[Ischemic lesions in kidneys after extracorporeal shock wave lithotripsy demonstrated by Proton NMR spectroscopy of urine samples]. / Évaluation par spectroscopie RMN des lésions rénales provoquées par la lithotripsie extracorporelle à partir d'échantillons d'urine.

PURPOSE: The aim of this prospective study was to investigate the effect of extracorporeal shock wave lithotripsy (SWL) on kidneys of patients with pyelic stone disease. The effects of SWL were assessed by high-resolution proton nuclear magnetic resonance (HNMR) spectroscopy of urine samples. METHODS: Twenty-three patients, aged 31-80years (mean: 55years), with pyelic stone disease were investigated before and after SWL. Multiparameter analysis was performed by HNMR spectroscopy of urine samples collected before and 5h after SWL (second miction post-SWL). RESULTS: The most relevant resonances determined by HNMR spectroscopy were acetate, lactate, trimethylamine N-oxide and amino acids. Excretion of these markers increased significantly in comparison with pre-SWL urinary samples. CONCLUSION: These results show that early ischemic damage occurs after SWL. Post-SWL. HNMR spectroscopy is an effective tool for noninvasive follow-up of renal damage.

'Desperate house genes': the dramatic example of hypoxia.

BACKGROUND: Microenvironmental conditions in normal or tumour tissues and cell lines may interfere on further biological analysis. To evaluate transcript variations carefully, it is common to use stable housekeeping genes (HKG) to normalise quantitative microarrays or real-time polymerase chain reaction results. However, recent studies argue that HKG fluctuate according to tissues and treatments. So, as an example of HKG variation under an array of conditions that are common in the cancer field, we evaluate whether hypoxia could have an impact on HKG expression. METHODS: Expression of 10 commonly used HKG was measured on four cell lines treated with four oxygen concentrations (from 1 to 20%). RESULTS: Large variations of HKG transcripts were observed in hypoxic conditions and differ along with the cell line and the oxygen concentration. To elect the most stable HKG, we compared the three statistical means based either on PCR cycle threshold coefficient of variation calculation or two specifically dedicated software. Nevertheless, the best HKG dramatically differs according to the statistical method used. Moreover, using, as a reference, absolute quantification of a target gene (here the proteinase activating receptor gene 1 (PAR1) gene), we show that the conclusions raised about PAR1 variation in hypoxia can totally diverge according to the selected HKG used for normalisation. CONCLUSION: The choice of a valid HKG will determine the relevance of the results that will be further interpreted, and so it should be seriously considered. The results of our study confirm unambiguously that HKG variations must be precisely and systematically determined before any experiment for each situation, to obtain reliable normalised results in the experimental setting that has been designed. Indeed, such assay design, functional for all in vitro systems, should be carefully evaluated before any extension to other experimental models including in vivo ones.

Prognostic value of prostate circulating cells detection in prostate cancer patients: a prospective study.

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<0.0001) than in group II (69.6 months) but did not significantly differ in group II and III (69.6 months vs 65.0). Such 5-year follow-up data show that preoperative circulating prostate cells are an independent prognosis factor of recurrence. Moreover, tumour handling induces cancer-cell seeding but surgical blood dissemination does not accelerate cancer evolution.

Comparative expression of Hedgehog ligands at different stages of prostate carcinoma progression.

Recent studies have revealed the potential involvement of Hedgehog (Hh) signalling in proliferation and invasive behaviour of prostate carcinoma (PCa). The aim of this study was to specify the role of Sonic Hh (Shh), Desert Hh (Dhh) and Indian Hh (Ihh) in the natural history of PCa. Hh ligands expression was compared in primary hormone-naive PCa (HNPC), hormone-treated PCa (HTPC) and hormone-refractory PCa (HRPC), using immunohistochemistry. Shh and Dhh were expressed by both epithelial and stromal cells of prostate tissues. Ihh was only expressed by stromal cells. For the three ligands, mRNA and immunostaining were not correlated. In HNPC, Shh epithelial expression was significantly associated with high Gleason scores (p = 0.03), metastatic lymph nodes (p = 0.004) and Dhh epithelial staining was associated with high pT stages (p = 0.003), seminal vesicle invasion (p = 0.03) and bladder neck invasion (p = 0.0008). Negative Shh staining in stromal cells was associated with high Gleason scores (p = 0.015), high pT stages (p = 0.01) and bladder neck invasion (p = 0.04). Concomitant absence of Shh and Dhh expression in stromal cells was an independent prognostic parameter for biological recurrence on multivariate analysis (p = 0.01). Epithelial expression of Shh and Dhh was increased in HTPC compared to HNPC (p = 0.02 and p = 0.04). Interestingly, in vitro, transcript analysis also showed increased expression of these 2 Hh ligands when androgen-sensitive LNCaP cells were maintained in androgen-free medium mimicking hormonal therapy. Epithelial expression of Dhh was increased (p < 0.0001) in HRPC compared to HNPC, while stromal expression of Shh and Dhh was decreased (p < 0.0001). In conclusion, the Hh signalling pathway is associated with pejorative pathological parameters in HNPC and is up-regulated in epithelial cells of HTPC and HRPC. Moreover, the lack of Hh molecules in stromal cells seems to be associated with invasive and hormone-refractory behaviours and suggests specific changes in stromal-epithelial crosstalks during PCa progression.

[Molecular pharmacogenetics in hospital laboratories in France: current data and future prospects]. / La pharmacogénétique moléculaire hospitalière en France: données actuelles et perspectives.

Molecular pharmacogenetic units have recently been established in several hospital laboratories in France. The clinical impact of these units is still limited and numerous problems of organizational, ethical, legal, technical, social and economical nature remain to be resolved. However, an increasing number of these units, a rise in their activities and an enlargement of their scope of application are foreseeable in the future. Ultimately, these units would significantly contribute to limit the public health problem caused by interindividual variabilities in drug effects. In view of these prospects, it seems essential that such hospital activity should be quickly recognised by the authorities and the various health sectors in France. It is also essential that the problems that arise from such pharmacogenetic activities should be considered by the authorities and would profit from the organization of a national network and from financial guarantees.

The use of intravenous catheterisation with a rest period is useful for determination of plasma cortisol levels but not plasma prolactin levels.

OBJECTIVE: The use of an intravenous catheter with a rest period has been recommended to avoid false-positive results for hyperprolactinaemia and false-negative results for hypocortisolaemia. We tested the relevance of this recommendation. DESIGN: Plasma cortisol and prolactin levels were determined before (T-15) and after a 15-min rest period (T0) in 119 patients, 38 males (M) and 81 females (F). 52 of the 119 patients were known (K; 30 females and 22 males) and 67 unknown (UK; 49 females and 18 males) to the unit. RESULTS: Prolactin was lower after rest in women (12.3+/-22.7 ng/l vs 11.7+/-22.5 ng/ml, P=0.03), but not in men (6.2+/-4.5 ng/ml at T-15 vs 5.8+/-3.2 ng/ml at T0, P=0.09), in the UK subgroup (10.6+/-20.7 ng/ml at T-15 vs 10.1+/-20.9 ng/ml at T0, P=0.06) and in the K subgroup (10.1+/-16.7 ng/ml at T-15 vs 9.7+/-15.8 ng/ml at T0, P=0.08). None of the patients with prolactin levels higher than 20 ng/ml at T-15 diminished its prolactin value below this cut-off value. Plasma cortisol levels were lower after rest in women (17.9+/-5.9 microg/dl at T-15 vs 16.5+/-6.1 microg/dl at T0, P<0.0001), in the UK subgroup (18+/-6.1 microg/dl at T-15 vs 16.6+/-6.4 microg/dl at T0, P=0.0003) but not in men (18+/-4.4 microg/dl at T-15 vs 17.5+/-5.8 microg/dl at T0, P=0.47) and in the K subgroup (17.8+/-4.6 microg/dl at T-15 vs 17+/-5.4 microg/dl at T0, P=0.13). At T0, 3.3% and 15% of patients presented values below the cut-off value of 10 microg/dl (276 nmol/l) and 17 microg/dl (470 nmol/l), respectively. CONCLUSION: These results don't justify intravenous catheterisation with a rest period for plasma prolactin determination in contrast with plasma cortisol determination.

Definition of an uptake pharmacophore of the serotonin transporter through 3D-QSAR analysis.

The serotonergic system plays a critical role in a wide variety of physiological and behavioral processes. Dysregulation of the tightly controlled extracellular concentration of serotonin (5-hydroxytryptamine, 5-HT) appears to be at the origin of a host of metabolic and psychiatric disorders. Since the plasma membrane 5-HT transporter (SERT) is the major protagonist in regulating extracellular 5-HT concentration, SERT is the target of most drugs interacting with the serotonergic system. Unfortunately, some of the drugs towards SERT (e.g. amphetamine derivatives) interfere with cell homeostasis leading to cell toxicity. Developing new SERT ligands devoid of any side-effect represents a major priority in the treatment of 5-HT-associated pathologies. Here, we report structure-activity relationships (SAR) and three-dimensional QSAR (3D-QSAR) studies of a library of 121 compounds including 5-HT analogs, harmanes, benzothiazoles, indanones, amphetamine derivatives and substrate-type 5-HT releasers, with the goal of identifying the structural determinants crucial for SERT uptake. In the absence of data about the bioactive form of 5-HT, conformational analysis of 5-HT was performed using quantum chemistry calculations. This led to three 5-HT stable conformers with anti, -gauche and +gauche side-chain conformation. These conformers, used as templates for superimposition with all the library compounds, enabled the design of a reliable 6-points pharmacophore representative of SERT uptake activity. Molecular dynamics (MD) simulations performed with compounds that are efficiently, moderately, poorly or not transported by SERT allowed to assess the validity of our pharmacophore. Altogether, our data provide for the first time a reliable pharmacophore of SERT uptake activity, which may help to the design of new drugs targeting SERT.

A histopathologic investigation of PGE(2) pathways as predictors of proliferation and invasion in urothelial carcinomas of the bladder.

INTRODUCTION AND OBJECTIVES: The pattern of arachidonate acid (AA) transformation in tumor cells has been shown to play a role in determining tumor cell invasiveness. AA is released from membrane phospholipids by cPLA(2). Then it is metabolized into prostaglandins and PGE(2) especially via cyclooxygenase pathways. PGE(2) production seems to be necessary for rendering the cells invasive. We aimed to characterize cPLA(2), cyclooxygenase 2 (COX2) and prostaglandine E synthase (PGES) expression in human transitional carcinoma (TCC) of the urinary bladder and correlate with the Ki-67 proliferating marker. METHODS: Formalin-fixed human TCC tissues (n=54) obtained from TURB or cystectomies were evaluated for cPLA(2), COX2, PGES and Ki-67 expression using specific antibodies. There were 6 CIS, 9 pTaG1, 9 pTaG3, 10 pT1G3 and 10 pT2G3. 10 normal bladder tissues were also evaluated. Control slides were incubated without primary antibodies and treated in a similar way. RESULTS: cPLA(2), COX2 and PGES were not expressed in the 10 normal tissues. In the same normal tissues, Ki-67 expression was observed only in 1% of the cells. However, cPLA(2) was expressed in 1/6 CIS, 1/9 pTaG1, 3/9 pTaG3, 6/10 pT1G3 and 2/10 pT2G3. COX2 was expressed in 0/6 CIS, 0/10 pTaG1, 2/9 pTaG3, 3/10 pT1G3 and 1/10 pT2G3. PGES was expressed in 4/6 CIS, 0/9 pTaG1, 4/9 pTaG3, 2/10 pT1G3 and 5/10 pT2G3. Ki-67 expression was 39.5% for CIS, 6.5% in pTaG1, 37% in pTaG3, 34.5% in pT1G3 and 55% in pT2G3. If we consider it a positive result when at least one enzyme was expressed, there were 5/6 CIS positive, 1/9 pTaG1 positive, 9/9 pTaG3 positive, 10/10 pT1G3 positive and 10/10 pT2G3 positive. Also the Ki-67 is more often expressed in cells with high grade tumor. CONCLUSIONS: These results suggest that (i). not only COX2 is involved in the tumorogenesis of the TCC but also cPLA(2) and PGES, (ii). there is relationship between the AA metabolic PGE(2) pathway expression and the aggressiveness of the TCC of the urinary bladder.

[RT-PCR in clinical diagnosis]. / La RT-PCR en diagnostic clinique.

Application fields of RT-PCR (reverse transcription-polymerase chain reaction) in clinical diagnosis comprises the assessment of viral load for RNA viruses and the analysis of gene transcription products. RT-PCR is also helpful when large genes have to be sequenced. Developments of quantitative approaches using real-time PCR recently led to a major widening of RT-PCR applications in clinical diagnosis. However, RT reaction is delicate due to its lack of reproducibility and to RNA lability and frequent contamination by DNA. In some cases additional difficulties come from the need to obtain a specific amplification in the presence of homologous sequences which might be present in higher amounts than the sequence of interest. These caveats have to be taken into account, when designing the RT protocol, and when choosing PCR primers and internal and/or external references. This review is aimed at helping the experimental setup of a RT-PCR based assay according to the objectives.

[RNA isolation and purification methods]. / Les méthodes d'extraction et de purification des ARN.

Recent advances in human, bacterial and viral genome projects and the development of quantitative real-time reverse transcription-polymerase chain reaction methods offer the possibility of analysing a large number of gene transcripts. These molecular developments represent an important advancein the field of genetics, cancer, virology, bacteriology and hematology. A limiting step remains the isolation of high quality mRNA purified from biological samples. This review describes the different methods used to isolate mRNA from biological samples and to verify RNA integrity and gives precise details about RNA storage conditions.

Abnormal E-cadherin expression and prostate cell blood dissemination as markers of biological recurrence in cancer.

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<0.0001). In logistic regression analysis, abnormal E-cadherin expression was a significant independent predictor for a later biological relapse. This impaired adhesion status was clearly correlated with a haematogenous spread of the primary tumour cells. It could therefore be an objective way to restrict the indications for radical surgery to patients not presenting with this feature.

Carcinoembryonic gene member 2 mRNA expression as a marker to detect circulating enterocytes in the blood of colorectal cancer patients.

BACKGROUND: The aim of this study was to report our experience with a new molecular tool to detect circulating enterocytes in the blood of patients with colorectal cancer. METHODS: The study included 193 individuals: 78 patients with colorectal cancer and 115 controls composed of patients with benign colorectal diseases (n = 16), patients with noncolorectal cancer (n = 31), healthy individuals (n = 62), and healthy bone marrow transplantation donors (n = 6). A nested reverse transcriptase-polymerase chain reaction with specific primers for the carcinoembryonic gene member 2 (CGM2) was used to detect circulating enterocytes in the peripheral blood of 78 patients with colorectal cancer. The blood (n = 109) or the bone marrow (n = 6) of the 115 controls was studied to test the absence of CGM2 illegitimate transcription in nucleated blood cells and nucleated blood cell progenitors. The assay sensitivity was effective in detecting 1 CGM2-positive cell per 10(6) nucleated blood cells. RESULTS: Fifty-nine percent (46/78) of patients with colorectal cancer were found positive whereas all negative controls remained negative. Positivity rates were 38% (3/8) in Dukes' A classification, 43% (9/21) in Dukes' B, 77% (23/30) in Dukes' C, and 58% (11/19) in Dukes' D. CONCLUSIONS: The clinical significance of enterocyte detection in the blood of colorectal cancer patients by means of this CGM2 messenger RNA assay needs further evaluation.

Blood-borne RT-PCR assay for prostasin- specific transcripts to identify circulating prostate cells in cancer patients.

OBJECTIVE: The aim of this study was to establish the specific detection of prostasin-expressing prostate cells in the blood of patients with prostate cancer. PATIENTS AND METHODS: A prostasin-specific RT-PCR assay was developed and optimized using limiting dilutions of cell line LNCaP mixed with normal blood specimens. Then, it was used to examine peripheral blood samples from 96 patients with prostate cancer (localized carcinoma, n = 69, metastatic, n = 27). Specificity was assessed by examination of 86 negative controls (healthy individuals, n = 47, benign prostate hyperplasia, n = 17, nonprostate cancer patients, n = 22). RESULTS: All 86 control samples failed to amplify the specific 546-bp prostasin PCR products. Blood samples from 35 out of 96 (36%) prostate cancer patients were found positive. In metastatic patients, 63% (17/27) scored positive whereas in localized adenocarcinoma prostasin primers detected prostate cells in 26% (18/69). CONCLUSION: Our results that approximately 30% of patients with localized prostate cancer scored positive for prostasin-specific RT-PCR confirm that the hematogenous spillage of prostate cells is an early event in the natural history of prostate cancer. As none of our negative controls were found positive, we conclude that blood-borne RT-PCR amplification of prostasin transcripts may lead to an earlier diagnosis of disseminated disease in patients with organ-confined carcinoma. The clinical significance of prostate cell detection and the potential applications of this new tool aside or along prostate-specific antigen or prostate-specific membrane antigen RT-PCR require longer-term follow-up.

Expression of prostate-specific membrane antigen in transitional cell carcinoma of the bladder: prognostic value?

The expression of Prostate-specific membrane antigen (PSMA) mRNA was assessed in the normal bladder urothelium (n = 9), transitional cell carcinoma (TCC) specimens (n = 52), TCC-derived cell lines (n = 3), and preoperative blood samples from TCC patients (n = 27). Specific PSMA mRNA was found in 100% of normal and malignant tissues and two cell lines. PSMA protein was detected in normal (n = 3) and malignant tissues (n = 4). Using a PSMA-specific substrate, PSMA enzymatic activity was found in two bladder cell lines and correlated with immunostaining. Seven of the 27 TCC preoperative blood samples were positive by reverse transcription-PCR. These preliminary results, obtained on a nonrandomized cohort of patients, correlated with tumor invasion (positive RT-PCR: 0% for pT < or = 2 versus 41% for pT > or = 3) and 2-year survival rate (81% in the PSMA-negative group versus 29% in the PSMA-positive group). Although the clinical usefulness of this assay requires confirmation in larger prospective randomized trials, current preliminary results suggest that a blood-borne PSMA mRNA PCR assay may be a useful tool to predict a poor outcome in TCC patients.

Regulation by neurotransmitter receptors of serotonergic or catecholaminergic neuronal cell differentiation.

The murine F9-derived 1C11 clone exhibits a stable epithelial morphology, expresses nestin, an early neuroectodermal marker, and expresses genes involved in neuroectodermal cell fate. Upon appropriate induction, 100% of 1C11 precursor cells develop neurite extensions and acquire neuronal markers (N-CAM, synaptophysin, gammagamma-enolase, and neurofilament) as well as the general functions of either serotonergic (1C11*(/5HT)) (5HT, 5-hydroxytryptamine) or noradrenergic (1C11**(/NE)) (NE, norepinephrine) neurons. The two programs are shown to be mutually exclusive. 1C11 thus behaves as a neuroepithelial cell line with a dual bioaminergic fate. 1C11*(/5HT) cells implement a functional 5-HT transporter and thereby a complete serotonergic phenotype within 4 days, whereas 5-HT(1B/D), 5-HT(2B), and 5-HT(2A) receptors are sequentially induced. The accurate time schedule of catecholaminergic differentiation was defined. Catecholamine synthesis, storage, and catabolism are acquired within 4 days; the noradrenergic phenotype is complete at day 12 and includes a functional norepinephrine transporter and an alpha(1D)-adrenoreceptor (day 8). The time-dependent onset of neurotransmitter-associated functions proper to either program is similar to in vivo observations. Along each pathway, the selective induction of serotonergic or adrenergic receptors is shown to be an essential part of the differentiation program, since they promote an autoregulation of the corresponding phenotype.