RESUMO
Sarcoidosis is a highly variable, systemic granulomatous disease of hitherto unknown aetiology. The GenPhenReSa (Genotype-Phenotype Relationship in Sarcoidosis) project represents a European multicentre study to investigate the influence of genotype on disease phenotypes in sarcoidosis.The baseline phenotype module of GenPhenReSa comprised 2163 Caucasian patients with sarcoidosis who were phenotyped at 31 study centres according to a standardised protocol.From this module, we found that patients with acute onset were mainly female, young and of Scadding type I or II. Female patients showed a significantly higher frequency of eye and skin involvement, and complained more of fatigue. Based on multidimensional correspondence analysis and subsequent cluster analysis, patients could be clearly stratified into five distinct, yet undescribed, subgroups according to predominant organ involvement: 1) abdominal organ involvement, 2) ocular-cardiac-cutaneous-central nervous system disease involvement, 3) musculoskeletal-cutaneous involvement, 4) pulmonary and intrathoracic lymph node involvement, and 5) extrapulmonary involvement.These five new clinical phenotypes will be useful to recruit homogenous cohorts in future biomedical studies.
Assuntos
Fenótipo , Sarcoidose/diagnóstico , Sarcoidose/fisiopatologia , Abdome , Doença Aguda , Adulto , Idoso , Europa (Continente) , Olho/fisiopatologia , Oftalmopatias/fisiopatologia , Feminino , Volume Expiratório Forçado , Genótipo , Humanos , Artropatias/fisiopatologia , Pulmão/fisiopatologia , Pneumopatias/fisiopatologia , Linfonodos/fisiopatologia , Masculino , Pessoa de Meia-Idade , Pele/fisiopatologia , Dermatopatias/fisiopatologia , Atenção Terciária à Saúde , População BrancaRESUMO
Patients with obstructive lung diseases commonly undergo bronchodilator reversibility testing during examination of their pulmonary function by spirometry. A positive response is defined by an increase in forced expiratory volume in 1 s (FEV1). FEV1 is a rather nonspecific criterion not allowing the regional effects of bronchodilator to be assessed. We employed the imaging technique of electrical impedance tomography (EIT) to visualize the spatial and temporal ventilation distribution in 35 patients with chronic obstructive pulmonary disease at baseline and 5, 10, and 20 min after bronchodilator inhalation. EIT scanning was performed during tidal breathing and forced full expiration maneuver in parallel with spirometry. Ventilation distribution was determined by EIT by calculating the image pixel values of FEV1, forced vital capacity (FVC), tidal volume, peak flow, and mean forced expiratory flow between 25 and 75% of FVC. The global inhomogeneity indexes of each measure and histograms of pixel FEV1/FVC values were then determined to assess the bronchodilator effect on spatial ventilation distribution. Temporal ventilation distribution was analyzed from pixel values of times needed to exhale 75 and 90% of pixel FVC. Based on spirometric FEV1, significant bronchodilator response was found in 17 patients. These patients exhibited higher postbronchodilator values of all regional EIT-derived lung function measures in contrast to nonresponders. Ventilation distribution was inhomogeneous in both groups. Significant improvements were noted for spatial distribution of pixel FEV1 and tidal volume and temporal distribution in responders. By providing regional data, EIT might increase the diagnostic and prognostic information derived from reversibility testing.
Assuntos
Albuterol/administração & dosagem , Broncodilatadores/administração & dosagem , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Administração por Inalação , Idoso , Idoso de 80 Anos ou mais , Impedância Elétrica , Feminino , Humanos , Pulmão , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Espirometria , Tomografia , Resultado do Tratamento , Capacidade VitalRESUMO
BACKGROUND: Inhaled corticosteroids (ICS) are widely used in the treatment of obstructive lung diseases. Recent data suggest a higher pneumonia risk in chronic obstructive pulmonary disease (COPD) patients treated with ICS. OBJECTIVE: Since non-typeable Haemophilus influenzae (NTHi) is the most common pathogen associated with acute exacerbations of COPD, we investigated the effects of budesonide (BUD) on NTHi-induced inflammation and invasive infection. METHODS: The alveolar epithelial cell line A549 and specimens of human lung tissue (HLT) were used in our experiments. Intracellular infection was determined by a lysis/culture assay of infected cells. Activated p38 mitogen-associated protein kinase (MAPK) was assessed using Western blotting and immunohistochemistry, expression of toll-like receptor 2 (TLR2) was determined by PCR, and CXCL-8 levels were measured using ELISA. Immunohistochemistry was used for detection of CXCL-8, platelet-activating factor receptor (PAF-R) and NTHi. RESULTS: BUD significantly reduced CXCL-8 secretion in A549 cells and lung tissue infected with NTHi. Furthermore, BUD decreased the expression of PAF-R in HLT and A549 cells. In A549 cells and HLT, BUD inhibited intracellular infection and - synergistically with NTHi - increased the expression of TLR2 (in A549 cells). TLR2 stimulation did not influence the intracellular infection of A549 cells, but p38 MAPK inhibition resulted in a significant reduction of infection. CONCLUSION: The present study adds new insights into the effects of glucocorticoids on pulmonary host defence after NTHi infection. Although the inflammatory response to infection is suppressed by BUD, interestingly, the intracellular infection is also inhibited. This effect seems to depend on the inhibition of p38 MAPK - a key enzyme in many pro-inflammatory pathways - as well as of PAF-R expression.
Assuntos
Budesonida/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Administração por Inalação , Anti-Inflamatórios/farmacologia , Western Blotting , Budesonida/efeitos adversos , Células Cultivadas , Meios de Cultivo Condicionados , Indução Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Infecções por Haemophilus/etiologia , Infecções por Haemophilus/fisiopatologia , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase , Doença Pulmonar Obstrutiva Crônica/microbiologia , Sensibilidade e Especificidade , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacosRESUMO
Alterations in the DNA methylome are characteristic for numerous diseases and a typical hallmark of cancer. Therefore, DNA methylation is currently under investigation in research labs and has also entered diagnostics. Recently, protocols like the BeadChip technology have become commercially available to study DNA methylation in an array format and semiquantitative fashion. However, it is known that fixation of the sample material with formalin prior to BeadChip analysis can affect the results. In this study we compared the influence of fixation on the outcome of BeadChip analysis. From six patients each a lung cancer tissue sample and a corresponding tumor-free lung tissue sample were collected. The samples were separated into three pieces. One piece of each sample was fixed with formalin, another one by the non-cross-linking HOPE technique (Hepes-glutamic acid buffer mediated Organic solvent Protection Effect). Subsequently, both became paraffin embedded. As a reference, the remaining third piece was cryopreserved. In addition we used three adenocarcinoma cell lines (H838, A549, and H1650) to validate the results from patient tissues. We show that using the HOPE technique instead of formalin largely prevents the introduction of formalin-fixation related artifacts. An ANOVA analysis significantly separated HOPE- and cryopreserved from formalin-fixed samples (FDR<0.05), while differences in the methylation data obtained from HOPE-fixed and cryopreserved material were minor. Consequently, HOPE fixation is superior to formalin fixation if a subsequent BeadChip analysis of paraffin-embedded sample material is intended.
Assuntos
Adenocarcinoma/metabolismo , Carcinoma de Células Escamosas/metabolismo , Metilação de DNA , DNA de Neoplasias/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Preservação de Tecido , Adenocarcinoma/patologia , Idoso , Artefatos , Soluções Tampão , Carcinoma Adenoescamoso/metabolismo , Carcinoma Adenoescamoso/patologia , Carcinoma de Células Grandes/metabolismo , Carcinoma de Células Grandes/prevenção & controle , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Criopreservação , DNA de Neoplasias/isolamento & purificação , Feminino , Ácido Glutâmico/química , HEPES/química , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Análise Serial de Tecidos , Fixação de TecidosAssuntos
Resistência das Vias Respiratórias , Asma/fisiopatologia , Exercício Físico , Pulmão/fisiopatologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Oscilometria , Pletismografia , Estudos Prospectivos , Doença Pulmonar Obstrutiva Crônica , EspirometriaRESUMO
BACKGROUND: Infection with Pseudomonas aeruginosa (PA) induces mucus hypersecretion in airways. Therapeutic options to attenuate excessive mucus expression are sparse. OBJECTIVE: To investigate the effect of steroids and N-acetyl-cysteine (NAC) on PA-induced mucus expression. MATERIAL AND METHODS: Calu-3 cells and explanted human mucosa from the upper airways were stimulated with either PA, lipopolysaccharide from alginate producing PA (smooth, sPA-LPS) or non-alginate producing PA (rough, rPA-LPS). Dexamethasone (DEX) and NAC were added in different concentrations. Expression of mucin (MUC5AC) gene and mucin protein expression was quantified using PAS (periodic acids Schiff) staining and real time PCR. RESULTS: PA, sPA-LPS or rPA-LPS significantly induced mucin protein and MUC5AC gene expression in Calu-3 cells and explanted mucosal tissue (P < 0.05). Both DEX and NAC significantly decreased PA-, sPA-LPS- and rPA-LPS-induced mucin protein expression both in vitro and ex vivo (P < 0.05). A significant reduction was also observed for MUC5AC gene expression with the two agents (P < 0.05) except for sPA-LPS-induced mucin gene expression in vitro (P > 0.05). DISCUSSION AND CONCLUSION: Our data show that both an anti-inflammatory drug (DEX) and an anti-oxidative agent (NAC) can attenuate PA-induced mucus expression in human airways. These results support the use of steroids and NAC in clinical practice to treat PA-induced mucus hypersecretion.
Assuntos
Acetilcisteína/farmacologia , Dexametasona/farmacologia , Muco/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Acetilcisteína/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Linhagem Celular , Dexametasona/administração & dosagem , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Expectorantes/administração & dosagem , Expectorantes/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mucina-5AC/genética , Muco/microbiologia , Reação em Cadeia da Polimerase , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologiaRESUMO
Intestinal edema remains a serious clinical problem, and novel approaches to study its pathophysiology are needed. It was our aim to develop a long-term stable isolated perfused rat small bowel preparation permitting analysis of vascular, luminal, interstitial, and lymphatic compartments and to demonstrate the utility of this model by studying the effects of the proinflammatory mediator platelet-activating factor (PAF). A temperature-controlled chamber with an integrated balance was designed to perfuse isolated intestines through the mesenteric artery and the gut lumen. Steroids or oxygen carriers were not needed. Functional and morphological integrity of the tissue was preserved for several hours as confirmed by oxygen consumption, venous lactate-to-pyruvate ratio, arterial and venous pH, lactose digestion and galactose uptake, intravascular and luminal pressures, maintained fluid homeostasis, gut motility, and quantitative light microscopic analysis. Administration of PAF caused typical effects such as vasoconstriction, gut atony, and loss of galactose uptake. PAF also elicited a transient loss of 20% of the perfusate liquid from the mesenteric vascular bed, two-thirds of which were transferred to the lumen. All these responses were entirely reversible. This new model provides detailed insights into the physiology of the small intestine and will allow to study fundamental processes such as fluid homeostasis, barrier functions, transport mechanisms, and immune responses in this organ. Using this model, here we show a dramatic and yet reversible response of the rat small bowel to PAF, suggesting luminal water clearance as a novel safety factor in the intestine that may be of clinical relevance.
Assuntos
Intestino Delgado/fisiologia , Técnicas de Cultura de Órgãos/métodos , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Feminino , Intestino Delgado/irrigação sanguínea , Músculo Liso/fisiologia , Técnicas de Cultura de Órgãos/normas , Perfusão , Peristaltismo/fisiologia , Fator de Ativação de Plaquetas/farmacologia , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
BACKGROUND: Nontypeable Haemophilus influenzae (NTHI) may play a role as an infectious trigger in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available regarding the influence of acute and persistent infection on tissue remodelling and repair factors such as transforming growth factor (TGF)-beta. METHODS: NTHI infection in lung tissues obtained from COPD patients and controls was studied in vivo and using an in vitro model. Infection experiments were performed with two different clinical isolates. Detection of NTHI was done using in situ hybridization (ISH) in unstimulated and in in vitro infected lung tissue. For characterization of TGF-beta signaling molecules a transcriptome array was performed. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-alpha and TGF-beta expression were evaluated in lung tissue and cell culture using ELISA. RESULTS: In 38% of COPD patients infection with NTHI was detected in vivo in contrast to 0% of controls (p < 0.05). Transcriptome arrays showed no significant changes of TGF-beta receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after in vitro infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-beta (p < 0.05) in combination with a strong proinflammatory response (p < 0.01). CONCLUSIONS: We show for the first time the expression of the TGF pseudoreceptor BAMBI in the human lung, which is upregulated in response to NTHI infection in COPD lung tissue in vivo and in vitro. The combination of NTHI-mediated induction of proinflammatory cytokines and inhibition of TGF-beta expression may influence inflammation induced tissue remodeling.
Assuntos
Haemophilus influenzae/isolamento & purificação , Pulmão/metabolismo , Pulmão/virologia , Proteínas de Membrana/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/virologia , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Pulmão/imunologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/imunologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad3/metabolismo , Técnicas de Cultura de Tecidos , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The gastrointestinal tract is a hostile biological environment, yet not all ingested materials are destroyed. The minute differences that determine whether a substance persists or is digested, liberated, adsorbed, excreted, or taken up are still poorly understood. Most attempts to investigate the events occurring during an orogastrointestinal passage rely on simplified in vitro systems where an analyte is exposed to artificial intestinal fluids. To closely mimic the events in the gastrointestinal tract, the exact intestinal fluid composition and the in vivo concentration of its constituents must be known. The widely used lavage procedures, however, dilute the intestinal fluids to an extent that precludes recalculation to the original concentrations. Thus, we developed procedures with which undiluted murine intestinal fluid can be harvested; determined the in vivo concentrations of the digestive enzymes trypsin, chymotrypsin, and elastase and the adsorbents mucin and immunoglobulin A in small intestinal fluid of fasted and unfasted female Balb/c mice; and identified chymotrypsin and immunoglobulin A as valid endogenous dilution markers for the recalculation of aqueous lavages. With these technologies and information at hand, more reliable investigations on the fate of allergens, pathogens, food, and anthropogenic xenobiotics in the gastrointestinal tract will be possible.
Assuntos
Bioquímica/métodos , Líquidos Corporais/metabolismo , Intestino Delgado/metabolismo , Proteínas/metabolismo , Animais , Digestão , Jejum/metabolismo , Feminino , Imunoglobulina A/metabolismo , Intestino Delgado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Mucinas/metabolismo , Peptídeo Hidrolases/metabolismo , Irrigação TerapêuticaRESUMO
BACKGROUND: While lipopolysaccharide (LPS) from Gram-negative bacteria has been shown to augment inflammation in ventilated lungs information on the effect of Gram-positive bacteria is lacking. Therefore the effect of LPS and a lipopetide from Gram-positive bacteria, PAM3, on ventilated lungs were investigated. METHODS: C57/Bl6 mice were mechanically ventilated. Sterile saline (sham) and different concentrations of LPS (1 microg and 5 microg) and PAM3 (50 nM and 200 nM) were applied intratracheally. Lung function parameters and expression of MIP-2 and TNFalpha as well as influx of neutrophils were measured. RESULTS: Mechanical ventilation increased resistance and decreased compliance over time. PAM3 but not LPS significantly increased resistance compared to sham challenge (P < 0.05). Both LPS and PAM3 significantly increased MIP-2 and TNFalpha mRNA expression compared to sham challenge (P < 0.05). The numbers of neutrophils were significantly increased after LPS at a concentration of 5 microg compared to sham (P < 0.05). PAM3 significantly increased the numbers of neutrophils at both concentrations compared to sham (P < 0.05). CONCLUSIONS: These data suggest that PAM3 similar to LPS enhances ventilator-induced inflammation. Moreover, PAM3 but not LPS increases pulmonary resistance in ventilated lungs. Further studies are warranted to define the role of lipopetides in ventilator-associated lung injury.
Assuntos
Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Respiração Artificial , Resistência das Vias Respiratórias/efeitos dos fármacos , Resistência das Vias Respiratórias/fisiologia , Animais , Quimiocina CXCL2/metabolismo , Relação Dose-Resposta a Droga , Lipopeptídeos/metabolismo , Lipopolissacarídeos/metabolismo , Pulmão/citologia , Pulmão/fisiologia , Complacência Pulmonar/efeitos dos fármacos , Complacência Pulmonar/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: A large number of studies have investigated the effects of high tidal volume ventilation in mouse models. In contrast data on very short term effects of low tidal volume ventilation are sparse. Therefore we investigated the functional and structural effects of low tidal volume ventilation in mice. METHODS: 38 Male C57/Bl6 mice were ventilated with different tidal volumes (Vt 5, 7, and 10 ml/kg) without or with application of PEEP (2 cm H2O). Four spontaneously breathing animals served as controls. Oxygen saturation and pulse rate were monitored. Lung function was measured every 5 min for at least 30 min. Afterwards lungs were removed and histological sections were stained for measurement of infiltration with polymorphonuclear leukocytes (PMN). Moreover, mRNA expression of macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)alpha in the lungs was quantified using real time PCR. RESULTS: Oxygen saturation did not change significantly over time of ventilation in all groups (P > 0.05). Pulse rate dropped in all groups without PEEP during mechanical ventilation. In contrast, in the groups with PEEP pulse rate increased over time. These effects were not statistically significant (P > 0.05). Tissue damping (G) and tissue elastance (H) were significantly increased in all groups after 30 min of ventilation (P < 0.05). Only the group with a Vt of 10 ml/kg and PEEP did not show a significant increase in H (P > 0.05). Mechanical ventilation significantly increased infiltration of the lungs with PMN (P < 0.05). Expression of MIP-2 was significantly induced by mechanical ventilation in all groups (P < 0.05). MIP-2 mRNA expression was lowest in the group with a Vt of 10 ml/kg + PEEP. CONCLUSIONS: Our data show that very short term mechanical ventilation with lower tidal volumes than 10 ml/kg did not reduce inflammation additionally. Formation of atelectasis and inadequate oxygenation with very low tidal volumes may be important factors. Application of PEEP attenuated inflammation.
Assuntos
Inflamação/fisiopatologia , Pulmão/fisiopatologia , Respiração com Pressão Positiva , Animais , Quimiocina CXCL2/metabolismo , Frequência Cardíaca/fisiologia , Hemodinâmica/fisiologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Neutrófilos/patologia , RNA Mensageiro/metabolismo , Volume de Ventilação Pulmonar/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The significance of human biorepositories for modern medical research, particularly for comprehensive population-based genetic analyses, is constantly growing. While large and centralized institutions are usually considered best suited to meet the increasing demand for high-quality "biobanks," most medical research institutions still host rather heterogeneous and fragmented biobanking activities, undertaken by clinical departments with oftentimes rather different scientific scope. Undoubtedly, most clinicians and medical researchers would appreciate infrastructural support in terms of the storage and handling of their biosamples, but they are also likely to expect access to their samples avoiding extensive formal requirements. We report on the establishment of the PopGen 2.0 Network (P2N), an overarching alliance of initially seven biobanks from Northern Germany which adopted a joint but lean governance structure and use-and-access policy for their samples and data. In addition, the members of P2N have pursued an intense collaboration on ethical, legal and social issues and maintain a common IT infrastructure. The implementation of P2N has substantially improved the prospects of biobank-based research at the participating institutions. The network may thus serve as a role model for similar initiatives geared at linking pre-existing biorepositories for the benefit of research quality, efficiency, and transparency.
RESUMO
Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia and is a main cause of infectious deaths. However, little is known about host-pathogen interaction in human lung tissue. We tested the hypothesis that human alveolar macrophages (AMs) and alveolar epithelial cells (AECs) are important for initiating the host response against S. pneumoniae, and we evaluated the role of Toll-like receptor (TLR) 2, TLR4, and p38 mitogen-activated protein kinase (MAPK) signaling in the inflammatory response after pneumococcal infection. We established a novel model of acute S. pneumoniae infection using vital human lung specimens. In situ hybridization analysis showed that S. pneumoniae DNA was detected in 80 to 90% of AMs and 15 to 30% of AECs after in vitro infection accompanied by increased expression of inflammatory cytokines. Enhanced phosphorylation of p38 MAPK and increased TLR2 and 4 mRNA expression were observed in infected lung tissue. Thirty to fifty percent of AMs and 10 to 20% of AECs showed evidence of apoptosis 24 hours after pneumococcal infection. After macrophage deactivation with Clodronate/liposomes, infected lung tissue exhibited a significantly decreased release of inflammatory mediators. Inhibition of p38 MAPK signaling markedly reduced inflammatory cytokine release from human lungs, whereas TLR2 blockade revealed only minor effects. AMs are central resident immune cells during S. pneumoniae infection and are the main source of early proinflammatory cytokine release. p38 MAPK holds a major role in pathogen-induced pulmonary cytokine release and is a potential molecular target to modulate overwhelming lung inflammation.
Assuntos
Pulmão/microbiologia , Pulmão/patologia , Modelos Biológicos , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/patologia , Streptococcus pneumoniae/imunologia , Doença Aguda , Apoptose , Antígeno CD11b/metabolismo , Caspase 3/metabolismo , DNA Bacteriano/genética , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Hibridização In Situ , Inflamação/metabolismo , Pulmão/imunologia , Macrófagos Alveolares/metabolismo , Pneumonia Pneumocócica/imunologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Colonisation of cystic fibrosis (CF) lungs with Pseudomonas aeruginosa is facilitated by two lectins, which bind to the sugar coat of the surface lining epithelia and stop the cilia beating. OBJECTIVES: We hypothesized that P. aeruginosa lung infection should be cleared by inhalation of fucose and galactose, which compete for the sugar binding site of the two lectins and thus inhibit the binding of P. aeruginosa. METHODS: 11 adult CF patients with chronic infection with P. aeruginosa were treated twice daily with inhalation of a fucose/galactose solution for 21 days (4 patients only received inhalation, 7 patients received inhalation and intravenous antibiotics). Microbial counts of P. aeruginosa, lung function measurements, and inflammatory markers were determined before and after treatment. RESULTS: The sugar inhalation was well tolerated and no adverse side effects were observed. Inhalation alone as well as combined therapy (inhalation and antibiotics) significantly decreased P. aeruginosa in sputum (P < 0.05). Both therapies also significantly reduced TNFalpha expression in sputum and peripheral blood cells (P < 0.05). No change in lung function measurements was observed. CONCLUSIONS: Inhalation of simple sugars is a safe and effective measure to reduce the P. aeruginosa counts in CF patients. This may provide an alternative therapeutical approach to treat infection with P. aeruginosa.
Assuntos
Fibrose Cística/complicações , Fucose/uso terapêutico , Galactose/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Administração por Inalação , Adulto , Aminoglicosídeos/administração & dosagem , Aminoglicosídeos/uso terapêutico , Proteína C-Reativa/metabolismo , Contagem de Células , Cefalosporinas/administração & dosagem , Cefalosporinas/uso terapêutico , Quimioterapia Combinada , Feminino , Fucose/administração & dosagem , Galactose/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Humanos , Isotipos de Imunoglobulinas/sangue , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Testes de Função Hepática , Masculino , Neutrófilos/citologia , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Testes de Função Respiratória , Escarro/citologia , Escarro/metabolismo , Escarro/microbiologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genéticaRESUMO
Gram-negative bacteria can stimulate mucin production, but excessive mucus supports bacterial infection and consequently leads to airway obstruction. Therefore, the effect of dexamethasone (DEX) and the antioxidant acetyl-cysteine (ACC) on bacteria-induced mucus expression was investigated. Explanted human airway mucosa and mucoepidermoid cells (Calu-3) were stimulated with lipopolysaccharide (LPS) or PAM3 (a synthetic lipoprotein). DEX or ACC were added to either LPS- or PAM3-stimulated airway mucosa or Calu-3 cells. Mucin mRNA expression (MUC5AC) and total mucus glycoconjugates (mucin protein) were quantified using real-time PCR and periodic acid Schiff staining. LPS and PAM3 significantly increased mucin expression in airway mucosa and Calu-3 cells (P < 0.05). DEX alone had no significant effect on mucin expression in airway mucosa or Calu-3 cells (P > 0.05). In contrast, DEX significantly reduced LPS- and PAM3-induced mucin expression in explanted mucosal tissue and mucin expression in Calu-3 cells (P < 0.05). In explanted human airway mucosa ACC alone significantly increased mucin expression (P < 0.05). In contrast, ACC significantly decreased LPS- and PAM3-induced mucin expression (P < 0.05). In Calu-3 cells ACC alone had no significant effect on mucin expression (P > 0.05). ACC decreased LPS- and PAM3-induced mucin expression, but this effect was not significant (P > 0.05). These data suggest that DEX can effectively reduce bacteria-induced mucin expression in the airways. ACC alone may increase mucin expression in noninfected mucosa, but it decreased bacteria-induced mucin expression. Further studies are warranted to evaluate whether the effect of DEX or ACC is clinically relevant.
Assuntos
Acetilcisteína/farmacologia , Anti-Inflamatórios/farmacologia , Dexametasona/farmacologia , Sequestradores de Radicais Livres/farmacologia , Mucinas/biossíntese , Mucosa Respiratória/efeitos dos fármacos , Salmonella/imunologia , Adulto , Anti-Inflamatórios não Esteroides/farmacologia , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/imunologia , Lipoproteínas/imunologia , Masculino , Mucinas/genética , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismoRESUMO
BACKGROUND: hCLCA1 is a member of the calcium-activated chloride channel family and is associated with disease-inducible mucus expression. Niflumic acid (NFA) and a closely related chemical structure are reported inhibitors of calcium-activated chloride channels and endotoxin-inducible mucus expression in the mouse. Therefore, we tested the hypothesis that hCLCA1 may be involved in lipopolysaccharide (LPS) induced mucin up-regulation in human airways. We also investigated the effect of NFA and MSI-2216 on LPS-induced mucin up-regulation. MATERIALS AND METHODS: Explanted human airways and the muco-epidermoid cell line Calu-3 were stimulated with LPS. Different concentrations of NFA or MSI-2216 were added to LPS-stimulated airway mucosa and Calu-3 cells. Expression of hCLCA1 and MUC5AC mRNA and protein was quantified in human airways using real-time PCR and PAS staining. In addition, immunohistochemistry was performed for quantification of inflammatory cells (lymphocytes, monocytes, eosinophils, and neutrophils) in the submucosa of the airways. Expression of hCLCA1 protein in Calu-3 cells was analysed by FACS. RESULTS: LPS significantly induced hCLCA1 and MUC5AC mRNA and protein expression in human airway mucosa (P < 0.05). NFA and MSI-2216 significantly decreased LPS-induced mucus expression in explanted airway mucosa in a dose-dependent manner (P < 0.05). In Calu-3 cells, LPS significantly increased hCLCA1 surface expression whereas intracellular expression was significantly decreased (P < 0.05). In Calu-3 cells, NFA and MSI-2216 also significantly reduced MUC5AC mRNA expression (P < 0.05). CONCLUSIONS: These data suggest that hCLCA1 may play a role in LPS-induced mucin expression in human airway mucosa. Calcium-activated chloride channel inhibitors significantly decreased LPS-induced mucus expression both ex vivo and in vitro . Therefore, blocking of hCLCA1 may offer a therapeutic approach to reduce bacterial-induced mucus hypersecretion.
Assuntos
Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/biossíntese , Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Mucinas/biossíntese , Muco/metabolismo , Mucosa Respiratória/metabolismo , Adulto , Linhagem Celular , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Imuno-Histoquímica , Inflamação/imunologia , Masculino , Pessoa de Meia-Idade , Mucina-5AC , Mucinas/genética , Ácido Niflúmico/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC. METHODS: In a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues) in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine) uptake as markers for proliferation and of cleaved (activated) effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines. RESULTS: Viability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC) and human cell lines (CPC-N, HEK) proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC). Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas. CONCLUSION: Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST provides a useful human model to study numerous aspects of mechanisms underlying tumor responsiveness towards improved anticancer treatment. The results presented here shall serve as a base for multiple functional tests of novel chemotherapeutic approaches to NSCLC in the future.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias Pulmonares/tratamento farmacológico , Antimetabólitos Antineoplásicos/uso terapêutico , Antineoplásicos/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Carboplatina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Caspase 3/química , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Resistência a Medicamentos , Feminino , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Sobrevivência de Tecidos/efeitos dos fármacos , Resultado do Tratamento , Vimblastina/análogos & derivados , Vimblastina/uso terapêutico , Vinorelbina , GencitabinaRESUMO
The causative agent of severe acute respiratory syndrome (SARS), which affected over 8000 individuals worldwide and was responsible for over 700 deaths in the 2002-2003 outbreak, is a coronavirus that was unknown before the outbreak. Although many different treatments were used during the outbreak, none were implemented in a controlled fashion. Thus, the optimal treatment for SARS is unknown. Since the outbreak, much work has been done testing new agents against SARS using in-vitro methods and animal models. In addition, global research efforts have focused on the development of vaccines against SARS. Efforts should be made to evaluate the most promising treatments and vaccines in controlled clinical trials, should another SARS outbreak occur.
Assuntos
Antivirais/uso terapêutico , Surtos de Doenças/prevenção & controle , Síndrome Respiratória Aguda Grave , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Animais , Humanos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Vacinas ViraisRESUMO
Severe acute respiratory syndrome (SARS) is a new infectious disease caused by a novel coronavirus that leads to deleterious pulmonary pathological features. Due to its high morbidity and mortality and widespread occurrence, SARS has evolved as an important respiratory disease which may be encountered everywhere in the world. The virus was identified as the causative agent of SARS due to the efforts of a WHO-led laboratory network. The potential mutability of the SARS-CoV genome may lead to new SARS outbreaks and several regions of the viral genomes open reading frames have been identified which may contribute to the severe virulence of the virus. With regard to the pathogenesis of SARS, several mechanisms involving both direct effects on target cells and indirect effects via the immune system may exist. Vaccination would offer the most attractive approach to prevent new epidemics of SARS, but the development of vaccines is difficult due to missing data on the role of immune system-virus interactions and the potential mutability of the virus. Even in a situation of no new infections, SARS remains a major health hazard, as new epidemics may arise. Therefore, further experimental and clinical research is required to control the disease.