Platelet glycoproteins IIb and IIIa as a calcium channel in liposomes

Human platelet membrane glycoproteins IIb and IIIa (GPIIb and IIIa) were incorporated into phospholipid vesicles by the reverse-phase technique to assess the ability of GPIIb and IIIa to function as a Ca2+ channel. Movement of Ca2+ across the lipid bilayer was quantitated by injection of proteoliposomes with encapsulated Fura-2 into Ca2+ buffers and measurement of Fura-2 fluorescence as an indicator of Ca2+ influx. Reciprocally, to assess the function of proteins in an inside-out orientation, Ca2+-loaded vesicles were injected into Ca2+-free buffer and Ca2+ efflux monitored by a calcium electrode. Incorporation of the IIb-IIIa complex produced significant facilitation of Ca2+ movement across the lipid bilayer. No net transmembrane Ca2+ movement was seen with dissociated IIb and IIIa. Movement of Ca2+ was proportional to the transmembrane Ca2+ gradient. Ca2+ movement into the vesicles was inversely proportional to extravesicular NaCl from 25 to 150 mmol/L, analogous to several studies in the intact platelet. Adenosine triphosphate had no effect on Ca2+ movement into or out of the vesicles. Specific inhibition of a Ca2+ shift into the vesicles was seen with M148, a monoclonal antibody to IIb/IIIa, while no inhibition was observed with a panel of other anti-IIb/IIIa monoclonal antibodies. This suggests that a specific site on the complex or orientation of the complex is essential for calcium channel function. These data demonstrate that the GPIIb/IIIa complex can serve as a passive Ca2+ channel across a phospholipid bilayer and has the potential to play a role in Ca2+ flux across the platelet plasma membrane.     

Evaluation of a recently described risk classification scheme for pancreatic fistulae development after pancreaticoduodenectomy without routine post-operative drainage

Background

Post-operative pancreatic fistula (POPF) formation occurs frequently after a pancreaticoduodenectomy (PD). Recently, a 10-point Fistula Risk Score (FRS) evaluating the likelihood of clinically relevant POPF (CR-POPF) development has been described and validated. This scheme has yet to be evaluated in PD patients managed without intra-operative drain placement.

Methods

Among patients undergoing PD at an academic centre since 2003, a retrospective analysis calculating FRS and its correlation with CR-POPF development was evaluated by logistic regression. Secondary analysis examined presentation and management of CR-POPF in undrained PD patients.

Results

FRS was calculated for 265 patients; 97.7% were managed without operative drains. The overall incidence of CR-POPF was 7.9%. Logistic regression revealed a 1.6-fold increase in CR-POPF risk per 1-point increase in FRS [95% confidence interval (CI) 1.2–2.0]. The negative predictive value in patients with FRS <3 was 100%, whereas the positive predictive value of FRS >6 was 16.7%. The median time to CR-POPF diagnosis was 18 days [interquartile range (IQR) 13–23]; 70.0% required readmission and 10.0% required a laparotomy.

Conclusions

Among patients without operative drainage, CR-POPF often has delayed presentations but most are managed non-operatively. The predictive value of high-risk FRS appears limited; conversely, a low-risk FRS accurately predicts the absence of CR-POPF and seems an appropriate metric for guiding care.     

The efficacy of hepatitis B treatments in achieving HBsAg seroclearance: A systematic review and meta‐analysis

Current therapies for chronic hepatitis B (CHB) include nucleos(t)ide analogues (NAs) and interferon (IFN), but their relative efficacy as monotherapy or in combination has not been examined systematically for HBsAg loss (functional cure). Hence, we systematically reviewed the evidence for HBsAg loss in CHB patients treated with IFN, NA or the combination. We searched PubMed, EMBASE and abstracts from EASL, Asia Pacific Association for study of the Liver and American Association for the Study of Liver Disease for randomized controlled trials of CHB patients, comparing NA, IFN or the combination. The Cochrane Risk of Bias tool v2.0 and GRADE method were used. Analyses were stratified by NA genetic barrier, cirrhosis, type of combination therapy, HBeAg, treatment naivety, IFN dosage/duration and outcome duration. Sensitivity analysis was performed for selected strata, and HBsAg loss was measured at the end‐of‐study (EOS), end‐of‐treatment (EOT) or end‐of‐follow‐up (EOF). Effects were reported as risk differences (RD) with 95% confidence intervals (CI) using a random‐effects model. Forty‐five studies were included, all with low risk of bias. For HBsAg loss at EOS, when comparing combination vs IFN, RD = 1%, 95%CI‐1%, 2%; combination vs NA, RD = 5%, 95%CI 3%,7%; IFN vs NA, RD = 3%, 95%CI 2%,5%. Subgroup analysis showed a significant effect of standard IFN dose vs nonstandard; IFN duration ≥48 weeks vs <48 weeks, and loss of efficacy >2 years of follow‐up. Similar findings were seen in HBsAg seroconversion, but only three studies reported HBsAg seroreversion. In conclusion, IFN monotherapy/combination had a small but significant increase in HBsAg loss over NA, associated with standard dose of IFN and ≥48 weeks of therapy, although this effect faded over time.     

FACTORS ASSOCIATED TO ADHERENCE TO DIFFERENT TREATMENT SCHEMES WITH MEGLUMINE ANTIMONIATE IN A CLINICAL TRIAL FOR CUTANEOUS LEISHMANIASIS

The favorable outcome of the treatment of a disease is influenced by the adherence to therapy. Our objective was to assess factors associated with adherence to treatment of patients included in a clinical trial of equivalence between the standard and alternative treatment schemes with meglumine antimoniate (MA) in the treatment of cutaneous leishmaniasis (CL), in the state of Rio de Janeiro. Between 2008 and 2011, 57 patients with CL were interviewed using a questionnaire to collect socioeconomic data. The following methods were used for adherence monitoring: counting of vial surplus, monitoring card, Morisky test and modified Morisky test (without the question regarding the schedule); we observed 82.1% (vial return), 86.0% (monitoring card), 66.7% (Morisky test) and 86.0% (modified Morisky test) adherence. There was a strong correlation between the method of vial counting and the monitoring card and modified Morisky test. A significant association was observed between greater adherence to treatment and low dose of MA, as well as with a lower number of people sleeping in the same room. We recommend the use of the modified Morisky test to assess adherence to treatment of CL with MA, because it is a simple method and with a good performance, when compared to other methods.     

Identifying patterns of diurnal blood pressure variation among ELSA‐Brasil participants

Ambulatory blood pressure monitoring (ABPM) is the gold standard method for the diagnosis of hypertension. ABPM provides a set of repeated measurements for blood pressure (BP), usually over 24 h. Traditional approaches characterize diurnal BP variation by single ABPM parameters such as average and standard deviation, regardless of the temporal nature of the data. In this way, information about the pattern of diurnal BP variation and relationship between parameters is lost. The objective of this study was to identify and characterize daily BP patterns considering the set of repeated measures from 24‐h ABPM. A total of 859 adult participants of the Brazilian Longitudinal Study of Adult Health (ELSA‐Brasil) performed a 24‐h ABPM record. Hypertension, sex, age, race/color, education, marital status, smoking, alcohol, physical activity, and BMI were the covariables analyzed. Techniques for longitudinal clustering, multinomial models, and models with mixed effects were used. Three daily BP patterns were identified. Daily BP patterns with high BP presented higher standard deviation and morning surge and lower nocturnal dipping. They showed greater systolic BP variability and faster rise than fall in diastolic BP during sleep. Hypertensive, “pardos,” and men had greater odds to present these patterns. Daily BP patterns with high BP presented the worst profile concerning ABPM parameters associated with cardiovascular risk. The daily BP patterns identified contribute to the characterization of diurnal BP variation.     

Differential effects of single versus repeated alcohol withdrawal on the expression of endocannabinoid system-related genes in the rat amygdala

Background: Endogenous cannabinoids such as anandamide and 2‐arachidonoylglycerol (2‐AG) exert important regulatory influences on neuronal signaling, participate in short‐ and long‐term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity, and affective control. Methods: The consequence of long‐term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT‐PCR analyses of amygdala tissue. Two groups of ethanol (EtOH)‐exposed rats were generated by maintenance on an EtOH liquid diet (10%): the first group received continuous access to EtOH for 15 days, whereas the second group was given intermittent access to the EtOH diet (5 d/wk for 3 weeks). Control subjects were maintained on an isocaloric EtOH‐free liquid diet. To provide an initial profile of acute withdrawal, amygdala tissue was harvested following either 6 or 24 hours of EtOH withdrawal. Results: Acute EtOH withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and 2‐AG (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL], respectively) were evident, as were reductions in mRNA expression for CB₁, CB₂, and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent EtOH exposure, alterations in MAGL and cannabinoid receptor‐related mRNA (e.g., CB₁, CB₂, GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal‐associated deficits in mRNA expression were evident following 24 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in 2‐AG biosynthesis (e.g., diacylglicerol lipase‐α/β) were found in any condition. Conclusions: These findings suggest that EtOH dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal‐related dysregulation of amygdalar neurotransmission.