Search

Objectives: Early diagnosis of rheumatoid arthritis (RA) is an unmet medical need in the field of rheumatology. Previously, we performed high-density transcriptomic studies on synovial biopsies from patients with arthritis, and found that synovial gene expression profiles were significantly different according to the underlying disorder. Here, we wanted to further explore the consistency of the gene expression signals in synovial biopsies of patients with arthritis, using low-density platforms. Methods: Low-density assays (cDNA microarray and microfluidics qPCR) were designed, based on the results of the high-density microarray data. Knee synovial biopsies were obtained from patients with RA, spondyloarthropathies (SA) or osteoarthritis (OA) (n = 39), and also from patients with initial undifferentiated arthritis (UA) (n = 49). Results: According to high-density microarray data, several molecular pathways are differentially expressed in patients with RA, SA and OA: T and B cell activation, chromatin remodelling, RAS GTPase activation and extracellular matrix regulation. Strikingly, disease activity (DAS28-CRP) has a significant influence on gene expression patterns in RA samples. Using the low-density assays, samples from patients with OA are easily discriminated from RA and SA samples. However, overlapping molecular patterns are found, in particular between RA and SA biopsies. Therefore, prediction of the clinical diagnosis based on gene expression data results in a diagnostic accuracy of 56.8%, which is increased up to 98.6% by the addition of specific clinical symptoms in the prediction algorithm. Similar observations are made in initial UA samples, in which overlapping molecular patterns also impact the accuracy of the diagnostic algorithm. When clinical symptoms are added, the diagnostic accuracy is strongly improved. Conclusions: Gene expression signatures are overall different in patients with OA, RA and SA, but overlapping molecular signatures are found in patients with these conditions. Therefore, an accurate diagnosis in patients with UA requires a combination of gene expression and clinical data. [ABSTRACT FROM AUTHOR]

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n = 5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5–8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n = 4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n = 7) or transversions (n = 9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time. [ABSTRACT FROM AUTHOR]

Separating CBRN mixed samples that contain both chemical and biological warfare agents (CB mixed sample) in liquid and solid matrices remains a very challenging issue. Parameters were set up to assess the performance of a simple filtration-based method first optimized on separate C- and B-agents, and then assessed on a model of CB mixed sample. In this model, MS2 bacteriophage, Autographa californica nuclear polyhedrosis baculovirus (AcNPV), Bacillus atrophaeus and Bacillus subtilis spores were used as biological agent simulants whereas ethyl methylphosphonic acid (EMPA) and pinacolyl methylphophonic acid (PMPA) were used as VX and soman (GD) nerve agent surrogates, respectively. Nanoseparation centrifugal devices with various pore size cut-off (30 kD up to 0.45 µm) and three RNA extraction methods (Invisorb, EZ1 and Nuclisens) were compared. RNA (MS2) and DNA (AcNPV) quantification was carried out by means of specific and sensitive quantitative real-time PCRs (qPCR). Liquid chromatography coupled to time-of-flight mass spectrometry (LC/TOFMS) methods was used for quantifying EMPA and PMPA. Culture methods and qPCR demonstrated that membranes with a 30 kD cut-off retain more than 99.99% of biological agents (MS2, AcNPV, Bacillus Atrophaeus and Bacillus subtilis spores) tested separately. A rapid and reliable separation of CB mixed sample models (MS2/PEG-400 and MS2/EMPA/PMPA) contained in simple liquid or complex matrices such as sand and soil was also successfully achieved on a 30 kD filter with more than 99.99% retention of MS2 on the filter membrane, and up to 99% of PEG-400, EMPA and PMPA recovery in the filtrate. The whole separation process turnaround-time (TAT) was less than 10 minutes. The filtration method appears to be rapid, versatile and extremely efficient. The separation method developed in this work constitutes therefore a useful model for further evaluating and comparing additional separation alternative procedures for a safe handling and preparation of CB mixed samples. [ABSTRACT FROM AUTHOR]