Conference report: The 13th Congress of the International Society of Developmental and Comparative Immunology

9 páginas.-- L. Courtney Smith ... et al.The 13th Congress of the International Society of Developmental and Comparative Immunology took place in Murcia Spain from June 28 to July 3, 2015 at the Victor Villegas Auditorium and Convention Center. There were two or three parallel sessions during the Congress that covered a wide range of immunological topics and brought researchers together from around the world who work in different areas of immunology. The Congress included three plenary presentations, 12 oral sessions, two poster sessions, and a special symposium. Here we report on some of the talks and a few of the posters that were presented at the meetingFunding from the US National Science Foundation (IOS-1461716) awarded to LCS and Louise Rollins-Smith (Vanderbilt University, USA) supported students and postdocs from laboratories in the US to attend the 13th Congress of ISDCIPeer reviewe

Nk-lysin, an antimicrobial peptide expressed in fish erythrocytes is involved in antiviral defense

2nd International Conference on Fish and Shellfish Immunology, June 26th – 30th, 2016 Portland, MaineErythrocytes or red cells are the most abundant cells in the blood of vertebrate organisms and their primary function is the transport of oxygen. Nevertheless, it is known that these cells also play a role in some immune responses. Nk-lysin is a cationic antimicrobial peptide mainly produced by natural killer cells and cytotoxic T lymphocytes and stored in cytolytic granules together with perforin and granzymes. In this work, Nk-lysin (nkl) was characterized for the first time in turbot (Scophthalmus maximus). Interestingly, immunofluorescence analysis of head kidney and blood cell populations by flow cytometry revealed the presence of the Nkl peptide in non-lymphoid lineages. Confocal images showed that nkl was highly expressed in lymphocytes but also in erythrocytes although, in this case, the presence of Nkl was mainly confined to spherical vacuolar structures. Using transmission electron microscopy (TEM) we observed that these structures corresponded to electron-dense double membrane vacuoles of different sizes and variable number. These vacuoles were positives for LysoTracker dye (retained in acidic subcellular compartments) and anti-LC3 immunofluorescence (used to monitor autophagy) revealing that, as was expected, these structures corresponded to autophagolysosomes. TEM images also revealed viral-like particles in some erythrocytes both in the cytoplasm and inside the autophagosomes. Therefore, Nkl could be involved in the autophagy process in erythrocytes as a defense mechanism against intracellular pathogens, such as viruses. Indeed, the hypothetical antiviral role of Nk-lysin is not fully understood. After a Viral Hemorrhagic Septicemia virus (VHSV) challenge, turbot nkl was overexpressed both in head kidney and spleen, suggesting again a certain function in the antiviral defense. Interestingly, the expression analysis of this gene in head kidney samples of VHSV-resistant and -susceptible selected families and the correlation between Nk-lysin expression in blood and susceptibility to VHSV, seem to indicate that higher basal levels of Nkl could induce higher resistance to the virusN

Pathogen-dependent role of turbot (Scophthalmus maximus) interferon-gamma

11 páginas, 7 figurasInterferon-gamma has been typically described as a pro-inflammatory cytokine playing an important role in the resolution of both viral and bacterial diseases. Nevertheless, some anti-inflammatory functions are also attributed to this molecule. In this work we have characterized for the first time the turbot (Scophthalmus maximus) interferon-gamma gene (ifng) and its expression pattern under basal conditions, after type I IFNs administration, and viral and bacterial infection. The intramuscular injection of an expression plasmid encoding turbot Ifng (pMCV1.4-ifng) was not able to affect the transcription of numerous immune genes directly related to the activity of IFN-gamma, with the exception of macrophage-colony stimulating factor (csf1). It was also unable to reduce the mortality caused by a Viral Hemorrhagic Septicemia Virus (VHSV) or Aeromonas salmonicida challenge. Interestingly, at 24 h post-infection, turbot previously inoculated with pMCV1.4-ifng and infected with VHSV showed an increase in the expression of pro-inflammatory cytokines and type I IFNs compared to those fish not receiving expression plasmid, indicating a synergic effect of Ifng and VHSV. On the other hand, some macrophage markers, such as the macrophage receptor with collagenous structure (marco), were down-regulated by Ifng during the viral infection. Ifng had the opposite effect in those turbot infected with the bacteria, showing a reduction in the transcription of pro-inflammatory and type I IFNs genes, and an increase in the expression of genes related to the activity of macrophagesThis work has been funded by the projects CSD2007-00002 “Aquagenomics”, 201230E057 (CSIC) and AGL2014-51773-C3 from the Spanish Ministerio de Economía y Competitividad. P. Pereiro received a predoctoral grant from the gs3:Ministerio de Educación [F.P.U. fellowship AP2010-2408]Peer reviewe

Conserved gene regulation during acute inflammation between zebrafish and mammals

9 páginas, 4 figuras, 1 tabla.-- This work is licensed under a Creative Commons Attribution 4.0 International LicenseZebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potentialThis work was funded by the projects CSD2007–00002 “Aquagenomics” and AGL2014-51773-C3 from the Spanish Ministerio de Economía y Competitividad, and 201230E057 “Proyecto Intramural Especial, PIE”, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC). P. Pereiro and M. Varela received predoctoral grants from the Ministerio de Educación (F.P.U. fellowship AP2010-2408) and the JAE Program (funded though the CSIC and European Social Funds), respectivelyPeer reviewe

DEVELOPMENT OF A ZEBRAFISH LARVAE INFECTION MODEL TO STUDY VIRULENCE FACTORS OF A. hydrophila

2nd International Conference of Fish & Shellfish Immunology, June 26th – 30th, 2016, Portland, MaineAeromonas hydrophila is a Gram negative bacteria widely distributed in aquatic environments. It is an opportunistic pathogen for fish and terrestrial animals including mammals. In fish it causes motile hemorrhagic septicemia leading to a high mortality and economic losses in aquaculture. In humans it is an emerging pathogen provoking gastroenteritis, septicemia, and skin and soft tissue infections. Some clinical conditions like cancer, hepatic diseases, diabetes and trauma increase the risk to develop a fatal A. hydrophila infection. The pathogenicity of A. hydrophila is multifactorial depending on several virulence factors, including surface polysaccharides (capsule, lipopolysaccharide, and glucan), S-layers, iron-binding systems, exotoxins and extracellular enzymes, secretion systems, fimbriae and other nonfilamentous adhesins, motility and flagella In this work two different models of experimental infection have been developed in zebrafish larvae (Danio rerio) to study the pathogenicity of A. hydrophila: microinjection and bath immersion. The transparency of zebrafish larvae and the availability of transgenic fish lines showing fluorescent immune cells or immune marker genes allow the evaluation of the inflammatory processes in vivo and the analysis of the host-microbe interactions.The infection model by bath immersion of injured zebrafish larvae is an alternative non-mammal model to study the implication of different virulence factors in the pathogenicity of A. hydrophila. This new model mimics the natural route of infection by water and an additional injury in the fins provides a natural portal of entry to the bacteria. The implication of the virulence factors in the pathogenesis of A. hydrophila was evaluated by experimental infections using the following specific mutant: AH-1::aopB lacking the T3SS, AH-1ΔrmlB which is devoid of the O-antigen LPS with a complete LPS-core, the AH-1ΔwahD which lacks the O-antigen LPS and part of the LPS outer-core, AH-1ΔvapA, lacking the gene coding for the S-layer protein, a non-polar flagellated mutant AH-1ΔFlaB-J unable to swim, and the AH-1::motX mutant, being non-motile but able to produce polar flagellum. All the results highlight the importance of the correct selection of the infection model to analyse the fish immune response and the implication of the virulence factors of A. hydrophilaN

Transcriptome profiles associated to VHSV infection or DNA vaccination in turbot (Scophthalmus maximus)

DNA vaccines encoding the viral G glycoprotein show the most successful protection capability against fish rhabdoviruses. Nowadays, the molecular mechanisms underlying the protective response remain still poorly understood. With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. Genes implicated in the Toll-like receptor signalling pathway, IFN inducible/regulatory proteins, numerous sequences implicated in apoptosis and cytotoxic pathways, MHC class I antigens, as well as complement and coagulation cascades among others were analyzed in the different experimental groups. Fish receiving the pMCV1.4-G860 vaccine showed transcriptomic patterns very different to the ones observed in pMCV1.4-injected turbot after 72 h. On the other hand, VHSV challenge in vaccinated and non-vaccinated turbot induced a highly different response at the transcriptome level, indicating a very relevant role of the acquired immunity in vaccinated fish able to alter the typical innate immune response profile observed in non-vaccinated individuals. This exhaustive transcriptome study will serve as a complete overview for a better understanding of the crosstalk between the innate and adaptive immune response in fish after viral infection/vaccination. Moreover, it provides interesting clues about molecules with a potential use as vaccine adjuvants, antiviral treatments or markers for vaccine efficiency monitoring

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

The mito-QC reporter for quantitative mitophagy assessment in primary retinal ganglion cells and experimental glaucoma models

This research was funded by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) PGC2018-098557-B-I00 and European Union’s Horizon 2020 research and innovation programme under grant agreement No 765912. BVZ is a recipient of PhD contract from the Fundación Tatiana Pérez de Guzmán el Bueno (Spain), PT from H2020-MSCA-ITN-2017, NRM a Juan de la Cierva Grant from Ministerio Ciencia e Innovación (Spain) and KB from DFG (Deutsche Forschungsgemeinschaft, Germany, 6619/1-1).Mitochondrial damage plays a prominent role in glaucoma. The only way cells can degrade whole mitochondria is via autophagy, in a process called mitophagy. Thus, studying mitophagy in the context of glaucoma is essential to understand the disease. Up to date limited tools are available for analyzing mitophagy in vivo. We have taken advantage of the mito-QC reporter, a recently generated mouse model that allows an accurate mitophagy assessment to fill this gap. We used primary RGCs and retinal explants derived from mito-QC mice to quantify mitophagy activation in vitro and ex vivo. We also analyzed mitophagy in retinal ganglion cells (RGCs), in vivo, using different mitophagy inducers, as well as after optic nerve crush (ONC) in mice, a commonly used surgical procedure to model glaucoma. Using mito-QC reporter we quantified mitophagy induced by several known inducers in primary RGCs in vitro, ex vivo and in vivo. We also found that RGCs were rescued from some glaucoma relevant stress factors by incubation with the iron chelator deferiprone (DFP). Thus, the mito-QC reporter-based model is a valuable tool for accurately analyzing mitophagy in the context of glaucoma.publishersversionpublishe

Repeatability and Reliability of the Rheumatoid Arthritis Foot Disease Activity Index in Spanish Patients : A Transcultural Adaptation

The Rheumatoid Arthritis Foot Disease Activity Index (RADAI-F5) questionnaire, based on five questions, is used to assess the severity of rheumatoid arthritis disease in the foot. Nowadays, RADAI-F5 has been validated in different languages; however a Spanish version was lacking. Therefore, the purpose of this research was to translate and validate the Spanish version (RADAI-F5-es). Methods: A cross-cultural translation of the RADAI-F5 questionnaire was performed from English to Spanish. To validate its use, 50 subjects with rheumatoid arthritis who responded to the translated questionnaire two times in an interval of less than 3 months were selected in order to verify the psychometric properties. Results: Excellent agreement between the two versions according to the Cronbach's α was shown. Five domains with regards to arthritis activity in foot joint tenderness and swelling, foot arthritis pain, general foot health and joint stiffness were added together to obtain the total score. Excellent retest reliability was shown for the total score. Test/retest reliability was excellent for joint stiffness on awakening and foot arthritis pain domains. There were no significant differences among any domains (p > 0.05). There were no statistically significant differences (p = 0.000) for the mean ± standard deviations (SD) between pre- and post-tests (98.09 ± 15.42) [93.75-102.43] and 97.96 ± 13.88 [94.5-101.86] points, respectively). Bland-Altman plots or clinically pertinent variations were not statistically significantly different. Conclusions: The RADAI-F5-es is considered a valid and strong tool with adequate repeatability in the Spanish communit

The coagulation system helps control infection caused by the ciliate parasite Philasterides dicentrarchi in the turbot Scophthalmus maximus (L.)

This is the accepted manuscript of the following article: Blanco-Abad, V., Noia, M., Valle, A., Fontenla, F., Folgueira, I., & De Felipe, A. et al. (2018). The coagulation system helps control infection caused by the ciliate parasite Philasterides dicentrarchi in the turbot Scophthalmus maximus (L.). Developmental & Comparative Immunology, 87, 147-156. doi: 10.1016/j.dci.2018.06.001Many studies have shown that coagulation systems play an important role in the defence against pathogens in invertebrates and vertebrates. In vertebrates, particularly in mammals, it has been established that the coagulation system participates in the entrapment of pathogens and activation of the early immune response. However, functional studies investigating the importance of the fish coagulation system in host defence against pathogens are scarce. In the present study, injection of turbot (Scopthalamus maximus) with the pathogenic ciliate Philasterides dicentrarchi led to the formation of macroscopic intraperitoneal clots in the fish. The clots contained abundant, immobilized ciliates, many of which were lysed. We demonstrated that the plasma clots immobilize and kill the ciliates in vitro. To test the importance of plasma clotting in ciliate killing, we inhibited the process by adding a tetrapeptide known to inhibit fibrinogen/thrombin clotting in mammals. Plasma tended to kill P. dicentrarchi slightly faster when clotting was inhibited by the tetrapeptide, although the total mortality of ciliates was similar. We also found that kaolin, a particulate activator of the intrinsic pathway in mammals, accelerates plasma clotting in turbot. In addition, PMA-stimulated neutrophils, living ciliates and several ciliate components such as cilia, proteases and DNA also displayed procoagulant activity in vitro. Injection of fish with the ciliates generated the massive release of neutrophils to the peritoneal cavity, with formation of large aggregates in those fish with live ciliates in the peritoneum. We observed, by SEM, numerous fibrin-like fibres in the peritoneal exudate, many of which were associated with peritoneal leukocytes and ciliates. Expression of the CD18/CD11b gene, an integrin associated with cell adhesion and the induction of fibrin formation, was upregulated in the peritoneal leukocytes. In conclusion, the findings of the present study show that P. dicentrarchi induces the formation of plasma clots and that the fish coagulation system may play an important role in immobilizing and killing this parasiteThis work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL), by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R and by grant GPC2014/069 from the Xunta de Galicia (Spain)S