cytokines interleukin (IL-)6 and IL-8 were assessed. Shorter heat stress applied for 1 h could strongly influence liver cell function by significantly increasing catabolic metabolism and extracellular H2O2 release, and by significantly decreasing HSP70, IL-6, and IL-8 production on both cell culture models. However, all these alterations were restored after 2 h heat exposure, indicating a fast recovery of liver cells. Hepatocyte mono-cultures and hepatocyte-nonparenchymal cell co-cultures responded to heat stress in a similar manner, but the higher metabolic rate of co-cultured cells may have contributed to a better capability of inflamed liver cells for accommodation to stress conditions. In conclusion, the established new primary cell culture models provide suitable tools for studying the hepatic inflammatory and stress response. The results of this study highlight the impact of short-term heat stress on the liver in chickens, underline the mediatory role of oxidative stress in acute stress response, and suggest a fast cellular adaptation potential in liver cells.

[Mechanism of action of modulated electro- hyperthermia (mEHT) induced tumor damage in colorectal adenocarcinoma models]. / A modulált elektrohipertermia (mEHT) indukálta tumorkárosodás mechanizmusa kolorektális karcinóma modellben.

Modulated electro-hyperthermia (mEHT) is a non-invasive treatment modality of cancer where electric field generated by 13.56 MHz radiofrequency can selectively accumulate in malignant tumors compared to adjacent normal tissues. This effect is based on the metabolic shift in cancer cells which upregulates glycolysis even under oxygenated conditions (Warburg effect), resulting in elevated lactate and ion concentration. The concomitant increased permittivity can induce dielectric polarization and rotational friction of dipole molecules resulting in elevated core temperature, which can be controlled at 42 °C with the treating instrument. Complementary application of loco-regional mEHT can improve the efficiency of chemo-, radio- and recently molecular targeted therapies based on increasing local perfusion and xenobiotic concentration, resolving tumor hypoxia and improved immune surveillance supported by high-fever range hyperthermia. We earlier showed that mEHT has its own tumor inhibiting/destructing effect, however, its mechanism had not been clarified. In this project we have investigated the molecular mechanism of action of mEHT treatment using in vitro and in vivo models of colorectal adenocarcinoma.

Modulated electro-hyperthermia induced loco-regional and systemic tumor destruction in colorectal cancer allografts.

Background: Modulated electro-hyperthermia (mEHT), a non-invasive intervention using 13.56 MHz radiofrequency, can selectively target cancers due to their elevated glycolysis (Warburg-effect), extracellular ion concentration and conductivity compared to normal tissues. We showed earlier that mEHT alone can provoke apoptosis and damage associated molecular pattern (DAMP) signals in human HT29 colorectal cancer xenografts of immunocompromised mice. Materials: Here we tested the mEHT induced stress and immune responses in C26 colorectal cancer allografts of immunocompetent (BALB/c) mice between 12-72 h post-treatment. The right side of the symmetrical tumors grown in both femoral regions of mice were treated for 30 minutes, while the left side tumors served for untreated controls. Results: Loco-regional mEHT treatment induced an ongoing and significant tumor damage with the blockade of cell cycle progression indicated by the loss of nuclear Ki67 protein. Nuclear shrinkage, apoptotic bodies and DNA fragmentation detected using TUNEL assay confirmed apoptosis. Cleaved/activated-caspase-8 and -caspase-3 upregulation along with mitochondrial translocation of bax protein and release of cytochrome-c were consistent with the activation of both the extrinsic and intrinsic caspase-dependent programmed cell death pathways. The prominent release of stress-associated Hsp70, calreticulin and HMGB1 proteins, relevant to DAMP signaling, was accompanied by the significant tumor infiltration by S100 positive antigen presenting dendritic cells and CD3 positive T-cells with only scant FoxP3 positive regulatory T-cells. In addition, mEHT combined with a chlorogenic acid rich T-cell promoting agent induced significant cell death both in the treated and the untreated contralateral tumors indicating a systemic anti-tumor effect. Conclusions: mEHT induced caspase-dependent programmed cell death and the release of stress associated DAMP proteins in colorectal cancer allografts can provoke major immune cell infiltration. Accumulating antigen presenting dendritic cells and T-cells are likely to contribute to the ongoing tumor destruction by an immunogenic cell death mechanism both locally and through systemic effect at distant tumor sites.

Demonstration of homologous recombination events in the evolution of bovine viral diarrhoea virus by in silico investigations.

Complete genome sequences of bovine viral diarrhoea virus types 1 and 2 (BVDV-1 and 2) deposited in the GenBank were submitted to bioinformatic analysis using a recombination-detecting software. The results indicate that recombination events are not rare in the case of BVDV, which frequently causes immunotolerance and, consequently, persistent infection in calves. The lack of specific immunity provides an ideal possibility for multiple infections by antigenically related but genetically different BVDV strains, and hence recombinations may occur. Among the 62 BVDV-1 genomes five recombinants and their possible parent strains, while among the 50 BVDV-2 genomes one simple recombinant and its parent strains were identified, which were supported by extremely strong probability values (P values varying between 1.26 × 10-4 and 1.58 × 10-310). Besides the newly identified recombinants, recombination events described previously were confirmed, but in some of these cases former information was completed with new data, or different parent(s) were suggested by the programme (RDP 4.46 BETA) used in this study.

Seroprevalence of bovine viral diarrhoea virus in Hungary - situation before launching an eradication campaign.

Bovine viral diarrhoea (BVD) is a viral disease appearing in various forms and causing high economic losses in the cattle stocks of Hungary. The aim of the present study was to determine the prevalence of bovine viral diarrhoea virus (BVDV) in Hungary through a monitoring survey carried out on samples collected in cattle-keeping units throughout the country. Since no such survey had been carried out in Hungary during the last thirty years, our study may serve as a basis for later monitoring investigations aimed at following the progress of an expected eradication campaign of BVD. The tests were carried out using an ELISA method, on a total of 1200 blood samples submitted from 54 cattle herds. The herds had not been vaccinated against BVDV before the sampling. Out of the 1200 samples, 521 proved to be positive (43.4%), 40 gave doubtful result (3.3%) and 639 were negative (53.3%). In some stocks the samples were collected from cows having completed several lactation periods, and therefore the seronegativity indicates the BVDV-free status of the given stock. Moreover, among the positive herds we found a few where the seropositivity rate was rather low (< 5%). According to the results of the survey, a rather high portion (about one third) of the cattle-keeping units of Hungary can be regarded as BVDV free, which ratio is much higher than had been expected on the basis of surveys carried out on a lower number of samples and in smaller regions of the country. Hence, the chances of an eradication campaign launched in the near future, or carried out parallel to the IBR eradication programme, are better than previously expected.

Changes in the distribution of type II transmembrane serine protease, TMPRSS2 and in paracellular permeability in IPEC-J2 cells exposed to oxidative stress.

The effect of oxidative stress on barrier integrity and localization of transmembrane serine proteinase 2 (TMPRSS2) were studied using porcine epithelial IPEC-J2 cells on membrane inserts. Increased paracellular permeability of FITC-dextran 4 kDa (fluorescence intensity 43,508 ± 2,391 versus 3,550 ± 759) and that of gentamicin (3.41 ± 0.06 % increase to controls) were measured parallel with the reduced transepithelial electrical resistance (23.3 ± 4.06 % decrease) of cell layers 6 h after 1 h 1 mM H2O2 treatment. The immunohistochemical localization of adherens junctional ß-catenin was not affected by reactive oxygen species (ROS) up to 4 mM H2O2. Peroxide-triggered enhanced paracellular permeability of IPEC-J2 cell layer was accompanied by predominantly cytoplasmic occurrence of TMPRSS2 embedded in cell membrane under physiological conditions. These results support that ROS can influence paracellular gate opening via multifaceted mode of action without involvement of ß-catenin redistribution in adherens junction. Altered distribution pattern of TMPRSS2 and relocalized transmembrane serine protease activity may contribute to weakening of epithelial barrier integrity under acute oxidative stress.

Upregulation of heat shock proteins and the promotion of damage-associated molecular pattern signals in a colorectal cancer model by modulated electrohyperthermia.

In modulated electrohyperthermia (mEHT) the enrichment of electric field and the concomitant heat can selectively induce cell death in malignant tumors as a result of elevated glycolysis, lactate production (Warburg effect), and reduced electric impedance in cancer compared to normal tissues. Earlier, we showed in HT29 colorectal cancer xenografts that the mEHT-provoked programmed cell death was dominantly caspase independent and driven by apoptosis inducing factor activation. Using this model here, we studied the mEHT-related cell stress 0-, 1-, 4-, 8-, 14-, 24-, 48-, 72-, 120-, 168- and 216-h post-treatment by focusing on damage-associated molecular pattern (DAMP) signals. Significant cell death response upon mEHT treatment was accompanied by the early upregulation (4-h post-treatment) of heat shock protein (Hsp70 and Hsp90) mRNA levels. In situ, the treatment resulted in spatiotemporal occurrence of a DAMP protein signal sequence featured by the significant cytoplasmic to cell membrane translocation of calreticulin at 4 h, Hsp70 between 14 and 24 h and Hsp90 between 24- and 216-h post-treatment. The release of high-mobility group box1 protein (HMGB1) from tumor cell nuclei from 24-h post-treatment and its clearance from tumor cells by 48 h was also detected. Our results suggest that mEHT treatment can induce a DAMP-related signal sequence in colorectal cancer xenografts that may be relevant for promoting immunological cell death response, which need to be further tested in immune-competent animals.

Genetic analysis and phylogenetic comparison of Black queen cell virus genotypes.

Phylogenetic analysis of 22 Black queen cell virus (BQCV) genotypes collected from honeybee colonies in Poland, Austria and Hungary was performed on a partial helicase enzyme coding region (ORF1) and on a partial structural polypeptide coding region (ORF2). While the phylogeny based on the ORF2 region showed--with the exception of one strain from Poland--clustering of the genotypes corresponding to their geographic origin, the ORF1-based tree exhibited a completely different distribution of the Polish strains: three of them clustered within a branch clearly separated from all other central European BQCVs, while four other Polish strains remained well within the central European BQCV genotypes. In order to investigate this discrepancy in more detail, the nearly complete genome sequences of the three differing Polish strains were determined, together with one Hungarian sample. The sequences were aligned to each other and to the reference strain from South-Africa. Comparison of the different genome regions revealed that the 5'-UTR and the intergenic regions of the BQCV genome are highly conserved with longer homologous sections. ORF1 (non-structural protein coding region) was found more variable compared to ORF2 (structural protein coding region). The 5'-proximal third of ORF1 was particularly variable and contained several deletions/insertions. The sudden changes in the similarity levels of BQCV strains in different genomic regions are indicative of preceding recombination events.

First detection and dominance of Nosema ceranae in Hungarian honeybee colonies.

Microsporidiosis (nosema disease) of the European honeybee ( Apis mellifera L.) is present in bee colonies worldwide. Until recently, Nosema apis had been regarded as the causative agent of the disease, which may have many negative effects on the colony and cause heavy economic losses in apicultures. Another microsporidium species, Nosema ceranae , was reported to infest the Asian honeybee ( Apis ceranae ), but both honeybee species are susceptible to both microsporidia. In the European honeybee N. ceranae was first detected in Spain in the year 2006. As it is difficult to distinguish N. ceranae and N. apis morphologically, a rapid and accurate assay has been developed to differentiate N. apis and N. ceranae based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the partial large subunit ribosomal RNA. The assay was tested on 38 Nosema -infested bee samples, which were collected from geographically distant Hungarian bee colonies representing all regions of the country. Only one sample contained N. apis , and in the other 37 samples N. ceranae was detected, which indicates the dominance of N. ceranae in Hungarian apiaries. This is the first report on the presence of N. ceranae in Hungary.

Location of parotid preganglionic neurons in the inferior salivatory nucleus and their relation to the superior salivatory nucleus of rat.

In major brain maps the location of the salivatory nuclei of the rat is depicted from the level of the root of the facial nerve to the level of the rostral tip of the nucleus of the solitary tract. Most published data deal with the superior salivatory nucleus (SSN). In the present study the topography of the parasympathetic preganglionic neurons of the inferior salivatory nucleus (ISN) that innervate the parotid gland through the otic ganglion was determined by means of a retrograde transneuronal labeling technique. Parasympathetic, sympathetic and sensory neurons were labeled following injection of the virus into the parotid gland. The majority of the ISN neurons were found dorsal to the facial motor nucleus, embedded in the parvocellular reticular formation. In addition to the ISN neurons, virus-labelled cells were present in the intermediolateral (IML) cell column of the thoracic spinal cord, in the brainstem catecholamine groups, and in medullary raphe neurons. The removal of the ipsilateral superior cervical ganglion prior to the virus injection into the parotid gland did not influence the labeling of the ISN neurons but labeled neurons were not observed in the IML and A5 catecholamine cell group. In our previous study we had defined the relationship between the lacrimal and submandibular subdivison of the SSN, while in the present study we defined the relationship between the ISN and the lacrimal subdivision of SSN: the later located ventrolaterally to the caudal portion of the ISN. On the basis of these data a three-dimensional topography is given suggesting the relationship between the ISN and SSN.