Xavier Busquets

Systemic effects of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterised by an inappropriate/excessive inflammatory response of the lungs to respiratory pollutants, mainly tobacco smoking. Recently, besides the typical pulmonary pathology of COPD (i.e. chronic bronchitis and emphysema), several effects occurring outside the lungs have been described, the so-called systemic effects of COPD. These effects are clinically relevant because they modify and can help in the classification and management of the disease. The present review discusses the following systemic effects of chronic obstructive pulmonary disease: 1) systemic inflammation; 2) nutritional abnormalities and weight loss; 3) skeletal muscle dysfunction; and 4) other potential systemic effects. For each of these, the potential mechanisms and clinical implications are discussed and areas requiring further research are highlighted.

Telomere shortening in smokers with and without COPD

Telomeres are complex DNA–protein structures located at the end of eukaryotic chromosomes. Telomere length shortens with age in all replicating somatic cells. It has been shown that tobacco smoking enhances telomere shortening in circulating lymphocytes. The present study investigated whether this effect was further amplified in smokers who develop chronic obstructive pulmonary disease. Telomere length was determined by fluorescence in situ hybridisation in circulating lymphocytes harvested from 26 never-smokers, 24 smokers with normal lung function and 26 smokers with moderate-to-severe airflow obstruction (forced expiratory flow in one second 48±4% predicted). In contrast to never-smokers, telomere length significantly decreased with age in smokers. There was also a dose–effect relationship between the cumulative long-life exposure to tobacco smoking (pack-yrs) and telomere length. The presence and/or severity of chronic airflow obstruction did not modify this relationship. The results of the current study confirm that smoking exposure enhances telomere shortening in circulating lymphocytes. It also demonstrates a dose–effect relationship between exposure to tobacco smoking and telomere length, but failed to show that this effect is amplified in smokers who develop chronic obstructive pulmonary disease.

Membranes: a meeting point for lipids, proteins and therapies

•  Introduction •  Membrane lipid composition •  Membrane lipid structure •  Membrane lipid organization ‐  Why so many different lipids? ‐  Lipid mixing and demixing ‐  Lateral pressure ‐  Surface electrostatics •  Role of lipids in cell functions •  Lipid influence in transmembrane protein function ‐  Prokaryotic potassium channel (KcsA) ‐  Mechanosensitive channels ‐  Voltage‐gated potassium channel (KvAP) ‐  Nicotinic acetylcholine receptor (nAcChR) ‐  G protein‐coupled receptors ‐  Other examples •  Non‐permanent proteins in membranes ‐  Proteins that interact reversibly with the bilayers ‐  Proteins that interact irreversibly with the bilayers ‐  Proteins that interact weakly with the membrane ‐  Proteins that interact strongly with the membrane ‐  G proteins and their interactions with membranes ‐  Small monomeric G proteins: the Ras and Ras‐like family ‐  Protein kinase C •  Membrane microdomains and lipid mediators in the control of heat‐shock protein response ‐  Stress sensing and signalling: the membrane sensor theory ‐  Hsp signalling in cancer and diabetes ‐  The role of membrane microdomains ‐  Lipid mediators of the stress response •  A subpopulation of Hsps can interact with and translocate through membranes ‐  Hsp90 in eukaryotic membranes ‐  Hsp70 in cell membranes ‐  Hsp27‐membrane interactions ‐  Secreted Hsps ‐  Representative cases where Hsps interact with membranes or release from the cells •  Concluding remarks

Enhanced neutrophil response in chronic obstructive pulmonary disease

BACKGROUND Neutrophils are likely to play a major role in the inflammatory response seen in chronic obstructive pulmonary disease (COPD). This study sought to address the hypothesis that an enhanced neutrophil response to proinflammatory agents in COPD may contribute to their recruitment and activation in the lungs. METHODS Circulating neutrophils were obtained from 10 patients with COPD, eight long term smokers with normal lung function, and eight healthy never smoking controls. The in vitro production of reactive oxygen species (ROS) was measured by the NADPH oxidase method (respiratory burst) and the surface expression of several adhesion molecules (Mac-1, LFA-1 andl-selectin) was measured by flow cytometry. Measurements were obtained under basal conditions and after stimulation with phorbol myristate acetate (PMA) and tumour necrosis factor alpha (TNFα). mRNA levels of p22-phox (a subunit of NADPH oxidase) and Mac-1 (CD11b) were also determined by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS Patients with COPD showed enhanced respiratory burst compared with smokers with normal lung function, both under basal conditions (mean (SE) fluorescence intensity (MFI) 15.1 (0.5) v 11.6 (0.5); mean difference –3.4 (95% CI of the difference –5.1 to –1.8), p<0.01) and after PMA stimulation (MFI 210 (7) v 133 (10); mean difference –77 (95% CI of the difference –102 to –52), p<0.01). Mac-1 surface expression was also enhanced in patients with COPD, both under basal conditions (MFI 91 (5)v 45 (3); mean difference –46 (95% CI of the difference –61 to –31), p<0.001) and after stimulation with TNFα (MFI 340 (15) v 263 (11); mean difference –77 (95% CI of the difference –119 to –34), p=0.001). These differences were also apparent when patients with COPD were compared with non-smokers (p<0.05). The mRNA levels of p22-phox and Mac-1 (CD11b) were similar in patients with COPD and smokers with normal lung function, suggesting that the observed differences were due to post-transcriptional regulation. CONCLUSIONS These results demonstrate an enhanced neutrophil response to proinflammatory agents in patients with COPD which may contribute to their enhanced recruitment and activation in the lungs of these patients. These findings support those of other studies which have indicated that the neutrophil is likely to play a major role in the pathogenesis of this disease.

NF-κB activation and iNOS upregulation in skeletal muscle of patients with COPD and low body weight

Background: Weight loss, mostly due to skeletal muscle atrophy, is a frequent and clinically relevant problem in patients with chronic obstructive pulmonary disease (COPD). The molecular mechanisms underlying this phenomenon are unclear. This study sought to investigate whether activation of the nuclear transcription factor NF-κB and upregulation of the inducible form of nitric oxide synthase (iNOS) occur in the skeletal muscle of patients with COPD and low body weight as potential molecular mechanisms leading to cachexia Methods: NF-κB DNA binding activity was determined by electromobility shift assay and the immunoreactivity of its inhibitory subunit IκB-κ and that of iNOS by Western blot analysis in biopsy specimens of the quadriceps femoris muscle of seven COPD patients with normal body mass index (BMI, 27.5 (1) kg/m2) and seven patients with low BMI (18.5 (1) kg/m2). Results: Compared with patients with normal body weight, those with low BMI showed a 30% increase in NF-κB DNA binding activity, a lower expression of IκB-α (3.37 (0.47) IOD v 5.96 (0.75) IOD, p<0.05; mean difference 2.59; 95% CI −4.53 to −0.65) and higher iNOS expression (1.51 (0.29) IOD v 0.78 (0.11) IOD, p<0.05; mean difference 0.74; 95% CI 0.04 to 1.42). Conclusions: NF-κB activation and iNOS induction occur in skeletal muscle of COPD patients with low body weight. These changes might contribute to the molecular pathogenesis of cachexia in COPD.

Modulation of immunoreactive protein kinase C-alpha and beta isoforms and G proteins by acute and chronic treatments with morphine and other opiate drugs in rat brain.

Abstract The abundance of protein kinase C-α and β isoforms (PKC-αβ), PKC-α messenger (m) RNA and guanine nucleotide-binding G protein subunits (Gαi1/2, Gαo and Gβ) were quantitated in the rat cerebral cortex after acute and chronic treatments with various opiate drugs. Acute (100mg/kg for 2h) and chronic (10 to 100mg/kg for 5 days) treatment with morphine decreased similarly the immunoreactivity of PKC-αβ (28% and 32%, respectively). Acute (2h) and chronic treatment (5 days) with other μ-agonists heroin (30mg/kg and 10 to 30mg/kg) and methadone (30mg/kg and 5 to 30mg/kg) also induced similar decreases of PKC-αβ (acute: 25% and 23%; chronic: 28% and 18%). After the chronic treatments, spontaneous (48h) or naloxone (2mg/kg)-precipitated opiate withdrawal (2h) resulted in up-regulation of PKC-αβ above control levels (30-38%), and in the case of morphine withdrawal in a concomitant marked increase in the expression of PKC-α mRNA levels (2.3-fold). Acute (2h) treatments with pentazocine (80mg/kg, mixed κ/δ-agonist and μ-antagonist), spiradoline (30mg/kg, selective κ-agonist) and [D-Pen2, D-Pen5] enkephalin (14nmol i.c.v., selective δ-agonist) induced significant decreases of PKC-αβ (19–33%). Chronic (5 days) treatment with pentazocine (10 to 80mg/kg), but not spiradoline (2 to 30mg/kg), also induced a similar decrease of PKC-αβ (35%). In pentazocine- or spiradoline-dependent rats, naloxone (2mg/kg) did not induce up-regulation of brain PKC-αβ. Acute (10mg/kg for 2h) and chronic (2×10mg/kg for 5 and 14 days) treatment with naloxone did not alter PKC-αβ immunoreactivity. Chronic, but not acute, treatment with μ-agonists (morphine, heroin and methadone) increased the immunoreactivities of Gαi1/2 (33-37%), Gαo (25-41%) and Gβ (10-33%) protein subunits. In heroin- and methadone-dependent rats naloxone (2mg/kg)-precipitated withdrawal (2h) did not modify the up-regulation of these G proteins induced by chronic μ-opiate treatment. In marked contrast to μ-agonists, chronic treatment with high doses of pentazocine and spiradoline or acute treatment with [D-Pen2, D-Pen5] enkephalin did not result in up-regulation of these G protein subunits. After chronic treatment with μ-agonists, significant negative correlations were found when the percentage changes in immunoreactivity of PKC-αβ were related to the percentage changes in immunoreactivity of Gαi1/2 (r =-0.53, n = 29) and Gβ (r =-0.41, n = 24) in the same brains. PKC-αβ abundance did not correlate significantly with the density of Gαo (r =-0.21, n = 28). Together the results indicate that the brain PKC-αβ system may play a major regulatory role in opiate tolerance and dependence. Moreover, the possible in vivo cross-communication between this regulatory enzyme and specific inhibitory G proteins may also be of relevance in the cellular and molecular processes of opiate addiction.

Mutations in TBX1 genocopy the 22q11.2 deletion and duplication syndromes: a new susceptibility factor for mental retardation

A screen for TBX1 gene mutations identified two mutations in patients with some features compatible with the 22q11.2-deletion syndrome but with no deletions. One is a de novo missense mutation and the other is a 5′ untranslated region (5′UTR) C>T change that affects a nucleotide with a remarkable trans-species conservation. Computer modelling shows that the 5′UTR change is likely to affect the mRNA structure and in vitro translation experiments demonstrate that it produces a twofold increase in translation efficiency. Recently, duplications in the 22q11.2 region were reported in patients referred for fragile-X determination because of cognitive and behavioural problems. Because the 5′UTR nucleotide change may be a functional equivalent of a duplication of the TBX1 gene, we decided to screen 200 patients who had been referred for fragile-X determination and 400 healthy control individuals. As a result, we found the 5′UTR mutation to be present in three patients with mental retardation or behavioural problems and absent in control individuals of the same ethnic background. This observation suggests that it may be reasonable to screen for such mutation among patients with unspecific cognitive deficits and we provide an easy and quick way to do it with an amplification refractory mutation system (ARMS) approach. To our knowledge, this is the first human mutation showing that TBX1 is a candidate causing mental retardation associated with the 22q11.2 duplication syndrome.

Decreased plasma levels of orexin-A in sleep apnea.

Background: Orexin-A, also known as hypocretin, is a neuropeptide implicated in appetite and sleep regulation. Because the obstructive sleep apnea syndrome (OSAS) is characterized by obesity and excessive daytime sleepiness, we hypothesized that orexin-A levels may be abnormal in patients with OSAS. Further, since treatment with continuous positive airway pressure (CPAP) in patients with OSAS is very effective in normalizing daytime sleepiness, we also hypothesized that the chronic use of CPAP may influence plasma levels of orexin-A in these patients. Objective: To evaluate plasma levels of orexin-A in patients with OSAS and the effect of CPAP treatment. Patients and Methods: We compared the plasma levels of orexin-A in 13 healthy controls, 27 untreated patients with OSAS and 14 patients treated with CPAP during at least 1 year (4.5 ± 0.5 h/night; mean ± SEM). All patients had severe OSAS (apnea-hypopnea index, 57 ± 4 h–1). Results: Orexin-A plasma levels were significantly lower in untreated (9.4 ± 1.9 pg·ml–1, p < 0.01) and treated patients with OSAS (4.2 ± 1.5 pg·ml–1, p < 0.001) than in healthy subjects (20.6 ± 4.5 pg·ml–1). In untreated patients, orexin-A levels were not significantly related to daytime somnolence assessed by Epworth scale (r = –0.18, p = 0.37) or the body mass index (r = –0.13, p = 0.52). Conclusions: Orexin-A plasma levels are abnormally low in patients with OSAS, independently of the level of somnolence and/or presence of obesity. These results suggest that these low orexin-A levels may be related to the pathogenesis of OSAS.

Inflamación sistémica durante las agudizaciones de la enfermedad pulmonar obstructiva crónica

OBJECTIVE: The circulating blood levels of several inflammatory cytokines and acute phase proteins are higher in patients with stable chronic obstructive pulmonary disease (COPD). However, whether or not these inflammatory markers increase during COPD exacerbation or are modified by corticosteroid treatment has not been investigated. The objective of this study was therefore 1) to describe changes in several inflammatory markers in systemic circulation during COPD exacerbation, and 2) to assess the potential effects of corticosteroid treatment during exacerbation. METHODS: Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), interleukin 8 (IL-8) and C-reactive protein (CRP) were determined for 10 patients (65 2 years old) with severe COPD (FEV1 35 4% reference) who were hospitalized for acute respiratory failure (PaO2 57 2 mm Hg; PaCO2 48 3 mm Hg). Blood samples were obtained in the emergency room (before starting intravenous corticosteroid treatment), during the first 24 hours of admission, upon discharge and two months later. Eight healthy non-smokers of a similar age (54 3 years) were also studied as control subjects. RESULTS: The COPD patients had higher concentrations of IL-6 (5.1 1.6 vs. 1.8 0.5 pg/mL, p < 0.05) and CRP (2.2 0.4 vs. 0.6 0.2 mg/dL, p < 0.005) than did controls, but the concentrations of IL-8 were similar (29 11.3 vs. 34.7 10.3 pg/mL, p = ns). No statistically significant changes were seen either during recovery, in spite of intravenous corticosteroid treatment, or two months after discharge.The ELISA test used was unable to detect TNF-alpha in any of the samples obtained from either patients or controls. CONCLUSION: The results show that 1) there is evidence of systemic inflammation during exacerbation of COPD, and 2) such systemic inflammation does not appear to be influenced significantly by intravenous corticosteroid treatment.

2-Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy

Despite recent advances in the development of new cancer therapies, the treatment options for glioma remain limited, and the survival rate of patients has changed little over the past three decades. Here, we show that 2-hydroxyoleic acid (2OHOA) induces differentiation and autophagy of human glioma cells. Compared to the current reference drug for this condition, temozolomide (TMZ), 2OHOA combated glioma more efficiently and, unlike TMZ, tumor relapse was not observed following 2OHOA treatment. The novel mechanism of action of 2OHOA is associated with important changes in membrane-lipid composition, primarily a recovery of sphingomyelin (SM) levels, which is markedly low in glioma cells before treatment. Parallel to membrane-lipid regulation, treatment with 2OHOA induced a dramatic translocation of Ras from the membrane to the cytoplasm, which inhibited the MAP kinase pathway, reduced activity of the PI3K/Akt pathway, and downregulated Cyclin D-CDK4/6 proteins followed by hypophosphorylation of the retinoblastoma protein (RB). These regulatory effects were associated with induction of glioma cell differentiation into mature glial cells followed by autophagic cell death. Given its high efficacy, low toxicity, ease of oral administration, and good distribution to the brain, 2OHOA constitutes a new and potentially valuable therapeutic tool for glioma patients.