Pavel Hyršl

Pathogen Entrapment by Transglutaminase-A Conserved Early Innate Immune Mechanism

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.

Clotting Factors and Eicosanoids Protect against Nematode Infections

We show that hemolymph clotting protects Drosophila melanogaster against infections with an entomopathogenic nematode and its symbiotic bacterium. We also provide biochemical and genetic evidence for an involvement of eicosanoids in the same infection model. Taken together, our results confirm the conserved nature of the immune function of clot formation.

Are fish immune systems really affected by parasites? an immunoecological study of common carp (Cyprinus carpio)

BackgroundThe basic function of the immune system is to protect an organism against infection in order to minimize the fitness costs of being infected. According to life-history theory, energy resources are in a trade-off between the costly demands of immunity and other physiological demands. Concerning fish, both physiology and immunity are influenced by seasonal changes (i.e. temporal variation) associated to the changes of abiotic factors (such as primarily water temperature) and interactions with pathogens and parasites. In this study, we investigated the potential associations between the physiology and immunocompetence of common carp (Cyprinus carpio) collected during five different periods of a given year. Our sampling included the periods with temporal variability and thus, it presented a different level in exposure to parasites. We analyzed which of two factors, seasonality or parasitism, had the strongest impact on changes in fish physiology and immunity.ResultsWe found that seasonal changes play a key role in affecting the analyzed measurements of physiology, immunity and parasitism. The correlation analysis revealed the relationships between the measures of overall host physiology, immunity and parasite load when temporal variability effect was removed. When analyzing separately parasite groups with different life-strategies, we found that fish with a worse condition status were infected more by monogeneans, representing the most abundant parasite group. The high infection by cestodes seems to activate the phagocytes. A weak relationship was found between spleen size and abundance of trematodes when taking into account seasonal changes.ConclusionsEven if no direct trade-off between the measures of host immunity and physiology was confirmed when taking into account the seasonality, it seems that seasonal variability affects host immunity and physiology through energy allocation in a trade-off between life important functions, especially reproduction and fish condition. Host immunity measures were not found to be in a trade-off with the investigated physiological traits or functions, but we confirmed the immunosuppressive role of 11-ketotestosterone on fish immunity measured by complement activity. We suggest that the different parasite life-strategies influence different aspects of host physiology and activate the different immunity pathways.

Genome-wide transcriptional analysis of Drosophila larvae infected by entomopathogenic nematodes shows involvement of complement, recognition and extracellular matrix proteins.

Heterorhabditis bacteriophora is an entomopathogenic nematode (EPN) which infects its host by accessing the hemolymph where it releases endosymbiotic bacteria of the species Photorhabdus luminescens. We performed a genome-wide transcriptional analysis of the Drosophila response to EPN infection at the time point at which the nematodes reached the hemolymph either via the cuticle or the gut and the bacteria had started to multiply. Many of the most strongly induced genes have been implicated in immune responses in other infection models. Mapping of the complete set of differentially regulated genes showed the hallmarks of a wound response, but also identified a large fraction of EPN-specific transcripts. Several genes identified by transcriptome profiling or their homologues play protective roles during nematode infections. Genes that positively contribute to controlling nematobacterial infections encode: a homolog of thioester-containing complement protein 3, a basement membrane component (glutactin), a recognition protein (GNBP-like 3) and possibly several small peptides. Of note is that several of these genes have not previously been implicated in immune responses.

Biological activities of selected polyphenol-rich fruits related to immunity and gastrointestinal health

Small fruits are a rich source of bioactive substances, including polyphenols, and are therefore suitable raw materials for the production of functional foods. In the current work, we studied the antioxidative properties of six fruits: rosehip, chokeberry, hawthorn, blackcurrant, blueberry and rowanberry via different methods (ORAC, TRAP, HORAC and inhibition of lipid peroxidation). Their effect on the production of reactive oxygen species (ROS) by phagocytes, antimicrobial properties against 11 human pathogens, and mitogenic effect on hamster spleen lymphocytes were also tested. Rosehip extract showed the highest antioxidant activity via ORAC, TRAP and HORAC assays, whereas blueberry extract was the most potent inhibitor of lipid peroxidation. All extracts inhibited ROS production of opsonized zymosan-activated phagocytes, indicating that extracts interfere with the signaling cascade of phagocyte activation upstream to the protein kinase C activation. Chokeberry, blackcurrant and rowanberry extracts revealed strong antimicrobial properties against a broad spectrum of microorganisms and also had the highest mitogenic activity.

Eicosanoids mediate hemolymph oxidative and antioxidative response in larvae of Galleria mellonella L.

Antioxidant enzymes play a major role in the defense against pro-oxidative effects of xenobiotics and pro-oxidant plant allelochemicals in insects. We posed the hypothesis that eicosanoids also mediate antioxidant enzymatic defense reactions to pro-oxidant challenge. To test this idea, we reared first-instar larvae of Galleria mellonella (L.) with the lypoxygenase inhibitor, esculetin (0.001%), the phospholipase A(2) inhibitor, dexamethasone (0.001%) and the dual inhibitor of cyclooxygenase and lipoxygenase, phenidone (0.1%) to seventh-instars. Newly ecdysed seventh-instars were then fed on artificial diet containing 0.05% xanthotoxin (XA) for 2 days. Treating seventh-instar larvae of G. mellonella with XA induced lipid peroxidation and protein carbonylation as evident from the increased content of malondialdehyde (MDA) and protein carbonyls respectively, and antioxidative enzymatic response in a dose-dependent manner. High dietary XA concentrations (0.005 and 0.1%) were associated with increasing MDA and carbonyl content (by 3-fold) and antioxidant enzyme activities, superoxide dismutase (SOD) (by 3-fold) and catalase (CAT) (by 4-fold), and glutathione-dependent enzymes, glutathione S-transferase (GST) (by 15-fold) and glutathione peroxidase (GPx) (by 7-fold). Relative to control, eicosanoid biosynthesis inhibitors (EBIs) esculetin, dexamethasone and phenidone also resulted in impaired MDA content and antioxidant enzyme activities. However, carbonyl content did not differ between control- and EBIs-feeding larvae. Finally, MDA and carbonyl content, and antioxidant enzymes SOD, GST and GPx activities exhibited an incremental increase while CAT activity was decreased in the experimental larvae that had been reared on media amended with esculetin, dexamethasone and phenidone and then challenged with our standard XA challenge dose. Two of the markers indicated that significantly higher levels of oxidative stress were produced in the hemolymph tissue of larvae fed diets supplemented with EBIs and then challenged with XA. This oxidative stress was associated with elicited antioxidative responses by increasing SOD, GST and GPx and decreasing CAT activities in hemolymph. We infer from these findings that eicosanoids mediate insect antioxidant enzymatic responses to dietary pro-oxidants.

Rank-based biomarker index to assess cadmium ecotoxicity on the earthworm Eisenia andrei.

A proper soil risk assessment needs to estimate the processes that affect the fate and the behaviour of a contaminant, which are influenced by soil biotic and abiotic components. For this reason, the measurement of biomarkers in soil bioindicator organisms, such as earthworms, has recently received increasing attention. In this study, the earthworm Eisenia andrei was used to assess the pollutant-induced stress syndrome after exposure to sublethal concentrations of Cd (10 or 100 μg g(-1)) in OECD soil, after 14 d of exposure. Cadmium bioaccumulation and potential biomarkers such as catalase (CAT), hydrogen peroxide (H2O2), glutathione-S-transferase (GST), malondialdehyde (MDA), phenoloxidase (PO), metallothioneins (MTs) and genotoxic damage were determined. Results suggested that the exposure to 10 and 100 μg g(-1) Cd significantly increased Cd bioaccumulation, MTs and MDA; 100 μg g(-1) Cd contamination evidenced significantly higher values of H2O2 content and PO activity; CAT activity was inhibited at the higher concentration while GST and Comet assay did not show any significant differences from the control. Rank-based biomarker index showed that both different contaminated soils had an effect on the earthworms and allowed to validate the ecotoxicological relevance of this battery of biomarkers for a promising integrated multi-marker approach in soil monitoring and assessment.

An improved method for nematode infection assays in Drosophila larvae

The infective juveniles (IJs) of entomopathogenic nematodes (EPNs) seek out host insects and release their symbiotic bacteria into their body cavity causing septicaemia, which eventually leads to host death. The interaction between EPNs and their hosts are only partially understood, in particular the host immune responses appears to involve pathways other than phagocytosis and the canonical transcriptional induction pathways. These pathways are genetically tractable and include for example clotting factors and lipid mediators. The aim of this study was to optimize the nematode infections in Drosophila melanogaster larvae, a well-studied and genetically tractable model organism. Here we show that two nematode species namely Steinernema feltiae and Heterorhabditis bacteriophora display different infectivity toward Drosophila larvae with the latter being less pathogenic. The effects of supporting media and IJ dosage on the mortality of the hosts were assessed and optimized. Using optimum conditions, a faster and efficient setup for nematode infections was developed. This newly established infection model in Drosophila larvae will be applicable in large scale screens aimed at identifying novel genes/pathways involved in innate immune responses.

Interactions between immunocompetence, somatic condition and parasitism in the chub Leuciscus cephalus in early spring

Relationships between immunocompetence, somatic condition, parasitism and water temperature in a wild population of chub Leuciscus cephalus were investigated. The effects of a rapid temperature increase in early spring were studied for both sexes as water temperature affects immunocompetence. Investment in gonads and activity of mucus lysozyme were negatively correlated; lysozyme activity decreased as temperature increased. No correlations were found between lysozyme activity and parasitism or intensity of infection by monogeneans, the most abundant metazoan parasite group in L. cephalus. There was a positive correlation, however, between respiratory burst intensity and parasitism. Indices of investment in gonads and spleen were correlated, showing that energetic reserves allowed either investment in gonads and spleen, or that spleen investment, even if often used in other studies in immunoecology, was not always a significant indicator of immunocompetence during this period. This last proposition is supported by the lack of correlation between spleen investment and other factors linked to immunocompetence.

A novel in situ silver/hyaluronan bio-nanocomposite fabrics for wound and chronic ulcer dressing: In vitro and in vivo evaluations

In-situ formed hyaluronan/silver (HA/Ag) nanoparticles (NPs) were used to prepare composite fibers/fabrics for the first time. Different concentrations of silver nitrate (1, 2mg/100ml) were added at ambient temperature to sodium hyaluronate solution (40mg/ml), then the pH was increased to 8 by adding sodium hydroxide. The in-situ formed HA/Ag-NPs were used to prepare fibers/nonwoven fabrics by wet-dry-spinning technique (WDST). UV/vis spectroscopy, SEM, TEM, DLS, XPS, XRD and TGA were employed to characterize the structure and composition of the nanocomposite, surface morphology of fiber/fabrics, particle size of Ag-NPs, chemical interactions of Ag0 and HA functional groups, crystallinity and thermal stability of the wound dressing, respectively. The resultant HA/Ag-NPs1 and HA/Ag-NPs2 composite showed uniformly dispersed throughout HA fiber/fabrics (SEM), an excellent distribution of Ag-NPs with 25±2, nm size (TEM, DLS) and acceptable mechanical properties. The XRD analysis showed that the in-situ preparation of Ag-NPs increased the crystallinity of the resultant fabrics as well as the thermal stability. The antibacterial performance of medical HA/Ag-NPs fabrics was evaluated against gram negative bacteria E. coli K12, exhibiting significant bactericidal activity. The fibers did not show any cytotoxicity against human keratinocyte cell line (HaCaT). In-vivo animal tests indicated that the prepared wound dressing has strong healing efficacy (non-diabetics/diabetics rat model) compared to the plain HA fabrics and greatly accelerated the healing process. Based on our results, the new HA/Ag-NPs-2mg nonwoven wound dressing fabrics can be used in treating wounds and chronic ulcers as well as cell carrier in different biological research and tissue engineering.