Incidence and predictors of single drug discontinuation according to the presence of HCV coinfection in HIV patients from the ICONA Foundation Cohort Study

To evaluate incidence rates of and predictors for any antiretroviral (ART) drug discontinuation by HCV infection status in a large Italian cohort of HIV infected patients. All patients enrolled in ICONA who started combination antiretroviral therapy (cART) containing abacavir or tenofovir or emtricitabine or lamivudine plus efavirenz or rilpivirine or atazanavir/r or darunavir/r (DRV/r) or lopinavir/r or dolutegravir or elvitegravir or raltegravir were included. Multivariate Poisson regression models were used to determine factors independently associated with single ART drug discontinuation. Inverse probability weighting method to control for potential informative censoring was applied. Data from 10,637 patients were analyzed and 1,030 (9.7%) were HCV-Ab positive. Overall, there were 15,464 ART discontinuations due to any reason in 82,415.9 person-years of follow-up (PYFU) for an incidence rate (IR) of 18.8 (95% confidence interval [95%CI] 18.5–19.1) per 100 PYFU. No difference in IR of ART discontinuation due to any reason between HCV-infected and -uninfected patients was found. In a multivariable Poisson regression model, HCV-infected participants were at higher risk of darunavir/r discontinuation due to any reason (adjusted incidence rate ratio?=?1.5, 95%CI 1.01–2.22, p value?=?0.045) independently of demographics, HIV-related, ART and life-style factors. Among DRV/r treated patients, we found that HCV-viremic patients had twice the risk of ART discontinuation due to any reason than HCV-aviremic patients. In conclusion, HIV/HCV coinfected patients had a marginal risk increase of DRV/r discontinuation due to any reason compared with those without coinfection.     

Synapsin III is a key component of α‐synuclein fibrils in Lewy bodies of PD brains

Lewy bodies (LB) and Lewy neurites (LN), which are primarily composed of α‐synuclein (α‐syn), are neuropathological hallmarks of Parkinson''s disease (PD) and dementia with Lewy bodies (DLB). We recently found that the neuronal phosphoprotein synapsin III (syn III) controls dopamine release via cooperation with α‐syn and modulates α‐syn aggregation. Here, we observed that LB and LN, in the substantia nigra of PD patients and hippocampus of one subject with DLB, displayed a marked immunopositivity for syn III. The in situ proximity ligation assay revealed the accumulation of numerous proteinase K‐resistant neuropathological inclusions that contained both α‐syn and syn III in tight association in the brain of affected subjects. Most strikingly, syn III was identified as a component of α‐syn‐positive fibrils in LB‐enriched protein extracts from PD brains. Finally, a positive correlation between syn III and α‐syn levels was detected in the caudate putamen of PD subjects. Collectively, these findings indicate that syn III is a crucial α‐syn interactant and a key component of LB fibrils in the brain of patients affected by PD.     

Understanding the frustration arising from the competition between function,misfolding, and aggregation in a globular protein

Folding and function may impose different requirements on the amino acid sequences of proteins, thus potentially giving rise to conflict. Such a conflict, or frustration, can result in the formation of partially misfolded intermediates that can compromise folding and promote aggregation. We investigate this phenomenon by studying frataxin, a protein whose normal function is to facilitate the formation of iron–sulfur clusters but whose mutations are associated with Friedreich’s ataxia. To characterize the folding pathway of this protein we carry out a Φ-value analysis and use the resulting structural information to determine the structure of the folding transition state, which we then validate by a second round of rationally designed mutagenesis. The analysis of the transition-state structure reveals that the regions involved in the folding process are highly aggregation-prone. By contrast, the regions that are functionally important are partially misfolded in the transition state but highly resistant to aggregation. Taken together, these results indicate that in frataxin the competition between folding and function creates the possibility of misfolding, and that to prevent aggregation the amino acid sequence of this protein is optimized to be highly resistant to aggregation in the regions involved in misfolding.Frustration is a general condition that arises in the presence of conflicting requirements. A system is frustrated when it is impossible to fully minimize its energy by optimizing simultaneously all of the possible interactions among its components (1). Although complex systems tend in general to exhibit frustration because of the large number and heterogeneity of their components, protein molecules are remarkable in that their folding process involves interactions that express a minimal level of frustration. According to the so-called principle of minimal frustration, the energy of proteins decreases as they explore conformations increasingly similar in structure to the native state (2). Consequently, the free energy landscape of proteins is characterized by the presence of a well-defined global minimum and very few other local minima, which are typically intermediate states along the folding pathway. This organization of conformational space normally ensures rapid and reliable folding (2–6).Proteins, however, have evolved not only to fold, but also to function. Because the evolutionary constraints that select for a given function may be in conflict with the folding process, it is possible that local frustration patterns may localize in specific regions of proteins, in particular in their active sites. Indeed, a statistical survey of different proteins has shown that frustrated interactions tend to cluster at binding sites and that such frustration decreases upon complex formation (7). Because frustration is associated with the presence of local minima in the free energy landscape, it is important to understand how proteins have evolved to minimize the possible effects associated with these local minima, which are likely to contain misfolded elements and thus to potentially give rise to aggregation.To address this question we studied frataxin, a mitochondrial protein that binds both Fe²⁺ and Fe³⁺ ions and forms a ternary complex with the two main components of the iron–sulfur cluster biogenesis machinery (8–11). This protein offers good opportunities for investigating the relationships between folding, misfolding, and disease. Indeed, its dysfunction is related to a neurodegenerative disease called Friedreich’s ataxia (12). Frataxin is also capable of binding different divalent and trivalent cations, whose recognition sites have been mapped (13). Furthermore, frataxin is involved in donating iron to ferrochelatease via direct interaction through an extended binding site involving some of the residues implicated in metal binding (14).We have previously shown that frataxin folds via a complex mechanism, which we described through a broad free energy barrier (15). This feature, which has been associated with frustration (16), allows the experimental characterization of both the early and late events of folding (16–19). In this work we explored the mechanistic details of the folding reaction of frataxin at residue-level resolution. This result was achieved by characterizing the structures of both the early and late events of folding using Φ-value analysis (20) and restrained molecular dynamics simulations (21). By analyzing the structures of the different states along the folding process we found an unexpected number of nonnative interactions that slow down folding and superpose with the highly frustrated regions, as detected by the frustratometer server (22). The nonnative regions, which display peculiar Φ values, either negative or greater than unity, were predicted on the basis of the transition state structures determined from the Φ values, and subsequently confirmed by a second round of amino acid substitutions rationally designed to probe misfolded regions along the folding pathway.The characterization of the folding pathway of frataxin and of its misfolded elements enables us to discuss the competition between folding and function and its consequences for misfolding and aggregation.     

Clinical efficacy and tolerability of an immune-stimulant<Superscript>*</Superscript> constituted by inactivated bacterial bodies in the prophylaxis of infectious episodes of airways: a double blind,placebo-controlled,randomized, multicentre study

Background

(Buccalin ®) is a Bacterial Lysates (BL) that belongs to a family of immune-stimulators, developed more than 30 years ago and it still has a role in the prophylaxis of Recurrent Respiratory Tract Infections (RRTI). However, original studies were conducted with an approach that does not seem to be aligned with the present methodologies. In addition, concomitant therapies substantially improved in the last decades. These two reasons strongly suggested to update our knowledge on the capacity of this bacterial lysate (Buccalin ®) to reduce the number of days with infectious episodes in patients with RRTI.

Methods

A double blind, placebo-controlled, randomized, multicentre study was programmed (EudraCT code: 2011-005187-25). The reduction of the number of days with infectious episodes (IE) was the primary endpoint. Secondary endpoints were the number of IE, the use of concomitant drugs, the efficacy on signs and symptoms of RRTI and the safety of the drug. Patients were treated according to the registered schedule and were followed up for a period of 6 months.

Results

From a cohort of 188 patients eligible for the study, 90 were included in the active group and 88 in the placebo group. The study was completed in 170 patients. A significant reduction of the number of days with IE was observed (6.57 days in the active group and 7.47 in the placebo group). Secondary endpoints were only partially achieved. No virtual adverse events related to the treatment were recorded.

Conclusion

The administration of bacterial lysate (Buccalin ®) in patients with RRTI had the capacity to significantly reduce the number of days with IE in a multicentre, randomized, placebo controlled, clinical study. The treatment was safe. Of note, all patients were free to be treated with the best concomitant therapies. In these conditions, the positive results observed demonstrated that this bacterial lysate has maintained its capacity of reducing the days with infections in patients with RRTI, also in association to the concomitant therapies available nowadays.

     

Clinical evaluation of an aerolysin-based screening test for paroxysmal nocturnal haemoglobinuria

BACKGROUND: Recently, a toxin produced by Aeromonas hydrophila was demonstrated to bind directly to the glycosyl-phosphatidyl-inositol (GPI) anchor. After coupling it to a fluorescent dye and applying it in fluorescence-activated cell scanning (FACS), this property was exploited to detect GPI-negative cells in the diagnosis of paroxysmal nocturnal haemoglobinuria (PNH). METHODS: We used this reagent according to a very simple staining protocol followed by single-colour FACS and compared the results in patients with PNH and normal controls with those obtained with antibody-mediated detection of cells lacking GPI-anchored proteins. RESULTS: We observed very good concordance between the two methods, with correlation coefficients (R2) of quantified GPI-deficient cell populations ranging from 0.952 to 0.969. The lower limit of detection was determined at 0.50% GPI-negative cells, which was in the range obtained with double-colour staining with antibodies (0.20-1.00%, depending on the antibody). A significant correlation was observed between the fraction of GPI-negative granulocytes and laboratory parameters of haemolysis, with the erythrocyte creatine having the best correlation (R2 = 0.671, P < 0.0001). CONCLUSIONS: Using this protocol, we were able to reliably diagnose PNH with a high sensitivity. The test allows the identification of GPI-negative granulocyte populations as small as 0.5%.     

Plasma renin activity in essential hypertension

Summary A study of the frequency distribution of plasma renin activity (PRA) in 123 patients with essential hypertension (EH) produced no evidence of a distinct subpopulation with low renin levels, whether the samples were taken from supine or upright patients. Applying an arbitraty classification criterion, however, low PRA levels were found in 30.1% of patients. There were no significant differences in mean blood pressure, 24-h sodium excretion, and age when groups with low, normal or high PRA levels were compared. The incidence of PRA hyporesponsiveness was similar in the three groups of patients, but increased with age. In the female there was a preponderance of low PRA levels. It is concluded that EH with low PRA levels is not a separate diagnostic entity and, when PRA is low in a hypertensive subject, the possible effects of age, blood pressure, and sex ought to be taken into account before other causes of low PRA are postulated.     

Middle cerebral artery peak systolic and ductus venosus velocity waveforms in the hydropic fetus.

OBJECTIVE: The purpose of this study was to assess whether Doppler assessment of the middle cerebral artery (MCA) peak systolic velocity (PSV) and ductus venosus (DV) velocity waveforms during sonography of hydropic fetuses may specify the cause of fetal hydrops. METHODS: A level II sonographic examination was performed in 16 hydropic fetuses, and the MCA PSV and DV velocity waveforms were assessed. The MCA PSV values divided hydropic fetuses into anemic (group 1) and nonanemic (group 2) fetuses. In group 2 fetuses, the DV was defined as normal or abnormal. Sonographic examination and Doppler assessment of these vessels specified the cause of hydrops and indicated the use of specific investigations for diagnosing the etiology of fetal hydrops. RESULTS: Seven of 16 fetuses had MCA PSV values greater than 1.50 multiples of the median (group 1). Nine of 16 fetuses had normal MCA PSV values (group 2); among them, 7 of 9 had either absent or reversed flow in the DV, and 2 had a normal DV. In group 1, the cause of fetal anemia was investigated by maternal serum tests, and 5 cordocentesis procedures were performed. In group 2, 7 of 9 fetuses had reversed flow in the DV, which suggested a cardiac abnormality confirmed by echocardiography. Five cordocentesis procedures were performed for fetal karyotype, and in 2 fetuses, the cause of hydrops was idiopathic. CONCLUSIONS: Our data suggest that assessment of the MCA PSV and DV velocity waveforms in the hydropic fetus may further our knowledge of the etiology of hydrops and may indicate which investigations among the many available should be used for diagnosing the cause of fetal hydrops.     

A new polysaccharidic gel matrix for drug delivery: preparation and mechanical properties.

A new type of hydrogel was prepared, under controlled conditions, by diffusion of Ca(II) ions into a solution of the carboxylated derivative of Scleroglucan (Sclerox). The obtained hydrogel was loaded with Theophylline (TPH) and Myoglobin (MGB), two model drugs of remarkably different steric hindrance, and also used, after freeze drying, for the preparation of tablets. Release studies were carried out on both the freshly prepared gel and on the tablets. As far as the gel systems experiments are concerned, the delivery profiles resulted to be deeply dependent on the molecular dimensions of the loaded molecules; TPH was easily released while the larger tested molecule (MGB) remained partially entrapped within the three-dimensional network. Furthermore, in the case of MGB, the release was dependent also on polymer concentration (c(p)): at the highest investigated c(p) value a corresponding lowest delivery of the guest molecule was observed. This effect of polymer concentration on the rate of delivery was studied applying three different mathematical approaches: the one that better fitted the experimental release profile allowed to support the explanation of the mechanism involved in the observed two-step delivery that has been related to the drug trapping inside the clusters of the gel network. The delivery profiles from the tablets showed how the release, in this case, could be related, essentially, to the molecular dimensions of the guest molecules, independently on the c(p) used to prepare the starting hydrogel. TPH was completely delivered in a few hours while the MGB was almost unable to diffuse out of the matrix and more than 80% resulted entrapped in the network for at least 24 h. The novel hydrogel, at different c(p), was also characterized by means of a texture analyzer to inspect its mechanical properties. According to the compression data, the hardness, the work of cohesion and the work of adhesion of the networks were estimated. Furthermore, by means of relaxation experiments, analysed applying the generalized Maxwell model, the gels can be classified as solid viscoelastic materials and the mechanical spectra indicated a predominance of the viscous behaviour, while the Young modulus, E0, as expected, was found to increase with polymer concentration.