RESUMEN
In this study, we isolated mesenchymal stromal (stem) cells (MSCs) from broncho-alveolar lavage fluid (BAL) of 2-6-week-old commercial pigs. BAL-MSCs displayed fibroblastic morphology and possessed self-renewal properties. Similar to bone-marrow MSCs, BAL-MSCs expressed mesenchymal markers and both cell types lacked the expression of hematopoetic markers. BAL-MSCs, when cultured in differentiation induction media, differentiated into adipocytes, osteocytes, and chondrocytes. Next, we examined if BAL-MSCs have the ability to treat lipopolysaccharide (LPS)-induced acute lung injury (ALI) in a pig model. Five-week-old commercial pigs were inoculated intra-tracheally with E. coli LPS (1 mg/kg body weight [b.wt.]). Twelve hours after the LPS inoculation, groups of pigs were inoculated intra-tracheally with BM-MSCs or BAL-MSCs (2 × 106 cells/kg b.wt.). Forty eight hours after the cells administration pigs were euthanized and neutrophils in BAL, lung lesions, and cytokines in lung lysates, and engraftment of MSCs in lungs were examined. Engraftment of BAL-MSCs in injured lungs was significantly higher than the BM-MSCs, however, both cell types were equally effective in attenuating LPS-induced ALI as evidenced by decreased inflammation, lung lesions, and proinflammatory cytokines in the lungs of pigs treated with BAL- or BM-MSCs. These data in a preclinical large animal model suggest that BAL-MSCs may be used in clinical settings to treat ALI in humans.
Asunto(s)
Lesión Pulmonar Aguda/terapia , Líquido del Lavado Bronquioalveolar/citología , Inflamación/terapia , Trasplante de Células Madre Mesenquimatosas , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Líquido del Lavado Bronquioalveolar/química , Diferenciación Celular/genética , Autorrenovación de las Células/efectos de los fármacos , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/patología , Lipopolisacáridos/toxicidad , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Neutrófilos/citología , PorcinosRESUMEN
We isolated stem/progenitor epithelial cells from the lungs of 4- to 6-week-old pigs. The epithelial progenitor colony cells were surrounded by mesenchymal stromal cells. The progenitor epithelial colony cells expressed stem cell markers such as octamer binding transcription factor 4 (Oct4) and stage-specific embryonic antigen 1 (SSEA-1), as well as the epithelial markers pancytokeratin, cytokeratin-18, and occludin, but not mesenchymal (CD44, CD29, and CD90) and hematopoietic (CD45) markers. The colony cells had extensive self-renewal potential and had the capacity to undergo differentiation to alveolar type I- and type II-like pneumocytes. Additionally, these cells expressed sialic acid receptors and supported the active replication of influenza virus, which was accompanied by cell lysis. The lysis of progenitor epithelial cells by influenza virus may cause a marked reduction in the potential of progenitor cells for self renewal and for their ability to differentiate into specialized cells of the lung. These observations suggest the possible involvement of lung stem/progenitor cells in influenza virus infection.
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Células Epiteliales/metabolismo , Virus de la Influenza A/fisiología , Gripe Humana/virología , Pulmón/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre/metabolismo , Replicación Viral , Animales , Diferenciación Celular , Células Epiteliales/citología , Células Epiteliales/virología , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Gripe Humana/metabolismo , Pulmón/citología , Pulmón/virología , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre/citología , Células Madre/virología , PorcinosRESUMEN
BACKGROUND: The genetic features that facilitate Campylobacter jejuni's adaptation to a wide range of environments are not completely defined. However, whole genome expression studies showed that respiratory proteins (RPs) were differentially expressed under varying conditions and stresses, suggesting further unidentified roles for RPs in C. jejuni's adaptation. Therefore, our objectives were to characterize the contributions of selected RPs to C. jejuni's i- key survival phenotypes under different temperature (37°C vs. 42°C) and oxygen (microaerobic, ambient, and oxygen-limited/anaerobic) conditions and ii- its interactions with intestinal epithelial cells from disparate hosts (human vs. chickens). RESULTS: C. jejuni mutant strains with individual deletions that targeted five RPs; nitrate reductase (ΔnapA), nitrite reductase (ΔnrfA), formate dehydrogenase (ΔfdhA), hydrogenase (ΔhydB), and methylmenaquinol:fumarate reductase (ΔmfrA) were used in this study. We show that only the ΔfdhA exhibited a decrease in motility; however, incubation at 42°C significantly reduced the deficiency in the ΔfdhA's motility as compared to 37°C. Under all tested conditions, the ΔmfrA showed a decreased susceptibility to hydrogen peroxide (H(2)O(2)), while the ΔnapA and the ΔfdhA showed significantly increased susceptibility to the oxidant as compared to the wildtype. Further, the susceptibility of the ΔnapA to H(2)O(2) was significantly more pronounced at 37°C. The biofilm formation capability of individual RP mutants varied as compared to the wildtype. However, the impact of the deletion of certain RPs affected biofilm formation in a manner that was dependent on temperature and/or oxygen concentration. For example, the ΔmfrA displayed significantly deficient and increased biofilm formation under microaerobic conditions at 37°C and 42°C, respectively. However, under anaerobic conditions, the ΔmfrA was only significantly impaired in biofilm formation at 42°C. Additionally, the RPs mutants showed differential ability for infecting and surviving in human intestinal cell lines (INT-407) and primary chicken intestinal epithelial cells, respectively. Notably, the ΔfdhA and the ΔhydB were deficient in interacting with both cell types, while the ΔmfrA displayed impairments only in adherence to and invasion of INT-407. Scanning electron microscopy showed that the ΔhydB and the ΔfdhA exhibited filamentous and bulging (almost spherical) cell shapes, respectively, which might be indicative of defects in cell division. CONCLUSIONS: We conclude that the RPs contribute to C. jejuni's motility, H(2)O(2) resistance, biofilm formation, and in vitro interactions with hosts' intestinal cells. Further, the impact of certain RPs varied in response to incubation temperature and/or oxygen concentration. Therefore, RPs may facilitate the prevalence of C. jejuni in a variety of niches, contributing to the pathogen's remarkable potential for adaptation.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Campylobacter jejuni/fisiología , Células Epiteliales/microbiología , Viabilidad Microbiana , Oxidorreductasas/metabolismo , Factores de Virulencia/metabolismo , Animales , Biopelículas/crecimiento & desarrollo , Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/patogenicidad , Campylobacter jejuni/efectos de la radiación , Pollos , Eliminación de Gen , Humanos , Peróxido de Hidrógeno/toxicidad , Locomoción , Microscopía Electrónica de Rastreo , Oxidorreductasas/genética , TemperaturaRESUMEN
Important roles played by invariant natural killer T (iNKT) cells in asthma pathogenesis have been demonstrated. We identified functional iNKT cells and CD1d molecules in pig lungs. Pig iNKT cells cultured in the presence of α-GalCer proliferated and secreted Th1 and Th2 cytokines. Like in other animal models, direct activation of pig lung iNKT cells using α-GalCer resulted in acute airway hyperreactivity (AHR). Clinically, acute AHR-induced pigs had increased respiratory rate, enhanced mucus secretion in the airways, fever, etc. In addition, we observed petechial hemorrhages, infiltration of CD4(+) cells, and increased Th2 cytokines in AHR-induced pig lungs. Ex vivo proliferated iNKT cells of asthma induced pigs in the presence of C-glycoside analogs of α-GalCer had predominant Th2 phenotype and secreted more of Th2 cytokine, IL-4. Thus, baby pigs may serve as a useful animal model to study iNKT cell-mediated AHR caused by various environmental and microbial CD1d-specific glycolipid antigens.
Asunto(s)
Hiperreactividad Bronquial/inmunología , Pulmón/inmunología , Células T Asesinas Naturales/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Antígenos CD1d/inmunología , Asma/inmunología , Hiperreactividad Bronquial/patología , Modelos Animales de Enfermedad , Glicósidos , Pulmón/patología , Activación de Linfocitos/efectos de los fármacos , Monosacáridos/farmacología , Células T Asesinas Naturales/efectos de los fármacos , Neumonía/inmunología , Neumonía/patología , PorcinosRESUMEN
Pigs are capable of generating reassortant influenza viruses of pandemic potential, as both the avian and mammalian influenza viruses can infect pig epithelial cells in the respiratory tract. The source of the current influenza pandemic is H1N1 influenza A virus, possibly of swine origin. This study was conducted to understand better the pathogenesis of H1N1 influenza virus and associated host mucosal immune responses during acute infection in humans. Therefore, we chose a H1N1 swine influenza virus, Sw/OH/24366/07 (SwIV), which has a history of transmission to humans. Clinically, inoculated pigs had nasal discharge and fever and shed virus through nasal secretions. Like pandemic H1N1, SwIV also replicated extensively in both the upper and lower respiratory tracts, and lung lesions were typical of H1N1 infection. We detected innate, proinflammatory, Th1, Th2, and Th3 cytokines, as well as SwIV-specific IgA antibody in lungs of the virus-inoculated pigs. Production of IFN-γ by lymphocytes of the tracheobronchial lymph nodes was also detected. Higher frequencies of cytotoxic T lymphocytes, γδ T cells, dendritic cells, activated T cells, and CD4+ and CD8+ T cells were detected in SwIV-infected pig lungs. Concomitantly, higher frequencies of the immunosuppressive T regulatory cells were also detected in the virus-infected pig lungs. The findings of this study have relevance to pathogenesis of the pandemic H1N1 influenza virus in humans; thus, pigs may serve as a useful animal model to design and test effective mucosal vaccines and therapeutics against influenza virus.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Enfermedades Pulmonares/virología , Infecciones por Orthomyxoviridae/inmunología , Enfermedades de los Porcinos/virología , Animales , Anticuerpos Antivirales , Modelos Animales de Enfermedad , Brotes de Enfermedades , Humanos , Inmunoglobulina A , Pulmón/inmunología , Pulmón/patología , Enfermedades Pulmonares/patología , Membrana Mucosa/inmunología , Membrana Mucosa/virología , Infecciones por Orthomyxoviridae/patología , Porcinos , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Infectious bursal disease (IBD) is an important immunosuppressive disease of chickens. The causative agent, infectious bursal disease virus (IBDV), consists of two serotypes, 1 and 2. Serotype 1 consists of classic IBDV (cIBDV) and variant IBDV (vIBDV). Both of these strains vary in antigenicity and pathogenesis. The goal of this study was to compare the immunopathogenesis of cIBDV and vIBDV. Three-week-old specific pathogen free chickens were inoculated intraocularly with standard challenge strain (STC) (cIBDV) and a variant strain Indiana (IN) (vIBDV). The cIBDV produced more pronounced bursal damage, inflammatory response and infiltration of T cells as compared to vIBDV. There were significant differences in the expression of innate (IFN-α and IFN-ß), proinflammatory cytokine and mediator (IL-6 and iNOS) in cIBDV- and vIBDV-infected bursas. The expression of chemokines genes, IL-8 and MIP-α was also higher in cIBDV-infected chickens during the early phase of infection. The expression of Toll like receptor 3 (TLR3) was downregulated at post inoculation days (PIDs) 3, 5, and 7 in the bursas of vIBDV-infected chickens whereas TLR3 was upregulated at PIDs 3 and 5 in cIBDV-infected bursas. In vIBDV-infected bursa, TLR7 expression was downregulated at PIDs 3 and 5 and upregulated at PID 7. However, TLR7 was upregulated at PIDs 3 and 7 in cIBDV-infected bursas. The expression of MyD88 was downregulated whereas TRIF gene expression was upregulated in cIBDV- and vIBDV-infected bursa. These findings demonstrate the critical differences in bursal lesions, infiltration of T cells, expression of cytokines, chemokines and TLRs in the bursa of cIBDV-and vIBDV-infected chickens.
Asunto(s)
Proteínas Aviares/genética , Infecciones por Birnaviridae/veterinaria , Pollos , Citocinas/genética , Regulación de la Expresión Génica , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Enfermedades de las Aves de Corral/inmunología , Receptores Toll-Like/genética , Animales , Antígenos Virales/metabolismo , Proteínas Aviares/metabolismo , Infecciones por Birnaviridae/genética , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/virología , Bolsa de Fabricio/inmunología , Bolsa de Fabricio/patología , Bolsa de Fabricio/virología , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/metabolismo , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Receptores Toll-Like/metabolismoAsunto(s)
Virus de la Diarrea Epidémica Porcina/fisiología , Replicación Viral , Animales , Línea Celular , Patos , PorcinosRESUMEN
The hemagglutinin (HA) protein of influenza virus mediates essential viral functions including the binding to host receptor and virus entry. It also has the antigenic sites required for virus neutralization by host antibodies. Here, we characterized an H3N2 triple reassortant (TR) influenza virus (A/turkey/Ohio/313053/04) with a mutation at the receptor binding domain (Asp190Ala) that occurred upon virus transmission from turkeys to pigs in an experimental infection study. The mutant virus replicated less efficiently than the parental virus in human, pig and turkey primary tracheal/bronchial epithelial cells, with more than 3-log10 difference in virus titer at 72 hours post infection. In addition, the mutant virus demonstrated lower binding efficiency to plasma membrane preparations from all three cell types compared to the parental virus. Antisera raised against the parental virus reacted equally to both homologous and heterlogous viruses, however, antisera raised against the mutant virus showed 4-8 folds lower reactivity to the parental virus.
Asunto(s)
Hemaglutininas Virales/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Mutación Missense , Infecciones por Orthomyxoviridae/veterinaria , Enfermedades de las Aves de Corral/virología , Virus Reordenados/genética , Enfermedades de los Porcinos/virología , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/virología , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Ohio , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , Unión Proteica , Estructura Terciaria de Proteína , Virus Reordenados/aislamiento & purificación , Receptores Virales , Porcinos , PavosRESUMEN
We exposed chicken embryos at embryonation day 18 (ED18) to a classical virulent infectious bursal disease virus (IBDV; cIBDV) and an attenuated strain of IBDV (aIBDV) and examined the response of embryonic lymphoid cells to these viruses. Embryos responded much more vigorously to cIBDV than to aIBDV. Following cIBDV exposure, embryonic thymus and bursa showed cellular destruction, enhanced rate of apoptosis and presence of viral proteins detectable by immunohistochemistry. At ED21, thymocytes from cIBDV-exposed embryos were severely deficient (P<0.05) in responding to stimulation in vitro with mitogens containing mouse anti-chicken CD28 mAb, PMA and ionomycin. Because purified CD3(+) T cells were also refractory to the mitogens, the mitogenic inhibition of embryonic thymocytes was not attributed to the presence of non-T cell suppressors. Cell suspensions prepared from embryonic thymus and spleen had upregulated gene expression of IFN-gamma and IL-6 cytokines and of chemokine IL-8. In sharp contrast to cIBDV, embryos exposed to aIBDV had minimal detectable changes in the thymus and bursa, although the rate of apoptosis was enhanced in the thymus. Viral antigen was not detectable in the bursa until after hatch. Thymocytes from these embryos responded vigorously to the mitogens, similar to the response of thymocytes from unexposed control embryos. In addition, aIBDV induced a modest gene upregulation of IFN-gamma, IL-6 and IL-8 in thymus and spleen. Relatively modest response of the embryo to aIBDV is significant because in ovo vaccination with aIBDV-type viruses and several other non-pathogenic viruses result in protective immunity that is well pronounced at hatch.
Asunto(s)
Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Linfocitos/inmunología , Animales , Apoptosis , Bolsa de Fabricio/citología , Bolsa de Fabricio/embriología , Bolsa de Fabricio/virología , Embrión de Pollo , Linfocitos/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Organismos Libres de Patógenos Específicos , Bazo/citología , Bazo/embriología , Bazo/virología , Timo/citología , Timo/embriología , Timo/virologíaRESUMEN
BACKGROUND: Mesenchymal stem (stromal) cells (MSCs) mediate their immunoregulatory and tissue repair functions by secreting paracrine factors, including extracellular vesicles (EVs). In several animal models of human diseases, MSC-EVs mimic the beneficial effects of MSCs. Influenza viruses cause annual outbreaks of acute respiratory illness resulting in significant mortality and morbidity. Influenza viruses constantly evolve, thus generating drug-resistant strains and rendering current vaccines less effective against the newly generated strains. Therefore, new therapies that can control virus replication and the inflammatory response of the host are needed. The objective of this study was to examine if MSC-EV treatment can attenuate influenza virus-induced acute lung injury in a preclinical model. METHODS: We isolated EVs from swine bone marrow-derived MSCs. Morphology of MSC-EVs was determined by electron microscopy and expression of mesenchymal markers was examined by flow cytometry. Next, we examined the anti-influenza activity of MSC-EVs in vitro in lung epithelial cells and anti-viral and immunomodulatory properties in vivo in a pig model of influenza virus. RESULTS: MSC-EVs were isolated from MSC-conditioned medium by ultracentrifugation. MSC-EVs were round-shaped and, similarly to MSCs, expressed mesenchymal markers and lacked the expression of swine leukocyte antigens I and II. Incubation of PKH-26-labeled EVs with lung epithelial cells revealed that MSC-EVs incorporated into the epithelial cells. Next, we examined the anti-influenza and anti-inflammatory properties of MSC-EVs. MSC-EVs inhibited the hemagglutination activity of avian, swine, and human influenza viruses at concentrations of 1.25-5 µg/ml. MSC-EVs inhibited influenza virus replication and virus-induced apoptosis in lung epithelial cells. The anti-influenza activity of MSC-EVs was due to transfer of RNAs from EVs to epithelial cells since pre-incubation of MSC-EVs with RNase enzyme abrogated the anti-influenza activity of MSC-EVs. In a pig model of influenza virus, intratracheal administration of MSC-EVs 12 h after influenza virus infection significantly reduced virus shedding in the nasal swabs, influenza virus replication in the lungs, and virus-induced production of proinflammatory cytokines in the lungs of influenza-infected pigs. The histopathological findings revealed that MSC-EVs alleviated influenza virus-induced lung lesions in pigs. CONCLUSIONS: Our data demonstrated in a relevant preclinical large animal model of influenza virus that MSC-EVs possessed anti-influenza and anti-inflammatory properties and that EVs may be used as cell-free therapy for influenza in humans.
Asunto(s)
Lesión Pulmonar Aguda/terapia , Vesículas Extracelulares/trasplante , Células Madre Mesenquimatosas/metabolismo , Infecciones por Orthomyxoviridae/patología , Mucosa Respiratoria/patología , Lesión Pulmonar Aguda/virología , Animales , Antiinflamatorios/metabolismo , Apoptosis , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/virología , Alphainfluenzavirus/metabolismo , Infecciones por Orthomyxoviridae/virología , ARN Viral/genética , Mucosa Respiratoria/citología , Mucosa Respiratoria/virología , Porcinos , Replicación ViralRESUMEN
We serially passaged classical infectious bursal disease virus (cIBDV) and antigenic variant IBDV (vIBDV) in an avian macrophage cell line, NCSU cells, referred as mcIBDV and mvIBDV respectively and examined the in vitro and in vivo characteristics of the macrophage-adapted viruses. NCSU adapted viruses caused earlier destruction of NCSU cells than the unadapted viruses. Nitric oxide (NO) was detected earlier in cultures infected with mcIBDV and mvIBDV than in cultures infected with cIBDV and vIBDV. cIBDV and vIBDV were able to infect DF-1 cells, a chicken embryo fibroblast cell line, only after one replication cycle in NCSU cells. The genetic basis of altered tropism of progeny virus from NCSU cells infected cultures was not identified. No aa substitutions were observed in hypervariable region of VP2 of cIBDV and vIBDV passaged 1 time in NCSU cells whereas both mcIBDV and mvIBDV had multiple aa substitutions. To assess protective efficacy of mcIBDV and mvIBDV, embryonated chicken eggs were inoculated with mcIBDV and mvIBDV at embryonation day 18 (ED 18) and challenged with a virulent cIBDV at 3 weeks of age. mcIBDV and mvIBDV were immunogenic and generated antibody responses and provided 100% protection against cIBDV.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Macrófagos/inmunología , Macrófagos/virología , Tropismo/inmunología , Replicación Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Variación Antigénica/genética , Variación Antigénica/inmunología , Apoptosis/inmunología , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/virología , Embrión de Pollo , Ensayo de Inmunoadsorción Enzimática/veterinaria , Técnica del Anticuerpo Fluorescente/veterinaria , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , ARN Viral/química , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Organismos Libres de Patógenos Específicos , Proteínas Virales/genética , Proteínas Virales/inmunología , Replicación Viral/genéticaRESUMEN
Turkeys exposed to avian metapneumovirus (aMPV) subtype C showed extensive lymphoid cell infiltrations in the nasal turbinates of the upper respiratory tract. The cellular infiltration occurred after the first virus exposure but not after re-exposure. Quantitation of the relative proportions of mucosal immunoglobulin (Ig)A+, IgG+, and IgM+ cells in controls and virus-exposed turkeys revealed that at 7 days after the first virus exposure, when mucosal infiltration was well pronounced, there was a significant increase (P < 0.05) in the numbers of infiltrating IgA+ but not of IgG+ and IgM+ cells. After the second virus exposure, although the overall numbers of mucosal lymphoid cells were similar in the virus-exposed and control turkeys, the relative proportions of IgA+ and IgG+ cells were significantly higher in the virus-exposed turkeys (P < 0.05) than in controls. Furthermore, elevated levels of aMPV-specific IgA were detected in the nasal secretions and the bile of virus-exposed birds after the second but not after the first virus exposure. These results suggest, for the first time, the possible involvement of local mucosal immunoglobulins in the pathogenesis of aMPV in turkeys.
Asunto(s)
Linfocitos B/fisiología , Metapneumovirus , Infecciones por Paramyxoviridae/veterinaria , Enfermedades de las Aves de Corral/patología , Sistema Respiratorio/citología , Pavos , Animales , Bilis , Inmunoglobulina A , Moco , Infecciones por Paramyxoviridae/patología , Enfermedades de las Aves de Corral/virología , Sistema Respiratorio/patología , Enfermedades Respiratorias/inmunología , Enfermedades Respiratorias/veterinaria , Enfermedades Respiratorias/virologíaRESUMEN
In the present study, we show that infection with infectious bursal disease virus (IBDV) causes activation of macrophages, the key cells involved in inflammatory and immune-regulatory functions. Exposure of cultured spleen macrophages (SM) from SPF chickens to IBDV resulted in the production of nitric oxide (NO). In addition, there was upregulation of mRNA expression of inducible nitric oxide synthase (iNOS), IL-8 and cyclooxygenase-2 (COX-2). The signal transduction pathways involved in macrophage activation were examined. The role of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) was tested by using specific pharmacological inhibitors. Addition of p38 MAPK inhibitor, SB-203580 and NF-kappaB inhibitor Bay 11-7082, suppressed IBDV-induced NO production and mRNA expression of iNOS, IL-8 and COX-2. The results suggest that IBDV uses cellular signal transduction machinery, in particular the p38 MAPK and NF-kappaB pathways, to elicit macrophage activation. The increased production of NO, IL-8 and COX-2 by macrophages may contribute to bursa inflammatory responses commonly seen during the acute IBDV infection.
Asunto(s)
Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Activación de Macrófagos , FN-kappa B/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Células Cultivadas , Pollos , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Imidazoles/farmacología , Inmunohistoquímica , Interleucina-8/biosíntesis , Interleucina-8/genética , Macrófagos/virología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Nitrilos/farmacología , Piridinas/farmacología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonas/farmacologíaRESUMEN
Pigs are considered as the source of some of the emerging human flu viruses. Inactivated swine influenza virus (SwIV) vaccine has been in use in the US swine herds, but it failed to control the flu outbreaks. The main reason has been attributed to lack of induction of strong local mucosal immunity in the respiratory tract. Invariant natural killer T (iNKT) cell is a unique T cell subset, and activation of iNKT cell using its ligand α-Galactosylceramide (α-GalCer) has been shown to potentiate the cross-protective immunity to inactivated influenza virus vaccine candidates in mice. Recently, we discovered iNKT cell in pig and demonstrated its activation using α-GalCer. In this study, we evaluated the efficacy of an inactivated H1N1 SwIV coadministered with α-GalCer intranasally against a homologous viral challenge. Our results demonstrated the potent adjuvant effects of α-GalCer in potentiating both innate and adaptive immune responses to SwIV Ags in the lungs of pigs, which resulted in reduction in the lung viral load by 3 logs compared to without adjuvant. Immunologically, in the lungs of pigs vaccinated with α-GalCer an increased virus specific IgA response, IFN-α secretion and NK cell-cytotoxicity was observed. In addition, iNKT cell-stimulation enhanced the secretion of Th1 cytokines (IFN-γ and IL-12) and reduced the production of immunosuppressive cytokines (IL-10 and TGF-ß) in the lungs of pigsâ In conclusion, we demonstrated for the first time iNKT cell adjuvant effects in pigs to SwIV Ags through augmenting the innate and adaptive immune responses in the respiratory tract.
Asunto(s)
Inmunidad Adaptativa/efectos de los fármacos , Galactosilceramidas/farmacología , Inmunidad Innata/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/veterinaria , Enfermedades de los Porcinos/inmunología , Inmunidad Adaptativa/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Citocinas/inmunología , Inmunidad Innata/inmunología , Inmunoglobulina A/sangre , Pulmón/virología , Células T Asesinas Naturales/química , Células T Asesinas Naturales/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/fisiopatología , Distribución Aleatoria , Porcinos , Enfermedades de los Porcinos/fisiopatología , Vacunas de Productos Inactivados/inmunología , Carga Viral , Aumento de Peso/efectos de los fármacosRESUMEN
In this study the effect of infectious bursal disease virus (IBDV) on bursal macrophages during the acute phase of the infection was examined. Specific-pathogen-free (SPF) chickens were exposed to virulent IBDV and bursal adherent cells were examined by immunohistochemisrty and RT-PCR for virus infection and by real-time quantitative RT-PCR (qRT-PCR) for mRNA transcripts of proinflammatory cytokines and iNOS. Viral genome was detected in bursal macrophages at 3, 5 and 7 days post-infection (dpi). Immuno-histochemical staining revealed double positive cells for KUL01 (macrophage marker) and intracellular viral proteins, showing viral replication in bursal macrophages of infected chickens. We noted a significant decrease in the total number of bursal macrophages in infected chickens, probably due to the lysis of infected cells. However, likely due to extensive necrosis of B cells, the relative proportion of bursal macrophages was significantly higher (P<0.05) in infected birds at 3 and 5 dpi than in controls. Among the cytokines examined, IL-6 showed the greatest upregulation (100-fold increase) at 3 dpi. Expression of IL-1beta was maximum at 3 dpi whereas IL-18 expression was highest at 1 dpi. Enhanced expression of iNOS mRNA was observed at 5 dpi. Increased expression of the proinflammatory cytokines and iNOS correlated well with the presence of the inflammatory response in the infected bursa. These data suggested that B cells may not be the sole targets for the virus; macrophages and possibly other cells may serve as host for IBDV.
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Infecciones por Birnaviridae/veterinaria , Bolsa de Fabricio/inmunología , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/virología , Enfermedades de las Aves de Corral/virología , Animales , Infecciones por Birnaviridae/inmunología , Bolsa de Fabricio/patología , Bolsa de Fabricio/virología , Recuento de Células , Células Cultivadas , Pollos , Perfilación de la Expresión Génica , Inmunohistoquímica , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Virus de la Enfermedad Infecciosa de la Bolsa/patogenicidad , Interleucina-1/genética , Interleucina-18/genética , Interleucina-6/genética , Enfermedades de las Aves de Corral/inmunología , ARN Mensajero/análisis , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/análisisRESUMEN
In this study, we describe the isolation and characterization of epithelial stem-like cells from the swine umbilical cord and their susceptibility to influenza virus infection. Swine umbilical cord epithelial stem cells (SUCECs) expressed stem cell and pluripotency associated markers such as SSEA-1, SSEA-4, TRA 1-60 and TRA 1-81 and Oct4. Morphologically, cells displayed polygonal morphology and were found to express epithelial markers; pancytokeratin, cytokeratin-18 and occludin; mesenchymal cell markers CD44, CD90 and haematopoietic cell marker CD45 were not detected on these cells. The cells had extensive proliferation and self- renewal properties. The cells also possessed immunomodulatory activity and inhibited the proliferation of T cells. Also, higher levels of anti-inflammatory cytokine IL-10 were detected in SUCEC-T cell co-cultures. The cells were multipotent and differentiated into lung epithelial cells when cultured in epithelial differentiation media. We also examined if SUCECs are susceptible to infection with influenza virus. SUCECs expressed sialic acid receptors, used by influenza virus for binding to cells. The 2009 pandemic influenza virus and swine influenza virus replicated in these cells. SUCECs due to their differentiation and immunoregulatory properties will be useful as cellular therapy in a pig model for human diseases. Additionally, our data indicate that influenza virus can infect SUCECs and may transmit influenza virus from mother to fetus through umbilical cord and transplantation of influenza virus-infected stem cells may transmit infection to recipients. Therefore, we propose that umbilical cord cells, in addition to other agents, should also be tested for influenza virus before cryopreservation for future use as a cell therapy for disease conditions.
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Células Epiteliales/virología , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Células Madre/virología , Cordón Umbilical/citología , Animales , Antígenos de Superficie/metabolismo , Biomarcadores/metabolismo , Biomarcadores de Tumor/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Epiteliales/citología , Interleucina-10/metabolismo , Antígeno Lewis X/metabolismo , Infecciones por Orthomyxoviridae , Mucosa Respiratoria/citología , Antígenos Embrionarios Específico de Estadio/metabolismo , Células Madre/citología , Porcinos , Cordón Umbilical/virologíaRESUMEN
INTRODUCTION: Mesenchymal stem (stromal) cells (MSCs) possess self-renewal, differentiation and immunoregulatory properties, and therefore are being evaluated as cellular therapy for inflammatory and autoimmune diseases, and for tissue repair. MSCs isolated from bone marrow are extensively studied. Besides bone marrow, MSCs have been identified in almost all organs of the body including the lungs. Lung-derived MSCs may be more effective as therapy for lung diseases as compared to bone marrow-derived MSCs. Pigs are similar to humans in anatomy, physiology and immunological responses, and thus may serve as a useful large animal preclinical model to study potential cellular therapy for human diseases. METHODS: We isolated MSCs from the lungs (L-MSCs) of 4-6-week-old germ-free pigs. We determined the self-renewal, proliferation and differentiation potential of L-MSCs. We also examined the mechanisms of immunoregulation by porcine L-MSCs. RESULTS: MSCs isolated from porcine lungs showed spindle-shaped morphology and proliferated actively in culture. Porcine L-MSCs expressed mesenchymal markers CD29, CD44, CD90 and CD105 and lacked the expression of hematopoietic markers CD34 and CD45. These cells were multipotent and differentiated into adipocytes, osteocytes and epithelial cells. Like human MSCs, L-MSCs possessed immunoregulatory properties and inhibited proliferation of T cells and interferon-γ and tumor necrosis factor-α production by T cells and dendritic cells, respectively, and increased the production of T-helper 2 cytokines interleukin (IL)-4 and IL-13 by T cells. L-MSCs induced the production of prostaglandin E2 (PGE2) in MSC-T cell co-cultures and inhibition of PGE2 significantly restored (not completely) the immune modulatory effects of L-MSCs. CONCLUSIONS: Here, we demonstrate that MSCs can be isolated from porcine lung and that these cells, similar to human lung MSCs, possess in vitro proliferation, differentiation and immunomodulatory functions. Thus, these cells may serve as a model system to evaluate the contribution of lung MSCs in modulating the immune response, interactions with resident epithelial cells and tissue repair in a pig model of human lung diseases.
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Diferenciación Celular , Inmunomodulación , Pulmón/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Animales , Antígenos de Diferenciación/biosíntesis , Proliferación Celular , Separación Celular , Células Dendríticas/metabolismo , Dinoprostona/inmunología , Femenino , Leucocitos Mononucleares/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Pluripotentes/metabolismo , PorcinosRESUMEN
Swine influenza is widely prevalent in swine herds in North America and Europe causing enormous economic losses and a public health threat. Pigs can be infected by both avian and mammalian influenza viruses and are sources of generation of reassortant influenza viruses capable of causing pandemics in humans. Current commercial vaccines provide satisfactory immunity against homologous viruses; however, protection against heterologous viruses is not adequate. In this study, we evaluated the protective efficacy of an intranasal Poly I:C adjuvanted UV inactivated bivalent swine influenza vaccine consisting of Swine/OH/24366/07 H1N1 and Swine/CO/99 H3N2, referred as PAV, in maternal antibody positive pigs against an antigenic variant and a heterologous swine influenza virus challenge. Groups of three-week-old commercial-grade pigs were immunized intranasally with PAV or a commercial vaccine (CV) twice at 2 weeks intervals. Three weeks after the second immunization, pigs were challenged with the antigenic variant Swine/MN/08 H1N1 (MN08) and the heterologous Swine/NC/10 H1N2 (NC10) influenza virus. Antibodies in serum and respiratory tract, lung lesions, virus shedding in nasal secretions and virus load in lungs were assessed. Intranasal administration of PAV induced challenge viruses specific-hemagglutination inhibition- and IgG antibodies in the serum and IgA and IgG antibodies in the respiratory tract. Importantly, intranasal administration of PAV provided protection against the antigenic variant MN08 and the heterologous NC10 swine influenza viruses as evidenced by significant reductions in lung virus load, gross lung lesions and significantly reduced shedding of challenge viruses in nasal secretions. These results indicate that Poly I:C or its homologues may be effective as vaccine adjuvants capable of generating cross-protective immunity against antigenic variants/heterologous swine influenza viruses in pigs.
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Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Poli I-C/administración & dosificación , Enfermedades de los Porcinos/prevención & control , Vacunación/métodos , Administración Intranasal , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Líquidos Corporales/inmunología , Europa (Continente) , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H2N2 del Virus de la Influenza A/inmunología , Pulmón/patología , Pulmón/virología , América del Norte , Sistema Respiratorio/inmunología , Porcinos , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Carga Viral , Esparcimiento de VirusRESUMEN
The methylmenaquinol:fumarate reductase (Mfr) of Campylobacter jejuni is a periplasmic respiratory (redox) protein that contributes to the metabolism of fumarate and displays homology to succinate dehydrogenase (Sdh). Since chemically oxidized redox-enzymes, including fumarate reductase and Sdh, contribute to the generation of oxidative stress in Escherichia coli, we assessed the role of Mfr in C. jejuni after exposure to hydrogen peroxide (H2 O2 ). Our results show that a Mfr mutant (∆mfrA) strain was less susceptible to H2 O2 as compared to the wildtype (WT). Furthermore, the H2 O2 concentration in the ∆mfrA cultures was significantly higher than that of WT after exposure to the oxidant. In the presence of H2 O2 , catalase (KatA) activity and katA expression were significantly lower in the ∆mfrA strain as compared to the WT. Exposure to H2 O2 resulted in a significant decrease in total intracellular iron in the ∆mfrA strain as compared to WT, while the addition of iron to the growth medium mitigated H2 O2 susceptibility and accumulation in the mutant. The ∆mfrA strain was significantly more persistent in RAW macrophages as compared to the WT. Scanning electron microscopy showed that infection with the ∆mfrA strain caused prolonged changes to the macrophages' morphology, mainly resulting in spherical-shaped cells replete with budding structures and craters. Collectively, our results suggest a role for Mfr in maintaining iron homeostasis in H2 O2 stressed C. jejuni, probably via affecting the concentrations of intracellular iron.