RESUMEN
BACKGROUND: In a mouse model of mild chronic asthma, both inflammation and remodelling can be suppressed by dexamethasone (a glucocorticoid) and roflumilast (a selective phosphodiesterase-4 inhibitor). OBJECTIVE: To better understand the underlying molecular mechanisms, we investigated the effects of treatment on airway expression of inflammation-related cytokines, as well as on epithelial expression of growth factors. METHODS: BALB/c mice systemically sensitized to ovalbumin were challenged with aerosolized antigen for 6 weeks and treated with roflumilast or dexamethasone during the final 2 weeks. Expression of mRNA, for a variety of cytokines and growth factors, was assessed in selectively dissected proximal airways or in airway epithelium obtained by laser capture microdissection. RESULTS: In the airway wall of vehicle-treated challenged animals, there was significantly elevated expression of mRNA for a variety of pro-inflammatory and T helper type 2 cytokines, as well as for IFN-gamma. All these cytokines were suppressed by dexamethasone. Treatment with roflumilast reduced expression of IL-17A, TNF-alpha, granulocyte-macrophage colony-stimulating factor and IL-6, but did not inhibit other cytokines. Both drugs suppressed the enhanced expression of mRNA for growth factors such as TGF-beta1 and FGF-2 in airway epithelium. CONCLUSIONS: Whereas dexamethasone non-specifically inhibits numerous mediators involved in inflammation and the immune response, roflumilast selectively inhibits a subset of pro-inflammatory cytokines and growth factors. These mediators and/or the cells that produce them may have critical roles in the pathogenesis of the lesions of chronic asthma.
Asunto(s)
Aminopiridinas/uso terapéutico , Antiasmáticos/uso terapéutico , Asma/tratamiento farmacológico , Benzamidas/uso terapéutico , Citocinas/antagonistas & inhibidores , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Inhibidores de Fosfodiesterasa/uso terapéutico , Aminopiridinas/farmacología , Animales , Antiasmáticos/farmacología , Asma/inmunología , Benzamidas/farmacología , Bronquios/efectos de los fármacos , Bronquios/patología , Enfermedad Crónica , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Citocinas/metabolismo , Dexametasona/farmacología , Femenino , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Inhibidores de Fosfodiesterasa 4 , Organismos Libres de Patógenos EspecíficosRESUMEN
OBJECTIVES: To investigate whether fetal cardiac axis is affected by the presence of an abdominal wall defect (AWD) independent of congenital heart disease (CHD). METHODS: Video ultrasound records from fetuses with AWDs identified from 1991-2004 were reviewed. Still images of the fetal cardiac four-chamber view were digitized and two independent examiners measured the cardiac axis. A cardiac axis of >65 degrees or <25 degrees was considered abnormal. Maternal charts were reviewed for fetal echocardiogram results and neonatal charts were reviewed for confirmation of CHD and type of AWD. RESULTS: Of 17 fetuses with omphalocele and 42 fetuses with gastroschisis, 16 (27%) fetuses had an abnormal cardiac axis, while only seven (12%) had CHD. Fifty-nine percent of fetuses with omphalocele had an abnormal cardiac axis and 35% had CHD. Fourteen percent of fetuses with gastroschisis had an abnormal cardiac axis and 2% had CHD. Of 43 fetuses with a normal cardiac axis, only one had CHD. CONCLUSIONS: Fetal cardiac axis is often affected by the presence of an AWD independent of CHD. A normal cardiac axis in fetuses with AWDs is an accurate predictor of the absence of CHD, the negative predictive value being 97.7%.
Asunto(s)
Pared Abdominal/anomalías , Cardiopatías Congénitas/diagnóstico por imagen , Ultrasonografía Prenatal/métodos , Pared Abdominal/diagnóstico por imagen , Ecocardiografía , Femenino , Gastrosquisis/diagnóstico por imagen , Hernia Umbilical/diagnóstico por imagen , Humanos , Embarazo , Resultado del Embarazo , Curva ROC , Estudios RetrospectivosRESUMEN
AIM: To evaluate the effectiveness of a programme of asthma clubs in improving quality of life in primary school children with asthma. METHODS: A cluster randomised intervention trial was undertaken in 22 primary schools within the urban area of south and east Belfast, Northern Ireland. Schools were randomised in pairs to immediate or delayed groups. The study subjects comprised 173 children aged 7-11 years whose parents had notified the school of their asthma diagnosis. Children attended school based weekly clubs over an 8 week period. The main outcome measures were the interview administered Paediatric Quality of Life Questionnaire scores, ranging from 1 (worst) to 7 (best), spirometry, and inhaler technique. RESULTS: Over 15 weeks, small but non-significant improvements in the overall quality of life score (mean 0.20; 95% confidence interval (CI) -0.20 to 0.61) and in each of its three components, activity limitation (0.20; -0.43 to 0.84), symptoms (0.23; -0.23 to 0.70), and emotional function (0.17; -0.18 to 0.52), were observed in the immediate compared with the delayed group. Inhaler technique at week 16 was markedly better in the immediate group, with 56% having correct technique compared with 15% in the delayed group. No significant effect of the intervention on spirometry results could be demonstrated. CONCLUSION: This primary school based asthma education programme resulted in sustained improvements in inhaler technique, but changes in quality of life scores were not significant.
Asunto(s)
Asma/terapia , Psicoterapia de Grupo , Calidad de Vida , Antiasmáticos/administración & dosificación , Asma/tratamiento farmacológico , Asma/psicología , Niño , Análisis por Conglomerados , Femenino , Humanos , Masculino , Nebulizadores y Vaporizadores , Educación del Paciente como Asunto , Instituciones Académicas , Sociedades , Resultado del TratamientoRESUMEN
BACKGROUND: Type 2 T helper lymphocytes (Th2 cells) and their cytokine products are important in the pathogenesis of asthma. OBJECTIVE: To examine the contribution of the signal transducer and activator of transcription (STAT) 6 pathway, involved in Th2 cytokine signalling, to the development of lesions of chronic asthma. METHODS: BALB/c mice sensitized to ovalbumin were chronically challenged by inhalational of low mass concentrations of antigen for 6 weeks. Airway lesions in wild-type mice were compared with those in STAT6-deficient mice and in IL-4/13 double-deficient mice by histomorphometry and immunohistochemistry. Airway responses to methacholine were evaluated by whole-body plethysmography. Cytokine production by peribronchial lymph node cells was quantified by enzyme immunoassay. RESULTS: STAT6-/- mice developed a variety of airway lesions that were at least equivalent to those in wild-type mice, including accumulation of intraepithelial eosinophils and of chronic inflammatory cells in the lamina propria, subepithelial fibrosis and epithelial thickening. In addition, STAT6-/- mice exhibited exaggerated airway hyper-reactivity (AHR) compared to wild-type animals. This was despite a shift from a Th2 to a Th1 pattern of immunoglobulin production by plasma cells in the inflammatory infiltrate and diminished mucous cell hyperplasia/metaplasia, together with increased production of IFN-gamma by peribronchial lymph node cells, consistent with absence of signalling via the STAT6 pathway. In contrast, gene-targeted IL-4/13-/- mice exhibited markedly diminished eosinophil recruitment and airway remodelling, as well as absence of AHR. CONCLUSIONS: In this model, the effects of STAT6 deficiency were in marked contrast to the suppression of inflammation and AHR described in models of allergic bronchopulmonary inflammation. These results, which provide evidence of STAT6-independent AHR in an inhalational challenge model of chronic asthma, emphasize the critical effector roles of IL-4 and IL-13, as well as the need to use appropriate models to understand cytokine signalling pathways that may be potential therapeutic targets in asthma.
Asunto(s)
Asma/inmunología , Citocinas/inmunología , Células Th2/inmunología , Transactivadores/inmunología , Animales , Asma/patología , Asma/fisiopatología , Pruebas de Provocación Bronquial , Enfermedad Crónica , Citocinas/biosíntesis , Femenino , Inmunoglobulina G/análisis , Interleucina-13/inmunología , Interleucina-4/inmunología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Mucosa Respiratoria/patología , Factor de Transcripción STAT6 , Transducción de SeñalRESUMEN
Asthma pathophysiology is intimately regulated by CD4(+) Th2 lymphocytes and the cytokines interleukin (IL)-4 and IL-13. However, the mechanisms by which these cytokines promote disease have not been fully elucidated. In order to identify novel molecular mediators of allergy, a comparison was made of the bronchoalveolar lavage, which demonstrated that the Ym2 protein was abundantly up-regulated in the lung during the development of allergy. Low levels of the Ym1 isomer were also detected. Importantly, neither Ym1 nor Ym2 has been characterized previously in the context of allergic pulmonary inflammation. Western immunoblot showed that enhanced expression of these proteins was dependent on CD4(+) T cells and IL-4 or IL-13 signaling via the IL-4Ralpha subunit. In addition, intratracheal instillation of IL-13 into naive mice was sufficient to induce expression. Ym1 is homologous to eosinophil chemotactic factor L. However, only weak eosinophil chemotaxis was observed in response to Ym protein in both in vitro and in vivo assays. By contrast, the homology of Ym1 and Ym2 to proteins associated with tissue remodeling, together with the previous findings that Ym1 is homologous to chitinase and binds heparin sulfate and GlcN oligomers (chitobiose, chitotriose, and chitotetraose), strongly suggests these proteins play an important role in airway wall remodeling in the allergic lung.
Asunto(s)
Hipersensibilidad/metabolismo , Interleucina-13/fisiología , Interleucina-4/fisiología , Lectinas/genética , Transducción de Señal , beta-N-Acetilhexosaminidasas , Secuencia de Aminoácidos , Animales , Líquido del Lavado Bronquioalveolar/química , Linfocitos T CD4-Positivos/fisiología , Eosinófilos/efectos de los fármacos , Eosinófilos/fisiología , Lectinas/análisis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Receptores de Interleucina-4/fisiologíaRESUMEN
Interleukin-13 (IL-13) plays a key role in immune responses and inflammation. A structural model of human IL-13 (HuIL-13) based on the nuclear magnetic resonance and X-ray structure of IL-4 is put forward. Unlike previous models, this model is based on new sequence alignments that take into account the formation of the two disulfide linkages that have been determined experimentally. The proposed structure of human IL-13 is similar to IL-4, consisting of a four helix bundle with hydrophobic residues lining the core of the molecule and surface polar residues showing a high degree of solvent accessibility. Regions of HuIL-13 that are critical for the interaction with its receptors are explored and discussed in relation to existing mutagenic studies. From these studies we predict that helices A and C of HuIL-13 interact with the IL-4 receptor alpha (IL-4Ralpha) region and helix D is responsible for the interaction with the IL-13 receptor alpha 1 (IL-13Ralpha1) receptor.
Asunto(s)
Interleucina-13/química , Interleucina-4/metabolismo , Receptores de Interleucina-4/metabolismo , Receptores de Interleucina/metabolismo , Secuencia de Aminoácidos , Animales , Simulación por Computador , Humanos , Interleucina-13/genética , Interleucina-13/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13 , Interleucina-4/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Interleucina-13 , Alineación de Secuencia , Programas InformáticosRESUMEN
Escherichia coli contains two major systems for transporting inorganic phosphate (P(i)). The low-affinity P(i) transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external P(i) concentrations by the pho regulon and is an ABC transporter. We isolated a third putative P(i) transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P(i) transporter that may be repressed at low P(i) levels by the pho regulon. While a pitB(+) cosmid clone allowed growth on medium containing 500 microM P(i), E. coli with wild-type genomic pitB (pitA Delta pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB Delta pstC345 triple mutant. The mutation Delta pstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB(+) pitA Delta pstC345 strain to utilize P(i), with P(i) uptake rates significantly higher than background levels. In addition, the apparent K(m) of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.
Asunto(s)
Proteínas Portadoras/química , Escherichia coli/metabolismo , Fosfatos/metabolismo , Transporte Biológico Activo , Proteínas Portadoras/genética , Codón Iniciador , Escherichia coli/genética , Cinética , Datos de Secuencia Molecular , Mutación , Proteínas de Unión a Fosfato , Plásmidos/metabolismo , RegulónRESUMEN
The potent spasmogenic properties of IL-13 have identified this molecule as a potential regulator of airways hyperreactivity (AHR) in asthma. Although IL-13 is thought to primarily signal through the IL-13Ralpha1-IL-4Ralpha complex, the cellular and molecular components employed by this cytokine to induce AHR in the allergic lung have not been identified. By transferring OVA-specific CD4(+) T cells that were wild type (IL-13(+/+) T cells) or deficient in IL-13 (IL-13(-/-) T cells) to nonsensitized mice that were then challenged with OVA aerosol, we show that T cell-derived IL-13 plays a key role in regulating AHR, mucus hypersecretion, eotaxin production, and eosinophilia in the allergic lung. Moreover, IL-13(+/+) T cells induce these features (except mucus production) of allergic disease independently of the IL-4Ralpha chain. By contrast, IL-13(+/+) T cells did not induce disease in STAT6-deficient mice. This shows that IL-13 employs a novel component of the IL-13 receptor signaling system that involves STAT6, independently of the IL-4Ralpha chain, to modulate pathogenesis. We show that this novel pathway for IL-13 signaling is dependent on T cell activation in the lung and is critically linked to downstream effector pathways regulated by eotaxin and STAT6.
Asunto(s)
Hiperreactividad Bronquial/inmunología , Quimiocinas CC , Interleucina-13/fisiología , Receptores de Interleucina-4/fisiología , Hipersensibilidad Respiratoria/inmunología , Traslado Adoptivo , Animales , Asma/genética , Asma/inmunología , Hiperreactividad Bronquial/etiología , Hiperreactividad Bronquial/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Quimiocina CCL11 , Citocinas/metabolismo , Citocinas/fisiología , Inmunofenotipificación , Interleucina-13/administración & dosificación , Interleucina-13/deficiencia , Interleucina-13/genética , Intubación Intratraqueal , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de Interleucina-4/deficiencia , Receptores de Interleucina-4/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Hipersensibilidad Respiratoria/etiología , Hipersensibilidad Respiratoria/genética , Factor de Transcripción STAT6 , Transducción de Señal/genética , Transducción de Señal/inmunología , Subgrupos de Linfocitos T , Células TH1/metabolismo , Células TH1/trasplante , Células Th2/metabolismo , Células Th2/trasplante , Transactivadores/deficiencia , Transactivadores/genética , Transactivadores/fisiologíaRESUMEN
In this review we identify the elemental signals that regulate eosinophil accumulation in the allergic lung. We show that there are two interwoven mechanisms for the accumulation of eosinophils in pulmonary tissues and that these mechanisms are linked to the development of airways hyperreactivity (AHR). Interleukin-(IL)-5 plays a critical role in the expansion of eosinophil pools in both the bone marrow and blood in response to allergen provocation of the airways. Secondly, IL-4 and IL-13 operate within the allergic lung to control the transmigration of eosinophils across the vascular bed into pulmonary tissues. This process exclusively promotes tissue accumulation of eosinophils. IL-13 and IL-4 probably act by activating eosinophil-specific adhesion pathways and by regulating the production of IL-5 and eotaxin in the lung compartment. IL-5 and eotaxin co-operate locally in pulmonary tissues to selectively and synergistically promote eosinophilia. Thus, IL-5 acts systemically to induce eosinophilia and within tissues to promote local chemotactic signals. Regulation of IL-5 and eotaxin levels within the lung by IL-4 and IL-13 allows Th2 cells to elegantly co-ordinate tissue and peripheral eosinophilia. Whilst the inhibition of either the IL-4/IL-13 or IL-5/eotaxin pathways resulted in the abolition of tissue eosinophils and AHR, only depletion of IL-5 and eotaxin concurrently results in marked attenuation of pulmonary inflammation. These data highlight the importance of targeting both IL-5 and CCR3 signalling systems for the resolution of inflammation and AHR associated with asthma.
Asunto(s)
Quimiocinas CC , Quimiotaxis de Leucocito , Citocinas/fisiología , Interleucina-5/fisiología , Eosinofilia Pulmonar/fisiopatología , Animales , Asma/inmunología , Asma/patología , Adhesión Celular , Quimiocina CCL11 , Eosinófilos , Humanos , Inmunoterapia , Inflamación , Interleucina-13/deficiencia , Interleucina-13/genética , Interleucina-13/fisiología , Interleucina-4/fisiología , Ratones , Ratones Noqueados , Modelos Inmunológicos , Eosinofilia Pulmonar/inmunología , Transducción de Señal , Células Th2/inmunologíaRESUMEN
T helper (Th)-2-derived cytokines and their involvement in the recruitment and activation of inflammatory cells crucially orchestrate asthma pathogenesis. A notable cellular component of this allergy-induced inflammation is the eosinophil. However, whether the eosinophil is an obligatory mediator for enhancing airways hyperreactivity (AHR) to cholinergic stimuli, a watershed of the asthmatic lung, is somewhat controversial. In this investigation we have endeavoured to define the spatial requirements for IL-4 and IL-13, and the downstream effector molecules, IL-5 and the CC chemokine eotaxin, for the recruitment of eosinophils and the development of AHR in a murine model of allergic pulmonary disease. These studies are of particular importance considering clinical trials, with either the soluble IL-4Ralpha subunit or a humanized anti-IL-5 antibody, are being conducted. Interestingly, our studies show that depletion of both IL-4 and IL-13 is necessary to ablate pulmonary eosinophilia and AHR, and that this may be attributed to the role these cytokines play in regulating the expression of the eosinophil- activating molecules, IL-5 and eotaxin. While it is clear that depletion of IL-5 diminishes pulmonary eosinophilia, we demonstrate in BALB/c mice that a deficiency in both IL-5 and eotaxin is necessary to abolish both the trafficking of eosinophils to the lung and AHR. However, in contrast to the neutrophil-rich inflammation observed in mice deficient in both IL-4 and IL-13, inflammation per se in mice deficient in both IL-5 and eotaxin is significantly attenuated. This suggests that asthma immunotherapy may be better directed towards the eosinophil- activating molecules IL-5 and eotaxin, rather than towards pleiotrophic molecules such IL-4 and IL-13, which are additionally important in modulating alternative inflammatory responses.
Asunto(s)
Asma/inmunología , Quimiocinas CC , Eosinófilos/inmunología , Pulmón/inmunología , Animales , Quimiocina CCL11 , Citocinas/inmunología , Humanos , Interleucina-13/inmunología , Interleucina-4/inmunología , Interleucina-5/inmunologíaRESUMEN
The proliferation of stent designs poses difficult problems to clinicians, who have to learn the relative merits of all stents to ensure optimal selection for each lesion, and also to regulatory authorities who have the dilemma of preventing the inappropriate marketing of substandard stents while not denying patients the benefits of advanced technology. Of the major factors influencing long-term results, those of patency and restenosis are being actively studied whereas the mechanical characteristics of devices influencing the technical results of stenting remain under-investigated. Each different stent design has its own particular features. A robust method for the independent objective comparison of the mechanical performance of each design is required. To do this by experimental measurement alone may be prohibitively expensive. A less costly option is to combine computer analysis, employing the standard numerical technique of the finite element method (FEM), with targeted experimental measurements of the specific mechanical behaviour of stents. In this paper the FEM technique is used to investigate the structural behaviour of two different stent geometries: Freedom stent geometry and Palmaz-Schatz (P-S) stent geometry. The effects of altering the stent geometry, the stent wire diameter and contact with (and material properties of) a hard eccentric intravascular lesion (simulating a calcified plaque) on stent mechanical performance were investigated. Increasing the wire diameter and the arterial elastic modulus by 150% results in the need to increase the balloon pressure to expand the stent by 10-fold. Increasing the number of circumferential convolutions increases the pressure required to initiate radial expansion of mounted stents. An incompressible plaque impinging on the mid portion of a stent causes a gross distortion of the Freedom stent and an hour-glass deformity in the P-S stent. These findings are of relevance for future comparative studies of the mechanical performance of stents, in designing newer stents and also in clinical practice.
Asunto(s)
Análisis de Elementos Finitos , Stents , Humanos , Diseño de PrótesisRESUMEN
In this investigation, we have examined the integrated relationship between IL-13, IL-4, and IL-5 for the development of airways hyperreactivity (AHR) in a model of asthma in BALB/c mice. Sensitization and aeroallergen challenge of both wild-type (WT) and IL-13 gene-targeted (IL-13-/-) mice induced allergic disease that was characterized by pulmonary eosinophilia and AHR to beta-methacholine. Although these responses in IL-13-/- mice were heightened compared with WT, they could be reduced to the level in nonallergic mice by the concomitant neutralization of IL-4. Mice in which both IL-4 and IL-13 were depleted displayed a marked reduction in tissue eosinophils, despite the development of a blood eosinophilia. Similar neutralization of IL-4 in WT mice only partially reduced AHR with no effect on tissue eosinophilia. In addition, neutralization of IL-5 in IL-13-/- mice, but not in WT mice, inhibited AHR, suggesting that tissue eosinophilia is linked to the mechanism underlying AHR only in the absence of IL-13. Additionally, mucus hypersecretion was attenuated in IL-13-/- mice, despite the persistence of AHR. Taken together, our data suggest both a modulatory role for IL-13 during sensitization and a proinflammatory role during aeroallergen challenge. The latter process appears redundant with respect to IL-4.
Asunto(s)
Hiperreactividad Bronquial/inmunología , Interleucinas/fisiología , Transducción de Señal/inmunología , Adyuvantes Inmunológicos/deficiencia , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Animales , Hiperreactividad Bronquial/etiología , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/metabolismo , Citocinas/biosíntesis , Sueros Inmunes/farmacología , Interleucina-13/deficiencia , Interleucina-13/genética , Interleucina-13/fisiología , Interleucina-4/antagonistas & inhibidores , Interleucina-4/inmunología , Interleucina-4/fisiología , Interleucina-5/antagonistas & inhibidores , Interleucina-5/inmunología , Interleucina-5/fisiología , Interleucinas/antagonistas & inhibidores , Interleucinas/genética , Interleucinas/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Eosinofilia Pulmonar/genética , Eosinofilia Pulmonar/inmunología , Eosinofilia Pulmonar/prevención & control , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patologíaRESUMEN
Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with otitis media and the exacerbation of chronic bronchitis. This study reports the vaccine potential of three peptides representing conserved regions of the NTHi P5 outer membrane protein which have been fused to a promiscuous measles virus F protein T-cell eptitope (MVF). The peptides correspond to a region in surface loop one (MVF/L1A), the central region of loop four (MVF/L4), and a C-terminal region homologous to peptide 10 of OprF from Pseudomonas aeruginosa (MVF/H3). Immunization of rats with MVF/H3 was the most efficacious in significantly reducing the number of viable NTHi in both the broncho-alveolar lavage fluid (74%) and lung homogenates (70%), compared to control rats. Importantly, despite significantly increased rates of clearance, immunization with MVF/H3 elicited poor antibody responses, suggesting that cell-mediated rather than humoral responses play an important role in the enhanced clearance of NTHi in this model.
Asunto(s)
Proteínas Bacterianas/genética , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/patogenicidad , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/microbiología , Péptidos/genética , Porinas/genética , Pseudomonas aeruginosa/genética , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/metabolismo , Especificidad de Anticuerpos , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Reacciones Cruzadas , Modelos Animales de Enfermedad , Vacunas contra Haemophilus/genética , Vacunas contra Haemophilus/inmunología , Haemophilus influenzae/inmunología , Inmunización , Datos de Secuencia Molecular , Péptidos/inmunología , Porinas/inmunología , Pseudomonas aeruginosa/inmunología , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de AminoácidoRESUMEN
Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen, and heterogeneity in the surface-exposed immunodominant domains of NTHI proteins is thought to be associated with the failure of an infection to stimulate an immune response that is cross-protective against heterologous NTHI strains. The aim of this study was to assess the vaccine potential of a surface-exposed component of the NTHI human transferrin receptor, TbpB, and to determine if the antibody response elicited was cross-reactive with heterologous strains of NTHI. The efficacy of immunization with a recombinant form of TbpB (rTbpB) was determined by assessing the pulmonary clearance of viable bacteria 4 h after a live challenge with NTHI. There was a significant reduction in the number of viable bacteria in both the bronchoalveolar lavage fluid (34% for the 20-microgram dose and 58% for the 40-microgram dose) and lung homogenates (26% for the 20-microgram dose and 60% for the 40-microgram dose) of rats immunized with rTbpB compared to the control animals. While rTbpB-specific antibodies from immunized rats were nonspecific in the recognition of TbpB from six heterologous NTHI strains on Western blots, these antibodies differed in their ability to block transferrin binding to heterologous strains and to cross-react in bactericidal assays. If bactericidal antibodies are key indicators of the efficacy of the immune response in eliminating NTHI, this data suggests that while immunization with rTbpB stimulates protective responses against the homologous isolate, variability in the recognition of TbpB from heterologous isolates may limit the potential of rTbpB as an NTHI vaccine component.
Asunto(s)
Proteínas Portadoras/inmunología , Haemophilus influenzae/inmunología , Pulmón/microbiología , Receptores de Transferrina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/inmunología , Vacunas Bacterianas/inmunología , Secuencia de Bases , Proteínas Portadoras/genética , Reacciones Cruzadas , Cartilla de ADN/genética , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/microbiología , Infecciones por Haemophilus/prevención & control , Haemophilus influenzae/clasificación , Haemophilus influenzae/patogenicidad , Humanos , Inmunidad Mucosa , Inmunización , Proteínas de Unión a Hierro , Masculino , Ratas , Ratas Wistar , Receptores de Transferrina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/prevención & control , Proteína B de Unión a Transferrina , Proteínas de Unión a TransferrinaRESUMEN
Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen, commonly associated with otitis media and exacerbations of chronic bronchitis. Studies concerning the pathogenesis of NTHi have proposed an important function for P5, an outer membrane protein believed to play a role in the initiation of infection by mediating adherence to respiratory mucin. P5 has also generated interest as a potential vaccine candidate. In a previous study, an NTHi library screen with antibodies raised against P5 purified from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified protein was contaminated with closely migrating proteins. Consequently, the aim of this study was to express P5 in a heterologous system to overcome potential contamination with NTHi proteins that may complicate analytical or vaccine studies. Recombinant P5, with an N terminal extension of 10 residues that included six histidines, was cloned and expressed in Escherichia coli. The rP5 was purified with the Talon metal affinity resin in a denatured form and then refolded by incorporation into mixed-detergent micelles of octylglucoside and SDS. Circular dichroism of the refolded rP5 demonstrated 55% beta-strand content, which is consistent with the beta-strand content of native P5 and the homologous E. coli protein OmpA.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Haemophilus influenzae/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Cromatografía de Afinidad , Dicroismo Circular , Clonación Molecular , Detergentes , Electroforesis en Gel de Poliacrilamida , Glucósidos , Haemophilus influenzae/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The sequence of the non-typable Haemophilus influenzae (NTHi) P5 outer-membrane protein from a range of clinical isolates is presented and represents the first analysis of the heterogeneity in P5 from NTHi isolates from diverse anatomical sites. The basis of the previously observed inter-strain variation in the electrophoretic mobility is attributed to heterogeneity in three hypervariable regions. Alignment of the P5 sequences identified regions which are highly conserved and align with the transmembrane region predicted for the homologous Escherichia coli protein, OmpA. Variable regions correspond to surface-exposed loops, of which the first loop falls into subclasses. However, these subclasses fail to correlate with anatomical predisposition. Although P5 has been proposed as a fimbrial protein composed of coiled coils, both structural analysis by circular dichroism of purified P5 and computer analysis of the multiply aligned sequences predict a high proportion of beta strand with no evidence of coiled coil structure. A detailed model of P5 is presented.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Variación Genética , Haemophilus influenzae/química , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Dicroismo Circular , Conjuntiva/microbiología , ADN Bacteriano/química , Oído/microbiología , Electroforesis en Gel de Poliacrilamida , Femenino , Haemophilus influenzae/genética , Haemophilus influenzae/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Senos Paranasales/microbiología , Faringe/microbiología , Estructura Secundaria de Proteína , Alineación de Secuencia , Esputo/microbiología , Vagina/microbiologíaRESUMEN
The purpose of this study was to evaluate and compare resistance exercise training with and without pelvic stabilization on the development of isolated lumbar extension strength. Isometric torque of the isolated lumbar extensor muscles was measured at seven positions through a 72 degree range-of-motion on 47 men and 30 women before and after 12 weeks of variable resistance lumbar extension training. Subjects were assigned to either a group that trained with pelvic stabilization (P-STAB, n = 21), a group that trained without pelvic stabilization (NO-STAB, n = 41), or a control group that did not train (n = 15). Subjects trained once a week with 8 to 12 repetitions to volitional exhaustion. The P-STAB and NO-STAB groups showed significant (p < or = 0.05) and similar increases in the weight load used for training (P-STAB = 24.1 +/- 9.4kg; NO-STAB = 19.4 +/- 11.0kg) during the 12-week training period. In contrast, posttraining isometric torque values describing isolated lumbar extension strength improved only for the P-STAB group (23.5%, p < or = 0.05) and not for the NO-STAB group (-1.2%, p > 0.05) relative to controls. These data indicate that pelvic stabilization is required to effectively train the lumbar extensor muscles. The increased training load for the NO-STAB group is probably the result of exercising the muscles involved in pelvic rotation (hamstring and buttock muscles).
Asunto(s)
Terapia por Ejercicio/métodos , Músculos/fisiología , Pelvis , Adulto , Análisis de Varianza , Fenómenos Biomecánicos , Femenino , Humanos , Dolor de la Región Lumbar/prevención & control , Dolor de la Región Lumbar/rehabilitación , MasculinoRESUMEN
A complex comprising the epsilon subunit of Escherichia coli F1-ATPase (ECF1-ATPase) and a glutathione-S-transferase gamma subunit (of ECF1-ATPase) fusion protein was formed in vivo and purified from cell extracts by binding to glutathione-agarose beads. The glutathione-S-transferase was released from the complex by digestion with thrombin and the gamma/epsilon complex purified by cation-exchange chromatography. Crystals of the complex were grown by vapour diffusion using PEG8000 as precipitant. The crystals are orthorhombic, space-group P2(1)2(1)2 with a = 161.9 A, b = 44.1 A and c = 63.4 A. The volume of the asymmetric unit is consistent with the presence of a complex of one gamma subunit and one epsilon subunit.
Asunto(s)
Escherichia coli/enzimología , ATPasas de Translocación de Protón/química , Cromatografía por Intercambio Iónico , Cristalización , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Glutatión Transferasa/genética , Plásmidos , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Fosfato , Prolina , Secuencia de Aminoácidos , Secuencia de Bases , Membrana Celular/metabolismo , Genotipo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Conformación Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
In 1980, SOFAR (sound fixing and ranging) floats were tracked acoustically in the western North Atlantic entirely by means of moored autonomous listening stations. During a 5-month period 17 float trajectories were obtained in the eastern (45 degrees to 65 degrees W) Gulf Stream and subtropical gyre interior at depths of 700 and 2000 meters. These mid-depth trajectories suggest a time-varying Gulf Stream with instances of both a narrow, swift, westward recirculation south of the stream and a northeastward penetration into the Newfoundland Basin. A hundredfold increase of eddy kinetic energy was observed at 2000 meters from the gyre interior (south of 30 degrees N) to the Gulf Stream.