Crystalline silica alters Sulfatase-1 expression in rat lungs which influences hyper-proliferative and fibrogenic effects in human lung epithelial cells.

Lung epithelial cells are the first cell-type to come in contact with hazardous dust materials. Upon deposition, they invoke complex reactions in attempt to eradicate particles from the airways, and repair damage. The cell surface is composed of a heterogeneous network of matrix proteins and proteoglycans, which act as scaffold and control cell-signaling networks. These functions are controlled, in part, by the sulfation patterns of heparin-sulfate proteoglycans (HSPGs), which are enzymatically regulated. Although there is evidence of altered HSPG-sulfation in idiopathic pulmonary fibrosis (IPF), this is not investigated in silicosis. Our previous studies revealed down-regulation of Sulfatase-1 (SULF1) in human bronchial epithelial cells (BECs) by crystalline silica (CS). In this study, CS-induced down-regulation of SULF1, and increases in Sulfated-HSPGs, were determined in human BECs, and in rat lungs. By siRNA and plasmid transfection techniques the effects of SULF1 expression on silica-induced fibrogenic and proliferative gene expression were determined. These studies confirmed down-regulation of SULF1 and subsequent increases in sulfated-HSPGs in vitro. Moreover, short-term exposure of rats to CS resulted in similar changes in vivo. Conversely, effects were reversed after long term CS exposure of rats. SULF1 knockdown, and overexpression alleviated and exacerbated silica-induced decrease in cell viability, respectively. Furthermore, overexpression of SULF1 promoted silica-induced proliferative and fibrogenic gene expression, and collagen production. These findings demonstrate that the HSPG modification enzyme SULF1 and HSPG sulfation are altered by CS in vitro and in vivo. Furthermore, these changes may contribute to CS-induced lung pathogenicity by affecting injury tolerance, hyperproliferation, and fibrotic effects.

Involvement of c-Jun N-Terminal Kinase in TNF-α-Driven Remodeling.

Lung tissue remodeling in chronic obstructive pulmonary disease (COPD) is characterized by airway wall thickening and/or emphysema. Although the bronchial and alveolar compartments are functionally independent entities, we recently showed comparable alterations in matrix composition comprised of decreased elastin content and increased collagen and hyaluronan contents of alveolar and small airway walls. Out of several animal models tested, surfactant protein C (SPC)-TNF-α mice showed remodeling in alveolar and airway walls similar to what we observed in patients with COPD. Epithelial cells are able to undergo a phenotypic shift, gaining mesenchymal properties, a process in which c-Jun N-terminal kinase (JNK) signaling is involved. Therefore, we hypothesized that TNF-α induces JNK-dependent epithelial plasticity, which contributes to lung matrix remodeling. To this end, the ability of TNF-α to induce a phenotypic shift was assessed in A549, BEAS2B, and primary bronchial epithelial cells, and phenotypic markers were studied in SPC-TNF-α mice. Phenotypic markers of mesenchymal cells were elevated both in vitro and in vivo, as shown by the expression of vimentin, plasminogen activator inhibitor-1, collagen, and matrix metalloproteinases. Concurrently, the expression of the epithelial markers, E-cadherin and keratin 7 and 18, was attenuated. A pharmacological inhibitor of JNK attenuated this phenotypic shift in vitro, demonstrating involvement of JNK signaling in this process. Interestingly, activation of JNK signaling was also clearly present in lungs of SPC-TNF-α mice and patients with COPD. Together, these data show a role for TNF-α in the induction of a phenotypic shift in vitro, resulting in increased collagen production and the expression of elastin-degrading matrix metalloproteinases, and provide evidence for involvement of the TNF-α-JNK axis in extracellular matrix remodeling.

Alteration of canonical and non-canonical WNT-signaling by crystalline silica in human lung epithelial cells.

Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damaging inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (ß-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells.

Exposure to common respiratory bacteria alters the airway epithelial response to subsequent viral infection.

BACKGROUND: Colonization of the airways with potential pathogenic bacteria is observed in a number of chronic respiratory diseases, such as COPD or cystic fibrosis. Infections with respiratory viruses are known triggers of exacerbations of these diseases. We here investigated if pre-exposure to bacteria alters the response of lung epithelial cells to subsequent viral infection. METHODS: Bronchial epithelial cells (BEAS-2B cells and primary bronchial epithelial cells) were exposed to heat-inactivated Haemophilus influenzae, Pseudomonas aeruginosa or Streptococcus pneumoniae and subsequently infected with respiratory syncytial virus (RSV), type 2 human adenovirus or influenza B. Levels of pro-inflammatory cytokines, viral replication and expression of pattern recognition receptors were determined in culture supernatants and/or cell lysates. RESULTS: Exposure of BEAS-2B cells to H. influenzae before and during RSV-infection synergistically increased the release of IL-6 (increase above calculated additive effect at 72 h: 56 % ± 3 %, mean ± SEM) and IL-8 (53 % ± 12 %). This effect was sustained even when bacteria were washed away before viral infection and was neither associated with enhanced viral replication, nor linked to increased expression of key pattern recognition receptors. P. aeruginosa enhanced the release of inflammatory cytokines to a similar extent, yet only if bacteria were also present during viral infection. S. pneumoniae did not enhance RSV-induced cytokine release. Surprisingly, adenovirus infection significantly reduced IL-6 release in cells exposed to either of the three tested bacterial strains by on average more than 50 %. Infection with influenza B on the other hand did not affect cytokine production in BEAS-2B cells exposed to the different bacterial strains. CONCLUSION: Pre-exposure of epithelial cells to bacteria alters the response to subsequent viral infection depending on the types of pathogen involved. These findings highlight the complexity of microbiome interactions in the airways, possibly contributing to the susceptibility to exacerbations and the natural course of airway diseases.

A comparative study of matrix remodeling in chronic models for COPD; mechanistic insights into the role of TNF-α.

Remodeling in chronic obstructive pulmonary disease (COPD) has at least two dimensions: small airway wall thickening and destruction of alveolar walls. Recently we showed comparable alterations of the extracellular matrix (ECM) compounds collagen, hyaluoran, and elastin in alveolar and small airway walls of COPD patients. The aim of this study was to characterize and assess similarities in alveolar and small airway wall matrix remodeling in chronic COPD models. From this comparative characterization of matrix remodeling we derived and elaborated underlying mechanisms to the matrix changes reported in COPD. Lung tissue sections of chronic models for COPD, either induced by exposure to cigarette smoke, chronic intratracheal lipopolysaccharide instillation, or local tumor necrosis factor (TNF) expression [surfactant protein C (SPC)-TNFα mice], were stained for elastin, collagen, and hyaluronan. Furthermore TNF-α matrix metalloproteinase (MMP)-2, -9, and -12 mRNA expression was analyzed using qPCR and localized using immunohistochemistry. Both collagen and hyaluronan were increased in alveolar and small airway walls of all three models. Interestingly, elastin contents were differentially affected, with a decrease in both alveolar and airway walls in SPC-TNFα mice. Furthermore TNF-α and MMP-2 and -9 mRNA and protein levels were found to be increased in alveolar walls and around airway walls only in SPC-TNFα mice. We show that only SPC-TNFα mice show changes in elastin remodeling that are comparable to what has been observed in COPD patients. This reveals that the SPC-TNFα model is a suitable model to study processes underlying matrix remodeling and in particular elastin breakdown as seen in COPD. Furthermore we indicate a possible role for MMP-2 and MMP-9 in the breakdown of elastin in airways and alveoli of SPC-TNFα mice.

Similar matrix alterations in alveolar and small airway walls of COPD patients.

BACKGROUND: Remodelling in COPD has at least two dimensions: small airway wall thickening and destruction of alveolar walls. Recent studies indicate that there is some similarity between alveolar and small airway wall matrix remodelling. The aim of this study was to characterise and assess similarities in alveolar and small airway wall matrix remodelling, and TGF-ß signalling in COPD patients of different GOLD stages. METHODS: Lung tissue sections of 14 smoking controls, 16 GOLD II and 19 GOLD IV patients were included and stained for elastin and collagens as well as hyaluronan, a glycosaminoglycan matrix component and pSMAD2. RESULTS: Elastin was significantly decreased in COPD patients not only in alveolar, but also in small airway walls. Interestingly, both collagen and hyaluronan were increased in alveolar as well as small airway walls. The matrix changes were highly comparable between GOLD stages, with collagen content in the alveolar wall increasing further in GOLD IV. A calculated remodelling index, defined as elastin divided over collagen and hyaluronan, was decreased significantly in GOLD II and further lowered in GOLD IV patients, suggesting that matrix component alterations are involved in progressive airflow limitation. Interestingly, there was a positive correlation present between the alveolar and small airway wall stainings of the matrix components, as well as for pSMAD2. No differences in pSMAD2 staining between controls and COPD patients were found. CONCLUSIONS: In conclusion, remodelling in the alveolar and small airway wall in COPD is markedly similar and already present in moderate COPD. Notably, alveolar collagen and a remodelling index relate to lung function.

Decreased plasma sRAGE levels in COPD: influence of oxygen therapy.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with systemic inflammation and oxidative stress. N(ε) -(carboxymethyl) lysine (CML), an advanced glycation end product (AGE) and the soluble decoy receptor, sRAGE, are exciting new molecules linked to oxidative stress and inflammation. Here the levels of plasma sRAGE and CML were determined and their variation in relation to lung function, external long-term oxygen therapy (LTOT) and plasma levels of inflammatory molecules in COPD evaluated. METHODS: Plasma sRAGE and CML levels were measured by ELISA in 146 patients with stable COPD and 81 healthy subjects, subgrouped from a larger case-control study and matched for age, gender and pack-years smoked. RESULTS: Decreased levels of plasma sRAGE and no significant difference in levels of plasma CML were found in patients with COPD in comparison with controls. In the total group, plasma sRAGE was positively associated with FEV(1) and forced vital capacity and negatively with pack-years smoked. In patients receiving LTOT, levels of plasma sRAGE were lower compared with those without LTOT. Only in controls, a weak correlation was found between plasma sRAGE and CML. sRAGE did not correlate with measured inflammatory markers, whereas CML was negatively correlated with fibrinogen. CONCLUSION: Plasma sRAGE levels are lower in patients with COPD compared with healthy control subjects, and even lower levels in patients receiving LTOT. Because sRAGE correlated with lung function only in the whole group, sRAGE can be considered a marker of COPD, but not of disease severity. A lack of clear association between sRAGE, CML and systemic inflammation is furthermore evident.

Smoking-Associated Exposure of Human Primary Bronchial Epithelial Cells to Aldehydes: Impact on Molecular Mechanisms Controlling Mitochondrial Content and Function.

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease primarily caused by exposure to cigarette smoke (CS). During the pyrolysis and combustion of tobacco, reactive aldehydes such as acetaldehyde, acrolein, and formaldehyde are formed, which are known to be involved in respiratory toxicity. Although CS-induced mitochondrial dysfunction has been implicated in the pathophysiology of COPD, the role of aldehydes therein is incompletely understood. To investigate this, we used a physiologically relevant in vitro exposure model of differentiated human primary bronchial epithelial cells (PBEC) exposed to CS (one cigarette) or a mixture of acetaldehyde, acrolein, and formaldehyde (at relevant concentrations of one cigarette) or air, in a continuous flow system using a puff-like exposure protocol. Exposure of PBEC to CS resulted in elevated IL-8 cytokine and mRNA levels, increased abundance of constituents associated with autophagy, decreased protein levels of molecules associated with the mitophagy machinery, and alterations in the abundance of regulators of mitochondrial biogenesis. Furthermore, decreased transcript levels of basal epithelial cell marker KRT5 were reported after CS exposure. Only parts of these changes were replicated in PBEC upon exposure to a combination of acetaldehyde, acrolein, and formaldehyde. More specifically, aldehydes decreased MAP1LC3A mRNA (autophagy) and BNIP3 protein (mitophagy) and increased ESRRA protein (mitochondrial biogenesis). These data suggest that other compounds in addition to aldehydes in CS contribute to CS-induced dysregulation of constituents controlling mitochondrial content and function in airway epithelial cells.

Enhanced deposition of low-molecular-weight hyaluronan in lungs of cigarette smoke-exposed mice.

Chronic obstructive pulmonary disease (COPD) is characterized by infiltration of inflammatory cells, destruction of lung parenchyma, and airway wall remodeling. Hyaluronan (HA) is a component of the extracellular matrix, and low-molecular-weight (LMW) HA fragments have proinflammatory capacities. We evaluated the presence of HA in alveolar and airway walls of C57BL/6 mice that were exposed to air or cigarette smoke (CS) for 4 weeks (subacute) or 24 weeks (chronic). We measured deposition of the extracellular matrix proteins collagen and fibronectin in airway walls and determined the molecular weight of HA purified from lung tissue. In addition, we studied the expression of HA-modulating genes by RT-PCR. HA staining in alveolar walls was significantly enhanced upon chronic CS exposure, whereas HA levels in the airway walls were already significantly higher upon subacute CS exposure and remained elevated upon chronic CS exposure. This differed from the deposition of collagen and fibronectin, which are only elevated at the chronic time point. In lungs of CS-exposed mice, the molecular weight of HA clearly shifted toward more LMW HA fragments. CS exposure significantly increased the mRNA expression of the HA synthase gene Has3 in total lung tissue, whereas the expression of Has1 was decreased. These in vivo studies in an experimental model of COPD show that CS exposure leads to enhanced deposition of (mostly LMW) HA in alveolar and bronchial walls by altering the expression of HA-modulating enzymes. This may contribute to airway wall remodeling and pulmonary inflammation in COPD.

IL6 and CRP haplotypes are associated with COPD risk and systemic inflammation: a case-control study.

BACKGROUND: Elevated circulating levels of C-reactive protein (CRP), interleukin (IL)-6 and fibrinogen (FG) have been repeatedly associated with many adverse outcomes in patients with chronic obstructive pulmonary disease (COPD). To date, it remains unclear whether and to what extent systemic inflammation is primary or secondary in the pathogenesis of COPD. The aim of this study was to examine the association between haplotypes of CRP, IL6 and FGB genes, systemic inflammation, COPD risk and COPD-related phenotypes (respiratory impairment, exercise capacity and body composition). METHODS: Eighteen SNPs in three genes, representing optimal haplotype-tagging sets, were genotyped in 355 COPD patients and 195 healthy smokers. Plasma levels of CRP, IL-6 and FG were measured in the total study group. Differences in haplotype distributions were tested using the global and haplotype-specific statistics. RESULTS: Raised plasma levels of CRP, IL-6 and fibrinogen were demonstrated in COPD patients. However, COPD population was very heterogeneous: about 40% of patients had no evidence of systemic inflammation (CRP < 3 mg/uL or no inflammatory markers in their top quartile). Global test for haplotype effect indicated association of CRP gene and CRP plasma levels (P = 0.0004) and IL6 gene and COPD (P = 0.003). Subsequent analysis has shown that IL6 haplotype H2, associated with an increased COPD risk (p = 0.004, OR = 4.82; 1.64 to 4.18), was also associated with very low CRP levels (p = 0.0005). None of the genes were associated with COPD-related phenotypes. CONCLUSION: Our findings suggest that common genetic variation in CRP and IL6 genes may contribute to heterogeneity of COPD population associated with systemic inflammation.

Effect of infliximab on local and systemic inflammation in chronic obstructive pulmonary disease: a pilot study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) with cachexia is characterized by inflammation reflected by increased levels of tumor necrosis factor-alpha (TNF-alpha). OBJECTIVES: In this study, infliximab, an anti-TNF-alpha antibody, was evaluated for its effects on systemic (plasma) and local (exhaled breath condensate, EBC) inflammation in cachectic patients with COPD. Also, baseline levels of new inflammatory markers were compared to control subjects. METHODS: Sixteen cachectic patients with moderate to severe COPD were examined for inflammatory status at baseline and compared to 25 control subjects. Patients were randomized (1:1) to receive infliximab (5 mg/kg) or placebo at weeks 0, 2 and 6. Patients were evaluated at weeks 8 and 12 and followed through week 26. RESULTS: EBC analysis revealed increased levels of several novel inflammatory markers, including macrophage migration inhibitory factor, IL-12, RANTES and sICAM-1, in patients with COPD compared to controls. EBC levels of inflammatory markers were unchanged in patients receiving infliximab. In addition, systemic levels of acute-phase proteins (C-reactive protein, fibrinogen and lipopolysaccharide-binding protein), IL-6 and soluble TNF receptor (sTNFR) 55 had not changed at weeks 8 or 12. Small increases in circulating levels of sTNFR75, myeloperoxidase and Clara cell protein 16 were seen at week 8, but not at week 12. CONCLUSIONS: In this small study, infliximab did not produce an observable decrease in local inflammation in cachectic patients with COPD and had minor effects on systemic inflammation. The detection of new inflammatory markers in EBC can help to further characterize local inflammatory processes in COPD.

Systemic effects of smoking.

Smoking is one of the major lifestyle factors influencing the health of human beings. Life-long cigarette smokers have a higher prevalence of common diseases such as atherosclerosis and COPD with significant systemic impact. The present review evaluates current knowledge concerning possible pathways through which cigarette smoking can affect human health, with special focus on extrapulmonary effects. Long-term smoke exposure can result in systemic oxidants-antioxidants imbalance as reflected by increased products of lipid peroxidation and depleted levels of antioxidants like vitamins A and C in plasma of smokers. A low-grade systemic inflammatory response is evident in smokers as confirmed by numerous population-based studies: elevated levels of C-reactive protein (CRP), fibrinogen, and interleukin-6, as well as increased counts of WBC have been reported. Furthermore, rheologic, coagulation and endothelial function markers like hematocrit, blood and/or plasma viscosity, fibrin d-dimer, circulating adhesion molecules (intracellular adhesion molecule-1, selectins), tissue plasminogen activator antigen, and plasminogen activator inhibitor type I are altered in chronic cigarette smokers. Although most of smoking-induced changes are reversible after quitting, some inflammatory mediators like CRP are still significantly raised in ex-smokers up to 10 to 20 years after quitting, suggesting ongoing low-grade inflammatory response persisting in former smokers. New longitudinal epidemiologic and genetic studies are required to evaluate the role of smoking itself and possible gene/environment interplay in initiation and development of smoking-induced common diseases affecting humans.

Longitudinal follow-up of systemic inflammation after acute exacerbations of COPD.

BACKGROUND: Acute exacerbations are important in the clinical course of COPD, yet the underlying mechanisms are poorly understood. Systemic inflammation is now considered as an important component in the disease process. In this study we evaluated longitudinally the systemic inflammation during hospital treatment for acute exacerbation and after clinical recovery. METHODS: Blood was collected on day 0, 1, 4 and 8 in 21 patients admitted for an acute exacerbation of COPD and at 1 month, 3 months and 6 months after discharge. Systemic inflammation was determined by measurement of the pro-inflammatory markers interleukin (IL)-6, soluble tumor necrosis factor (TNF) receptors sTNFR55 and sTNFR75, the anti-inflammatory mediator sIL-1RII, and bactericidal permeability increasing protein (BPI) as a marker of neutrophil activation. In addition, plasma level of Trolox antioxidant capacity (TEAC) was determined. Healthy age-matched controls were measured for the same markers at one time-point. RESULTS: All inflammatory markers analyzed were elevated on first day of admission for exacerbation of COPD, as compared to healthy controls. During treatment, levels of IL-6, and sTNFR75 rapidly decreased, whereas sTNFR55 and BPI remained elevated. Moreover, sIL-1RII and TEAC increased during first 8 days of treatment. In the stable condition all inflammatory markers returned to values comparable to healthy controls, with the exception of BPI, which remained persistently elevated compared to healthy controls. CONCLUSION: This study clearly demonstrates upregulation of systemic inflammation in acute exacerbations of COPD. Attenuation of systemic inflammation may offer new perspectives in the management of COPD patients to reduce the burden of exacerbations.

Human bancroftian filariasis: immunological markers of morbidity and infection.

Induction of host cytokines plays a critical role in infection as well as disease in human filariasis. Measurements of such molecules in plasma could be used as windows of markers both for understanding the pathogenesis of the disease and for identifying markers of morbidity. Eight inflammatory and non-inflammatory host molecules in circulation were quantified in 207 subjects in filariasis endemic area of Orissa, India. IL-6, IL-8, IL-10, TNF-alpha, TNFR-I, TNFR-II, LBP and sICAM-1 were quantified by immunoassays and were analyzed by multivariate exploratory data analysis methods followed by multivariate analysis of variance. Raised levels of IL-6 and IL-8 emerged as markers of acute as well as chronic disease, while increased TNF-alpha was a feature found only in acute filariasis. Decreased sICAM-1 was a feature found only in asymptomatic subjects with filarial infection. There was a dichotomy in plasma levels of two TNF receptors between infected subjects and patients with filarial disease. Since plasma levels of these cytokines are often determined by host genetics, studies on cytokine genetic polymorphisms could offer new insights into the relationship between infection and disease in human lymphatic filariasis.

Patterns of inflammation and the use of reversibility testing in smokers with airway complaints.

BACKGROUND: Although both smoking and respiratory complaints are very common, tools to improve diagnostic accuracy are scarce in primary care. This study aimed to reveal what inflammatory patterns prevail in clinically established diagnosis groups, and what factors are associated with eosinophilia. METHOD: Induced sputum and blood plasma of 59 primary care patients with COPD (n = 17), asthma (n = 11), chronic bronchitis (CB, n = 14) and smokers with no respiratory complaints ('healthy smokers', n = 17) were collected, as well as lung function, smoking history and clinical work-up. Patterns of inflammatory markers per clinical diagnosis and factors associated with eosinophilia were analyzed by multiple regression analyses, the differences expressed in odds ratios (OR) with 95% confidence intervals. RESULTS: Multivariately, COPD was significantly associated with raised plasma-LBP (OR 1.2 [1.04-1.37]) and sTNF-R55 in sputum (OR 1.01 [1.001-1.01]), while HS expressed significantly lowered plasma-LBP (OR 0.8 [0.72-0.95]). Asthma was characterized by higher sputum eosinophilic counts (OR 1.3 [1.05-1.54]), while CB showed a significantly higher proportion of sputum lymphocytic counts (OR 1.5 [1.12-1.9]). Sputum eosinophilia was significantly associated with reversibility after adjusting for smoking, lung function, age, gender and allergy. CONCLUSION: Patterns of inflammatory markers in a panel of blood plasma and sputum cells and mediators were discernable in clinical diagnosis groups of respiratory disease. COPD and so-called healthy smokers showed consistent opposite associations with plasma LBP, while chronic bronchitics showed relatively predominant lymphocytic inflammation compared to other diagnosis groups. Only sputum eosinophilia remained significantly associated with reversibility across the spectrum of respiratory disease in smokers with airway complaints.

Lipopolysaccharide-binding protein is produced in the epididymis and associated with spermatozoa and prostasomes.

Lipopolysaccharide-binding protein (LBP) is an acute phase protein known to play a central role in the defense against Gram-negative bacteria. It binds lipopolysaccharides of Gram-negative bacteria and, after binding to CD14, the complex signals through Toll-like receptor (TLR)-4, eliciting host-defense responses, such as cytokine production, in inflammatory cells. The present study demonstrates constitutive expression of the gene encoding lipopolysaccharide-binding protein in the epithelium of the human epididymis by in situ hybridization. Using immunohistochemistry lipopolysaccharide-binding protein was shown to be present in the same cells and also attached to the heads and tails of spermatozoa. Cell-free seminal plasma, lysed spermatozoa and lysed prostasomes were subjected to Western blot; all showed immunoreactive bands corresponding to the size of lipopolysaccharide-binding protein. Gel filtration demonstrated that lipopolysaccharide-binding protein colocalizes with prostasomes. The concentration of lipopolysacharide-binding protein in seminal plasma was 127+/-42ng/mL (mean+/-S.D.; range 73-215ng/mL). Taken together, our results suggest roles for lipopolysaccharide-binding protein during human reproduction.

Tumor necrosis factor-alpha +489G/A gene polymorphism is associated with chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by a chronic inflammatory process, in which the pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-alpha is considered to play a role. In the present study the putative involvement of TNF-alpha gene polymorphisms in pathogenesis of COPD was studied by analysis of four TNF-alpha gene polymorphisms in a Caucasian COPD population. METHODS: TNF-alpha gene polymorphisms at positions -376G/A, -308G/A, -238G/A, and +489G/A were examined in 169 Dutch COPD patients, who had a mean forced expiratory volume in one second (FEV1) of 37 +/- 13%, and compared with a Dutch population control group of 358 subjects. RESULTS: The data showed that the TNF-alpha +489G/A genotype frequency tended to be different in COPD patients as compared to population controls, which was due to an enhanced frequency of the GA genotype. In line herewith, carriership of the minor allele was associated with enhanced risk of development of COPD (odds ratio = 1.9, p = 0.009). The other TNF-alpha gene polymorphisms studied revealed no discrimination between patients and controls. No differences in the examined four TNF-alpha polymorphisms were found between subtypes of COPD, which were stratified for the presence of radiological emphysema. However, comparison of the COPD subtypes with controls showed a significant difference in the TNF-alpha +489G/A genotype in patients without radiological emphysema (chi2-test: p < 0.025 [Bonferroni adjusted]), while no differences between COPD patients with radiological emphysema and controls were observed. CONCLUSION: Based on the reported data, it is concluded that COPD, and especially a subgroup of COPD patients without radiological emphysema, is associated with TNF-alpha +489G/A gene polymorphism.

Childhood asthma: exhaled markers of airway inflammation, asthma control score, and lung function tests.

Exhaled markers of airway inflammation become increasingly important in the management of childhood asthma. The aims of the present study are: 1) to compare exhaled markers of inflammation (nitric oxide, carbon monoxide, and acidity of breath condensate) with conventional asthma measures (lung function tests and asthma control score) in childhood asthma; and 2) to investigate the detectability of albumin, CRP, IL-6, IL-8, TNF-alpha, sICAM-1, and sTNF-R75 in the exhaled breath condensate (EBC) of asthmatic children. Thirty-two children with mild to moderate persistent asthma and healthy controls aged 6-12 years were studied. We measured exhaled NO and CO, and subsequently EBC was collected. Inflammatory mediators in EBC were measured using an enzyme-linked immunosorbent assay. Respiratory symptoms and asthma control were assessed using the asthma control questionnaire (ACQ) of Juniper et al. (Eur Respir J 1999;14:902-907). Exhaled NO showed a significant correlation with exhaled CO (r = 0.59, P < 0.05) and FEV1 (r = -0.59, P < 0.05), but not with ACQ score (r = 0.48, P = 0.06). Exhaled CO was correlated with prebronchodilator FEV1 (r = -0.45, P < 0.05), but not with asthma control (r = 0.18, P = 0.35). Acidity of EBC was significantly lower in asthmatic children than in healthy controls (P < 0.05), but did not correlate with any of the conventional asthma measures. We were not able to demonstrate the presence of CRP, IL-6, IL-8, TNF-alpha, sICAM-1, and sTNF-R75 in EBC. Albumin was found in two EBC samples of asthmatic children. We conclude that exhaled NO had a better correlation with lung function parameters and asthma control than exhaled CO and acidity of EBC, in mild to moderate persistent childhood asthma. However, exhaled NO, CO, and deaerated pH of EBC did not differ between asthmatic children and controls, possibly because of a too homogeneous and well-controlled study population. To further evaluate the clinical utility of exhaled markers in monitoring childhood asthma, more studies are required on a wider range of asthma severity, and preferably with repeated measurements of markers and of asthma control.