Marc Conti

Urinary cystatin C as a specific marker of tubular dysfunction.

Abstract Background: Cystatin C (CST3), a strong inhibitor of cysteine proteinases, is freely filtered by the kidney glomerulus and is reabsorbed by the tubules, where it is almost totally catabolized, with the remainder then eliminated in urine. In tubular diseases, it seems sensible to postulate that CST3 degradation would be reduced and consequently an increase in its urinary elimination would be observed. Methods: We report here the development of an automatic quantitative assay to measure CST3 concentrations in urine using a Behring N-Latex Cystatin C kit on a BNII laser nephelometer. We tested its clinical relevance on several kidney disease patients. Results: This assay is sensitive (limit of detection 0.008mg/L) and precise (within- and between-day CVs <4%). Reference values for freshly collected urine samples range from 0.03 to 0.18mg/L. Mean urine CST3 concentrations obtained from 52 patients with kidney tubular disease (4.31±3.85mg/L) were significantly higher than those for 60 controls (0.096±0.044mg/L; p<0.0001) and 47 glomerular disease patients (0.106±0.133mg/L; p<0.0001). Conclusion: Increased urinary CST3 concentrations allow the accurate detection of tubular dysfunction among pure and mixed nephropathies. Because of its ability to be processed on automated clinical chemistry analyzers, this assay could easily be used as an adjunct to the standard panel used to screen kidney pathologies, even in emergency situations.

Increase in liver antioxidant enzyme activities in non‐alcoholic fatty liver disease

Abstract: Aims: Steatosis may increase oxidative stress, which is counteracted by cellular enzymatic (cytosolic and mitochondrial superoxide dismutases (Cu/Zn‐SOD and Mn‐SOD), glutathione peroxidase (GPx), catalase) and non‐enzymatic antioxidant systems. We aimed to determine, in patients with non‐alcoholic fatty liver disease (NAFLD), the level of antioxidant defenses (1) in liver biopsies, to demonstrate the existence of oxidative stress; (2) in erythrocytes and plasma, to determine whether their antioxidant defenses reflect liver oxidative stress.

'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.

Postresuscitation syndrome: Potential role of hydroxyl radical-induced endothelial cell damage

Objective:After out of hospital cardiac arrest, it has been reported that endothelium dysfunction may occur during the postresuscitation syndrome. However, the consequences of the reperfusion phase on endothelial reactive oxygen species production and redox homeostasis have not been explored in out of hospital cardiac arrest patients. Design:Prospective, observational study. Setting:Medical intensive care unit in a university hospital. Patients:Twenty successfully resuscitated out of hospital cardiac arrest patients, seven septic shock patients, and ten healthy volunteers. Intervention:Plasma was collected from patients at admission and 12, 24, 36, 48, and 72 hrs after cardiac arrest. We studied the production of reactive oxygen species and cell survival during plasma perfusion using perfused endothelial cells (human umbilical vein endothelial cells) as a model. Cell antioxidant response was studied by measuring superoxide dismutase, glutathione peroxidase, and glutathione reductase activities and reduced and oxidized glutathione levels. Mitochondrial respiratory chain activity was assessed by measuring complex I, II, III, and IV activities and anaerobic glycolysis by measuring glucose-6-phosphate dehydrogenase activity. Measurements and Main Results:Using perfused endothelial cells as a model, we demonstrate that plasma from out of hospital cardiac arrest patients induced on naive human umbilical vein endothelial cells a significant and massive cell death compared to plasma from septic shock patients and healthy volunteers. An increase of reactive oxygen species production with a decrease in antioxidant defenses (superoxide dismutase, glutathione peroxidase, and glutathione reductase activities, reduced and oxidized glutathione levels) was observed. The metabolic consequence of plasma exposure showed that mitochondrial respiratory chain activity was significantly impaired and anaerobic glycolysis was significantly increased. Inhibiting hydroxyl radical production significantly decreased cell death, suggesting that plasma from out of hospital cardiac arrest induced significant cell death by triggering the Fenton reaction. Conclusion:Plasma from out of hospital cardiac arrest induces major endothelial toxicity with an acute pro-oxidant state in the cells and impairment of mitochondrial respiratory chain activity. This toxicity could be due to hydroxyl radical production by activation of the Fenton reaction.

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.

Induction of the CD23/nitric oxide pathway in endothelial cells downregulates ICAM-1 expression and decreases cytoadherence of Plasmodium falciparum -infected erythrocytes

Cytoadherence of parasitized red blood cells (PRBCs) to postcapillary venules and cytokine production are clearly involved in the pathogenesis of cerebral malaria. Nitric oxide and TNF‐α have been proposed as major effector molecules both in protective and physiopathological processes during malaria infections. Nitric oxide production has been shown to be induced by engagement of CD23 antigen. This study aimed to investigate the potential role of the CD23/nitric oxide pathway in the control of the cytoadherence of PRBCs on human endothelial cells. We demonstrate that normal human lung endothelial cells (HLECs) are able to express the low affinity receptor for IgE (Fc∈RII/CD23), following cell incubation with interleukin 4 or PRBCs. Ligation of the CD23 antigen by a specific anti‐CD23 monoclonal antibody at the cell surface of HLECs was found to induce iNOS mRNA and protein expression, NO release and P. falciparum killing. In addition, the specific CD23‐engagement on these cells also induced a significant decrease in ICAM‐1 expression, an adhesion molecule implicated in PRBCs cytoadherence. These data not only described for the first time the expression of a CD23 antigen at the cell surface of endothelial cells but also suggest a possible new regulatory mechanisms via the CD23/NO pathway during malaria infection.

Renoprotective Potency of Heme Oxygenase-1 Induction in Rat Renal Ischemia-Reperfusion

PURPOSE Renal injury caused by ischemia-reperfusion (IR) can lead to acute renal failure or delayed graft function. Renal ischemia-reperfusion (RIR) induces inflammatory disorders via activation of arachidonic acid metabolism into prostaglandin E(2) (PGE(2)). Two inducible enzymes, COX-2 and microsomal prostaglandin E synthase (mPGES), regulate PGE(2) production. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated during cellular stress. Overexpression of HO-1 is beneficial in transplantation models including antigen-independent IR injury, acute and chronic allograft rejection. MATERIALS AND METHODS We investigated the effect of HO-1 induction on the COX pathway, antioxidant enzyme activities, malondialdehyde (MDA) levels, and apoptosis in rat kidneys subjected to 45 min ischemia and 1 h or 24 h reperfusion. Rats were injected intraperitoneally with either: 50 mg/kg hemin (HO-1 inducer groups: H1, H2); 50 micromol/kg ZnPP (HO-1 inhibitor groups: Hz1, Hz2); or 0.9% saline (control groups: r1, r2). Sham animals (Sh) did not undergo RIR. RESULTS Serum creatinine increased significantly after RIR (r vs Sh; p <0.05). Hemin treatment induced a significant decrease in serum creatinine after RIR (H vs r; p <0.05) whereas ZnPP treatment significantly increased serum creatinine levels (Sh vs Hz; p <0.05). Hemin reduced the severity of acute tubular necrosis and significantly reduced COX-2 and mPGES expression (p <0.05). Hemin did not alter depleted antioxidant enzyme activity but did decrease levels of MDA (p <0.05). Hemin also reduced caspase-3 expression. CONCLUSIONS HO-1 decreased the degree and severity of tubular damage after IR, probably by attenuating the cytotoxic effects of inflammatory infiltrates and apoptosis.

Specific real-time PCR vs. fluorescent dyes for serum free DNA quantification.

Abstract Background: Detecting and quantifying circulating free DNA in patient serum has become a major challenge. New methods using conventional or automated DNA amplification have been developed. As quantitative real-time PCR (QPCR) remains expensive and requires dedicated automated instrumentation, we questioned whether simple quantification using fluorescent dyes is efficient for determination of free DNA levels in serum. Methods: Serum samples from 180 cancer patients and 58 healthy volunteers were used for DNA quantification according to three methods: (i) using an exonic part of the β-globin gene as the amplifying target; (ii) amplifying a 105-bp intron 1 part of the housekeeping cyclophilin A gene, both referring to specific standard curves; and (iii) using a PicoGreen DNA quantification kit without amplification. Results: The 58 samples from healthy controls showed a reference limit of (95th percentile) <160 cyclophilin gene copies/mL. The 180 cancer samples displayed values ranging between 300 and 215,000 copies/mL. The cyclophilin method showed a high level of correlation with both the β-globin (r=0.911, p<0.0001) and PicoGreen (r=0.915, p<0.0001) methods. Conclusions: Aside from the disadvantage that the QPCR assays can only be used in clinical biochemistry laboratories that possess QPCR apparatus, the use of direct PicoGreen quantification displays major advantages in a routine context: it is less time-consuming and is quite inexpensive, but is still correlated with QPCR. Clin Chem Lab Med 2007;45:993–5.

Should kidney tubular markers be adjusted for urine creatinine? The example of urinary cystatin C.

Abstract Background: Evaluation of specific urinary markers with respect to urine creatinine (uCreat) is common. However, as uCreat is a function of both glomerular filtration and tubular secretion, using uCreat for specific tubular markers, suggests that glomerular function is normal, and there is no tubular secretion. Thus, adjusting values of any tubular marker to uCreat, especially in patients with acute or even moderate chronic renal failure, can be misleading. Methods: Using urine cystatin-C (uCST3) as a model tubular marker for following 120 kidney graft recipients daily, we evaluated the utility of either uCST3 alone or the uCST3/uCreat ratio to detect tubular damage. All positive kidney biopsies were always associated with a uCST3>0.18 mg/L. Results: Using the uCST3/uCreat ratio, discrepancies regarding biopsy status were observed in nine patients (4 false positive, 5 false negative results). In two patients, variability of uCreat appeared to be the most important factor causing inconsistent uCST3/uCreat ratios. With a negative predictive value (NPV) of 85.7%, uCST3/uCreat can lead to errors in clinical interpretation. These errors can be avoided when estimates of tubular damage are based on uCST3 concentrations alone (NPV=100%). Conclusions: We recommend using the uCST3 value to evaluate the extent of renal tubular damage. Indeed, our conflicting results on uCST3/uCreat can be extended to every marker of tubular function. Evaluating a urine marker specific for renal tubular damage to a second urine marker that is itself strongly dependent upon glomerular or other renal or non-renal conditions, impairs its clinical relevance and may lead to incorrect interpretations. Correction with uCreat can be performed only in pure glomerulopathy, when specific markers of glomerular function are measured (i.e., urinary albumin). In all other cases of renal diseases, such correction is inappropriate and should be avoided. Clin Chem Lab Med 2009;47:1553–6.

Cation-π Interactions in Serotonin: Conformational, Electronic Distribution, and Energy Decomposition Analysis.

An adiabatic conformational analysis of serotonin (5-hydroxytryptamine, 5-HT) using quantum chemistry led to six stable conformers that can be either +gauche (Gp), -gauche (Gm), and anti (At) depending upon the value taken by ethylamine side chain and 5-hydroxyl group dihedral angles φ1, φ2, and φ4, respectively. Further vibrational frequency analysis of the GmGp, GmGm, and GmAt conformers with the 5-hydroxyl group in the anti position revealed an additional red-shifted N-H stretch mode band in GmGp and GmGm that is absent in GmAt. This band corresponds to the 5-HT side-chain N-H bond involved in an intramolecular nonbonded interaction with the 5-hydroxy indole ring. The influence of this nonbonded interaction on the electronic distribution was assessed by analysis of the spin-spin coupling constants of GmGp and GmGm that show a marked increase for C2-C3 and C8-C9 bonds in GmGm and GmGp, respectively, with a decrease of their double bond character and an increase of their length. The Atoms in Molecules (AIM), Natural Bond Orbital (NBO), and fluorescence and CD spectra (TDDFT method) analyses confirmed the existence in GmGp and GmGm of a through-space charge-transfer between the HOMO and the HOMO-1 π-orbital of the indole ring and the LUMO σ* N-H antibonding orbital of the ammonium group. The strength of the cation-π interaction was determined by calculating binding energies of the NH4(+)/5-hydroxyindole complexes extracted from stable conformers. The energy decomposition analysis indicated that cationic-π interactions in the GmGp and GmGm conformers are governed by the electrostatic term with significant contributions from polarization and charge transfer. The lower stability of the GmGm over the GmGp comes from a higher exchange repulsion and a weaker polarization contributions. Our results provide insight into the nature of intramolecular forces that influence the conformational properties of 5-HT.