La sostituzione amminoacidica spontanea Ala147Thr nel dominio carbossil-terminale del Traslocatore Proteico (TSPO): influenza sulla produzione di pregnenolone in linfomonociti umani di individui sani.

Lo scopo della presente tesi è stato quello di indagare l’influenza della sostituzione amminoacidica spontanea (147 Ala→Thr), presente nel dominio carbossil-terminale del TSPO, sulla produzione di pregnenolone in soggetti sani. Al fine di caratterizzare la distribuzione genotipica del polimorfismo nella popolazione, è stata effettuata la sua genotipizzazione in 232 soggetti, ai quali è stato prelevato un campione di saliva da cui è stato estratto il DNA, quindi tramite la tecnica della PCR, analisi RFLP ed infine elettroforesi su gel d’agarosio è stato possibile determinarne il genotipo. E’ stata poi effettuata la quantificazione della concentrazione di pregnenolone in linfomonociti mediante test ELISA. I valori di pregnenolone ottenuti sono stati stratificati nei tre gruppi genotipici, quindi è stata effettuata un’analisi statistica evidenziando che sia gli individui eterozigoti che gli individui omozigoti Thr 147 mostrano livelli di pregnenolone significativamente più bassi rispetto ad individui omozigoti Ala 147. Sono stati inoltre valutati i livelli di LDL, HDL, colesterolo totale e trigliceridi ed in questo caso si è osservata una significatività statistica solo in riferimento ai livelli delle LDL. In particolare è emerso che gli eterozigoti presentano livelli di LDL significativamente più bassi rispetto agli individui omozigoti Ala 147. In conclusione, questi risultati dimostrano una correlazione tra il polimorfismo rs6971 e la produzione di pregnenolone in individui sani ed inoltre un’associazione del polimorfismo con i livelli di LDL. The purpose of this thesis was to inquire the influence of spontaneous amino acid substitution (147 Ala → Thr), present in the carboxy-terminal domain of the TSPO, into production of pregnenolone in healthy subjects. In order to define the genotypic distribution of polymorphism in the population, its genotyping was performed in 232 subjects, who were taken a saliva sample from which DNA was extracted, and, consequently, it was possible to determine the genotype by using the PCR technique, the RFLP analysis and, at the end, the agarose gel electrophoresis. The concentration of pregnenolone in lymphomonocytes was quantified by the ELISA test. The values of pregnenolone were stratified into three genotypic groups, and the statistical analysis performed have shown that both heterozygous individuals and homozygous individuals Thr147 reveal significantly lower levels of pregnenolone compared with homozygous individuals Ala147. Moreover, levels of LDL, HDL, total cholesterol and triglycerides were assessed and in this case there was a statistical significance only in reference to the levels of LDL. In particular, it was discovered that heterozygote individuals have significantly lower LDL levels than homozygous individuals Ala 147. In conclusion, these results demonstrate a correlation between the polymorphism rs6971 and the production of pregnenolone in healthy individuals and also an association of polymorphism with levels of LDL

Conjunctivally Applied BDNF Protects Photoreceptors from Light-Induced Damage

Purpose: To test whether the topical eye treatment with BDNF prevents the effects of continuous light exposure (LE) in the albino rat retina. Methods: Two groups of albino rats were used. The first group of rats received an intraocular injection of BDNF (2 lL, 1 lg/lL) before LE, while the second group was treated with one single drop of BDNF (10 lL, 12 lg/lL) dissolved in different types of solutions (physiological solution, the polysaccharide fraction of Tamarind gum, TSP, and sodium carboxy methyl cellulose), at the level of conjunctival fornix before LE. The level of BDNF in the retina and optic nerve was determined by enzyme-linked immunosorbent assay. We recorded the flash electroretinogram (fERG) in dark adapted rats 1 week after LE. At the end of the recording session, the retinas were removed and labeled so that the number of photoreceptors nuclear rows and thickness of the outer nuclear layer was analyzed. Results: Intravitreal injection of BDNF before LE prevented fERG impairment. Different ophthalmic preparations were used for topical eye application; the TSP resulted the most suitable vehicle to increase BDNF level in the retina and optic nerve. Topical eye application with BDNF/TSP before LE partially preserved both fERG response and photoreceptors. Conclusions: Topical eye treatment with BDNF represents a suitable, noninvasive tool to increase the retinal content of BDNF up to a level capable of exerting neuroprotection toward photoreceptors injured by prolonged LE. Translational Relevance: A collyrium containing BDNF may serve as an effective, clinically translational treatment against retinal degeneration

Comparison and combination of a hemodynamics/biomarkers-based model with simplified PESI score for prognostic stratification of acute pulmonary embolism: findings from a real world study

Background: Prognostic stratification is of utmost importance for management of acute Pulmonary Embolism (PE) in clinical practice. Many prognostic models have been proposed, but which is the best prognosticator in real life remains unclear. The aim of our study was to compare and combine the predictive values of the hemodynamics/biomarkers based prognostic model proposed by European Society of Cardiology (ESC) in 2008 and simplified PESI score (sPESI).Methods: Data records of 452 patients discharged for acute PE from Internal Medicine wards of Tuscany (Italy) were analysed. The ESC model and sPESI were retrospectively calculated and compared by using Areas under Receiver Operating Characteristics (ROC) Curves (AUCs) and finally the combination of the two models was tested in hemodinamically stable patients. All cause and PE-related in-hospital mortality and fatal or major bleedings were the analyzed endpointsResults: All cause in-hospital mortality was 25% (16.6% PE related) in high risk, 8.7% (4.7%) in intermediate risk and 3.8% (1.2%) in low risk patients according to ESC model. All cause in-hospital mortality was 10.95% (5.75% PE related) in patients with sPESI score ≥1 and 0% (0%) in sPESI score 0. Predictive performance of sPESI was not significantly different compared with 2008 ESC model both for all cause (AUC sPESI 0.711, 95% CI: 0.661-0.758 versus ESC 0.619, 95% CI: 0.567-0.670, difference between AUCs 0.0916, p=0.084) and for PE-related mortality (AUC sPESI 0.764, 95% CI: 0.717-0.808 versus ESC 0.650, 95% CI: 0.598-0.700, difference between AUCs 0.114, p=0.11). Fatal or major bleedings occurred in 4.30% of high risk, 1.60% of intermediate risk and 2.50% of low risk patients according to 2008 ESC model, whereas these occurred in 1.80% of high risk and 1.45% of low risk patients according to sPESI, respectively. Predictive performance for fatal or major bleeding between two models was not significantly different (AUC sPESI 0.658, 95% CI: 0.606-0.707 versus ESC 0.512, 95% CI: 0.459-0.565, difference between AUCs 0.145, p=0.34). In hemodynamically stable patients, the combined endpoint in-hospital PE-related mortality and/or fatal or major bleeding (adverse events) occurred in 0% of patients with low risk ESC model and sPESI score 0, whilst it occurred in 5.5% of patients with low-risk ESC model but sPESI ≥1. In intermediate risk patients according to ESC model, adverse events occurred in 3.6% of patients with sPESI score 0 and 6.65% of patients with sPESI score ≥1.Conclusions: In real world, predictive performance of sPESI and the hemodynamic/biomarkers-based ESC model as prognosticator of in-hospital mortality and bleedings is similar. Combination of sPESI 0 with low risk ESC model may identify patients with very low risk of adverse events and candidate for early hospital discharge or home treatment.

2-Acetyl-5-tetrahydroxybutyl imidazole (THI) protects 661W cells against oxidative stress

Retinal degeneration and in particular retinitis pigmentosa (RP) is associated to ceramide (Cer) accumulation and cell death induction. Cer and sphingosine-1-phosphate (S1P) belong to the sphingolipids class and exert a pro-apoptotic and pro-survival activity, respectively. Our aim is to target sphingolipid metabolism by inhibiting S1P lyase that regulates one of the S1P degradation pathways, to reduce retinal photoreceptor damage. The murine 661W cone-like cell line was pretreated with THI, an inhibitor of S1P lyase and exposed to H2O2-induced oxidative stress. 661W cell viability and apoptosis were evaluated by Trypan Blue and TUNEL assay, respectively. Protein expression of mediators of the survival/death pathway (ERK1/2, Akt, Bcl-2, Bax) was analyzed by Western blotting. RT-PCR was performed to establish HO-1 transcript changes and LC-MS analysis to measure Cer intracellular content. THI rescues inhibitory H2O2-effect on 661W cell viability and impairs H2O2-induced apoptosis by increasing Bcl-2/Bax ratio. THI administration counteracts the oxidative stress effects of H2O2 on 661W cells by activating the Nrf2/HO-1 pathway, regulating ERK and Akt phosphorylation levels, and decreasing Cer intracellular content. We conclude that sphingolipid metabolism manipulation can be considered a therapeutic target to promote photoreceptor survival. © 2017 Springer-Verlag Berlin HeidelbergPeer reviewe

Rescue of retinal function by BDNF in a mouse model of glaucoma.

Vision loss in glaucoma is caused by progressive dysfunction of retinal ganglion cells (RGCs) and optic nerve atrophy. Here, we investigated the effectiveness of BDNF treatment to preserve vision in a glaucoma experimental model. As an established experimental model, we used the DBA/2J mouse, which develops chronic intraocular pressure (IOP) elevation that mimics primary open-angle glaucoma (POAG). IOP was measured at different ages in DBA/2J mice. Visual function was monitored using the steady-state Pattern Electroretinogram (P-ERG) and visual cortical evoked potentials (VEP). RGC alterations were assessed using Brn3 immunolabeling, and confocal microscope analysis. Human recombinant BDNF was dissolved in physiological solution (0.9% NaCl); the effects of repeated intravitreal injections and topical eye BDNF applications were independently evaluated in DBA/2J mice with ocular hypertension. BDNF level was measured in retinal homogenate by ELISA and western blot. We found a progressive decline of P-ERG and VEP responses in DBA/2J mice between 4 and 7 months of age, in relationship with the development of ocular hypertension and the reduction of Brn3 immunopositive RGCs. Conversely, repeated intravitreal injections (BDNF concentration = 2 µg/µl, volume = 1 µl, for each injection; 1 injection every four days, three injections over two weeks) and topical eye application of BDNF eye-drops (12 µg/µl, 5 µl eye-drop every 48 h for two weeks) were able to rescue visual responses in 7 month DBA/2J mice. In particular, BDNF topical eye treatment recovered P-ERG and VEP impairment increasing the number of Brn3 immunopositive RGCs. We showed that BDNF effects were independent of IOP reduction. Thus, topical eye treatment with BDNF represents a promisingly safe and feasible strategy to preserve visual function and diminish RGC vulnerability to ocular hypertension

Effects of topical eye treatment with BDNF on RGCs expressing Brn3.

<p>Topical eye treatment was performed as already described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115579#pone-0115579-g004" target="_blank">Fig. 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115579#pone-0115579-g005" target="_blank">5</a>; at the end of the P-ERG recording session mice were sacrificed for immunocytochemistry. <b>A</b>. Magnifications from the central (top row) and peripheral (bottom row) field from flat mounted retinas showing Brn3 immunopositive RGCs in BDNF treated eye (left column) and contralateral vehicle treated eye (right column) of a 7 month DBA/2J mouse; scale bar = 40 µm. <b>B</b>. All immunopositive RGCs were counted independently from the level of Brn3 expression. The histogram reports the mean cell density of RGCs expressing Brn3 (cells/mm²) in the central and peripheral retina from BDNF treated eye (12 µg/µl); Brn3 immunopositive RGC cell density was significantly higher in both the central and peripheral retina of BDNF treated eye respect to vehicle treated eye, *p<0.05 (one-way ANOVA). <b>C</b>. Summary of Brn3 results obtained in 7 month DBA/2J untreated eyes, 7 month DBA/2J BDNF treated and vehicle treated eyes; BDNF treated eyes <i>versus</i> vehicle treated eyes and untreated eyes, *p<0.05 (one-way ANOVA).</p

Effects of topical eye treatment with BDNF on VEP of DBA/2J mice.

<p>Topical eye treatment consisted of 1 drop of 5 µl containing BDNF (12 µg/µl) or physiological solution (vehicle) for two weeks, one application every 48 h, in 7 month DBA/2J mice with ocular hypertension. VEPs were recorded through a screw inserted in the occipital bone contralateral to the stimulated eye. <b>A</b>. Examples of VEPs recorded for stimulation of BDNF treated eye (top row) and contralateral vehicle treated eye (bottom row); VEP was evoked by 0.05 and 0.2 c/deg, 1^st and 2^nd column, respectively. Calibration horizontal bar = 100 msec, vertical bar = 1 µV. <b>B</b>. The histogram reports the mean VEP amplitudes at 0.05 and 0.2 c/deg in 12 µg/µl BDNF treated eyes and vehicle treated eyes of 7 month DBA/2J mice; BDNF treated eyes <i>versus</i> vehicle treated eyes, *p<0.05 (one-way ANOVA). <b>C</b>. The cortical spatial resolution limit was calculated by linear extrapolation to noise level as in the example from a 7 month DBA/2J mouse. VEP amplitudes were plotted as a function of spatial frequencies, semilog coordinates, for each eye, i.e. the eye treated with topical applications of BDNF (12 µg/µl; black circles, solid line indicates the linear fit) and the contralateral eye treated with vehicle (empty circles, dashed line indicates the linear fit); cortical spatial resolution limit for each eye (BDNF treated and vehicle treated eye) is indicated by an arrow. <b>D</b>. The averaged cortical spatial resolution limit (cortical acuity) in the BDNF treated eyes was significantly higher than that in the vehicle treated eyes; *p<0.05. Error bars indicate SEM.</p

VEP in DBA/2J mice.

<p><b>A</b>. Examples of VEP recordings from C57BL/6J (1^st column), 4 month (middle column) and 7 month DBA/2J mice (3^rd lateral column); VEPs were recorded through a screw inserted in the occipital bone contralateral to the stimulated eye in response to stimuli of 0.05 (1^st row) and 0.2 c/deg (2^nd row), 90% contrast (1 Hz frequency of alternation). VEPs show a prominent positive component characterized by latency around 107 msec in C57BL/6J mouse. Horizontal calibration bar = 100 msec, vertical calibration bar = 5 µV. <b>B</b>. The averaged amplitudes of VEP in C57BL/6J, 4 month and 7 month DBA/2J mice were plotted as a function of the spatial frequencies (semi-logarithmic scale); the mean noise level (responses recorded at different spatial frequencies with contrast = 0) is visible as a dotted line. Mean VEP amplitudes were reduced at all spatial frequencies in 7 month DBA/2J mice with respect to 4 month DBA/2J and C57BL/6J mice. <b>C</b>. Mean VEP amplitudes at 0.05 and 0.2 c/deg in C57BL/6J, 4 month and 7 month DBA/2J mice; 7 month DBA/2J <i>versus</i> C57BL/6J and 4 month DBA/2J, *p<0.05 (one-way ANOVA). Error bars indicate SEM. <b>D</b>. Scatter plot of VEP amplitudes vs. IOP in 4 month and 7 month DBA/2J mice (for spatial frequencies of 0.05 and 0.2 c/deg). At both frequencies VEP amplitudes were inversely correlated to IOP in DBA/2J mice. The dashed line is the best linear fit for 0.2 c/deg. The solid line is the best linear fit for the 0.05 c/deg. The black square indicates the mean VEP amplitude ± SEM at 0.05 c/deg in C57BL/6J mice and the empty square indicates the mean VEP amplitude ± SEM at 0.2 c/deg in C57BL/6J mice.</p

Effects of eye treatments with BDNF on P-ERG of DBA/2J mice.

<p><b>A, B</b>. Intravitreal treatment consisted of three intravitral injections over two weeks (BDNF = 2 µg/µl, volume = 1 µl for each injection) in 7 month DBA/2J mice (n = 5, n eyes = 5) with ocular hypertension; the contralateral eye was injected with the physiological solution (vehicle). <b>A</b>. Examples of P-ERG recorded from BDNF injected eye (1^st column) and contralateral vehicle treated eye (2^nd column); P-ERG was evoked by 0.05 and 0.2 c/deg spatial frequencies, 1^st and 2^nd row, respectively. Horizontal calibration bar = 50 msec, vertical calibration bar = 1 µV. <b>B</b>. Mean P-ERG amplitude at spatial frequency of 0.05 c/deg and 0.2 c/deg for BDNF and vehicle injected eyes in 7 month DBA/2J mice; * p<0.05. <b>C–F</b>. Topical eye treatment consisted of 1 drop of 5 µl containing BDNF at different concentrations or vehicle (physiological solution) for two weeks, one application every 48 h, in 7 month DBA/2J mice with ocular hypertension. <b>C</b>. Examples of P-ERG recorded from BDNF treated eye (12 µg/µl, 1^st column) and contralateral vehicle treated eye (2^nd column); P-ERG was evoked by 0.05 and 0.2 c/deg spatial frequencies, 1^st and 2^nd row, respectively. Horizontal calibration bar = 50 msec, vertical calibration bar = 1 µV. <b>D</b> and <b>E</b>. Mean P-ERG amplitude at spatial frequency of 0.05 c/deg (D) and 0.2 c/deg (E) from the eyes of 7 month DBA/2J mice treated with BDNF at concentrations of 1, 5 and 12 µg/µl; the averaged P-ERG amplitude in the contralateral eye treated with vehicle is reported for statistical comparison; BDNF (12 µg/µl) <i>versus</i> vehicle treated eyes, 1 and 5 µg/µl BDNF, * p<0.05 (one-way ANOVA). <b>F</b>. IOP measured before and after treatment in BDNF treated eyes (12 µg/µl, n eyes = 7) and vehicle (n eyes = 7). Error bars indicate SEM.</p