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Rationale: Patients with chronic obstructive pulmonary disease (COPD) experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury. Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD. Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterized by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage was assessed. Histotoxic protein concentrations were measured in normoxic versus hypoxic neutrophil supernatants and plasma from patients experiencing COPD exacerbation and healthy control subjects. Measurements and Main Results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from patients with COPD. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage, and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia, and hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of patients with COPD had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers. Conclusions: Hypoxia drives a destructive "hypersecretory" neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of neutrophil degranulation and vascular injury identified in plasma of patients with COPD. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic opportunities for endothelial damage in COPD.

Rationale: Augmentation therapy with intravenous AAT (alpha-1 antitrypsin) is the only specific therapy for individuals with pulmonary disease from AAT deficiency (AATD). The recommended standard dose (SD; 60 mg/kg/wk) elevates AAT trough serum levels to around 50% of normal; however, outside of slowing emphysema progression, its effects in other clinical outcomes have not been rigorously proven. Objectives: To evaluate the biological effects of normalizing AAT trough levels with double-dose (DD) therapy (120 mg/kg/wk) in subjects with AATD already receiving SD therapy. Methods: Clinically stable subjects were evaluated after 4 weeks of SD therapy, followed by 4 weeks of DD therapy, and 4 weeks after return to SD therapy. At the end of each phase, BAL fluid (BALF) and plasma samples were obtained. Measurements and Main Results: DD therapy increased trough AAT levels to normal and, compared with SD therapy, reduced serine protease activity in BALF (elastase and cathepsin G), plasma elastase footprint (Aα-Val360), and markers of elastin degradation (desmosine/isodesmosine) in BALF. DD therapy also further downregulated BALF ILs and cytokines including Jak-STAT (Janus kinases–signal transducer and activator of transcription proteins), TNFα (tumor necrosis factor-α), and T-cell receptor signaling pathways, cytokines involved in macrophage migration, eosinophil recruitment, humoral and adaptive immunity, neutrophil activation, and cachexia. On restarting SD after DD treatment, a possible carryover effect was seen for several biological markers. Conclusions: Subjects with AATD on SD augmentation therapy still exhibit inflammation, protease activity, and elastin degradation that can be further improved by normalizing AAT levels. Higher AAT dosing than currently recommended may lead to enhanced clinical benefits and should be explored further. Clinical trial registered with www.clinicaltrials.gov (NCT 01669421).

Introduction Asthma is a common, chronic lung disease marked by reversible airflow obstruction driven by inflammation. There is increasing evidence for a neutrophil-dominant inflammatory endotype1 with clear mechanistic potential for neutrophil elastase (NE) to cause clinical manifestations of disease. However, the relationship between neutrophil proteinases and asthma pathophysiology remains uncertain. We hypothesised that patients with a neutrophilic-endotype would have increased NE activity compared to other asthma endotypes and healthy smokers. Methods Patients with respiratory physician-confirmed asthma diagnosis were recruited. Controls without asthma were age-matched with normal lung function and no symptoms suggestive of asthma/alternative respiratory disease Asthma patients were divided into endotypes by analysis of sputum cellular composition as previously described.1 NE activity was measured in plasma using an ELISA based on specific fibrinogen-cleavage product Aα–Val360.2 Results Forty-five asthma patients (Mean (SD): age 60.6 years (10.3); 40% male) and 45 controls (age 60.2 years (10.1); 51% male) were recruited. Asthma patients had evidence of airflow obstruction (mean (SD): FEV1/FVC% predicted asthma 64.3 (11.7) versus controls 77.5 (6.3), p When considered together, patients with asthma had a significantly higher NE activity footprint than controls (mean (SD): asthma 12.7 nM (5.7), control 9.4 nM (3.1), p=0.0011) When divided into phenotypes, patients with neutrophil-dominant asthma (n=15) had increased activity footprint compared to a specifically age-matched sub-group of controls (n=15, Mean (SD): asthma 15.0 nM (5.9), control 8.9 nM (2.9), p=0.0034). There were no differences between the NE activity footprint comparing patients with eosinophilic (n=11, 11.4 nM (5.5) p>0.9999) or paucigranulocytic (n=14, 10.3 nM (5.4) p>0.9999) endotypes to controls. In neutrophilic asthma, there was no correlation between NE activity and neutrophils in sputum (%) (R2=0.02677, p=0.5601), age (R2=0.08626, p=0.2880), FEV1/FVC% (R2=0.04410, p=0.4911), or smoking exposure (R2=0.5515, p=0.4766). Conclusions These findings suggest that there is a differing systemic proteolytic enzyme activity in neutrophil–dominant asthma patients compared to controls and between neutrophilic asthma and paucigranulocytic asthma, which may influence disease pathophysiology. References Simpson JL, et al. ERJ 2014. Carter RI, et al. Thorax 2015.

AIM To investigate associations between periodontitis and chronic obstructive pulmonary disease (COPD) with and without alpha-1 antitrypsin deficiency (AATD), including neutrophil functions implicated in tissue damage. METHODS The presence and severity of periodontitis (using two international criteria) and lung disease were assessed in 156 COPD patients with and without AATD accounting for common confounding factors. Saliva and systemic inflammatory markers were measured by ELISA together with neutrophil migration. RESULTS COPD and AATD patients exhibited higher prevalence of periodontitis (COPD 95%; AATD 88%) than reported in unselected community-dwelling populations even when risk factors (age, smoking history, socio-economic status and dental habits) were considered. Periodontitis severity associated with lung disease severity (AATD, periodontitis versus no periodontitis; FEV1 = 56% versus 99% predicted; TLCO = 59% versus 81% predicted, p

Introduction Chronic obstructive pulmonary disease (COPD) is a progressive, irreversible inflammatory disease caused by excess neutrophilic inflammation resulting in an enhanced inflammatory response in the airways. It is widely accepted that the inflammation and tissue damage relates to a proteinase/anti-proteinase imbalance with both neutrophil elastase (NE) and proteinase 3 (PR3) implicated.1 2 PR3 may be more important especially in alpha-1 antitrypsin (AAT) deficiency (AATD), where mutations in SERPINA1 results in little/no production of the anti-proteinase AAT preventing effective inhibition of both NE and especially PR3.3 The relationship of the SZ form of AAT to increase susceptibility to develop COPD remains uncertain. We hypothesised that patients with non-deficient COPD would have increased PR3 activity compared to healthy smokers, but similar to patients with the intermediate PiSZ AATD indicating no increased susceptibility. Methods PR3 activity was measured using a novel indirect ELISA based on a specific fibrinogen cleavage product (Aα–Val541). Plasma samples from patients with non-deficient COPD (n=31) and PiSZ AATD (n=95) were tested alongside healthy smoking controls (n=52) for the presence of this marker. Results Patients with usual COPD had an average FEV1/FCV ratio of 0.47 (SD ±0.17), compared to 0.63 (±0.21) in PiSZ AATD patients, and 0.77 (±0.07) in healthy patients. The Aα-Val541 concentration was increased (p 0.99). Conclusions These findings build a proteolytic enzyme activity profile in COPD patients, supporting the role of the proteinase/anti-proteinase imbalance in the pathophysiology of COPD. The data does not provide evidence to support an increased role in patients with PiSZ AATD. References Sinden NJ, et al. ERJ2013. Sinden NJ, et al. Am J Physiol Lung Cell Mol Physiol2015. Zorzetto M, et al. Clin Chem2008.

Background Common autoimmune disorders tend to coexist in the same subjects and to cluster in families. Methods We performed a cross-sectional multicenter study of 3286 Caucasian subjects (2791 with Graves' disease; 495 with Hashimoto's thyroiditis) attending UK hospital thyroid clinics to quantify the prevalence of coexisting autoimmune disorders. All subjects completed a structured questionnaire seeking a personal and parental history of common autoimmune disorders, as well as a history of hyperthyroidism or hypothyroidism among parents. Results The frequency of another autoimmune disorder was 9.67% in Graves' disease and 14.3% in Hashimoto's thyroiditis index cases (P = .005). Rheumatoid arthritis was the most common coexisting autoimmune disorder (found in 3.15% of Graves' disease and 4.24% of Hashimoto's thyroiditis cases). Relative risks of almost all other autoimmune diseases in Graves' disease or Hashimoto's thyroiditis were significantly increased (>10 for pernicious anemia, systemic lupus erythematosus, Addison's disease, celiac disease, and vitiligo). There was relative “clustering” of Graves' disease in the index case with parental hyperthyroidism and of Hashimoto's thyroiditis in the index case with parental hypothyroidism. Relative risks for most other coexisting autoimmune disorders were markedly increased among parents of index cases. Conclusion This is one of the largest studies to date to quantify the risk of diagnosis of coexisting autoimmune diseases in more than 3000 index cases with well-characterized Graves' disease or Hashimoto's thyroiditis. These risks highlight the importance of screening for other autoimmune diagnoses if subjects with autoimmune thyroid disease present with new or nonspecific symptoms.

Objective: The protein-tyrosine-phosphate nonreceptor 22 gene (PTPN22) has recently been identified as a susceptibility locus for a number of autoimmune diseases including Graves' disease (GD). PTPN21 is another member of the PTPN family and its gene PTPN21 maps to the first reported region of genetic linkage to GD, GD-1, on chromosome 14q31. The aim of this study was to determine whether PTPN21 is acting as a GD susceptibility locus in UK Caucasian subjects. Design: A case control association study of seven Tag single nucleotide polymorphisms (SNPs) (rs1469602, rs8007288, rs1998670, rs11622270, rs2274736, rs2295136 and rs366476) selected to predict 51 un-genotyped polymorphisms present within PTPN21. Patients: Unrelated Caucasian patients of UK origin with GD and ethnically and gender matched control subjects with no family history of autoimmune disease were recruited. In total, DNA was obtained from 768 GD patients and 768 control subjects. Results: No association of any of the seven Tag SNPs was detected with GD. Preliminary evidence of association of rs2274736 was found with younger age of GD onset (0-30 years) (OR = 1. 48 95% CI = 1.11-1.97). No other correlations with clinical phenotype or previously established susceptibility loci were detected. Conclusions: Using a Tag SNP approach we screened PTPN21 as a susceptibility locus for GD and found no evidence for association with disease. Preliminary evidence for association of rs2274736 with younger age of GD onset requires replication in similar sized data sets to exclude a false positive result. Methods such as the Tag SNP approach significantly reduce the amount of genotyping required when screening candidate loci, including those within regions of chromosomal linkage. © 2006 Blackwell Publishing Ltd.

Objective- The Fc receptor-like 3 (FCRL3) molecule, involved in controlling B-cell signalling, may contribute to the autoimmune disease process. Recently, a genome-wide screen detected association of neighbouring gene FCRL5 with Graves' disease (GD). To determine whether FCRL5 represents a further independent B-cell signalling GD susceptibility loci, we screened 12 tag SNPs, capturing all known common variation within FCRL5, in 5192 UK Caucasian GD index cases and controls. Design- A case-control association study investigating twelve tag SNPs within FCRL5 which captured the majority of known common variation within this gene region. Patients- A data set comprising 2504 UK Caucasian patients with GD and 2688 geographically matched controls taken from the 1958 British Birth cohort. Measurements- We used the chi-squared test and haplotype analysis to investigate the association between the tag SNPs and GD before performing logistic regression analysis to determine whether association at FCRL5 was independent of the known FCRL3 association. Results- Three of the FCRL5 tag SNPs, rs6667109, rs3811035 and rs6692977 showed association with GD (P = 0.015-0.001, OR = 1�15-1�16). Logistic regression performed on all FCRL5 and, previously screened, FCRL3 tag SNPs revealed that association with FCRL5 was secondary to linkage disequilibrium with the FCRL3, rs11264798 and rs10489678 SNPs. Conclusions- FCRL5 does not appear to be exerting an independent effect on the development of GD in the UK. Fine mapping of the entire FCRL region is required to determine the exact location of the aetiological variant/s present. © 2010 Blackwell Publishing Ltd.

A recent association scan using a genome-wide set of nonsynonymous coding single-nucleotide polymorphisms (nsSNPs) conducted in four diseases including Graves' disease (GD), identified nine novel possible regions of association with GD. We used a case-control approach in an attempt to replicate association of these nine regions in an independent collection of 1578 British GD patients and 1946 matched Caucasian controls. Although none of these loci showed evidence of association with GD in the independent data set, when combined with the original Wellcome Trust Case-Control Consortium study group, minor differences in allele frequencies (P>or=10(-3)) remained in the combined collection of 5924 subjects for four of the nsSNPs, present within HDLBP, TEKT1, JSRP1 and UTX. An additional 29 Tag SNPs were screened within these four gene regions to determine if further associations could be detected. Similarly, minor differences only (P=0.042-0.002) were detected in two HDLBP and two TEKT1 Tag SNPs in the combined UK GD collection. In conclusion, it is unlikely that the SNPs selected in this replication study have a significant effect on the risk of GD in the United Kingdom. Our study confirms the need for large data sets and stringent analysis criteria when searching for susceptibility loci in common diseases.

Graves' disease (GD) and Hashimoto's thyroiditis (HT) represent the commonest forms of autoimmune thyroid disease (AITD) each presenting with distinct clinical features. Progress has been made in determining association of HLA class II DRB1, DQB1 and DQA1 loci with GD demonstrating a predisposing effect for DR3 (DRB1(*)03-DQB1(*)02-DQA1(*)05) and a protective effect for DR7 (DRB1(*)07-DQB1(*)02-DQA1(*)02). Small data sets have hindered progress in determining HLA class II associations with HT. The aim of this study was to investigate DRB1-DQB1-DQA1 in the largest UK Caucasian HT case control cohort to date comprising 640 HT patients and 621 controls. A strong association between HT and DR4 (DRB1(*)04-DQB1(*)03-DQA1(*)03) was detected (P=6.79 x 10(-7), OR=1.98 (95% CI=1.51-2.59)); however, only borderline association of DR3 was found (P=0.050). Protective effects were also detected for DR13 (DRB1(*)13-DQB1(*)06-DQA1(*)01) (P=0.001, OR=0.61 (95% CI=0.45-0.83)) and DR7 (P=0.013, OR=0.70 (95% CI=0.53-0.93)). Analysis of our unique cohort of subjects with well characterized AITD has demonstrated clear differences in association within the HLA class II region between HT and GD. Although HT and GD share a number of common genetic markers this study supports the suggestion that differences in HLA class II genotype may, in part, contribute to the different immunopathological processes and clinical presentation of these related diseases.

Background: Single nucleotide polymorphisms (SNPs) of family with sequence familiarity 13, member A (FAM13A), hedgehog interacting protein (HHIP) and iron regulatory binding protein 2 (IREB2) are associated with emphysema on computed tomography scan in alpha-1 antitrypsin deficiency (AATD) [Khiroya et al. Am J Respir Crit Care Med 189; 2014 A5783]. Along with emphysema, rate of lung function decline also enables us to identify patients who are likely to have poorer outcomes. Aim: To determine if there is a relationship between these SNPs and lung function decline in the AATD population. Methods: 662 PiZZ and PiZnull AATD subjects were genotyped for rs2568494 ( IREB2 ), rs7671167 and rs1903003 ( FAM13A ), and rs1542725 and rs13118928 ( HHIP ). Genotyping was carried out using TaqMan technologies (Applied Biosystems, UK). Linear regressions were performed to look for genetic associations with KCO and post-bronchodilator FEV1 at baseline assessment. Logistic regressions were performed to look for associations with rapid decline in KCO and FEV1. Rapid decline was defined as loss of ≥1% in the % predicted value of these parameters per year. Results: All SNPs were in Hardy-Weinburg equilibrium. rs1903003 ( FAM13A ) showed a trend towards KCO at baseline assessment ( p =0.062). None of the SNPs were significantly associated with post-bronchodilator FEV1 or rapid FEV1 or KCO decline. Conclusions: rs7671167 and rs1903003 ( FAM13A ) are in significant linkage disequilibrium, as are rs1542725 and rs13118928 ( HHIP ). Therefore it is difficult to say whether one SNP is responsible for emphysema and KCO, or if it is a combined effect. Future work should target these SNPs to understand their pathological role with a view to determining new therapies.

CONTEXT: A number of small data sets have suggested a potential role for skewed X chromosome activation (XCI), away from the expected 50:50 parent of origin ratio, as an explanation for the strong female preponderance seen in the common autoimmune thyroid diseases (AITD), Graves' disease (GD), and Hashimoto's thyroiditis (HT). OBJECTIVE: The objective of the study was to confirm a role for XCI skewing as a potential explanation for the strong female preponderance seen in AITD. DESIGN: The design of the study was to screen XCI in the largest GD, HT, and control case-control cohort and family cohort to date and undertake a meta-analysis of previous AITD XCI reports. SETTING: The study was conducted at a research laboratory. PATIENTS: Three hundred and nine GD, 490 HT, and 325 female UK Caucasians controls, 273 UK Caucasian GD families, and a meta-analysis of 454 GD, 673 HT, and 643 female Caucasian controls were included in the study. MAIN OUTCOME MEASURES: Case-control and family-based association studies and meta-analysis were measured. RESULTS: Skewed XCI was observed with GD [odds ratio (OR) 2.17 [95% confidence interval (CI) 1.43-3.30], P=2.1×10(-4)] and a trend toward skewing with HT (P=.08) compared with the control cohort. A meta-analysis of our UK data and that of four previous non-UK Caucasian studies confirmed significant skewing of XCI with GD [OR 2.54 (95% CI 1.58-4.10), P=1.0×10(-4), I2=30.2%] and HT [OR 2.40 (95% CI 1.10-5.26), P=.03, I2=74.3%]. CONCLUSIONS: Convincing evidence exists to support a role for skewed XCI in female subjects with AITD, which may, in part, explain the strong female preponderance observed in this disease.

Background Vitamin D-binding protein (DBP) genetic polymorphisms have been associated with chronic obstructive pulmonary disease (COPD). DBP has an indirect role in macrophage activation; thus it was hypothesised that DBP is present in the airway and contributes to lung disease by this mechanism. Methods 471 PiZZ subjects with a1-antitrypsin deficiency (AATD) were genotyped for tag single nucleotide polymorphisms (SNPs) covering the DBP gene (GC), together with known functional variants, prior to seeking association with COPD phenotypes. 140 subjects with usual COPD and 480 controls were available for replication. Vitamin D and DBP levels were measured by tandem mass spectrometry and ELISA, respectively, in serum and DBP in the sol phase of sputum in a subset of 60 patients. Concentrations were related to phenotype and to alveolar macrophage activation. Results rs2070741 was associated with airway bacterial colonisation (p¼0.04) and bronchiectasis (p¼0.01), as was rs7041 (p¼0.03) which also influenced vitamin D concentrations (p¼0.01). The GC2 variant predisposed to bronchiectasis in AATD (p¼0.04) and protected against COPD (p¼0.05); the latter association was replicated in usual COPD versus controls (p¼0.04). Circulating DBP related inversely to forced expiratory volume in 1 s (FEV1 )( p ¼0.02), in direct contrast to vitamin D, where deficiency related to low FEV1 (p¼0.04). Sol DBP related directly to alveolar macrophage activation (p¼0.004). Conclusions The genetic association of DBP with COPD may be mediated by effects on macrophage activation, since DBP relates to FEV1, and affects macrophage activation. Vitamin D effects may be independent of this, relating more strongly to innate immunity.

Objective Although autoantibody production is a key feature of autoimmunity, it is not known whether variation in autoantibody production and clearance pathways is involved in disease susceptibility. The Fc Gamma Receptor IIa (FcGRIIa) molecule is involved in the clearance of autoantibodies and a functional single nucleotide polymorphism (SNP), rs1801274, which has been shown to alter autoantibody clearance, has been associated with a number of autoimmune diseases (AIDs) including systemic lupus erythematosus and type 1 diabetes. This study aimed to determine whether FcGRIIa is associated with Graves' disease (GD) in the UK Caucasian population by Tag SNP screening common polymorphisms within the FcGRIIa region. Design A case control association study investigating nine Tag SNPs within FcGRIIa, which captured the majority of known common variation within this gene region. Patients A dataset comprising 2504 UK Caucasian GD patients and 2784 geographically matched controls taken from the 1958 British Birth cohort. Measurements We used the Io2-test to investigate association between the Tag SNPs and GD. Results Association between the rs1801274 (P= 0·003, OR = 1·12 95% CI = 1·03-1·22 and rs6427598 (P = 0·012, OR = 0·90 95% CI = 0·83-0·98) SNPs and GD was observed. No other SNPs showed association with GD. No associations were seen between any of the SNPs investigated and specific GD clinical phenotypes. Conclusions This study suggests that variation in FcGRIIa predisposes to GD and further supports the role of FcGRIIa as a susceptibility locus for AIDs in general. © 2010 Blackwell Publishing Ltd.

To the Editors: We read with interest the article by Zhu et al. 1 describing the association of two single nucleotide polymorphisms (SNPs; rs231775 and rs3087243) within the CTLA4 gene and chronic bronchitis. Both of these polymorphisms may be risk factors for autoimmunity 2, and are often referred to as A49G and CT60, respectively. As alluded to briefly by the authors in their discussion, there has been recent interest in a possible role for autoimmunity in chronic obstructive pulmonary disease (COPD), hence shared genetic susceptibility with other autoimmune diseases would support this hypothesis. Although the authors report their association in two independent cohorts, general acceptance of the importance of this locus would require further validation in other COPD populations. We would …

Graves' disease (GD) is a common autoimmune disease (AID) that shares many of its susceptibility loci with other AIDs. The thyroid stimulating hormone receptor (TSHR) represents the primary autoantigen in GD, in which autoantibodies bind to the receptor and mimic its ligand, thyroid stimulating hormone, causing the characteristic clinical phenotype. Although early studies investigating the TSHR and GD proved inconclusive, more recently we provided convincing evidence for association of the TSHR region with disease. In the current study, we investigated a combined panel of 98 SNPs, including 70 tag SNPs, across an extended 800 kb region of the TSHR to refine association in a cohort of 768 GD subjects and 768 matched controls. In total, 28 SNPs revealed association with GD (P

Alpha-1-antitrypsin deficiency is associated with variable development of airflow obstruction and emphysema. Index patients have greater airflow obstruction than subjects detected by screening, but it is unclear if this reflects smoking differences and/or ascertainment bias, or is due to additional genetic factors. In this study 72 sibling pairs with alpha-1-antitrypsin deficiency were compared using lung function measurements and HRCT chest. Tag single nucleotide polymorphisms to cover all common variation in four genes involved in relevant inflammatory pathways (Tumour necrosis factor alpha, Transforming growth Factor beta, Surfactant protein B and Vitamin D binding protein) were genotyped using TaqMan technology and compared between pairs for their frequency and relationship to lung function. 63.5% of non-index siblings had airflow obstruction and 59.5% an FEV(1) < 80% predicted. Index siblings had lower FEV(1) and FEV(1)/FVC ratio, a higher incidence of emphysema (all P

Background Genetic variation may underlie phenotypic variation in chronic obstructive pulmonary disease (COPD) in subjects with and without alpha 1 antitrypsin deficiency (AATD). Genotype specific sub-phenotypes are likely and may underlie the poor replication of previous genetic studies. This study investigated subjects with AATD to determine the relationship between specific phenotypes and TNFα polymorphisms. Methods 424 unrelated subjects of the PiZZ genotype were assessed for history of chronic bronchitis, impairment of lung function and radiological presence of emphysema and bronchiectasis. A subset of subjects with 3 years consecutive lung function data was assessed for decline of lung function. Four single nucleotide polymorphisms (SNPs) tagging TNFα were genotyped using TaqMan® genotyping technologies and compared between subjects affected by each phenotype and those unaffected. Plasma TNFα levels were measured in all PiZZ subjects. Results All SNPs were in Hardy-Weinberg equilibrium. A significant difference in rs361525 genotype (p = 0.01) and allele (p = 0.01) frequency was seen between subjects with and without chronic bronchitis, independent of the presence of other phenotypes. TNFα plasma level showed no phenotypic or genotypic associations. Conclusion Variation in TNFα is associated with chronic bronchitis in AATD.

Objective: The Programmed Cell Death 1 gene (PDCD1) on chromosome 2q37.3 encodes PD-1 which is involved in providing a negative signal to activated T cells. Large case-control studies have shown association of PDCD1 with several autoimmune diseases although, to date, no such studies have been performed for Graves' disease (GD). The objective of our study was to investigate eight tag SNPs representing the majority of common variation in PDCD1 within a well-characterized large UK Caucasian GD dataset. Design: A case control association study of eight polymorphisms. Patients: 2671 Graves' disease patients and 864 controls. Measurements: Tests for association with disease. Results: No association with disease was seen for any of the +4163, +5049, +5318, +5640, +5678 and +7078 SNPs genotyped in this study. Association was detected between the +2375 SNP (P = 0.021, OR = 1.14 95% CI = 1.01-1.29) and GD and a small protective effect was seen with the +6799 SNP genotypes (P = 0.028, OR = 0.77 95% CI = 0.58-1.03). Conclusions: This study has, for the first time, shown that small effects within PDCD1 may contribute towards the development of GD, supporting the hypothesis that much of the currently unknown genetic contribution to GD could be due to several small genetic effects with ORs 1.2. Replication of this result is now needed to confirm our findings and justify more detailed fine mapping of a primary aetiological variant in this gene region. © 2007 The Authors.

Introduction There is growing evidence that iron plays an important role within the lung cancer, the leading cause of cancer-related mortality worldwide. As a result of this, iron homeostasis has potential as a new avenue for targeting and treatment of lung cancer. In this study, the effect of iron loading on cellular proliferation and iron homeostasis gene expression was investigated. In addition, the effect of the chelator deferasirox on cellular iron levels and proliferation rates was studied. Methods Cellular proliferation was assessed by the BrdU assay and cellular iron levels were assessed using the ferrozine assay. Manipulation of IREB2 gene expression was achieved using short interfering RNA (siRNA) and subsequent expression of this and other iron homeostasis genes was assessed using real time PCR. All experiments were carried out on both the A549 adenocarcinoma and QG56 squamous cell carcinoma cell lines in triplicate. Primary bronchial epithelia cells (PBEC) were used as reference of normal behaviour. Results A dose of 150uM of iron was seen to cause a significant increase in proliferation in both the A549 (50% increase) and QG56 (40% increase) cell lines (P = 0.002 and 0.03 respectively) whilst no change was seen in the PBECs. A corresponding increase in cellular iron was also seen.When the cancer cell lines were treated with deferasirox, cellular iron loading decreased by roughly 25% in each cell line (P = 0.001 and 0.01 respectively) and cellular proliferation decreased below levels seen in unstimulated cells. Deferasirox was also seen to effect unstimulated cancer cells, reducing their proliferation by 50% (P = 0.02 and 0.03 respectively). Conclusion Iron exposure was shown to have a significant effect on cellular proliferation within lung cancer cell lines, although the underlying mechanism is not yet fully understood. This iron mediated cellular proliferation could be reversed using the chelator deferasirox. Down-regulated expression of IREB2 may cause the cancer cell lines to exhibit similar behaviour to the PBECs when stimulated with iron. These finding show that iron may provide a potential new target and deferasirox a potential new therapeutic agent for lung cancer.

Background There are many genetic influences documented on both lung function and susceptibility to COPD. In GWAS of pulmonary function several hits in the region of the MHC on chromosome 6 have been found, and we have shown previously that HLADR3 positive individuals have lower FEV1 than those without this HLA type. This is an HLA type classically associated with autoimmunity. Interactions between HLA type and cigarette smoke are recognised in autoimmune diseases. Hypothesis HLA type influences differentiation of monocytes in the presence of cigarette smoke. Methods 15 ex and never smokers with COPD and 5 healthy controls were studied. PBMCs were isolated and exposed to varying concentrations of cigarette smoke extract (CSE) for 90 min. CD14 and CD16 markers were used in flow cytometry to ascertain relative expression and absolute cell counts for each monocyte subpopulation, defined as CD14++CD16- (classical), CD14++CD16+ (anti-inflammatory) and CD14+CD16++ (non-classical). Within the patient group differences in baseline profile and response to CSE were compared between ex-smokers and those that had never smoked. Patients were HLA class II typed as described previously1 and the same comparisons made between DR3 positive and negative patients. Results At baseline the MFI for CD14 was lower in COPD than health (p=0.04), although no clear differences in cell counts were seen. Counts were generally higher in ex-smokers, although no clear differences in subpopulations were seen. On exposure to cigarette smoke there was a dose dependent rise in classical monocytes, which was more marked in DR3+ patients and never smokers. Conclusions CSE induces a pro-inflammatory phenotype of monocytes, and this occurs most in HLADR3+ individuals. This could be the mechanism behind lower FEV1 in DR3+ individuals.