RESUMO
In neuropathic pain clinical trials, the patient's perspective is often insufficiently reflected focusing mainly on pain intensity. Comparability of outcome assessment is limited due to heterogenous patient reported outcome measures (PROMs). The MEDLINE, CENTRAL, and Embase databases and reference lists of published meta-analyses were searched. Randomized controlled studies assessing treatment efficacy of drugs for chronic neuropathic pain were included. PROMs were assigned to recommended IMMPACT/NeuPSIG domains: pain intensity, pain other aspects, physical functioning, emotional functioning, global improvement and satisfaction, adverse events, participant disposition. Domains and PROMs were compared regarding the publication year and methodological quality of the studies. Within the 251 included studies 200 PROMs were used with 27 being recommended by IMMPACT/NeuPSIG. The number of domains was higher in high/moderate quality studies. The (sub-) domains 'physical functioning', 'global improvement and satisfaction', and 'neuropathic pain quality' were assessed more frequently in high/moderate quality studies and those published after 2011. Recent studies and those of better quality more often used the recommended PROMs. Although neuropathic assessment via PROMs has improved, there is still a high heterogeneity. A standardized core set of outcome domains and should be defined to improve neuropathic pain treatment and to achieve better comparability of clinical trials. Perspective: This systematic literature review assesses the use of patient reported outcome measures (PROMs) in chronic neuropathic pain. The results show that there is still a high heterogeneity, highlighting the need for a standardized core set of outcome domains and PROMs to improve comparability of clinical trials and neuropathic pain treatment.
Assuntos
Dor Crônica , Neuralgia , Humanos , Neuralgia/tratamento farmacológico , Resultado do Tratamento , Avaliação de Resultados em Cuidados de Saúde , Medidas de Resultados Relatados pelo Paciente , Dor Crônica/tratamento farmacológicoRESUMO
BACKGROUND: Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects. METHODS: SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide. RESULTS: Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space. CONCLUSIONS: We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.
Assuntos
Células Epiteliais/efeitos dos fármacos , Doenças Pulmonares Intersticiais/genética , Chaperonas Moleculares/genética , Alvéolos Pulmonares/efeitos dos fármacos , Proteína C Associada a Surfactante Pulmonar/genética , Surfactantes Pulmonares/metabolismo , Animais , Azatioprina/farmacologia , Linhagem Celular , Ciclofosfamida/farmacologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Hidroxicloroquina/farmacologia , Doenças Pulmonares Intersticiais/imunologia , Metilprednisolona/farmacologia , Camundongos , Chaperonas Moleculares/metabolismo , Mutação , Fosfolipídeos/análise , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Proteína C Associada a Surfactante Pulmonar/imunologiaRESUMO
BACKGROUND: ABCA3 transporter (ATP-binding cassette transporter of the A subfamily) is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII). It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD) of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER). METHODS: Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level. RESULTS: We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. CONCLUSION: Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Apoptose , Retículo Endoplasmático/patologia , Células Epiteliais/patologia , Pulmão/patologia , Mutação , Estresse Fisiológico , Transportadores de Cassetes de Ligação de ATP/metabolismo , Processamento Alternativo , Anexina A5/metabolismo , Apoptose/genética , Caspase 3/metabolismo , Caspases Iniciadoras/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Separação Celular/métodos , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Células Epiteliais/metabolismo , Citometria de Fluxo , Imunofluorescência , Glutationa/metabolismo , Glicosilação , Proteínas de Choque Térmico/metabolismo , Humanos , Pulmão/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfolipídeos/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição de Fator Regulador X , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Transfecção , Proteína 1 de Ligação a X-BoxRESUMO
BACKGROUND: Heterozygous mutations of SFTPC, the gene encoding surfactant protein C (SP-C), cause sporadic and familial interstitial lung disease (ILD) in children and adults. The most frequent SFTPC mutation in ILD patients leads to a threonine for isoleucine substitution at position 73 (I73T) of the SP-C preprotein (proSP-C), however little is known about the cellular consequences of SP-CI73T expression. RESULTS: To address this, we stably expressed SP-CI73T in cultured MLE-12 alveolar epithelial cells. This resulted in increased intracellular accumulation of proSP-C processing intermediates, which matched proSP-C species recovered in bronchial lavage fluid from patients with this mutation. Exposure of SP-CI73T cells to drugs currently used empirically in ILD therapy, cyclophosphamide, azathioprine, hydroxychloroquine or methylprednisolone, enhanced expression of the chaperones HSP90, HSP70, calreticulin and calnexin. SP-CI73T mutants had decreased intracellular phosphatidylcholine level (PC) and increased lyso-PC level without appreciable changes of other phospholipids. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CI73T cells secreted into the medium soluble factors that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine influence of SP-CI73T on neighboring cells in the alveolar space. CONCLUSION: We show that I73T mutation leads to impaired processing of proSP-C in alveolar type II cells, alters their stress tolerance and surfactant lipid composition, and activates cells of the immune system. In addition, we show that some of the mentioned cellular aspects behind the disease can be modulated by application of pharmaceutical drugs commonly applied in the ILD therapy.
Assuntos
Células Epiteliais/metabolismo , Proteína C Associada a Surfactante Pulmonar/metabolismo , Substituição de Aminoácidos , Animais , Azatioprina/uso terapêutico , Líquido da Lavagem Broncoalveolar , Linfócitos T CD4-Positivos/imunologia , Calnexina/metabolismo , Calreticulina/metabolismo , Linhagem Celular , Ciclofosfamida/uso terapêutico , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Hidroxicloroquina/uso terapêutico , Doenças Pulmonares Intersticiais/tratamento farmacológico , Metilprednisolona/uso terapêutico , Camundongos , Mutação , Fosfatidilcolinas/metabolismo , Proteína C Associada a Surfactante Pulmonar/análise , Proteína C Associada a Surfactante Pulmonar/genética , Receptores CCR2/metabolismo , Receptores de Interleucina-8A/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: The surfactant proteins B (SP-B) and C (SP-C) are important for the stability and function of the alveolar surfactant film. Their involvement and down-regulation in inflammatory processes has recently been proposed, but their level during neutrophilic human airway diseases are not yet known. METHODS: We used 1D-electrophoresis and Western blotting to determine the concentrations and molecular forms of SP-B and SP-C in bronchoalveolar lavage (BAL) fluid of children with different inflammatory airway diseases. 21 children with cystic fibrosis, 15 with chronic bronchitis and 14 with pneumonia were included and compared to 14 healthy control children. RESULTS: SP-B was detected in BAL of all 64 patients, whereas SP-C was found in BAL of all but 3 children; those three BAL fluids had more than 80% neutrophils, and in two patients, who were re-lavaged later, SP-C was then present and the neutrophil count was lower. SP-B was mainly present as a dimer, SP-C as a monomer. For both qualitative and quantitative measures of SP-C and SP-B, no significant differences were observed between the four evaluated patient groups. CONCLUSION: Concentration or molecular form of SP-B and SP-C is not altered in BAL of children with different acute and chronic inflammatory lung diseases. We conclude that there is no down-regulation of SP-B and SP-C at the protein level in inflammatory processes of neutrophilic airway disease.
Assuntos
Bronquite Crônica/metabolismo , Líquido da Lavagem Broncoalveolar , Fibrose Cística/metabolismo , Pneumonia/metabolismo , Proteína B Associada a Surfactante Pulmonar/metabolismo , Proteína C Associada a Surfactante Pulmonar/metabolismo , Doença Aguda , Adolescente , Adulto , Bronquite Crônica/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Doença Crônica , Fibrose Cística/patologia , Regulação para Baixo , Feminino , Humanos , Lactente , Masculino , Neutrófilos/patologia , Pneumonia/patologia , Proteína B Associada a Surfactante Pulmonar/genética , Proteína C Associada a Surfactante Pulmonar/genéticaRESUMO
The mutation (g.1286T>C) of the pulmonary surfactant-associated protein C gene (SFTPC) leads to the I73T substitution in the precursor protein (pro-SP-C) and results in interstitial lung disease with the histological pattern of non-specific interstitial pneumonia and pulmonary alveolar proteinosis. Central for the disease is the abnormal processing of the SP-C pro-protein to mature SP-C; however little is known about the nature of intermediates and processing products. We report here the application of high resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry to the characterization of processing intermediates of hydrophobic pulmonary surfactant proteins SP-B and SP-C in intra- alveolar surfactant material of a patient with I73T mutation. SP-C and SP-B processing forms were separated from broncho-alveolar lavage fluid using chloroform/methanol extraction and sodium dodecyl sulfate poly acrylamide gel electrophoreis, detected by Western blot and identified by electrospray- and matrix-assisted laser desorption/ionization-FT-ICR mass spectrometry. The mass spectrometric and immuno-analytical results show the intra-alveolar accumulation of an aberrant C-terminal SP-C processing products in which the mature SP-C protein part is missing and aberrant processing intermediates of SP-B.
Assuntos
Espectrometria de Massas/métodos , Proteína B Associada a Surfactante Pulmonar/química , Proteína C Associada a Surfactante Pulmonar/química , Sequência de Aminoácidos , Líquido da Lavagem Broncoalveolar/química , Análise de Fourier , Humanos , Dados de Sequência Molecular , Proteína B Associada a Surfactante Pulmonar/genética , Proteína C Associada a Surfactante Pulmonar/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was developed and applied to the proteome analysis of bronchoalveolar lavage fluid (BALF) from a patient with pulmonary alveolar proteinosis. With use of 1-D and 2-D gel electrophoresis, surfactant protein A (SP-A) and other surfactant-related lung alveolar proteins were efficiently separated and identified by matrix-assisted laser desorption/ionization FTICR mass spectrometry . Low molecular mass BALF proteins were separated using a gradient 2-D gel. An efficient extraction/precipitation system was developed and used for the enrichment of surfactant proteins. The result of the BALF proteome analysis show the presence of several isoforms of SP-A, in which an N-non-glycosylierte form and several proline hydroxylations were identified. Furthermore, a number of protein spots were found to contain a mixture of proteins unresolved by 2-D gel electrophoresis, illustrating the feasibility of high-resolution mass spectrometry to provide identifications of proteins that remain unseparated in 2-D gels even upon extended pH gradients.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Proteômica/métodos , Proteinose Alveolar Pulmonar/metabolismo , Alvéolos Pulmonares/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Substituição de Aminoácidos , Ciclotrons , Eletroforese em Gel Bidimensional/métodos , Análise de Fourier , Hemoglobinas/análise , Hemoglobinas/metabolismo , Humanos , Complexo Antígeno L1 Leucocitário/análise , Complexo Antígeno L1 Leucocitário/metabolismo , Dados de Sequência Molecular , Prolina/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/análise , Proteoma/metabolismo , Proteína A Associada a Surfactante Pulmonar/análise , Proteína A Associada a Surfactante Pulmonar/metabolismoRESUMO
Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.
Assuntos
Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Neutrófilos/metabolismo , Receptores de Interleucina-8A/fisiologia , Animais , Glicosilação , Humanos , Interleucina-8/metabolismo , Pulmão/microbiologia , Camundongos , Modelos Biológicos , Neutrófilos/microbiologia , Receptores de Interleucina-8A/metabolismo , Receptor 2 Toll-Like/metabolismo , alfa 1-Antitripsina/farmacologiaRESUMO
Mitochondrial pseudogenes in the human nuclear genome have been previously described, mostly as a source of artifacts during the analysis of the mitochondrial genome. With the availability of the complete human genome sequence, we performed a comprehensive analysis of mtDNA insertions into the nucleus. We found 612 independent integrations that are evenly distributed among all chromosomes as well as within each individual chromosome. The identified pseudogenes account for a content of at least 0.016% of the human nuclear DNA. Up to 30% of a chromosome's mtDNA pseudogene content is composed of fragments that encompass two or more adjacent mitochondrial genes, and we found no correlation between the abundance of mitochondrial transcripts and the multiplicity of integrations. These observations indicate that the migrations of mitochondrial DNA sequences to the nucleus were predominantly DNA mediated. Phylogenetic analysis of the mtDNA pseudogenes and mtDNA sequences of primates indicate a continuous transfer into the nucleus. Because of the limited window of opportunity for mtDNA transfer to the germline, sperm mtDNA, which is released from degenerating mitochondria after fertilization, could be an important source of nuclear mtDNA pseudogenes.
Assuntos
Núcleo Celular/genética , Genoma Humano , Mitocôndrias/genética , Pseudogenes/genética , Transporte Biológico/genética , Mapeamento Cromossômico/métodos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Bases de Dados Genéticas , Ordem dos Genes , Humanos , Filogenia , Recombinação Genética/genéticaRESUMO
Members of the BCL-2-related antiapoptotic family of proteins have been shown previously to regulate ATP/ADP exchange across the mitochondrial membranes and to prevent the loss of coupled mitochondrial respiration during apoptosis. We have found that BCL-2/BCL-x(L) can also improve mitochondrial oxidative phosphorylation in cells harboring pathogenic mutations in mitochondrial tRNA genes. The effect of BCL-2 overexpression in mutated cells was independent from apoptosis and was presumably associated with a modulation of adenine nucleotide exchange between mitochondria and cytosol. These results suggest that BCL-2 can regulate respiratory functions in response to mitochondrial distress by regulating the levels of adenine nucleotides.