RESUMEN
Our objective was to determine the effects of nondigestible oligosaccharides (NDO) on lung health and performance. Three hundred male Holstein-Friesian calves aged 18.0 ± 3.6 d received 1 of 6 treatments for 8.5 wk (period 1). Treatments included a negative control (CON), galacto-oligosaccharides (GOS) administered as a spray via the nose once daily (SPR), GOS administered via the milk replacer (MR) at 1% (GOS-L) and 2% (GOS-H), fructo-oligosaccharides administered via the MR at 0.25% (FOS) and a combination of GOS and fructo-oligosaccharides administered via the MR at 1% and 0.25%, respectively (GOS-FOS). Milk replacer was fed twice daily. Feeding levels were equal between calves and increased progressively in time. Body weight was measured every 4 wk and clinical health was scored weekly. Blood and broncho-alveolar lavage fluid (BALF) samples were collected bi-weekly from a subset of calves (n = 120). After period 1, all calves received the same control MR for 18 wk until slaughter (period 2), during which general performance and clinical health were measured. Generally, infection pressure was high, with clinical scores and BALF proinflammatory TNFα concentrations increasing with time in period 1, which resulted in a high number of required group antimicrobial treatments (6 group antimicrobial treatments in 13 wk, supplied to all calves). Average daily gain adjusted to equal solid feed intake was increased for GOS-L (+61 g/d) compared with CON calves from experimental wk 1 to 5. Plasma white blood cell concentration tended to be lowered by GOS-L, plasma IL-8 concentration was reduced by all orally supplemented NDO, plasma IL-6 was reduced by all NDO treatments except GOS-FOS and plasma IL-1ß was reduced by all NDO treatments compared with CON, although this differed per time point for SPR. The neutrophil percentage in BALF was reduced by GOS-L in wk 6, which was associated with a relative increase in macrophages. The BALF concentration of TNFα and IL-8 was reduced or tended to be reduced by GOS-L and GOS-H, while IL-6 was or tended to be reduced by SPR, GOS-L, GOS-H, and GOS-FOS, and IL-1ß was reduced by SPR, GOS-L, GOS-H, and FOS. Generally, feeding the combination of GOS and FOS was not more effective than feeding GOS or FOS alone, because feeding GOS-FOS resulted in higher concentrations of plasma and BALF cytokine and chemokine concentrations compared with feeding GOS-L alone, and resulted in higher plasma cytokine concentrations compared with feeding FOS alone. None of the BALF and plasma cytokine or chemokine concentrations differed between the GOS-L and GOS-H treatment. Performance and clinical scores in period 2 did not differ among treatments. Altogether, all tested NDO reduced systemic and lung inflammation in calves under high natural infection pressure and for GOS-fed calves, this increased performance during the first 4 wk. Combining GOS and FOS did not have a synergistic effect. The intranasal administration of GOS also lowered systemic and lung inflammation, but tended to negatively affect performance. Overall, this study demonstrates the potential of NDO to alleviate systemic and respiratory inflammation in calves.
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Antiinfecciosos , Enfermedades de los Bovinos , Neumonía , Animales , Bovinos , Masculino , Dieta/veterinaria , Factor de Necrosis Tumoral alfa , Interleucina-6 , Interleucina-8 , Alimentación Animal/análisis , Leche , Inflamación/veterinaria , Oligosacáridos/farmacología , Peso Corporal , Pulmón , Neumonía/veterinaria , Destete , Enfermedades de los Bovinos/tratamiento farmacológicoRESUMEN
Background: Early life provides a window of opportunity to prevent allergic diseases. With a prevalence of 0.5-2% in infants, hen's egg allergy is one of the most common food allergies. The immunomodulatory effects of human milk oligosaccharides (HMOs), 2'-fucosyllactose (2'FL), and 3-fucosyllactose (3FL) were studied in an in vitro mucosal immune model and an in vivo murine model for hen's egg (ovalbumin) allergy. Methods: Intestinal epithelial cell (IEC)/dendritic cell (DC) and DC/T cell cocultures were used to expose IECs to ovalbumin (OVA) in an in vitro mucosal immune model. The effects of epithelial pre-incubation with 0.1% 2'FL or 3FL and/or 0.5 mM butyrate were studied. Three- to four-weeks-old female C3H/HeOuJ mice were fed AIN93G diets containing 0.1-0.5% 2'FL or 3FL 2 weeks before and during OVA sensitization and challenge. Allergic symptoms and systemic and local immune parameters were assessed. Results: Exposing IECs to butyrate in vitro left the IEC/DC/T cell cross-talk unaffected, while 2'FL and 3FL showed differential immunomodulatory effects. In 3FL exposed IEC-DC-T cells, the secretion of IFNγ and IL10 was enhanced. This was observed upon pre-incubation of IECs with 2'FL and butyrate as well, but not 2'FL alone. The presence of butyrate did not affect OVA activation, but when combined with 3FL, an increase in IL6 release from DCs was observed (p < 0.001). OVA allergic mice receiving 0.5% 3FL diet had a lower %Th2 cells in MLNs, but the humoral response was unaltered compared to control mice. OVA-allergic mice receiving 0.1 or 0.5% 2'FL diets had lower serum levels of OVA-IgG2a (p < 0.05) or the mast cell marker mMCP1, in association with increased concentration of cecal short-chain fatty acids (SCFAs) (p < 0.05). Conclusion: In vitro butyrate exposure promotes the development of a downstream type 1 and regulatory response observed after 2'FL exposure. 2'FL and 3FL differentially modulate ovalbumin-induced mucosal inflammation predominantly independent of butyrate. Mice receiving dietary 3FL during ovalbumin sensitization and challenge had lowered Th2 activation while the frequency of Treg cells was enhanced. By contrast, 2'FL improved the humoral immune response and suppressed mast cell activation in association with increased SCFAs production in the murine model for hen's egg allergy.
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PURPOSE: The incidence and severity of allergic asthma is rising, and novel strategies to prevent or treat this disease are needed. This study investigated the effects of different mixtures of non-digestible oligosaccharides combined with Bifidobacterium breve M-16V (BB) on the development of allergic airway inflammation in an animal model for house dust mite (HDM)-induced allergic asthma. METHODS: BALB/c mice were sensitized intranasally (i.n.) with HDM and subsequently challenged (i.n.) with PBS or HDM while being fed diets containing different oligosaccharide mixtures in combination with BB or an isocaloric identical control diet. Bronchoalveolar lavage fluid (BALF) inflammatory cell influx, chemokine and cytokine concentrations in lung homogenates and supernatants of ex vivo HDM-restimulated lung cells were analyzed. RESULTS: The HDM-induced influx of eosinophils and lymphocytes was reduced by the diet containing the short-chain and long-chain fructo-oligosaccharides and BB (FFBB). In addition to the HDM-induced cell influx, concentrations of IL-33, CCL17, CCL22, IL-6, IL-13 and IL-5 were increased in supernatants of lung homogenates or BALF and IL-4, IFN-γ and IL-10 were increased in restimulated lung cell suspensions of HDM-allergic mice. The diet containing FFBB reduced IL-6, IFN-γ, IL-4 and IL-10 concentrations, whereas the combination of galacto-oligosaccharides and long-chain fructo-oligosaccharides with BB was less potent in this model. CONCLUSION: These findings show that synbiotic dietary supplementation can affect respiratory allergic inflammation induced by HDM. The combination of FFBB was most effective in the prevention of HDM-induced airway inflammation in mice.
Asunto(s)
Asma/terapia , Bifidobacterium breve , Hipersensibilidad/terapia , Inflamación/terapia , Oligosacáridos/farmacología , Simbióticos/administración & dosificación , Animales , Asma/inmunología , Líquido del Lavado Bronquioalveolar/microbiología , Quimiocina CCL17/metabolismo , Quimiocina CCL22/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/metabolismo , Hipersensibilidad/inmunología , Inflamación/inmunología , Interferón gamma/metabolismo , Interleucinas/metabolismo , Pulmón/citología , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Pyroglyphidae/inmunologíaRESUMEN
Although interest in using probiotics to prevent and treat intestinal diseases is increasing, the effects of specific probiotic strains still remain unclear. Here, we assess the therapeutic effects of two probiotic strains, Lactobacillus rhamnosus NutRes 1 and Bifidobacterium breve NutRes 204 on a dextran sodium sulphate (DSS)-induced chronic murine colitis model. The chronic colitis was induced by two DSS treatment cycles with a rest period of 10 days (the remission or resolution phase). The probiotic supplementation was started during the resolution phase, after the first DSS treatment cycle, and continued until the end of the experiment. In addition to clinical observations made during the experiment, cellular infiltration was measured along with mRNA expression of pro-inflammatory cytokines, T cell-associated cytokines, and Toll like receptors (TLR) in the inflamed colon after second DSS treatment cycle. L. rhamnosus, but not B. breve, rapidly and effectively improved the DSS-induced bloody diarrhoea during the resolution phase. However, a contradictory effect by both probiotic strains on the faecal condition was found after re-induction of colitis. The worsening of the faecal condition was accompanied by a reduced number of neutrophils and increased expression of interferon-γ in the colons of DSS-treated mice. Furthermore, an increased expression of TLR2, TLR6 and pro-inflammatory markers including chemokine (C-C motif) ligand 2, interleukin (IL)-1ß, tumour necrosis factor α and IL-6 was found in DSS-treated mice with L. rhamnosus supplementation. These results indicate that therapeutic administration of specific probiotics might be beneficial during the resolution phase of colitis. However, caution should be taken as specific probiotic treatments reduce neutrophil influx, which may be the reason of exacerbation of chronic colitis.
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Bifidobacterium/fisiología , Colitis/tratamiento farmacológico , Lacticaseibacillus rhamnosus/fisiología , Probióticos/administración & dosificación , Animales , Enfermedad Crónica/terapia , Colitis/genética , Colitis/inmunología , Colitis/patología , Colon/microbiología , Colon/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Ratones , Ratones Endogámicos C57BL , RecurrenciaRESUMEN
AIM: Blocking of lysophosphatidic acid (LPA) receptor (LPAR) 1 may be a novel therapeutic option for bronchopulmonary dysplasia (BPD) by preventing the LPAR1-mediated adverse effects of its ligand (LPA), consisting of lung inflammation, pulmonary arterial hypertension (PAH) and fibrosis. METHODS: In Wistar rats with experimental BPD, induced by continuous exposure to 100% oxygen for 10 days, we determined the beneficial effects of LPAR1 deficiency in neonatal rats with a missense mutation in cytoplasmic helix 8 of LPAR1 and of LPAR1 and -3 blocking with Ki16425. Parameters investigated included survival, lung and heart histopathology, fibrin and collagen deposition, vascular leakage and differential mRNA expression in the lungs of key genes involved in LPA signalling and BPD pathogenesis. RESULTS: LPAR1-mutant rats were protected against experimental BPD and mortality with reduced alveolar septal thickness, lung inflammation (reduced influx of macrophages and neutrophils, and CINC1 expression) and collagen III deposition. However, LPAR1-mutant rats were not protected against alveolar enlargement, increased medial wall thickness of small arterioles, fibrin deposition and vascular alveolar leakage. Treatment of experimental BPD with Ki16425 confirmed the data observed in LPAR1-mutant rats, but did not reduce the pulmonary influx of neutrophils, CINC1 expression and mortality in rats with experimental BPD. In addition, Ki16425 treatment protected against PAH and right ventricular hypertrophy. CONCLUSION: LPAR1 deficiency attenuates pulmonary injury by reducing pulmonary inflammation and fibrosis, thereby reducing mortality, but does not affect alveolar and vascular development and, unlike Ki16425 treatment, does not prevent PAH in neonatal rats with experimental BPD.
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Displasia Broncopulmonar/metabolismo , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Receptores del Ácido Lisofosfatídico/deficiencia , Animales , Animales Recién Nacidos , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Hiperoxia/complicaciones , Isoxazoles/farmacología , Propionatos/farmacología , Ratas , Ratas Mutantes , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Severe asthma (SA) is associated with neutrophil recruitment and T helper (TH )17 chemokine overexpression in bronchial biopsies. We aimed to evaluate IL-17A and IL-17F expression in nasal/bronchial lamina propria of atopic mild-to-severe asthmatics and controls in relation to neutrophilia and asthma exacerbations. Cryostat sections of nasal/bronchial biopsies obtained from 14 SA and 14 mild asthma (MA) stable atopic patients with rhinitis, and seven healthy controls were analyzed by immunohistochemistry for neutrophils, IL-17A and IL-17F expression. Atopic SA showed an increase in asthma exacerbations number, IL-17F and IL-17A expression in nasal/bronchial lamina propria compared to MA and controls, and a higher expression of bronchial neutrophils in SA compared to MA and controls. In all asthmatics, significant relationships were found between bronchial IL-17F and neutrophils/FEV1 , nasal IL-17F and bronchial neutrophil/IL-17 markers and between the latter and exacerbations, suggesting that nasal IL-17F might be informative on bronchial IL17-driven neutrophilia in atopic SA.
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Asma/diagnóstico , Asma/metabolismo , Hipersensibilidad Inmediata/diagnóstico , Hipersensibilidad Inmediata/metabolismo , Interleucina-17/metabolismo , Neutrófilos/metabolismo , Adulto , Biopsia , Bronquios/metabolismo , Bronquios/patología , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Volumen Espiratorio Forzado , Humanos , Masculino , Persona de Mediana Edad , Mucosa Nasal/metabolismo , Infiltración Neutrófila , Nariz/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Factores de RiesgoRESUMEN
T-helper 1 and 17 (Th1/Th17) responses are important in inflammatory bowel disease (IBD), and research indicates that Toll-like receptor 6 (TLR6) stimulation leads to Th17 cell development within the lung. The gastrointestinal tract, like the lung, is a mucosal surface that is exposed to bacterially derived TLR6 ligands. Thus, we looked at the effects of TLR6 stimulation on the expression of Th17-, Th1-, and regulatory T-cell-associated transcription factors; RORγt, T-bet, and Foxp3, respectively; in CD4+ T cells within gut-associated lymphoid tissue (GALT) in vitro and in vivo. Cells from GALT and spleen were stimulated with anti-CD3 and TLR ligands for TLR1/2 and TLR2/6 (Pam3CSK4 and FSL-1, respectively). FSL-1 was more effective than Pam3CSK4 at inducing Th1 and Th17 responses in the GALT while Pam3CSK4 rivaled FSL-1 in the spleen. TLR6 was further explored in vivo using experimental colitis. Tlr6-/- mice were resistant to colitis, and oral FSL-1 led to more severe colitis in wild-type mice. Similar pro-inflammatory reactions were seen in human peripheral blood mononuclear cells, and TLR6 expression was directly correlated with RORC mRNA levels in inflamed intestines of IBD patients. These results demonstrate that TLR6 supports Th1- and Th17-skewed responses in the GALT and might be an important target for the development of new medical interventions in IBD.
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Colitis/prevención & control , Tracto Gastrointestinal/inmunología , Tejido Linfoide/inmunología , Células TH1/inmunología , Células Th17/inmunología , Receptor Toll-Like 6/fisiología , Animales , Células Cultivadas , Colitis/fisiopatología , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Receptor Toll-Like 6/genética , Factores de Transcripción/genéticaRESUMEN
Chronic obstructive pulmonary disease (COPD) is a multicomponent disease characterized by emphysema and/or chronic bronchitis. COPD is mostly associated with cigarette smoking. Cigarette smoke contains over 4,700 chemical compounds, including free radicals and LPS (a Toll-Like Receptor 4 agonist) at concentrations which may contribute to the pathogenesis of diseases like COPD. We have previously shown that short-term exposure to cigarette smoke medium (CSM) can stimulate several inflammatory cells via TLR4 and that CSM reduces the degranulation of bone-marrow-derived mast cells (BMMCs). In the current study, the effect of CSM on mast cells maturation and function was investigated. Coculturing of BMMC with CSM during the development of bone marrow progenitor cells suppressed the granularity and the surface expression of c-kit and Fc ε RI receptors. Stimulation with IgE/antigen resulted in decreased degranulation and release of Th1 and Th2 cytokines. The effects of CSM exposure could not be mimicked by the addition of LPS to the culture medium. In conclusion, this study shows that CSM may affect mast cell development and subsequent response to allergic activation in a TLR4-independent manner.
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Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de IgE/metabolismo , Humo/efectos adversos , Fumar/efectos adversos , Animales , Células Cultivadas , Citometría de Flujo , Masculino , RatonesRESUMEN
Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. Cigarette smoke has been considered a major player in the pathogenesis of COPD. The inflamed airways of COPD patients contain several inflammatory cells. Vitamin A metabolites have been implicated in the repair of lung damage. Exposure to cigarette smoke has been shown to depress levels of retinol in lungs of rats. The purpose of this study was to investigate if a low, but not deficient, vitamin A status potentiated susceptibility to the development of cigarette smoke-induced lung emphysema in mice. Mice were bred that were the offspring's of 3 generations of mice that were fed a purified diet containing low levels of vitamin A and exposed to cigarette smoke for 3 months, every weekday. Then, levels of 9-cis, 13-cis, and all-trans retinoic acid, retinol and retinyl palmitate were measured in plasma, liver and right lung lobe. The left lung lobe was used to assess mean linear intercept (Lm), as a measure of smoke-induced lung damage. Average feed intakes were not different between treatment groups. We show that both retinol and retinyl palmitate levels were dramatically decreased in the storage organs of mice on the low vitamin A diet (retinol 2-fold in both lung and liver, and retinyl palmitate 5- fold in lung) which shows that the depletion was successful. However, this treatment did not result in the development of lung emphysema. However, smoke exposure led to a significant increase in Lm in mice with a low vitamin A status compared to the room air-breathing controls. Lung levels of acid retinoids were similar in all mice, irrespective of diet or smoke exposure. Concluding, a low vitamin A status increases the susceptibility to the development of cigarette smoke-induced lung emphysema, possibly because of decreased anti-oxidant capacity in the lungs due to locally reduced retinol and retinyl palmitate levels. These observations indicate that human populations with a low vitamin A status and a high prevalence of smoking may be at increased risk of developing lung emphysema.
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Enfisema Pulmonar/etiología , Enfisema Pulmonar/metabolismo , Fumar/efectos adversos , Deficiencia de Vitamina A/complicaciones , Deficiencia de Vitamina A/metabolismo , Vitamina A/administración & dosificación , Animales , Susceptibilidad a Enfermedades , Femenino , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Retinoides/metabolismo , Fumar/metabolismoRESUMEN
COPD is a chronic airway disease associated with inflammation and cigarette smoking. Airway epithelial cells are the first cells exposed to cigarette smoke (CS) and can release CXCL-8 and IL-1ß. These cytokines are involved in acute and chronic inflammatory processes in COPD. The aim of this study was to investigate whether toll-like receptors (TLRs) located in/on epithelial cells were involved in cigarette smoke-induced cytokine production. Here we demonstrate that CS induces the release of CXCL-8 and IL-1ß from human bronchial epithelial cells (HBE-14o). CS-induced CXCL-8 production was inhibited by an antibody against TLR4 and by inhibitory ODN suggesting the involvement of TLR4 and TLR9. In addition, exposure of HBE-14o cells to TLR4 or TLR9 ligands resulted in the release of CXCL-8 and IL1ß. TLR4 and also TLR9 were present on the cell surface and the expression of both receptors decreased after CS exposure. The molecular mechanism of the CS-induced CXCL-8 production by the epithelial cells was further investigated. It was found that P2X7 receptors and reactive oxygen species were involved. Interestingly, the inflammasome activator monosodium urate crystals (MSU) induced the release of CXCL-8 and IL-1ß and the caspase-1 inhibitor Z-VADDCB suppressed the CS-induced release of CXCL-8. In addition, CS, CpGODN, lipopolysaccharide and MSU all increased the expression of caspase-1 and IL-1ß. In conclusion, our results demonstrate that CS releases CXCL-8 from HBE-14o cells via TLR4 and TLR9 and inflammasome activation. Therefore, inflammasome signaling in airway epithelial cells may play an important role in pathogenesis of diseases like COPD.
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Células Epiteliales/metabolismo , Interleucina-8/metabolismo , Humo , Fumar , Receptores Toll-Like/fisiología , Bronquios/citología , Caspasa 1/metabolismo , Línea Celular , Humanos , Inflamasomas/fisiología , Interleucina-1beta/biosíntesis , Interleucina-8/biosíntesis , Oligopéptidos/farmacología , Fumar/metabolismo , Nicotiana , Receptor Toll-Like 4/biosíntesis , Receptor Toll-Like 9/biosíntesisRESUMEN
Since an allergen-induced early asthmatic reaction is likely to be accompanied by oxidative stress and since levels of the endogenous antioxidant glutathione can be enhanced by a whey-based diet (undenatured whey protein concentrate, UWPC), it was investigated whether UWPC could alleviate allergen-induced lung contractions. Guinea pigs were fed water or UWPC twice a day starting at day - 3 up to day 20. The animals were sensitised to ovalbumin or received saline on day 0. Serum samples were taken at several days after sensitisation to measure allergen-specific IgG. On day 20, lungs were isolated and perfused with buffer containing the allergen ovalbumin. Airway contractions were assessed, and mediators and indicators for oxidative stress were measured in the lung effluent. Moreover, glutathione levels were determined in the liver. The indicator of oxidative stress and airway contractile mediator, 8-iso-PGF(2α), was increased upon ovalbumin challenge in ovalbumin-sensitised groups. Furthermore, thiobarbituric acid-reactive substances (TBARS) were increased as well. Sensitisation with ovalbumin increased IgG levels from day 12 up to day 20, which were not influenced by the UWPC diet. In contrast, the UWPC diet significantly enhanced glutathione levels in the liver. Moreover, the UWPC diet significantly reduced the ovalbumin-induced anaphylactic response by 45 % and decreased PGE2 levels by 55 % in the effluent fluid. We show for the first time that during anaphylaxis, there is acute oxidative stress in the respiratory tract. The UWPC diet did not influence the sensitisation response to the allergen but did increase endogenous glutathione levels. The UWPC diet profoundly reduces allergen-induced airway constrictions, which opens new avenues for dietary management of allergic diseases.
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Asma/dietoterapia , Broncoconstricción , Modelos Animales de Enfermedad , Glutatión/administración & dosificación , Proteínas de la Leche , Animales , Cobayas , Masculino , Estrés Oxidativo , Proteína de Suero de LecheRESUMEN
Emphysema is characterized by enlargement of the alveoli, which is the most important parameter to assess the presence and severity of this disease. Alveolar enlargement is primarily defined on morphological criteria; therefore, characterization of this disease with morphological parameters is a prerequisite to study the pathogenesis. For this purpose, different methods of lung fixation were evaluated in a murine model of LPS-induced lung emphysema. Five different methods of lung fixation were evaluated: intratracheal instillation of fixatives, in situ fixation, fixed-volume fixation, vascular whole body perfusion, and vacuum inflation. In addition, the effects of three different fixatives (10% formalin, Carnoy's, and agarose/10% formalin solution) and two embedding methods (paraffin and plastic) were investigated on the murine lung morphology. Mice received intranasal administration of LPS to induce alveolar wall destruction. Quantification of air space enlargement was determined by mean linear intercept analysis, and the histological sections were analyzed for the most optimal fixation method. Additionally, routine immunohistological staining was performed on lung tissue of PBS-treated mice. Intratracheal instillation of formalin or agarose/formalin solution, in situ fixation, and fixed-volume fixation provided a normal lung architecture, in contrast to the lungs fixed via whole body perfusion and vacuum inflation. Formalin-fixed lungs resulted in the most optimal lung morphology for lung emphysema analysis when embedded in paraffin, while for Carnoy's fixed lungs, plastic embedding was preferred. The histological findings, the mean linear intercept measurement, and the immunohistochemistry data demonstrated that fixation by intratracheal instillation of 10% formalin or in situ fixation with 10% formalin are the two most optimal methods to fix lungs for alveolar enlargement analysis to study lung emphysema.
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Fijadores/farmacología , Técnicas de Preparación Histocitológica , Pulmón , Enfisema Pulmonar/patología , Animales , Modelos Animales de Enfermedad , Lipopolisacáridos/farmacología , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/patología , Enfisema Pulmonar/inducido químicamenteRESUMEN
The prevalence of asthma has increased worldwide. The reasons for this rise remain unclear. Oxidative stress plays an important role in the pathogenesis of asthma. Glutathione (GSH) is the major representative of the class of nonprotein thiols and plays a pivotal role in a variety of enzymatic and nonenzymatic reactions that protect tissues against oxidative stress. In antioxidative reactions, GSH is converted into its oxidized form, glutathione disulfide (GSSG) that in its turn is enzymatically reduced into GSH to maintain a physiological redox balance. We used a guinea pig model of asthma to assess whether the early asthmatic reaction is associated with decreased lung levels of glutathione, and whether decreased glutathione is implicated in the increased airway smooth muscle reactivity that is associated with exposure of the lungs to allergen. Lung glutathione levels were decreased immediately after the onset of the early asthmatic reaction in vivo and associated with the release of 8-iso-PGF(2alpha), an indicator for oxidative stress. Glutathione ethylester, a glutathione precursor, blunted the airway obstruction during an early asthmatic reaction in a perfusion model and glutathione depletion rendered the airways hyperreactive. Glutathione ethyl ester in the buffer prevented this hyperreactivity. These results indicate that glutathione can modulate the early asthmatic reaction as well as the airway hyperresponsiveness.
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Asma/metabolismo , Asma/prevención & control , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/prevención & control , Glutatión/fisiología , Animales , Asma/fisiopatología , Hiperreactividad Bronquial/fisiopatología , Modelos Animales de Enfermedad , Glutatión/antagonistas & inhibidores , Cobayas , Histamina/farmacología , Masculino , Ovalbúmina/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Factores de TiempoRESUMEN
BACKGROUND/AIM: Evidence demonstrating an important role of the intestinal microbiota in the incidence of allergic disorders has led to the concept of using probiotics as possible antiallergic therapy. This study aimed to select a bacterial strain with the best antiallergic treatment effects from a panel of 6 bacterial strains in a mouse model of ovalbumin(OVA)-allergic asthma. METHODS: OVA-sensitized BALB/c mice were orally administered the bacterial strains Bifidobacterium breve M-16V, B. infantis NumRes251, B. animalis NumRes252 and NumRes253, Lactobacillus plantarum NumRes8 and L. rhamnosus NumRes6. After challenge by OVA inhalation in the lungs, the response to methacholine was measured. Pulmonary inflammation was assessed by analyzing bronchoalveolar lavage fluid for the presence of eosinophils, neutrophils, macrophages and lymphocytes and for interleukin 4, interleukin 5, interleukin 10 and interferon-gamma. OVA-specific IgE, IgG1 and IgG2a were measured in serum. Next, the effect on acute allergic skin reaction was measured after treatment with B. breve M-16V and L. plantarum NumRes8. RESULTS: Of the panel of 6 strains, B. breve M-16V and L. plantarum NumRes8 inhibited (1) the response to methacholine, (2) reduced the number of eosinophils in the bronchoalveolar lavage fluid, (3) reduced both OVA-specific IgE and (4) OVA-specific IgG1, whereas the other strains did not affect all these parameters simultaneously. B. breve M-16V but not L. plantarum NumRes8 reduced interleukin 4, interleukin 5 and interleukin 10. Furthermore, B. breve M-16V but not L. plantarum NumRes8 reduced acute allergic skin reactions to OVA. CONCLUSION: B. breve M-16V was identified as the most potent antiallergic strain.
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Asma/dietoterapia , Ovalbúmina/inmunología , Probióticos/administración & dosificación , Probióticos/uso terapéutico , Administración Oral , Animales , Asma/inmunología , Asma/patología , Asma/fisiopatología , Bifidobacterium , Hiperreactividad Bronquial/fisiopatología , Pruebas de Provocación Bronquial , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Recuento de Células , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Lactobacillus plantarum , Lacticaseibacillus rhamnosus , Linfocitos/patología , Macrófagos Alveolares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Neutrófilos/patología , Eosinofilia Pulmonar/patología , Pruebas Cutáneas , Organismos Libres de Patógenos Específicos , VacunaciónRESUMEN
Chronic obstructive pulmonary disease (COPD) is a major health problem and cigarette smoke is the main risk factor for the development of COPD. The characteristic changes in airway morphology, inflammatory cell infiltration and mediator expression in COPD may result from direct effects of cigarette smoke on airway cells. Toll-like receptors (TLRs) are key elements in pathogen recognition by the host immune system. Although TLRs have been intensely studied in innate immunity and infection, their critical role in noninfectious challenges has only recently emerged. Here we investigate whether cigarette smoke induces TLR9 signalling in human neutrophils. Human neutrophils were isolated from buffy coat and exposed to cigarette smoke extract. The production of CXC chemokine ligand (CXCL)8 was measured as a functional readout and the role of TLR9 signalling was investigated. Cigarette smoke extract induced CXCL8 release via TLR9 activation in neutrophils, which was confirmed in TLR9 stably transfected human embryonic kidney 293 cells. Moreover, cigarette smoke extract upregulated the expression of TLR9 and the upregulated expression was suppressed by N-acetylcysteine. TLR9 mediates cigarette smoke-induced release of CXCL8 and this may contribute to the accumulation of neutrophils and inflammation within the airways of smokers.
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Interleucina-8/metabolismo , Neutrófilos/metabolismo , Neumonía/inmunología , Fumar/inmunología , Receptor Toll-Like 9/metabolismo , Células HEK293 , Humanos , Interleucina-8/genética , Interleucina-8/inmunología , FN-kappa B/genética , FN-kappa B/metabolismo , Neutrófilos/citología , Óxido Nítrico/metabolismo , Neumonía/epidemiología , Neumonía/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Riesgo , Transducción de Señal/inmunología , Fumar/epidemiología , Fumar/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunología , TransfecciónRESUMEN
Inflammation is a stereotypical physiological response to infections and tissue injury; it initiates pathogen killing as well as tissue repair processes and helps to restore homeostasis at infected or damaged sites. Acute inflammatory reactions are usually self-limiting and resolve rapidly, due to the involvement of negative feedback mechanisms. Thus, regulated inflammatory responses are essential to remain healthy and maintain homeostasis. However, inflammatory responses that fail to regulate themselves can become chronic and contribute to the perpetuation and progression of disease. Characteristics typical of chronic inflammatory responses underlying the pathophysiology of several disorders include loss of barrier function, responsiveness to a normally benign stimulus, infiltration of inflammatory cells into compartments where they are not normally found in such high numbers, and overproduction of oxidants, cytokines, chemokines, eicosanoids and matrix metalloproteinases. The levels of these mediators amplify the inflammatory response, are destructive and contribute to the clinical symptoms. Various dietary components including long chain omega-3 fatty acids, antioxidant vitamins, plant flavonoids, prebiotics and probiotics have the potential to modulate predisposition to chronic inflammatory conditions and may have a role in their therapy. These components act through a variety of mechanisms including decreasing inflammatory mediator production through effects on cell signaling and gene expression (omega-3 fatty acids, vitamin E, plant flavonoids), reducing the production of damaging oxidants (vitamin E and other antioxidants), and promoting gut barrier function and anti-inflammatory responses (prebiotics and probiotics). However, in general really strong evidence of benefit to human health through anti-inflammatory actions is lacking for most of these dietary components. Thus, further studies addressing efficacy in humans linked to studies providing greater understanding of the mechanisms of action involved are required.
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Inflamación/fisiopatología , Fenómenos Fisiológicos de la Nutrición/fisiología , Artritis Reumatoide/dietoterapia , Artritis Reumatoide/fisiopatología , Enfermedades Cardiovasculares/dietoterapia , Enfermedades Cardiovasculares/fisiopatología , Enfermedad Celíaca/dietoterapia , Enfermedad Celíaca/fisiopatología , Humanos , Inflamación/dietoterapia , Enfermedades Inflamatorias del Intestino/dietoterapia , Enfermedades Inflamatorias del Intestino/fisiopatología , Obesidad/dietoterapia , Obesidad/fisiopatología , Hipersensibilidad Respiratoria/dietoterapia , Hipersensibilidad Respiratoria/fisiopatología , Enfermedades de la Piel/dietoterapia , Enfermedades de la Piel/fisiopatologíaRESUMEN
BACKGROUND: Mast cells are important effector cells in innate or acquired immunity that contribute to host defence. Excessive activation of mast cells can result in the development of allergic diseases, including atopic asthma. Mast cell activation by IgE and specific antigen induces the cells to release spasmogenic, vasoactive and pro-inflammatory mediators, which enhance airway smooth muscle contraction, vascular permeability and inflammatory cell recruitment. Recently, we have demonstrated that exposure of mast cells to cigarette smoke medium (CSM) triggered mast cells to produce chemokines. On the other hand, smoking may decrease the risk of allergic sensitization, which could be explained by a reduced IgE production or a diminished response of mast cells to activation of the IgE receptor. OBJECTIVE: In this study, we investigated the effect of CSM on the allergic activation of mast cells through IgE and antigen. METHODS: Primary cultured murine mast cells were exposed to CSM and activated with IgE and antigen or lipopolysaccharide (LPS). The release of granules, production of leukotrienes, chemokines and cytokines was determined in the supernatants by ELISA. The effect of CSM exposure on intracellular signalling, especially the nuclear factor (NF)-kappaB and extracellular signal-regulated kinase (Erk)1/2 pathways, was analysed by Western blotting. RESULTS: CSM suppressed IgE-mediated degranulation and cytokine release, but no effect was observed on leukotriene release. CSM induced phosphorylation of Erk1/2 in mast cells. In CSM-exposed mast cells, activating transcription factor (ATF)-1 was phosphorylated after stimulation with IgE/Ag. LPS-activated mast cells were not influenced by CSM. CONCLUSION: Our study suggests that exposure to cigarette smoke may lead to a reduced allergic activation of mast cells without affecting their response to activation via e.g. bacterial-derived LPS.
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Degranulación de la Célula/inmunología , Inmunoglobulina E/sangre , Mastocitos/inmunología , Nicotiana/inmunología , Humo , Factor de Transcripción Activador 1/inmunología , Factor de Transcripción Activador 1/metabolismo , Animales , Células Cultivadas , Citocinas/efectos de los fármacos , Citocinas/inmunología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Leucotrienos/biosíntesis , Leucotrienos/inmunología , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Fosforilación/efectos de los fármacos , Fosforilación/inmunologíaRESUMEN
Specific mixtures of prebiotic oligosaccharides showed immune modulatory effects in previous murine vaccination experiments, suggesting a shift towards T-helper 1 (Th1) immunity. These mixtures consisted of short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharides (lcFOS) in a 9:1 ratio (Immunofortis), with or without pectin-derived acidic oligosaccharides (pAOS). To investigate whether these mixtures could suppress Th2-related responses, they were tested in an ovalbumin (OVA)-induced model for experimental allergic asthma in BALB/c mice. Supplementation with two mixtures of scGOS/lcFOS and scGOS/lcFOS/pAOS at approximately 1% (w/w% net oligosaccharides) in the diet, starting two weeks before OVA sensitization and lasting until the end of the experiment, decreased of several parameters of allergic asthma. The OVA-induced airway inflammation and hyperresponsiveness was significantly suppressed by both mixtures. Moreover, OVA-specific IgE titers were decreased by more than 25%, although this effect was not significant. The effects of the oligosaccharide mixture with pAOS appeared to be more pronounced than the effects of the scGOS/lcFOS mixture without pAOS, but a direct comparison between the mixtures was not made. Overall, the results further support the hypothesis that specific mixtures of oligosaccharides modulate the Th1/Th2 balance by enhancing Th1-related and suppressing Th2-related parameters.
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Asma/prevención & control , Carbohidratos de la Dieta/farmacología , Suplementos Dietéticos , Oligosacáridos/farmacología , Animales , Asma/sangre , Asma/inmunología , Pruebas de Provocación Bronquial , Líquido del Lavado Bronquioalveolar/citología , Recuento de Células , Carbohidratos de la Dieta/administración & dosificación , Fructanos/administración & dosificación , Fructanos/farmacología , Galactanos/administración & dosificación , Galactanos/farmacología , Inmunización , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/farmacología , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Masculino , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Oligosacáridos/administración & dosificación , Ovalbúmina/inmunología , Pectinas/química , Pectinas/farmacología , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/fisiopatología , Hipersensibilidad Respiratoria/prevención & controlAsunto(s)
Asma/inmunología , Bloqueadores de los Canales de Calcio/farmacología , Calmodulina/inmunología , Proteínas Portadoras/farmacología , Oligopéptidos/farmacología , Péptidos/farmacología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Animales , Calmodulina/análogos & derivados , Calmodulina/antagonistas & inhibidores , Células Epiteliales/inmunología , Cobayas , Péptidos y Proteínas de Señalización Intercelular , Macrófagos/inmunología , Mastocitos/inmunología , Modelos AnimalesRESUMEN
BACKGROUND: Exposure of patients with atopic asthma to allergens produces a long term increase in exhaled nitric oxide (FENO), probably reflecting inducible NO synthase (NOS) expression. In contrast, bradykinin (BK) rapidly reduces FENO. It is unknown whether BK suppresses increased FENO production after allergen exposure in asthma, and whether it modulates FENO via NOS inhibition. METHODS: Levels of FENO in response to aerosolised BK were studied before (day 3) and 48 hours after (day 10) randomised diluent (diluent/placebo/BK (Dil/P/BK)), allergen (allergen/placebo/BK (All/P/BK), and allergen/L-NMMA/BK (All/L/BK)) challenges (day 8) in 10 atopic, steroid naïve, mild asthmatic patients with dual responses to inhaled house dust mite extract. To determine whether BK modulates FENO via NOS inhibition, subjects performed pre- and post-allergen BK challenges after pretreatment with the NOS inhibitor L-NMMA in the All/L/BK period. RESULTS: Allergen induced a fall in FENO during the early asthmatic reaction (EAR) expressed as AUC(0-1) (ANOVA, p=0.04), which was followed by a rise in FENO during the late asthmatic reaction (LAR) expressed as AUC(1-48) (ANOVA, p=0.008). In the Dil/P/BK period, FENO levels after BK on pre- and post-diluent days were lower than FENO levels after placebo (difference 23.5 ppb (95% CI 6.2 to 40.9) and 22.5 ppb (95% CI 7.3 to 37.7), respectively; p<0.05). Despite the long lasting increase in FENO following allergen challenge in the LAR, BK suppressed FENO levels at 48 hours after allergen challenge in the All/P/BK period, lowering the increased FENO (difference from placebo 54.3 ppb (95% CI 23.8 to 84.8); p=0.003) to the baseline level on the pre-allergen day (p=0.51). FENO levels were lower after L-NMMA than after placebo on pre-allergen (difference 10.85 ppb (95% CI 1.3 to 20.4); p=0.03) and post-allergen (difference 36.2 ppb (95% CI 5.5 to 66.9); p=0.03) days in the All/L/BK and All/P/BK periods, respectively. L-NMMA did not significantly potentiate the pre- and post-allergen reduction in BK induced FENO. CONCLUSIONS: Bradykinin suppresses the allergen induced increase in exhaled NO in asthma; this is not potentiated by L-NMMA. Bradykinin and L-NMMA may follow a common pathway in reducing increased NO production before and after experimental allergen exposure. Reinforcement of this endogenous protective mechanism should be considered as a therapeutic target in asthma.