Variazione dei parametri di funzionalita piastrinica durante la conservazione di buffy-coat

Introduzione: In questi ultimi 10 anni i metodi per la raccolta, la lavorazione e la conservazione dei concentrati piastrinici si sono rapidamente evoluti mentre, di pari passo, si è ampliato il numero delle patologie che si giovano di tale trattamento cosicché, a partire dai primi anni ottanta ad oggi, l'uso dei concentrati piastrinici è aumentato del 400% . Inizialmente i concentrati piastrinici per uso terapeutico erano allestiti come pool di plasma ricco di piastrine ottenuto da una singola donazione di sangue intero. Sono poi state introdotte nuove metodologie produttive, ad esempio l'allestimento di concentrati piastrinici random a partire da pool di buffy-coat; la conservazione di buffy-coat ha però diversi punti critici, tra i quali quanto tempo possono essere conservati. Attualmente la conservazione dei buffy-coat prima dell’assemblaggio non è definita per cui, alla luce dell’aumentata richiesta di concentrati piastrinici sviluppatasi negli ultimi anni, sarebbe utile conoscere il tempo massimo di conservazione di questi ultimi. Scopo di questo lavoro è valutare come variano i parametri funzionalità e morfologia piastrinica nel concentrato ottenuto da un pool di buffy-coat conservato fino a 5 giorni. Materiali e Metodi: da 15 buffy coat di gruppo A è stato preparato un pool conservato in agitazione a 20-24°C. A partire dal giorno della raccolta dei buffy coat (T1), dopo 3 (T3) e 5 giorni (T5) è stato ottenuto il concentrato piastrinico. Su quest’ultimo è stato effettuata l’analisi emocromocitometrica, e determinati alcuni parametri biochimici quali il glucosio, lattato, lattato deidrogenasi (LDH) e pH. È stata inoltre valutata la funzionalità piastrinica mediante test di aggregazione (usando come agonisti l’ADP e il collagene), il grado di attivazione delle piastrine in base all’espressione di P-selectina sulla superficie mediante citofluorimetria e la concentrazione di serotonina mediante HPLC. Risultati: I risultati ottenuti hanno dimostrato una diminuzione progressiva della concentrazione di glucosio da T1 a T5 accompagnata da un aumento sia della concentrazione del lattato che dell’attività catalitica di LDH. Il pH diminuisce da 6,5 a 6,2 dopo 5 giorni. Anche i parametri relativi alla morfologia piastrinica si modificano nel tempo con un aumento progressivo sia dell’MPV che del PDW. L’espressione della P-selectina va dal 10,3% il primo giorno dopo la raccolta (T1) al 29,4% il terzo giorno (T3) fino ad arrivare al 44,1% a T5. Insieme all’aumentata espressione della P-selectina è stato riscontrato anche un aumento dei livelli di serotonina. Conclusioni: Da questi primi risultati è possibile concludere che già dopo tre giorni di conservazione i concentrati piastrinici presentano alterazioni biochimiche del mezzo, ed alterazioni morfologiche e di funzionalità delle piastrine. Questo studio preliminare suggerisce che non è consigliabile la conservazione dei buffy-coat oltre i tre giorni

Circulating gamma-glutamyltransferase fractions in cirrhosis.

Background: Four GGT fractions (b-, m-, s-, and f-GGT) have been identified in human plasma and their concentrations and ratios vary in different pathological conditions. Aim: To assess the behavior of fractional GGT in cirrhotic patients evaluated for liver transplantation. Methods: This was a single-center, cross-sectional study; GGT fractions were determined by gel-filtration chromatography. Results: 264 cirrhotic patients (215 males; median age 54.5 years) were included and compared against a group of 200 healthy individuals (100 males; median age 41.5). Median (25th-75th percentile) total and fractional GGT were higher in cirrhotics, with s-GGT showing the greatest increase [36.6 U/L (21.0-81.4) vs. 5.6 U/L (3.2-10.2), (p<0.0001)], while the median b-GGT/s-GGT ratio was lower in cirrhotics than in healthy controls [0.06 (0.04-0.10)] vs. 0.28 (0.20-0.40), p<0.0001]. The ratio showed higher diagnostic accuracy (ROC-AUC, 95% CI: 0.951, 0.927-0.969) then either s-GGT (0.924, 0.897-0.947; p<0.05) or total GGT (0.900, 0.869-0.925; p<0.001). The diagnostic accuracy of the ratio was maintained (0.940, 0.907-0.963) in cirrhotic patients (n=113) with total GGT values within the reference range. The s-GGT fraction consisted of two components, with one (s2-GGT) showing a significant positive correlation with serum AST, ALT, LDH, ALP and bilirubin, and negative with albumin. The b-GGT fraction showed a positive correlation with albumin, fibrinogen, and platelet counts, and negative with INR, bilirubin and LDH. Conclusions: The ratio performs as a sensitive biomarker of the liver parenchymal rearrangement, irrespective of etiology of cirrhosis and presence of hepatocellular carcinoma, even in patients with total GGT values within the reference range

Event-Based Power/Performance-Aware Thermal Management for High-Density Microprocessors

The density of modern microprocessors is so high, that operating all their units at full power would destroy them by thermal runaway. Hence, thermal control is vital, but at the same time has to integrate with power/performance management, to not unduly limit computational speed. In addition, the controller must be simple and computationally light, as millisecond-scale response is required. Finally, since microprocessors face a variety of operating conditions, postsilicon tuning is an issue. We here present a solution, by exploiting event-based control and a hardware/software partition to maximize efficiency, lightness, and flexibility. We show experiments on real hardware, evidencing the obtained advantages over the state of the art

Forme molecolari della γ-glutammiltransferasi: Caratteristiche e biogenesi

Four GGT fractions (b-, m-, s- and f-GGT) have been described in plasma. The aim of this study was to characterize their molecular nature in human plasma and bile. Plasma was obtained from healthy volunteers and primary bile was collected from patients undergoing liver transplant. For each GGT fraction we determined MW, density, sedimentation conditions in centrifugation assays, and the sensitivity to detergent [deoxycholic acid (DOC)] and protease (papain). A partial purification of b-GGT for immunogold analysis was obtained by ultracentrifugation. Plasma b-GGT showed a MW of 2000 kDa and a density between 1.063-1.210 g/mL. Treatment with 1% DOC converted b-GGT into s-GGT fraction, while b-GGT was not sensible to papain treatment. Plasma m-GGT and s-GGT showed a MW of 1000 and 200 kDa, and their densities were between 1.006-1.063 g/mL and 1.063-1.210 g/mL, respectively. Both fractions were unaffected by DOC treatment, while GGT activity was completely recovered in f-GGT peak after their incubation with papain. Plasma f-GGT showed a MW of 70 kDa and a density >1.21 g/mL. In human hepatic bile we identified two peaks showing the same characteristics of plasma b- and f-GGT fractions. Collected data showed that b-GGT is constituted by membrane microvesicles both in bile and plasma, as confirmed by immunogold; m-GGT and s-GGT might be constituted by bile-acid micelles, while f-GGT represents the free-soluble form of the enzyme. The understanding of the nature and properties of plasma GGT fractions may allow a better clinical utilization of GGT as a clinical biomarker

The β-adrenergic system as a possible new target for pharmacologic treatment of neovascular retinal diseases

Retinal neovascular pathologies, such as diabetic retinopathy, retinopathy of prematurity (ROP) and agerelated macular degeneration, may be treated with intravitreal injections of drugs targeting vascular endothelial growth factor (VEGF), the main inducer of neoangiogenesis; however further improvements and alternative strategies are needed. In the last few years, an intense research activity has focused on the b-adrenergic system. The results indicate that, in different experimental models, a decrease of the badrenergic function may result either in reduction or in exacerbation of the vascular changes, thus suggesting possible dual effects of b-adrenoreceptor (b-AR) modulation depending on the experimental setting. In in vivo models of proliferative retinopathies, most of the data point to a strong inhibitory role against vascular changes exerted by the blockade of specific b-ARs. In particular, the b2-AR seems to be the mostly involved in these responses, and the b1-/b2-AR blocker propranolol results highly effective in inhibiting both the increase of VEGF expression caused by a hypoxic insult and the consequent neovascular response. These observations have prompted clinical trials in preterm infants with ROP, where oral administrations of propranolol produced positive results in terms of efficacy, although safety problems were also reported. In addition, the possibility of using topical propranolol administrations in the form of eye drops opens new potential routes of drug administration in humans. A further point that should be considered is that there are data demonstrating significant antiapoptotic effects exerted by b- ARs, therefore if b-AR blockers were used to inhibit aberrant neovascularization, there may be a burden to pay in terms of impaired neuronal viability

Acetyl-11-keto-<beta>-boswellic acid reduces retinal angiogenesis in a mouse model of oxygen-induced retinopathy

Retinal diseases characterized by pathologic retinal angiogenesis are the leading causes of blindness worldwide. Although therapies directed toward vascular endothelial growth factor (VEGF) represent a significant step forward in the treatment of proliferative retinopathies, further improvements are needed. In the last few years, an intense research activity has focused around the use of herbal and traditional natural medicines as an alternative for slowing down the progression of proliferative retinopathies. In the present study, we investigated the antiangiogenic effects of acetyl-11-keto-β-boswellic acid (AKBA), one of the active principles derived from the plant Boswellia serrata, used in Ayurvedic systems of medicine. We studied the antiangiogenic properties of AKBA using the mouse model of oxygen-induced retinopathy (OIR), which mimics the neovascular response seen in human retinopathy of prematurity. We first evaluated the effects of subcutaneously administered AKBA on the expression/activity of proteins which are known to play a role in the OIR model. In the retina, AKBA increased expression and activity of Src homology region 2 domain-containing phosphatase 1 and reduced the phosphorylation of the transcription factor signal transducer and activator of transcription 3 (STAT3) as well as VEGF expression and VEGF receptor (VEGFR)-2 phosphorylation. Likely as a result of these effects, AKBA significantly reduced retinal neovascularization in OIR mice without affecting retinal cell survival and retinal function. Using retinal explants cultured in hypoxia and an activator of STAT3 phosphorylation, we showed that the AKBA-induced inhibition of VEGFR-2 phosphorylation is likely to be mediated by a mechanism depending on an SHP-1/STAT3/VEGF axis. In the OIR model, neovascularization results from the activation of retinal endothelial cells, therefore we evaluated whether AKBA affected the angiogenic response of human retinal microvascular endothelial cells (HRMECs). We observed that AKBA reduced proliferation, migration and tube formation in HRMECs stimulated with exogenous VEGF, while it reduced migration and tube formation in untreated HRMECs. Taken together, our results demonstrate the antiangiogenic effects of AKBA in a model of pathologic neovascularization, providing a rationale for further investigation of AKBA as a promising therapeutic agent to reduce the impact of proliferative retinopathies

Plasma gamma-glutamyltransferase (GGT) activity in inflammatory bowel disease: Is the clinical laboratory plasma GGT assay sensitive enough for gastroenterology?

BACKGROUND

β3 adrenergic receptor activity modulates melanoma cell proliferation and survival through nitric oxide signaling.

We have recently shown in B16F10 melanoma cells that blockade of β3-adrenergic receptors (β3-ARs) reduces cell proliferation and induces apoptosis, likely through the involvement of nitric oxide (NO) signaling. Here, we tested the hypothesis that the effects of β3-AR blockade on melanoma cells are mainly mediated by a decrease in the activity of the NO pathway, possibly due to reduced expression of inducible NO synthase (iNOS). B16F10 cells were used. Nitrite production, iNOS expression, cell proliferation and apoptosis were evaluated. β3-AR blockade with L-748,337 reduced basal nitrite production, while β3-AR stimulation with BRL37344 increased it. The effects of β3-AR blockade were prevented by NOS activation, while the effects of β3-AR activation were prevented by NOS inhibition. Treatments increasing nitrite production also increased iNOS expression, while treatments decreasing nitrite production reduced iNOS expression. Among the different NOS isoforms, experiments using L-748,337 or BRL37344 with activators or inhibitors targeting specific NOS isoforms demonstrated a prominent role of iNOS in nitrite production. β3-AR blockade decreased cell proliferation and induced apoptosis, while β3-AR activation had the opposite effects. The effects of β3-AR blockade/activation were prevented by iNOS activation/inhibition, respectively. Taken together, these results demonstrate that iNOS-produced NO is a downstream effector of β3-ARs and that the beneficial effects of β3-AR blockade on melanoma B16F10 cells proliferation and apoptosis are functionally linked to reduced iNOS expression and NO production. Although it is difficult to extrapolate these data to the clinical setting, the targeted inhibition of the β3-AR-NO axis may offer a new therapeutic perspective to treat melanomas

Gamma-glutamyltransferase fractions in human plasma and bile: characteristic and biogenesis.

Total plasma gamma-glutamyltransferase (GGT) activity is a sensitive, non-specific marker of liver dysfunction. Four GGT fractions (b-, m-, s-, f-GGT) were described in plasma and their differential specificity in the diagnosis of liver diseases was suggested. Nevertheless fractional GGT properties have not been investigated yet. The aim of this study was to characterize the molecular nature of fractional GGT in both human plasma and bile. Plasma was obtained from healthy volunteers; whereas bile was collected from patients undergoing liver transplantation. Molecular weight (MW), density, distribution by centrifugal sedimentation and sensitivity to both detergent (deoxycholic acid) and protease (papain) were evaluated. A partial purification of b-GGT was obtained by ultracentrifugation. Plasma b-GGT fraction showed a MW of 2000 kDa and a density between 1.063-1.210 g/ml. Detergent converted b-GGT into s-GGT, whereas papain alone did not produce any effect. Plasma m-GGT and s-GGT showed a MW of 1,000 and 200 kDa, and densities between 1.006-1.063 g/ml and 1.063-1.210 g/ml respectively. Both fractions were unaffected by deoxycholic acid, while GGT activity was recovered into f-GGT peak after papain treatment. Plasma f-GGT showed a MW of 70 kDa and a density higher than 1.21 g/ml. We identified only two chromatographic peaks, in bile, showing similar characteristics as plasma b- and f-GGT fractions. These evidences, together with centrifugal sedimentation properties and immunogold electronic microscopy data, indicate that b-GGT is constituted of membrane microvesicles in both bile and plasma, m-GGT and s-GGT might be constituted of bile-acid micelles, while f-GGT represents the free-soluble form of the enzyme