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Salmon farming may face stress due to the intensive culture conditions with negative impacts on overall performance. In this aspect, functional feed improves not only the basic nutritional requirements but also the health status and fish growth. However, to date no studies have been carried out to evaluate the effect of functional diets in salmon subjected to crowding stress. Thus, the aim of this study was to evaluate the effect of yeast extract (Xanthophyllomyces dendrorhous; diet A) and the combination of plant extracts (common Saint John's wort, lemon balm, and rosemary; diet B) on the antioxidant and immune status of Atlantic salmon grown under normal cultured conditions and then subjected to crowding stress. Fish were fed with functional diets during 30 days (12â¯kg/m3) and then subjected to crowding stress (20â¯kg/m3) for 10 days. The lipid peroxidation in gut showed that both diets induced a marked decrease on oxidative damage when fish were subjected to crowding stress. The protein carbonylation in muscle displayed at day 30 a marked decrease in both functional diets that was more marked on the stress condition. The expression of immune markers (IFNγ, CD4, IL-10, TGF-ß, IgMmb, IgMsec, T-Bet, and GATA-3) indicated the upregulation of those associated to humoral-like response (CD4, IL-10, GATA-3) when fish were subjected to crowding stress. These results were confirmed with the expression of secreted IgM. Altogether, these functional diets improved the antioxidant status and increased the expression of genes related to Th2-like response suggesting a protective role on fish subjected to crowding stress.
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Basidiomycota/química , Aglomeração , Hypericum/química , Melissa/química , Rosmarinus/química , Salmo salar/fisiologia , Ração Animal/análise , Animais , Antioxidantes/metabolismo , Dieta/veterinária , Suplementos Nutricionais/análise , Imunidade Inata/efeitos dos fármacos , Extratos Vegetais/química , Estresse FisiológicoRESUMO
This study reports the isolation and functional characterization of rainbow trout (Oncorhynchus mykiss) CD4-1(+) T cells and the establishment of an IL-15-dependent CD4-1(+) T cell line. By using Abs specific for CD4-1 and CD3ε it was possible to isolate the double-positive T cells in spleen and head kidney. The morphology and the presence of transcripts for T cell markers in the sorted CD4-1(+)CD3ε(+) cells were studied next. Cells were found to express TCRα, TCRß, CD152 (CTLA-4), CD154 (CD40L), T-bet, GATA-3, and STAT-1. The sorted CD4-1(+) T cells also had a distinctive functional attribute of mammalian T lymphocytes, namely they could undergo Ag-specific proliferation, using OVA as a model Ag. The OVA-stimulated cells showed increased expression of several cytokines, including IFN-γ1, IL-4/13A, IL-15, IL-17D, IL-10, and TGF-ß1, perhaps indicating that T cell proliferation led to differentiation into distinct effector phenotypes. Using IL-15 as a growth factor, we have selected a lymphoid cell line derived from rainbow trout head kidney cells. The morphology, cell surface expression of CD4-1, and the presence of transcripts of T cell cytokines and transcription factors indicated that this is a CD4-1(+) T cell line. To our knowledge, this is the first demonstration of the presence of CD4-1(+)CD3ε(+) T cells in salmonids. As in mammals, CD4-1(+) T cells may be the master regulators of immune responses in fish, and therefore these findings and the new model T cell line developed will contribute to a greater understanding of T cell function and immune responses in teleost fish.
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Linfócitos T CD4-Positivos/fisiologia , Rim Cefálico/imunologia , Oncorhynchus mykiss/imunologia , Baço/imunologia , Animais , Complexo CD3/imunologia , Complexo CD3/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Proliferação de Células , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Interleucina-15/metabolismo , Ativação Linfocitária , Mamíferos/imunologia , Filogenia , Receptores de Antígenos de Linfócitos T/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
Infectious Pancreatic Necrosis Virus (IPNV) is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species but also persistent infection in surviving fish after outbreaks. Because viral persistence of susceptible mammal hosts appears to be associated with the modulation of anti-inflammatory cytokine expression, in this study we examined the expression levels of key pro- and anti-inflammatory cytokines in kidney and spleen of trout, as well as humoral immune response (IgM and IgT) during experimental persistent viral infection and in the acute phase of infection as a comparison. IPNV infection in rainbow trout resulted in a distinct profile of cytokine expression depending on the type of infection, acute or persistent. Levels of early pro-inflammatory cytokines, IL-1ß and IL-8, did not increase in the head kidney of the fish with persistent asymptomatic infection but increased in some of the symptomatic infected fish. The antiviral cytokine IFNα was not significantly induced in any of the infected fish groups. The level of expression of the Th1-related cytokine IL-12 was significantly higher in trout with persistent asymptomatic infection than in symptomatic fish. This was also accompanied by an increase in IFNγ. The anti-inflammatory cytokines IL-10 and TGF-ß1 had distinct expression profiles. While IL-10 expression increased in all infected fish, TGF-ß1 was only up-regulated in fish with persistent infection. All infected fish had significantly lower total IgM levels than the non-infected fish whereas IgT levels did not change. Specific and neutralizing antibodies against IPNV were not observed in acute and persistent infection except in the group of fish with the lowest degree of clinical signs. Interestingly, the lack of humoral immune response could be associated with the high expression of anti-inflammatory cytokines, which might inhibit antibody production. The balance between pro-inflammatory Th1 type cytokines and the regulatory cytokines could explain the high percentage of survival and the resolution of the inflammatory response in the IPNV-infected fish but also the establishment of viral persistence.
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Infecções por Birnaviridae/veterinária , Citocinas/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Vírus da Necrose Pancreática Infecciosa/imunologia , Mediadores da Inflamação/metabolismo , Oncorhynchus mykiss , Animais , Infecções por Birnaviridae/imunologia , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Estimativa de Kaplan-Meier , Rim/metabolismo , Testes de Neutralização , Reação em Cadeia da Polimerase em Tempo Real , Baço/metabolismoRESUMO
Background: As the COVID-19 pandemic persists, infections continue to surge globally. Presently, the most effective strategies to curb the disease and prevent outbreaks involve fostering immunity, promptly identifying positive cases, and ensuring their timely isolation. Notably, there are instances where the SARS-CoV-2 virus remains infectious even after patients have completed their quarantine. Objective: Understanding viral persistence post-quarantine is crucial as it could account for localized infection outbreaks. Therefore, studying and documenting such instances is vital for shaping future public health policies. Design: This study delves into a unique case of SARS-CoV-2 persistence in a 60-year-old female healthcare worker with a medical history of hypertension and hypothyroidism. The research spans 55 days, marking the duration between her initial and subsequent diagnosis during Chile's first COVID-19 wave, with the analysis conducted using RT-qPCR. Results: Genomic sequencing-based phylogenetic analysis revealed that the SARS-CoV-2 detected in both Nasopharyngeal swab samples (NPSs) was consistent with the 20B clade of the Nextstrain classification, even after a 55-day interval. Conclusion: This research underscores the need for heightened vigilance concerning cases of viral persistence. Such instances, albeit rare, might be pivotal in understanding sporadic infection outbreaks that occur post-quarantine.
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Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world. N. gonorrhoeae does not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. N. gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whether N. gonorrhoeae directly conditions the phenotype of RAW 264.7 murine macrophage cell line and its response. We established that gonococcus was effectively phagocytosed by the RAW 264.7 cells and upregulates production of immunoregulatory cytokines (IL-10 and TGF- ß 1) but not the production of proinflammatory cytokine TNF- α , indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover, N. gonorrhoeae did not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed that N. gonorrhoeae infected macrophage cell line fails to elicit proliferative CD4+ response. This implies that macrophage that can phagocytose gonococcus do not display proper antigen-presenting functions. These results indicate that N. gonorrhoeae induces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Neisseria gonorrhoeae/patogenicidade , Animais , Linfócitos B/imunologia , Antígeno B7-2/metabolismo , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Gonorreia/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Sistema Imunitário , Interleucina-10/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Fenótipo , Fator de Crescimento Transformador beta1/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: The respiratory microbiome is dynamic, varying between anatomical niches, and it is affected by various host and environmental factors, one of which is lifestyle. Few studies have characterized the upper respiratory tract microbiome profile according to lifestyle. We explored the association between lifestyles and microbiota profiles in the upper respiratory tract of healthy adults. METHODS: We analyzed nasal samples from 110 healthy adults who were living in Santiago, Chile, using 16S ribosomal RNA gene-sequencing methods. Volunteers completed a structured questionnaire about lifestyle. RESULTS: The composition and abundance of taxonomic groups varied across lifestyle attributes. Additionally, multivariate models suggested that alpha diversity varied in the function of physical activity, nutritional status, smoking, and the interaction between nutritional status and smoking, although the significant impact of those variables varied between women and men. Although physical activity and nutritional status were significantly associated with all indexes of alpha diversity among women, the diversity of microbiota among men was associated with smoking and the interaction between nutritional status and smoking. CONCLUSIONS: The alpha diversity of nasal microbiota is associated with lifestyle attributes, but these associations depend on sex and nutritional status. Our results suggest that future studies of the airway microbiome may provide a better resolution if data are stratified for differences in sex and nutritional status.
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Piscirickettsia salmonis is the most important health problem facing Chilean Aquaculture. Previous reports suggest that P. salmonis can survive in salmonid macrophages by interfering with the host immune response. However, the relevant aspects of the molecular pathogenesis of P. salmonis have been poorly characterized. In this work, we evaluated the transcriptomic changes in macrophage-like cell line SHK-1 infected with P. salmonis at 24- and 48-hours post-infection (hpi) and generated network models of the macrophage response to the infection using co-expression analysis and regulatory transcription factor-target gene information. Transcriptomic analysis showed that 635 genes were differentially expressed after 24- and/or 48-hpi. The pattern of expression of these genes was analyzed by weighted co-expression network analysis (WGCNA), which classified genes into 4 modules of expression, comprising early responses to the bacterium. Induced genes included genes involved in metabolism and cell differentiation, intracellular transportation, and cytoskeleton reorganization, while repressed genes included genes involved in extracellular matrix organization and RNA metabolism. To understand how these expression changes are orchestrated and to pinpoint relevant transcription factors (TFs) controlling the response, we established a curated database of TF-target gene regulatory interactions in Salmo salar, SalSaDB. Using this resource, together with co-expression module data, we generated infection context-specific networks that were analyzed to determine highly connected TF nodes. We found that the most connected TF of the 24- and 48-hpi response networks is KLF17, an ortholog of the KLF4 TF involved in the polarization of macrophages to an M2-phenotype in mammals. Interestingly, while KLF17 is induced by P. salmonis infection, other TFs, such as NOTCH3 and NFATC1, whose orthologs in mammals are related to M1-like macrophages, are repressed. In sum, our results suggest the induction of early regulatory events associated with an M2-like phenotype of macrophages that drives effectors related to the lysosome, RNA metabolism, cytoskeleton organization, and extracellular matrix remodeling. Moreover, the M1-like response seems delayed in generating an effective response, suggesting a polarization towards M2-like macrophages that allows the survival of P. salmonis. This work also contributes to SalSaDB, a curated database of TF-target gene interactions that is freely available for the Atlantic salmon community.
Assuntos
Salmo salar , Animais , Salmo salar/genética , Perfilação da Expressão Gênica , Macrófagos/metabolismo , Fatores de Transcrição/metabolismo , RNA/metabolismo , MamíferosRESUMO
Reactive oxygen species (ROSs) affect several macromolecules and cellular components in eukaryotic and prokaryotic cells. In this work, the effect of various ROS-generating compounds on the Escherichia coli membrane was studied. Membrane fatty acid profiles, oxidative damage levels and bacterial resistance to these toxicants were determined. Studies included wild-type cells as well as a strain exhibiting a modified monounsaturated fatty acid (MUFA) profile (accomplished by overexpressing the ß-hydroxyacyl acyl carrier protein dehydratase-encoding gene, fabA). Levels of membrane MUFAs and oxidative damage markers decreased slightly upon toxicant exposure with a concomitant increase in cell resistance to these ROS-generating compounds. A direct relationship between MUFAs and lipid peroxidation was observed. The lower the MUFA the lower the peroxide levels, suggesting that MUFAs are targets for membrane lipid oxidation.
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Escherichia coli/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Hidroliases/metabolismo , Peroxidação de Lipídeos , Carbonilação ProteicaRESUMO
IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1ß, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1ß and IL-8 did not rise in the spleen although an increase of IL-1ß, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1ß and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1ß and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion.
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Infecções por Birnaviridae/veterinária , Citocinas/metabolismo , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica , Vírus da Necrose Pancreática Infecciosa/fisiologia , Salmo salar , Animais , Infecções por Birnaviridae/imunologia , Linhagem Celular , Doenças dos Peixes/virologia , Perfilação da Expressão Gênica , Brânquias/imunologia , Rim Cefálico/imunologiaRESUMO
Anthropogenic pollution has a huge impact on the water quality of marine ecosystems. Heavy metals and antibiotics are anthropogenic stressors that have a major effect on the health of the marine organisms. Although heavy metals are also associate with volcanic eruptions, wind erosion or evaporation, most of them come from industrial and urban waste. Such contamination, coupled to the use and subsequent misuse of antimicrobials in aquatic environments, is an important stress factor capable of affecting the marine communities in the ecosystem. Bivalves are important ecological components of the oceanic environments and can bioaccumulate pollutants during their feeding through water filtration, acting as environmental sentinels. However, heavy metals and antibiotics pollution can affect several of their physiologic and immunological processes, including their microbiome. In fact, heavy metals and antibiotics have the potential to select resistance genes in bacteria, including those that are part of the microbiota of bivalves, such as Vibrio spp. Worryingly, antibiotic-resistant phenotypes have been shown to be more tolerant to heavy metals, and vice versa, which probably occurs through co- and cross-resistance pathways. In this regard, a crucial role of heavy metal resistance genes in the spread of mobile element-mediated antibiotic resistance has been suggested. Thus, it might be expected that antibiotic resistance of Vibrio spp. associated with bivalves would be higher in contaminated environments. In this review, we focused on co-occurrence of heavy metal and antibiotic resistance in Vibrio spp. In addition, we explore the Chilean situation with respect to the contaminants described above, focusing on the main bivalves-producing region for human consumption, considering bivalves as potential vehicles of antibiotic resistance genes to humans through the ingestion of contaminated seafood.
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Bivalves , Metais Pesados , Microbiota , Agricultura , Animais , Antibacterianos/farmacologia , Bactérias/genética , ChileRESUMO
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many countries have reported the experience of at least two contagion waves, describing associated mortality rates and population behavior. The analysis of the effect of this pandemic in different localities can provide valuable information on the key factors to consider in the face of future massive infectious diseases. This work describes the first retrospective and comparative study about behavior during the first and second waves of the COVID-19 pandemic in Chile from a primary Healthcare Center. From 19,313 real-time quantitative PCR (RT-qPCR) tests assessed, the selected 1,694 positive diagnostics showed a decrease in mortality rate in the second wave (0.6%) compared with the first (4.6%). In addition, we observed that infections in the second wave were mainly in young patients with reduced comorbidities. The population with a complete vaccination schedule shows a decrease in the duration of symptoms related to the disease, and patients with more comorbidities tend to develop severe illness. This report provides evidence to partially understand the behavior and critical factors in the severity of the COVID-19 pandemic in the population of Santiago of Chile.
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COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , Chile/epidemiologia , Humanos , Estudos Longitudinais , Pandemias , Atenção Primária à Saúde , Estudos RetrospectivosRESUMO
The identification and tracking of SARS-CoV-2 infected patients in the general population are essential components of the global strategy to limit the COVID-19 viral spread, specifically for maintaining traceability and suppressing the resurgence of local outbreaks. Public health programs that include continuous RT-qPCR testing for COVID-19 in the general population, viral sequencing, and genomic surveillance for highly contagious forms of the virus have allowed for the identification of SARS-CoV-2 infections and reinfections. This work identified SARS-CoV-2 reinfection in a homeless person, which occurred 58 days after the first COVID-19 diagnosis. Genomic sequencing identified a different Nextstrain classification clade (20A and 20B) and PANGO lineage, with a divergence of 4 single nucleotide variants (SNVs) in S and ORF1ab genes, suggesting reinfection by different viral variants. This study is the first from the great metropolitan area of Santiago, Chile, one of the top ten countries in the world to live during the COVID-19 pandemic. We support the importance of performing intensive genomic surveillance programs in the whole population and high-risk groups, such as homeless people, nearly 20 thousand people in Chile, and have limited access to health care services and poor viral traceability.
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COVID-19 , Pessoas Mal Alojadas , COVID-19/epidemiologia , Teste para COVID-19 , Chile/epidemiologia , Humanos , Pandemias , Reinfecção , SARS-CoV-2/genéticaRESUMO
A key characteristic of Human immunodeficiency virus type 1 (HIV-1) infection is the generation of latent viral reservoirs, which have been associated with chronic immune activation and sustained inflammation. Macrophages play a protagonist role in this context since they are persistently infected while being a major effector of the innate immune response through the generation of type-I interferons (type I IFN) and IFN-stimulated genes (ISGs). The balance in the IFN signaling and the ISG induction is critical to promote a successful HIV-1 infection. Classically, the IFNs response is fine-tuned by opposing promotive and suppressive signals. In this context, it was described that HIV-1-infected macrophages can also synthesize some antiviral effector ISGs and, positive and negative regulators of the IFN/ISG signaling. Recently, epitranscriptomic regulatory mechanisms were described, being the N6-methylation (m6A) modification on mRNAs one of the most relevant. The epitranscriptomic regulation can affect not only IFN/ISG signaling, but also type I IFN expression, and viral fitness through modifications to HIV-1 RNA. Thus, the establishment of replication-competent latent HIV-1 infected macrophages may be due to non-classical mechanisms of type I IFN that modulate the activation of the IFN/ISG signaling network.
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Infecções por HIV/metabolismo , Interferon Tipo I/metabolismo , Interferons/metabolismo , Macrófagos/metabolismo , Latência Viral/fisiologia , Animais , Infecções por HIV/virologia , Humanos , Transdução de Sinais/fisiologiaRESUMO
Piscirickettsia salmonis, the etiological agent of the Salmon Rickettsial Septicemia (SRS), is one the most serious health problems for the Chilean salmon industry. Typical antimicrobial strategies used against P. salmonis include antibiotics and vaccines, but these applications have largely failed. A few years ago, the first attenuated-live vaccine against SRS (ALPHA JECT LiVac® SRS vaccine) was released to the market. However, there is no data about the agents involved in the activation of the immune response induced under field conditions. Therefore, in this study we evaluated the expression profile of a set of gene markers related to innate and adaptive immunity in the context of a cellular response in Atlantic salmon (Salmo salar) reared under productive farm conditions and immunized with a live-attenuated vaccine against P. salmonis. We analyzed the expression at zero, 5-, 15- and 45-days post-vaccination (dpv). Our results reveal that the administration of the attenuated live SRS LiVac vaccine induces a short-term upregulation of the cellular-mediated immune response at 5 dpv modulated by the upregulation of ifnα, ifnγ, and the cd4 and cd8α T cell surface markers. In addition, we also registered the upregulation of il-10 and tgfß. Altogether, the results suggest that a balanced activation of the immune response took place only at early times post-vaccination (5 dpv). The scope of this short-term upregulation of the cellular-mediated immune response against a natural outbreak in fish subjected to productive farm conditions deserves further research.
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Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.
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Anticorpos Antibacterianos/imunologia , Doenças dos Peixes/imunologia , Lisossomos/imunologia , Macrófagos/imunologia , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/imunologia , Salmão/imunologia , Animais , Doenças dos Peixes/microbiologia , Lisossomos/microbiologia , Macrófagos/microbiologia , Infecções por Piscirickettsiaceae/veterinária , Salmão/microbiologiaRESUMO
Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis is able to survive in host macrophages, localized within a vacuole like-compartment which prevents lysosomal degradation. However, the relevant aspects of the pathogenesis of P. salmonis as the host modulation that allow its intracellular survival have been poorly characterized. In this study, we evaluated the role of lysosomes in the response to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A gentamicin protection assay was performed to recover intracellular bacteria and the 16S rDNA copy number was quantified through quantitative polymerase chain reaction in order to determine the replication of P. salmonis within macrophages. Lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon macrophage-enriched cell cultures, accompanied by an increase in the detection of the 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and replication appears to be favored by a perturbation of the lysosomal degradation system. We observed a modulation in the total number of lysosomes and lysosomal acidification following infection with P. salmonis. Collectively, the results of this study showed that infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal response which may be associated with host immune evasion mechanisms of P. salmonis that have not been previously reported.
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Doenças dos Peixes/imunologia , Macrófagos/imunologia , Piscirickettsia , Infecções por Piscirickettsiaceae/imunologia , Salmo salar/imunologia , Animais , Células Cultivadas , DNA Ribossômico , Rim Cefálico/citologia , Rim Cefálico/imunologia , Lisossomos/imunologia , Macrófagos/microbiologia , Piscirickettsia/genética , Infecções por Piscirickettsiaceae/veterináriaRESUMO
Single-nucleotide polymorphisms (SNPs) are single genetic code variations considered one of the most common forms of nucleotide modifications. Such SNPs can be located in genes associated to immune response and, therefore, they may have direct implications over the phenotype of susceptibility to infections affecting the productive sector. In this study, a set of immune-related genes (cc motif chemokine 19 precursor [ccl19], integrin ß2 (itß2, also named cd18), glutathione transferase omega-1 [gsto-1], heat shock 70 KDa protein [hsp70], major histocompatibility complex class I [mhc-I]) were analyzed to identify SNPs by data mining. These genes were chosen based on their previously reported expression on infectious pancreatic necrosis virus (IPNV)-infected Atlantic salmon phenotype. The available EST sequences for these genes were obtained from the Unigene database. Twenty-eight SNPs were found in the genes evaluated and identified most of them as transition base changes. The effect of the SNPs located on the 5'-untranslated region (UTR) or 3'-UTR upon transcription factor binding sites and alternative splicing regulatory motifs was assessed and ranked with a low-medium predicted FASTSNP score risk. Synonymous SNPs were found on itß2 (c.2275G > A), gsto-1 (c.558G > A), and hsp70 (c.1950C > T) with low FASTSNP predicted score risk. The difference in the relative synonymous codon usage (RSCU) value between the variant codons and the wild-type codon (ΔRSCU) showed one negative (hsp70 c.1950C > T) and two positive ΔRSCU values (itß2 c.2275G > A; gsto-1 c.558G > A), suggesting that these synonymous SNPs (sSNPs) may be associated to differences in the local rate of elongation. Nonsynonymous SNPs (nsSNPs) in the gsto-1 translatable gene region were ranked, using SIFT and POLYPHEN web-tools, with the second highest (c.205A > G; c484T > C) and the highest (c.499T > C; c.769A > C) predicted score risk possible. Using homology modeling to predict the effect of these nonsynonymous SNPs, the most relevant nucleotide changes for gsto-1 were observed for the nsSNPs c.205A > G, c484T > C, and c.769A > C. Molecular dynamics was assessed to analyze if these GSTO-1 variants have significant differences in their conformational dynamics, suggesting these SNPs could have allosteric effects modulating its catalysis. Altogether, these results suggest that candidate SNPs identified may play a crucial potential role in the immune response of Atlantic salmon.
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Neisseria gonorrhoeae is a gram-negative diplococcus that in human beings produces gonorrhea. Much clinical evidence has led to the conclusion that gonococcus has important mechanisms to evade host immune functions; however, these mechanisms are only now beginning to be elucidated. In this study, we determined that the BALB/c mouse is a good animal model to study gonococcus infection and examined the immune response against the bacteria. We determined that after intravaginal inoculation of mice with Neisseria gonorrhoeae, the bacteria reached and invaded the upper female reproductive tissues and elicited a T-cell-specific immune response associated with a very weak humoral response, altogether resembling gonococcus infection and disease in women. Remarkably, in the draining lymph nodes of the genital tracts of infected mice, we found an increase of regulatory T lymphocytes, namely, transforming growth factor beta1-positive CD4(+) T cells and CD4(+) CD25(+) Foxp3(+) T cells. Altogether, results indicate that N. gonorrhoeae induces regulatory T cells, which might be related to the local survival of the pathogen and the establishment of a chronic asymptomatic infection.
Assuntos
Gonorreia/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Gonorreia/veterinária , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Neisseria gonorrhoeae , Fator de Crescimento Transformador beta1/biossínteseRESUMO
Infectious salmon anemia virus (ISAV) has caused great losses to the Chilean salmon industry, and the success of prevention and treatment strategies is uncertain. The use of RNA interference (RNAi) is a promising approach because during the replication cycle, the ISAV genome must be transcribed to mRNA in the cytoplasm. We explored the capacity of E. coli transformed with plasmids that produce double-stranded RNA (dsRNA) to induce antiviral activity when added to infected ASK cells. We transformed the non-pathogenic Escherichia coli HT115 (DE3) with plasmids that expressed highly conserved regions of the ISAV genes encoding the nucleoprotein (NP), fusion (F), hemagglutinin (HE), and matrix (M) proteins as dsRNA, which is the precursor of the RNAi mechanism. The inactivated transformed bacteria carrying dsRNA were tested for their capacity to silence the target ISAV genes, and the dsRNA that were able to inhibit gene expression were subsequently tested for their ability to attenuate the cytopathic effect (CPE) and reduce the viral load. Of the four target genes tested, inactivated E. coli transformed with plasmids producing dsRNA targeting HE showed antiviral activity when added to infected ASK cells.
RESUMO
This study aims to identify at the expression level the immune-related genes associated with IPN-susceptible and resistant phenotypes in Atlantic salmon full-sibling families. We have analyzed thirty full-sibling families infected by immersion with IPNV and then classified as resistant or susceptible using a multivariate survival analysis based on a gamma-Cox frailty model and the Kaplan-Meier mortality curves. In four families within each group head kidneys were pooled for real-time PCR and one-color salmon-specific oligonucleotide microarray (21K) analysis at day 1 and 5 post-infection. Transcripts involved in innate response (IL-6, IFN-α), antigen presentation (HSP-70, HSP-90, MHC-I), TH1 response (IL-12, IFN-γ, CRFB6), immunosuppression (IL-10, TGF-ß1) and leukocyte activation and migration (CCL-19, CD18) showed a differential expression pattern between both phenotypes, except in IL-6. In susceptible families, except for IFN-γ, the expressions dropped to basal values at day 5 post-infection. In resistant families, unlike susceptible families, levels remained high or increased (except for IL-6) at day 5. Transcriptomic analysis showed that both families have a clear differential expression pattern, resulting in a marked down-regulation in immune related genes involved in innate response, complement system, antigen recognition and activation of immune response in IPN-resistant. Down-regulation of genes, mainly related to tissue differentiation and protein degradation metabolism, was also observed in resistant families. We have identified an immune-related gene patterns associated with susceptibility and resistance to IPNV infection of Atlantic salmon. This suggests that a limited immune response is associated with resistant fish phenotype to IPNV challenge while a highly inflammatory but short response is associated with susceptibility.