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1.
Fish Shellfish Immunol ; 148: 109512, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38499216

RESUMO

The global aquaculture industry has significant losses each year due to disease outbreaks. Antibiotics are one of the common methods to treat fish infections, but prolonged use can lead to the emergence of resistant strains. Aeromonas spp. Infections are a common and problematic disease in fish, and members of this genera can produce antibiotic resistant strains. Antimicrobial peptides (AMPs) have emerged as an alternative method to treat and prevent infections and pituitary adenylate cyclase activating polypeptide (PACAP) is a prominent member of this family. The objective of this research was to study PACAP's direct antimicrobial activity and its toxicity in fish cells. Four synthetic variants of the natural PACAP from Clarias gariepinus were tested in addition to the natural variant. The experimental results show a different antimicrobial activity against A. salmonicida and A. hydrophila of each PACAP variant, and for the first time show dependence on the culture broth used. Furthermore, the results suggest that the underlying mechanism of PACAP antimicrobial activity includes a bacterial membrane permeabilizing effect, classifying PACAP as a membrane disruptive AMP. This study also demonstrated that the five PACAP variants evaluated showed low toxicity in vitro, at concentrations relevant for in vivo applications. Therefore, PACAP could be a promising alternative to antibiotics in the aquaculture sector.


Assuntos
Anti-Infecciosos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Animais , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Bactérias , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Aquicultura
2.
Fish Shellfish Immunol ; 115: 150-159, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34146673

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide that belongs to the secretin/glucagon/GHRH/VIP superfamily. Some of these molecules have antimicrobial activity and they are capable of stimulating the immune system. The present work studied the antibacterial and immunostimulatory activity of PACAP-38 from African catfish Clarias gariepinus against the Gram-negative bacterium Pseudomonas aeruginosa in an in vivo test. PACAP-38 improved antimicrobial activity of skin mucus molecules against P. aeruginosa. The peptide modulates the gene expression profile of TLR-1, TLR-5, MyD88, IL-1ß, TNF-ɑ, IL-8, pardaxin, hepcidin and G/C-type lysozymes in skin, spleen and head kidney. The influenced exerted depended on the time after infection and tissue analyzed. This study provides the first evidence of a link between PACAP and antimicrobial peptides hepcidin and pardaxin. Our results suggest further use of PACAP as antimicrobial agent that could potentially be used to control disease in aquaculture.


Assuntos
Anti-Infecciosos/imunologia , Peixes-Gato/genética , Peixes-Gato/imunologia , Proteínas de Peixes/genética , Imunidade Inata/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Transdução de Sinais/genética , Animais , Proteínas de Peixes/imunologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Transdução de Sinais/imunologia , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/imunologia , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/imunologia
3.
Fish Shellfish Immunol ; 110: 44-54, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33348037

RESUMO

Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3-CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.


Assuntos
Anticorpos Monoclonais/imunologia , Ciclídeos/imunologia , Proteínas de Peixes/imunologia , Imunidade Humoral , Cadeias Pesadas de Imunoglobulinas/imunologia , Imunoglobulina M/imunologia , Sequência de Aminoácidos , Animais , Alinhamento de Sequência
4.
Fish Shellfish Immunol ; 103: 58-65, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32334130

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide belonging to the glucagon/secretin superfamily. In teleost fish, PACAP has been demonstrated to have an immunomodulatory role. Although previous studies have shown that viral/bacterial infections can influence the transcription of PACAP splicing variants and associated receptors in salmonids, the antiviral activity of PACAP has never been studied in teleost. Thus, in the present work, we investigated in vitro the influence of synthetic Clarias gariepinus PACAP-38 on the transcription of genes related to viral immunity using the rainbow trout monocyte/macrophage-like cell line RTS11 as a model. Positive transcriptional modulation of interferon gamma (IFNγ), interferon alpha (FNα1,2), interleukin 8 (IL-8), Mx and Toll-like receptor 3 (TLR3) genes was found in a dose and time dependent manner. We also explored how a pre-treatment with PACAP could enhance antiviral immune response using poly (I:C) as viral mimic. Interferons and IL-8 transcription levels were enhanced when PACAP was added 24 h previous to poly (I:C) exposure. With these evidences, we tested in vivo how PACAP administration by immersion bath affected the survival of rainbow trout fry to a challenge with viral hemorrhagic septicemia virus (VHSV). After challenge, PACAP-treated fish had increased survival compared to non-treated/challenge fish. Furthermore, PACAP was able to decrease the viral load in spleen/kidney and stimulate the transcription of IFNs and Mx when compared to untreated infected fish. Altogether, the results of this work provide valuable insights regarding the role of teleost PACAP in antiviral immunity and point to a potential application of this peptide to reduce the impact of viral infections in aquaculture.


Assuntos
Antivirais/imunologia , Peixes-Gato/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Imunidade Inata , Oncorhynchus mykiss , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Animais , Proteínas de Peixes/imunologia , Novirhabdovirus/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Poli I-C/farmacologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária
5.
Fish Shellfish Immunol ; 92: 322-330, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31200071

RESUMO

The development of vaccines employing conserved protein antigens, for instance ribosomal protein P0, has as disadvantage the high degree of identity between pathogen and host proteins due to possible induction of tolerance or auto antibodies in the host organism. To overcome this drawback, peptide-based vaccines have been designed with a proved high efficacy. The use of defined peptides as antigens has the problem that they are generally poor immunogenic unless coupled to a carrier protein. Several studies have established the potential for promiscuous T cell epitopes incorporated into chimeric peptides to enhance the immunogenicity in mammals. On the contrary, studies about the role of these epitopes on teleost immune system are scarce. Therefore, the main objective of our present study was to evaluate the potential of promiscuous T cell epitopes to boost specific IgM immune response in teleost fish against a peptide antigen. With this aim, we used a peptide of 35 amino acids from the ribosomal P0 protein of Lepeophtheirus salmonis, an important parasite in salmon aquaculture. We fused this peptide to the C-terminal of T cell epitopes from tetanus toxin and measles virus and produced the chimeric protein in Escherichia coli. Following vaccination, IgM antibody production was monitored in different immunization schemes in Tilapia, African catfish and Atlantic salmon. The results demonstrated for first time that the addition of T cell epitopes at the N-terminal of a target peptide increased IgM specific response in different teleost species, revealing the potential of this approach to develop peptide-based vaccines for aquaculture. The results are also of great importance in the context of vaccine development against sea lice using ribosomal protein P0 as antigen taking into account the key role of P0 in protein synthesis and other essential physiological processes.


Assuntos
Copépodes/imunologia , Ectoparasitoses/veterinária , Epitopos de Linfócito T/imunologia , Doenças dos Peixes/imunologia , Imunidade Inata/efeitos dos fármacos , Imunoglobulina M/imunologia , Animais , Proteínas de Artrópodes/imunologia , Peixes-Gato/imunologia , Ciclídeos/imunologia , Ectoparasitoses/imunologia , Peptídeos/imunologia , Proteínas Ribossômicas/imunologia , Salmo salar/imunologia , Vacinas de Subunidades Antigênicas/imunologia
6.
Fish Shellfish Immunol ; 71: 275-285, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29017941

RESUMO

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 was also induced in gills 24 h after Edwardsiella tarda infection suggesting its important role in immunity against intracellular bacteria. The recombinant protein produced in Escherichia coli induced Mx gene transcription in head kidney primary culture cells. These results are the first steps to characterize the role of tilapia IFNγ in the defense against pathogens in tilapia. Furthermore, the isolation of this molecule provides a new tool to characterize the cellular immune response to various stimuli in this organism.


Assuntos
Ciclídeos/genética , Ciclídeos/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interferon gama/genética , Interferon gama/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Interferon gama/química , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária
7.
Fish Shellfish Immunol ; 50: 50-5, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26804662

RESUMO

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In previous studies, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide in vitro e in vivo. Hyperimmune serum was obtained from rabbits immunized with a P. argus -NOS fragment of 31 kDa produced in Escherichia coli, which specifically detected the recombinant polypeptide and the endogenous NOS from lobster hemocytes by western blotting and immunofluorescence. In the present work, we demonstrate that the hyperimmune serum obtained against P. argus NOS also recognizes Litopenaeus vannamei NOS in hemocytes by western blotting and immunofluorescence. Our data also show that while the hemolymph of L. vannamei has a strong antibacterial activity against the Gram negative bacteria Aeromonas hydrophila, the administration of the anti NOS serum reduce the natural bacterial clearance. These results strongly suggest that NOS is required for the shrimp immune defense toward Gram negative bacteria. Therefore, the monitoring of induction of NOS could be an important tool for testing immunity in shrimp farming.


Assuntos
Aeromonas hydrophila/fisiologia , Proteínas de Artrópodes/metabolismo , Imunidade Inata , Óxido Nítrico Sintase/metabolismo , Penaeidae/genética , Penaeidae/imunologia , Animais , Anti-Infecciosos/metabolismo , Hemolinfa/imunologia , Penaeidae/microbiologia
8.
Fish Shellfish Immunol ; 47(2): 923-32, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26481517

RESUMO

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Septicemia Hemorrágica Viral/imunologia , Fragmentos de Peptídeos/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Precursores de Proteínas/genética , Receptores de Peptídeo Intestinal Vasoativo/genética , Truta , Yersiniose/veterinária , Animais , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Septicemia Hemorrágica Viral/genética , Septicemia Hemorrágica Viral/virologia , Rim/microbiologia , Rim/virologia , Dados de Sequência Molecular , Novirhabdovirus/fisiologia , Fragmentos de Peptídeos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Análise de Sequência de DNA/veterinária , Organismos Livres de Patógenos Específicos , Baço/microbiologia , Baço/virologia , Transcriptoma , Yersinia/fisiologia , Yersiniose/genética , Yersiniose/imunologia , Yersiniose/microbiologia
9.
Fish Shellfish Immunol ; 35(6): 1788-96, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24036332

RESUMO

The high conservation of the pituitary adenylate cyclase activating polypeptide (PACAP) sequence indicates that this peptide fulfills important biological functions in a broad spectrum of organisms. However, in invertebrates, little is known about its presence and its functions remain unclear. Up to now, in non-mammalian vertebrates, the majority of studies on PACAP have focused mainly on the localization, cloning and structural evolution of this peptide. As yet, little is known about its biological functions as growth factor and immunomodulator in lower vertebrates. Recently, we have shown that PACAP, apart from its neuroendocrine role, influences immune functions in larval and juvenile fish. In this work, we isolated for the first time the cDNA encoding the mature PACAP from a crustacean species, the white shrimp Litopenaeus vannamei, corroborating its high degree of sequence conservation, when compared to sequences reported from tunicates to mammalian vertebrates. Based on this, we have evaluated the effects of purified recombinant Clarias gariepinus PACAP administrated by immersion baths on white shrimp growth and immunity. We demonstrated that PACAP improves hemocyte count, superoxide dismutase, lectins and nitric oxide synthase derived metabolites in treated shrimp related with an increase in total protein concentration and growth performance. From our results, PACAP acts as a regulator of shrimp growth and immunity, suggesting that in crustaceans, as in vertebrate organisms, PACAP is an important molecule shared by both the endocrine and the immune systems.


Assuntos
Proteínas de Artrópodes/genética , Penaeidae/genética , Penaeidae/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/genética , Larva/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Dados de Sequência Molecular , Penaeidae/crescimento & desenvolvimento , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Reação em Cadeia da Polimerase/veterinária , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterinária
10.
Exp Parasitol ; 135(2): 188-99, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23850998

RESUMO

Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination could be an environmentally friendly alternative for sea lice control; however, research on the development of such vaccines is still at an early stage of development. Recent results have suggested that subolesin/akirin/my32 are good candidate antigens for the control of arthropod infestations, including sea lice, but background knowledge about these genes in crustaceans is limited. Herein, we characterize the my32 gene/protein from two important sea lice species, Caligus rogercresseyi and Lepeophtheirus salmonis, based on cDNA sequence isolation, phylogenetic relationships, three dimensional structure prediction and expression analysis. The results show that these genes/proteins have the main characteristics of akirins from invertebrates. In addition, immunization with purified recombinant my32 from L. salmonis elicited a specific antibody response in mice and fish. These results provide an improvement to our current knowledge about my32 proteins and their potential use as vaccine candidates against sea lice in fish.


Assuntos
Antígenos/imunologia , Copépodes/imunologia , Doenças dos Peixes/prevenção & controle , Salmo salar/parasitologia , Vacinas , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Antígenos/química , Antígenos/genética , Aquicultura , Sequência de Bases , Chile , Clonagem Molecular , Copépodes/química , Copépodes/genética , DNA Complementar/química , Feminino , Doenças dos Peixes/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Noruega , Filogenia , Conformação Proteica , RNA/genética , RNA/isolamento & purificação , Alinhamento de Sequência , Tilápia
11.
Fish Shellfish Immunol Rep ; 5: 100116, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37753327

RESUMO

Recent work has identified pituitary adenylate cyclase activating polypeptide (PACAP) as a potential antimicrobial and immune stimulating agent which may be suitable for use in aquaculture. However, its effects on teleost immunity are not well studied and may be significantly different than what has been observed in mammals. In this study we examined the effects of PACAP on the Atlantic salmon macrophage cell line SHK-1. PACAP was able to increase the expression of LPS-induced il-1ß in at concentrations of 1 uM when administered 24h prior to LPS stimulation. Furthermore, concentrations as low as 40nM had an effect when administered both 24h prior and in tandem with LPS. PACAP was also capable of increasing the expression of il-1ß and tnf-α in SHK-1 cells challenged with a low dose of heat-killed Flavobacterium columnare. We attempted to get a better understanding of the mechanism underlying this enhancement of il-1ß expression by manipulating downstream signaling of PACAP with inhibitors of phosphodiesterase and phospholipase C activity. We found that inducing cAMP accumulation with phosphodiesterase inhibitors failed to recapitulate the effect of PACAP administration on LPS-mediated il-1ß expression by PACAP, while use of a phospholipase C inhibitor caused a PACAP-like enhancement in LPS-mediated il-1ß expression. Interestingly, the VPAC1 receptor inhibitor PG97-269, but not the PAC1 inhibitor max.d.4, also was capable of causing a PACAP-like enhancement in LPS-mediated il-1ß expression. This suggests that fish do not utilize the PACAP receptors in the same manner as mammals, but that it still exerts an immunostimulatory effect that make it a good immunostimulant for use in aquaculture.

12.
Biomedicines ; 11(11)2023 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-38002050

RESUMO

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein.

13.
Antibiotics (Basel) ; 12(10)2023 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-37887185

RESUMO

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a multifunctional neuropeptide that is widely distributed and conserved across species. We have previously shown that in teleost fish, PACAP not only possesses direct antimicrobial properties but also immunomodulatory effects against the bacterial pathogens Flavobacterium psychrophilum and Pseudomonas aeruginosa using in vitro and in vivo experiments. These previous results suggest PACAP can be used as an alternative to antibiotics to prevent and/or treat bacterial infections in the aquaculture industry. To accomplish this goal, more studies are needed to better understand the effect of PACAP on pathogens affecting fish in live infections. In the present study, the transcripts PACAP, PRP/PACAP, and VPAC2 receptor were examined in rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri, which exhibited an increase in their expression in the spleen when compared to healthy fish. Synthetic Clarias gariepinus PACAP-38 has direct antimicrobial activity on Y. ruckeri and inhibits up to 60% of the bacterial growth when the peptide is at concentrations between 50 and 100 µM in TSB. The growth inhibition increased up to 90% in the presence of 12.5 µM of PACAP-38 when salt-free LB broth was used instead of TSB. It was also found to inhibit Y. ruckeri growth in a dose-dependent manner when the rainbow trout monocyte/macrophage-like cell line (RTS11) was pre-treated with lower concentrations of the peptide (0.02 and 0.1 µM) before going through infection. Differential gene expression was analyzed in this in vitro model. Overall, the results revealed new evidence to support the role of PACAP as an antimicrobial and immunomodulatory peptide treatment in teleosts.

14.
PLoS One ; 18(9): e0288006, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37751460

RESUMO

Despite that more than one hundred vaccines against SARS-CoV-2 have been developed and that some of them were evaluated in clinical trials, the latest results revealed that these vaccines still face great challenges. Among the components of the virus, the N-protein constitutes an attractive target for a subunit vaccine because it is the most abundant, highly conserved and immunogenic protein. In the present work, a chimeric protein (N-CD protein) was constructed by the fusion of the N-protein to the extracellular domain of human CD154 as the molecular adjuvant. HEK-293 cells were transduced with lentiviral vector bearing the N-CD gene and polyclonal cell populations were obtained. The N-CD protein was purified from cell culture supernatant and further characterized by several techniques. Immunogenicity studies in mice and non-human primates showed the N-CD protein induced high IgG titers in both models after two doses. Moreover, overall health monitoring of non-human primates demonstrated that animals were healthy during 228 days after first immunization. Data obtained support further investigation in order to develop this chimeric protein as vaccine candidate against COVID-19 and other coronavirus diseases.


Assuntos
COVID-19 , Vacinas , Humanos , Animais , Camundongos , SARS-CoV-2/genética , COVID-19/prevenção & controle , Células HEK293 , Vacinas contra COVID-19 , Nucleocapsídeo , Ligante de CD40/genética , Proteínas Recombinantes de Fusão/genética
15.
Fish Shellfish Immunol Rep ; 4: 100093, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37122444

RESUMO

Teleost IgT/Z plays a principal role in the defense mechanisms against infectious agents in the mucosal compartments and in systemic immunity. Previously, Nile tilapia (Oreochromis niloticus) IgT was discovered and characterized at transcription level. In this work, we generated a monoclonal antibody (mAb) that specifically recognized the Nile tilapia IgT. BALB/c mice were immunized with three synthetic peptides conjugated to KLH. The sequences of these peptides derived from the constant region of the Nile tilapia IgT heavy chain. ELISA and Western blotting confirmed the specificity of the polyclonal sera and the culture supernatant from a positive hybridoma clone. We observed immunoreactivity against a recombinant IgT fragment and native IgT in skin mucus. The anti-IgT mAb did not cross-react with purified tilapia IgM. Direct ELISA analysis allowed the quantification of skin mucus IgM and IgT concentrations. Flow cytometry analysis revealed differences in the percentage of IgT+ B cell populations between juveniles and adults in peripheral blood, head kidney and spleen lymphocytes and among the tissues analyzed. For further validation of the anti-IgT mAb utility, a recombinant vaccine candidate against sea lice (TT-P0 Ls) was injected into juvenile tilapia. Direct ELISA results revealed a differential secretion of skin mucus IgT and IgM after immunostimulation. In addition, the percentages of IgT+ B cells were determined at 7 days after booster and ex-vivo stimulation by flow cytometry. This mAb constitutes an important immunological tool to study the biological function and structural characteristics of tilapia IgT.

16.
Vaccines (Basel) ; 10(6)2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35746505

RESUMO

COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host's cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose.

17.
Nitric Oxide ; 25(4): 396-406, 2011 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-21971212

RESUMO

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In a previous study, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide (LPS) in vitro. In the present work, lobster hemocytes and gills exposed to Escherichia coli O55:B5 LPS showed an increase in both NOS activity and NOS gene expression in vivo. This response was dose and time dependent. The 3D NOS structure was predicted by comparative modeling showing the oxygenase and reductase domains. These domains contain the conserved binding motifs of NOS already found in a variety of organisms. The 3D structure prediction analysis allowed the selection of a fragment of 666bp that was cloned and subsequently expressed in E. coli BL21, in which a recombinant product of around 31KDa was obtained. Hyperimmune serum obtained from immunized rabbits was tested and employed to specifically detect the recombinant polypeptide or the endogenous NOS from lobster hemocytes by western blot and immunofluorescence. This study contributes to enlarge the existing knowledge related to NOS structure and NOS participation in the immune response in lobsters. The evaluation of an antibody capable to recognize NOS from lobsters constitutes a novel and interesting tool for the implementation of further studies on NOS functions in crustaceans.


Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase/metabolismo , Palinuridae/enzimologia , Palinuridae/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , Relação Dose-Resposta Imunológica , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Imunofluorescência , Brânquias/citologia , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Hemócitos/citologia , Hemócitos/efeitos dos fármacos , Hemócitos/enzimologia , Soros Imunes , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/imunologia , Palinuridae/genética , Conformação Proteica , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo
18.
Fish Shellfish Immunol ; 29(3): 513-20, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20510368

RESUMO

To date, published in-vivo studies on the action of the PACAP in fish are few and these are concerned with reproduction, brain development and feeding behavior. Recently, we demonstrated for the first time that PACAP, apart from its neuroendocrine role, influences immune functions in fish larvae. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on important immune parameters in juvenile fish. We observed that a single injection of the recombinant peptide (0.1 microg per g of body weight) was able to increase the nitric oxide synthase-derived metabolites (NOS) and total immunoglobulin M (IgM) concentration in serum of juvenile catfish C. gariepinus and tilapia Orechromis niloticus respectively, after 24 h of its administration. In addition, our results showed that recombinant PACAP increases IgM, NOS and lysozyme in serum correlated with its ability to enhance growth performance in juvenile fish. Finally, the PACAP mRNA expression and PACAP immunoreactivity detected in peripheral blood leucocytes from juvenile catfish suggest a direct autocrine or/and paracrine mechanism of regulation of this peptide to mediate immune functions in fish.


Assuntos
Peixes-Gato/imunologia , Sistema Imunitário/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Peso Corporal , Peixes-Gato/sangue , Peixes-Gato/crescimento & desenvolvimento , Ciclídeos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Crescimento/efeitos dos fármacos , Imunoglobulina M/sangue , Leucócitos/imunologia , Tecido Linfoide/imunologia , Muramidase/sangue , Óxido Nítrico Sintase/sangue , Óxido Nítrico Sintase/metabolismo , Proteínas Recombinantes/farmacologia
19.
Fish Shellfish Immunol ; 29(3): 469-79, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20580828

RESUMO

Nitric oxide (NO) is a free radical gas involved in a variety of physiological processes in invertebrates, such as neuromodulation, muscle contraction and host defense. Surprisingly, little is known about the involvement of NO synthase (NOS) in the immune system of crustaceans. This work is focused on the study of the NOS gene of the spiny lobster Panulirus argus, a crustacean with commercial interest, and its relationship with the immune response to a microbial elicitor. A NOS full-length DNA was isolated from hemocytes by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers. The open reading frame (ORF) encodes a protein of 1200 amino acids, with an estimated molecular mass of 135.9 kDa, which contains the conserved domains and binding motifs of NOS found in a variety of organisms. NOS gene expression in lobster gills, heart, stomach, digestive gland, abdominal muscle, gut and hemocytes was studied by Real Time quantitative PCR (Real Time qPCR). The expression was higher in hemocytes, heart and gills. In addition, when lobster hemocytes were exposed in vitro to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase in the NOS activity and also in the NOS gene expression evaluated by Real Time qPCR was observed, thus demonstrating the presence of an inducible crustacean NOS by a microbial elicitor of the immune response. The information is relevant in providing basic knowledge for further studies of crustacean defense mechanisms.


Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/imunologia , Palinuridae/enzimologia , Palinuridae/genética , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Hemócitos/enzimologia , Hemócitos/imunologia , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Palinuridae/classificação , Palinuridae/imunologia , Alinhamento de Sequência
20.
PLoS One ; 15(10): e0239827, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33006991

RESUMO

Infection with parasitic copepod salmon louse Lepeophtheirus salmonis, represents one of the most important limitations to sustainable Atlantic salmon (Salmo salar L.) farming today in the North Atlantic region. The parasite exerts negative impact on health, growth and welfare of farmed fish as well as impact on wild salmonid populations. It is therefore central to ensure continuous low level of salmon lice with the least possible handling of the salmon and drug use. To address this, vaccination is a cost-effective and environmentally friendly control approach. In this study, efficacy of a vaccine candidate, containing a peptide derived from ribosomal protein P0, was validated post infestation with L. salmonis, at the lab-scale. The sampling results showed good potential of the vaccine candidate when administered intraperitoneally in the host, in reducing the ectoparasite load, through reduction of adult female lice counts and fecundity and with greater presumptive effect in F1 lice generation. The sampling results correlated well with the differential modulation of pro-inflammatory, Th1, Th2 and T regulatory mediators at the transcript level at different lice stages. Overall, the results supports approximately 56% efficacy when administered by intraperitoneal injection. However, additional validation is necessary under large-scale laboratory trial for further application under field conditions.


Assuntos
Copépodes/imunologia , Doenças dos Peixes/prevenção & controle , Proteínas Ribossômicas/imunologia , Salmo salar/imunologia , Vacinas/uso terapêutico , Animais , Doenças dos Peixes/parasitologia , Interações Hospedeiro-Parasita , Vacinação/veterinária
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