NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fu... more NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun Nterminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NKlysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.
Proliferative enteropathy, caused by Lawsonia intracellularis, represents a threat for swine indu... more Proliferative enteropathy, caused by Lawsonia intracellularis, represents a threat for swine industry. Current vaccines are effective but difficult to obtain and scaled up, because of demanding bacterial culture conditions. In this work, a subunit vaccine candidate against L. intracellularis was developed and its efficacy was evaluated in vivo, alone or co-formulated with pig recombinant IFN-a. The vaccine formulation contains three chimeric antigens: two outer membrane proteins and a secreted one, which were engineered by adding T epitopes using bioinformatics tools. After simultaneously expressing the three antigens in E. coli, its immunogenicity was tested in mice and pigs. Antigens co-formulated with porcine IFN-a were also assayed in the last species. Immune response was assessed by ELISA and qPCR, and histopathological studies of intestinal epithelial tissue were performed after challenge. Mice and pigs showed an increased IgG response against chimeric antigens. Particularly, there were significant differences in the antibody response when porcine IFN-a was co-administrated with L. intracellularis antigens. Besides, mRNAs from il12 and cd4 marker were detected during the first week after immunization of pigs, suggesting a Th1-type cellular immune response. The significant enhancement of oas2 gene expression indicates the effect exerted by porcine IFN-a. Post-mortem histopathological analysis post-challenge revealed damage only into epithelial cells of the gastrointestinal tract from animals of the negative control group. Injuries were related to atrophy of the intestinal villi, where a decrease of globet cells and a greater migration of lymphocytes were observed. Overall, our results demonstrated that the vaccine candidate elicited significant humoral and cellular immune responses. Besides, histopathological analysis suggested that vaccinated animals were protected against experimental L. intracellularis infection. This research constitutes a step forward to the generation of the first recombinant chimeric vaccine against L. intracellularis, representing a faster, easier and cost effective approach to counteract the porcine proliferative enteropathy.
Naturally occurring antimicrobial peptides take place in the first line of host defense against p... more Naturally occurring antimicrobial peptides take place in the first line of host defense against pathogen as part of the humoral innate immune response. b-defensins are among the most abundant antimicrobial peptides in mammals, and thought to be solely found in vertebrates until a recent report describing the cloning and sequencing of defensin like peptides in the spiny lobster Panulirus japonicus. In the current study, we cloned and sequenced two genes from the hemocytes of the spiny lobster Panulirus argus encoding for two isoforms of defensin-like peptides, thus confirming the presence of this protein in the Panulirus genus. The 44 amino acids mature peptides showed the conservation of cysteine pattern characterizing the b-defensins, as well as known amino acids residues critical to exert their antimicrobial activity. They are also amphipathics, hydrophobics, and display an overall positive charge (þ1) located at the C-terminus. The tertiary structure obtained by homology modeling indicated that likely conformations of lobster peptides are highly similar to b-defensins from vertebrates. The phylogenetic study carried out by probabilistic methods confirmed the relation with ancestral b-defensin from vertebrates. The finding of a putative defensin-like peptide in the expressed sequence tag (EST) of the lobster Homarus americanus with high homology with those of P. argus described in this study, would indicate the presence of this peptides in Palinuridae family. Taking into account all similarities between these peptides with b-defensins from vertebrates, it is conceivable to further support the finding of a new family of b-defensins in invertebrate.
As an important disulfide reductase of the intracellular antioxidant system, Thioredoxin (Trx) pl... more As an important disulfide reductase of the intracellular antioxidant system, Thioredoxin (Trx) plays an important role in maintaining oxidative stress balance and protecting cells from oxidative damage. In recent years, there is increasing evidence that Trx is a key molecule in the pathogenesis of various diseases and a potential therapeutic target for major diseases including lung, colon, cervical, gastric and pancreatic cancer. However, few knowledge is known about the function of Trx in virus infection. In this study, we reported the cloning and functional investigation of a Trx homologue gene, named MjTrx, in shrimp Marsupenaeus japonicus suffered white spot syndrome virus (WSSV) infection. MjTrx is a 105-amino acid polypeptide with a conservative Cys-Gly-Pro-Cys motif in the catalytic center. Phylogenetic trees analysis showed that MjTrx has a higher relationship with Trx from other invertebrate and clustered with Trx1 from arthropod. MjTrx transcripts is abundant in the gill and intestine tissues and can be detected in the hemocytes, heart, stomach, and hepatopancreas tissues. The transcription levels of MjTrx in hemocytes, gills and intestine tissues of shrimp were significantly up-regulated after white spot syndrome virus infection. MjTrx was recombinant expressed in vitro and exhibited obvious disulfide reductase activity. In addition, overexpression MjTrx in shrimp resulted in the increase of hydrogen peroxide (H 2 O 2) concentration in vivo. All these results strongly suggested that MjTrx functioned in redox homeostasis regulating and played an important role in shrimp antiviral immunity.
Continuous photoperiod is extensively used in fish farming, to regulate the reproductive cycle, d... more Continuous photoperiod is extensively used in fish farming, to regulate the reproductive cycle, despite evidence suggesting that artificial photoperiods can act as a stressor and impair the immune system. We evaluated the potential effects of an artificial photoperiod on mucus components: lysozyme and mucin, in juvenile rainbow trout (Oncorhynchus mykiss) after exposure for one month to natural photoperiod (LD12:12) or constant light (LD24:0) artificial photoperiod. For each treatment, we assessed changes in peripheral blood cells (erythrocytes and leukocytes) and skin mucus component concentrations. Our results show a decrease in lysozyme concentration, while mucin levels are increased. Similarly, we find elevated monocytes and polymorphonuclears under constant light photoperiod. These findings suggest that LD24:0 regulates lysozyme, mucin, and leukocytes, implying that artificial photoperiods could be a stressful.
The membrane-anchored and soluble Toll-like Receptor 5 -TLR5M and TLR5S, respectively-from teleos... more The membrane-anchored and soluble Toll-like Receptor 5 -TLR5M and TLR5S, respectively-from teleost recognize bacterial flagellin and induce the pro-inflammatory cytokines expression in a MyD88-dependent manner such as the TLR5 mammalian orthologous receptor. However, it has not been demonstrated whether the induced signaling pathway by these receptors activate innate effector mechanisms MyD88-dependent in salmonids. Therefore, in this work we study the MyD88 dependence on the induction of TLR5M/TLR5S signaling pathway mediated by flagellin as ligand on the activation of some innate effector mechanisms. The intracellular and extracellular Reactive Oxygen Species (ROS) production and conditioned supernatants production were evaluated in RTS11 cells, while the challenge with Piscirickettsia salmonis was evaluated in SHK-1 cells. Our results demonstrate that flagellin directly stimulates ROS production and indirectly stimulates it through the production of conditioned supernatants, both in a MyD88-dependent manner. Additionally, flagellin stimulation prevents the cytotoxicity induced by infection with P. salmonis in a MyD88-dependent manner. In conclusion we demonstrate that MyD88 is an essential adapter protein in the activation of the TLR5M/TLR5S signaling pathway mediated by flagellin in salmonids, which leads downstream to the induction of innate effector mechanisms, promoting immuno-protection against a bacterial challenge with P. salmonis.
Currently, the generation of cell lines for the production of recombinant proteins has the limita... more Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transd...
TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune diso... more TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformedE. coli.Protein detection was assessed by SDS-PAGE and western blot. The KDvalues for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis. Molecular sizes of the complexes were characterized by SEC-HPLC. Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to lineal epitopes and to one protomer of rhTNFα. Binding curves of two phases with low and high KDvalues (<10−9M and >10−8M) were observed during thermophoresis experiments, suggesting the gener...
Developmental and comparative immunology, Nov 20, 2018
Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebr... more Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebrates, by protecting the organism from a wide variety of pathogens. Nile tilapia (Oreochromis niloticus) is extensively cultivated worldwide, with a strong established market demand. It constitutes one of the model species for the study of fish immunology and its genome is currently fully sequenced. The presence of the immunoglobulin M gene in this species is well documented, as well as its major role in systemic immunity. To date, the IgT gene from O. niloticus has not been identified and, therefore, no information is available on the role of this immunoglobulin isotype in the immune response in tilapia. In the present work, novel secreted and membrane immunoglobulin T isotypes and a fragment of IgM were isolated from tilapia head kidney lymphocytes. Their transcriptional profiles were analyzed by quantitative PCR in larval development and in different tissues of healthy or lipopolysacch...
Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. This c... more Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. This cytokine plays a very important role in defining Th1 immune response in all vertebrates. In the present study, we identified and isolated for the first time the gene coding for Nile tilapia (Oreochromis niloticus) IFNγ from spleen lymphocytes. The isolated tilapia IFNγ has between 24 and 62% of amino acid identity as compared to reported sequences for other teleost fishes. It has close phylogenetic relationships with IFNγ molecules belonging to the group of Perciforms and presents the typical structural characteristics of gamma interferon molecules. The tissue expression analysis showed that IFNγ is expressed constitutively in head kidney, skin, intestine, muscle and brain. Its expression was not detected in gills by conventional RT-PCR. However, under conditions of stimulation with Poly I:C and LPS, IFNγ expression was up-regulated in gills after 24 h post-stimulation. IFNγ expression wa...
Growth hormone-releasing peptide-6 (GHRP-6) is one of the earliest developed synthetic peptidyl g... more Growth hormone-releasing peptide-6 (GHRP-6) is one of the earliest developed synthetic peptidyl growth hormone secretagogue receptor agonists. These compounds mimic the effect of the endogenous ligand ghrelin. In vertebrates, ghrelin is a potent circulating orexigenic hormone with functional roles in controlling food intake, energy expenditure, adiposity, growth hormone secretion and immunity. Ghrelin has been studied mainly in vertebrates; thus, little is known about its role in invertebrates, including crustaceans. We first evaluated the effect of GHRP-6 injection over feed intake in shrimp and its effects on shrimp growth when the peptide was administrated by successive immersion baths. GHRP-6 increased feed intake, body weight and size, the number of rostral spines and gill branches, protein concentration and haemocyte number in treated shrimps. We also evaluated the peptide uptake and clearance in a pharmacokinetics, using [H 3 ]GHRP-6 administered to postlarvae. Given a limited exposure and efficient clearance of the peptide-associated radioactivity from larvae, our findings suggested that GHRP-6-treated Litopenaeus vannamei can be consumed safely by humans after aquaculture applications. These results propose that GHRP-6 could be an additional tool to study growth physiology in crustaceans and also a promising candidate for development into a new biotechnology product for improving shrimp growth and quality.
It was recently discovered that double-stranded RNA (dsRNA) was a potent and specific inhibitor o... more It was recently discovered that double-stranded RNA (dsRNA) was a potent and specific inhibitor of gene transcrip- tion in the Caenorhabditis elegans nematode. Similar results have been reported in Drosophila melanogaster, planaria, mice, zebrafish, among other species. The interfering RNA (RNAi) phenomena occurs when a dsRNA is processed by a protein complex, specifically generating small size molecules that anneal to a target sequence of messenger RNA and promote its degradation, while also blocking its expression. The dsRNA-mediated interference is in most cases a post-transcriptional gene silencing mechanism. This paper, demonstrates that gene function can be studied in aquatic organisms by using RNA interference. Here, we describe the application of this technique in two aquatic organisms: - Zebrafish (Danio rerio), a currently relevant animal model in the field of genetics, developmental biology and biomedicine. - White shrimp (Litopenaeus schimitti), uneconomically relevant s...
Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon ind... more Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry in the last 30 years. Caligus rogercresseyi is the most important species affecting Chile's salmon industry. Vaccines against caligid copepods have the potential to be a cost-effective means of controlling the infestation and avoid many of the disadvantages of medicine treatments. However, research in the development of such vaccines has begun only recently and approaches used thus far have met with little or no success. In the present study, we characterized a novel gene (denoted as my32) from C. rogercresseyi which has the highest identity with the Lepeophtheirus salmonis gene akirin-2. To assess the function of the gene an RNA interference experiment was developed and a reduction in the number of ectoparasites on fish in the my32-dsRNA treated group was observed. The recombinant my32 protein was used in a vaccinationchallenge trial to evaluate its ability to protect against sea lice infestations. A significant reduction in the number of parasites per fish was observed at 24 days post-challenge. These results, together with the delay observed in the development of parasites from the vaccinated group suggest that the major effect of immunization was on the second parasite generation. The results of these experiments suggest that the my32 protein may be a promising target for vaccine development to control sea lice infestations in fish.
Swimming respirometry was employed to compare inactive metabolic rate (R r), maximum metabolic ra... more Swimming respirometry was employed to compare inactive metabolic rate (R r), maximum metabolic rate (R max), resultant aerobic scope and maximum sustainable (critical) swimming speed (U crit), in growth hormone transgenic (GHT) and wild-type (W) tilapia Oreochromis sp. hybrids. Although the R r of GHT tilapia was significantly (58%) higher than their W conspecifics, there were no significant differences in their net aerobic scope because GHT tilapia exhibited a compensatory increase in R max that was equal to their net increase in R r. As a consequence, the two groups had the same U crit. The GHT and W tilapia also exhibited the same capacity to regulate oxygen uptake during progressive hypoxia, despite the fact that the GHT fish were defending a higher demand for O 2. The results indicate that ectopic expression of GH raises metabolic rate in tilapia, but the fish compensate for this metabolic load and preserve such physiological determinants of fitness as aerobic scope, swimming performance and tolerance of hypoxia.
Nowadays, the studies of pituitary adenylate cyclase-activating polypeptide (PACAP)-related pepti... more Nowadays, the studies of pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) and PACAP in non-mammalian vertebrates, especially in fish, have paid attention mainly to the localization, cloning, and structural evolution of the peptides, but very little is known about its biological functions as growth-promoting factors in low vertebrates. In this work, we have cloned and characterized the PRP/PACAP cDNA from the commercially important North African catfish Clarias gariepinus. The sequence obtained agrees with the higher conservation of PACAP than of PRP peptide sequences. We have reported for the first time the recombinant expression of fish PRP and PACAP in mammalian cells and bacteria and also demonstrated that the growth rate of fish is enhanced by both PRP and PACAP recombinant peptides. The results obtained in vivo in three different fish species, catfish (C. gariepinus), tilapia (Oreochromis niloticus), and carp (Cyprinus carpio) support the finding...
Myostatin is a member of the transforming growth factor- (TGF-) family that functions as a nega... more Myostatin is a member of the transforming growth factor- (TGF-) family that functions as a negative regulator of skeletal muscle development and growth. Recently, it has been reported that the transgenic zebrafish expressing myostatin prodomain exhibited an increased number of fiber in skeletal muscle. Other novel results suggest that myostatin plays a mayor role during myogenesis, apart from inhibition of proliferation as well as differentiation. We have investigated the ability of double-stranded RNA (dsRNA) to inhibit myostatin function in the zebrafish. By microinjection dsRNA, corresponding to biologically active C-terminal domain from aminoacid 268 to end codon of tilapia myostatin protein, we produced an increased body mass in treated fish. The dsRNA injection in early development stage in zebrafish produced hyperplasia or hypertrophy. In addition, the interference of gene function showed a strong dependence on the amount of dsRNA.
NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fu... more NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun Nterminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NKlysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.
Proliferative enteropathy, caused by Lawsonia intracellularis, represents a threat for swine indu... more Proliferative enteropathy, caused by Lawsonia intracellularis, represents a threat for swine industry. Current vaccines are effective but difficult to obtain and scaled up, because of demanding bacterial culture conditions. In this work, a subunit vaccine candidate against L. intracellularis was developed and its efficacy was evaluated in vivo, alone or co-formulated with pig recombinant IFN-a. The vaccine formulation contains three chimeric antigens: two outer membrane proteins and a secreted one, which were engineered by adding T epitopes using bioinformatics tools. After simultaneously expressing the three antigens in E. coli, its immunogenicity was tested in mice and pigs. Antigens co-formulated with porcine IFN-a were also assayed in the last species. Immune response was assessed by ELISA and qPCR, and histopathological studies of intestinal epithelial tissue were performed after challenge. Mice and pigs showed an increased IgG response against chimeric antigens. Particularly, there were significant differences in the antibody response when porcine IFN-a was co-administrated with L. intracellularis antigens. Besides, mRNAs from il12 and cd4 marker were detected during the first week after immunization of pigs, suggesting a Th1-type cellular immune response. The significant enhancement of oas2 gene expression indicates the effect exerted by porcine IFN-a. Post-mortem histopathological analysis post-challenge revealed damage only into epithelial cells of the gastrointestinal tract from animals of the negative control group. Injuries were related to atrophy of the intestinal villi, where a decrease of globet cells and a greater migration of lymphocytes were observed. Overall, our results demonstrated that the vaccine candidate elicited significant humoral and cellular immune responses. Besides, histopathological analysis suggested that vaccinated animals were protected against experimental L. intracellularis infection. This research constitutes a step forward to the generation of the first recombinant chimeric vaccine against L. intracellularis, representing a faster, easier and cost effective approach to counteract the porcine proliferative enteropathy.
Naturally occurring antimicrobial peptides take place in the first line of host defense against p... more Naturally occurring antimicrobial peptides take place in the first line of host defense against pathogen as part of the humoral innate immune response. b-defensins are among the most abundant antimicrobial peptides in mammals, and thought to be solely found in vertebrates until a recent report describing the cloning and sequencing of defensin like peptides in the spiny lobster Panulirus japonicus. In the current study, we cloned and sequenced two genes from the hemocytes of the spiny lobster Panulirus argus encoding for two isoforms of defensin-like peptides, thus confirming the presence of this protein in the Panulirus genus. The 44 amino acids mature peptides showed the conservation of cysteine pattern characterizing the b-defensins, as well as known amino acids residues critical to exert their antimicrobial activity. They are also amphipathics, hydrophobics, and display an overall positive charge (þ1) located at the C-terminus. The tertiary structure obtained by homology modeling indicated that likely conformations of lobster peptides are highly similar to b-defensins from vertebrates. The phylogenetic study carried out by probabilistic methods confirmed the relation with ancestral b-defensin from vertebrates. The finding of a putative defensin-like peptide in the expressed sequence tag (EST) of the lobster Homarus americanus with high homology with those of P. argus described in this study, would indicate the presence of this peptides in Palinuridae family. Taking into account all similarities between these peptides with b-defensins from vertebrates, it is conceivable to further support the finding of a new family of b-defensins in invertebrate.
As an important disulfide reductase of the intracellular antioxidant system, Thioredoxin (Trx) pl... more As an important disulfide reductase of the intracellular antioxidant system, Thioredoxin (Trx) plays an important role in maintaining oxidative stress balance and protecting cells from oxidative damage. In recent years, there is increasing evidence that Trx is a key molecule in the pathogenesis of various diseases and a potential therapeutic target for major diseases including lung, colon, cervical, gastric and pancreatic cancer. However, few knowledge is known about the function of Trx in virus infection. In this study, we reported the cloning and functional investigation of a Trx homologue gene, named MjTrx, in shrimp Marsupenaeus japonicus suffered white spot syndrome virus (WSSV) infection. MjTrx is a 105-amino acid polypeptide with a conservative Cys-Gly-Pro-Cys motif in the catalytic center. Phylogenetic trees analysis showed that MjTrx has a higher relationship with Trx from other invertebrate and clustered with Trx1 from arthropod. MjTrx transcripts is abundant in the gill and intestine tissues and can be detected in the hemocytes, heart, stomach, and hepatopancreas tissues. The transcription levels of MjTrx in hemocytes, gills and intestine tissues of shrimp were significantly up-regulated after white spot syndrome virus infection. MjTrx was recombinant expressed in vitro and exhibited obvious disulfide reductase activity. In addition, overexpression MjTrx in shrimp resulted in the increase of hydrogen peroxide (H 2 O 2) concentration in vivo. All these results strongly suggested that MjTrx functioned in redox homeostasis regulating and played an important role in shrimp antiviral immunity.
Continuous photoperiod is extensively used in fish farming, to regulate the reproductive cycle, d... more Continuous photoperiod is extensively used in fish farming, to regulate the reproductive cycle, despite evidence suggesting that artificial photoperiods can act as a stressor and impair the immune system. We evaluated the potential effects of an artificial photoperiod on mucus components: lysozyme and mucin, in juvenile rainbow trout (Oncorhynchus mykiss) after exposure for one month to natural photoperiod (LD12:12) or constant light (LD24:0) artificial photoperiod. For each treatment, we assessed changes in peripheral blood cells (erythrocytes and leukocytes) and skin mucus component concentrations. Our results show a decrease in lysozyme concentration, while mucin levels are increased. Similarly, we find elevated monocytes and polymorphonuclears under constant light photoperiod. These findings suggest that LD24:0 regulates lysozyme, mucin, and leukocytes, implying that artificial photoperiods could be a stressful.
The membrane-anchored and soluble Toll-like Receptor 5 -TLR5M and TLR5S, respectively-from teleos... more The membrane-anchored and soluble Toll-like Receptor 5 -TLR5M and TLR5S, respectively-from teleost recognize bacterial flagellin and induce the pro-inflammatory cytokines expression in a MyD88-dependent manner such as the TLR5 mammalian orthologous receptor. However, it has not been demonstrated whether the induced signaling pathway by these receptors activate innate effector mechanisms MyD88-dependent in salmonids. Therefore, in this work we study the MyD88 dependence on the induction of TLR5M/TLR5S signaling pathway mediated by flagellin as ligand on the activation of some innate effector mechanisms. The intracellular and extracellular Reactive Oxygen Species (ROS) production and conditioned supernatants production were evaluated in RTS11 cells, while the challenge with Piscirickettsia salmonis was evaluated in SHK-1 cells. Our results demonstrate that flagellin directly stimulates ROS production and indirectly stimulates it through the production of conditioned supernatants, both in a MyD88-dependent manner. Additionally, flagellin stimulation prevents the cytotoxicity induced by infection with P. salmonis in a MyD88-dependent manner. In conclusion we demonstrate that MyD88 is an essential adapter protein in the activation of the TLR5M/TLR5S signaling pathway mediated by flagellin in salmonids, which leads downstream to the induction of innate effector mechanisms, promoting immuno-protection against a bacterial challenge with P. salmonis.
Currently, the generation of cell lines for the production of recombinant proteins has the limita... more Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transd...
TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune diso... more TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformedE. coli.Protein detection was assessed by SDS-PAGE and western blot. The KDvalues for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis. Molecular sizes of the complexes were characterized by SEC-HPLC. Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to lineal epitopes and to one protomer of rhTNFα. Binding curves of two phases with low and high KDvalues (<10−9M and >10−8M) were observed during thermophoresis experiments, suggesting the gener...
Developmental and comparative immunology, Nov 20, 2018
Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebr... more Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebrates, by protecting the organism from a wide variety of pathogens. Nile tilapia (Oreochromis niloticus) is extensively cultivated worldwide, with a strong established market demand. It constitutes one of the model species for the study of fish immunology and its genome is currently fully sequenced. The presence of the immunoglobulin M gene in this species is well documented, as well as its major role in systemic immunity. To date, the IgT gene from O. niloticus has not been identified and, therefore, no information is available on the role of this immunoglobulin isotype in the immune response in tilapia. In the present work, novel secreted and membrane immunoglobulin T isotypes and a fragment of IgM were isolated from tilapia head kidney lymphocytes. Their transcriptional profiles were analyzed by quantitative PCR in larval development and in different tissues of healthy or lipopolysacch...
Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. This c... more Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. This cytokine plays a very important role in defining Th1 immune response in all vertebrates. In the present study, we identified and isolated for the first time the gene coding for Nile tilapia (Oreochromis niloticus) IFNγ from spleen lymphocytes. The isolated tilapia IFNγ has between 24 and 62% of amino acid identity as compared to reported sequences for other teleost fishes. It has close phylogenetic relationships with IFNγ molecules belonging to the group of Perciforms and presents the typical structural characteristics of gamma interferon molecules. The tissue expression analysis showed that IFNγ is expressed constitutively in head kidney, skin, intestine, muscle and brain. Its expression was not detected in gills by conventional RT-PCR. However, under conditions of stimulation with Poly I:C and LPS, IFNγ expression was up-regulated in gills after 24 h post-stimulation. IFNγ expression wa...
Growth hormone-releasing peptide-6 (GHRP-6) is one of the earliest developed synthetic peptidyl g... more Growth hormone-releasing peptide-6 (GHRP-6) is one of the earliest developed synthetic peptidyl growth hormone secretagogue receptor agonists. These compounds mimic the effect of the endogenous ligand ghrelin. In vertebrates, ghrelin is a potent circulating orexigenic hormone with functional roles in controlling food intake, energy expenditure, adiposity, growth hormone secretion and immunity. Ghrelin has been studied mainly in vertebrates; thus, little is known about its role in invertebrates, including crustaceans. We first evaluated the effect of GHRP-6 injection over feed intake in shrimp and its effects on shrimp growth when the peptide was administrated by successive immersion baths. GHRP-6 increased feed intake, body weight and size, the number of rostral spines and gill branches, protein concentration and haemocyte number in treated shrimps. We also evaluated the peptide uptake and clearance in a pharmacokinetics, using [H 3 ]GHRP-6 administered to postlarvae. Given a limited exposure and efficient clearance of the peptide-associated radioactivity from larvae, our findings suggested that GHRP-6-treated Litopenaeus vannamei can be consumed safely by humans after aquaculture applications. These results propose that GHRP-6 could be an additional tool to study growth physiology in crustaceans and also a promising candidate for development into a new biotechnology product for improving shrimp growth and quality.
It was recently discovered that double-stranded RNA (dsRNA) was a potent and specific inhibitor o... more It was recently discovered that double-stranded RNA (dsRNA) was a potent and specific inhibitor of gene transcrip- tion in the Caenorhabditis elegans nematode. Similar results have been reported in Drosophila melanogaster, planaria, mice, zebrafish, among other species. The interfering RNA (RNAi) phenomena occurs when a dsRNA is processed by a protein complex, specifically generating small size molecules that anneal to a target sequence of messenger RNA and promote its degradation, while also blocking its expression. The dsRNA-mediated interference is in most cases a post-transcriptional gene silencing mechanism. This paper, demonstrates that gene function can be studied in aquatic organisms by using RNA interference. Here, we describe the application of this technique in two aquatic organisms: - Zebrafish (Danio rerio), a currently relevant animal model in the field of genetics, developmental biology and biomedicine. - White shrimp (Litopenaeus schimitti), uneconomically relevant s...
Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon ind... more Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry in the last 30 years. Caligus rogercresseyi is the most important species affecting Chile's salmon industry. Vaccines against caligid copepods have the potential to be a cost-effective means of controlling the infestation and avoid many of the disadvantages of medicine treatments. However, research in the development of such vaccines has begun only recently and approaches used thus far have met with little or no success. In the present study, we characterized a novel gene (denoted as my32) from C. rogercresseyi which has the highest identity with the Lepeophtheirus salmonis gene akirin-2. To assess the function of the gene an RNA interference experiment was developed and a reduction in the number of ectoparasites on fish in the my32-dsRNA treated group was observed. The recombinant my32 protein was used in a vaccinationchallenge trial to evaluate its ability to protect against sea lice infestations. A significant reduction in the number of parasites per fish was observed at 24 days post-challenge. These results, together with the delay observed in the development of parasites from the vaccinated group suggest that the major effect of immunization was on the second parasite generation. The results of these experiments suggest that the my32 protein may be a promising target for vaccine development to control sea lice infestations in fish.
Swimming respirometry was employed to compare inactive metabolic rate (R r), maximum metabolic ra... more Swimming respirometry was employed to compare inactive metabolic rate (R r), maximum metabolic rate (R max), resultant aerobic scope and maximum sustainable (critical) swimming speed (U crit), in growth hormone transgenic (GHT) and wild-type (W) tilapia Oreochromis sp. hybrids. Although the R r of GHT tilapia was significantly (58%) higher than their W conspecifics, there were no significant differences in their net aerobic scope because GHT tilapia exhibited a compensatory increase in R max that was equal to their net increase in R r. As a consequence, the two groups had the same U crit. The GHT and W tilapia also exhibited the same capacity to regulate oxygen uptake during progressive hypoxia, despite the fact that the GHT fish were defending a higher demand for O 2. The results indicate that ectopic expression of GH raises metabolic rate in tilapia, but the fish compensate for this metabolic load and preserve such physiological determinants of fitness as aerobic scope, swimming performance and tolerance of hypoxia.
Nowadays, the studies of pituitary adenylate cyclase-activating polypeptide (PACAP)-related pepti... more Nowadays, the studies of pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) and PACAP in non-mammalian vertebrates, especially in fish, have paid attention mainly to the localization, cloning, and structural evolution of the peptides, but very little is known about its biological functions as growth-promoting factors in low vertebrates. In this work, we have cloned and characterized the PRP/PACAP cDNA from the commercially important North African catfish Clarias gariepinus. The sequence obtained agrees with the higher conservation of PACAP than of PRP peptide sequences. We have reported for the first time the recombinant expression of fish PRP and PACAP in mammalian cells and bacteria and also demonstrated that the growth rate of fish is enhanced by both PRP and PACAP recombinant peptides. The results obtained in vivo in three different fish species, catfish (C. gariepinus), tilapia (Oreochromis niloticus), and carp (Cyprinus carpio) support the finding...
Myostatin is a member of the transforming growth factor- (TGF-) family that functions as a nega... more Myostatin is a member of the transforming growth factor- (TGF-) family that functions as a negative regulator of skeletal muscle development and growth. Recently, it has been reported that the transgenic zebrafish expressing myostatin prodomain exhibited an increased number of fiber in skeletal muscle. Other novel results suggest that myostatin plays a mayor role during myogenesis, apart from inhibition of proliferation as well as differentiation. We have investigated the ability of double-stranded RNA (dsRNA) to inhibit myostatin function in the zebrafish. By microinjection dsRNA, corresponding to biologically active C-terminal domain from aminoacid 268 to end codon of tilapia myostatin protein, we produced an increased body mass in treated fish. The dsRNA injection in early development stage in zebrafish produced hyperplasia or hypertrophy. In addition, the interference of gene function showed a strong dependence on the amount of dsRNA.
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Papers by Jannel Acosta