Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Macrophages are key targets of HIV-1 infection. We have previously described that the expressionof CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is furtherup-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication ininfected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibitsHIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restrictionfactors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoproteinB mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members.Results:CCL2 neutralization potently reduced the number of p24 Gag+cells during the course of either productive orsingle cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thusdemonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viralDNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates ofHIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of themodulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression,to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replicationmediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was typeI IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expressionrevealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in thedefence response to viruses.Conclusions:Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primaryMDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which mightcontribute to regulate the expression of innate intracellular viral antagonistsin vivo. Thus, our study may potentially leadto the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection

The Role of Patient-Specific Morphological Features of the Left Atrial Appendage on the Thromboembolic Risk Under Atrial Fibrillation

Background: A large majority of thrombi causing ischemic complications under atrial fibrillation (AF) originate in the left atrial appendage (LAA), an anatomical structure departing from the left atrium, characterized by a large morphological variability between individuals. This work analyses the hemodynamics simulated for different patient-specific models of LAA by means of computational fluid–structure interaction studies, modeling the effect of the changes in contractility and shape resulting from AF. Methods: Three operating conditions were analyzed: sinus rhythm, acute atrial fibrillation, and chronic atrial fibrillation. These were simulated on four patient-specific LAA morphologies, each associated with one of the main morphological variants identified from the common classification: chicken wing, cactus, windsock, and cauliflower. Active contractility of the wall muscle was calibrated on the basis of clinical evaluations of the filling and emptying volumes, and boundary conditions were imposed on the fluid to replicate physiological and pathological atrial pressures, typical of the various operating conditions. Results: The LAA volume and shear strain rates were analyzed over time and space for the different models. Globally, under AF conditions, all models were well aligned in terms of shear strain rate values and predicted levels of risk. Regions of low shear rate, typically associated with a higher risk of a clot, appeared to be promoted by sudden bends and focused at the trabecule and the lobes. These become substantially more pronounced and extended with AF, especially under acute conditions. Conclusion: This work clarifies the role of active and passive contraction on the healthy hemodynamics in the LAA, analyzing the hemodynamic effect of AF that promotes clot formation. The study indicates that local LAA topological features are more directly associated with a thromboembolic risk than the global shape of the appendage, suggesting that more effective classification criteria should be identified

Residues of 165 pesticides in citrus fruits using LC-MS/MS: a study of the pesticides distribution from the peel to the pulp

Abstract A sensitive LC–ESI-MS/MS method was developed for the determination of 165 pesticides in 50 citrus fruit samples collected in Sicily. Moreover, an evaluation of pesticides levels in the citrus layers (peel, albedo, and pulp) was carried out. The method presented acceptable trueness, precision, and linearity with LOQ of 5 μg/kg. The results obtained showed a high frequency of fungicides class pesticides in all the citrus samples examined (>95%) with the highest concentrations in the peel (4468 µg/Kg). A significant difference of concentrations was found between the layers of the citrus fruits analysed (p < 0.05). In particular, the peel and albedo present higher pesticides significantly higher than the pulp. Our findings confirming the widespread use of these substances in citrus cultivation and suggesting the importance of pesticides analysis in all the citrus fruit layers separately, considering the different interactions between the physicochemical characteristics of the matrices and the pesticides

Novel Mechanisms of Tumor Promotion by the Insulin Receptor Isoform A in Triple-Negative Breast Cancer Cells

The insulin receptor isoform A (IR-A) plays an increasingly recognized role in fetal growth and tumor biology in response to circulating insulin and/or locally produced IGF2. This role seems not to be shared by the IR isoform B (IR-B). We aimed to dissect the specific impact of IR isoforms in modulating insulin signaling in triple negative breast cancer (TNBC) cells. We generated murine 4T1 TNBC cells deleted from the endogenous insulin receptor (INSR) gene and expressing comparable levels of either human IR-A or IR-B. We then measured IR isoform-specific in vitro and in vivo biological effects and transcriptome in response to insulin. Overall, the IR-A was more potent than the IR-B in mediating cell migration, invasion, and in vivo tumor growth. Transcriptome analysis showed that approximately 89% of insulin-stimulated transcripts depended solely on the expression of the specific isoform. Notably, in cells overexpressing IR-A, insulin strongly induced genes involved in tumor progression and immune evasion including chemokines and genes related to innate immunity. Conversely, in IR-B overexpressing cells, insulin predominantly induced the expression of genes primarily involved in the regulation of metabolic pathways and, to a lesser extent, tumor growth and angiogenesis

Frailty, psychological well-being, and social isolation in older adults with cognitive impairment during the SARS-CoV-2 pandemic: data from the GeroCovid initiative

Background: The containment measures linked to the COVID-19 pandemic negatively affected the phyco-physical well-being of the population, especially older adults with neurocognitive disorders (NCDs). This study aims to evaluate whether the frailty of NCD patients was associated with different changes in multiple health domains, in particular in relation to loneliness and social isolation, pre- and post-lockdown. Materials and methods: Patients were recruited from 10 Italian Centers for Cognitive Disorders and Dementia. Data were collected in the pre-pandemic period (T0), during the pandemic lockdown (T1), and 6-9 months post-lockdown (T2). The UCLA Loneliness Scale-3, Activities of Daily Living (ADL), Instrumental ADL (IADL), Mini-Mental State Examination, and Neuropsychiatric Inventory (NPI) were administered. Caregivers' burden was also tested. Patients were categorized as non-frail, pre-frail, and frail according to the Fatigue, Resistance, Ambulation, Illness, and Loss of Weight scale. Results: The sample included 165 subjects (61.9% women, mean age 79.5 ± 4.9 years). In the whole sample, the ADL, IADL, and NPI scores significantly declined between T0 and T2. There were no significative variations in functional and cognitive domains between the frail groups. During lockdown we recorded higher Depression Anxiety Stress Scales and Perceived Stress Scale scores in frail people. In multivariable logistic regression, frailty was associated with an increase in social isolation, and a loss of IADL. Conclusions: We observed a global deterioration in functional and neuro-psychiatric domains irrespective of the degree of frailty. Frailty was associated with the worsening of social isolation during lockdown. Frail patients and their caregivers seemed to experience more anxiety and stress disorders during SARS-CoV-2 pandemic

Toxic mineral elements in Mytilus galloprovincialis from Sicilian coasts (Southern Italy)

We assessed the relationship between V, Cr, Mn, Hg, As, Cd, Sn, Sb and Pb concentrations in Mytilus galloprovincialis samples from the coasts of Sicily and the expression of metallothioneins. Toxic mineral elements assessment was carried out by A.A. Spectrometry and ICP-MS. The metallothioneins expression was performed by q-PCR method. Low metals' levels were found in the mussel samples examined, in comparison with what was reported in literature. The highest mean values of toxic mineral elements were found in Gela (Cr 0.178 ± 0.03 mg/Kg, Mn 4.325 ± 0.012 mg/Kg, As 3.706 ± 0.009 mg/Kg, Sn 0.148 ± 0.014 mg/Kg, Sb 0.009 ± 0.004 mg/Kg e Pb 0.364 ± 0.01 mg/Kg). Significant levels of Hg were found in samples from Catania (0.014 ± 0.005 mg/Kg). Only vanadium and lead concentrations showed significant differences between sampling areas (p < 0.05). Molecular analysis verified a basal expression of Mt1 and the absence of over-expression of Mt2, confirming the low mineral's concentrations found in the samples examined

Genetic Discrimination Between LADA and Childhood-Onset Type 1 Diabetes Within the MHC

OBJECTIVE The MHC region harbors the strongest loci for latent autoimmune diabetes in adults (LADA); however, the strength of association is likely attenuated compared with that for childhood-onset type 1 diabetes. In this study, we recapitulate independent effects in the MHC class I region in a population with type 1 diabetes and then determine whether such conditioning in LADA yields potential genetic discriminators between the two subtypes within this region. RESEARCH DESIGN AND METHODS Chromosome 6 was imputed using SNP2HLA, with conditional analysis performed in type 1 diabetes case subjects (n = 1,985) and control subjects (n = 2,219). The same approach was applied to a LADA cohort (n = 1,428) using population-based control subjects (n = 2,850) and in a separate replication cohort (656 type 1 diabetes case, 823 LADA case, and 3,218 control subjects). RESULTS The strongest associations in the MHC class II region (rs3957146, beta [SE] = 1.44 [0.05]), as well as the independent effect of MHC class I genes, on type 1 diabetes risk, particularly HLA-B*39 (beta [SE] = 1.36 [0.17]), were confirmed. The conditional analysis in LADA versus control subjects showed significant association in the MHC class II region (rs3957146, beta [SE] = 1.14 [0.06]); however, we did not observe significant independent effects of MHC class I alleles in LADA. CONCLUSIONS In LADA, the independent effects of MHC class I observed in type 1 diabetes were not observed after conditioning on the leading MHC class II associations, suggesting that the MHC class I association may be a genetic discriminator between LADA and childhood-onset type 1 diabetes.Peer reviewe

The Implementation of Managed Entry Agreements in Central and Eastern Europe : Findings and Implications

Funding Information: In Bosnia and Herzegovina, both The Federation of Bosnia and Herzegovina and the Republic of Srpska, also have special funds and budgets in place for the financing of expensive medicines, which are innovative and under patent. Similar earmarked funds are available in Scotland (the New Medicines Fund funded by the Pharmaceutical Price Regulation Scheme [PPRS] rebates) [35] and England (the Cancer Drugs Fund) [36]. However, support for such earmarked funds is mixed. While they facilitate access, critics raised issues about fairness towards other disease areas and patient groups that are not eligible for special funding [3, 39]. Further, the views of a Patient and Clinician Engagement meeting in Scotland [37] and the end-of-life criteria in England [38] offer opportunities for special considerations affecting medicines for end-of-life and very rare conditions to be taken into account in the health technology assessment process. Funding Information: The authors would like to acknowledge Dr. Jan Jones from the Scottish Medicines Consortium, Scotland, for contributing to the discussion with information on Scotland, Drs. Lyudmila Bezmelnitsyna and Anastasia Isaeva for contributing to data collection in Russia and Dr. Kate?ina Podrazilov? from SZP ?R for providing information on the Czech Republic. Alessandra Ferrario was a Research Officer at the LSE Health at the time this research was conducted. She is now a postdoctoral Research Fellow at the Department of Population Medicine, Harvard Medical School and Harvard Pilgrim Health Care Institute, Boston, USA. Email: [email protected] No sources of funding were used for this study. The authors declare they have no conflicts of interest. However, Di?na Ar?ja, Maria Dimitrova, Jurij F?rst, Ieva Grei?i?t?-Kuprijanov, Iris Hoxha, Arianit Jakupi, Erki Laidm?e, Vanda Markovic-Pekovic, Dmitry Meshkov, Guenka Petrova, Maciej Pomorski and Patricia Vella Bonanno work directly for national health authorities or are advisers to them. Alessandra Ferrario, Tomasz Bochenek, Ileana Mardare, Dominik Tomek, Luka Voncina, Alan Haycox, Panos Kanavos,?Olga L?blov?, and Brian Godman are academics and independent researchers also working with national and regional health authorities and others to improve the quality and efficiency of prescribing, and Tarik Catic, D?vid Dank?,and Tanja Novakovic are involved with pharmaceutical, pharmacoeconomics and outcomes research groups in their countries. Olga L?blov? has also carried out remunerated consultancy activities for A&R Partners, Baxter AG and Instytut Arcana and Ileana Mardare has signed a consulting contract with Ewopharma A.G. Romania. The content of the paper and the conclusions are those of each author and may not necessarily reflect those of any organisation that employs them. Publisher Copyright: © 2017, The Author(s).Background: Managed entry agreements (MEAs) are a set of instruments to facilitate access to new medicines. This study surveyed the implementation of MEAs in Central and Eastern Europe (CEE) where limited comparative information is currently available. Method: We conducted a survey on the implementation of MEAs in CEE between January and March 2017. Results: Sixteen countries participated in this study. Across five countries with available data on the number of different MEA instruments implemented, the most common MEAs implemented were confidential discounts (n = 495, 73%), followed by paybacks (n = 92, 14%), price-volume agreements (n = 37, 5%), free doses (n = 25, 4%), bundle and other agreements (n = 19, 3%), and payment by result (n = 10, >1%). Across seven countries with data on MEAs by therapeutic group, the highest number of brand names associated with one or more MEA instruments belonged to the Anatomical Therapeutic Chemical (ATC)-L group, antineoplastic and immunomodulating agents (n = 201, 31%). The second most frequent therapeutic group for MEA implementation was ATC-A, alimentary tract and metabolism (n = 87, 13%), followed by medicines for neurological conditions (n = 83, 13%). Conclusions: Experience in implementing MEAs varied substantially across the region and there is considerable scope for greater transparency, sharing experiences and mutual learning. European citizens, authorities and industry should ask themselves whether, within publicly funded health systems, confidential discounts can still be tolerated, particularly when it is not clear which country and party they are really benefiting. Furthermore, if MEAs are to improve access, countries should establish clear objectives for their implementation and a monitoring framework to measure their performance, as well as the burden of implementation.publishersversionPeer reviewe

Country, Sex, EDSS Change and Therapy Choice Independently Predict Treatment Discontinuation in Multiple Sclerosis and Clinically Isolated Syndrome

We conducted a prospective study, MSBASIS, to assess factors leading to first treatment discontinuation in patients with a clinically isolated syndrome (CIS) and early relapsing-remitting multiple sclerosis (RRMS). The MSBASIS Study, conducted by MSBase Study Group members, enrols patients seen from CIS onset, reporting baseline demographics, cerebral magnetic resonance imaging (MRI) features and Expanded Disability Status Scale (EDSS) scores. Follow-up visits report relapses, EDSS scores, and the start and end dates of MS-specific therapies. We performed a multivariable survival analysis to determine factors within this dataset that predict first treatment discontinuation. A total of 2314 CIS patients from 44 centres were followed for a median of 2.7 years, during which time 1247 commenced immunomodulatory drug (IMD) treatment. Ninety percent initiated IMD after a diagnosis of MS was confirmed, and 10% while still in CIS status. Over 40% of these patients stopped their first IMD during the observation period. Females were more likely to cease medication than males (HR 1.36, p = 0.003). Patients treated in Australia were twice as likely to cease their first IMD than patients treated in Spain (HR 1.98, p = 0.001). Increasing EDSS was associated with higher rate of IMD cessation (HR 1.21 per EDSS unit, p<0.001), and intramuscular interferon-β-1a (HR 1.38, p = 0.028) and subcutaneous interferon-β-1a (HR 1.45, p = 0.012) had higher rates of discontinuation than glatiramer acetate, although this varied widely in different countries. Onset cerebral MRI features, age, time to treatment initiation or relapse on treatment were not associated with IMD cessation. In this multivariable survival analysis, female sex, country of residence, EDSS change and IMD choice independently predicted time to first IMD cessation