Mulero Méndez, Victoriano Francisco

Person:
Mulero Méndez, Victoriano Francisco

Name

Mulero Méndez, Victoriano Francisco

publication.page.department

Universidad de Murcia. Departamento de Biología Celular e Histología

Full item page

Search Results

Now showing 1 - 10 of 11

Restricted
The colony-stimulating factor-1 receptor is a specific marker of macrophages from the bony fish gilthead seabream
(Elsevier, 2005-08-30) Lopez-Castejón, Gloria; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
We report the molecular cloning of the colony-stimulating factor-1 receptor gene from the bony fish gilthead seabream (sbCSF-1R). The deduced sbCSF-1R shows a predicted signal sequence, a transmembrane domain and a tyrosine kinase domain, all in conserved positions. A transcript showing a premature stop codon that predicted the removal of 84 C-terminal amino acids was also found. RT-PCR expression studies demonstrate that, although the sbCSF-1R transcripts are found in different immune tissues, including gill, liver, spleen, blood, peritoneal exudate, thymus and head-kidney (HK), their expression is confined to the monocyte/macrophage lineage. Furthermore, the expression of sbCSF-1R might be modulated by the activation stage of the macrophages, since both the infection of fish and the in vitro activation of leukocytes resulted in the down-regulation of gene expression. These data indicate that the CSF-1R may be used as a specific probe for cells of the monocyte/macrophage lineage in the gilthead seabream, an immunological tractable fish model. In addition, the functional characterisation of the CSF-1R and its ligand may shed light into the mechanisms of proliferation and the pathways of differentiation of macrophages in bony fish.
Restricted
Evolution of Lipopolysaccharide (LPS) Recognition and Signaling: Fish TLR4 Does Not Recognize LPS and Negatively Regulates NF-κB Activation
(American Association of Immunologists Oxford University Press, 2009-02-15) Alcaraz-Perez, Francisca; López-Muñoz, Azucena; Meseguer Peñalver, J.; Cayuela Fuentes, Maria Luisa; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
It has long been established that lower vertebrates, most notably fish and amphibians, are resistant to the toxic effect of LPS. Furthermore, the lack of a TLR4 ortholog in some fish species and the lack of the essential costimulatory molecules for LPS activation via TLR4 (i.e., myeloid differentiation protein 2 (MD-2) and CD14) in all the fish genomes and expressed sequence tag databases available led us to hypothesize that the mechanism of LPS recognition in fish may be different from that of mammals. To shed light on the role of fish TLRs in LPS recognition, a dual-luciferase reporter assay to study NF-κB activation in whole zebrafish embryos was developed and three different bony fish models were studied: 1) the gilthead seabream (Sparus aurata, Perciformes), an immunological-tractable teleost model in which the presence of a TLR4 ortholog is unknown; 2) the spotted green pufferfish (Tetraodon nigroviridis, Tetraodontiformes), which lacks a TLR4 ortholog; and 3) the zebrafish (Danio rerio, Cypriniformes), which possesses two TLR4 orthologs. Our results show that LPS signaled via a TLR4- and MyD88-independent manner in fish, and, surprisingly, that the zebrafish TLR4 orthologs negatively regulated the MyD88-dependent signaling pathway. We think that the identification of TLR4 as a negative regulator of TLR signaling in the zebrafish, together with the absence of this receptor in most fish species, explains the resistance of fish to endotoxic shock and supports the idea that the TLR4 receptor complex for LPS recognition arose after the divergence of fish and tetrapods.
Restricted
New insights into the evolution of IFNs: zebrafish group II IFNs induce a rapid and transient expression of IFN-dependent genes and display powerful antiviral activities
(American Association of Immunologists, Oxford University Press, 2009-03-15) López-Muñoz, Azucena; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
The IFNs and their receptors have existed in early chordates for ∼500 million years and represent the early elements in innate and adaptive immunity. Both types I and II IFNs have been discovered in fish, and type I has recently been classified into two groups based on their primary protein sequences. However, the biological activities of fish IFNs and their roles in infection are largely unknown. Using the zebrafish and manageable bacterial (Streptococcus iniae) and viral (spring viremia of carp virus) infection models, we are reporting in this study that zebrafish IFN (zfIFN) γ failed to induce antiviral and proinflammatory genes when administered in vivo, which correlates with its inability to protect the fish against bacterial and viral infections. We also found that, although both group I (i.e., zfIFN1) and group II zfIFNs (i.e., zfIFN2 and zfIFN3) displayed strong in vivo antiviral activities, only group I zfIFN was able to protect the fish against bacterial infection, which may reflect the different patterns and kinetics of immune-related genes elicited by these two groups of IFNs. Thus, group II zfIFNs induced a rapid and transient expression of antiviral genes, whereas group I zfIFN exerted a slow but more powerful induction of several antiviral and proin-flammatory genes. Collectively, our results suggest nonredundant, complementary roles of type I zfIFNs in viral infections and provide evidence for a pivotal role of the recently identified group II IFN of fish in the early stages of viral infections.
Open Access
Interleukin-1β isolated from a marine fish reveals up-regulated expression in macrophages following activation with lipopolysaccharide and lymphokines
(Elsevier, 2001) García-Castillo, Jesús; Meseguer, José; Mulero Méndez, Victoriano Francisco; Pelegrín Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
The gilthead seabream IL-1β gene consists of five exons/four introns. The complete coding sequence contains a 102 bp 5’ untraslated region (UTR), a single open reading frame of a 762 bp which translates into a 253 amino acid molecule, and a 407 bp 3’UTR with a polyadenilation signal 14 nucleotides upstream of the poly(A)tail. The seabream sequence has highest degree of nucleotide (61.7%) and amino acid (53%) identity with the trout IL-1β sequences. The IL-1β message was detected by RT-PCR in head-kidney, blood, spleen, liver, gill and peritoneal exudate of both non-infected and Vibrio anguillarum-challenged fish. More importantly, IL-1β was highly expressed by purified macrophage monolayers and was up-regulated by lipopolysaccharide and lymphocyte-derived macrophage-activating factor stimulation.
Restricted
Characterization of macrophages from the bony fish gilthead seabream using an antibody against the macrophage colony-stimulating factor receptor
(Elsevier, 2008-04-07) Mulero Méndez, Iván; Meseguer Peñalver, J.; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr+) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr+ cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.
Open Access
Production and mechanism of secretion of interleukin-1b from the marine fish gilthead seabream
(2004) Elena, Chaves-Pozo; José, Meseguer; Mulero Méndez, Victoriano Francisco; Pelegrín Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
Interleukin-1b (IL-1b) is a secretory cytokine lacking a signal peptide, and does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1b-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. No information is available on the production and release of fish IL-1b, but the IL-1b gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor (MAF)/bacterial DNA (VaDNA)-primed immune cells of fish accumulate intracellular IL-1b as a ~30 kDa polypeptide (proIL-1b). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor (PRR). More importantly, addition of extracellular ATP does not promote IL-1b secretion by immune cells and fails to induce phosphatidylserine (PS) flip. In contrast, fish SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1b form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1b by SAF-1 cells is abrogated by a specific ICE inhibitor.
Restricted
Evolution of the inflammatory response in vertebrates: fish TNF-α is a powerful activator of endothelial cells but hardly activates phagocytes
(2008-10-01) Mulero Méndez, Iván; López-Muñoz, Azucena; Renshaw, Stephen A.; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
TNF-α is conserved in all vertebrate classes and has been identified in all taxonomic groups of teleost fish. However, its biological activities and its role in infection are largely unknown. Using two complementary fish models, gilthead seabream and zebrafish, we report here that the main proinflammatory effects of fish TNF-α are mediated through the activation of endothelial cells. Thus, TNF-α promotes the expression of E-selectin and different CC and CXC chemokines in endothelial cells, thus explaining the recruitment and activation of phagocytes observed in vivo in both species. We also found that TLR ligands, and to some extent TNF-α, were able to increase the expression of MHC class II and CD83 in endothelial cells, which might suggest a role for fish endothelial cells and TNF-α in Ag presentation. Lastly, we found that TNF-α increases the susceptibility of the zebrafish to viral (spring viremia of carp virus) and bacterial (Streptococcus iniae) infections. Although the powerful actions of fish TNF-α on endothelial cells suggest that it might facilitate pathogen dissemination, it was found that TNF-α increased antiviral genes and, more importantly, had little effect on the viral load in early infection. In addition, the stimulation of ZF4 cells with TNF-α resulted in increased viral replication. Together, these results indicate that fish TNF-α displays different sorts of bioactivity to their mammalian counterparts and point to the complexity of the evolution that has taken place in the regulation of innate immunity by cytokines.
Open Access
Acidophilic granulocytes of the marine fish gilthead seabream (Sparus aurata L.) produce interleukin-1b following infection with Vibrio anguillarum
(Springer, 2004) Chaves-Pozo, Elena; García-Castillo, Jesús; Meseguer, José; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Pelegrín Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1b (IL-1b) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1b. We found that gilthead seabream head- kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1b intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1b following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1b were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed.
Restricted
Post-transcriptional regulation of cytokine genes in fish: A role for conserved AU-rich elements located in the 3′-untranslated region of their mRNAs
(Elsevier, 2006-04-03) Cayuela Fuentes, Maria Luisa; Secombes, Chris. J.; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
The overproduction of cytokines, such us interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3'UTR of IL-1beta and TNFalpha from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3'UTR of the IL-1beta from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1beta and TNFalpha mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45 min in control and activated cells.
Restricted
Turbot TNFα gene: molecular characterization and biological activity of the recombinant protein
(Elsevier, 2006-04-17) Ordas, M. C.; Costa, Maria del Mar; Lopez-Castejón, Gloria; Meseguer Peñalver, J.; Figueras, Antonio; Novoa, Beatriz; Mulero Méndez, Victoriano Francisco; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
The tumor necrosis factor (TNF) superfamily is composed by several proteins with similar structure and functions. One of the main representatives of this family is TNF-alpha (TNFα), a proinflammatory cytokine which is produced by different immune cells and presents a wide variety of activities. Using the RACE technique, we have cloned and sequenced the turbot TNF cDNA. The analysis of its sequence showed several conserved motifs characteristic of members of the TNFα family. A phylogenetic tree constructed with different TNFs of fish and mammals grouped our sequence within the fish TNFα cluster. Therefore, the turbot TNF here studied was identified as TNFα. The complete TNFα gene was obtained by gene walking, and, similarly to the other known fish TNFα genes, presented three introns and four exons. A PCR was designed to study the turbot TNFα expression in vivo using as stimulus the bacteria Vibrio pelagius strain Hq222 and virus VHSV. The expression of the cytokine happened early after injection, and it was dependent on the pathogen injected and organ analyzed. Virus induced a higher TNFα expression, but this response was shorter in time than that induced by bacteria. In addition, TNFα expression was in general higher in kidney than in liver, as expected since the former is the haematopoietic organ of fish. The turbot recombinant TNFα (rTNFα) was obtained by IPTG induction of bacteria transformed with the pET15b-TNFα construct, and it was purified in native conditions. The recombinant protein was approximately 20 kDa in size, and its biological activity was assessed in vitro. No effect of the rTNFα neither alone nor in combination with LPS was observed on the chemiluminescence activity of turbot macrophages at any time tested. However, NO production was enhanced by the recombinant protein alone or with LPS 72 h after the addition of the treatments. Finally, turbot rTNFα was able to recruit and activate inflammatory cells when injected in gilthead seabream, although to a lesser extent than gilthead seabream rTNFα.