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We have previously demonstrated that systemic AMP-activated protein kinase α1 (AMPKα1) invalidation enhanced adverse LV remodelling by increasing fibroblast proliferation, while myodifferentiation and scar maturation were impaired. We thus hypothesised that fibroblastic AMPKα1 was a key signalling element in regulating fibrosis in the infarcted myocardium and an attractive target for therapeutic intervention. The present study investigates the effects of myofibroblast (MF)-specific deletion of AMPKα1 on left ventricular (LV) adaptation following myocardial infarction (MI), and the underlying molecular mechanisms. MF-restricted AMPKα1 conditional knockout (cKO) mice were subjected to permanent ligation of the left anterior descending coronary artery. cKO hearts exhibit exacerbated post-MI adverse LV remodelling and are characterised by exaggerated fibrotic response, compared to wild-type (WT) hearts. Cardiac fibroblast proliferation and MF content significantly increase in cKO infarcted hearts, coincident with a significant reduction of connexin 43 (Cx43) expression in MFs. Mechanistically, AMPKα1 influences Cx43 expression by both a transcriptional and a post-transcriptional mechanism involving miR-125b-5p. Collectively, our data demonstrate that MF-AMPKα1 functions as a master regulator of cardiac fibrosis and remodelling and might constitute a novel potential target for pharmacological anti-fibrotic applications. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00846-y.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, mainly due to cigarette smoking, which represents the third cause of mortality worldwide. The mechanisms driving its epithelial salient features remain largely elusive. We aimed to evaluate the activation and the role of the canonical, β-catenin-dependant WNT pathway in the airway epithelium from COPD patients. METHODS: The WNT/β-catenin pathway was first assessed by WNT-targeted RNA sequencing of the air/liquid interface-reconstituted bronchial epithelium from COPD and control patients. Airway expression of total and active β-catenin was assessed in lung sections, as well as WNT components in laser-microdissected airway epithelium. Finally, we evaluated the role of WNT at the bronchial epithelial level by modulating the pathway in the reconstituted COPD epithelium. FINDINGS: We show that the WNT/β-catenin pathway is upregulated in the COPD airway epithelium as compared with that of non-smokers and control smokers, in targeted RNA-sequencing of in vitro reconstituted airway epithelium, and in situ in lung tissue and laser-microdissected epithelium. Extrinsic activation of this pathway in COPD-derived airway epithelium inhibited epithelial differentiation, polarity and barrier function, and induced TGF-β-related epithelial-to-mesenchymal transition (EMT). Conversely, canonical WNT inhibition increased ciliated cell numbers, epithelial polarity and barrier function, whilst inhibiting EMT, thus reversing COPD features. INTERPRETATION: In conclusion, the aberrant reactivation of the canonical WNT pathway in the adult airway epithelium recapitulates the diseased phenotype observed in COPD patients, suggesting that this pathway or its downstream effectors could represent a future therapeutic target. FUNDING: This study was supported by the Fondation Mont-Godinne, the FNRS and the WELBIO. ispartof: EBIOMEDICINE vol:61 ispartof: location:Netherlands status: published

Background Over the past recent years, Vibrio cholerae has been associated with outbreaks in sub-Saharan Africa, notably in Democratic Republic of the Congo (DRC). This study aimed to determine the genetic relatedness of isolates responsible for cholera outbreaks in eastern DRC between 2014 and 2017, and their potential spread to bordering countries. Methods/Principal findings Phenotypic analysis and whole genome sequencing (WGS) were carried out on 78 clinical isolates of V. cholerae associated with cholera in eastern provinces of DRC between 2014 and 2017. SNP-based phylogenomic data show that most isolates (73/78) were V. cholerae O1 biotype El Tor with CTX-3 type prophage. They fell within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and were contained in the introduction event (T)10 in East Africa. These isolates clustered in two sub-clades corresponding to Multiple Locus Sequence Types (MLST) profiles ST69 and the newly assigned ST515, the latter displaying a higher genetic diversity. Both sub-clades showed a distinct geographic clustering, with ST69 isolates mostly restricted to Lake Tanganyika basin and phylogenetically related to V. cholerae isolates associated with cholera outbreaks in western Tanzania, whereas ST515 isolates were disseminated along the Albertine Rift and closely related to isolates in South Sudan, Uganda, Tanzania and Zambia. Other V. cholerae isolates (5/78) were non-O1/non-O139 without any CTX prophage and no phylogenetic relationship with already characterized non-O1/non-O139 isolates. Conclusions/Significance Current data confirm the association of both DRC O1 7PET (T)10 sub-clades ST69 and ST515 with recurrent outbreaks in eastern DRC and at regional level over the past 10 years. Interestingly, while ST69 is predominantly a locally endemic sequence type, ST515 became adaptable enough to expand across DRC neighboring countries., Author summary Cholera is a severe diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. After originating in Asia, the disease spread across sub-Saharan Africa, notably Democratic Republic of the Congo. The aim of our study was to assess the transmission pattern of V. cholerae strains prevailing in eastern DRC, and determine their genetic relatedness to strains from other African countries and other parts of the world. Between 2014 and 2017, we isolated V. cholerae from fecal samples of patients with acute diarrhea in eastern DRC, and subsequently examined the DNA of the bacteria. The results show that they all clustered in two genetic groups (ST69 and ST515) falling within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and T10 introduction event in East Africa. The genetic signature of ST515 may be involved in its adaptation to environmental conditions found in eastern DRC, and contribute to its extended geographic distribution. Indeed, unlike the locally endemic ST69, ST515 is spreading extensively through DRC cross-border countries such as South Sudan, Tanzania, Uganda and Zambia. This plainly justifies a regional strategy to strengthen the fight against cholera in eastern Africa.

Background Nanotechnologies provide new opportunities for improving the safety, quality, shelf life, flavor and appearance of foods. The most common nanoparticles (NPs) in human diet are silver metal, mainly present in food packaging and appliances, and silicon and titanium dioxides used as additives. The rapid development and commercialization of consumer products containing these engineered NPs is, however, not well supported by appropriate toxicological studies and risk assessment. Local and systemic toxicity and/or disruption of the gut microbiota (GM) have already been observed after oral administration of NPs in experimental animals, but results are not consistent and doses used were often much higher than the estimated human intakes. In view of the strong evidence linking alterations of the GM to cardiometabolic (CM) diseases, we hypothesized that dietary NPs might disturb this GM-CM axis. Materials and methods We exposed male C57BL/6JRj mice (n = 13 per dose group) to dietary NPs mixed in food pellets at doses relevant for human exposure: Ag (0, 4, 40 or 400 μg/kg pellet), SiO2 (0, 0.8, 8 and 80 mg/kg pellet) or TiO2 (0, 0.4, 4 or 40 mg/kg pellet). After 24 weeks of exposure, we assessed effects on the GM and CM health (n = 8 per dose group). The reversibility of the effects was examined after 8 additional weeks without NPs exposure (recovery period, n ≤ -5 per dose group). Results No overt toxicity was recorded. The GM β-diversity was dose-dependently disrupted by the three NPs, and the bacterial short chain fatty acids (SCFAs) were dose-dependently reduced after the administration of SiO2 and TiO2 NPs. These effects disappeared completely or partly after the recovery period, strengthening the association with dietary NPs. We did not observe atheromatous disease or glucose intolerance after NP exposure. Instead, dose-dependent decreases in the expression of IL-6 in the liver, circulating triglycerides (TG) and urea nitrogen (BUN) were recorded after administration of the NPs. Conclusion We found that long-term oral exposure to dietary NPs at doses relevant for estimated human intakes disrupts the GM composition and function. These modifications did not appear associated with atheromatous or deleterious metabolic outcomes.

Background:During the West Africa Ebola virus disease (EVD) outbreak, a Belgian laboratory was deployed for supporting the Ebola treatment unit (ETU) of N’Zerekore, Guinea. Besides diagnosis of EVD and malaria, biochemical parameters were tested and used to guide supportive treatment of EVD.Methods:To preserve analytes stability, lithium-heparin blood samples were analyzed using the i-STATResults:Providing the medical staff with real-time biochemical data modified their therapeutic attitude, shifting from empiric to a semi-intensive laboratory-guided treatment of hydro-electrolytic disturbances, metabolic acidosis and/or impaired kidney function. As illustrated with representative EVD cases (n=8), optimized supportive treatment with intravenous fluid therapy and electrolyte replacement often helped correct these abnormalities. However, the harsh operating conditions, especially the use of bleach decontamination inside the glovebox, caused several technical failures and the final breakdown of the POCT device.Conclusions:POCT availability resulted in a paradigm shift in laboratory practice and care delivery at the N’Zerekore ETU. We conclude that there is urgent need for novel well-designed and validated POCT devices usable by non-expert operators in high ambient temperature and limited space. These devices should withstand regular and thorough decontamination by the personnel working on-site with life-threatening pathogens and be compatible with high biosafety level procedures. Such specific users’ requirements need a European validation and standardization process of proposed solutions led by the EU Standardization Committee (CEN).

BACKGROUND Regorafenib is the first small-molecule multikinase inhibitor which showed survival benefits in pretreated metastatic colorectal cancer (mCRC) patients. Beside classical adverse events of this drug class, hepatotoxicity has been described as a frequent side effect. MATERIAL AND METHODS Patients with refractory mCRC treated with regorafenib in our institution were reviewed. Severe treatment-related liver toxicity was investigated. Clinical history, liver histology and genetic assessment (sequence analysis) of cytochrome P 3A4 (CYP3A4) and uridine diphosphate-glucuronosyltransferase 1A9 (UGT1A9) involved in regorafenib metabolization were here reported for patients with severe hepatotoxicity. RESULTS Among the 93 reviewed patients, 3 presented severe and icteric toxic hepatitis which was fatal for 1 patient. Histopathological liver lesions were different depending on the onset of hepatotoxicity (acute or subacute): acinar zone 3 necrosis in case of acute symptoms and portal tract inflammation with porto-central bridging and fibrosis in the delayed presentation. None patients had CYP3A4 gene mutations. Similar polymorphisms in UGT1A9 gene promoter region (UGT1A9 variant -118T9>10 (rs3832043)) were found in both patients who presented acute hepatitis. Moreover, it appears retrospectively that both of them already experienced significant toxicity under irinotecan-based chemotherapy. CONCLUSION This is the first report of severe hepatotoxicity with available liver histology and genetic assessment of enzymes involved in regorafenib metabolization. This report also reminds the importance of a close liver tests monitoring during regorafenib treatment. This article is protected by copyright. All rights reserved.

Background Ebola virus disease (EVD) is a highly lethal condition for which no specific treatment has proven efficacy. In September 2014, while the Ebola outbreak was at its peak, the World Health Organization released a short list of drugs suitable for EVD research. Favipiravir, an antiviral developed for the treatment of severe influenza, was one of these. In late 2014, the conditions for starting a randomized Ebola trial were not fulfilled for two reasons. One was the perception that, given the high number of patients presenting simultaneously and the very high mortality rate of the disease, it was ethically unacceptable to allocate patients from within the same family or village to receive or not receive an experimental drug, using a randomization process impossible to understand by very sick patients. The other was that, in the context of rumors and distrust of Ebola treatment centers, using a randomized design at the outset might lead even more patients to refuse to seek care. Therefore, we chose to conduct a multicenter non-randomized trial, in which all patients would receive favipiravir along with standardized care. The objectives of the trial were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak, and to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD. The trial was not aimed at directly informing future guidelines on Ebola treatment but at quickly gathering standardized preliminary data to optimize the design of future studies. Methods and Findings Inclusion criteria were positive Ebola virus reverse transcription PCR (RT-PCR) test, age ≥ 1 y, weight ≥ 10 kg, ability to take oral drugs, and informed consent. All participants received oral favipiravir (day 0: 6,000 mg; day 1 to day 9: 2,400 mg/d). Semi-quantitative Ebola virus RT-PCR (results expressed in “cycle threshold” [Ct]) and biochemistry tests were performed at day 0, day 2, day 4, end of symptoms, day 14, and day 30. Frozen samples were shipped to a reference biosafety level 4 laboratory for RNA viral load measurement using a quantitative reference technique (genome copies/milliliter). Outcomes were mortality, viral load evolution, and adverse events. The analysis was stratified by age and Ct value. A “target value” of mortality was defined a priori for each stratum, to guide the interpretation of interim and final analysis. Between 17 December 2014 and 8 April 2015, 126 patients were included, of whom 111 were analyzed (adults and adolescents, ≥13 y, n = 99; young children, ≤6 y, n = 12). Here we present the results obtained in the 99 adults and adolescents. Of these, 55 had a baseline Ct value ≥ 20 (Group A Ct ≥ 20), and 44 had a baseline Ct value < 20 (Group A Ct < 20). Ct values and RNA viral loads were well correlated, with Ct = 20 corresponding to RNA viral load = 7.7 log10 genome copies/ml. Mortality was 20% (95% CI 11.6%–32.4%) in Group A Ct ≥ 20 and 91% (95% CI 78.8%–91.1%) in Group A Ct < 20. Both mortality 95% CIs included the predefined target value (30% and 85%, respectively). Baseline serum creatinine was ≥110 μmol/l in 48% of patients in Group A Ct ≥ 20 (≥300 μmol/l in 14%) and in 90% of patients in Group A Ct < 20 (≥300 μmol/l in 44%). In Group A Ct ≥ 20, 17% of patients with baseline creatinine ≥110 μmol/l died, versus 97% in Group A Ct < 20. In patients who survived, the mean decrease in viral load was 0.33 log10 copies/ml per day of follow-up. RNA viral load values and mortality were not significantly different between adults starting favipiravir within, In the context the recent Ebola outbreak, Xavier Anglaret and colleagues test an experimental treatment, favipiravir, for Ebola virus disease in a multicenter non-randomized trial., Editors' Summary Background In 2014 and 2015, an Ebola virus outbreak larger than any known before occurred in West Africa. Ebola virus disease (EVD) is highly contagious, and many infected people die. Central to the emergency response to the recent outbreak were local Ebola treatment centers where patients were diagnosed, were isolated, and received supportive care. With thousands of patients dying and many health workers contracting the disease, fear was ubiquitous and distrust abundant. While conducting research in this environment was extremely challenging, the urgent need for treatments and the opportunity to conduct studies that could bring such treatments closer to reality was also recognized. In September 2014, WHO released a short list of existing drugs that were candidates for clinical trials among patients infected in the outbreak. Favipiravir, an antiviral drug developed in Japan for patients with severe influenza, was on the list. Why Was This Study Done? Because of the urgent need to find drugs that could reduce deaths caused by Ebola, the researchers decided to conduct a clinical trial using favipiravir in patients with EVD in Guinea. In view of the circumstances, they decided against a randomized controlled trial and instead designed a study where all participants would receive the same treatment. In randomized controlled trials only some participants receive the treatment in addition to standard care, while others serve as a control group and receive standard care only, or standard care plus a placebo. Such studies allow stronger conclusions to be drawn about whether a treatment is safe and whether it works or not. The researchers had two main reasons for this decision. First, patients from the same family or village often sought EVD treatment at the same time, and the researchers felt that it was ethically unacceptable to randomize such groups, with only some of them receiving the experimental drug. Second, the strict isolation procedures imposed to interrupt virus transmission had intensified fear in affected communities and fueled rumors of illicit drug experimentation and organ theft at the treatment centers. In this context, the researchers worried that a randomized study might increase distrust among the community and the reluctance of patients to seek care. Rather than seeking definitive answers about the safety and efficacy of favipiravir in patients with EVD, the objectives of the study as it was designed were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak and to learn lessons from the experience. In addition, the researchers planned to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD in the hope that their preliminary findings could improve the design of subsequent trials and the chance to provide conclusive answers. What Did the Researchers Do and Find? After 13 weeks of preparation, the trial took place from December 2014 to April 2015 at four separate Ebola treatment centers, three in rural areas and one in an urban setting. In addition to standard care (which included rehydration, antimalarial and antibacterial therapies, and medication to reduce fever, pain, and nausea), all participants were given favipiravir by mouth for ten days, at doses substantially higher than those recommended for patients with influenza. Outcomes measured were mortality, viral load changes over time (based on blood samples), and adverse events. EVD was confirmed with an assay that used patient blood and provided an estimate of the viral load, that is, of how much virus the blood contained. Because viral load was known to influence the course of EVD, the researchers analyzed the participants in two groups, namely, those with a viral load estimate above a certain threshold and those with viral load estimate below the threshold. They also used existing data from Guinean patients diagnosed with Ebola earlier in the outbreak who had received only standard care and calculated an expected mortality rate for patients above and below the viral load threshold. The researchers were able to enroll 126 participants in the trial. Of these, 111 were included in the final analysis. Of 99 adult and adolescent participants 13 years and older, 55 were in the lower viral load group and 44 in the higher viral load group. Mortality was 20% in the former and 91% in the latter. Neither mortality rate was significantly different from that of earlier patients who had received only standard care. The researchers also found that favipiravir was well tolerated. None of the patients stopped the course of treatment, vomiting following drug intake was rare, and no severe adverse events were attributed to the drug. The researchers did not see a difference in mortality between patients who reported onset of symptoms less than three days before the start of treatment and those whose symptoms had started more than three days the start of treatment. What Do these Findings Mean? The report shows that it is possible to conduct an emergency trial during an outbreak in a low-resource setting. In fact, at the time of its acceptance, this paper reported on an Ebola treatment trial larger than any other yet published. The experience described should be useful for similar undertakings in the future. The following conditions contributed to the success of the trial: close collaboration between researchers, local health officials, and affected communities on one hand, and flexibility in design, conduct, and analysis based on close monitoring and interim assessments on the other. Besides using interim results to influence the conduct and analysis of their own trial, the researchers also shared these results with the scientific community in real time, and this feedback influenced other research during the outbreak. The trial could not answer definitively whether favipiravir treatment was safe or reduced mortality in patients with EVD. The results suggest that the drug is unlikely to be beneficial for patients with very high viral loads, at least when given by itself. They also suggest that favipiravir is safe in patients with lower viral loads, and that in such patients additional efficacy studies are warranted. Intermediate analysis of various measurements in trial participants showed that the estimate of viral load from the field EVD diagnosis test is a good proxy for the actual viral load (determined after the samples were shipped to and analyzed in a reference laboratory in France) and suitable as a surrogate marker. The results also confirm that viral load is a strong predictor of mortality. Additional Information This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001967. The World Health Organization has pages on Ebola virus disease, trials of Ebola treatments and vaccines, and the current update of the list of suitable drugs for testing or use in patients infected with Ebola (originally compiled in September 2014) US Centers for Disease Control and Prevention has information on the Ebola outbreak in West Africa The European Centre for Disease Prevention and Control also has information on the Ebola outbreak in West Africa

TPMT deficiency is associated with azathioprine (AZA)-induced myelosuppression (MS). However, in one previous study, only about ¼ of MS episodes in Crohn's Disease patients under AZA can be attributed to TPMT deficiency. Recently, new TPMT mutations have been described and our aim is to investigate their clinical relevance before and after a first MS episode on thiopurine therapy.Clinical data from 61 IBD patients having developed MS during AZA therapy were collected. Sequencing analysis was carried out on TPMT cDNA for the presence of all currently known mutations.Only TPMT *2, *3A and *3C mutations were found in this cohort. TPMT mutations were observed in 15 out of 61 patients (25%). Four out of 15 were homozygous for a TPMT mutation (low methylator, LM genotype) and 11 were heterozygous (intermediate methylator, IM genotype). Median delays of MS onset were 2, 2.75 and 6months in the LM, IM and HM (high methylator, wild type TPMT) groups, respectively. After the first MS episode, 36 patients resumed thiopurine treatment of which 13 experienced a second MS episode. This second episode was also rarely associated with TPMT mutations.One quarter of MS episodes during AZA were associated with TPMT deficient genotype. After a first leucopenia episode, thiopurine therapy may be resumed in a majority of patients independently of their TPMT genotype.

This study aimed at investigating the contribution of CYP2C9 and VKORC1 genetic polymorphisms to inter-individual variability of acenocoumarol pharmacokinetics and pharmacodynamics in Black Africans from Benin. Fifty-one healthy volunteers were genotyped for VKORC1 1173CT polymorphism. All of the subjects had previously been genotyped for CYP2C9*5, CYP2C9*6, CYP2C9*8, CYP2C9*9 and CYP2C9*11 alleles. Thirty-six subjects were phenotyped with a single 8 mg oral dose of acenocoumarol by measuring plasma concentrations of (R)- and (S)-acenocoumarol 8 and 24 h after the administration using chiral liquid-chromatography tandem mass-spectrometry. International normalized ratio (INR) values were determined prior to and 24 h after the drug intake. The allele frequency of VKORC1 variant (1173CT) was 1.96% (95% CI 0.0-4.65%). The INR values did not show statistically significant difference between the CYP2C9 genotypes, but were correlated with body mass index and age at 24 h post-dosing (P0.05). At 8 h post dose, the (S)-acenocoumarol concentrations in the CYP2C9*5/*8 and CYP2C9*9/*11 genotypes were about 1.9 and 5.1 fold higher compared with the CYP2C9*1/*1 genotype and 2.2- and 6.0-fold higher compared with the CYP2C9*1/*9 group, respectively. The results indicated that pharmacodynamic response to acenocoumarol is highly variable between the subjects. This variability seems to be associated with CYP2C9*5/*8 and *9/*11 variant and demographic factors (age and weight) in Beninese subjects. Significant association between plasma (S)-acenocoumarol concentration and CYP2C9 genotypes suggested the use of (S)-acenocoumarol for the phenotyping purpose. Larger number of subjects is needed to study the effect of VKORC1 1173CT variant due to its low frequency in Beninese population.

Microbiological cultures are moderately sensitive for diagnosing prosthetic joint infection (PJI). This study was conducted to determine whether amplification-based DNA methods applied on intraoperative samples could enhance PJI diagnosis compared with culture alone in routine surgical practice. Revision arthroplasty was performed for suspected PJI (n = 41) and osteoarthrosis control (n = 28) patients, and a diagnosis of PJI was confirmed in 34 patients. Amplification by polymerase chain reaction was performed on both 16S ribosomal DNA universal target genes and femA Staphylococcus-specific target genes. Species identification was achieved through amplicon sequencing. Amplification of the femA gene led to subsequent testing for methicillin resistance by amplification of the mecA gene. Microbiological and molecular assays identified a causative organism in 22 of 34 patients (64.7%) and in 31 of 34 patients (91.2%), respectively. In 18 of the 22 culture-positive patients, molecular and microbiological results were concordant for bacterial genus, species, and/or methicillin resistance. Bacterial agents were identified only by molecular methods in nine PJI patients, including seven who were receiving antibiotics at the time of surgery and one with recent but not concomitant antibiotherapy. DNA-based methods were found to effectively complement microbiological methods, without interfering with existing procedures for sample collection, for the identification of causative pathogens from intraoperative PJI samples, especially in patients with recent or concomitant antibiotherapy.

Background Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. Methods In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). Results All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ∼229 US$ /patient to ∼64 US$ /patient). Conclusions This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while providing results as reliable as uniplex analysis. Using this combined multiplex approach also substantially reduced the production of waste material. These genotyping assays appear therefore to be biologically, economically and ecologically highly relevant, being worth to be integrated in genetic-based predictive strategies for better selecting patients at risk for prostate cancer. In addition, the same approach could now equally be transposed to other clinical/research applications relying on the computation of MGRS based on multi-SNP genotyping.

Lauwerys BR, Hernández-Lobato D, Gramme P, Ducreux J, Dessy A, Focant I, et al. (2015) Heterogeneity of Synovial Molecular Patterns in Patients with Arthritis. PLoS ONE 10(4): e0122104. doi:10.1371/journal.pone.0122104, 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., This work was supported by grants from the Région Wallonne (BioWin), and the Fondation Saint-Luc (Cliniques Universitaires Saint-Luc, Brussels, Belgium). JD is funded by a unrestricted UCB grant (« Chaire UCB sur les rhumatismes inflammatoires et systémiques »). BRL is a part-time research fellow (« Clinicien-chercheur ») of the Fonds National de la Recherche Scientifique (Communauté française de Belgique). DNAlytics provided support in the form of salaries for authors PG and TH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.

Purpose: To investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21 pathway alteration and as a predictor of recurrence in superficial transitional cell carcinomas.Experimental Design: p53 transcriptional activity was prospectively analyzed in 52 newly diagnosed transitional cell carcinoma using FASAY competent for the transactivation of p21 and bax promoters. TP53 and p21 gene expression was quantified by real-time PCR, and expression of corresponding proteins was assessed by immunohistochemistry. In addition to tumor stage and grade, the predictive value of FASAY, real-time PCR, and immunohistochemistry for tumor recurrence was assessed by Cox survival analysis.Results: A total (p21 and bax) or partial (bax only) loss of transcriptional activity was observed in 15 of 52 (29%) and 4 of 52 (7.7%) cases, respectively, a partial loss being consistently associated with R283H mutation. p53 nuclear overexpression grossly overestimated (∼40%) or underestimated (∼10%) the true incidence of p53 transcriptional abnormalities, especially in Ta-T1 grade 1 to 2 tumors. Loss of p21 transactivation significantly correlated with decreased p21 gene expression and lack of expression of p21 (P = 0.001). FASAY had a better predictive value for recurrence than p53 immunohistochemistry (Cox hazard ratio, 6.57 versus 3.95; P = 0.0002 versus 0.019, respectively), whereas neither p21 immunohistochemistry (hazard ratio, 1.9; P = 0.29) nor TP53 or p21 gene expression were significant predictors of recurrence. The prognostic difference between FASAY and p53 immunohistochemistry was maintained in the subgroup of Ta-T1 grade 3 tumors.Conclusions: FASAY is a valuable surrogate marker for assessing p53/p21 pathway alteration and predicts transitional cell carcinoma recurrence better than p53 immunohistochemistry.

OBJECTIVE: Blood cultures and cultures of disc material are required to identify and treat bacterial agents responsible for septic spondylodiscitis, but these methods have limited sensitivities. We undertook this study to compare nonculture amplification-based DNA analysis with conventional culture of disc aspirate. METHODS: Nineteen patients with spondylodiscitis, including 11 with a history of spinal surgery, presented with negative blood cultures and underwent percutaneous disc or epidural abscess puncture for bacterial diagnosis. Amplification by polymerase chain reaction was performed on 16S ribosomal DNA universal target genes and femA staphylococci-specific target genes in all patients, and on the upstream p34 mycobacterial gene in 1 patient. Species identification relied on amplicon sequencing and comparison with templates from GenBank. Amplification of the femA gene led to subsequent testing for methicillin resistance by amplification of the mecA gene. Further assessment using a staphylococci- and methicillin resistance-specific DNA array was performed on 3 samples. RESULTS: Microbiologic and molecular assays identified the causative organism in 14 of 19 patients (74%) and 19 of 19 patients (100%), respectively. In culture-positive patients, DNA-based and microbiologic results were highly correlated. Five agents (Staphylococcus simulans, Staphylococcus sciuri, Brucella species, Actinomyces israelii, and Mycobacterium tuberculosis complex) were identified only by DNA-based methods. In 1 sample, Corynebacterium jeikeium and coagulase-negative Staphylococcus were both cultured, whereas DNA analysis identified only Staphylococcus hominis. CONCLUSION: DNA-based methods are highly sensitive and specific. They can usefully complement standard microbiologic methods for identifying the cause of infectious spondylodiscitis and contribute to species-specific therapeutic orientation in patients with negative blood and disc aspirate cultures.

BACKGROUND: To predict poor outcome in patients with a biochemical recurrence (rising PSA) after radical prostatectomy (RP), urologists rely primarily on Gleason score, PSA doubling time, and time from surgery to biochemical (i.e., PSA) recurrence. In the present study, we assess the value of RT-PCR detection circulating prostate cells in blood of patients with a rising PSA. METHODS: RNA from blood samples was obtained from 55 patients with a rising PSA and from 45 patients without evidence of biochemical failure (PSA < 0.1 ng/ml). Both groups were matched for age, Gleason score, pT stage, and interval between radical prostatectomy and PCR testing. RESULTS: PSA positive cells were detected in 1/45 (2%) patients without a PSA recurrence and 19/55 (34%) patients with a PSA recurrence. In the rising PSA group, mean PSA doubling time was significantly shorter in patients with positive RT-PCR (5 months) than in patients with negative RT-PCR (16 months; P = 0.001). An earlier onset of recurrence was also detected in patients with a positive RT-PCR (31 months for positive RT-PCR vs. 50 months for negative RT-PCR) but this result did not achieve statistical significance (P = 0.102). Salvage radiation therapy was administered in 15 patients. Three of the five patients with a positive RT-PCR progressed during radiotherapy whereas 7 of the 10 patients with a negative RT-PCR obtained a complete response and none have progressed. CONCLUSIONS: These preliminary results suggest that RT-PCR detection of prostate cells in blood of patients after RP correlates with rapidly progressing biochemical failure after RP.

To compare the relapse-free survival (RFS) in Vietnamese (n=141) and white (n=94) children living in Vietnam and Belgium, respectively, and treated in their own country for acute lymphoblastic leukemia according to the same FRALLE 2000 protocol.RFS was significantly worse in Vietnamese children (hazards ratio=4.48; 95% confidence interval [CI], 2.16-9.3; P0.01). The 5-year RFS was 83.8% (95% CI, 76.3%-92.0%) and 47.8% (95% CI, 35.6%-64.2%) for white and Vietnamese children, respectively. In the latter group, relapses occurred in bone marrow and cerebrospinal fluid at a much earlier stage. The outcome was compared at first relapse only because of different treatments afterward, according to the country. Both series were similar for sex, age at diagnosis, initial white blood cell count, cytogenetic abnormalities, and corticosensitivity at day 8. Higher frequency of L2-acute lymphoblastic leukemia (P0.001) but lower frequency of T-acute lymphoblastic leukemia (P=0.004) were observed in Vietnamese children.Several factors may contribute to the poor RFS in Vietnamese children, which include the time interval before the first intrathecal therapy and differences in the management of drug-related toxicity. However, additional contribution of socioeconomic factors and/or variations in pharmacogenetic polymorphisms in Vietnamese patients cannot currently be ruled out.

In clinically organ-confined prostate cancer patients, bloodstream tumour cell dissemination generally occurs, and may be enhanced by surgical prostate manipulation. To evaluate cancer-cell seeding impact upon patient recurrence-free survival, 155 patients were prospectively enrolled then followed. Here, 57 patients presented blood prostate cell shedding preoperatively and intraoperatively (group I). Of the 98 preoperatively negative patients, 53 (54%) remained negative (group II) and 45 (46%) became intraoperatively positive (group III). Median biological and clinical recurrence-free time was far shorter in group I (36.2 months, P

The sensitive and specific detection of circulating tumor cells holds great promise for more accurate staging of cancer patients. Several reverse transcription-PCR (RT-PCR) procedures based on tissue-specific mRNA expression are now able to detect one cell derived from a given tissue among >10 peripheral nucleated blood cells [PNBCs; for a review, see (1)]. However, even for a single marker, highly discrepant results have been observed among the available clinical studies; e.g., the frequencies of positive prostate-specific antigen (PSA) RT-PCR results range from 25% (2) to 80% (3) in patients with metastatic prostate cancer (CaP), blurring the clinical relevance of these assays. Standardization and quality control in molecular diagnosis are crucial to the solution of this issue. We have previously studied factors potentially affecting RT-PCR results (4), and this current work focuses on the approaches for harvesting prostate cells among nucleated blood cells. Since the first 1992 clinical report describing the RT-PCR detection of circulating prostatic cells in CaP, the majority of reported assays have used gradient separations to recover nucleated cells from the peripheral blood (1). We (5) and others (6) rather choose overnight hypo-osmotic red blood cell lysis as an easier and more cost-effective protocol. Because the approach used to harvest PNBCs may account for discrepancies in clinical results, we have compared a panel of nucleated blood cell separation methods. Fresh blood from four healthy blood donors (total volume, 400 mL) was sampled in 40 × 10-mL EDTA-treated tubes (Becton-Dickinson). The LNCaP cell line, derived from a metastatic prostate carcinoma, was cultured as described (7). After trypsinization (2.5 mL/L trypsin), 10 confluent LNCaP cells were added into each of the 40 tubes, which were gently mixed. Six tubes were processed immediately by overnight hypoosmotic red blood cell lysis, as described (5)(6): Two volumes of ammonium chloride (9 …

The presence of the 20210A allele of the prothrombin gene has recently been shown to be a risk factor of venous thromboembolism, probably mediated through increased prothrombin levels. The aim of the study was to determine the frequency of the prothrombin 20210A allele in 193 consecutive unselected patients with venous thromboembolism and 100 healthy controls and to analyze the clinical profile associated with this new inherited thrombophilic factor. In agreement with previous reports, we found a frequency of 7.3% of heterozygous carriers of the 20210A allele among patients and 1% among controls. We confirm that plasma prothrombin levels are more elevated in the individuals bearing the prothrombin 20210A allele compared with those who do not. We did not find any relationship between the presence of the prothrombin 20210A allele and either a family history of thromboembolism, the rate of recurrences or the age at disease onset. However, the co-inheritance in the same individual of both prothrombin 20210A allele and factor V Leiden was associated with a significantly lower age at disease onset suggesting a synergistic contribution of both abnormalities.

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with higher risk of prostate cancer (PCa). This study aimed to evaluate whether published SNPs improve the performance of a clinical risk-calculator in predicting prostate biopsy result.Three hundred forty-six patients with a previous prostate biopsy (191 positive, 155 negative) were enrolled. After literature search, nine SNPs were selected for their statistically significant association with increased PCa risk. Allelic odds ratios were computed and a new logistic regression model was built integrating the clinical risk score (i.e., prior biopsy results, PSA level, prostate volume, transrectal ultrasound, and digital rectal examination) and a multilocus genetic risk score (MGRS). Areas under the receiver operating characteristic (ROC) curves (AUC) of the clinical score alone versus the integrated clinic-genetic model were compared. The added value of the MGRS was assessed using the Integrated Discrimination Improvement (IDI) and Net Reclassification Improvement (NRI) statistics.Predictive performance of the integrated clinico-genetic model (AUC = 0.781) was slightly higher than predictive performance of the clinical score alone (AUC = 0.770). The prediction of PCa was significantly improved with an IDI of 0.015 (P-value = 0.035) and a continuous NRI of 0.403 (P-value 0.001).The predictive performance of the clinical model was only slightly improved by adding MGRS questioning the real clinical added value with regards to the cost of genetic testing and performance of current inexpensive clinical risk-calculators.

Contains fulltext : 116540.pdf (Publisher’s version ) (Open Access) BACKGROUND: Angiotensin-converting enzyme (ACE) (EC 4.15.1) metabolizes many biologically active peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels are associated with different cardiovascular and respiratory diseases. METHODS AND RESULTS: Two Belgian families with a 8-16-fold increase in blood ACE level were incidentally identified. A novel heterozygous splice site mutation of intron 25 - IVS25+1G>A (c.3691+1G>A) - cosegregating with elevated plasma ACE was identified in both pedigrees. Messenger RNA analysis revealed that the mutation led to the retention of intron 25 and Premature Termination Codon generation. Subjects harboring the mutation were mostly normotensive, had no left ventricular hypertrophy or cardiovascular disease. The levels of renin-angiotensin-aldosterone system components in the mutated cases and wild-type controls were similar, both at baseline and after 50 mg captopril. Compared with non-affected members, quantification of ACE surface expression and shedding using flow cytometry assay of dendritic cells derived from peripheral blood monocytes of affected members, demonstrated a 50% decrease and 3-fold increase, respectively. Together with a dramatic increase in circulating ACE levels, these findings argue in favor of deletion of transmembrane anchor, leading to direct secretion of ACE out of cells. CONCLUSIONS: We describe a novel mutation of the ACE gene associated with a major familial elevation of circulating ACE, without evidence of activation of the renin-angiotensin system, target organ damage or cardiovascular complications. These data are consistent with the hypothesis that membrane-bound ACE, rather than circulating ACE, is responsible for Angiotensin II generation and its cardiovascular consequences. 12 p.

Bcl-2 expression and its prognostic value were evaluated using the APAAP technique in 40 ALL patients. Because of a possible synergy between c-myc and bcl-2 proteins in cell lines, we compared 40 ALL of either T or B lineage with seven Burkitt's ALL or sporadic Burkitt's lymphomas (BL). We found the same high expression of bcl-2 in adult (Ad) and childhood (Ch) malignancies examined at presentation, regardless of the T or B phenotype. No bcl-2 expression was detectable in BL cases, except for two investigated at relapse. Bcl-2 staining intensity was also not related to the stage of blast differentiation, except in Ad T-ALL, or to the relapse rate. Ki-67, a marker of proliferation, was used to investigate the possible correlation between bcl-2 expression and the proliferative activity. An inverse correlation was found only in BL at presentation. We confirm that bcl-2 expression is the rule in ALL, regardless of the immunophenotypic characteristics at presentation, and that high expression is not correlated with a bad prognosis. Sporadic BL may represent a particular type of tumor, with bcl-2 expression in relapse, but not at presentation.

BACKGROUND: The purpose of this study was to investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21 pathway alteration in head and neck squamous cell carcinoma (HNSCC). METHODS: The p53 transcriptional activity was prospectively analyzed in 82 newly diagnosed patients with HNSCC. FASAY and p53 immunostaining were carried out on paired tumoral and histologically normal tissues. The predictive value of FASAY for locoregional recurrence was assessed by Cox survival analysis. RESULTS: Loss of p53/p21 transcriptional activity was encountered in 88% tumoral and 18% histologically normal samples, associated with mutations (79%) and insertions/deletions (21%). The p53 overexpression underestimated p53 transcriptional abnormalities. FASAY-positive histologically normal mucosa was significantly associated with locoregional recurrence. CONCLUSION: FASAY positivity indicates field cancerization in a subgroup of patients with HNSCC, in which nonfunctional p53 was significantly associated with locoregional recurrence. This prompted us to pursue the study on the p53 functional status of normal mucosa in patients with HNSCC. © 2011 Wiley Periodicals, Inc. Head Neck, 2011.

Introduction: Recent approval and introduction into clinical practice of epidermal growth factor receptor inhibitors such as the chimeric monoclonal antibody cetuximab and the fully human monoclonal antibody panitumumab have provided new treatment options for chemotherapy-refractory patients. Here, we report a case of a 47-year-old man with metastatic, chemotherapy-refractory colorectal cancer who achieved long-term partial remission during panitumumab therapy. Case presentation: A 41-year-old male patient presented with a 24-hour history of abdominal pain and fever. A computed tomography (CT) scan revealed a voluminous and perforated abscess with a suspected tumour lesion in the sigmoid colon. The patient underwent sigmoidectomy and was diagnosed with a poorly differentiated necrotic carcinoma of the sigmoid colon with invasion in 13 of 19 tested lymph nodes. A colonoscopy revealed multiple tubular adenomas and a positron emission tomography CT scan showed multiple and bilateral hyperfixating lumbar-aortic lymph nodes leading to a final tumour classification of T4 N2 M1. Carcinoembryonic antigen (CEA) was elevated. The patient achieved a partial response following six cycles of FOLFIRI (irinotecan, 5-fluorouracil, leucovorin), then progressed and was enrolled in a trial where he received treatment with FOLFOX4 (oxaliplatin, leucovorin and 5-fluorouracil) with or without a vascular endothelial growth factor inhibitor (PTK787/ZK 222584 [valatinib]). Eight months later he progressed again and was included in a panitumumab (6 mg/kg every 2 weeks) monotherapy trial. A partial response was noted after 8 weeks of therapy along with a rapid CEA reduction and decrease in lymph node size. The patient is continuing panitumumab treatment and is still in partial remission after 65 months' treatment. He has non-mutated KRAS and no human-anti-human antibodies have been detected. During treatment the patient has on occasion experienced grade 1-2 diarrhoea as well as folliculitis and acne-like rash up to grade 3 in severity. Cutaneous toxicity was managed with a combination dose interruption/reduction and the use of topical agents. No eye or nail toxicities occurred. Conclusion: This case shows that long-term responses are possible during panitumumab therapy and that this agent may be an effective long-term treatment option for selected patients with metastatic colorectal cancer. The associated skin toxicities can be successfully managed. (C) 2010 Elsevier Ltd. All rights reserved.

A 31-year-old patient presented with a diagnosis of granulomatous dermatophytosis based on the clinical aspect of the lesions and the rare presence of hyphae on direct microscopic examination of clinical material. A chronic evolution and progression of the disease, its resistance to a wide range of antifungal agents, the occasional presence of hyphae on direct examination but consistently negative cultures over a 5-year period prompted the use of amplification-based DNA analyses of several successive swab samples or skin biopsies. DNA was extracted using a combination of two semi-automated DNA isolation methods (FastPrep(R) preparation and NucliSENS(R) lysis magnetic extraction method). Identification relied both on sequence analysis of amplicons after SYBR(R) Green real-time PCR of the panfungal internal transcribed spacer 1 (ITS1) genetic target, as well as the unique amplicon melting curve profile of positive samples. Accordingly, Trichophyton rubrum was unambiguously identified in several clinical samples collected over a 7-month period. This case illustrates the contribution of DNA-based assays applied directly to sample biopsies for identifying causative agents in cases in which fungal pathogens are highly suspected but culture are repeatedly negative. It also pinpoints the benefit of combining semi-automated DNA preparation methods, analysis of ITS1 amplicon melting curve profiles and sequence analysis on repeated skin biopsy samples for unambiguous identification of the causative fungal species.

We report a case of life-threatening intoxication and a controlled re-exposure study to dextromethorphan. A 60-year-old man developed postsurgical neuropathic cervical pain treated by hydromorphone, gabapentin, clonazepam, and amitriptyline. He received a dextromethorphan preparation for a catarrhal syndrome. Two days later, he was admitted into an emergency department in a profound coma. Thirty-six hours later, after withdrawal of all drugs, the situation normalized. A genotyping for UDP-glucuronyltransferase 1A1 and CYP2D6 was followed by a re-exposure study. During the three days, vital parameters and side effects of drugs were prospectively recorded. The second day, dextromethorphan was introduced. No significant impairment in parameters nor influence on analgesic efficacy were noted. Dextromethorphan concentrations suggest an accumulation without reaching any steady state. Somnolence was noted for plasma concentrations around 100ng/mL. The CYP2D6*4 variant leading to a poor metabolizer phenotype was founded. Moreover, this phenotype was potentially aggravated by amitriptyline intake. This study allowed the identification and the confirmation of the cause of the coma. In conclusion, it is probably wise to recommend avoiding dextromethorphan in patients taking tricyclic antidepressants or another inhibitor of CYP2D6. Drug-drug interactions are probably underdiagnosed and underreported, and drugs considered as safe may induce serious complications.

The genetically polymorphic cytochrome P450 2C9 (CYP2C9) metabolizes many important drugs. Among them, phenytoin has been used as a probe to determine CYP2C9 phenotype by measuring the urinary excretion of its major metabolite, S-enantiomer of 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). Phenytoin pharmacokinetic is also dependent on the activity of CYP2C19 and p-glycoprotein (ABCB1). To determine the influence of CYP2C9, CYP2C19 and ABCB1 genetic polymorphisms on phenytoin metabolism in a Black population, 109 healthy Beninese subjects received a single 300 mg oral dose of phenytoin. Blood was drawn 4 h after drug intake and urine was collected during the first 8 h. Plasma phenytoin and urine S- and R-enantiomers of p-HPPH were determined by high-performance liquid chromatography. Urinary excretion of (S)-p-HPPH [defined as urinary volumex(S)-p-HPPH urinary concentration] and PMR (defined as the ratio of p-HPPH in urine to 4 h phenytoin plasma concentration), both markers of CYP2C9 activity, were used to determine the functional relevance of new variants of CYP2C9 (*5, *6, *8, *9 and *11) in this population. Plasma phenytoin concentration was significantly associated with ABCB1 haplotype/genotype (P=0.05, Kruskal-Wallis test) and levels increased significantly in the genotype order: wild-type, T3421A and Block-2 genotypes (P=0.015, Jonckheere-Terpstra test). Urinary excretion of (S)-p-HPPH and PMR were significantly associated with the CYP2C9 genotype (P=0.001, analysis of variance (ANOVA) and P

Conclusive identification of RB1 mutations in retinoblastoma is predicted to improve the clinical management of affected children and relatives. However, despite clear clinical benefits, RB1 screening remains difficult, most of the alterations being unique and randomly distributed throughout the entire coding sequence. In this report, we present the results of a constitutional RB1 analysis undertaken in our institution over the last four years. The detection of RB1 gene deletion or mutation was performed by Southern blot and sequence analyses in 73 patients (including three families with 2, 3 and 3 probands, respectively). Complementary constitutional chromosome and fluorescent in situ hybridization (FISH) analyses of RB1 gene were applied in cases where hereditary retinoblastoma was suspected despite negative detection. Altogether, germline abnormalities were found in 11% (4/36 patients) of sporadic unilateral retinoblastoma (median age, 21.5 months) and 86% (32/37 patients) of sporadic bilateral or positive familial history retinoblastoma (median age, 5 months). The spectrum of germline alterations found in 31 distinct families included 12 nonsense mutations (39%); 10 point insertions or deletions with frameshift (32%); 4 mutations and 1 deletion affecting splice sites (16%); 2 missense mutations (6%); and 2 large deletions (6%). A total of 15 mutations have not been previously reported. In this small series, splicing mutations were associated with bilateral disease whilst most of the frameshift mutations were identified in patients with an early age at diagnosis, bilateral disease or hereditary forms of the disease. This study confirms that screening for constitutional RB1 mutation should become an integral part of current management of any patient affected by retinoblastoma irrespective of the tumour laterality and familial background.

The molecular basis of beta-thalassemia was investigated at the DNA level in 28 Belgians from 14 unrelated families. All the patients were heterozygous for beta-thalassaemia. Seven different mutations were identified using a combination of dot-blot hybridization with allele-specific oligonucleotide probes and direct automated fluorescence-based DNA sequencing. Among these mutations, four are commonly found in the Mediterraneans - codon 8 (-AA), IVS-I-1 (G --> A), IVS-I-6 (T --> C) and codon 39 (C --> T)-and two have occasionally been reported-initiation codon (T --> C) and codon 35 (C --> A). The last mutation, a -CC deletion at codons 38/39, appears to be a novel mutation and can routinely be investigated by AvaII restriction on amplified DNA. We report our findings, discuss the diversity of the mutations found in Belgium and show the usefulness of direct DNA sequencing in a population in which the molecular defects of beta-thalassaemia have yet to be characterized and in which screening is hampered by the wide range of potential mutations.

OBJECTIVE: The aim of the study was to determine the prevalence of the C677T mutation in a cohort of type 2 diabetic patients with and without elevated total plasma homocysteine (tHcy). METHODS: 80 type 2 diabetic patients with hyperhomocysteinaemia (group 1, tHcy: 21.3 +/- 6.7 micromol/L) and 50 subjects with normal levels (group 2, tHcy 11.2 +/- 2.3 micromol/L) were studied. C677T mutation was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Homozygosity was present in 23% of patients in group 1 and 8% in group 2 (P

Background and aim Previous data indicate that the urinary losartan/E-3174 ratio is a marker for cytochrome P450 (CYP) 2C9 activity in vivo. The functional impact of CYP2C9*5, *6, *8, and *11 polymorphisms in vivo has not been investigated previously in humans.A single oral dose of losartan (25 mg) was given to 19 Beninese subjects with CYP2C9*1/*1 (n = 9), *1/*5 (n = 1), *1/*6 (n = 1), *1/*8 (n = 2), *1/*11 (n = 3), *5/*6 (n = 1), *5/*8 (n = 1), and *8/*11 (n = 1) genotypes. Concentrations of losartan and its active metabolite E-3174 were determined in urine from 0 to 8 hours by HPLC. The losartan/E-3174 metabolic ratio was used as a measure of losartan oxidation in vivo.The urinary losartan/E-3174 ratio in the various genotypes was as follows: 1.85 +/- 2.4 (mean +/- SD) for CYP2C9*1/*1, 14.6 for CYP2C9*1/*5, 4.2 for CYP2C9*1/*6, 188 for CYP2C9*5/*6, 11.6 for CYP2C9*5/*8, 0.44 +/- 0.13 (mean +/- SD) for CYP2C9*1/*8, 2.2 for CYP2C9*8/*11, and 5.72 +/- 4.5 (mean +/- SD) for CYP2C9*1/*11. Compared with the CYP2C9*1/*1 genotypes, the losartan/E-3174 ratio was significantly different in the CYP2C9*5 allele carriers (CYP2C9*1/*5, CYP2C9*5/*8, and CYP2C9*5/*6 genotypes) (P =.01, Mann-Whitney) but was not different in CYP2C9*1/*8 (P =.16) and CYP2C9*1/*11 (P =.11) carriers. The urinary losartan/E-3174 ratio of the single CYP2C9*1/*6 subject was higher than the 95% confidence interval of the mean of the CYP2C9*1/*1 group (0.0-3.7), whereas the metabolic ratio of the CYP2C9*8/*11 carrier was inside the 95% confidence interval of the means of the CYP2C9*1/*1 and CYP2C9*1/*11 groups (0.0-18).The CYP2C9*5 and *6 alleles are associated with decreased enzyme activity in vivo compared with the wild-type variant, whereas the CYP2C9*8 and *11 variants did not appear to have large in vivo effects.

A case of invasive drug resistant thymoma, expressing P-glycoprotein, which showed noticeable clinical response to chemotherapy and the multidrug resistance modulating agents cyclosporin and quinine is reported. A 46 year old man presented with severe left shoulder pain and a diagnosis of invasive lymphoepithelial thymoma was made following chest x ray and a computed tomography scan. The patient underwent extensive chemotherapy without resolution of the tumour. More than 90% of the malignant epithelial cells were strongly positive for P-glycoprotein and based on this observation, cyclosporin and quinine were added to the chemotherapy regimen. The mediastinal mass completely resolved and the size of the pleural metastasis decreased substantially. The patient, however, died of an intercurrent infection. This case report highlights the feasibility and efficacy of using cyclosporin and quinine in combination with VAD chemotherapy in the treatment of invasive thymoma.

Fasting plasma homocysteine level and the related clinical findings were analysed in 240 consecutive patients with venous thromboembolism. Hyperhomocysteinemia, defined as a plasma level above 20 micromol/l (corresponding to the percentile 95th in the controls), was present in 11.2% of the patients. Plasma homocysteine level was similar in patients presenting with either deep venous thrombosis, pulmonary embolism or both conditions. It was significantly higher in patients with primary (unprovoked) VTE than in patients with secondary disease (associated with at least one risk factor): 12.3 vs. 9.55 micromol/l (p < 0.005). Mean homocysteine was higher in male than in female patients (14.51 vs. 12.9 micromol/l, p < 0.05) and increased significantly with age. Hyperhomocysteinemia was more frequent in patients with relapsing disease (14 of 76, 18.4%) than in those presenting with a single episode (13 of 164, 7.9%) (p = 0.034). Furthermore, hyperhomocysteinemia was correlated with reduced protein C level (p = 0.013). In a multivariate analysis, two factors were significantly associated with hyperhomocysteinemia: older age (p < 0.0001) and idiopathic occurrence (p < 0.02). Since the frequency of homozygous MTHFR thermolabile variant was rather similar in patients and controls, testing for C677T mutation was not helpful in screening VTE patients. However, the homozygous mutation was significantly more prevalent among hyperhomocysteinemia patients, confirming its role in the genesis of hyperhomocysteinemia. According to its prevalence, to the putative role in venous and arterial disease and the availability of an effective and low-cost corrective therapy, hyperhomocysteinemia deserves interest, especially in the elderly and in the patients with idiopathic VTE disease.

Until now, no molecular parameter has been available for predicting the metastatic potential of prostate tumours, which leaves their outcome uncertain despite an apparent benign histology or early stage. Abnormal expression of adhesion molecules, such as E-cadherin, can be contributing factors for increased invasiveness and metastatic potential. Histological analysis for E-cadherin expression was carried out on paraffin-embedded tumour tissues. Tumour metastatic potential was indirectly evaluated by detecting circulating prostate cells (CPC), using reverse transciptase-polymerase chain reaction (RT-PCR) and prostate-specific membrane antigen (PSMA) as a target. Patients were followed-up for a median of 14 months (range 10--19 months) after surgery with serum prostate-specific antigen (PSA) level measurement. Interestingly, 23 of 44 localised tumours exhibited aberrant E-cadherin expression. Prior to primary surgery, PSMA RT-PCR detected the spread of prostate cells to the blood in 24 patients. Statistical analysis showed that abnormal E-cadherin expression in the tumours was the only variable that was independently correlated with prostate cell dissemination in the blood (P

Assessing the activity of a particular drug-metabolizing enzyme (i.e., its phenotypic status) has practical implications in the fields of therapeutic drug monitoring and clinical toxicology as well as for the biological monitoring of environmental and/or occupational exposure to chemical agents. This report describes a novel, convenient, and rapid analytical method for phenotyping cytochrome P4502E1 (CYP2E1), an important drug-metabolizing enzyme in industrial toxicology (1). The measurement of CYP2E1 expression also has applications for monitoring alcoholic patients (2). Although the most accurate assessment of CYP2E1 activity is based on direct hepatic measurement, which requires a liver biopsy, alternative noninvasive approaches have been developed. Until now, the “gold standard” method for CYP2E1 phenotyping has used a probe drug, chlorzoxazone (CZX), administered orally (500 or 250 mg) to fasting individuals and metabolized to 6-hydroxychlorzoxazone (HCZX) by CYP2E1. Various CZX pharmacokinetic measures have been proposed to reflect CYP2E1 activity, as reviewed recently (3): plasma CZX t 1/2β, HCZX renal excretion, CZX oral clearance, fractional clearance to HCZX, and HCZX area under the concentration curve (AUC). However, these procedures are time-consuming, laborious (e.g., urine collection), and require subject compliance and catheterization. Furthermore, recent in vitro studies suggest that CYP1A1, CYP1A2, and possibly CYP3A are involved in CZX 6-hydroxylation, limiting the specificity of CZX for CYP2E1 phenotyping (3). CYP2E1 phenotyping for screening or follow-up of large numbers of individuals, in epidemiological studies, or even as an adjunct to improve biological monitoring programs requires the implementation of more convenient methods. Circulating lymphocytes have been used as surrogates because CYP2E1 expression in these cells appears to parallel the concentration of the hepatic enzyme, suggesting that both cell types are influenced by the same factors, including xenobiotics and physiological state (4). In a recent report involving alcoholics and controls (2), pharmacokinetic parameters for CZX hydroxylation (CZX clearance …

Human prostate-specific membrane antigen (PSMA), a 100-kDa integral transmembrane glycoprotein, is considered to be a highly specific marker of the prostate gland, and has successfully been used as a marker of circulating prostatic epithelial cells. Extended PSMA homology has been demonstrated with a cDNA found in rat cerebral and renal tissues. In this study, we aimed to evaluate the expression of PSMA mRNA in a variety of human renal cancer tissues (n = 20) and cell lines (n = 12). Using reverse transcriptase-polymerase chain reaction, DNA sequencing, blottings, and specific anti-PSMA labelling with CYT 351 antibody, we identified PSMA mRNA and protein in normal and in neoplastic renal tissue. The sequence of the polymerase-chain-reaction products is identical to that of PSMA cDNA derived from prostate tissue. Immunological staining with the CYT 351 reveals that PSMA is expressed mainly in tubular cells. Since PSMA does not appear to be restricted to prostatic tissue, this novel biomarker may prove useful in the staging of renal cancer and in the search for the hematogenous spread of renal cells.

Although not in routine use, molecular techniques appear able to detect small numbers of solid tumor cells in blood, lymph nodes, and bone marrow (for review see 1 ). Provided that a given mRNA is expressed exclusively in tumor tissue and not by nucleated blood cells, lymph node cells, or bone marrow cells, its presence outside the organ identifies disseminated metastatic cells. In prostate cancer (CaP), early detection of metastases by molecular staging could help in selecting which patients would benefit most from surgical therapy (2). Several reverse transcriptase-polymerase chain reaction (RT-PCR) protocols are able to amplify the mRNAs encoding prostate-specific antigen (PSA) or prostate-specific membrane antigen (PSMA) in blood (1), lymph nodes (3), or bone marrow (4). Nevertheless, the results reported have varied so widely (e.g., positive PSA RT-PCR results ranging from 25% (5) to 80% (6) for metastatic CaP patients) that definitive conclusions about the clinical relevance of this new molecular tool have not yet been established. The heterogeneity of both preanalytical and analytical steps of PCR protocols may explain the discrepancies observed between studies. To our knowledge, no systematic studies of the successive steps of the RT-PCR method have yet been performed. To better standardize the assay, we have assessed the effects of the storage conditions of blood samples and delay before processing. To compare the effect of delayed vs immediate sample processing, we collected 250-mL blood samples in 25 10-mL EDTA-treated tubes (Becton Dickinson) from 4 …

Multiplex amplification of femA and mecA genetic determinants allowed an early and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) in endotracheal aspirates of mechanically ventilated patients. femA and/or mecA amplification and bacteriological results were concordant in 57 of 60 samples. In all three discrepant cases, complementary bacteriological tests confirmed the presence of MRSA first identified by molecular analysis. These results underline the value and rapidity of this molecular diagnosis for MRSA infection and control surveillance in intensive care units. Rapid MRSA detection is expected to have a significant clinical impact not only on patient outcome but also on the costs for isolation and treatment.

Mycobacterium simiae is commonly found in nature and its role as a pathogen has been controversial. A case of disseminated M. simiae infection with blood, pulmonary and cutaneous localization is reported here. The pathogenic role of M. simiae was clearly demonstrated as it was the only organism isolated from sputum, broncho-alveolar lavage fluid, as well as blood and skin tissue. Identification of M. simiae by conventional testing may be difficult. Analysis of fatty and mycolic acid patterns, as performed in this case, is necessary to confirm its identification.