The Immune System of Marine Organisms as Source for Drugs against Infectious Diseases.

The high proliferation of microorganisms in aquatic environments has allowed their coevolution for billions of years with other living beings that also inhabit these niches. Among the different existing types of interaction, the eternal competition for supremacy between the susceptible species and their pathogens has selected, as part of the effector division of the immune system of the former ones, a vast and varied arsenal of efficient antimicrobial molecules, which is highly amplified by the broad biodiversity radiated, above any others, at the marine habitats. At present, the great recent scientific and technological advances already allow the massive discovery and exploitation of these defense compounds for therapeutic purposes against infectious diseases of our interest. Among them, antimicrobial peptides and antimicrobial metabolites stand out because of the wide dimensions of their structural diversities, mechanisms of action, and target pathogen ranges. This revision work contextualizes the research in this field and serves as a presentation and scope identification of the Special Issue from Marine Drugs journal "The Immune System of Marine Organisms as Source for Drugs against Infectious Diseases".

Effect of ß-1/3,1/6-glucan upon immune responses and bacteria in the gut of healthy common carp (Cyprinus carpio).

ß-glucans are frequently included in the diet of healthy common carp Cyprinus carpio as a pre-emptive measure for combatting disease. In order to study the effect this has on the relationship between the gut bacteria and host immune response, carp were maintained on either a ß-glucan free diet or feed containing 0.1% MacroGard®, a ß-1/3, 1/6-glucan, for up to 7 weeks and analysis of innate immune gene expression and molecular analysis of the gut bacteria was performed. The data reveals feeding of MacroGard® to healthy carp does not induce bactericidal innate immune gene expression in the gut but does appear to alter bacterial species richness that did not have a negative effect on overall health. Analysis of innate immune gene expression within the upper midgut revealed that there were significant changes over time in the expression of Interleukin (il)-1ß, inducible nitric oxide synthase (inos), mucin (muc2) and C-reactive protein (crp2). Diet did not affect the number of copies of the bacterial 16s rDNA gene in the gut, used as a as a measure of total bacteria population size. However, PCR-denaturing gradient gel electrophoresis (DGGE) analysis revealed a shift in bacterial species richness with MacroGard feeding. Bactericidal immune gene expression of crp2, muc2 and il-1ß was weakly correlated with gut bacteria population size indicating a potentially limited role of these genes in interacting with the gut bacteria in healthy carp in order to maintain gut homeostatic conditions. These findings highlight the importance of considering both host immunity and the microbiome together in order to fully elucidate the effeect of immunomodulants, such as ß-glucans, upon gut health.

ß-Glucan-supplemented diets increase poly(I:C)-induced gene expression of Mx, possibly via Tlr3-mediated recognition mechanism in common carp (Cyprinus carpio).

We have previously observed that in common carp (Cyprinus carpio), administration of ß-glucan (MacroGard®) as feed additive leads to a lower expression of pro-inflammatory cytokines suggesting that this immunostimulant may be preventing an acute and potentially dangerous response to infection, particularly in the gut. However, in general, mechanisms to detect and eliminate pathogens must also be induced in order to achieve an efficient clearance of the infection. Protection against viral diseases acquired through ß-glucan-supplemented feed has been extensively reported for several experimental models in fish but the underlining mechanisms are still unknown. Thus, in order to better characterize the antiviral action induced by ß-glucans in fish, MacroGard® was administered daily to common carp in the form of supplemented commercial food pellets. Carp were fed for a period of 25 days prior to intra-peritoneal injection with polyinosinic:polycytidylic acid (poly(I:C)), a well-known double-stranded RNA mimic that triggers a type-I interferon (IFN) response. Subsequently, a set of immune related genes, including mx, were analysed by real-time PCR on liver, spleen, head kidney and mid gut tissues. Results obtained confirmed that treatment with ß-glucan alone generally down-regulated the mRNA expression of selected cytokines when compared to untreated fish, while mx gene expression remained stable or was slightly up-regulated. Injection with poly(I:C) induced a similar down-regulated gene expression pattern for cytokines in samples from ß-glucan fed fish. In contrast, poly(I:C) injection markedly increased mx gene expression in samples from ß-glucan fed fish but hardly in samples from fish fed control feed. In an attempt to explain the high induction of mx, we studied Toll-like receptor 3 (TLR3) gene expression in these carp. TLR3 is a prototypical pattern recognition receptor considered important for the binding of viral double-stranded RNA and triggering of a type-I IFN response. Through genome data mining, two sequences for carp tlr3 were retrieved (tlr3.1 and tlr3.2) and characterized. Constitutive gene expression of both tlr3.1 and tlr3.2 was detected by real-time PCR in cDNA of all analysed carp organs. Strikingly, 25 days after ß-glucan feeding, very high levels of tlr3.1 gene expression were observed in all analysed organs, with the exception of the liver. Our data suggest that ß-glucan-mediated protection against viral diseases could be due to an increased Tlr3-mediated recognition of ligands, resulting in an increased antiviral activity of Mx.

Towards virtual knowledge broker services for semantic integration of life science literature and data sources.

Research in the life sciences requires ready access to primary data, derived information and relevant knowledge from a multitude of sources. Integration and interoperability of such resources are crucial for sharing content across research domains relevant to the life sciences. In this article we present a perspective review of data integration with emphasis on a semantics driven approach to data integration that pushes content into a shared infrastructure, reduces data redundancy and clarifies any inconsistencies. This enables much improved access to life science data from numerous primary sources. The Semantic Enrichment of the Scientific Literature (SESL) pilot project demonstrates feasibility for using already available open semantic web standards and technologies to integrate public and proprietary data resources, which span structured and unstructured content. This has been accomplished through a precompetitive consortium, which provides a cost effective approach for numerous stakeholders to work together to solve common problems.

Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with ß-glucan supplements.

The objective of the present study was to determine the action of ß-glucans as feed additives on the gene expression profile of some inflammatory-related cytokines from common carp (Cyprinus carpio L.) during the early stages of a non-lethal bacterial infection with Aeromonas salmonicida. ß-glucan (MacroGard(®)), was administered daily to carp (6 mg per kg body weight) in the form of supplemented commercial food pellets for 14 days prior to infection. Control and treated fish were then intraperitoneally injected with PBS or 4×10(8) bacteria per fish and were sampled at time 0 and 6h, 12h, 1 day, 3 days and 5 days post-injection. Head kidney and gut were collected and the gene expression patterns for tnfα1, tnfα2, il1ß, il6 and il10 were analyzed by quantitative PCR. Results obtained showed that treatment with ß-glucans generally down-regulated the expression of all measured genes when compared to their corresponding controls. After injection, highest changes in the gene expression levels were obtained at 6h; particularly, in head kidney there was higher up-regulation of tnfa1 and tnfa2 in infected fish fed ß-glucans in comparison to control feed; however, in gut there was a significant down-regulation of tnfα1, tnfα2, il1ß and il6 in infected fish fed ß-glucans. Analysis of carp specific antibodies against A. salmonicida 30 days after injection revealed their levels were reduced in the infected ß-glucan group. In conclusion, a diet supplemented with ß-glucan (MacroGard(®)) reduced the gene expression levels of some inflammation-related cytokines in common carp. Such a response appears to be dependent of organ studied and therefore the immunostimulant may be preventing an acute and potential dangerous response in gut, whilst enhancing the inflammatory response in head kidney when exposed to A. salmonicida.

Molecular characterization and expression analysis of two new C-reactive protein genes from common carp (Cyprinus carpio).

C-Reactive protein (CRP) plays an important role in the acute phase response. Transcripts encoding two new CRP-like molecules (ccCRP1 and ccCRP2) from European common carp have been characterized which has enabled seven CRP-like genes to be identified in zebrafish. 79.3% (ccCRP1) and 74.5% (ccCRP2) identity to CRP from East-Asian common carp occurs and fish CRP genes form a distinct clade. ccCRP2 gene organization comprises four exons and three introns, in contrast to the two exons/one intron organization of mammalian CRP genes. Gene expression assays showed both ccCRP-like molecules are constitutively expressed in liver, skin, gill, gut, muscle, kidney, spleen and blood. Protein levels of ccCRP in serum and spleen were significantly different from other organs analyzed, and levels were greatest in the liver. It is proposed that the two carp CRP genes defined differ in their expression profiles which may suggest differences in their biological activities.

Characterisation of a carp cell line for analysis of apoptosis.

Teleost fish in general, and common carp in particular, are excellent genetic models for bridging the gap in knowledge between invertebrate models such as C. elegans and D. melanogaster, on one hand, and higher vertebrates on the other hand, although, until now, there have been few well characterised fish cell lines shown to be suitable for studies on apoptosis. The present study describes the suitability of a permanent, nonleukemic, nonvirally infected carp cell line for apoptotic studies. A traditional approach using known apoptotic inducers such as UV-light combined with RNA interference, the latest ready-to-use technology widely used in higher vertebrates, was tested in the carp leucocyte cell line (CLC). This study was designed as a first step towards a better knowledge of fish macrophages and their fate after different types of apoptotic insults.

Serum CRP-like protein profile in common carp Cyprinus carpio challenged with Aeromonas hydrophila and Escherichia coli lipopolysaccharide.

The potential of C-reactive protein (CRP)-like proteins to be used as a biomarker of health status in cultured carp obtained from various European fish lines has been assessed. Varying CRP-like protein levels in the serum of carp were monitored using an indirect competitive enzyme-linked immunosorbent assay. CRP-like protein basal levels in normal fish varied between carp lines, ranging on average from 2.9+/-0.15 to 12.57+/-1.19 microg ml(-1). Serum levels of CRP-like protein in carp were observed to increase several fold in fish infected with the pathogen Aeromonas hydrophila. However, carp injected with Escherichia coli lipopolysaccharide (LPS) serotype 0111:B4 did not exhibit an increase in CRP-like proteins levels.

Isolation and characterisation of pentraxin-like serum proteins from the common carp Cyprinus carpio.

There is increasing economic and ecological interest in the development of assays for the early detection of infection, disease activity and environmental stress in marine and freshwater animals. In humans the serum pentraxin C-reactive protein (CRP) is universally used as a clinical indicator of inflammation and underlying infection. As a first step towards assessing the potential of an immunoassay for CRP in fish, we have isolated and characterised common carp Cyprinus carpio CRP and a highly specific and sensitive anti-carp CRP polyclonal antibody has been raised. The results show levels of CRP in healthy fish similar to those found in healthy humans. A protein of unknown function, which displays the characteristic calcium-dependent phosphate monoester binding exhibited by CRP and some similarity to the known fish pentraxin sequences, has also been identified.