ZNF385B and VEGFA Are Strongly Differentially Expressed in Serous Ovarian Carcinomas and Correlate with Survival

Background

The oncogenesis of ovarian cancer is poorly understood. The aim of this study was to identify mRNAs differentially expressed between moderately and poorly differentiated (MD/PD) serous ovarian carcinomas (SC), serous ovarian borderline tumours (SBOT) and superficial scrapings from normal ovaries (SNO), and to correlate these mRNAs with clinical parameters including survival.

Methods

Differences in mRNA expression between MD/PD SC, SBOT and SNO were analyzed by global gene expression profiling (n = 23), validated by RT-qPCR (n = 41) and correlated with clinical parameters.

Results

Thirty mRNAs differentially expressed between MD/PD SC, SBOT and SNO were selected from the global gene expression analyses, and 21 were verified (p<0.01) by RT-qPCR. Of these, 13 mRNAs were differentially expressed in MD/PD SC compared with SNO (p<0.01) and were correlated with clinical parameters. ZNF385B was downregulated (FC = −130.5, p = 1.2×10⁻⁷) and correlated with overall survival (p = 0.03). VEGFA was upregulated (FC = 6.1, p = 6.0×10⁻⁶) and correlated with progression-free survival (p = 0.037). Increased levels of TPX2 and FOXM1 mRNAs (FC = 28.5, p = 2.7×10⁻¹⁰ and FC = 46.2, p = 5.6×10⁻⁴, respectively) correlated with normalization of CA125 (p = 0.03 and p = 0.044, respectively). Furthermore, we present a molecular pathway for MD/PD SC, including VEGFA, FOXM1, TPX2, BIRC5 and TOP2A, all significantly upregulated and directly interacting with TP53.

Conclusions

We have identified 21 mRNAs differentially expressed (p<0.01) between MD/PD SC, SBOT and SNO. Thirteen were differentially expressed in MD/PD SC, including ZNF385B and VEGFA correlating with survival, and FOXM1 and TPX2 with normalization of CA125. We also present a molecular pathway for MD/PD SC.     

Target renal damage: the microvascular associations of increased aortic stiffness in patients with COPD

Background

Although renal impairment has been described in COPD, there is opportunity to evaluate further to determine nature and consider optimal management. Increased aortic stiffness, as seen in COPD, leads to reduced buffering of pulsatile flow. We hypothesised that urinary albumin creatinine ratio (UACR) would reflect glomerular damage related to aortic stiffness.

Methods

Patients with COPD and controls underwent spirometry, blood pressure, arterial stiffness - aortic pulse wave velocity (PWV) and provided a spot urine sample for UACR, with other renal biomarkers measured.

Results

The UACR was increased in patients (n = 52): 0.80 mg/mmol compared to controls (n = 34): 0.46 mg/mmol, p < 0.05. Aortic PWV was related to log₁₀ UACR in all subjects (r = 0.426, p < 0.001) and COPD patients alone. Aortic PWV was a significant variable for UACR with oxygen saturations, after accounting for potential confounders. Eight subjects (7 patients) reached a defined clinical microalbuminuria threshold, with aortic PWV greater in these patients compared to those patients without, although albuminuria is a continuum. Proximal tubular damage biomarkers, unlike the glomerular marker, were not different between patients and controls.

Conclusions

There is glomerular damage in patients with COPD evidenced by increased UACR, related to increased aortic stiffness. Besides the macrovascular prognostic implications of increased aortic stiffness, the microvascular state in COPD management should be considered.     

Associations between COPD related manifestations: a cross-sectional study

Background

Cardiovascular disease, osteoporosis and emphysema are associated with COPD. Associations between these factors and whether they predict all-cause mortality in COPD patients are not well understood. Therefore, we examined associations between markers of cardiovascular disease (coronary artery calcification [CAC], thoracic aortic calcification [TAC] and arterial stiffness), bone density (bone attenuation of the thoracic vertebrae), emphysema (PI-950 and 15th percentile) and all-cause mortality in a COPD cohort.

Methods

We assessed CAC, TAC, bone attenuation of the thoracic vertebrae, PI-950 and 15th percentile on low-dose chest computed tomography in COPD subjects. We measured arterial stiffness as carotid-radial pulse wave velocity (PWV), and identified deaths from the national register.

Results

We studied 119 COPD subjects; aged 67.8 ±7.3, 66% were males and mean FEV₁% predicted was 46.0 ±17.5. Subjects were classified into three pre-specificed groups: CAC = 0 (n = 14), 0 < CAC ≤ 400 (n = 41) and CAC > 400 (n = 64). Subjects with higher CAC were more likely to be older (p < 0.001) and male (p = 0.03), and more likely to have higher systolic blood pressure (p = 0.001) and a history of hypertension (p = 0.002) or ischemic heart disease (p = 0.003). Higher CAC was associated with higher PWV (OR 1.62, p = 0.04) and lower bone attenuation (OR 0.32, p = 0.02), but not with 15th percentile, after adjustment for age, sex and pack-years of smoking. In a Cox proportional hazards model, CAC, TAC and 15th percentile predicted all-cause mortality (HR 2.01, 2.09 and 0.66, respectively).

Conclusions

Increased CAC was associated with increased arterial stiffness and lower bone density in a COPD cohort. In addition, CAC, TAC and extent of emphysema predicted all-cause mortality.

Trial registration

Lothian NHS Board, Lothian Research Ethics Committee, LREC/2003/8/28.     

Increased plasma mannose binding lectin levels are associated with bronchiolitis obliterans after lung transplantation

Background

Long-term lung allograft survival is limited by bronchiolitis obliterans syndrome (BOS). Mannose binding lectin (MBL) belongs to the innate immune system, participates in complement activation, and may predispose to graft rejection. We investigated mannose binding (MBL) during cold ischemia and in tissue samples from explanted lungs with BOS, and assessed MBL and complement proteins in plasma post-lung transplantation relative to BOS staging.

Methods

MBL was detected by immunohistochemistry lung tissue at the time of cold ischemia and in samples with BOS. MBL was assayed in the peripheral blood of 66 lung transplant patients transplanted between 1990–2007.

Results

MBL localized to vasculature and basement membrane during cold ischemia and BOS. Patients further out post-lung transplant > 5 years (n = 33), had significantly lower levels of MBL in the blood compared to lung transplant patients < 5 years with BOS Op-3 (n = 17), 1738 ± 250 ng/ml vs 3198 ± 370 ng/ml, p = 0.027, and similar levels to lung transplant patients < 5 years with BOS 0 (n = 16), 1738 ± 250 ng/ml vs 1808 ± 345 ng/ml. MBL levels in all BOS 0 (n = 30) vs. all BOS Op-3 (n = 36) were 1378 ± 275 ng/ml vs. 2578 ± 390 ng/ml, p = 0.001, respectively. C3 plasma levels in BOS 0 (n = 30) vs. BOS Op-3 (n = 36) were 101 ± 19.8 mg/ml vs. 114 ± 25.2 mg/ml, p = 0.024, respectively.

Conclusions

MBL localizes within the lung during graft ischemia and BOS, higher levels of plasma MBL are associated with BOS Op-3 and < 5 years post-transplant, and higher level of plasma complement protein C3 was associated with BOS Op-3 clinical status. MBL may serve as a biomarker for poorer outcome post-lung transplantation.     

Increased human Ca2+-activated Cl- channel 1 expression and mucus overproduction in airway epithelia of smokers and chronic obstructive pulmonary disease patients

Background

The mechanisms underlying the association between smoking and mucus overproduction remain unknown. Because of its involvement in other airway diseases, such as asthma, we hypothesized that Ca²⁺-activated Cl^- channel 1 (CLCA1) was associated with overproduction of mucus in the airways of smokers and COPD patients.

Methods

Using real-time quantitative PCR analyses, we compared the CLCA1 mRNA expression levels in induced-sputum cells from COPD patients (n = 20), smokers without COPD (n = 5), and non-smokers (n =13). We also examined the relationship between CLCA1 protein expression and mucus production in lung airway epithelia of COPD patients (n = 6), smokers without COPD (n = 7), and non-smokers (n = 7).

Results

CLCA1 mRNA expression was significantly up-regulated in the induced-sputum cells of COPD patients compared with cells of non-smokers (p = 0.02), but there was no significant difference compared with cells of smokers without COPD. Using immunostaining with an anti-CLCA1 antibody, semi-quantitative image analyses of airway epithelium demonstrated significantly increased CLCA1 expression in smokers without COPD (p = 0.02) and in COPD patients (p = 0.002) compared with non-smokers. There were significant negative correlations between CLCA1 protein expression and FEV₁/FVC (r = −0.57, p = 0.01) and %predicted FEV₁ (r = −0.56, p = 0.01). PAS staining for mucus showed that there was a significant positive correlation between CLCA1 protein expression and mucus production (r = 0.67, p = 0.001). These markers were significantly increased in smokers without COPD (p = 0.04) and in COPD patients (p = 0.003) compared with non-smokers (non-smokers < smokers ≤ COPD).

Conclusions

CLCA1 expression is significantly related to mucus production in the airway epithelia of smokers and COPD patients, and may contribute to the development and pathogenesis of COPD by inducing mucus production.     

Leptin modulates electrophysiological characteristics and isoproterenol-induced arrhythmogenesis in atrial myocytes

Background

Obesity is an important risk factor for atrial fibrillation (AF). Leptin is an important adipokine. However, it is not clear whether leptin directly modulates the electrophysiological characteristics of atrial myocytes.

Results

Whole cell patch clamp and indo-1 fluorescence were used to record the action potentials (APs) and ionic currents in isolated rabbit left atrial (LA) myocytes incubated with and without (control) leptin (100 nM) for 1 h to investigate the role of leptin on atrial electrophysiology. Leptin-treated LA myocytes (n = 19) had longer 20% of AP duration (28 ± 3 vs. 21 ± 2 ms, p < 0.05), but similar 50% of AP duration (51 ± 4 vs. 50 ± 3 ms, p > 0.05), and 90% of AP duration (89 ± 5 vs. 94 ± 4 ms, p > 0.05), as compared to the control (n = 22). In the presence of isoproterenol (10 nM), leptin-treated LA myocytes (n = 21) showed a lower incidence (19% vs. 54.2%, p < 0.05) of delayed afterdepolarization (DAD) than the control (n = 24). Leptin-treated LA myocytes showed a larger sodium current, but a smaller ultra-rapid delayed rectifier potassium current, and sodium-calcium exchanger current than the control. Leptin-treated and control LA myocytes exhibited a similar late sodium current, inward rectifier potassium current, transient outward current and L-type calcium current. In addition, the leptin-treated LA myocytes (n = 38) exhibited a smaller intracellular Ca²⁺ transient (0.21 ± 0.01 vs. 0.26 ± 0.01 R410/485, p < 0.05) and sarcoplasmic reticulum Ca²⁺ content (0.35 ± 0.02 vs. 0.43 ± 0.03 R410/485, p < 0.05) than the control LA myocytes (n = 42).

Conclusions

Leptin regulates the LA electrophysiological characteristics and attenuates isoproterenol-induced arrhythmogenesis.     

Diverging trends of chronic bronchitis and smoking habits between 1998 and 2010

Background

No study has been carried out on the time trend in the prevalence of chronic bronchitis (CB) in recent years, despite its clinical and epidemiological relevance. We evaluated the trend in CB prevalence during the past decade among young Italian adults.

Methods

A screening questionnaire was mailed to general population samples of 20–44 year-old subjects in two cross-sectional surveys: the Italian Study on Asthma in Young Adults (ISAYA) (1998/2000; n = 18,873, 9 centres) and the screening stage of the Gene Environment Interactions in Respiratory Diseases (GEIRD) study (2007/2010; n = 10,494, 7 centres). CB was defined as having cough and phlegm on most days for a minimum of 3 months a year and for at least 2 successive years. The prevalence rates and the risk ratios (RRs) for the association between CB and each potential predictor were adjusted for gender, age, season of response, type of contact, cumulative response rate, and centre.

Results

CB prevalence was 12.5% (95% CI: 12.1-12.9%) in 1998/2000 and 12.6% (95% CI: 11.7-13.7%) in 2007/2010; it increased among never smokers (from 7.6 to 9.1%, p = 0.003), current light smokers (<15 pack-years; from 15.1 to 18.6%, p < 0.001), and unemployed/retired subjects (from 14.3 to 19.1%, p = 0.001). In this decade, the prevalence of current smoking decreased (from 33.6 to 26.9%, p < 0.001), whereas the prevalence of unemployment/premature retirement (from 5.3 to 6.0%, p = 0.005), asthma (from 5.0 to 6.2%, p = 0.003), and allergic rhinitis (from 19.5 to 24.5%, p < 0.001) increased. In both 1998/2000 and 2007/2010, the likelihood of having CB was significantly higher for women, current smokers, asthmatic patients, and subjects with allergic rhinitis. During this period, the strength of the association between CB and current heavy smoking (≥15 pack-years) decreased (RR: from 4.82 to 3.57, p = 0.018), whereas it increased for unemployment/premature retirement (from 1.11 to 1.53, p = 0.019); no change was observed for gender, asthma, and allergic rhinitis.

Conclusions

Despite the significant reduction in current smoking, CB prevalence did not vary among young Italian adults. The temporal pattern of CB prevalence can only be partly explained by the increase of unemployment/premature retirement, asthma and allergic rhinitis, and suggests that other factors could have played a role.     

Serum tryptase detected during acute coronary syndrome is significantly related to the development of major adverse cardiovascular events after 2?years

Background

One of the greatest challenges in cardiovascular medicine is to define the best tools for performing an accurate risk stratification for the recurrence of ischemic events in acute coronary syndrome (ACS) patients.

Methods

We followed 65 ACS patients enrolled in a previous pilot study for 2 years after being discharged, focusing on the occurrence of major adverse cardiovascular events (MACE).The relationship between serum tryptase levels on admission, SYNergy between percutaneous coronary intervention with the TAXUS drug-eluting stent and the cardiac surgery score (SX-score), cardiovascular complexity and MACE at 2 years follow-up were analyzed.

Results

The ACS population was divided in two groups: patients with MACE (n = 23) and patients without MACE (n = 42).The tryptase measurement at admission (T0) and at discharge (T3) and SX-score were higher in patients who experienced MACE than in those without (p = 0.0001, p < 0.0001 and p = 0.006, respectively). Conversely, we found no significant association between MACE and C-reactive protein (CRP), and between MACE and maximum level of high-sensitivity troponin (hs-Tn) values.Among all patients with MACE, 96% belonged to the group that presented with cardiovascular complexity at the beginning of ACS index admission (p < 0.0001).The predictive accuracy of serum tryptase for MACE at follow up set at the cut-off point of 4.95 ng/ml at T0 and of 5.2 ng/ml at T3. Interestingly, patients with both the above cut-off tryptase values at T0 and at T3 presented a 1320% increase in the odds of developing MACE (p < 0.0001).

Conclusion

In ACS patients, serum tryptase measured during index admission is significantly correlated to the development of MACE up to 2 years, demonstrating a possible long-term prognostic role of this biomarker.

Electronic supplementary material

The online version of this article (doi:10.1186/s12948-015-0013-0) contains supplementary material, which is available to authorized users.     

Phenotyping community-acquired pneumonia according to the presence of acute respiratory failure and severe sepsis

Background

Acute respiratory failure (ARF) and severe sepsis (SS) are possible complications in patients with community-acquired pneumonia (CAP). The aim of the study was to evaluate prevalence, characteristics, risk factors and impact on mortality of hospitalized patients with CAP according to the presence of ARF and SS on admission.

Methods

This was a multicenter, observational, prospective study of consecutive CAP patients admitted to three hospitals in Italy, Spain, and Scotland between 2008 and 2010. Three groups of patients were identified: those with neither ARF nor SS (Group A), those with only ARF (Group B) and those with both ARF and SS (Group C) on admission.

Results

Among the 2,145 patients enrolled, 45% belonged to Group A, 36% to Group B and 20% to Group C. Patients in Group C were more severe than patients in Group B. Isolated ARF was correlated with age (p < 0.001), COPD (p < 0.001) and multilobar infiltrates (p < 0.001). The contemporary occurrence of ARF and SS was associated with age (p = 0.002), residency in nursing home (p = 0.007), COPD (p < 0.001), multilobar involvement (p < 0.001) and renal disease (p < 0.001). 4.2% of patients in Group A died, 9.3% in Group B and 26% in Group C, p < 0.001. After adjustment, the presence of only ARF had an OR for in-hospital mortality of 1.85 (p = 0.011) and the presence of both ARF and SS had an OR of 6.32 (p < 0.001).

Conclusions

The identification of ARF and SS on hospital admission can help physicians in classifying CAP patients into three different clinical phenotypes.     

Circulating levels of copeptin predict outcome in patients with pulmonary arterial hypertension

Objective

To determine the levels of circulating copeptin in patients with pulmonary arterial hypertension (PAH), and to evaluate its relation with disease severity, outcome and response to treatment.

Background

Vasopressin is a key regulator of body fluid homeostasis. The co-secreted protein copeptin serves as surrogate for plasma vasopressin levels and increases in acute and chronic left ventricular dysfunction. Copeptin has not been studied in PAH.

Methods

Serum copeptin levels were evaluated in a retrospective cohort of 92 treatment-naïve patients with PAH, 39 patients with normal right ventricular hemodynamics (diseased controls) and 14 apparently healthy individuals (healthy controls). In a second prospective cohort of 15 patients with PAH, serial changes of copeptin levels after initiation of PAH treatment were measured. Copeptin levels were compared with clinical, biochemical and hemodynamic parameters as well as response to treatment and clinical outcome.

Results

Circulating copeptin levels were elevated in PAH patients compared to diseased controls (20.1 pmol/l vs. 5.1 pmol/l; p = 0.001). Baseline levels of copeptin correlated with NYHA functional class (r = 0.46; p = 0.01), 6 minute walking distance (r = -0.26; p = 0.04), NT-proBNP (r = 0.49, p = 0.01), creatinine (r = 0.39, p = 0.01) and estimated glomerular filtration rate (r = -0.32, p = 0.01). Copeptin levels did not correlate with hemodynamics but decreased after initiation of PAH therapy (p = 0.001). Elevated copeptin levels were associated with shorter survival (p < 0.001) and independent predictors of mortality in a multiple Cox regression analysis (HR1.4; 95% confidence interval 1.1-2.0; p = 0.02).

Conclusions

Patients with PAH had elevated copeptin levels. High circulating levels of copeptin were independent predictors of poor outcome, which makes copeptin a potentially useful biomarker in PAH.     

Tiotropium might improve survival in subjects with COPD at high risk of mortality

Background

Inhaled therapies reduce risk of chronic obstructive pulmonary disease (COPD) exacerbations, but their effect on mortality is less well established. We hypothesized that heterogeneity in baseline mortality risk influenced the results of drug trials assessing mortality in COPD.

Methods

The 5706 patients with COPD from the Understanding Potential Long-term Impacts on Function with Tiotropium (UPLIFT®) study that had complete clinical information for variables associated with mortality (age, forced expiratory volume in 1 s, St George’s Respiratory Questionnaire, pack-years and body mass index) were classified by cluster analysis. Baseline risk of mortality between clusters, and impact of tiotropium were evaluated during the 4-yr follow up.

Results

Four clusters were identified, including low-risk (low mortality rate) patients (n = 2339; 41%; cluster 2), and high-risk patients (n = 1022; 18%; cluster 3), who had a 2.6- and a six-fold increase in all-cause and respiratory mortality compared with cluster 2, respectively. Tiotropium reduced exacerbations in all clusters, and reduced hospitalizations in high-risk patients (p < 0.05). The beneficial effect of tiotropium on all-cause mortality in the overall population (hazard ratio, 0.87; 95% confidence interval, 0.75–1.00, p = 0.054) was explained by a 21% reduction in cluster 3 (p = 0.07), with no effect in other clusters.

Conclusions

Large variations in baseline risks of mortality existed among patients in the UPLIFT® study. Inclusion of numerous low-risk patients may have reduced the ability to show beneficial effect on mortality. Future clinical trials should consider selective inclusion of high-risk patients.     

Impact of precise modulation of reactive oxygen species levels on spermatozoa proteins in infertile men

Background

Elevated levels of reactive oxygen species (ROS) are detected in 25% to 80% of infertile men. They are involved in the pathology of male infertility. Understanding the effect of increasing levels of ROS on the differential expression of sperm proteins is important to understand the cellular processes and or/pathways that may be implicated in male infertility. The aim of this study was to examine differentially expressed proteins (DEPs) in spermatozoa from patients with low, medium and high ROS levels.

Methods

A total of 42 infertile men presenting for infertility and 17 proven fertile men were enrolled in the study. ROS levels were measured by chemiluminescence assay. Infertile men were divided into Low (0- < 93 RLU/s/10⁶ sperm) (n = 11), Medium (>93-500 RLU/s/10⁶ sperm) (n = 17) and High ROS (>500 RLU/s/10⁶ sperm) group (n = 14). All fertile men had ROS levels between 4-50 RLU/s/10⁶ sperm. 4 subjects from fertile group and 4 each from the Low, Medium and High ROS were pooled. Protein extraction, protein estimation, gel separation of the proteins, in-gel digestion, LTQ-orbitrap elite hybrid mass spectrometry system was conducted. The DEPs, the cellular localization and pathways of DEPs involved were examined utilizing bioinformatics tools.

Results

1035 proteins were identified in the 3 groups by global proteomic analysis. Of these, 305 were DEPs. 51 were unique to the Low ROS group, 47 Medium ROS group and 104 were unique to the High ROS group. 6 DEPs were identified by Uniprot and DAVID that had distinct reproductive functions and they were expressed only in 3 ROS groups but not in the control.

Conclusions

We have for the first time demonstrated the presence of 6 DEPs with distinct reproductive functions only in men with low, medium or high ROS levels. These DEPs can serve as potential biomarkers of oxidative stress induced male infertility.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-12-4) contains supplementary material, which is available to authorized users.     

Clinical utility of diagnostic guidelines and putative biomarkers in lymphangioleiomyomatosis

Background

Lymphangioleiomyomatosis is a rare disease occurring almost exclusively in women. Diagnosis often requires surgical biopsy and the clinical course varies between patients with no predictors of progression. We evaluated recent diagnostic guidelines, clinical features and serum biomarkers as diagnostic and prognostic tools.

Methods

Serum vascular endothelial growth factor-D (VEGF-D), angiotensin converting enzyme (ACE), matrix metalloproteinases (MMP) -2 and -9, clinical phenotype, thoracic and abdominal computerised tomography, lung function and quality of life were examined in a cohort of 58 patients. 32 healthy female controls had serum biomarkers measured.

Results

Serum VEGF-D, ACE and total MMP-2 levels were elevated in patients. VEGF-D was the strongest discriminator between patients and controls (median = 1174 vs. 332 pg/ml p < 0.0001 with an area under the receiver operating characteristic curve of 0.967, 95% CI 0.93-1.01). Application of European Respiratory Society criteria allowed a definite diagnosis without biopsy in 69%. Adding VEGF-D measurement to ERS criteria further reduced the need for biopsy by 10%. VEGF-D was associated with lymphatic involvement (p = 0.017) but not the presence of angiomyolipomas.

Conclusions

Combining ERS criteria and serum VEGF-D reduces the need for lung biopsy in LAM. VEGF-D was associated with lymphatic disease but not lung function.     

Screening and Identification of Biomarkers in Ascites Related to Intrinsic Chemoresistance of Serous Epithelial Ovarian Cancers

Objective

The ability to predict responses to chemotherapy for serous epithelial ovarian cancer (EOC) would be valuable since intrinsically chemoresistant EOC patients (persistent or recurrent disease within 6 months) gain little benefit from standard chemotherapy. The aim of this study was to screen and identify distinctive biomarkers in ascites of serous EOC associated with intrinsic chemoresistance.

Methods

Protein samples from ascites of 12 chemosensitive and 7 intrinsically chemoresistant serous EOC patients were analyzed using two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Furthermore, the identified proteins were validated by ELISA in ascites samples from 19 chemosensitive and 9 intrinsically chemoresistant EOC patients.

Results

The number of spots detected in all 2-D DIGE gels ranged from 1523–1711 using DeCyder software analysis. Thirty-four spots were differentially expressed based on the criteria of an average ratio of more than 1.5 and a student t-test P value <0.05. After MALDI-TOF/TOF MS analysis, 11 differentially expressed proteins, including 3 up-regulated and 8 down-regulated proteins, in ascites of chemoresistant tumors were successfully identified. Of the four selected proteins (ceruloplasmin, apoliprotein A-IV, transthyretin and haptoglobin) in ascites tested by ELISA, only ceruloplasmin was present at significantly different levels between the chemoresistant and chemosensitive ascites samples with average concentrations of 192.2 µg/ml and 157.5 µg/ml, respectively (P = 0.001).

Conclusion

The significantly up-regulated level of ceruloplasmin in the ascites fluid of intrinsic chemoresistant serous EOC patients suggests its potential as a prognostic biomarker for responses to chemotherapy. This finding prompts further investigation with a larger study in order to validate the clinical utility of ceruloplasmin.     

Increased levels of soluble interleukin-6 receptor and CCL3 in COPD sputum

Background

COPD patients have increased numbers of macrophages and neutrophils in the lungs. Interleukin-6 (IL-6) trans-signaling via its soluble receptor sIL-6R, governs the influx of innate immune cells to inflammatory foci through regulation of the chemokine CCL3. We hypothesized that there would be enhanced levels of IL-6, sIL-6R and CCL3 in COPD sputum.

Methods

59 COPD patients, 15 HNS and 15 S underwent sputum induction and processing with phosphate buffered saline to obtain supernatants for IL-6, sIL-6R and CCL3 analysis. Cytoslides were produced for differential cell counting and immunocytochemistry (COPD; n = 3) to determine cell type surface expression of the CCL3 receptors CCR5 and CCR1.

Results

COPD patients expressed higher levels (p < 0.05) of sIL-6R and CCL3 compared to controls (sIL-6R medians pg/ml: COPD 166.4 vs S 101.1 vs HNS 96.4; CCL3 medians pg/ml: COPD 117.9 vs S 0 vs HNS 2.7). COPD sIL-6R levels were significantly correlated with sputum neutrophil (r = 0.5, p < 0.0001) and macrophage (r = 0.3, p = 0.01) counts. Immunocytochemical analysis revealed that CCR5 and CCR1 were exclusively expressed on airway macrophages.

Conclusion

Enhanced airway generation of sIL-6R may promote IL-6 trans-signaling in COPD. Associated upregulation of CCL3 may facilitate the recruitment of macrophages into the airways by ligation of CCR1 and CCR5.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0103-4) contains supplementary material, which is available to authorized users.     

Preliminary characterizations of a serum biomarker for sarcoidosis by comparative proteomic approach with tandem-mass spectrometry in ethnic Han Chinese patients

Background

The diagnosis of sarcoidosis is still a significant challenge in China because of the need to exclude other diseases including granulomatous infections and malignancies that may be clinically and radiographically similar. The specific aim of the study is to search for serum protein biomarkers of sarcoidosis and to validate their clinical usefulness in differential diagnosis.

Methods

Serum samples were collected from patients with sarcoidosis (n = 37), and compared to those from patients with tuberculosis (n = 20), other pulmonary diseases (n = 20), and healthy volunteers (n = 20) for determination of sarcoidosis-specific or -associated protein expression profiles. The first part of this study focused on proteomic analysis of serum from patients with sarcoidosis to identify a pattern of peptides capable of differentiating the studied populations using the ClinProt profiling technology based on mass spectrometry. Enzyme Linked Immunosorbent Assay (ELISA) was then used to verify corresponding elevation of the serum protein concentration of the potential biomarkers in the same patients sets. Receiver operating characteristic curve (ROC) analyses was performed to determine the optimal cutoff value for diagnosis. Immunohistochemistry was carried out to further confirm the protein expression patterns of the biomarkers in lung tissue.

Results

An unique protein peak of M/Z 3,210 Daltons (Da) was found to be differentially expressed between the sarcoidosis and control groups and was identified as the N-terminal peptide of 29 amino acids (94-122) of serum amyloid A (SAA). ELISA confirmed that the serum SAA level was significantly higher in the sarcoidosis group than that of the other 3 control groups (p < 0.05). The cutoff for serum SAA concentration determined by ROC analysis was 101.98 ng/ml, with the sensitivity and specificity of 96.3% and 52.5%, respectively. Immunohistochemical staining showed that the SAA depositions in lung tissue of the sarcoidosis patients were also significantly more intense than in non-sarcoid lung tissue (p < 0.05).

Conclusion

This is the first study to investigate serum protein markers in Chinese subjects with sarcoidosis. This study shows that the serum SAA expression profiles were different between the sarcoidosis and non-sarcoidosis groups. SAA may be a potential serum biomarker for ruling-out the diagnosis of sarcoidosis in Chinese subjects.     

The association of plasma biomarkers with computed tomography-assessed emphysema phenotypes

Rationale

Chronic obstructive pulmonary disease (COPD) is a phenotypically heterogeneous disease. In COPD, the presence of emphysema is associated with increased mortality and risk of lung cancer. High resolution computed tomography (HRCT) scans are useful in quantifying emphysema but are associated with radiation exposure and high incidence of false positive findings (i.e., nodules). Using a comprehensive biomarker panel, we sought to determine if there was a peripheral blood biomarker signature of emphysema.

Methods

114 plasma biomarkers were measured using a custom assay in 588 individuals enrolled in the COPDGene study. Quantitative emphysema measurements included percent low lung attenuation (%LAA) ≤ −950 HU, ≤ − 910 HU and mean lung attenuation at the 15^th percentile on lung attenuation curve (LP15A). Multiple regression analysis was performed to determine plasma biomarkers associated with emphysema independent of covariates age, gender, smoking status, body mass index and FEV₁. The findings were subsequently validated using baseline blood samples from a separate cohort of 388 subjects enrolled in the Treatment of Emphysema with a Selective Retinoid Agonist (TESRA) study.

Results

Regression analysis identified multiple biomarkers associated with CT-assessed emphysema in COPDGene, including advanced glycosylation end-products receptor (AGER or RAGE, p < 0.001), intercellular adhesion molecule 1 (ICAM, p < 0.001), and chemokine ligand 20 (CCL20, p < 0.001). Validation in the TESRA cohort revealed significant associations with RAGE, ICAM1, and CCL20 with radiologic emphysema (p < 0.001 after meta-analysis). Other biomarkers that were associated with emphysema include CDH1, CDH 13 and SERPINA7, but were not available for validation in the TESRA study. Receiver operating characteristics analysis demonstrated a benefit of adding a biomarker panel to clinical covariates for detecting emphysema, especially in those without severe airflow limitation (AUC 0.85).

Conclusions

Our findings, suggest that a panel of blood biomarkers including sRAGE, ICAM1 and CCL20 may serve as a useful surrogate measure of emphysema, and when combined with clinical covariates, may be useful clinically in predicting the presence of emphysema compared to just using covariates alone, especially in those with less severe COPD. Ultimately biomarkers may shed light on disease pathogenesis, providing targets for new treatments.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0127-9) contains supplementary material, which is available to authorized users.     

Heart Failure Induces Significant Changes in Nuclear Pore Complex of Human Cardiomyocytes

Aims

The objectives of this study were to analyse the effect of heart failure (HF) on several proteins of nuclear pore complex (NPC) and their relationship with the human ventricular function.

Methods and Results

A total of 88 human heart samples from ischemic (ICM, n = 52) and dilated (DCM, n = 36) patients undergoing heart transplant and control donors (CNT, n = 9) were analyzed by Western blot. Subcellular distribution of nucleoporins was analysed by fluorescence and immunocytochemistry. When we compared protein levels according to etiology, ICM showed significant higher levels of NDC1 (65%, p<0.0001), Nup160 (88%, p<0.0001) and Nup153 (137%, p = 0.004) than those of the CNT levels. Furthermore, DCM group showed significant differences for NDC1 (41%, p<0.0001), Nup160 (65%, p<0.0001), Nup153 (155%, p = 0.006) and Nup93 (88%, p<0.0001) compared with CNT. However, Nup155 and translocated promoter region (TPR) did not show significant differences in their levels in any etiology. Regarding the distribution of these proteins in cell nucleus, only NDC1 showed differences in HF. In addition, in the pathological group we obtained good relationship between the ventricular function parameters (LVEDD and LVESD) and Nup160 (r = −0382, p = 0.004; r = −0.290, p = 0.033; respectively).

Conclusions

This study shows alterations in specific proteins (NDC1, Nup160, Nup153 and Nup93) that compose NPC in ischaemic and dilated human heart. These changes, related to ventricular function, could be accompanied by alterations in the nucleocytoplasmic transport. Therefore, our findings may be the basis for a new approach to HF management.     

Decreased expression of the endothelial cell-derived factor EGFL7 in systemic sclerosis: potential contribution to impaired angiogenesis and vasculogenesis

Introduction

Microvascular damage and defective angiogenesis and vasculogenesis have a major role in the pathogenesis of systemic sclerosis (SSc). Epidermal growth factor-like domain 7 (EGFL7) is a proangiogenic molecule which is predominantly expressed and secreted by endothelial cells and their progenitors and controls vascular development and integrity. In this study, we investigated the possible involvement of EGFL7 in SSc.

Methods

Serum EGFL7 levels from 60 patients with SSc and 35 age- and sex-matched healthy controls were examined by colorimetric sandwich enzyme-linked immunosorbent assay. The expression of EGFL7 in forearm skin biopsies (n = 16 SSc, n = 10 controls), cultured dermal microvascular endothelial cells (MVECs) (n = 3 SSc, n = 3 controls) and late-outgrowth peripheral blood endothelial progenitor cell (EPC)-derived endothelial cells (n = 15 SSc, n = 8 controls) was investigated by immunofluorescence and Western blotting.

Results

Serum EGFL7 levels were detectable in 68.6% of healthy controls and 45% of SSc cases (P < 0.05). Circulating levels of EGFL7 were significantly decreased in SSc patients compared with healthy controls (P = 0.01). Serum levels of EGFL7 were significantly lower in both limited cutaneous SSc and diffuse cutaneous SSc patients than in controls (P = 0.02 and P = 0.04, respectively). In SSc, decreased serum EGFL7 levels were significantly correlated with the severity of nailfold capillary abnormalities. Patients with the most severe capillary changes and digital ulcers had serum EGFL7 levels significantly lower than healthy controls, while the EGFL7 levels did not differ significantly between controls and SSc patients with less capillary damage and lack of digital ulcers. Endothelial EGFL7 expression was strongly downregulated or even almost completely undetectable in SSc-affected dermis compared with controls (P < 0.001). In cultured SSc dermal MVECs and late-outgrowth peripheral blood EPC-derived endothelial cells, EGFL7 was significantly downregulated compared with cells obtained from healthy subjects (P < 0.01 and P < 0.001, respectively).

Conclusions

Our findings suggest that the loss of EGFL7 expression in endothelial cells and their progenitors might play a role in the development and progression of peripheral microvascular damage and the defective vascular repair process characteristic of SSc.     

Circulating microRNAs have a sex-specific association with metabolic syndrome

Background

The microRNAs let-7 g and miR-221 have been demonstrated to be related to the glucose metabolism. This study assessed the serum levels of these two microRNAs in subjects with and without metabolic syndrome (MetS).

Results

The serum microRNA levels were detected in 102 subjects aged 40 to 80 years who were recruited from the general population. The status of MetS was defined by the Adult Treatment Panel III (ATP III) criteria modified for Asians. Subjects with histories of cardiovascular diseases or who were receiving treatment with hypoglycemic or lipid-lowering agents were excluded. The levels of both circulating microRNAs (let-7 g and miR-221) were higher in subjects with MetS (p = 0.004 and p = 0.01, respectively). The sex-specific analysis showed that the difference was more prominent in women (for both miRNAs, p < 0.05 in women and p > 0.1 in men). In the female subjects, increased expression of both microRNAs was associated with an increased number of MetS risk components (p = 0.002 for let-7 g and p = 0.022 for miR-221). Moreover, the elevation of serum let-7 g was significantly associated with a low level of high-density lipoprotein cholesterol (p = 0.022) and high blood pressure (p = 0.023). In contrast, the miR-221 level was not associated with any individual MetS risk component.

Conclusions

The circulating levels of let-7 g and miR-221 displayed a female-specific elevation in individuals with metabolic syndrome.