Pathology and immune response of the blue mussel (Mytilus edulis L.) after an exposure to the harmful dinoflagellate Prorocentrum minimum

The harmful dinoflagellate Prorocentrum minimum has different effects upon various species of grazing bivalves, and these effects also vary with life-history stage. Possible effects of this dinoflagellate upon mussels have not been reported; therefore, experiments exposing adult blue mussels, Mytilus edulis, to P. minimum were conducted. Mussels were exposed to cultures of toxic P. minimum or benign Rhodomonas sp. in glass aquaria. After a short period of acclimation, samples were collected on day 0 (before the exposure) and after 3, 6, and 9 days of continuous-exposure experiment. Hemolymph was extracted for flow-cytometric analyses of hemocyte, immune-response functions, and soft tissues were excised for histopathology. Mussels responded to P. minimum exposure with diapedesis of hemocytes into the intestine, presumably to isolate P. minimum cells within the gut, thereby minimizing damage to other tissues. This immune response appeared to have been sustained throughout the 9-day exposure period, as circulating hemocytes retained hematological and functional properties. Bacteria proliferated in the intestines of the P. minimum-exposed mussels. Hemocytes within the intestine appeared to be either overwhelmed by the large number of bacteria or fully occupied in the encapsulating response to P. minimum cells; when hemocytes reached the intestine lumina, they underwent apoptosis and bacterial degradation. This experiment demonstrated that M. edulis is affected by ingestion of toxic P. minimum; however, the specific responses observed in the blue mussel differed from those reported for other bivalve species. This finding highlights the need to study effects of HABs on different bivalve species, rather than inferring that results from one species reflect the exposure responses of all bivalves.

Ecological and physiological variance in T-cell mediated immune response in Cory's shearwaters

T-cell mediated immune response (CMI) hasbeen widely studied in relation to individual andfitness components in birds. However, few studieshave simultaneously examined individual and socialfactors and habitat-mediated variance in theimmunity of chicks and adults from the samepopulation and in the same breeding season. Weinvestigated ecological and physiological variancein CMI of male and female nestlings and adults in abreeding population of Cory's Shearwaters(Calonectrisdiomedea) in theMediterranean Sea. Explanatory variables includedindividual traits (body condition, carbon andnitrogen stable isotope ratios, plasma totalproteins, triglycerides, uric acid, osmolarity,β-hydroxy-butyrate, erythrocyte meancorpuscular diameter, hematocrit, andhemoglobin) and burrow traits(temperature, isolation, and physicalstructure). During incubation, immune responseof adult males was significantly greater than thatof females. Nestlings exhibited a lower immuneresponse than adults. Ecological and physiologicalfactors affecting immune response differed betweenadults and nestlings. General linear models showedthat immune response in adult males was positivelyassociated with burrow isolation, suggesting thatmales breeding at higher densities suffer immunesystem suppression. In contrast, immune response inchicks was positively associated with bodycondition and plasma triglyceride levels.Therefore, adult immune response appears to beassociated with social stress, whereas a trade-offbetween immune function and fasting capability mayexist for nestlings. Our results, and those fromprevious studies, provide support for anasymmetrical influence of ecological andphysiological factors on the health of differentage and sex groups within a population, and for theimportance of simultaneously considering individualand population characteristics in intraspecificstudies of immune response.

Ecological and physiological variance in T-cell mediated immune response in Cory's shearwaters

T-cell mediated immune response (CMI) hasbeen widely studied in relation to individual andfitness components in birds. However, few studieshave simultaneously examined individual and socialfactors and habitat-mediated variance in theimmunity of chicks and adults from the samepopulation and in the same breeding season. Weinvestigated ecological and physiological variancein CMI of male and female nestlings and adults in abreeding population of Cory's Shearwaters(Calonectrisdiomedea) in theMediterranean Sea. Explanatory variables includedindividual traits (body condition, carbon andnitrogen stable isotope ratios, plasma totalproteins, triglycerides, uric acid, osmolarity,β-hydroxy-butyrate, erythrocyte meancorpuscular diameter, hematocrit, andhemoglobin) and burrow traits(temperature, isolation, and physicalstructure). During incubation, immune responseof adult males was significantly greater than thatof females. Nestlings exhibited a lower immuneresponse than adults. Ecological and physiologicalfactors affecting immune response differed betweenadults and nestlings. General linear models showedthat immune response in adult males was positivelyassociated with burrow isolation, suggesting thatmales breeding at higher densities suffer immunesystem suppression. In contrast, immune response inchicks was positively associated with bodycondition and plasma triglyceride levels.Therefore, adult immune response appears to beassociated with social stress, whereas a trade-offbetween immune function and fasting capability mayexist for nestlings. Our results, and those fromprevious studies, provide support for anasymmetrical influence of ecological andphysiological factors on the health of differentage and sex groups within a population, and for theimportance of simultaneously considering individualand population characteristics in intraspecificstudies of immune response.

High-throughput sequence analysis of turbot (Scophthalmus maximus) transcriptome using 454-pyrosequencing for the discovery of antiviral immune genes

Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations.

Development and Validation of Single Nucleotide Polymorphisms (SNPs) Markers from Two Transcriptome 454-Runs of Turbot (Scophthalmus maximus) Using High-Throughput Genotyping

The turbot (Scophthalmus maximus) is a commercially valuable flatfish and one of the most promising aquaculture species in Europe. Two transcriptome 454-pyrosequencing runs were used in order to detect Single Nucleotide Polymorphisms (SNPs) in genesrelated to immune response and gonad differentiation. A total of 866 true SNPs were detected in 140 different contigs representing 262,093 bp as a whole. Only one true SNP was analyzed in each contig. One hundred and thirteen SNPs out of the 140 analyzed were feasible (genotyped), while Ш were polymorphic in a wild population. Transition/transversion ratio (1.354) was similar to that observed in other fish studies. Unbiased gene diversity (He) estimates ranged from 0.060 to 0.510 (mean = 0.351), minimum allele frequency (MAF) from 0.030 to 0.500 (mean = 0.259) and all loci were in Hardy-Weinberg equilibrium after Bonferroni correction. A large number of SNPs (49) were located in the coding region, 33 representing synonymous and 16 non-synonymous changes. Most SNP-containing genes were related to immune response and gonad differentiation processes, and could be candidates for functional changes leading to phenotypic changes. These markers will be useful for population screening to look for adaptive variation in wild and domestic turbot

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Bacterial lipopolysaccharide induces apoptosis in the trout ovary

BACKGROUND: In mammals it is well known that infections can lead to alterations in reproductive function. As part of the innate immune response, a number of cytokines and other immune factors is produced during bacterial infection or after treatment with lipopolysaccharide (LPS) and acts on the reproductive system. In fish, LPS can also induce an innate immune response but little is known about the activation of the immune system by LPS on reproduction in fish. Therefore, we conducted studies to examine the in vivo and in vitro effects of lipopolysaccharide (LPS) on the reproductive function of sexually mature female trout. METHODS: In saline- and LPS -injected brook trout, we measured the concentration of plasma steroids as well as the in vitro steroidogenic response (testosterone and 17alpha-hydroxyprogesterone) of ovarian follicles to luteinizing hormone (LH), the ability of 17alpha,20beta-dihydroxy-4-pregnen-3-one to induce germinal vesicle breakdown (GVBD) in vitro, and that of epinephrine to stimulate follicular contraction in vitro. We also examined the direct effects of LPS in vitro on steroid production, GVBD and contraction in brook trout ovarian follicles. The incidence of apoptosis was evaluated by TUNEL analysis. Furthermore, we examined the gene expression pattern in the ovary of saline- and LPS-injected rainbow trout by microarray analysis. RESULTS: LPS treatment in vivo did not affect plasma testosterone concentration or the basal in vitro production of steroids, although a small but significant potentiation of the effects of LH on testosterone production in vitro was observed in ovarian follicles from LPS-treated fish. In addition, LPS increased the plasma concentration of cortisol. LPS treatment in vitro did not affect the basal or LH-stimulated steroid production in brook trout ovarian follicles. In addition, we did not observe any effects of LPS in vivo or in vitro on GVBD or follicular contraction. Therefore, LPS did not appear to impair ovarian steroid production, oocyte final maturation or follicular contraction under the present experimental conditions. Interestingly, LPS administration in vivo induced apoptosis in follicular cells, an observation that correlated with changes in the expression of genes involved in apoptosis, as evidenced by microarray analysis. CONCLUSION: These results indicate that female trout are particularly resistant to an acute administration of LPS in terms of ovarian hormone responsiveness. However, LPS caused a marked increase in apoptosis in follicular cells, suggesting that the trout ovary could be sensitive to the pro-apoptotic effects of LPS-induced inflammatory cytokines.

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout

Background: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). Methods: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses. Results: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha. Conclusions In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.

The effects of cocoa on the immune system

Cocoa is a food relatively rich in polyphenols, which makes it a potent antioxidant. Due to its activity as an antioxidant, as well as through other mechanisms, cocoa consumption has been reported to be beneficial for cardiovascular health, brain functions, and cancer prevention. Furthermore, cocoa influences the immune system, in particular the inflammatory innate response and the systemic and intestinal adaptive immune response. Preclinical studies have demonstrated that a cocoa-enriched diet modifies T-cell functions that conduce to a modulation of the synthesis of systemic and gut antibodies. In this regard, it seems that a cocoa diet in rats produces changes in the lymphocyte composition of secondary lymphoid tissues and the cytokines secreted by T cells. These results suggest that it is possible that cocoa could inhibit the function of Th2 cells, and in line with this, the preventive effect of cocoa on IgE synthesis in a rat allergy model has been reported, which opens up new perspectives when considering the beneficial effects of cocoa compounds. On the other hand, cocoa intake modifies the functionality of gut-associated lymphoid tissue by means of modulating IgA secretion and intestinal microbiota. The mechanisms involved in these influences are discussed here. Further research may elucidate the cocoa compounds involved in such an effect and also the possible medical approaches to these repercussions

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases.

Inorganic Nanoparticles and the Immune System: Detection, Selective Activation and Tolerance

The immune system is the responsible for body integrity and prevention of external invasion. On one side, nanoparticles are no triggers that the immune system is prepared to detect, on the other side it is known that foreign bodies, not only bacteria, viruses and parasites, but also inorganic matter, can cause various pathologies such as silicosis, asbestosis or inflammatory reactions. Therefore, nanoparticles entering the body, after interaction with proteins, will be either recognized as self-agents or detected by the immune system, encompassing immunostimulation or immunosuppression responses. The nature of these interactions seems to be dictated not specially by the composition of the material but by modifications of NP coating (composition, surface charge and structure). Herein, we explore the use of gold nanoparticles as substrates to carry multifunctional ligands to manipulate the immune system in a controlled manner, from undetection to immunostimulation. Murine bone marrow macrophages can be activated with artificial nanometric objects consisting of a gold nanoparticle functionalized with peptides. In the presence of some conjugates, macrophage proliferation was stopped and pro-inflammatory cytokines were induced. The biochemical type of response depended on the type of conjugated peptide and was correlated with the degree of ordering in the peptide coating. These findings help to illustrate the basic requirements involved in medical NP conjugate design to either activate the immune system or hide from it, in order to reach their targets before being removed by phagocytes. Additionally, it opens up the possibility to modulate the immune response in order to suppress unwanted responses resulting from autoimmunity, or allergy or to stimulate protective responses against pathogens.