High Sensitivity ELISA Research Articles

Abstract 3279: Circulating inflammatory biomarker C-reactive protein and radiotherapy-related pain in breast cancer patients

Abstract Background: Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer death in American women. Post-surgery adjuvant radiotherapy (RT) significantly improves clinical outcomes. However, many patients develop treatment-related pain that negatively impacts quality of life. Methods: We evaluated an inflammatory biomarker, C-reactive protein (CRP) in RT-associated pain in a prospective study of 366 breast cancer patients (64% Hispanic whites, 20% African Americans, 16% non-Hispanic whites) undergoing RT. In each patient, we measured pre- and post-RT plasma CRP levels using a highly-sensitive ELISA kit. Pain score was assessed at pre- and post-RT as the mean of four pain severity items (i.e., pain at its worst, least, average, and now) from the Brief Pain Inventory. Pain scores of 4-10 were considered clinically-relevant pain. Association between pain and CRP levels was evaluated using logistic regression models after adjusting for potential confounders. Results: Overall, 16% patients at pre-RT and 31% patients at post-RT had pain. In multivariable analyses, RT-related pain score change >4 was significantly associated with CRP change >1 mg/L (OR=3.24, 95%CI=1.33, 7.88), age <50 (OR=3.27, 95%CI=1.20, 8.92), and 3+ comorbidity conditions (OR=5.91, 95%CI=1.33, 26.82). Conclusion: This is the first study to suggest that increased inflammatory biomarker, CRP level was associated with RT-related pain. Impact: Future larger studies are warranted to validate our findings and facilitate the discovery and development of anti-inflammatory agents to protect patients from RT-related pain and improve quality of life in breast cancer patients undergoing RT. Citation Format: Eunkyung Lee, Omar L. Nelson, Carolina Puyana, Cristiane Takita, Jean L. Wright, Wei Zhao, Isildinha M. Reis, Rick Lin, Jennifer J. Hu. Circulating inflammatory biomarker C-reactive protein and radiotherapy-related pain in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3279. doi:10.1158/1538-7445.AM2017-3279

Abstract 1615: C-Reactive protein and radiotherapy-induced skin toxicity in breast cancer

Abstract Background: Post-surgery adjuvant radiotherapy (RT) for breast cancer significantly reduced the local recurrence rate. However, many patients develop early adverse skin reactions (EASRs) that impact quality of life. Methods: In a large prospective study of 1,000 breast cancer patients undergoing RT, we evaluated an inflammatory biomarker, C-reactive protein (CRP) in predicting RT-induced EASRs. In each patient, we measured pre- and post-RT plasma CRP levels using a highly-sensitive ELISA CRP assay. RT-induced EASRs were assessed using the Oncology Nursing Society Skin Toxicity Criteria. Association between EASRs and CRP were assessed using logistic regression models after adjusting for potential confounders. Results: The study population includes 405 non-Hispanic White, 280 African Americans, 218 Hispanic Whites, 52 Asians, and 45 others. RT-induced grade 3+ and 4+ skin toxicity at the end of RT were observed in 42% and 15% patients, respectively. CRP levels differ significantly by race/ethnicity at baseline and at the end of RT. RT-induced grade 4+ skin toxicity was significantly associated with: obesity and pre-RT CRP > 2mg/L (OR=3.27; 95%CI=1.88, 5.68), obesity and post-RT CRP > 2mg/L (OR=4.42; 95%CI=2.38, 8.23), or obesity and change of CRP > 1mg/L (OR=3.58; 95%CI=2.00, 6.39). Conclusion: The current data validate our previous findings that the inflammatory biomarker CRP is associated with RT-induced EASRs, particularly combined with obesity. Impact: Our current findings support the discovery and development of anti-inflammatory agents to protect normal tissue from RT-induced EASRs and improve quality of life in breast cancer patients undergoing RT. Citation Format: Jennifer J. Hu, Doug Case, Mark O. Lively, Eunkyung Lee, Cristiane Takita, James J. Urbanic, Glenn J. Lesser, Edward G. Shaw. C-Reactive protein and radiotherapy-induced skin toxicity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1615. doi:10.1158/1538-7445.AM2017-1615

High-sensitive CRP as a predictive marker of long-term outcome in juvenile idiopathic arthritis.

To evaluate whether C-reactive protein (CRP), including variation within the normal range, is predictive of long-term disease outcome in Juvenile Idiopathic Arthritis (JIA). Consecutive patients with newly diagnosed JIA were included prospectively from defined geographic areas of the Nordic countries from 1997 to 2000. Inclusion criteria were availability of a baseline serum sample within 12 months after disease onset and 8-year clinical assessment data. Systemic onset JIA was not included. CRP was measured by high-sensitive ELISA (detection limit of 0.2mg/l). One hundred and thirty participants with a median follow-up time of 97 months (range 95-100) were included. At follow-up, 38% of the patients were in remission off medication. Absence of remission was associated with elevated level of CRP at baseline (odds ratio (OR) 1.33, confidence interval (CI) 1.08-1.63, p = 0.007). By applying a cutoff at the normal upper limit (>10mg/l), the risk of not achieving remission was increased to an OR of 8.60 (CI 2.98-24.81, p < 0.001). Variations of CRP within the normal range had no predictive impact on disease activity at follow-up. Baseline levels of ESR were available in 80 patients (61%) and elevated ESR was associated with absence of remission in a multivariable logistic regression analysis (OR 2.32, CI 1.35-4.00, p = 0.002). This results of this study indicate that baseline CRP concentrations above 10mg/l are predictive of a poor outcome at 8-year follow-up. We could not demonstrate any predictive value of CRP variations within the normal range.

Relationship between polymorphisms in the CRP, LEP and LEPR genes and high sensitivity C-reactive protein levels in Spanish children.

We investigated the association of single nucleotide polymorphisms (SNPs) in the C-reactive protein (CRP), leptin (LEP) and leptin receptor (LEPR) genes with high sensitivity CRP (hs-CRP) levels in two independent cohorts of healthy Spanish children. We measured hs-CRP levels in 646 6-8-year-old and 707 12-16-year-old children using a high-sensitivity C-Reactive Protein ELISA kit. Four SNPs in the CRP gene (rs1205, rs1130864, rs2794521 and rs1800947), one SNP in the LEP gene (rs7799039) and two SNPs in the LEPR (rs1137100 and rs1137101) gene were determined by TaqMan® allelic discrimination assays. The four CRP SNPs studied were significantly (p<0.05) associated with hs-CRP levels in both cohorts. Furthermore, two common CRP haplotypes (constructed using the SNPs in order: rs1205, rs1130864, rs1800947, rs2794521) ACGA and GCGG were associated with significantly lower CRP levels (p<0.05) at both ages. The LEPR SNPs rs1137100 (K109R) and rs1137101 (Q223R), and LEP SNP rs7799039 (G2548A) were also associated to hs-CRP levels (p<0.05) in both cohorts. hs-CRP levels in healthy Spanish children, besides being associated to common polymorphisms in the CRP gene, are associated to polymorphisms in the LEP and LEPR genes, which suggests that other loci, in addition the CRP gene, may have a role determining CRP levels in children.

Modulatory Effects of Chemoradiation on Angiogenic Factors and Laminin in Cervical Cancer: Link with Treatment Response

Objective:Carcinoma of the uterine cervix is either the first or second most common malignancy in Indian women, depending on the registry. Tumor growth and metastasis primarily are determined by angiogenesis and parameters of the molecular environment including extracellular matrix elements, growth factors and cytokines. Effects of chemo-irradiation on biomarkers like vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2) and laminin in patients with carcinoma cervix therefore need to be explored.Methods:Circulatory and mRNA levels of VEGF, Ang-2 and laminin in patients with stage III carcinoma cervix (n=40) were compared with those of normal healthy women (n=20). Measurement was prior to treatment, and after chemotherapy and teleradiation, using high sensitivity ELISA kits and Q-PCR. Clinical response was evaluated as per WHO criteria and was assessed for correlation with the biochemical markers.Results:Levels of all the studied molecules were significantly (p<0.001) higher in patients than in controls. After treatment significant decline (p<0.001) was noted. Out of 40 patients, 33 were complete responders and 7 were non-responders on clinical assessment. On comparison of before and after treatment levels of these molecules complete responders showed significant decline whereas non-responders showed non-significant decrease. Follow-up of the responders for 3 years, revealed 28 of 33 patients to still be disease free, the other 5 demonstrating recurrence.Conclusions:Higher levels of angiogenic factors along with laminin indicate roles played in disease progression aiding angiogenesis. These markers may serve as useful tools in post treatment disease mapping, for which available imaging methods may not provide a true picture.

Abstract # 1783 Metabolic dysfunction, inflammation, and task-induced emotional stress reactivity: Neural and cognitive correlates in 331 middle-aged adults

Pre-diabetes and type 2 diabetes (T2D) etiology is characterized by insulin resistance (IR) and systemic inflammation. These factors can cause deficits in cognitive and affective processing, particularly psychological stress reactivity. The neural correlates of these deficits are unknown. We wished to determine how T2D, IR, and interleukin-6 (IL-6) were related to psychophysiological, behavioral, and hormonal measures of psychological stress reactivity and negative affect predisposition. Among 331 adults (36–84 years old) from the Midlife in the United States (MIDUS) study, we reviewed startle eye-blink response (EBR) to “unpleasant” versus “pleasant” international affect picture system (IAPS) stimuli. Using resting electroencephalography (EEG), we analyzed right frontal alpha asymmetry, a well-established biomarker of negative affect predisposition. Finally, cognitive performance and cortisol output were gauged during an arithmetic stressor task. Fasting glucose and insulin characterized T2D or the homeostatic model assessment of IR (HOMA-IR). High-sensitivity ELISA was used to derive IL-6. For EBR startle magnitude, higher HOMA-IR strongly corresponded to greater EBR for “unpleasant” versus “pleasant” stimuli [R2 = 0.218, p = .020]. For resting EEG, IL-6 [p = 0.030], or T2D showed greater right frontal alpha asymmetry [p = .011]. During the stressor task, participants with T2D scored 26 ± 2% worse than those with euglycemia [p = .040]. Surprisingly, cortisol output at baseline was lower in participants with T2D and pre-diabetes [p = .025]. These results suggest that dysmetabolism and inflammation may increase stress sensitization both at the neuronal and behavioral level.

Serum Immune System Biomarkers Neopterin and Interleukin-10 Are Strongly Related to Tryptophan Metabolism in Healthy Young Adults

Background: Changes in tryptophan metabolism through the vitamin B-6–dependent kynurenine pathway have been linked to activation of the immune system.Objective: We hypothesized that blood concentrations of tryptophan and its catabolites were associated with biomarkers relevant to inflammatory processes in healthy noninflamed subjects.Methods: Healthy young adults (n = 737) aged 18–28 y without any known diseases or clinical evidence of inflammation provided blood samples for analysis of serum tryptophan/kynurenine metabolites, neopterin, C-reactive protein (CRP), and plasma pyridoxal 5′-phosphate (PLP) with LC–tandem mass spectrometry methodologies. A panel of cytokines was measured in serum by using high-sensitivity ELISA assays. Anthropometric and lifestyle data were collected by questionnaire. Multiple linear regression analysis to determine the effect of measured serum cytokine concentrations as predictors of tryptophan metabolites was performed on inverse normal-rank transformations of the data, adjusted for sex, body mass index, smoking, alcohol intake, and contraceptive use in women.Results: Median serum CRP and neopterin concentrations were well below established clinical cutoffs for inflammation. We observed significant positive associations between serum interleukin-10 (IL-10) and serum kynurenine (P = 0.0002), the kynurenine-to-tryptophan ratio (KTR) (P = 0.003), 3-hydroxykynurenine (P = 0.01), and 3-hydroxyanthranilic acid (P = 0.04). Serum neopterin was positively associated with kynurenine, the KTR (both P < 0.0001), and anthranilic acid (P = 0.004), and was negatively associated with serum tryptophan (P = 0.01) and PLP (P < 0.0001). Serum tumor necrosis factor α was also negatively associated with tryptophan (P < 0.001).Conclusions: In healthy young adults with no apparent inflammatory conditions, serum tryptophan metabolites are significantly associated with key immune system biomarkers. The observed association between IL-10 and kynurenine is unexpected and suggests that kynurenine-linked mechanisms promoting negative regulation of inflammatory responses are associated with normal immune homeostasis.

Evaluation of highly sensitive immunoassay technologies for quantitative measurements of sub-pg/mL levels of cytokines in human serum

A comprehensive cross-platform and cross-assay evaluation using nine technology platforms and four cytokine immunoassays (IL-6, TNFα, IL-17a, IL-2) was performed by comparing assay precision, sensitivity, parallelism and data correlation between platforms. The precision was acceptable for most evaluated assays. In addition to comparing the analytical assay sensitivity using a spiked recombinant analyte in buffer, forty serum samples from both normal controls and multiple sclerosis patients were used to measure the frequency of endogenous analyte detection (FEAD) as a parameter of each assay's ability to detect the endogenous analyte. The highest FEAD measurements were observed on the Simoa™, Erenna®, Milliplex® and Imperacer® platforms. However, only Simoa and Erenna results showed a high correlation across all evaluated cytokine assays, followed by a more moderate correlation of results across platforms for the V-plex™, high sensitivity ELISA and the Ella™ IL-6 and TNFα assays. In contrast, results from the evaluated cytokine assays on the Milliplex, AMMP™ ViBE® and Imperacer platforms did not correlate to each other nor to other evaluated assays. Acceptable parallelism was observed for the Simoa, Erenna, V-plex and Ella assays but not for the Milliplex, AMMP ViBE and Imperacer assays. In conclusion, the Simoa, Erenna,V-plex and Ella platforms performed well in one or more evaluated cytokine assays. Among those, the Simoa and Erenna assays had the highest sensitivity for detection of cytokines present at sub-pg/mL levels in human serum. In addition, the cross-platform and cross-assay comparisons demonstrated that different immunoassays may yield different results, which underscores the importance of performing such comparative evaluations, especially in the absence of reliable reference standards for the quantitative assessments of biomarkers in immunoassays.

High-Sensitivity Sandwich ELISA for Plasma NT-proUcn2: Plasma Concentrations and Relationship to Mortality in Heart Failure.

Urocortin 2 (Ucn2) has powerful hemodynamic, renal, and neurohormonal actions and likely participates in normal circulatory homeostasis and the compensatory response to heart failure (HF). A validated assay for endogenous circulating Ucn2 would facilitate investigations into Ucn2 physiology and elucidate its derangement and potential as a biomarker in heart disease. We developed a chemiluminescence-based sandwich ELISA to measure plasma N-terminal (NT)-proUcn2 in non-HF patients (control; n = 160) and HF patients with reduced (HFREF; n = 134) and preserved (HFPEF; n = 121) left ventricular ejection fraction (LVEF). The ELISA had a limit of detection of 8.47 ng/L (1.52 pmol/L) and working range of 23.8-572 ng/L. Intra- and interassay CV and total error were 4.8, 16.2, and 17.7%, respectively. The median (interquartile range) plasma NT-proUcn2 concentration in controls was 112 (86-132) ng/L. HFREF, HFPEF, and all HF plasma concentrations were significantly increased [117 (98-141) ng/L, P = 0.0007; 119 (93-136) ng/L, P = 0.0376, and 119 (97-140) ng/L, P = 0.001] compared with controls but did not differ significantly between HFREF and HFPEF. NT-proUcn2 was modestly related to age (r = 0.264, P = 0.001) and cardiac troponin T (r = 0.258, P = 0.001) but not N-terminal pro-B-type natriuretic peptide, body mass index, LVEF, or estimated glomerular filtration rate. On multivariate analysis, plasma NT-proUcn2 was independently and inversely related to 2-year mortality in HF. The validated ELISA measured human NT-proUcn2 in plasma and showed modest but significant increases in HF patients compared with controls. In HF, the unusual inverse relationship between plasma NT-proUcn2 and 2-year mortality portends potential prognostic value but requires further corroboration.

86 A novel, highly-sensitive ELISA assay can detect Pseudomonas aeruginosa virulence factors in CF sputum

86 A novel, highly-sensitive ELISA assay can detect Pseudomonas aeruginosa virulence factors in CF sputum

New insights into immune mechanisms underlying response to Rituximab in patients with membranous nephropathy: A prospective study and a review of the literature.

Idiopathic membranous nephropathy (MN) is a common immune-mediated glomerular disease and the main cause of nephrotic syndrome (NS) in Caucasian adults. Rituximab (RTX) has been reported to safely reduce proteinuria in patients with primary MN and severe NS. However, the effects of RTX treatment on T-cells including regulatory T-cells (Treg) in MN have not been fully determined. Seventeen patients [mean age 67 (29-86) years, 6 women, 11 men] with biopsy-proven MN, and persistent proteinuria >3.5 g/24h were prospectively enrolled and received RTX, 375 mg/m(2) (iv) on days 1, 8, 15 and 22. Changes in circulating B and T cell homeostasis were examined in the peripheral blood by flow-cytometry studies; serum levels of IL-35 were measured using a high-sensitivity ELISA kits (baseline, at month 3, 6, 9 and 12). Patients had been followed-up for a mean of 36.3 months (24-48). Proteinuria decreased from 5.6 (3.5-8) g/24h to 2.4 (0.06-13) g/24h at 6 months (p<0.05) and to 1.3 (0.06-8) at 12 months (p<0.01), respectively after therapy with RTX. Four patients received a 2nd course of RTX (one at 6 months because of persistent NS, and three at 12, 18, or 30 months for relapse). The three relapsing patients became proteinuria-free (<0.5 g/24h) in the following 6 months. Serum creatinine remained stable during the follow-up: median 1mg/dl (0.7-1.6) at 12 months and 1.1 (0.7-1.7) at 24 months as compared to 1 (0.5-2.4) at baseline. At 6 months after RTX, complete remission (CR) was observed in 7 patients, partial remission (PR) in 4, while 6 were non responders (NR) non responder (NR). At the end of the follow-up, 14 patients were in CR, 1 in PR, while 2 were NR. In the T-cell compartment, upon detection of B cell depletion, there was an increase in Treg up to 10-fold when comparing baseline and at month 12 (mean ± SD 1.2 ± 0.6%, and 5.8 ± 0.7% p=0.02, respectively). When stratifying patients in responders (CR+PR) and NRs at month 12, we observed a significant increase in Treg cells from month 6 which persisted till 12 months only in the responder group (5.5 ± 0.6% and 1.1 ± 0.6%, p=0.04, respectively in responders and NRs). A statistically significant decrease in the levels of active T-lymphocytes (HLA-DR+CD8+ cells) was observed, with a maximum reached at 12 months after treatment with RTX [6 ± 1.1% baseline, 4.7 ± 1.7% at 6 months (p=0.043) and 1.5 ± 1.4% at 12 months (p=0.05)]. A marked increase in IL-35 levels [defined as delta >40% (serum values at 6 months minus baseline values)] was seen in 68% of the patients who achieved clinical response (CR or PR) at 12 month, but in none of the patients who failed to respond (p=0.034). Our findings and data from literature support the idea that RTX can be envisaged as a first-line therapy for patients at risk of progression because of persistent NS due to idiopathic MN. Insights into the putative T cell-related mechanisms of action have been discussed.

Inflammation markers are associated with metabolic syndrome and ventricular arrhythmia in patients with coronary artery disease

Inflammation plays a major role in the development and progression of atherosclerosis and coronary artery disease (CAD). Inflammation markers, including white blood cell (WBC) count, C-reactive protein (CRP) and interleukin-6 (IL-6), are widely used for cardiovascular risk prediction. The aim of the study was to establish factors associated with WBC, CRP and IL-6 in patients with CAD. Two functional polymorphisms in genes encoding enzymes participating in adenosine metabolism were analyzed (C34T AMPD1, G22A ADA). Plasma concentrations of IL-6 were measured using high-sensitivity ELISA kits, and the nephelometric method was used for high-sensitivity CRP (hs-CRP) measurement in 167 CAD patients. Presence of metabolic syndrome (MS) and its components, presence of heart failure, severity of CAD symptoms, severe past ventricular arrhythmia (sustained ventricular tachycardia [sVT] or ventricular fibrillation [VF]), lower left ventricle ejection fraction, higher left ventricle mass index, higher end-diastolic volume and higher number of smoking pack-years were significantly associated with higher WBC, CRP and IL-6. Strong associations with arrhythmia were observed for IL-6 (median 3.90 vs 1.89 pg/mL, p<0.00001) and CRP concentration (6.32 vs 1.47 mg/L, p=0.00009), while MS was associated most strongly with IL-6. CRP and IL-6 were independent markers discriminating patients with sVT or VF. There were no associations between AMPD1 or ADA genotypes and inflammation markers. WBC, CRP and IL-6 are strongly associated with components of the metabolic syndrome. Their strong association with life-threatening ventricular arrhythmia emphasizes the proarrhythmic role of inflammation in the increased cardiovascular risk of CAD patients.

Reduction of Amyloid-β Plasma Levels by Hemodialysis: An Anti-Amyloid Treatment Strategy?

Cognitive impairment in hemodialysis patients is common, but the underlying pathogenesis remains unclear. Alzheimer's disease is the most common cause of dementia in the general elderly population. Histopathological hallmarks are, among others, senile plaques, which consist of amyloid-β (Aβ). To measure plasma levels of Aβ42 and Aβ40 during hemodialysis and to examine potential associations with cognitive performance in cognitively impaired hemodialysis patients. Plasma samples of 26 hemodialysis patients were collected shortly before, after 50% of dialysis time, and at the end of a dialysis session. Aβ42 and Aβ40 levels were measured by a high-sensitivity ELISA for human amyloid-β. Cognition was tested under standardized conditions using the Montreal Cognitive Assessment (MoCA) as proposed previously. Clearance rates of both peptides during one dialysis session were 22% and 35% for Aβ42 and Aβ40, respectively. Aβ42 but not Aβ40 baseline levels were significantly associated with MoCA test results (r = 0.654, p = 0.001). In cognitively impaired hemodialysis patients plasma Aβ42 levels were associated with cognitive performance and both Aβ42 and Aβ40 plasma levels could be effectively reduced by dialysis. By inducing peripheral Aβ sink, hemodialysis may be considered as an anti-amyloid treatment strategy.

Correlation of Oxidative Damage with Pro-Inflammatory Markers (IL-6, TNF-α) in Meningocele.

Oxidative damage induces alteration in the status of pro-inflammatory markers like IL-6 and TNF-α in meningocele. The study was performed with estimation of the levels of MDA (Malonyldialdehyde), SOD (Superoxide dismutase) taken as oxidative damage markers and IL-6 (interleukin 6) and TNF-α (Tumour necrosis factor alpha) taken as inflammatory markers, in the serum of meningocele patients and age, sex matched normal neonates. Correlation among the different serum levels of MDA, SOD, IL-6 and TNF-α was determined. It is a case-control study, comprising of 153 participants: 101 newborns with meningocele and 52 healthy newborns. The study was conducted in the Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, in collaboration with the Department of Paediatric Surgery and Department of Obstetrics and Gynecology, Sir Sunderlal Hospital, Banaras Hindu University, Varanasi. The study was conducted during the period of 2012 to 2014. Serum was extracted from blood collected from both groups i.e. meningocele patient group and healthy neonatal control group. The levels of MDA and SOD were determined by spectrophotometric method. IL-6 was determined by the Human IL-6 High Sensitivity ELISA Kit and TNF-α was determined by the Human TNF-α ELISA KIT. The levels of MDA, TNF-α and IL-6 were found to be much higher and level of SOD was found lower in the patients with meningocele as compared to the normal healthy neonates. Increased MDA (oxidative damage product), IL-6, and TNF-α (inflammatory marker) and low level of SOD shows an increased inflammatory response in Meningocele. Our study shows Negative Correlation between MDA and SOD in case & control groups, while a Positive Correlation between TNF alpha and IL-6 in control & case groups.

Prognostic and diagnostic potential of local and circulating levels of pentraxin 3 in lung cancer patients.

There is a well-established link between inflammation and cancer of various organs, but little data are available on inflammation-associated markers of diagnostic and prognostic clinical utility in pulmonary malignancy. Blood samples were prospectively collected from 75 resectable lung cancer patients before surgery and in a cohort of 1,358 high-risk subjects. Serum levels of long pentraxin 3 (PTX3) were determined by high-sensitivity ELISA. PTX3 immunostaining was evaluated by immunohistochemistry in cancer tissue. Serum PTX3 levels in the high-risk population were not predictive of developing subsequent lung cancer or any other malignancy; however, serum PTX3 values in patients with lung cancer were significantly higher compared with cancer-free heavy smokers. With a cutoff of 4.5 ng/ml, specificity was 0.80, sensitivity 0.69, positive predictive value 0.15 and negative predictive value 0.98. The receiver operating curve (ROC) for serum PTX3 had an area under the curve (AUC) of 83.52%. Preoperative serum PTX3 levels in lung cancer patients did not correlate with patient outcome, but high interstitial expression of PTX3 in resected tumor specimens was a significant independent prognostic factor associated with shorter survival (p < 0.001). These results support the potential of serum PTX3 as a lung cancer biomarker in high-risk subjects. Furthermore, PTX3 immunohistochemistry findings support the role of local inflammatory mechanisms in determining clinical outcome and suggest that local expression of PTX3 may be of prognostic utility in lung cancer patients.

Red blood cell long-chain polyunsaturated fatty acid (LC-PUFA) levels in pregnant women predict length of gestation via effects on sleep quality and inflammation

Objective Mechanistic pathways linking maternal long-chain polyunsaturated fatty acid (LC-PUFA) status with gestational length are poorly delineated. Key LC-PUFAs implicated in this association are the antiinflammatory omega-3 docosahexaenoic acid (DHA) and proinflammatory omega-6 arachidonic acid (AA). We hypothesized that (1) lower red blood cell (RBC) DHA:AA ratios would predict shorter gestation and (2) this effect would be mediated by both inflammation and sleep quality. Methods Pregnant women (n = 135) provided blood and completed the Pittsburg Sleep Quality Index at 20–27 weeks gestation. RBC fatty acids were determined by gas chromatography and serum inflammatory markers (IL-6, IL-8, TNF- α , IL-1 β , CRP) were determined by high-sensitivity ELISA. Results Lower DHA:AA ratios predicted higher serum IL-8 (r = −0.20, p = 0.02) and poorer sleep quality (b = −13.8, p = 0.02). These relationships remained after adjusting for depressive symptoms, age, BMI, income, race, and smoking. Mediational analyses demonstrated indirect effects linking lower RBC DHA:AA with shorter gestation via higher IL-8 (95% CI = 0.10, 3.84) and poor sleep (95% CI = 0.35, 3.99). A significant race X DHA:AA interaction in predicting preterm birth (PTB) was observed (p = 0.049); among African Americans only, the odds of PTB decreased significantly as DHA:AA increased (OR for 0.1 unit increase = 0.25 (95% CI = 0.06, 0.99, p = 0.048). Discussion These data support both inflammatory pathways and sleep quality as significant mediators linking maternal DHA:AA status and length of gestation. African Americans may be at greater risk for PTB related to suboptimal DHA levels.

Suppression of nutrient-induced inflammation with a nonsteroidal anti-inflammatory agent ameliorates ovarian dysfunction in lean insulin-sensitive women with polycystic ovary syndrome (PCOS)

early follicular phase. TNFa, IL-27, IL-35, IL-37, a-1 acid glycoprotein were measured by a high sensitivity ELISA, while CRP, testosterone (T), androstenedione (AE), SHBG, DHEA-S levels were measured by RIA. Free androgen index (FAI) was calculated as percentage ratio of total testosterone to SHBG values. RESULTS: Levels of inflammatory mediators (CRP, TNFa, a-1 acid glycoprotein) were significantly increased in obese PCOS patients compared to obese controls and in lean PCOS patients compared to lean controls (all p<0.001). Levels of cytokines with anti-inflammatory properties (IL-27, IL-35, IL-37) were significantly decreased in obese PCOS patients compared to obese controls and in lean PCOS patients compared to lean controls (all p<0.001). Levels of T, androstenedione, DHEA-S were significantly increased, while level of SHBG was significantly increased in obese PCOS patients compared to obese controls and in lean PCOS patients compared to lean controls (all p<0.001). When obese and lean PCOS patients were compared, levels of IL-27, IL-35, IL-37 were significantly lower, while level of a-1 acid glycoprotein was significantly higher in obese PCOS patients compared lean patients (all p<0.001). FAI was higher in obese PCOS patients compared to obese controls, PCOS leans patients compared to lean controls (both p<0.001). BMI had a strong negative correlation with IL-27, IL-25, IL-37 and a positive correlation with a-1 acid glycoprotein (all p<0.001). FAI had a strong positive correlation with CRP, TNFa, a-1 acid glycoprotein and a negative correlation with IL-27, IL-25, IL-37 (all p<0.001). BMI had a positive correlation with FAI (p<0.05). CONCLUSIONS: The findings of the present study further confirm the pro-inflammatory state of PCOS.Moreover, obesity along with PCOS significantly elevates the inflammatory status and hyperandrogenism. Supported by: This research received funding from the Scientific Research Projects Coordination Unit of Istanbul University (grant 31834).

Handgrip strength, functionality and plasma levels of IL-6 in elderly women

AbstractIntroduction Aging is associated with a chronic low-grade inflammatory process characterized by an increased production of inflammatory mediators. These elevated levels are predictors for muscle function (sarcopenia) and deterioration of physical performance in the elderly.Objective To investigate the correlation between sarcopenia, functional capacity, and interleukin-6 levels.Materials and methods This cross-sectional study assessed a convenience sample of elderly individuals (n = 130) using a handgrip dynamometer (JAMAR™), and the functional capacity test was performed by sitting and standing. Interleukin-6 levels were determined using high sensitivity ELISA kits. Spearman correlation coefficient was used to assess the correlation between these variables, at a significance level (α) of 0.05.Results There was significant inverse correlation between strength and function (rS = -0.273, p = 0.002) such that elderly individuals with lower peak force took longer to perform the sit-to-stand test, in contrast with the relationship between plasma interleukin-6 levels and strength (rS = -0.043, p = 0.624) or functionality (rS = -0.060, p = 0.501).Conclusion The outcome measures analyzed could predict the decline in muscle strength and functional capacity, and may be indicative of sarcopenia in elderly individuals. There was no correlation between the interleukin-6 levels and the grip strength and functional capacity of the sample investigated.

A comparison of two commercially available ELISA methods for the quantification of human plasma heat shock protein 70 during rest and exercise stress.

This study compared resting and exercise heat/hypoxic stress-induced levels of plasma extracellular heat shock protein 70 (eHSP70) in humans using two commercially available enzyme-linked immunosorbent assay (ELIS)A kits. EDTA plasma samples were collected from 21 males during two separate investigations. Participants in part A completed a 60-min treadmill run in the heat (HOT70; 33.0 ± 0.1 °C, 28.7 ± 0.8 %, n = 6) at 70 % V̇O2max. Participants in part B completed 60 min of cycling exercise at 50 % V̇O2max in either hot (HOT50; 40.5 °C, 25.4 relative humidity (RH)%, n = 7) or hypoxic (HYP50; fraction of inspired oxygen (FIO2) = 0.14, 21 °C, 35 % RH, n = 8) conditions. Samples were collected prior to and immediately upon termination of exercise and analysed for eHSP70 using EKS-715 high-sensitivity HSP70 ELISA and new ENZ-KIT-101 Amp'd(™) HSP70 high-sensitivity ELISA. ENZ-KIT was superior in detecting resting eHSP70 (1.54 ± 3.27 ng · mL(-1); range 0.08 to 14.01 ng · mL(-1)), with concentrations obtained from 100 % of samples compared to 19 % with EKS-715 assay. The ENZ-KIT requires optimisation prior to running samples in order to ensure participants fall within the standard curve, a step not required with EKS-715. Using ENZ-KIT, a 1:4 dilution allowed for quantification of resting HSP70 in 26/32 samples, with a 1:8 (n = 3) and 1:16 (n = 3) dilution required to determine the remaining samples. After exercise, eHSP70 was detected in 6/21 and 21/21 samples using EKS-715 and ENZ-KIT, respectively. eHSP70 was increased from rest after HOT70 (p < 0.05), but not HOT50 (p > 0.05) or HYP50 (p > 0.05) when analysed using ENZ-KIT. It is recommended that future studies requiring the precise determination of resting plasma eHSP70 use the ENZ-KIT (i.e. HSP70 Amp'd(®) ELISA) instead of the EKS-715 assay, despite additional assay development time and cost required.

Increased circulating interleukin-17 levels in preeclampsia

Increasing evidence suggests that an exaggerated maternal systemic inflammatory response and an angiogenic imbalance might play a central role in the pathogenesis of preeclampsia. We determined circulating levels of interleukin-17 (IL-17) along with those of angiogenic factors in healthy nonpregnant and pregnant women and preeclamptic patients, and examined whether serum IL-17 levels of preeclamptic patients were related to their clinical features and angiogenic factor concentrations. Fifty-nine preeclamptic patients, 60 healthy pregnant women and 56 healthy nonpregnant women were involved in this case–control study. Serum levels of IL-17A were measured using a high-sensitivity ELISA. Serum total soluble fms-like tyrosine kinase-1 (sFlt-1) and biologically active placental growth factor (PlGF) levels were determined by electrochemiluminescence immunoassay. For statistical analyses, nonparametric methods were applied. Serum IL-17 levels were significantly higher in preeclamptic patients than in healthy nonpregnant and pregnant women. We did not find any relationship between serum IL-17 concentrations of preeclamptic patients and their clinical features and serum sFlt-1 and PlGF levels or sFlt-1/PlGF ratios. However, elevated serum IL-17 level and sFlt-1/PlGF ratio were found to have an additive effect on the risk of preeclampsia, as shown by the substantially higher odds ratios of a combination of the two than of either alone. In conclusion, serum IL-17 levels are increased in preeclampsia, which may contribute to the development of the excessive systemic inflammatory response characteristic of the maternal syndrome of the disease. In addition, elevated serum IL-17 level and sFlt-1/PlGF ratio had an additive (joint) effect on the risk of preeclampsia.