Taurine acts as antioxidant, cell osmolyte, modulator of glucose metabolism, and plays a role in the retinal function. It is 10(3)-fold more concentrated in the intracellular than in the extracellular milieu due to a specific taurine-Na-dependent transporter (TauT), which is upregulated by hypertonicity, low extracellular taurine, or oxidative stress and acutely downregulated 'in vitro' by high glucose concentrations. Aim of this study was to investigate whether TauT expression was modified in mononuclear peripheral blood cells (MPC) of type 2 diabetic patients with or without micro/macrovascular complications. Plasma taurine, as well as other sulphur-containing aminoacids (assayed by HPLC) and TauT gene expression (assayed by real-time PCR analysis) were measured in MPC of 45 controls and of 81 age-and-sex matched type 2 diabetic patients with or without micro/macrovascular complications. Median value (interquartile range) of plasma taurine was significantly lower in diabetic patients than in controls [28.7 (13.7) μmol/l vs. 46.5 (20.3) μmol/l; P<0.05], while median TauT expression, in arbitrary units, was significantly higher in diabetics than in controls [3.8 (3.9) vs. 1 (1.3); P<0.05) and was related to HbA1c only in controls (r=0.34; P<0.05). Patients with retinopathy (n=25) had lower TauT expression than those who were unaffected [3.1 (2.8) vs. 4.1 (3.4); P<0.05], while persistent micro/macroalbuminuria was associated with unchanged TauT expression. A trend toward reduction in TauT expression was observed in patients with macroangiopathy [n=27; 3.3 (2.5) vs. 4 [3.7]; P=NS]. In conclusion, TauT gene is overexpressed in MPC of type 2 diabetic patients, while presence of retinopathy is specifically associated with a drop in TauT overexpression, suggesting its possible involvement in this microangiopathic lesion.
Background. Taurine transporter gene expression (RNA-TauT) has a role in retinal cell function and is modulated in vitro and in vivo by hyperglycemia and/or oxidative stress. This study was aimed at testing whether RNA-TauT gene expression is modified in blood mononuclear peripheral cells (MPCs) of type 1 diabetic patients, is related to plasma markers of oxidative stress or endothelial dysfunction, or, finally, is related to presence of retinopathy. Methods. RNA-TauT was measured in MPCs by real-time PCR-analysis in 35 type 1 diabetic patients and in 33 age- and sex-matched controls, additionally measuring plasma and cell taurine and markers of oxidative stress and endothelial dysfunction. Results. RNA-TauT, expressed as 2−ΔΔCt, was significantly higher in MPCs of type 1 diabetic patients than in controls [median (interquartile range): 1.32(0.31) versus 1.00(0.15); P = 0.01]. In diabetic patients RNA-TauT was related to HbA1c (r = 0.42; P = 0.01) and inversely to plasma homocysteine (r = −0.39; P = 0.02) being additionally significantly higher in MPCs of patients without retinopathy [(n = 22); 1.36(0.34)] compared to those with retinopathy [(n = 13); 1.16(0.20)], independently from HbA1c or diabetes duration. Conclusions. RNA-TauT gene expression is significantly upregulated in MPCs of type 1 diabetes patients and is related to HbA1c levels and inversely to plasma homocysteine. Finally, in diabetes patients, RNA-TauT upregulation seems to be blunted in patients with retinopathy independently of their metabolic control or longer diabetes duration.
INTRODUZIONELe infezioni sessualmente trasmesse (MST) hanno un'incidenza di circa 340 milioni di nuovi casi ogni anno (48); se non trattate, molte di esse possono produrre infezioni con gravi complicanze, quali infertilità, aborti precoci e malattie neonatali di grado severo (18,24). I costi socio-economici di queste infezioni e delle loro complicanze sono elevati; lo screening per la prevenzione primaria del cancro della cervice e gli interventi per il trattamento e follow-up delle neoplasie cervicali intraepiteliali di grado lieve (CIN 1), moderato (CIN 2) e grave (CIN 3), il trattamento dell'infertilità, la cura della cecità e delle infezioni polmonari nei neonati, rappresentano una larga porzione dei costi sanitari complessivi. Quantificare l'incidenza e la distribuzione di questi patogeni nella popolazione, mediante l'investimento in opportune strategie di controllo e prevenzione, è importante per ridurre la morbilità e la mortalità correlata a questo tipo di infezioni con ricadute, sicuramente, in termini di qualità di vita per i pazienti ma anche in termini di spesa sanitaria più complessa da calcolare. Numerosi sono i test di amplificazione degli acidi nucleici (NAATs) in Real-Time polymerase chain reaction (RT-PCR) per la rivelazione di Chlamydia trachomatis e Neisseria gonorrhoeae (11,23,32,45) mentre poco implementati quelli per la rilevazione di Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum e Ureaplasma parvum, nonostante numerosi studi abbiano dimostrato una maggiore sensibilità delle metodiche 2,28,41,44). Diversi sono i laboratori che stanno implementando la diagnostica molecolare e impiegano le metodiche colturali solo quando è necessario l'antibiogramma. Praticamente inesistenti i test di diagnostica quantitativa in RT-PCR che hanno un'importanza nel follow-up del trattamento. Per la diagnostica del Papillomavirus umano ad alto rischio oncogeno (HR-HPV), numerose sono invece le metodiche NAATs per la rilevazione e la genotipizzazione basate sull'amplificazione del DNA virale delle
<em>Background</em> <em>and</em> <em>Aims</em>. Bacterial meningitis and sepsis are medical emergencies where tests with a high sensitivity and short turn around time (TAT) are crucial for an early targeted therapy. Aim of this study was the evaluation of an optimal diagnostic strategy for infectious meningitis/sepsis management, assessing seven methods: cerebrospinal fluid (CSF) physical-chemical examination, CSF cultural tests (CCT), Gram stained smears (GSS), CSF latex agglutination test (CLAT), blood culture (BC), Real-Time (RT)-PCR and FilmArray Technology (FAT) performed directly on CSF/blood. <br /><em>Materials</em> <em>and</em> <em>Methods</em>. Samples of CSF (240), blood (180) and cavitary fluids (9) were tested by commercial RT-PCR (Eurospital and Liferiver kits) and traditional methods. Positive samples (BC and RT-PCR) were tested by FAT (Blood Culture Identification Panel, Biofire, Salt Lake City, UT, USA) performed directly on CSF, blood and cavitary fluids. <br /><em>Results</em>. In CSF, GSS, CLAT, CCT, RT-PCR and FAT sensitivity was 41%, 35%, 41%, 100% and 62,5%, respectively. In blood, BC, RT-PCR and FAT sensitivity was 96%, 70% and 44%, respectively. TAT was 48-96 hrs, 3 hrs and 1 hr and NPV was 98%, 89% and 57%, respectively. <br /><em>Conclusions</em>. For sepsis management, RT-PCR is faster than BC (3 hrs vs 24-72 hrs), but limited by a low overall sensitivity (70%), due to the low number of detectable pathogens; FAT, performed directly on positive BC should replace biochemical identification (Vitek 2, Biomérieux Marcy-l’Étoile, France) reducing TAT (1 hr <em>vs</em> 12 hrs). For meningitis management, RT-PCR is the most sensitive and rapid method used in routine and emergency regimen. It is cost effective and it represents the gold standard for diagnosis and follow-up of patients under treatment. For meningitis management, FAT, with a higher sensitivity and rapidity and an easier and objective interpretation, should replace CLAT and GSS in emergency regimen.
SummaryPre-analytical and post-analytical evaluation in the era of molecular diagnosis of sexually transmitted diseases: cellularity control and internal control.Background. Increase of molecular tests performed on DNA extracted from various biological materials should not be carried out without an adequate standardization of the pre-analytical and postanalytical phase.Materials and Methods. Aim of this study was to evaluate the role of internal control (IC) to standardize pre-analytical phase and the role of cellularity control (CC) in the suitability evaluation of biological matrices, and their influence on false negative results. 120 cervical swabs (CS) were pre-treated and extracted following 3 different protocols. Extraction performance was evaluated by amplification of: IC, added in each mix extraction; human gene HPRT1 (CC) with RT-PCR to quantify sample cellularity; L1 region of HPV with SPF10 primers. 135 urine, 135 urethral swabs, 553 CS and 332 ThinPrep swabs (TP) were tested for C. trachomatis (CT) and U. parvum (UP) with RT-PCR and for HPV by endpoint-PCR. Samples were also tested for cellularity.Results. Extraction protocol with highest average cellularity (Ac)/sample showed lowest number of samples with inhibitors; highest HPV positivity was achieved by protocol with greatest Ac/PCR. CS and TP under 300.000 cells/sample showed a significant decrease of UP (P<0.01) and HPV (P<0.005) positivity. Female urine under 40.000 cells/mL were inadequate to detect UP (P<0.05).Conclusions. Our data show that IC and CC allow optimization of pre-analytical phase, with an increase of analytical quality. Cellularity/sample allows better sample adequacy evaluation, crucial to avoid false negative results, while cellularity/PCR allows better optimization of PCR amplification. Further data are required to define the optimal cut-off for result normalization.
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