Delayed luminescence to monitor programmed cell death induced by berberine on thyroid cancer cells

Correlation between apoptosis and UVA-induced ultraweak photon emission delayed luminescence (DL) from tumor thyroid cell lines was investigated. In particular, the effects of berberine, an alkaloid that has been reported to have anticancer activities, on two cancer cell lines were studied. The FTC-133 and 8305C cell lines, as representative of follicular and anaplastic thyroid human cancer, respectively, were chosen. The results show that berberine is able to arrest cell cycle and activate apoptotic pathway as shown in both cell lines by deoxyribonucleic acid fragmentation, caspase-3 cleavage, p53 and p27 protein overexpression. In parallel, changes in DL spectral components after berberine treatment support the hypothesis that DL from human cells originates mainly from mitochondria, since berberine acts especially at the mitochondrial level. The decrease of DL blue component for both cell lines could be related to the decrease of intra-mitochondrial nicotinamide adenine dinucleotide and may be a hallmark of induced apoptosis. In contrast, the response in the red spectral range is different for the two cell lines and may be ascribed to a different iron homeostasis

Dynamic changes in cytoskeleton proteins of olfactory ensheathing cells induced by radiofrequency electromagnetic fields.

Several evidences have suggested the ability of radio frequency electromagnetic fields to influence biological systems, even if the action mechanisms are not well understood. Only few data have reported about the effect of radio frequency electromagnetic fields on self-renewal of neural progenitor cells. A particular glial type, which shows characteristics of stem cells, are Olfactory Ensheathing Cells (OECs). Herein, we assessed the non-thermal effects induced on Olfactory Ensheathing Cells through radio frequency electromagnetic fields changing the envelope of the electromagnetic wave. Primary OEC cultures were exposed to continuous or amplitude modulated 900 MHz electromagnetic fields, in far field condition and at different exposition times (10, 15, 20 min). The expression of Olfactory Ensheathing Cells markers (S-100 and Nestin), cytoskeletal proteins (GFAP and Vimentin), apoptotic pathway activation by Caspase-3 cleavage and cell viability were evaluated. Our results highlight that 20 min of exposure to continuous or amplitude modulated 900 MHz electromagnetic fields induced a different and significant decrease in cell viability. In addition, according to the electromagnetic fields waveform, diverse dynamic changes in the expression of the analysed markers in Olfactory Ensheathing Cells and activation of apoptotic pathway were observed. The data suggest that radio frequency electromagnetic fields might play different and important role in the self-renewal of OEC stem cells, which are involved in nervous system repair

Use of the T-spot.TB test for the diagnosis of latent tuberculosis infection

Background:Tuberculosis (TB) represents a major health problem both in developing and both in industrialized countries.The identification of individuals latently infected with Mycobacterium tuberculosis (Mtb) play a key role for the efficacy of TB control. These individuals with a latent tuberculosis infection (LTBI), especially those with high risk of reactivation (e.g. HIV + / AIDS-infected individuals, patients undergoing immunosuppressive therapy and children younger than 5 years) could benefit from a preventive treatment with isoniazid reducing the risk of progression from LTBI to active TB. Until recently, detection of LTBI has relied on the tuberculin skin test (TST), but despite the widespread use in clinical practice,TST does not reliably diagnose LTBI because several drawbacks, e.g. lacking in specificity, particularly in who were exposed to non-tuberculous mycobacteria (NTM) or were vaccinated with Bacille Calmette-Guerin (BCG) In addition, in young subjects,TST sensitivity is hampered by impaired T cell function leading frequently to false negative results.These several drawbacks limit the use of TST for the diagnose an LTBI in patients who may benefit from preventive chemotherapy. On the other hand, an accurate diagnosis of LTBI avoid the over-treatment of those patients with a positive TST results but not latently infected with Mtb. Recently, new tests based on the detection of interferon-gamma (IFN-γ) after stimulation with Mtb-specific antigens: Early secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10) have been proposed for the diagnosis of active TB and LTBI. Methods: During the period from January 2009 to June 2009, in our laboratory 70 patients were tested with T-SPOT.TB (Oxford Immunotech, Abingdon, United Kingdom).We enrolled transplant patients and subjects ongoing transplant, patients immigrants from high prevalence TB countries, patients screened for immunosuppressive treatment, HIV / AIDS – infected individuals.We also tested 3 patients with clinical / radiological suspicion of active TB and 3 patients with positive tuberculin skin test and with a positive direct examination for mycobacteria in the urinary sediment. Results: In 2 patients with symptoms suggestive of TB in place,T-SPOT.TB showed a higher response of (IFN-g), more than 100 spots.Among individuals ongoing renal transplant, 6 patients tested T-SPOT.TB positive and 4 subjects were T.SPOT.TB -negative. Two patients with an autoimmune disease showed an high response to Mtb-specific antigens with T-SPOT.TB test tested before to start any treatment.T-SPOT.TB test tested strongly negative in 4 paediatric patients and in one HIV-infected individuals, regardless a positive response to a internal positive response (phytohaemagglutinin (PHA), suggesting a normal immune response. Conclusions:This preliminary data suggest that the T.SPOT.TB showed high sensitivity and specificity, producing a strongly negative response to Mtb-specific antigens in subjects who had a history of previous BCG-vaccination. In addition, T-SPOT.TB test provides, unlike the TST, indication about the potential immunosuppression of tested patient with an internal positive control that can highlight the production of IFN- γ by lymphocytes resulting in the application of this test in immunocompromised patients, e.g. children and transplantated patients and others

Mitochondrial respiratory complex I probed by delayed luminescence spectroscopy.

The role of mitochondrial complex I in ultraweak photon-induced delayed photon emission (delayed luminescence (DL)) of human leukemia Jurkat T cells was probed by using complex I targeting agents like rotenone, menadione, and quercetin. Rotenone, a complex I-specific inhibitor, dose-dependently increased the mitochondrial level of reduced nicotinamide adenine dinucleotide (NADH), decreased clonogenic survival, and induced apoptosis. A strong correlation was found between the mitochondrial levels of NADH and oxidized flavin mononucleotide (FMNox) in rotenone-, menadione- and quercetin-treated cells. Rotenone enhanced DL dose-dependently, whereas quercetin and menadione inhibited DL as well as NADH or FMNox. Collectively, the data suggest that DL of Jurkat cells originates mainly from mitochondrial complex I, which functions predomi- nantly as a dimer and less frequently as a tetramer. In individual monomers, both pairs of pyridine nucleotide (NADH/reduced nicotinamide adenine dinucleotide phosphate) sites and flavin (FMN-a/FMN-b) sites appear to bind cooperatively their specific ligands. Enhancement of delayed red-light emission by rotenone suggests that the mean time for one-electron reduction of ubiquinone or FMN-a by the terminal Fe/S center (N2) is 20 or 284 μs, respectively. All these findings suggest that DL spectroscopy could be used as a reliable, sensitive, and robust technique to probe electron flow within complex I in situ. © 2013 Society of Photo-Optical Instrumentatio

Prognostic Significance of Myocardial Fibrosis in Hypertrophic Cardiomyopathy

ObjectivesWe investigated the significance of fibrosis detected by late gadolinium enhancement cardiovascular magnetic resonance for the prediction of major clinical events in hypertrophic cardiomyopathy (HCM).BackgroundThe role of myocardial fibrosis in the prediction of sudden death and heart failure in HCM is unclear with a lack of prospective data.MethodsWe assessed the presence and amount of myocardial fibrosis in HCM patients and prospectively followed them for the development of morbidity and mortality in patients over 3.1 ± 1.7 years.ResultsOf 217 consecutive HCM patients, 136 (63%) showed fibrosis. Thirty-four of the 136 patients (25%) in the fibrosis group but only 6 of 81 (7.4%) patients without fibrosis reached the combined primary end point of cardiovascular death, unplanned cardiovascular admission, sustained ventricular tachycardia or ventricular fibrillation, or appropriate implantable cardioverter-defibrillator discharge (hazard ratio [HR]: 3.4, p = 0.006). In the fibrosis group, overall risk increased with the extent of fibrosis (HR: 1.18/5% increase, p = 0.008). The risk of unplanned heart failure admissions, deterioration to New York Heart Association functional class III or IV, or heart failure-related death was greater in the fibrosis group (HR: 2.5, p = 0.021), and this risk increased as the extent of fibrosis increased (HR: 1.16/5% increase, p = 0.017). All relationships remained significant after multivariate analysis. The extent of fibrosis and nonsustained ventricular tachycardia were univariate predictors for arrhythmic end points (sustained ventricular tachycardia or ventricular fibrillation, appropriate implantable cardioverter-defibrillator discharge, sudden cardiac death) (HR: 1.30, p = 0.014). Nonsustained ventricular tachycardia remained an independent predictor of arrhythmic end points after multivariate analysis, but the extent of fibrosis did not.ConclusionsIn patients with HCM, myocardial fibrosis as measured by late gadolinium enhancement cardiovascular magnetic resonance is an independent predictor of adverse outcome. (The Prognostic Significance of Fibrosis Detection in Cardiomyopathy; NCT00930735