Diagnostic plasma miRNA-profiles for ovarian cancer in patients with pelvic mass.

BACKGROUND: Ovarian cancer is the fifth most common cancer in women worldwide. Moreover, there are no reliable minimal invasive tests to secure the diagnosis of malignant pelvic masses. Cell-free, circulating microRNAs have the potential as diagnostic biomarkers in cancer. Here, we performed and validated a miRNA panel with the potential to distinguish OC from benign pelvic masses. METHODS: The profile of plasma microRNA was determined with a panel of 46 candidates in a discovery group and a validation group, each consisting of 190 pre-surgery plasma samples from age-matched patients with malignant (n = 95) and benign pelvic mass (n = 95), by real time RT-qPCR. RESULTS: Four up-regulated (miR-200c-3p, miR-221-3p, miR-21-5p, and miR-484) and two down-regulated (miR-195-5p and miR-451a) microRNAs were discovered. From those, miR-200c-3p and miR-221-3p were further confirmed in a validation cohort. A combination of these 2 microRNAs together with CA-125 yielded an overall diagnostic accuracy of AUC = 0.96. CONCLUSIONS: We showed consistent plasma microRNA profiles that provide independent diagnostic information of late stage OC.

Exercise as a potential modulator of inflammation in patients with Alzheimer's disease measured in cerebrospinal fluid and plasma.

BACKGROUND: Neuroinflammation is recognized as part of the pathological progression of Alzheimer's disease (AD), but the molecular mechanisms are still not entirely clear. Systemically, physical exercise has shown to have a positive modulating effect on markers of inflammation. It is not known if this general effect also takes place in the central nervous system in AD. The aim of this study was to investigate the effect of 16 weeks of moderate to high-intensity physical exercise on selected biomarkers of inflammation both systemically and in the CNS, in patients with AD. METHODS: Plasma and cerebrospinal fluid (CSF) from 198 patients with Alzheimer's disease participating in the Preserving Cognition, Quality of Life, Physical Health and Functional Ability in Alzheimer's Disease: The Effect of Physical Exercise (ADEX) study were analyzed for concentrations of 8­isoprostane, soluble trigger receptor expressed on myeloid cells 2 (sTREM2), and the MSD v-plex proinflammation panel 1 human containing interferon gamma (IFNγ), Interleukin-10 (IL10), IL12p70, IL13, IL1ß, IL2, IL4, IL6, IL8, and tumor necrosis factor alpha (TNFα), before and after a 16-week intervention with physical exercise, and we studied whether changes were modulated by the patients' APOE genotype. RESULTS: Most inflammatory markers remained unchanged after exercise. We found an increasing effect of 16 weeks of physical exercise on sTREM2 measured in CSF. Further, IL6 in plasma increased in the exercise group after physical exercise (mean relative change 41.03, SD 76.7), compared to controls (-0.97, SD 49.4). In a sub-analysis according to APOE genotype, we found that in ε4 carriers, exercise had a stabilizing effect on IFNγ concentration with a mean relative change of 7.84 (SD 42.6), as compared to controls (114.7 (SD 188.3), p = 0.038. CONCLUSION: Our findings indicate an effect of physical exercise on markers of neuroinflammation in CSF measured by an increase in sTREM2 in patients with AD. Further, there may be a small inflammatory systemic effect related to physical exercise in patients with AD.

Value of anti-plasminogen binding peptide, anti-carbonic anhydrase II, immunoglobulin G4, and other serological markers for the differentiation of autoimmune pancreatitis and pancreatic cancer.

The diagnosis of autoimmune pancreatitis (AIP) and its differential diagnosis from pancreatic cancer (PC) can be challenging. In this retrospective study, we aimed to evaluate the value of anti-plasminogen binding peptide (a-PBP), immunoglobulin G4 (IgG4), and anti-carbonic anhydrase-II (a-CA-II), together with other serological markers whose value is not fully elucidated.The serum levels of a-PBP, IgG4, IgG, anti-nuclear antibodies (ANA), anti-lactoferrin (a-LF), a-CA-II, and rheumatoid factor (RF) were evaluated in patients with AIP (n = 29), PC (n = 17), pancreatic neuroendocrine neoplasm (P-NEN, n = 12), and alcoholic chronic pancreatitis (ACP, n = 41). ANCA were measured in the AIP patients.There was no statistically significant difference in mean a-PBP values in AIP compared with PC. A ROC curve showed that, when using a cut-off of 38.3 U, low values of a-PBP had a sensitivity and specificity of 45% and 71% for differentiating AIP from PC. The sensitivity and specificity of IgG4 (cut-off 1.4 g/L) for differentiating AIP from PC was 45% and 88%, but rose to 52% and 88% when using a cut-off of 1.09 g/L. When using this cut-off, the sensitivity and specificity for differentiating type 1 AIP from PC was 68% and 88%. None of the other markers were significantly changed in AIP versus PC. For differentiation of type 1 and type 2 AIP, the only significant differences were IgG4 in type 1 AIP (P < .01), with a sensitivity of 68% and a specificity of 80%, and c-ANCA elevations found in some type 2 AIP patients (P < .05).The only serological marker for which we found a statistically significant difference in mean values between AIP and PC was IgG4. However, the value of IgG4 for the distinction of AIP from PC was limited, probably in part due to the relatively high number of type 2 AIP patients in our study. In accord with recent publications, our data do not support a role of increased serum a-PBP for the diagnosis of AIP.

Plasma MicroRNA Profiles in Patients with Early Rheumatoid Arthritis Responding to Adalimumab plus Methotrexate vs Methotrexate Alone: A Placebo-controlled Clinical Trial.

OBJECTIVE: The aim was to identify plasma (i.e., cell-free) microRNA (miRNA) predicting antitumor necrosis and/or methotrexate (MTX) treatment response in patients enrolled in an investigator-initiated, prospective, double-blinded, placebo-controlled trial (The OPERA study, NCT00660647). METHODS: We included 180 disease-modifying antirheumatic drug-naive patients with early rheumatoid arthritis (RA) randomized to adalimumab (ADA; n = 89) or placebo (n = 91) in combination with MTX. Plasma samples before and 3 months after treatment initiation were analyzed for 91 specific miRNA by quantitative reverse transcriptase-polymerase chain reaction on microfluidic dynamic arrays. A linear mixed-effects model was used to test for associations between pretreatment miRNA and changes in miRNA expression and American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) Boolean (28 joints) remission at 3 and 12 months, applying false discovery rate correction for multiple testing. Using leave-one-out cross validation, we built predictive multivariate miRNA models and estimated classification performances using receiver-operating characteristics (ROC) curves. RESULTS: In the ADA group, a higher pretreatment level of miR-27a-3p was significantly associated with remission at 12 months. The level decreased in remitting patients between pretreatment and 3 months, and increased in nonremitting patients. No associations were found in the placebo group receiving only MTX. Two multivariate miRNA models were able to predict response to ADA treatment after 3 and 12 months, with 63% and 82% area under the ROC curves, respectively. CONCLUSION: We identified miR-27a-3p as a potential predictive biomarker of ACR/EULAR remission in patients with early RA treated with ADA in combination with MTX. We conclude that pretreatment plasma-miRNA profiles may be of predictive value, but the results need confirmation in independent cohorts.

A review of studies of the proteomes of circulating microparticles: key roles for galectin-3-binding protein-expressing microparticles in vascular diseases and systemic lupus erythematosus.

Subcellular microvesicles (MVs) have attracted increasing interest during the past decades. While initially considered as inert cellular debris, several important roles for MVs in physiological homeostasis, cancer, cardiovascular, and autoimmune diseases have been uncovered. Although still poorly understood, MVs are involved in trafficking of information from cell-to-cell, and are implicated in the regulation of immunity, thrombosis, and coagulation. Different subtypes of extracellular MVs exist. This review focuses on the cell membrane-derived shedded MVs (ranging in size from 200 to 1000 nm) typically termed microparticles (MPs). The numbers and particularly the composition of MPs appear to reflect the state of their parental cells and MPs may therefore carry great potential as clinical biomarkers which can be elucidated and developed by proteomics in particular. Determination of the identity of the specific proteins and their quantities, i.e. the proteome, in complex samples such as MPs enables an in-depth characterization of the phenotypical changes of the MPs during disease states. At present, only a limited number of proteomic studies of circulating MPs have been carried out in healthy individuals and disease populations. Interestingly, these studies indicate that a small set of MP-proteins, in particular, overexpression of galectin-3-binding protein (G3BP) distinguish MPs in patients with venous thromboembolism and the systemic autoimmune disease, systemic lupus erythematosus (SLE). G3BP is important in cell-cell adhesion, clearance, and intercellular signaling. MPs overexpressing G3BP may thus be involved in thrombosis and hemostasis, vascular inflammation, and autoimmunity, further favoring G3BP as a marker of "pathogenic" MPs. MPs expressing G3BP may also hold a potential as biomarkers in other conditions such as cancer and chronic viral infections. This review highlights the methodology and results of the proteome studies behind these discoveries and places them in a pathophysiological and biomarker perspective.

Circulating Extracellular microRNA in Systemic Autoimmunity.

MicroRNAs (miRNAs) are differentially regulated in healthy, activated, inflamed, neoplastic, or otherwise pathological cells and tissues. While their main functions are executed intracellularly, many miRNAs can reproducibly be detected extracellularly in plasma and serum. This circulating, extracellular miRNA is protected against degradation by complexation with carrier proteins and/or by being enclosed in subcellular membrane vesicles. This, together with their tissue- and disease-specific expression, has fuelled the interest in using circulating microRNA profiles as harbingers of disease, i.e., as diagnostic analytes and as clues to dysregulated pathways in disease. Many studies show that inflammation and immune dysregulation, e.g., in autoimmune diseases, are associated with distinct miRNA expression changes in targeted tissues and in innate and adaptive immunity cells such as lymphocytes, natural killer cells, neutrophil granulocytes, and monocyte-macrophages. Exploratory studies (only validated in a few cases) also show that specific profiles of circulating miRNAs are associated with different systemic autoimmune diseases including systemic lupus erythematosus (SLE), systemic sclerosis, and rheumatoid arthritis. Even though the link between cellular alterations and extracellular profiles is still unpredictable, the data suggest that circulating miRNAs in autoimmunity may become diagnostically useful. Here, we review important circulating miRNAs in animal models of inflammation and in systemic autoimmunity and summarize some proposed functions of miRNAs in immune regulation and dysregulation. We conclude that the studies suggest new hypotheses and additional experiments, and that further diagnostic development is highly dependent on analytical method development and on obtaining sufficient numbers of uniformly processed samples from clinically well-characterized patients and controls.

Genetic Variations in Pattern Recognition Receptor Loci Are Associated with Anti-TNF Response in Patients with Rheumatoid Arthritis.

OBJECTIVES: To determine whether genetic variation within genes related to the Toll-like receptor, inflammasome and interferon-γ pathways contributes to the differences in treatment response to tumour necrosis factor inhibitors (anti-TNF) in patients with rheumatoid arthritis (RA). METHODS: In a retrospective case-case study, we assessed 23 functional single nucleotide polymorphisms (SNPs) in 15 genes. We included 538 anti-TNF naïve Danish RA patients from the nationwide DANBIO database. Multivariable logistic regression analyses were performed to detect associations (p-value<0.05) between genotypes and European League Against Rheumatism (EULAR) treatment responses. False Discovery Rate corrections for multiple testing (q-value) and stratified analyses were performed to investigate association with individual therapies and IgM-rheumatoid factor (RF) status. RESULTS: Six of twenty successfully genotyped polymorphisms were nominally associated with EULAR treatment response. Three of these were in weak to moderate linkage disequilibrium with polymorphisms previously reported associated with anti-TNF treatment response. TLR5(rs5744174) variant allele carriers (odds ratio(OR) = 1.7(1.1-2.5),p = 0.010,q = 0.46) and TLR1(rs4833095) homozygous variant carriers (OR = 2.8(1.1-7.4),p = 0.037,q = 0.46) had higher odds for a positive treatment response. NLRP3(rs10754558) variant allele carriers (odds ratio(OR) = 0.6(0.4-1.0),p = 0.045,q = 0.46) were more likely to have a negative treatment response. The association in TLR5(rs5744174) remained significant after correction for multiple comparisons among patients negative for RF (OR = 6.2(2.4-16.3),p = 0.0002,q = 0.024). No other association withstood correction for multiple testing. Post hoc analyses showed that change in Patient Global score on a visual analogue scale (VAS) and change in pain VAS were the main factors responsible for the association. CONCLUSIONS: We reproduced previously reported associations between genetic variation in the TLR10/1/6 gene cluster, TLR5, and NLRP3 loci and response to anti-TNF treatment in RA. Changes in VAS pain and patient global scores were the main contributors to the association found for TLR5. Furthermore, we identified other candidate genes that require replication in independent cohorts.

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Anti-TNF treatment response in rheumatoid arthritis patients is associated with genetic variation in the NLRP3-inflammasome.

OBJECTIVE: Many patients with rheumatoid arthritis (RA) benefit from tumor necrosis factor-α blocking treatment (anti-TNF), but about one third do not respond. The objective of this study was to replicate and extend previously found associations between anti-TNF treatment response and genetic variation in the TNF-, NF-κB- and pattern recognition receptor signalling pathways. METHODS: Forty-one single nucleotide polymorphisms (SNPs), including 34 functional, in 28 genes involved in inflammatory pathways were assessed in 538 anti-TNF naive Danish RA patients with clinical data. Multivariable logistic regression analyses were performed to test associations between genotypes and treatment response at 3-6 months using the European League Against Rheumatism (EULAR) response criterion. American College of Rheumatology treatment response (ACR50) and relative change in 28-joint disease activity score (relDAS28) were used as secondary outcomes. Subgroup analyses were stratified according to smoking status, type of anti-TNF drug and IgM-Rheumatoid Factor (IgM-RF) status. False discovery rate (FDR) controlling was used to adjust for multiple testing. RESULTS: Statistically significant associations with EULAR response were found for two SNPs in NLRP3(rs4612666) (OR (odds ratio) for good/moderate response = 0.64 (95% confidence interval: 0.44-0.95), p = 0.025, q = 0.95) and INFG(rs2430561) (OR = 0.40 (0.21-0.76), p = 0.005, q = 0.18) and among IgM-RF positive patients for TNFRS1A(rs4149570) (0.59 (0.36-0.98), p = 0.040, q = 0.76). Current smokers who carried the NLRP3(rs4612666) variant allele were less likely to benefit from anti-TNF treatment (OR = 0.24 (0.10-0.56), p = 0.001, q = 0.04). CONCLUSIONS: In a population of Danish RA patients, we confirm the NLRP3 gene as associated with EULAR anti-TNF response as previously reported. The NLRP3 variant (T) allele is associated with lower treatment response, in particular among current smokers. Furthermore, we find that a functional polymorphism in the interferon-γ gene is associated with anti-TNF response. All findings should be tested by replication in independent validation cohorts and augmented by assessing cytokine levels and activities of the relevant gene products.

Cell-free plasma microRNA in pancreatic ductal adenocarcinoma and disease controls.

OBJECTIVES: There are no tumor-specific biochemical markers for pancreatic ductal adenocarcinoma (PDAC). Tissue-specific gene expression including microRNA (miRNA) profiling, however, identifies specific PDAC signatures. This study evaluates associations between circulating, cell-free plasma-miRNA profiles and PDAC in a disease and disease-control cohort. METHODS: We performed a microarray profiling of 847 different mature miRNAs from plasma in an exploratory cohort of 20 patients with PDAC or other pancreatic diseases, profiling of 45 miRNAs in plasma samples from PDAC (n = 48) and disease controls (n = 47), and evaluation of associations of data with diagnosis, survival, and CA-19-9. RESULTS: We find 7 significantly deregulated miRNAs in PDAC using univariate statistics. At a false-discovery rate of 5%, miRNA-375 remained significantly elevated in PDAC. MicroRNA-375 did not improve diagnosis of PDAC in this cohort (70% accuracy) and did not correlate with survival. However, 3 controls (other gastrointestinal cancers) with increased CA-19-9 did not show increased miRNA-375. CONCLUSIONS: In the plasma-miRNA population, we find miRNA-375, which is selectively expressed in the endocrine pancreas under normal conditions, increased in PDAC cases compared with patients with other pancreatic or gastrointestinal diseases. The miRNA-375 does not outperform CA-19-9 diagnostically in the present cohort. However, it shows promising specificity and should be examined in larger prospective studies.

Neprilysin-like activity correlates with CSF-Tau and phospho-tau in patients with Alzheimer's disease.

A relation between amyloid-ß peptide (Aß) accumulation and neprilysin (NEP), an Aß degrading enzyme, has been proposed but studies of NEP and the levels of the pathological hallmarks of Alzheimer's disease (AD), Aß and tau, in cerebrospinal fluid (CSF) are scarce. In this study, we measured the level and enzyme activity of NEP in serum and CSF, using a sandwich enzyme-linked immunosorbent assay and a fluorescence resonance energy transfer assay, respectively, in patients with AD, frontotemporal dementia (FTD), Creutzfeldt-Jakob disease (CJD), and depression. Results were correlated with the levels of CSF AD biomarkers Aß42, hyperphosphorylated tau (p-tau), and total tau (t-tau). In serum, we found no differences in NEP-like activity or concentration between the groups and there were no correlations between NEP and AD biomarkers. In CSF, no influence of age or gender on NEP levels or enzyme activity was seen. However, NEP concentration was lower and the specific activity was higher in FTD compared to AD. Aß42 levels in CSF did not correlate with NEP concentration or activity in the AD, CJD, or depression groups, but NEP-like activity and Aß42 levels correlated significantly in the FTD group. In AD and depression, the NEP-like activity in CSF correlated with levels of p-tau, and, in the AD group, it also was correlated with t-tau levels. Our results suggest that the relation between the specific activity of NEP and t-tau and p-tau is a characteristic trait of AD. The correlation between NEP concentration and Aß42 in FTD is unexpected and warrants further investigation.

Pre-analytical factors influencing the stability of cerebrospinal fluid proteins.

Cerebrospinal fluid (CSF) is a potential source for new biomarkers due to its proximity to the brain. This study aimed to clarify the stability of the CSF proteome when undergoing pre-analytical factors. We investigated the effects of repeated freeze/thaw cycles, protease inhibitors and delayed storage for 4h, 24h or 14 days at -20°C, 4°C and room temperature (RT) after centrifugation compared with our standard practice of two hours at RT before placing the samples in an -80°C environment. The results were obtained using immunoassays for amyloid-beta 1-42 (Aß42), tau, phosphorylated tau (P-tau) and cystatin C and using surface-enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) mass spectrometry for proteomic profiling. Tau and P-tau were susceptible to repeated freeze/thaw cycles while SELDI-TOF analysis produced eight significant peaks and additional artefact peaks from samples with added protease inhibitors. Delayed storage for different durations and in different temperatures produced six significant SELDI-TOF peaks. Aß42 and tau were susceptible to increased temperatures and the duration before storage, whereas P-tau and cystatin C were not. Transthyretin and several of its isoforms were found using SELDI-TOF and were susceptible to freeze/thaw cycles and to increased temperature and length of time prior to storage. We recommend that CSF should be collected and centrifuged immediately after sampling and prior to storage at -80°C without the addition of protease inhibitors. Freeze/thawing should be avoided because of the instability of tau, P-tau and transthyretin. Standardised CSF sampling, handling and storage for biomarker research are essential for accurately comparing the results obtained by different studies and institutions.

ß(2)-microglobulin amyloidosis.

Dialysis-related amyloidosis (DRA) is a clinical syndrome of pain, loss of function and other symptoms due to the deposition of amyloid consisting of ß(2)-microglobulin (ß(2)m) in the musculoskeletal system. The condition is seen in patients who suffer from chronic kidney disease and are treated with hemodialysis for a long time. Even though ß(2)m easily can be manipulated to form amyloid in laboratory experiments under non-physiological conditions the precise mechanisms involved in the formation of ß(2)m-amyloid in patients with DRA have been difficult to unravel. The current knowledge which is reviewed here indicates that conformational fluctuations centered around the D-strand, the DE-loop, and around the cis-configured Pro32 peptide bond are involved in ß(2)m amyloidosis. Also required are highly increased concentrations of circulating ß(2)m and possibly various post-translational modifications mediated by the pro-inflammatory environment in uremic blood, together with the influence of divalent metal ions (specifically Cu(2 +)), uremic toxins, and dialysis-enhanced redox-processes. It seems plausible that domain-swapped ß(2)m dimers act as building blocks of ß-spine cross-ß -sheet fibrils consisting of otherwise globular, roughly natively folded protein. An activated complement system and cellular activation perpetuate these reactions which due to the affinity of ß(2)m-amyloid for the collagen of synovial surfaces result in the DRA syndrome.

MicroRNAs in liver disease.

MicroRNAs are small noncoding RNA molecules that regulate gene expression posttranscriptionally through complementary base pairing with thousands of messenger RNAs. They regulate diverse physiological, developmental, and pathophysiological processes. Recent studies have uncovered the contribution of microRNAs to the pathogenesis of many human diseases, including liver diseases. Moreover, microRNAs have been identified as biomarkers that can often be detected in the systemic circulation. We review the role of microRNAs in liver physiology and pathophysiology, focusing on viral hepatitis, liver fibrosis, and cancer. We also discuss microRNAs as diagnostic and prognostic markers and microRNA-based therapeutic approaches for liver disease.

Circulating antinuclear antibodies in patients with pelvic masses are associated with malignancy and decreased survival.

BACKGROUND: Circulating autoantibodies occur more frequently in cancer patients than in patients without cancer. METHODS AND FINDINGS: We examined sera from patients referred for pelvic mass symptoms to a tertiary university clinic. A total of 127 were diagnosed with epithelial ovarian cancer while 386 had a benign condition. A screen for IgG anti-nuclear antibodies (ANA) by indirect immunofluorescence on HEp-2 cells confirmed a highly significant overrepresentation of ANA in the cancer group where 40% had detectable (i.e., a titer ≥160) ANA compared with less than 12% in the benign group. The overrepresentation of ANA in the cancer group persisted (p<0.0001) after matching the age-profile of the benign group with the ovarian cancer group. Only 19 out of 127 patients in the age-matched benign subgroup were positive for ANA corresponding to an 85% specificity at 40% sensitivity of ANA as the only marker for malignancy. No correlation of ANA positivity in either group with specific bands in immunoblots could be demonstrated even though immunoblot positivity was clearly increased in the malignant group (41% vs. 3%). The presence, strength, and type of ANA did not correlate with serum CA-125 values or with staging, and ANA outcome did not contribute with independent diagnostic information. However, survival was significantly shorter in ANA-positive compared with ANA-negative cancer patients and patients with CA-125 below the median CA-125 value in the cancer group had a significantly decreased survival when positive for ANA. ANA status made no difference in the group with CA-125 values above the median. Also, there was a significant correlation between speckled ANA-strength and histological tumor grade. CONCLUSIONS: Circulating antibodies are a promising source for new biomarkers in cancer. Characterization of epitope specificities and measurements of consecutive samples will be important for further elucidating the role of ANA in evaluating ovarian cancer patients.

Characterization and stability of transthyretin isoforms in cerebrospinal fluid examined by immunoprecipitation and high-resolution mass spectrometry of intact protein.

Post-translational modifications (PTMs) contribute significantly to the complexity of proteins. PTMs may vary in certain patterns according to diseases and microenviroments making them potential markers for pathological processes. Human transthyretin (TTR) is a transporter of thyroxine and retinol in blood and cerebrospinal fluid (CSF). A single free cysteine thiol group in TTR possesses the ability to form mixed disulfides potentially related to diseases such as TTR amyloidosis and Alzheimer's disease (AD). Additionally, TTR-Cys10 S-thiolations might mirror the oxidative stress and redox balance of CSF. Here we describe a quick and gentle method for immunoprecipitating (IP) TTR from CSF with minimal introduction of sample-handling artifacts. A high-resolution mass spectrometer (LTQ-Orbitrap XL) was used in a simple setup with direct infusion that generates data suitable for confident assignment of TTR isoforms and validation of the protocol. Moreover, we demonstrate how simple storage of CSF at 4°C induces major oxidative modifications of TTR. Using the optimized method, we show data from a limited number of mild cognitive impairment (MCI) and AD patients. The protocol controls and minimizes the introduction of sample-handling artifacts during purification of TTR isoforms for high-resolution MS analysis.

Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer.

Circulating micro-RNA (miR) profiles have been proposed as promising diagnostic and prognostic biomarkers for cancer, including lung cancer. We have developed methods to accurately and reproducibly measure micro-RNA levels in serum and plasma. Here, we study paired serum and plasma samples from 220 patients with early stage nonsmall cell lung cancer (NSCLC) and 220 matched controls. We use qRT-PCR to measure the circulating levels of 30 different miRs that have previously been reported to be differently expressed in lung cancer tissue. Duplicate RNA extractions were performed for 10% of all samples, and micro-RNA measurements were highly correlated among those duplicates. This demonstrates high reproducibility of our assay. The expressions of miR-146b, miR-221, let-7a, miR-155, miR-17-5p, miR-27a and miR-106a were significantly reduced in the serum of NSCLC cases, while miR-29c was significantly increased. No significant differences were observed in plasma of patients compared with controls. Overall, expression levels in serum did not correlate well with levels in plasma. In secondary analyses, reduced plasma expression of let-7b was modestly associated with worse cancer-specific mortality in all patients, and reduced serum expression of miR-223 was modestly associated with cancer-specific mortality in stage IA/B patients. MiR profiles also showed considerable differences comparing African American and European Americans. In summary, we found significant differences in miR expression when comparing cases and controls and find evidence that expression of let-7b is associated with prognosis in NSCLC.