RNA Profiles of Tear Fluid Extracellular Vesicles in Patients with Dry Eye-Related Symptoms.

Currently, diagnosing and stratifying dry eye disease (DED) require multiple tests, motivating interest in a single definitive test. The purpose of this study was to investigate the potential for using tear fluid extracellular vesicle (EV)-RNA in DED diagnostics. With a role in intercellular communication, nanosized EVs facilitate the protected transport of diverse bioactive molecules in biofluids, including tears. Schirmer strips were used to collect tears from 10 patients presenting with dry eye-related symptoms at the Norwegian Dry Eye Clinic. The samples comprised two groups, five from patients with a tear film break-up time (TBUT) of 2 s and five from patients with a TBUT of 10 s. Tear fluid EV-RNA was isolated using a Qiagen exoRNeasy Midi Kit, and the RNA was characterized using Affymetrix ClariomTM D microarrays. The mean signal values of the two groups were compared using a one-way ANOVA. A total of 26,639 different RNA transcripts were identified, comprising both mRNA and ncRNA subtypes. Approximately 6% of transcripts showed statistically significant differential abundance between the two groups. The mRNA sodium channel modifier 1 (SCNM1) was detected at a level 3.8 times lower, and the immature microRNA-130b was detected at a level 1.5 times higher in the group with TBUT 2 s compared to the group with TBUT 10 s. This study demonstrates the potential for using tear fluid EV-RNA in DED diagnostics.

Non-Coding RNA in Salivary Extracellular Vesicles: A New Frontier in Sjögren's Syndrome Diagnostics?

Sjögren's syndrome is an autoimmune rheumatic disease characterized by inflammation of the salivary and lacrimal glands, often manifesting as dry mouth and dry eyes. To simplify diagnostics of primary Sjögren's syndrome (pSS), a non-invasive marker is needed. The aim of the study was to compare the RNA content of salivary extracellular vesicles (EVs) between patients with pSS and healthy controls using microarray technology. Stimulated whole saliva was collected from 11 pSS patients and 11 age-matched controls. EV-RNA was isolated from the saliva samples using a Qiagen exoRNeasy Midi Kit and analyzed using Affymetrix Clariom D™ microarrays. A one-way ANOVA test was used to compare the mean signal values of each transcript between the two groups. A total of 9307 transcripts, coding and non-coding RNA, were detected in all samples. Of these transcripts, 1475 showed statistically significant differential abundance between the pSS and the control groups, generating two distinct EV-RNA patterns. In particular, tRNAs were downregulated in pSS patients, with the transcript tRNA-Ile-AAT-2-1 showing a 2-fold difference, and a promise as a potential biomarker candidate. This study therein demonstrates the potential for using salivary EV-RNA in pSS diagnostics.

Global miRNA expression analysis of serous and clear cell ovarian carcinomas identifies differentially expressed miRNAs including miR-200c-3p as a prognostic marker.

BACKGROUND: Improved insight into the molecular characteristics of the different ovarian cancer subgroups is needed for developing a more individualized and optimized treatment regimen. The aim of this study was to a) identify differentially expressed miRNAs in high-grade serous ovarian carcinoma (HGSC), clear cell ovarian carcinoma (CCC) and ovarian surface epithelium (OSE), b) evaluate selected miRNAs for association with clinical parameters including survival and c) map miRNA-mRNA interactions. METHODS: Differences in miRNA expression between HGSC, CCC and OSE were analyzed by global miRNA expression profiling (Affymetrix GeneChip miRNA 2.0 Arrays, n = 12, 9 and 9, respectively), validated by RT-qPCR (n = 35, 19 and 9, respectively), and evaluated for associations with clinical parameters. For HGSC, differentially expressed miRNAs were linked to differentially expressed mRNAs identified previously. RESULTS: Differentially expressed miRNAs (n = 78) between HGSC, CCC and OSE were identified (FDR < 0.01%), of which 18 were validated (p < 0.01) using RT-qPCR in an extended cohort. Compared with OSE, miR-205-5p was the most overexpressed miRNA in HGSC. miR-200 family members and miR-182-5p were the most overexpressed in HGSC and CCC compared with OSE, whereas miR-383 was the most underexpressed. miR-205-5p and miR-200 members target epithelial-mesenchymal transition (EMT) regulators, apparently being important in tumor progression. miR-509-3-5p, miR-509-5p, miR-509-3p and miR-510 were among the strongest differentiators between HGSC and CCC, all being significantly overexpressed in CCC compared with HGSC. High miR-200c-3p expression was associated with poor progression-free (p = 0.031) and overall (p = 0.026) survival in HGSC patients. Interacting miRNA and mRNA targets, including those of a TP53-related pathway presented previously, were identified in HGSC. CONCLUSIONS: Several miRNAs differentially expressed between HGSC, CCC and OSE have been identified, suggesting a carcinogenetic role for these miRNAs. miR-200 family members, targeting EMT drivers, were mostly overexpressed in both subgroups, among which miR-200c-3p was associated with survival in HGSC patients. A set of miRNAs differentiates CCC from HGSC, of which miR-509-3-5p and miR-509-5p are the strongest classifiers. Several interactions between miRNAs and mRNAs in HGSC were mapped.

Distinct microRNA and protein profiles of extracellular vesicles secreted from myotubes from morbidly obese donors with type 2 diabetes in response to electrical pulse stimulation.

Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from six morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these four were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions. In conclusion, we have used a combined transcriptome-proteome approach to describe how EPS affected the cargo of EVs derived from myotubes from morbidly obese patients with T2D, and revealed several new factors, both miRs and proteins, that might act as exercise factors.

Long-term occupational outcomes of endotoxin exposure and the effect of exposure cessation.

OBJECTIVE: To study the possible respiratory and haematological effects of endotoxin exposure to bacterial single-cell protein (BSCP) in workers during a follow-up period of 5 years including 4 years of exposure and 1 year without exposure. METHODS: The study included 28 workers examined in 2002-2005 and 1 year after exposure termination in 2007. The arithmetic mean endotoxin exposure was 5800-11,000 EU/m(3) among the high exposure group and 390 EU/m(3) in the low exposure group. Assessment of lung function included spirometry and gas diffusion in 2003, 2004 and 2007. Rhinometry was performed in 2004 and 2007. Blood analysis included leukocyte cell count and measurement of the acute phase proteins: C-reactive protein, interleukin-6, eosinophilic cationic protein, macrophage inflammatory protein-1α, monocyte chemoattractant protein-1, chemoattractant protein RANTES, platelet-derived growth factor BB, fibrinogen and D-dimer. RESULTS: In the low exposure group, but not in the high exposure group, there were significant improvements in both forced vital capacity (FVC) (290 ml) and forced expiratory volume in 1 s (FEV(1)) (180-210 ml) (p=0.004-0.03) 1 year after the end of exposure. The number of leukocytes and eosinophilic cationic protein and D-dimer levels increased significantly with increasing endotoxin exposures and decreased significantly 1 year after exposure termination. Changes in acute phase proteins suggested exposure-related tolerance. CONCLUSIONS: An inflammatory tendency during an exposure period of 4 years seems to reverse 1 year after cessation of exposure to endotoxins from a single species. Lung function improved significantly among workers exposed to low levels of endotoxin but not among the highly exposed workers.

Genetic variation of VKORC1 and CYP4F2 genes related to warfarin maintenance dose in patients with myocardial infarction.

The aim of this study was to investigate whether the VKORC1*3 (rs7294/9041 G > A), VKORC1*4 (rs17708472/6009 C > T), and CYP4F2 (rs2108622/1347 C > T) polymorphisms were associated with elevated warfarin maintenance dose requirements in patients with myocardial infarction (n = 105) from the Warfarin Aspirin Reinfarction Study (WARIS-II). We found significant associations between elevated warfarin dose requirements and VKORC1*3 and VKORC1*4 polymorphisms (P = .001 and P = .004, resp.), whereas CYP4F2 (1347 C > T) showed a weak association on higher warfarin dose requirements (P = .09). However, analysing these variant alleles in a regression analysis together with our previously reported data on VKORC1*2, CYP2C9*2 and CYP2C9*3 polymorphisms, gave no significant associations for neither VKORC1*3, VKORC1*4 nor CYP4F2 (1347 C > T). In conclusion, in patients with myocardial infarction, the individual contribution to warfarin dose requirements from VKORC1*3, VKORC1*4, and CYP4F2 (1347 C > T) polymorphisms was negligible. Our results indicate that pharmacogenetic testing for VKORC1*2, CYP2C9*2 and CYP2C9*3 is more informative regarding warfarin dose requirements than testing for VKORC1*3, VKORC1*4, and CYP4F2 (1347 C > T) polymorphisms.

Uptake of circulating extracellular vesicles from rectal cancer patients and differential responses by human monocyte cultures.

Extracellular vesicles (EVs) released by tumor cells can directly or indirectly modulate the phenotype and function of the immune cells of the microenvironment locally or at distant sites. The uptake of circulating EVs and the responses by human monocytes in vitro may provide new insights into the underlying biology of the invasive and metastatic processes in cancer. Although a mixed population of vesicles is obtained with most isolation techniques, we predominantly isolated exosomes (small EVs) and microvesicles (medium EVs) from the SW480 colorectal cancer cell line (established from a primary adenocarcinoma of the colon) by sequential centrifugation and ultrafiltration, and plasma EVs were prepared from 22 patients with rectal adenoma polyps or invasive adenocarcinoma by size-exclusion chromatography. The EVs were thoroughly characterized. The uptake of SW480 EVs was analyzed, and small SW480 EVs were observed to be more potent than medium SW480 EVs in inducing monocyte secretion of cytokines. The plasma EVs were also internalized by monocytes; however, their cytokine-releasing potency was lower than that of the cell line-derived vesicles. The transcriptional changes in the monocytes highlighted differences between adenoma and adenocarcinoma patient EVs in their ability to regulate biological functions, whereas the most intriguing changes were found in monocytes receiving EVs from patients with metastatic compared with localized cancer.

No Association Between Risk of Anterior Cruciate Ligament Rupture and Selected Candidate Collagen Gene Variants in Female Elite Athletes From High-Risk Team Sports.

BACKGROUND: Several single-nucleotide variants (SNVs) in collagen genes have been reported as predisposing factors for anterior cruciate ligament (ACL) tears. However, the evidence is conflicting and does not support a clear association between genetic variants and risk of ACL ruptures. PURPOSE: To assess the association of previously identified candidate SNVs in genes encoding for collagen and the risk of ACL injury in a population of elite female athletes from high-risk team sports. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: A total of 851 female Norwegian and Finnish elite athletes from team sports were included from 2007 to 2011. ACL injuries acquired before inclusion in the cohort were registered by interview. The participants were followed prospectively through 2015 to record new complete ACL injuries. Six selected SNVs were genotyped ( COL1A1: rs1800012, rs1107946; COL3A1: rs1800255; COL5A1: rs12722, rs13946; COL12A1: rs970547). RESULTS: No associations were found between ACL rupture and the SNVs tested. CONCLUSION: The study does not support a role of the 6 selected SNVs in genes encoding for collagen proteins as risk factors for ACL injury. CLINICAL RELEVANCE: Genetic profiling to identify athletes at high risk for ACL rupture is not yet feasible.

Effect of CYP4F2, VKORC1, and CYP2C9 in Influencing Coumarin Dose: A Single-Patient Data Meta-Analysis in More Than 15,000 Individuals.

The cytochrome P450 (CYP)4F2 gene is known to influence mean coumarin dose. The aim of the present study was to undertake a meta-analysis at the individual patients level to capture the possible effect of ethnicity, gene-gene interaction, or other drugs on the association and to verify if inclusion of CYP4F2*3 variant into dosing algorithms improves the prediction of mean coumarin dose. We asked the authors of our previous meta-analysis (30 articles) and of 38 new articles retrieved by a systematic review to send us individual patients' data. The final collection consists of 15,754 patients split into a derivation and validation cohort. The CYP4F2*3 polymorphism was consistently associated with an increase in mean coumarin dose (+9% (95% confidence interval (CI) 7-10%), with a higher effect in women, in patients taking acenocoumarol, and in white patients. The inclusion of the CYP4F2*3 in dosing algorithms slightly improved the prediction of stable coumarin dose. New pharmacogenetic equations potentially useful for clinical practice were derived.

Identification of genes particularly sensitive to lipopolysaccharide (LPS) in human monocytes induced by wild-type versus LPS-deficient Neisseria meningitidis strains.

Lipopolysaccharide (LPS) in the outer membrane of Neisseria meningitidis plays a dominant role as an inflammation-inducing molecule in meningococcal disease. We have used microarray analysis to study the global gene expression after exposure of human monocytes for 3 h to wild-type N. meningitidis (10(6)), LPS-deficient N. meningitidis (10(6) and 10(8)), and purified N. meningitidis LPS (1 ng [33 endotoxin units]/ml) to identify LPS-inducible genes. Wild-type N. meningitidis (10(6)) induced 4,689 differentially expressed genes, compared with 72 differentially expressed genes induced by 10(6) LPS-deficient N. meningitidis organisms. However, 10(8) LPS-deficient N. meningitidis organisms induced 3,905 genes, indicating a dose-response behavior of non-LPS cell wall molecules. A comparison of the gene expression patterns from 10(6) wild-type N. meningitidis and 10(8) LPS-deficient N. meningitidis organisms showed that 2,401 genes in human monocytes were not strictly LPS dependent. A list of "particularly LPS-sensitive" genes (2,288), differentially induced by 10(6) wild-type N. meningitidis but not by 10(8) LPS-deficient N. meningitidis organisms, showed an early expression of beta interferon (IFN-beta), most likely through the Toll-like receptor-MyD88-independent pathway. Subsequently, IFN-beta may activate the type I IFN signaling pathway, and an unknown number of IFN-beta-inducible genes, such as those for CXCL9, CXCL10, CXCL11, IFIT1, IFIT2, IFIT3, and IFIT5, are transcribed. Supporting this, human monocytes secreted significantly higher levels of CXCL10 and CXCL11 when stimulated by 10(6) wild-type N. meningitidis organisms than when stimulated by 10(8) LPS-deficient N. meningitidis organisms. Plasma CXCL10, but not CXCL11, was positively correlated (r = 0.67; P < 0.01) to LPS in patients (n = 24) with systemic meningococcal disease. Thus, new circulating biomarkers in meningococcal disease may be suggested through LPS-induced gene expression changes in human monocytes.

The human ABC transporter pseudogene family: Evidence for transcription and gene-pseudogene interference.

BACKGROUND: Pseudogenes are an integral component of the human genome. Little attention, however, has so far been paid to the phenomenon that some pseudogenes are transcriptionally active. Recently, we demonstrated that the human ortholog of the rodent testis-specific ATP-binding cassette (ABC) transporter Abca17 is a ubiquitously transcribed pseudogene (ABCA17P). The aim of the present study was to establish a complete inventory of all ABC transporter pseudogenes in the human genome and to identify transcriptionally active ABC transporter pseudogenes. Moreover, we tested the hypothesis that a regulatory interdependency exists between ABC transporter pseudogenes and their parental protein coding equivalents. RESULTS: Systematic bioinformatic analysis revealed the existence of 22 ABC transporter pseudogenes within the human genome. We identified two clusters on chromosomes 15 and 16, respectively, which harbor almost half of all pseudogenes (n = 10). Available information from EST and mRNA databases and RT-PCR expression profiling indicate that a large portion of the ABC transporter pseudogenes (45%, n = 10) are transcriptionally active and some of them are expressed as alternative splice variants. We demonstrate that both pseudogenes of the pseudoxanthoma elasticum gene ABCC6, ABCC6P1 and ABCC6P2, are transcribed. ABCC6P1 and ABCC6 possess near-identical promoter sequences and their tissue-specific expression profiles are strikingly similar raising the possibility that they form a gene-pseudogene dual transcription unit. Intriguingly, targeted knockdown of the transcribed pseudogene ABCC6P1 resulted in a significant reduction of ABCC6 mRNA expression levels. CONCLUSION: The human genome contains a surprisingly small number of ABC transporter pseudogenes relative to other known gene families. They are unevenly distributed across the chromosomes. Importantly, a significant portion of the ABC transporter pseudogenes is transcriptionally active. The downregulation of ABCC6 mRNA levels by targeted suppression of the expression of its pseudogene ABCC6P1 provides evidence, for the first time, for a regulatory interdependence of a transcribed pseudogene and its protein coding counterpart in the human genome.

Warfarin dose and INR related to genotypes of CYP2C9 and VKORC1 in patients with myocardial infarction.

BACKGROUND: Warfarin treatment has a narrow therapeutic range, requiring meticulous monitoring and dosage titration. Individual dosage requirement has recently partly been explained by genetic variation of the warfarin metabolizing enzyme CYP2C9 and the Vitamin K-activating enzyme VKORC1. In the WARIS-II study, comparing three different antithrombotic regimens after myocardial infarction, warfarin treatment reduced thrombotic events, but was associated with more frequent bleeding than use of acetylsalisylic acid (ASA) alone. AIMS: The primary aim of the present study was to investigate the relation between genotypes of CYP2C9 and VKORC1 and warfarin maintenance dose in myocardial infarction. The secondary aim was to relate the genotypes to international normalized ratio (INR). METHODS: Genotyping was performed in 212 myocardial infarction patients from the WARIS-II study by robotic isolation of DNA from EDTA whole blood (MagNa Pure LC) before PCR amplification (LightCycler) and melting point analysis. RESULTS: The 420 C>T substitution of CYP2C9*2, the 1075 A>C substitution of CYP2C9*3 and the 1173 C>T substitution of VKORC1 had minor allele frequencies of, 11.3%, 5.7% and 36.6% respectively. Warfarin weekly dose varied between 17 mg and 74 mg among the patients. INR did not vary between genotypes. Warfarin dosage requirement was significantly associated with CYP2C9 and VKORC1 genotypes, treatment group and age. The VKORC1 genotype contributed 24.5% to the interindividual variation in warfarin dosage, whereas the combined CYP2C9 genotypes were only responsible for 7.2% of the dose variation. CONCLUSION: CYP2C9 and VKORC1 genotype frequencies in myocardial infarction patients appear similar to other patient groups and have similar impact on warfarin maintenance dose.

The factor V Leiden, prothrombin gene 20210GA, methylenetetrahydrofolate reductase 677CT and platelet glycoprotein IIIa 1565TC mutations in patients with acute ischemic stroke and atrial fibrillation.

BACKGROUND AND PURPOSE: We wanted to investigate whether common prothrombotic mutations are more prevalent in patients with atrial fibrillation who have had a stroke than in healthy controls. We also wanted to assess whether early recurrent ischemic cerebrovascular events were more frequent among carriers of the factor V Leiden or the prothrombin gene mutations than among others. METHODS: We used a case-control design with 367 patients with acute ischemic stroke and atrial fibrillation (cases) and 482 healthy blood donors (controls). All mutations were detected with conventional polymerase-chain reaction protocols. RESULTS: The odds ratios for carriers of the factor V Leiden, prothrombin gene 20210GA, methylenetetrahydrofolate reductase 677CT, or platelet glycoprotein IIIa 1565TC (Pl(A2)) mutation were 0.91, (95% CI, 0.51 to 1.59), 2.25 (95% CI, 0.61 to 8.90), 0.83 (0.61 to 1.13), and 0.79 (0.57 to 1.10), respectively. Early recurrent ischemic stroke and total recurrent ischemic cerebrovascular events were slightly more frequent among carriers of the factor V Leiden mutation than among noncarriers: odds ratio 1.45 (95% CI, 0.41 to 5.1), and 1.59 (0.61 to 4.1), respectively. None of the patients with recurrent ischemic cerebrovascular events had the prothrombin gene mutation. CONCLUSIONS: These mutations are not important risk factors for thromboembolic stroke associated with atrial fibrillation. Carriers of the factor V Leiden mutation had a small, nonsignificantly higher risk of early recurrent ischemic cerebrovascular events.

[ABCA-transporters: regulators of cellular lipid transport]. / ABCA-transportører - regulatorer av den cellulaere lipidtransporten.

BACKGROUND: The transport of lipids, which is orchestrated by a multitude of molecular factors, is a key feature of the physiology of living cells. A new group of transporter protein, the A-subclass of ATP-binding cassette (ABC) transporters, was recently discovered. ABCA-transporters play pivotal roles in cellular lipid transport and their discovery has brought important new insights into the molecular basis of this process. This review article presents the biology of ABCA-transport proteins and their implication for clinical medicine. MATERIAL AND METHODS: Literature retrieved from Pubmed, including own research results, formed the basis for the article. RESULTS AND INTERPRETATION: Mutations in ABCA-transporter genes have been shown to result in hereditary diseases involving major physiologicical processes in the cardiovascular, respiratory, visual and integumentary systems. Accumulated evidence suggests that ABCA-transporters play critical roles in the pathogenesis of complex multifactorial disorders with a high incidence; such as atherosclerosis, age-related macula degeneration and Alzheimer's disease.

The human ortholog of the rodent testis-specific ABC transporter Abca17 is a ubiquitously expressed pseudogene (ABCA17P) and shares a common 5' end with ABCA3.

BACKGROUND: During the past years, we and others discovered a series of human ATP-binding cassette (ABC) transporters, now referred to as ABC A-subfamily transporters. Recently, a novel testis-specific ABC A transporter, Abca17, has been cloned in rodent. In this study, we report the identification and characterization of the human ortholog of rodent Abca17. RESULTS: The novel human ABC A-transporter gene on chromosome 16p13.3 is ubiquitously expressed with highest expression in glandular tissues and the heart. The new ABC transporter gene exhibits striking nucleotide sequence homology with the recently cloned mouse (58%) and rat Abca17 (51%), respectively, and is located in the syntenic region of mouse Abca17 indicating that it represents the human ortholog of rodent Abca17. However, unlike in the mouse, the full-length ABCA17 transcript (4.3 kb) contains numerous mutations that preclude its translation into a bona fide ABC transporter protein strongly suggesting that the human ABCA17 gene is a transcribed pseudogene (ABCA17P). We identified numerous alternative ABCA17P splice variants which are transcribed from two distinct transcription initiation sites. Genomic analysis revealed that ABCA17P borders on another ABC A-subfamily transporter - the lung surfactant deficiency gene ABCA3. Surprisingly, we found that both genes overlap at their first exons and are transcribed from opposite strands. This genomic colocalization and the observation that the ABCA17P and ABCA3 genes share significant homologies in several exons (up to 98%) suggest that both genes have evolved by gene duplication. CONCLUSION: Our results demonstrate that ABCA17P and ABCA3 form a complex of overlapping genes in the human genome from which both non-coding and protein-coding ABC A-transporter RNAs are expressed. The fact that both genes overlap at their 5' ends suggests interdependencies in their regulation and may have important implications for the functional analysis of the disease gene ABCA3. Moreover, this is the first demonstration of the expression of a pseudogene and its parent gene from a common overlapping DNA region in the human genome.

LPS from Neisseria meningitidis is crucial for inducing monocyte- and microparticle-associated tissue factor activity but not for tissue factor expression.

Neisseria meningitidis causes sepsis with coagulopathy. The present study evaluated the tissue factor (TF)-inducing capacity of bacterial LPS in different presentation forms, i.e. membrane-bound LPS versus purified LPS, and of non-LPS components of N. meningitidis. By using a wild-type N. meningitidis, a mutant N. meningitidis lacking LPS (LPS-deficient N. meningitidis), purified LPS from N. meningitidis and Escherichia coli, we measured TF-expression and TF-activity on human monocytes and microparticles (MPs). The effect of TF-modulators, such as phosphatidylserine (PS), tissue factor pathway inhibitor (TFPI) and recombinant IL-10 (rhIL-10) was investigated. In plasmas from meningococcal patients, fibrinopeptide A (FPA), LPS and IL-10 were quantified. Monocytes and MPs exposed to purified LPS or wild-type N. meningitidis had much higher TF-activity than monocytes and MPs exposed to LPS-deficient N. meningitidis (clot formation assay). Incubation with wild-type N. meningitidis, but also LPS-deficient N. meningitidis, resulted in TF-expression on monocytes (flow cytometry, qRT-PCR). Increased cellular TF-activity is associated with coincident surface-exposure of PS and the number of monocytes positive for both PS and TF was significantly higher for monocytes exposed to wild-type N. meningitidis (7.6%) compared with monocytes exposed to LPS-deficient N. meningitidis (1.8%). Treatment with rhIL-10 reduced monocyte- and MP-associated TF-activity, the number of monocytes positive for both TF and PS, and microvesiculation. Patients with meningococcal septicemia had significantly higher levels of LPS, FPA and IL-10 than patients with distinct meningitis. Our results indicate that LPS from N. meningitidis is crucial for inducing TF-activity, but not for monocyte- and MP-associated TF-expression. TF-activity seems to require coincident expression of TF and PS on monocytes, and LPS induces such double-positive monocytes.

POLD2 and KSP37 (FGFBP2) correlate strongly with histology, stage and outcome in ovarian carcinomas.

BACKGROUND: Epithelial ovarian cancer (EOC) constitutes more than 90% of ovarian cancers and is associated with high mortality. EOC comprises a heterogeneous group of tumours, and the causes and molecular pathology are essentially unknown. Improved insight into the molecular characteristics of the different subgroups of EOC is urgently needed, and should eventually lead to earlier diagnosis as well as more individualized and effective treatments. Previously, we reported a limited number of mRNAs strongly upregulated in human osteosarcomas and other malignancies, and six were selected to be tested for a possible association with three subgroups of ovarian carcinomas and clinical parameters. METHODOLOGY/PRINCIPAL FINDINGS: The six selected mRNAs were quantified by RT-qPCR in biopsies from eleven poorly differentiated serous carcinomas (PDSC, stage III-IV), twelve moderately differentiated serous carcinomas (MDSC, stage III-IV) and eight clear cell carcinomas (CCC, stage I-IV) of the ovary. Superficial scrapings from six normal ovaries (SNO), as well as biopsies from three normal ovaries (BNO) and three benign ovarian cysts (BBOC) were analyzed for comparison. The gene expression level was related to the histological and clinical parameters of human ovarian carcinoma samples. One of the mRNAs, DNA polymerase delta 2 small subunit (POLD2), was increased in average 2.5- to almost 20-fold in MDSC and PDSC, respectively, paralleling the degree of dedifferentiation and concordant with a poor prognosis. Except for POLD2, the serous carcinomas showed a similar transcription profile, being clearly different from CCC. Another mRNA, Killer-specific secretory protein of 37 kDa (KSP37) showed six- to eight-fold higher levels in CCC stage I compared with the more advanced staged carcinomas, and correlated positively with an improved clinical outcome. CONCLUSIONS/SIGNIFICANCE: We have identified two biomarkers which are markedly upregulated in two subgroups of ovarian carcinomas and are also associated with stage and outcome. The results suggest that POLD2 and KSP37 might be potential prognostic biomarkers.

Quantification of relative changes in specific mRNAs from frozen whole blood - methodological considerations and clinical implications.

BACKGROUND: Based on quantification of relative changes in lipopolysaccharide (LPS)-regulated mRNA transcripts, the present study aimed to establish a robotic method to isolate RNA from stabilized frozen whole blood suitable for gene expression analysis. METHODS: Whole blood (+/-LPS) was stored in EasyLyse solution or PAXgene tubes (room temperature and -70 degrees C) for comparison of storage methods, then subjected to robotic isolation of total RNA. Yield, quality and relative changes in 11 selected mRNA transcripts were examined. Method precision (% coefficient of variation) for a longitudinal control was established. The influence of globin mRNA from reticulocytes in quantitative RT-PCR was examined. RESULTS: All storage methods gave a similar high-quality RNA yield. The differences in the 11 specific mRNA quantities stored in EasyLyse or PAXgene at -70 degrees C were small: mean -0.01 (95% CI -0.19 to 0.17). The CV for mRNAs in the longitudinal control ranged from 3% to 150%. Thus, the number of replicates necessary to detect a 20% difference (power 0.8) ranged from 2-50. Globin mRNA had no influence on quantitative RT-PCR. CONCLUSIONS: Based on measuring the relative changes in specific mRNAs in LPS-exposed whole blood, we conclude that PAXgene tubes stored at -70 degrees C could preferentially be used. This may open opportunities for monitoring gene expression changes in clinical settings.