201 research outputs found
Fish Red Blood Cells Modulate Immune Genes in Response to Bacterial Inclusion Bodies Made of TNFa and a G-VHSV Fragment
Fish Red-Blood Cells (RBCs) are nucleated cells that can modulate the expression of
different sets of genes in response to stimuli, playing an active role in the homeostasis
of the fish immune system. Nowadays, vaccination is one of the main ways to control
and prevent viral diseases in aquaculture and the development of novel vaccination
approaches is a focal point in fish vaccinology. One of the strategies that has recently
emerged is the use of nanostructured recombinant proteins. Nanostructured cytokines
have already been shown to immunostimulate and protect fish against bacterial
infections. To explore the role of RBCs in the immune response to two nanostructured
recombinant proteins, TNFa and a G-VHSV protein fragment, we performed different in
vitro and in vivo studies. We show for the first time that rainbow trout RBCs are able
to endocytose nanostructured TNFa and G-VHSV protein fragment in vitro, despite not
being phagocytic cells, and in response to nanostructured TNFa and G-VHSV fragment,
the expression of different immune genes could be modulated.This work was supported by the European Research
Council fund to MO-V (ERC Starting Grant GA639249)and by grants from the Spanish Ministry of Science,
European commission and AGAUR funds to NR
(AGL2015-65129-R MINECO/FEDERand 2014SGR- 345 AGAUR). RT holds a pre-doctoral scholarship from
AGAUR (Spain)
Evaluation of the outcomes of newly diagnosed patients with high-risk myelodysplastic syndrome according to the initial therapeutical strategies chosen in usual clinical practice
Clinical practice; Myelodysplastic syndrome; TreatmentPrĂ ctica clĂnica; SĂndrome mielodisplĂ stica; TractamentPráctica clĂnica; SĂndrome mielodisplásico; TratamientoMyelodysplastic syndromes (MDS) are a heterogeneous group of diseases without a care standard and show variability in treatment outcomes. This Spanish, observational, prospective study ERASME (CEL-SMD-2012-01) assessed the evolution of newly diagnosed and treatment-naĂŻve high-risk MDS patients (according to IPPS-R). 204 patients were included: median age 73.0 years, 54.4% males, 69.6% 0-1 ECOG, and 94.6% with comorbidities. Active treatment was the most common strategy (52.0%) vs. stem cell transplantation (25.5%) and supportive care/watchful-waiting (22.5%). Overall (median) event-free survival was 7.9 months (9.1, 8.3, and 5.3); progression-free survival: 10.1 months (12.9, 12.8, and 4.3); and overall survival: 13.8 months (15.4, 14.9; 8.4), respectively, with significant differences among groups. Adverse events (AEs) of ≥3 grade were reported in 72.6% of patients; serious AEs reported in 60.6%. 33.1% of patients died due to AEs. Three patients developed second primary malignant neoplasms (median: 8.2 months). Our study showed better outcomes in patients receiving active therapy early after diagnosis
The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences
A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5′ upstream sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp β-actin gene (pAE6-GVHSV) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-GVHSV). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-GVHSV. However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation
In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling
Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in
several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated
signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out
for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly
was conducted, in order to create a transcriptome database for RBCs. For proteome profiling,
we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane
fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel
step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray
Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined
transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune
functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and
adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation,
antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation
and regulation of leukocyte activation were identified. These preliminary findings further implicate
nucleated RBCs in immune function, such as antigen presentation and leukocyte activationThis work was supported by the European Research Council (ERC Starting Grant GA639249The proteomic analyses were performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB2-ISCIII, supported by Grant PT13/000
Rainbow Trout Erythrocytes ex vivo Transfection With a DNA Vaccine Encoding VHSV Glycoprotein G Induces an Antiviral Immune Response
Fish red blood cells (RBCs), are integral in several biologic processes relevant to immunity,
such as pathogen recognition, pathogen binding and clearance, and production of
effector molecules and cytokines. So far, one of the best strategies to control and
prevent viral diseases in aquaculture is DNA immunization. DNA vaccines (based on
the rhabdoviral glycoprotein G [gpG] gene) have been shown to be effective against
fish rhabdoviruses. However, more knowledge about the immune response triggered
by DNA immunization is necessary to develop novel and more effective strategies. In
this study, we investigated the role of fish RBCs in immune responses induced by
DNA vaccines. We show for the first time that rainbow trout RBCs express gpG of
viral hemorrhagic septicaemia virus (VHSV) (GVHSV) when transfected with the DNA
vaccine ex vivo and modulate the expression of immune genes and proteins. Functional
network analysis of transcriptome profiling of RBCs expressing GVHSV revealed changes
in gene expression related to G-protein coupled receptor (GPCR)-downstream signaling,
complement activation, and RAR related orphan receptor a (RORA). Proteomic profile
functional network analysis of GVHSV-transfected RBCs revealed proteins involved in
the detoxification of reactive oxygen species, interferon-stimulated gene 15 (ISG15)
antiviral mechanisms, antigen presentation of exogenous peptides, and the proteasome.
Conditioned medium of GVHSV-transfected RBCs conferred antiviral protection and
induced ifn1 and mx gene expression in RTG-2 cells infected with VHSV. In summary,
rainbow trout nucleated RBCs could be actively participating in the regulation of the fish
immune response to GVHSV DNA vaccine, and thus may represent a possible carrier
cells for the development of new vaccine approaches.This work was supported by the European Research Council (ERC Starting Grant GA639249)The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology
(CNB-CSIC) belonging to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019This work was supported by the European Research Council
(ERC Starting Grant GA639249).The proteomic analysis was
performed in the Proteomics Facility of The Spanish National
Center for Biotechnology (CNB-CSIC) of ProteoRed, PRB3-
ISCIII, supported by grant PT17/001
Potential Role of Rainbow Trout Erythrocytes as Mediators in the Immune Response Induced by a DNA Vaccine in Fish
In recent years, fish nucleated red blood cells (RBCs) have been implicated in the response
against viral infections. We have demonstrated that rainbow trout RBCs can express the antigen
encoded by a DNA vaccine against viral hemorrhagic septicemia virus (VHSV) and mount an immune
response to the antigen in vitro. In this manuscript, we show, for the first time, the role of RBCs in the
immune response triggered by DNA immunization of rainbow trout with glycoprotein G of VHSV
(GVHSV). Transcriptomic and proteomic profiles of RBCs revealed genes and proteins involved in
antigen processing and presentation of exogenous peptide antigen via MHC class I, the Fc receptor
signaling pathway, the autophagy pathway, and the activation of the innate immune response, among
others. On the other hand, GVHSV-transfected RBCs induce specific antibodies against VHSV in
the serum of rainbow trout which shows that RBCs expressing a DNA vaccine are able to elicit a
humoral response. These results open a new direction in the research of vaccination strategies for
fish since rainbow trout RBCs actively participate in the innate and adaptive immune response in
DNA vaccination. Based on our findings, we suggest the use of RBCs as target cells or carriers for the
future design of novel vaccine strategiesThis research was funded by the European Research Council, grant number GA639249 (ERC
Starting Grant)We would like to thank The Spanish National Center for Biotechnology (CNB-CSIC) of ProteoRed,
PRB3-ISCIII for the proteomic analysis supported by grant PT17/001
Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective [version 2; referees: 2 approved]
Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection have not been studied yet.
Methods: Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques.
Results: IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line.
Conclusions: Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs.This work was supported by the European Research Council (ERC starting grant 2014 GA639249
Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules
It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year
Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?
Primitive nucleated erythroid cells in the bloodstream have long been suggested to be
more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and
adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo
morphological changes, especially when exposed to stress, and enter a new cell stage that we have
coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission
electron microscopy (TEM) micrographs, Wright–Giemsa staining, cell marker immunostaining,
and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm,
hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte
molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive
pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine
(PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout
bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic
studies resulted in the identification of proteins involved in pathways related to the regulation of cell
morphogenesis involved in differentiation, cellular response to stress, and immune system process.
In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 _ (IL1_), interferon 8888 (IFN8888),
and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic
septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in
roles related to immune response mediation, homeostasis, and the differentiation and development
of blood cells.This work was supported by the European Research Council (ERC Starting Grant GA639249The proteomic analyses were performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC)
that belongs to ProteoRed, PRB2-ISCIII, supported by Grant PT13/0001
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