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Background: Obesity and diabetes mellitus are independently associated with the development of heart failure. In this study, we determined the respective effects of obesity, insulin resistance, and diabetes mellitus on the intrinsic contraction and mitochondrial function of the human myocardium before the onset of cardiomyopathy., Methods and Results: Right atrial myocardium was obtained from 141 consecutive patients presenting no sign of cardiomyopathy. We investigated ex vivo isometric contraction, mitochondrial respiration and calcium retention capacity, and respiratory chain complex activities and oxidative stress status. Diabetes mellitus was associated with a pronounced impairment of intrinsic contraction, mitochondrial dysfunction, and increased myocardial oxidative stress, regardless of weight status. In contrast, obesity was associated with less pronounced contractile dysfunction without any significant perturbation of mitochondrial function or oxidative stress status. Tested as continuous variables, glycated hemoglobin A1C, but neither body mass index nor the insulin resistance index (homeostasis model assessment-insulin resistance), was independently associated with cardiac mitochondrial function. Furthermore, diabetes mellitus was associated with cardiac mitochondrial network fragmentation and significantly decreased expression of the mitochondrial fusion related protein MFN1. Myocardial MFN1 content was inversely proportional to hemoglobin A1C., Conclusion: Worsening of intrinsic myocardial contraction in the transition from obesity to diabetes mellitus is likely related to worsening of cardiac mitochondrial function because impaired mitochondrial function and dynamics and contractile dysfunction are observed in diabetic patients but not in "metabolically healthy" obese patients at early stage in insulin resistance., (© 2014 American Heart Association, Inc.)

Objectives: Amylase concentration in respiratory secretions was reported to be a potentially useful marker for aspiration and pneumonia. The aim of this study was to determine accuracy of α-amylase in diagnosing microaspiration in critically ill patients., Methods: Retrospective analysis of prospectively collected data collected in a medical ICU. All patients requiring mechanical ventilation for at least 48 h, and included in a previous randomized controlled trial were eligible for this study, provided that at least one tracheal aspirate was available for α-amylase measurement. As part of the initial trial, pepsin was quantitatively measured in all tracheal aspirates during a 48-h period. All tracheal aspirates were frozen, allowing subsequent measurement of α-amylase for the purpose of the current study. Microaspiration was defined as the presence of at least one positive tracheal aspirate for pepsin (>200 ng.mL-1). Abundant microaspiration was defined as the presence of pepsin at significant level in >74% of tracheal aspirates., Results: Amylase was measured in 1055 tracheal aspirates, collected from 109 patients. Using mean α-amylase level per patient, accuracy of α-amylase in diagnosing microaspiration was moderate (area under the receiver operator curve 0.72±0.05 [95%CI 0.61-0.83], for an α-amylase value of 1685 UI.L-1). However, when α-amylase levels, coming from all samples, were taken into account, area under the receiver operator curve was 0.56±0.05 [0.53-0.60]. Mean α-amylase level, and percentage of tracheal aspirates positive for α-amylase were significantly higher in patients with microaspiration, and in patients with abundant microaspiration compared with those with no microaspiration; and similar in patients with microaspiration compared with those with abundant microaspiration. α-amylase and pepsin were significantly correlated (r2 = 0.305, p = 0.001)., Conclusion: Accuracy of mean α-amylase in diagnosing microaspiration is moderate. Further, when all α-amylase levels were taken into account, α-amylase was inaccurate in diagnosing microaspiration, compared with pepsin.

Loss of TP53 function through gene mutation is a critical event in the development and progression of colorectal cancer (CRC). Here we examined 51 primary CRC tumors from Tunisia for mutations in TP53 exons 4-9 using PCR-direct sequencing. TP53 status and mutation site/type were than correlated with nuclear protein accumulation, familial and clinicopathologic variables and data on KRAS mutations and microsatellite instability (MSI-H). The TP53 mutation analysis was possible in the tumor of 47 patients and a deleterious somatic mutation has been detected in 59.6% of the patients (28/47) including 20 (71.4%) missense mutations, 7 nonsense mutations (25%) and 1 (3.6%) frameshift mutation. 89.3% (25/28) of the detected mutations were in exons 5-8, whereas 10.7% (3/28) were in exon 4. Among the 27 non frameshift mutations, 89% (24/27) were transitions and 11% (3/27) were transversions. 64.3% (18/27) of the altered amino acids corresponded to arginine. 74% (20/27) were G>C to A>T transitions, and more than half (14/27) occur at hotspots codons with CpG sites. TP53 mutations correlated closely with TP53 accumulation (p = 0.0090) and inversely with MSI phenotype (p = 0.0658). A KRAS somatic mutation was identified in 25% (7/28) of the TP53 mutated tumors. All these mutations were G>A transitions in codon 12 and all the tumors with combined alterations but one were distally located and MSS. In conclusion, frequency and types of TP53 mutations and correlations with TP53 protein accumulation, and MSI were as reported for non-Tunisian patients. However, no significant associations have been detected between TP53 mutations and clinicopathological data in Tunisian patients as previously reported.

Background: The primary objective of this study was to determine the efficiency of a pneumatic device in controlling cuff pressure (Pcuff) in patients intubated with polyurethane-cuffed tracheal tubes. Secondary objectives were to determine the impact of continuous control of Pcuff, and cuff shape on microaspiration of gastric contents., Methods: Prospective randomized controlled study. All patients requiring intubation and mechanical ventilation ≥48 h were eligible. The first 32 patients were intubated with tapered polyurethane-cuffed, and the 32 following patients were intubated with cylindrical polyurethane-cuffed tracheal tubes. Patients randomly received 24 h of continuous control of Pcuff using a pneumatic device (Nosten®), and 24 h of routine care of Pcuff using a manometer. Target Pcuff was 25 cmH2O. Pcuff was continuously recorded, and pepsin was quantitatively measured in all tracheal aspirates during these periods., Results: The pneumatic device was efficient in controlling Pcuff (med [IQ] 26 [24, 28] vs 22 [20, 28] cmH2O, during continuous control of Pcuff and routine care, respectively; p = 0.017). In addition, percentage of patients with underinflation (31% vs 68%) or overinflation (53% vs 100%) of tracheal cuff, and percentage of time spent with underinflation (0.9 [0, 17] vs 14% [4, 30]) or overinflation (0 [0, 2] vs 32% [9, 54]) were significantly (p < 0.001) reduced during continuous control of Pcuff compared with routine care.No significant difference was found in microaspiration of gastric content between continuous control of Pcuff compared with routine care, or between patients intubated with tapered compared with cylindrical polyurethane-cuffed tracheal tubes., Conclusion: The pneumatic device was efficient in controlling Pcuff in critically ill patients intubated with polyurethane-cuffed tracheal tubes., Trial Registration: The Australian New Zealand Clinical Trials Registry (NCT01351259).

Mutations in KRAS gene are among the critical transforming alterations occurring during CRC tumorigenesis. Here we screened 51 primary CRC tumors from Tunisia for mutations in KRAS (codons 12 and 13) using PCR-direct sequencing. Our aim was to analyze tumor mutation frequencies and spectra in Tunisian patients with CRC. KRAS status and mutation site/type were than correlated with familial and clinicopathologic variables and data on TP53 mutations and nuclear protein accumulation and microsatellite instability (MSI). A KRAS somatic mutation has been detected in the CRC tumor of 31.5 % (16/51) of the patients. 81.2 % had a single mutation at codon 12 and 23 % had a single mutation at codon 13. The most common single mutation (50 %) was a G>A transition in codon 12 (c.35G>A; p.Gly12Asp). 81.25 % of the KRAS mutations were transitions and 23 % were transversions. All the mutations in codon 13 were a c.38G>A transition, whereas both G>A transitions and G>T and G>C transversions were found in codon 12. The mutation spectrum was different between MSS and MSI-H tumors and more varied mutations have been detected in MSS tumors. Some amino acid changes were detected only in MSS tumors, i.e. p.Gly12Ser, p.Gly12Cys and p.Gly12Ala. Whereas, the KRAS mutation p.Gly13Asp have been detected only in MSI-H. 43.75 % of the patients harboured combined mutations in KRAS and TP53 genes and the tumor of 71.42 % of them showed TP53 overexpression. In conclusion, the frequency and types of KRAS mutations were as reported for non-Tunisian patients. However, no significant associations have been detected between KRAS mutations and clinicopathologic variables and MSI in Tunisian patients as previously reported.

Objectives: This study sought to provide bedside evidence of the potential link between cardiac mitochondrial dysfunction and arrhythmia as reported in bench studies., Background: Atrial fibrillation (AF) is a frequent complication of cardiac surgery. Underlying mechanisms of post-operative atrial fibrillation (POAF) remain largely unknown. Because cardiac mitochondrial dysfunction has been reported in clinical conditions with a high risk of POAF, we investigated whether a causal link exists between POAF onset and pre-operative function of cardiac mitochondria., Methods: Pre-operative mitochondrial respiration and calcium retention capacity, respiratory complex activity, and myocardial oxidative stress were quantified in right atrial tissue from 104 consecutive patients with metabolic syndrome, in sinus rhythm, and undergoing coronary artery bypass graft surgery., Results: In this high-risk population, POAF occurred in 44% of patients. Decreased pre-operative mitochondrial respiration and increased sensitivity to calcium-induced mitochondrial permeability transition pore opening were significantly associated with POAF. Adenosine diphosphate-stimulated mitochondrial respiration supported by palmitoyl-l-carnitine was significantly lower in POAF patients and remained independently associated with AF onset after adjustment for age, body mass index, heart rate, beta-blocker use, and statin medication (multivariate logistic regression coefficient per unit = -0.314 ± 0.144; p = 0.028). Gene expression profile analysis identified a general downregulation of the mitochondria/oxidative phosphorylation gene cluster in pre-operative atrial tissue of patients in whom AF developed., Conclusions: Our prospective study identifies an association between pre-operative mitochondrial dysfunction of the atrial myocardium and AF occurrence after cardiac surgery in patients with metabolic disease, providing novel insights into the link between mitochondria and arrhythmias in patients., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: Basophils and eosinophils represent less than 1 and 5% of white blood cells, respectively. Their role in asthma and allergic inflammation remains incompletely defined. The present review addresses recent advances regarding the role of these two cell populations in allergic inflammation and asthma regarding both biological and genetic point of view., Recent Findings: Regarding eosinophils, the role of interleukin(IL)-25, IL-33 and thymic stromal lymphoprotein (TSLP) have been evidenced, and activation states of eosinophil β1 and β2 integrins have been found to correlate with the measurement of eosinophil recruitment and pulmonary function in asthma. New insights into the biology of basophils concern their role as regulators of Th2 cell response through IL-4 expression or the differentiation of monocytes to macrophages, and their population heterogeneity in human. The transcription factor PU.1 was reported to be involved in controlling transcription of specific genes both in eosinophils and basophils. Candidate genetic studies on eosinophils have explored genes involved in the intracellular calcium influx and apoptosis. At the genome-wide level, studies identified genetic variants belonging to IL1RL1, TSLP and IL-33, and four loci with pleiotropic effects on eosinophil and basophil counts [GATA2 (3q21), MHC (6p21), HBS1L-MYB (6q23), and ERG (21q22)]., Summary: Recent findings from biological and genetic studies on eosinophils and basophils highlight the role of epithelial cell-derived cytokines such as TSLP and IL-33 in asthma and allergic diseases.

Aim: To determine correlations between family history, clinical features and mutational status of genes involved in the progression of colorectal cancer (CRC)., Methods: Histo-pathological features and molecular changes [KRAS, BRAF and CTNNB1 genes mutations, microsatellite instability (MSI) phenotype, expression of mismatch repair (MMR) and mucin (MUC) 5AC proteins, mutation and expression analysis of TP53, MLH1 promoter hypermethylation analysis] were examined in a series of 51 unselected Tunisian CRC patients, 10 of them had a proven or probable hereditary disease, on the track of new tumoral markers for CRC susceptibility in Tunisian patients., Results: As expected, MSI and MMR expression loss were associated to the presence of familial CRC (75% vs 9%, P < 0.001). However, no significant associations have been detected between personal or familial cancer history and KRAS (codons 12 and 13) or TP53 (exons 4-9) alterations. A significant inverse relationship has been observed between the presence of MSI and TP53 accumulation (10.0% vs 48.8%, P = 0.0335) in CRC tumors, suggesting different molecular pathways to CRC that in turn may reflect different environmental exposures. Interestingly, MUC5AC expression was significantly associated to the presence of MSI (46.7% vs 8.3%, P = 0.0039), MMR expression loss (46.7% vs 8.3%, P = 0.0039) and the presence of familial CRC (63% vs 23%, P = 0.039)., Conclusion: These findings suggest that MUC5AC expression analysis may be useful in the screening of Tunisian patients with high risk of CRC.

This study evaluated the associations between biological markers in the nitrate-nitrite-NO pathway and four environmental exposures among subjects examined in the second survey (2003-2007) of the French Epidemiological study on Genetics and Environment of Asthma (EGEA). Total nitrite and nitrate (NO(2)(-) /NO(3)(-)) levels were measured both in plasma and in exhaled breath condensate (EBC) in 949 adults. Smoking, diet and exposure to chlorine products were assessed using standardized questionnaires. Exposure to air pollutants was estimated by using geostatistical models. All estimates were obtained with generalized estimating equations for linear regression models. Median levels of NO(2)(-)/NO(3)(-) were 36.3 μM (1st-3rd quartile: 25.7, 51.1) in plasma and 2.0 μmol/mg proteins (1st-3rd quartile 0.9, 3.9) in EBC. After adjustment for asthma, age, sex and menopausal status, plasma NO(2)(-)/NO(3)(-) level increased with leafy vegetable consumption (above versus below median=0.04 (95%CI: 0.001, 0.07)) and decreased in smokers (versus non/ex-smokers=-0.08 (95%CI: -0.11, -0.04). EBC NO(2)(-)/NO(3)(-) level decreased in smokers (-0.08 (95%CI: -0.16, -0.001)) and with exposure to ambient O(3) concentration (above versus below median=-0.10 (95%CI: -0.17, -0.03)). Cured meat, chlorine products, PM(10) and NO(2) concentrations were not associated with NO(2)(-)/NO(3)(-) levels. Results suggest that potential modifiable environmental and behavioral risk factors may modify NO(2)(-)/NO(3)(-) levels in plasma and EBC according to the route of exposure., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Microaspiration of contaminated oropharyngeal secretions and gastric contents frequently occurs in intubated critically ill patients, and plays a major role in the pathogenesis of ventilator-associated pneumonia. Risk factors for microaspiration include impossible closure of vocal cords, longitudinal folds in high-volume low-pressure polyvinyl chloride cuffs, and underinflation of tracheal cuff. Zero positive end expiratory pressure, low peak inspiratory pressure, tracheal suctioning, nasogastric tube and enteral nutrition increase the risk for microaspiration. Other patient related factors include supine position, coma, sedation, and hyperglycemia. Technetium 99 labelled enteral feeding is probably the most accurate marker of microaspiration in critically ill patients. However, use of this radioactive marker is restricted to nuclear medicine departments. Blue methylene is a reliable qualitative marker of microaspiration. However, fiberoptic bronchoscopy is required to diagnose microaspiration of blue dye in ICU patients. Quantitative pepsin measurement in tracheal aspirates is accurate in diagnosing microaspiration of gastric contents in critically ill patients. In addition, this marker is easy to use in routine practice. However, pepsin should be detected rapidly after aspiration. In vitro, and clinical studies suggested that semirecumbent position, polyurethane cuffs, positive end expiratory pressure, low-volume low-pressure cuff, and continuous control of cuff pressure were efficient in reducing microaspiration in ICU patients. Other preventive measures such as subglottic aspiration, tapered shape cuff, guayule latex cuff, lateral horizontal patient position, gastrostomy tube, and postpyloric feeding require further investingation.

Objectives: We checked the efficiency of a new dialysis device adapted to small volumes to remove glycerol from cryopreserved red blood cells., Design and Methods: Dialysis was performed on D-Tube96™ Dialyzer Mini device. In a preliminary trial, we measured the residual glycerol before, and 2, 4 and 24 h after dialysis. Glycerol and hemoglobin concentrations and antioxidant enzymes activities were measured in three samples with or without glycerolization/deglycerolization procedure. The mini dialysis was then applied to 96 samples from the French Epidemiological study on the Genetics and Environment of Asthma., Results: Ninety-two percent of glycerol was removed after 24 h of dialysis. Hemoglobin content and activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase were recovered. No significant loss of volume was observed. Results obtained for the 96 samples perfectly fitted with reference values of our laboratory., Conclusion: This new dialysis method seems to be particularly adapted for processing a large number of samples of RBCs cryoconserved in small volumes from epidemiological studies., (Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Purpose: High rates of early colorectal cancers (CRC) are observed in Tunisia suggesting genetic susceptibility. Nevertheless, up to now, no molecular study has been performed in the Tunisian population. In our research, we evaluated the clinical characteristics of Tunisian families suspected of Lynch syndrome and the contribution of DNA mismatch repair (MMR) genes., Methods: Thirty-one unrelated families suspected of Lynch syndrome were studied. Probands were tested for the presence of germline mutations in the MMR genes MLH1, MSH2, MSH6 and in MUTYH. Available tumours were analysed for microsatellite instability and expression of MMR proteins. Detailed family and medical histories were collected., Results: A total of 134 cancers were noted in the 31 families, the most frequent type of cancer corresponding to CRC (69%), followed by uterine cancer (7.5%). Germline mutations were identified in 11 (35.5%) families (six MSH2, five MLH1, including seven novel mutations), seven of which fulfilled the Amsterdam criteria (sensitivity, 63.6%; positive predictive value, 58.3%). Noteworthy, germline mutations were detected in 52.6% of male patients tested, but in only 8.3% of females (p = 0.02). Moreover, CRC were essentially left sided in families without detected mutation (p = 0.017). Ages of onset of cancers and tumour spectrum were very similar in families with or without MMR germline mutation, contrasting with previous studies performed in other populations., Conclusions: MMR genes contribute significantly to CRC susceptibility in the Tunisian population. However, the cause of early CRC susceptibility remains unknown in most cases, especially in women and in patients with early left colon or rectal cancer.

Objective: To determine the impact of polyurethane (PU) on variations in cuff pressure (P (cuff)) in intubated critically ill patients., Methods: Prospective observational before-after study performed in a ten-bed ICU. Cuff pressure was continuously recorded for 24 h in 76 intubated patients, including 26 with polyvinyl chloride (PVC), 22 with cylindrical polyurethane (CPU), and 28 with tapered polyurethane (TPU)-cuffed tracheal tubes. P (cuff) was manually adjusted every 8 h by nurses and was maintained around 25 cmH(2)O. Time spent with cuff underinflation and overinflation was continuously measured. In addition, pepsin, a proxy for microaspiration of gastric contents, was quantitatively measured in tracheal secretions at the end of recording period., Results: A total of 1,824 h of continuous recording of cuff pressure was analyzed. Patient characteristics were similar in the three groups. No significant difference was found in percentage of time spent with underinflation (mean +/- SD, 26 +/- 22, 28 +/- 12, 30 +/- 13% in PVC, CPU, and TPU groups, respectively) and overinflation [median (IQR), 7 (2-14), 6 (3-14), 11% (5-20)] among the three groups. However, a significant difference was found in the coefficient of variation of P (cuff) (mean +/- SD, 82 +/- 48, 92 +/- 47, 135 +/- 67, p = 0.002). While the coefficient of P (cuff) variation was significantly (p < 0.017) higher in the TPU compared to CPU and PVC groups, no significant difference was found between the CPU and PVC groups. The pepsin level was significantly different among the three groups (408 +/- 282, 217 +/- 159, 178 +/- 126 ng/ml; p < 0.001). In fact, the pepsin level was significantly lower in the CPU and TPU groups compared with the PVC group., Conclusion: PU does not impact variations in P (cuff) in critically ill patients.

Unlabelled: We assessed comparatively the diagnostic value of two potential malignant pleural mesothelioma (MPM) markers: hyaluronic acid (HA) and soluble mesothelin., Materials and Method: We measured serum and pleural fluid values of mesothelin and hyaluronic acid in 76 patients with MPM, 33 patients with pleural metastases of carcinomas (Mets group) and 27 patients with benign pleural effusion related to asbestos exposure (BPLAE)., Results: Using a serum HA cut-off of 100 microg/L, 8 patients/33 (24.2%) were positive in the Mets group versus 20/76 (26.3%) in the MPM group and only 1/27 BPLAE patients. The area under ROC curve for serum HA in MPM versus Mets or BPLAE groups was only 0.617 while it was 0.755 for mesothelin. In pleural fluid, both markers had similar diagnostic values., Conclusions: Serum mesothelin is more sensitive than hyaluronic acid in diagnosing MPM and there is no benefit in combining both markers.

Background: Although E-cadherin and beta-catenin are key regulators in tumor invasion and proliferation, few studies have been undertaken on the expression of these genes at the messenger ribonucleic acid (mRNA) level in relation to the progression of colon cancer., Patients and Methods: In this study, tissue samples from colectomy (n = 37) or hepatectomy (n = 23) were collected in both tumor and adjacent normal tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction was used to quantify E-cadherin and beta-catenin mRNAs in reference to 18S RNA., Results: E-cadherin and beta-catenin levels in colon carcinomas were not statistically different compared with adjacent normal mucosa and were not correlated with tumor, nodes, and metastases (TNM) stage. Conversely, E-cadherin and beta-catenin levels were significantly higher in liver metastases than in adjacent normal tissue. Interestingly, we found that E-cadherin level in liver metastases was correlated to the TNM stage of the related primary tumor: a higher E-cadherin level was found for State I-II TNM. In addition, a high expression of E-cadherin in liver metastases was associated with a lower occurrence of extra-hepatic metastases after resection of liver metastases., Conclusion: Taken together, these data show that E-cadherin and beta-catenin expressions are regulated throughout colon cancer progression.

The glycocalyx constitutes the first line of the blood tissue interface and is thus involved in many physiological processes, deregulation of which may lead to microvascular dysfunction. Because administration of LPS is accompanied by severe microvascular dysfunction, the purpose of the study was to investigate microvascular glycocalyx function during endotoxemia. Bolus infusion of LPS (10 mg kg(-1)) to male Sprague-Dawley rats elicited the development of hyporeactivity to vasoactive agents and microvascular derangements, including decreased capillary density and significant increases in intermittent and stopped flow capillaries in the small intestine muscularis layer compared with controls. LPS elicited plasma hyluronan release and reduction in endothelial surface thickness, indicative of glycocalyx degradation. Because endothelial glycocalyx is extremely sensitive to free radicals, oxidative stress was evaluated by oxidation of dihydrorhodamine in microvascular beds and levels of heart malondialdehyde and plasma carbonyl proteins, which were all increased in LPS-treated rats. Activated protein C (240 microg kg(-1) h(-1)) enhanced systemic arterial pressure response to norepinephrine in LPS-treated rats. Activated protein C (240 microg kg(-1) h(-1)) prevented capillary perfusion deficit in the septic microvasculature that were associated with reduced oxidative stress and preservation of glycocalyx. Our findings support the conclusion that LPS induces major microcirculation dysfunction accompanied by microvascular oxidative stress and glycocalyx degradation that may be limited by activated protein C treatment.

Background: Laboratory evaluation of alpha 1-antitrypsin (A1AT) deficiency is generally performed by determination of A1AT concentrations and identification of specific allelic variants by phenotyping. For this purpose, we evaluated a new Hydragel 18 A1AT Isofocusing kit on the semi-automatic Hydrasys System (Sebia) for the determination of A1AT phenotypes by isoelectrofocusing on ready-to-use agarose gels with specific immunological detection., Methods: Serum samples from 66 patients were analysed with this new kit in comparison with the conventional and manually performed isoelectrofocusing method on polyacrylamide gels with Coomassie Blue staining., Results: A1AT phenotypes showed comparable iso-electrofocusing patterns in both systems. The good within-gel reproducibility of this kit was demonstrated using two normal serum samples (M1 and M1M2 phenotypes) and six pathological serum samples with different phenotypes (MS, SS, SZ, MZ, ZZ). A sensitivity study was undertaken by performing serial dilutions on a serum with a ZZ phenotype containing 0.27 g/L A1AT. The detection limit was 0.050 g/L., Conclusions: This new method is highly specific, rapid and simple to perform. It improves identification of not only the most common but also various rare A1AT phenotypes. It appears to be suitable for routine analysis and screening applications in a clinical laboratory setting.

A successful prevention of renal diseases induced by occupational exposure to lead (Pb) and/or cadmium (Cd) largely relies on the capability to detect nephrotoxic effects at a stage when they are still reversible or at least not yet compromising renal function. Hence, the aim of this cross-sectional study was to evaluate the usefulness of a set of early biological markers of oxidative stress or nephrotoxicity for the biomonitoring of workers occupationally exposed to Pb and/or Cd in a non-ferrous metal smelter, and gender, age, socioeconomic status, smoking habits, and drug use-matched control individuals. In exposed subjects, mean levels of Pb in blood and urine were also 387.1+/-99.1 microg Pb/L (1.868+/-0.478 micromol Pb/L) and 217.7+/-117.7 microg Pb/g creatinine (1.051+/-0.568 micromol Pb/g creatinine), and mean levels of Cd in blood and urine were 3.26+/-2.11 microg Cd/L (0.029+/-0.019 micromol Cd/L) and 2.51+/-1.89 microg Cd/g creatinine (0.022+/-0.017 micromol Cd/g creatinine), suggesting thereby relatively low occupational exposure levels. Statistically significant variations in zinc protoporphyrin, malondialdehyde, retinol binding protein, alpha-glutathione S-transferase, and urinary protein levels were reported between the two groups, and were closely correlated with Pb and/or Cd exposure levels. Variations in alphaGST levels were closely associated with Pb exposure. Taken together, these results suggest the use of alpha-glutathione S-transferase excretion in urine as a hallmark of early changes in the proximal tubular integrity.

The rapid identification of pathogenic yeasts is a crucial step in ensuring that effective antifungal treatment is started as early as possible. CandiSelect 4 (CS4; Bio-Rad) is a new chromogenic medium for the isolation of fungi, the direct identification of Candida albicans and the presumptive identification of the major pathogenic Candida species. The performance of CS4 was compared with that of another chromogenic medium, CHROMagar Candida (CA; Becton Dickinson). For primary cultures, 502 of the 1549 (32 %) samples were culture-positive. A total of 542 yeasts were isolated including 465 monomicrobial and 37 mixed cultures: 392 C. albicans, 60 Candida glabrata, 25 Candida tropicalis, 12 Candida krusei and 53 other Candida species. The percentage of C. albicans isolates that could be identified directly after 24, 48 and 72 h culture was 31.6, 82.9 and 92.1 %, respectively, for CS4, and 32.9, 82.9 and 91.1 % for CA. The presumptive identification of C. glabrata, C. tropicalis and C. krusei was evaluated after 48 h incubation. The percentage of strains with morphologically typical colonies was 80, 68 and 84.6 %, respectively, for CS4 compared with 75, 76 and 76.9 % for CA. For pure subcultures, from 24 h, all isolates of C. albicans (n=21) were directly identifiable on the two chromogenic media CA and CS4. At 48 h, the proportion of typical strains observed on the two chromogenic media was identical for C. glabrata (85 %) and C. krusei (100 %). A slight difference in favour of CS4 was observed for C. tropicalis (100 vs 95 %). CS4 also allowed the growth of several other fungi. CS4 can be recommended as a primary isolation medium for the identification of C. albicans, and for the rapid and effective differentiation of the major pathogenic Candida species.

Aim: To investigate the expression of the four secreted gel-forming mucins (MUC2, MUC5AC, MUC5B and MUC6) in a series of gastric carcinomas, classified according Lauren's, Mulligan's, WHO and Goseki's classifications, with special attention to all the different components (major and minor) present in tumors and to follow up clinical data., Methods: Expression of MUC2, MUC5AC, MUC5B and MUC6 was investigated using immunohistochemistry and in situ hybridization., Results: Expression of secreted gel-forming mucins in gastric carcinoma was particularly complex, each mucin being not restricted to any histopathological type even considering all components (major and minor) present in a given tumor. There was a worst survival in patients with a higher content of mucus (Goseki II or IV) and high positive MUC2 expression., Conclusion: Complexity of mucin gene expression patterns in gastric cancer may reflect a precise state of differentiation at the cell level not recognized in used morphologic classification systems. High expression of MUC2 was nevertheless associated with mucinous subtype of the WHO classification and with group II of Goseki's classification identified by the major component of a particular tumor. The quantity and quality of mucus were related to survival.

Exposure to urban airborne particulate matter (PM) has been associated with adverse health effects. In this work, we focused our attention on the capacity of air pollution PM to induce cytotoxic, oxidative stress, and inflammatory responses in human epithelial lung cells (L132) in culture. PM were collected in Dunkerque, a French seaside city, and their physical and chemical characteristics were carried out. Their size distribution showed that 92.15% of the PM were equal or smaller than 2.5 and their specific surface area was 1 m2/g. Inorganic (i.e. Fe, Al, Ca, Na, K, Mg, Pb, etc.) and organic (i.e. VOC, PAH, etc.) chemicals were found in PM. Physical and chemical properties of Dunkerque City's PM suggested that much of the collected PM derived from wind-borne dust from the industrial complex and the heavy motor vehicle traffic. Their cytotoxicity, as evaluated by survival rate determination, lactate dehydrogenase activity, and mitochondrial dehydrogenase activity showed concentration and time-dependent effects in L132 cells (LC10 = 18.84 microg PM/ml; LC50 = 75.36 microg PM/ml). Moreover, in PM-exposed L132 cells, there were concentration- and time-dependent changes in lipid peroxidation, superoxide dismutase activity, 8-hydroxy-2'-deoxyguanosine formation, and poly(ADP-ribosyl)ation, on the one hand, and in tumor necrosis factor-alpha secretion, inducible nitric oxide synthase activity, and nitric oxide release, on the other hand. Taken together, these findings suggested that oxidative stress and inflammatory responses proceeded cytotoxicity in PM-exposed L132 cells.

Background: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a role in the processes of extracellular matrix degradation. Changes in their expression levels have been observed in various tumor types, including lung carcinoma. However, their clinical significance and their prognostic importance in the progression of nonsmall cell lung carcinoma (NSCLC) remain to be specified. In this study, mRNA expression levels of MMP-1, MMP-9, TIMP-1, and TIMP-2 were evaluated in patients with resected NSCLC, and their associations with disease progression and prognosis were determined., Methods: Between June 1996 and December 1999, 116 patients underwent resection for NSCLC. Expression levels of MMPs and TIMPs were evaluated using Northern blot analysis in these NSCLC tissue samples and in 39 matched samples of normal lung tissue., Results: MMP-1, MMP-9, and TIMP-1 expression levels were increased in tumor samples compared with matched, corresponding normal tissues. In contrast, TIMP-2 expression was decreased in tumor samples. MMP-1 tumor expression was correlated significantly with the evolution of lymph node status and tumor-lymph node-metastasis (TNM) stage. In contrast, MMP-9 tumor expression was correlated significantly with increased T stage. TIMP-1 overexpression was an independent predictor of worse survival in patients with NSCLC that was not associated with other prognosis factors, such as TNM stage., Conclusions: The overexpression of TIMP-1 was an independent prognostic marker in patients with NSCLC, and evaluating TIMP-1 may be important for identifying patients who are at greater risk of disease recurrence., ((c) 2005 American Cancer Society.)

Objectives: In this work, we studied impregnation levels of workers occupationally exposed to lead (Pb) and cadmium (Cd), usefulness of early urinary markers of nephrotoxicity, and occurrence of oxidative stress as the underlying mechanism involved in Pb- or Cd-induced adverse effects. Thirty-five men were recruited from a nonferrous metal smelter. Pb and Cd in blood (B-Pb, B-Cd) and urine (U-Pb, U-Cd) were measured. Relations between oxidative stress markers (malondialdehyde, superoxide dismutase, glutathione peroxidase, selenium, glutathione reductase, glutathione status, 8-hydroxy-2'-deoxyguanosine) and exposure levels, on the one hand, and early urinary markers (alpha-1-microprotein, beta-2-microglobulin, retinol binding protein, alpha and pi-glutathione S-transferases) and exposure levels, on the other hand, were evaluated., Results: Mean exposure levels were moderate (B-Pb = 395.71 microg Pb/L; U-Pb = 95.19 microg Pb/g creatinine; B-Cd = 5.83 microg Cd/L; U-Cd = 4.67 microg Cd/g creatinine). Changes in malondialdehyde, glutathione status, 8-hydroxy-2'-deoxyguanosine, and alpha-glutathione S-transferases were closely correlated with exposure levels and did not depend on tobacco consumption. We showed that these workers showed moderate Pb and Cd exposure levels., Conclusions: Taken together, the data suggests the use of alpha-glutathione S-transferases excretion in urine as a hallmark of early changes in the proximal tubular integrity that could later lead to clinical disease if exposure is not reduced.

Any influence of iron in polycyclic aromatic hydrocarbon (PAH)/iron oxide mixtures on the capacity of PAHs to induce metabolizing enzymes will be one of the ways that iron oxides can affect PAH carcinogenicity. Because cytochromes P450 (CYPs) are haemoproteins, it will be of interest to investigate the possible involvement of Fe(2)O(3) in benzo[a]pyrene (BaP)/Fe(2)O(3) mixtures on the induction of CYP1A1 enzymes in the lung. Male Sprague-Dawley rats were instilled intratracheally with haematite ((56)Fe(2)O(3) or (54)Fe(2)O(3), 3 mg), BaP (3 mg) or BaP (3 mg) coated onto haematite ((56)Fe(2)O(3) or (54)Fe(2)O(3)) particles (3 mg). Firstly, mRNA expressions of cyp1a1 were studied. Secondly, protein concentrations and catalytic activities (7-ethoxyresorufin O-deethylase: EROD) of CYP1A1 were determined. Thirdly, (54)Fe from BaP/(54)Fe(2)O(3) mixtures in microsomal proteins was studied using time-of- flight laser microprobe mass spectrometry (ToF-LMMS). Statistically significant increases in mRNA expressions, protein concentrations and catalytic activities of CYP1A1 were observed in animals exposed to BaP, to BaP coated onto (56)Fe(2)O(3) particles or to BaP coated onto (54)Fe(2)O(3) particles versus controls. Both of the BaP/Fe(2)O(3) mixtures induced higher CYP1A1 protein levels and EROD activities than BaP alone. Iron oxide particles per se did not affect mRNA levels of cyp1a1 but only enhanced BaP-mediated increases of CYP1A1 protein levels and activity. The ToF-LMMS spectrum pro fi les showed that the (54)Fe/(56)Fe ratio in the microsomes of BaP coated onto (54)Fe(2)O(3) particle-instilled animals was 1.3 instead of the theoretical ratio (i.e. 0.063) observed in BaP coated onto (56)Fe(2)O(3) particle-instilled animals. Taken together, these novel data support the hypothesis that the Fe(2)O(3)-induced increases of the metabolic activation of BaP might rely on the property of Fe(2)O(3) particles to enhance the BaP-induced translation rate of the cyp1a1 gene into functional haemoproteins., (Copyright 2004 John Wiley & Sons, Ltd.)

In this work, the question that needs to be answered was whether concurrent exposure to iron oxides and polycyclic aromatic hydrocarbons (PAHs) could affect the induction of PAH-metabolizing enzymes. Male Sprague-Dawley rats were intratracheally instilled with hematite (Fe(2)O(3); 3mg), benzo(a)pyrene (B(a)P; 3mg), or B(a)P (3mg)-coated onto Fe(2)O(3) particles (3mg). Forty-eight hours later, we investigated mRNA expressions of cytochrome p4501a1 (cyp1a1), microsomal epoxide hydrolase (meh), and glutathione-S-transferase-ya and -yc (gst-ya and gst-yc, respectively), protein concentrations of CYP1A1, and 7-ethoxyresorufin O-deethylase (EROD) activities in lungs. Exposure to B(a)P alone or coated-onto Fe(2)O(3) particles induced cyp1a1 gene transcription (P < 0.01) and increased both the CYP1A1 protein levels (P < 0.01) and the EROD activities (P < 0.001). However, in this work, we focused our attention on the potential of Fe(2)O(3) in B(a)P/Fe(2)O(3) mixtures to affect the capacity of B(a)P to induce PAH-metabolizing enzymes. Exposure to B(a)P-coated onto Fe(2)O(3) particles increased meh mRNA expressions (1.15-fold, P < 0.05), CYP1A1 protein concentrations (1.85-fold, P < 0.05), and EROD activities (1.95-fold, P < 0.01), versus exposure to B(a)P alone. Hence, animal short-term exposure to B(a)P-coated onto Fe(2)O(3) particles favored dramatically the induction of PAH-bioactivating enzymes to the detriment of PAH-inactivating enzymes in lungs. Taken together, these results support the hypothesis that the Fe(2)O(3)-induced increase of the metabolic activation of B(a)P might rely on several properties of Fe(2)O(3), including its capacity to enhance the rate of CYP1A1 hemoprotein elaboration. The influence of Fe(2)O(3) in B(a)P/Fe(2)O(3) mixtures on the ability of B(a)P to induce PAH-metabolizing enzymes will also be one of the fundamental ways that Fe(2)O(3) can affect B(a)P carcinogenicity in lungs.

Objectives: To investigate, by real-time quantitative reverse transcriptase-polymerase chain reaction, the expression of MUC3 and vascular endothelial growth factor (VEGF) to correlate them with histologic parameters and with prognosis. Human mucins are large O-glycoproteins expressed in epithelial tissues. Deregulation of mucin genes has been demonstrated in several epithelial neoplasms. In the kidney, MUC3 is expressed in normal convoluted tubules and in renal clear cell carcinoma., Methods: Twenty-six renal clear cell carcinoma specimens were studied. For all tumors, samples of normal and tumor kidney were frozen. After RNA extraction, using ultracentrifugation through a cesium chloride cushion, VEGF and MUC3 mRNA were analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction. The pathologic parameters included Fuhrman nuclear grade and TNM stage. All follow-up data were available., Results: The median level of MUC3 and VEGF expression was greater in tumor areas compared with normal areas (P < 0.002 and P < 0.001, respectively). The MUC3 tumor/normal tissue expression ratio was greater in nuclear grade 3 tumor than in low grades (grade 1-2; P < 0.005). No statistically significant relationship was found with the prognosis for MUC3 and VEGF in our study., Conclusions: The results of this study demonstrate that MUC3 and VEGF are overexpressed in renal clear cell carcinoma, and the MUC3 expression ratio is greater in nuclear grade 3 than in grades 1 and 2 (low grades) tumor. These findings suggest the implication of MUC3 in renal carcinogenesis.

Papillary renal cell carcinoma is now a well-established entity with distinct histological and cytogenetic features. A subdivision has been proposed in correlation with prognosis. Type 1 is the most frequent subtype and appears to have a better prognosis than Type 2. The subdivision is based on microscopic criteria. To investigate these 2 types of papillary renal cell carcinoma, we have compared the clinical features, ancillary factors (TNM stage, Fuhrman grade), survival and MUC1 expression in 25 Type 1 and 12 Type 2 papillary renal cell carcinomas. Type 2 tumors were significantly associated with a higher Fuhrman grade (Grade III frequent; P <.001). Type 2 tumors were also associated with a poorer prognosis than Type 1 (P <.005). Fuhrman grade was significantly associated with prognosis (P <.005). The type and the prognosis were not correlated with the TNM stage. We have shown a differential expression of MUC1 between Type 1 and Type 2 with a polarized expression in Type 1 and a rare expression in Type 2. In conclusion we confirm that the morphologic sub-typing and Fuhrman grade are valuable factors of outcome of papillary renal cell carcinomas and that MUC1 immunostaining is useful in differentiating Type 1 and Type 2 tumors.

We studied, by immunohistochemical analysis, the expression of MUC1 and epithelial membrane antigen in 44 stage pT1 renal cell carcinomas (RCCs). Six patients had a metastatic evolution. The percentage of stained cells was determined for each tumor. All tumors and normal adjacent renal parenchyma were stained. In normal kidney, distal convoluted tubules and collecting ducts stained strongly with an apical distribution. In tumors, there was a significant statistical correlation of the MUC1 expression level with the nuclear grade and with tumor progression. High-grade tumors had more stained cells than did low-grade tumors. Metastatic tumors also were more stained than nonmetastatic lesions. By using the Kaplan-Meier method and the log-rank test, we observed that patients with fewer than 10% of stained cells had no metastatic evolution. In contrast, patients with 70% or more stained cells had significantly lower metastasis-free survival rates. We conclude that MUC1 is expressed in RCC and is associated with tumor progression in pT1 RCC.

Inhaled nitric oxide (NO) has been shown to have some protective effect in the peripheral distal inflamed vasculature. The objective of the study was to determine whether inhaled NO would reduce endotoxin-induced leukocyte activation and myocardial contractile dysfunction. Rats were treated with either saline or endotoxin (10 mg/kg iv) and then allowed to breathe (4 h) either air or air plus NO (10 ppm). In endotoxemic rats, mesenteric venular endothelium leukocyte firm adhesion increased compared with control rats (1.15 +/- 0.32 vs. 4.08 +/- 0.96 leukocytes/100 microm; P < 0.05). Inhaled NO significantly attenuated endotoxin-induced venular endothelium leukocyte adhesion (4.08 +/- 0.96 vs. 1.86 +/- 0.76 leukocytes/100 microm; P < 0.05) and FITC-conjugated anti-intercellular adhesion molecule-1 fluorescence intensity. Endotoxin-induced myocardial dysfunction and leukocyte content increases were reduced in inhaled NO-treated rats. These observations suggest that inhaled NO reduces the degree of cardiovascular dysfunction and inflammation in endotoxemic rats.

Proteases contribute to tumor invasion and metastasis via their potential to degrade basement membranes and extracellular matrix. Our aim was to compare the level of several proteases: urokinase-type plasminogen activator (u-PA), matrix metalloproteinase 2 (MMP-2; 72-kDa type IV collagenase, also known as gelatinase A), MMP-11 [also known as stromelysin 3 (STR3)], and cathepsins B and L in resected non-small cell lung cancer. Between June 1996 and March 1998, samples of lung tumor tissues were taken from 119 surgically treated patients. Thirty out of the 119 tumor samples were matched with corresponding adjacent normal tissue. u-PA was measured by a commercially available immunoluminometric assay. Metalloproteinases and cathepsins have been evaluated at the RNA level by Northern blot and quantified with a PhosphorImager. Expression of these proteases was compared to the following clinicopathological parameters: pathological diagnosis, tumor size, exposure to asbestos, radiotherapy, neo-adjuvant chemotherapy, tumor-node-metastasis stage, lymph node involvement, presence of metastasis. u-PA, MMP-2, MMP-11/STR3, and cathepsin B were significantly increased in tumor (the tumor:normal ratio was on average increased by 5.4-, 2.2-, 83.5-, and 2.2-fold, respectively). The tumor:normal ratio of MMP-11/ STR3 was found to be significantly linked to the lymph node involvement (P < 0.05). Our results suggest that several proteases are involved in the invasive potential of non-small cell lung cancer and that the quantification of MMP-11/ STR3 could represent an useful prognostic marker.

Several matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were studied in highly invasive (MDA-MB-231) and slightly invasive (MCF-7, T47D, BT-20) breast cancer cell lines. Investigations were carried out at the protein level and/or at the mRNA level, either in cells cultured as monolayers on plastic, or in cells seeded on a thin layer of Matrigel basement membrane matrix. Analysis of MMP expression by RT-PCR showed expression of MMP-1. MMP-3, and MMP-13 in highly invasive MDA-MB-231 cells, but not in slightly invasive cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516, by 25% and 50% at the concentrations of 2 x 10(-6) M and 10(-5) M, respectively. In conclusion, our data show that highly invasive MDA-MB-231 cells but not slightly invasive T47D, MCF-7 and BT-20 cells express MMP-1, MMP-3, MMP-9 and MMP-13. MMP-9 which is specifically up-regulated by cell contact to Matrigel, may play a key role in the invasiveness of MDA-MB-231 cells through basement membranes.

The in vitro O-glycosylation reaction of the MUC5AC mucin motif peptide, TTSAPTTS (in one-letter code), was achieved with human gastric microsomal homogenates. The analyses using capillary electrophoresis online coupled with electrospray mass spectrometry and further Edman degradation of the purified products (obtained by capillary electrophoresis at preparative scale) allowed us to distinguish two components at close masses: the addition of a mass of 202 corresponded to an N-terminal elongation of the peptide TTSAPTTS with the dipeptide (TT) and the addition of a mass of 203 corresponded to an N-acetylgalactosamine O-linkage. Using different peptidase inhibitors, a dipeptidyl peptidase/transferase activity was further characterized. A thiol dependence and an inhibition by H-Gly-PheCHN2 (specific to cathepsin C activity) were found. Moreover, besides TTSAPTTS, other MUC5AC motif peptides (GTTPSPVP, TSAPTTS) were also dipeptide donors (GT and TS, respectively) and our results suggested the involvement of a single dipeptidyl peptidase/transferase activity. Finally, this latter activity modified the in vitro GalNAc incorporation rates when using our selected MUC5AC motif peptides. Our study therefore shows that caution must be taken to prevent peptidic substrate elongation while performing in vitro O-glycosylation with microsomal preparations as the enzyme source. In fact, the results of the N-acetylgalactosamine incorporation rates and thus the microsomal N-acetylgalactosamine transferase affinity can be misinterpreted if dipeptidyl peptidase/transferase activity is not inhibited by the thiol inhibitor E-64 or the cathepsin C inhibitor H-Gly-PheCHN2.

We have studied the cellular content and the extracellular release of cathepsins B and D, and of plasminogen activator, in 2 different tumor cell populations before confluence and after late confluence: the HT-29 colon carcinoma cell line, which contains primarily undifferentiated cells, and a subpopulation derived from this cell line, which contains cells committed to differentiation into mucus-secreting goblet cells after confluence. In both populations, cellular cathepsin-B activity increased after confluence, and latent cathepsin B was found in all culture media. In the parental cell line, cellular cathepsin D activity decreased after confluence; however, cathepsin D was secreted at high levels into the extracellular medium. In contrast, in the subpopulation of cells committed to differentiation, cellular cathepsin D activity increased after confluence, and cathepsin D was not secreted into the extracellular medium, but was immunolocalized in the apical brush border of the differentiated cells. Plasminogen activator of urokinase type was identified by immunocytochemistry. Both subconfluent cell populations, and the post-confluent undifferentiated cell population, produced plasminogen activator activity at similar levels. In contrast, in the differentiated postconfluent cells, the production of plasminogen activator activity was markedly lower. Our data show that the differentiation of HT-29 colon carcinoma cells into mucus-secreting cells impairs the secretion of plasminogen activator and cathepsin D, but does not affect cathepsin B.