Search

Immunity to M.tuberculosis (MTB) infection consists of interactions between various T-cell subsets that control the infection and prevent further reactivation. We analysed the effector/memory T-cell dynamics and cytokines production in the peripheral blood of patients with pulmonary tuberculosis (TB). We observed that the frequency of CD4+ T-cell effectors was significantly increased during active TB, confirming a major role of this T-cell subset in TB immunity. Pre-terminally differentiated CD8+ T-lymphocytes were increased in the peripheral blood as well. In contrast, we observed a reduced number of effector mycobacteria-reactive γδ+ T-lymphocytes with a specific defects in reacting to mycobacterial nonpeptidic antigens, suggesting that this innate response is rapidly lost during TB infection. Nevertheless, the frequency of γδ+ T-cells effectors in TB patients was higher than the αβ+ T-cell response to peptide from MTB-ESAT-6 protein and quantitatively similar to PPD reactivity. Thus, αβ+and γδ+ T-cell differentiation and function are differently triggered by active TB infection.

Therapeutic drug monitoring is pivotal to improve the management of HIV infection. Here, a new HPLC–UV method to quantify simultaneously maraviroc and raltegravir levels in human plasma is reported. Remarkably, this is the first method for maraviroc determination in human plasma. The volume of the plasma sample was 600 μL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (30 mg divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 200 μL 50/50 of mobile-phase solution (0.01 M KH2PO4 and acetonitrile). Twenty microliters of these samples were injected into a HPLC–UV system, the analytes were eluted on an analytical dC18 Atlantis column (150 mm × 4.6 mm I.D.) with a particle size of 5 μm. The mobile phase (0.01 M KH2PO4 and acetonitrile) was delivered at 1.0 mL/min with isocratic elution. The total run time for a single analysis was 10 min; maraviroc and raltegravir were detected by UV at 197 and 300 nm. The calibration curves were linear up to 2,500 ng/mL. The absolute recovery ranged between 93 and 100%. The HPLC–UV method reported here has been validated and is currently applied to monitor plasma levels of maraviroc and raltegravir in HIV-infected patients. © 2009 IUBMB IUBMB Life, 61(4):470–475, 2009

The BDProbeTec MTB assay for direct detection of Mycobacterium tuberculosis was evaluated in comparison with the AMTD-II assay on 94 samples from different patients with clinical suspicion of tuberculosis. Using a combination of culture on Lowenstein–Jensen medium (with or without preculture in BACTEC 9000) and clinical diagnosis as the standard, BDProbeTec MTB showed high sensitivity and specificity (96.1% and 100%, respectively), similar to AMTD-II (96.1% and 97.1%, respectively), with significantly higher sensitivity than the Ziehl–Neelsen stain for acid-fast bacilli (73%, p < 0.05).

The nucleoside reverse transcriptase inhibitors lamivudine and zidovudine and the protease inhibitors lopinavir and ritonavir are currently used in anti-human immunodeficiency virus (HIV) therapy. Here, a high-performance liquid chromatography- mass spectrometry (HPLC-MS/MS) method, using a hybrid quadrupole time-of-flight mass analyzer, is reported for the simultaneous quantification of lamivudine, lopinavir, ritonavir, and zidovudine in plasma of HIV-infected patients. The volume of plasma sample was 600 μL. Plasma samples were extracted by solid-phase using 1 cc Oasis HLB Cartridge (divinylbenzene and N-vinylpyrrolidone) and evaporated in a water bath under nitrogen stream. The extracted samples were reconstituted with 100-μL methanol. Five microliters of the reconstituted samples were injected into a HPLC-MS/MS apparatus, and the analytes were eluted on a Vydac column (250 x 1.0 mm i.d.) filled with 3-μm C 18 particles. The mobile phase was delivered at 70 μL/ min with a linear gradient elution, both acetonitrile and ultrapure water solvents contained 0.2% formic acid. The calibration curves were linear from 0.47 to 20 ng/mL. The absolute recovery ranged between 91 and 107%. The minimal concentration of lamivudine, lopinavir, ritonavir, and zidovudine detectable by HPLC-MS/MS is 0.47, 0.28, 0.30, and 0.66 ng/mL, respectively. The great advantage of the new HPLC-MS/MS method here reported is the possibility to achieve a very high specificity toward the selected anti-HIV drugs, despite the simple and rapid sample preparation. Moreover, this method is easily extendible to the analysis of co-administrated drugs.

""The rate-limiting step of cholesterol biosynthetic. pathway is catalyzed by 3-hydroxy-3-methylglutaryl. coenzyme reductase (HGMR), whose inhibitors,. the statins, widely used in clinical practice to treat. hypercholesterolemia, often cause myopathy, and. rarely rhabdomyolysis. All studies to date are limited to. the definition of statin-induced myotoxicity omitting to. investigate whether and how HMGR inhibition influences. muscle functions. To this end, 3-mo-old male rats. (Rattus norvegicus) were treated for 3 wk with a daily. intraperitoneal injection of simvastatin (1.5 mg\\\/kg\\\/d),. and biochemical, morphological, mechanical, and functional. analysis were performed on extensor digitorum. longus (EDL) muscle. Our results show that EDL. muscles from simvastatin-treated rats exhibited reduced. HMGR activity; a 15% shift from the fastest. myosin heavy-chain (MHC) isoform IIb to the slower. IIa\\\/x; and reduced power output and unloaded shortening. velocity, by 41 and 23%, respectively, without any. change in isometric force and endurance. Moreover,. simvastatin-treated rats showed a decrease of maximum. speed reached and the latency to fall off the rotaroad. (30%). These results indicate that the molecular. mechanism of the impaired muscle function following. statin treatment could be related to the plasticity of fast. MHC isoform expression.—Trapani, L., Melli, L., Segatto,. M., Trezza, V., Campolongo, P., Jozwiak, A.,. Swiezewska, E., Pucillo, L.P., Moreno, S., Fanelli, F.,. Linari, M., Pallottini, V. Effects of myosin heavy chain. (MHC) plasticity induced by HMGCoA-reductase inhibition. on skeletal muscle functions.""

Therapeutic drug monitoring allows to determine the best dosage regimen adapted to each patient optimizing the therapeutic benefits, while minimizing the risk for side effects. Here, the first methodological approach based on matrix-assisted laser desorption/ionization source equipped with tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry for the determination of the antituberculosis (anti-TB) drugs ethambutol, pyrazinamide, rifampicin, and streptomycin concentration in the plasma of tuberculosis-infected patients is reported. The volume of the plasma sample was 200 microL. Plasma samples were cleaned-up by protein precipitation and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with 200 microL of 50% methanol-0.03% formic acid solution (v/v), spiked with known amounts of anti-TB drugs, mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid solution; v/v), and spotted onto the MALDI-TOF/TOF sample target plate. The anti-TB drug concentration was determined by standard additions analysis. Regression of standard additions was linear over the whole anti-TB drug concentration range explored (the final anti-TB drug concentration ranged from 0.20 to 200 pmol/microL). The absolute recovery of the anti-TB drugs ranged between 87 and 110%. The minimal ethambutol, pyrazinamide, rifampicin, and streptomycin concentration detectable by MALDI-TOF/TOF is 0.08, 0.20, 0.12, and 0.15 pmol/microL, respectively.