The role of human immunodeficiency virus in lymphocytic alterations and in Leishmania coinfection

Tra le proteine codificate da HIV-1, Tat sembrerebbe essere responsabile della regolazione dei linfociti B del centro germinativo mediante l’attivazione costitutiva del fattore NF-kB, coinvolto nella linfomagenesi. Per cercare di definire questa interazione, nella prima parte del progetto è stata ricercata la presenza di un’eventuale correlazione tra Tat e NF-kB in colture di cellule germinal center (GCs), isolate da organi linfoidi. L’espressione di NF-kB è stata valutata mediante Western blot a partire da colture di GCs attivate e trattate con Tat. I dati ottenuti indicano che l'espressione di NF-kB nelle cellule GCs è finemente regolata nel tempo e la sua modulazione è connessa alla presenza di Tat. In futuro, l’analisi micro-array dei profili di espressione genica in presenza/assenza di Tat è necessaria per determinare i pathways di trasduzione del segnale e i fattori di trascrizione coinvolti. Dal momento che durante l’infezione da HIV si verificano numerose infezioni, nella seconda parte dello studio, è stata analizzata la coinfezione Leishmania/HIV. La diagnosi precoce di infezione da Leishmania è indispensabile per poter introdurre una terapia mirata, ma viene raramente considerata negli algoritmi diagnostici con conseguente sotto-diagnosi. Recentemente, in Emilia Romagna è stato registrato un aumento di casi di leishmaniosi, pertanto, è stato condotto uno studio su donatori sani in Valsamoggia (BO) per dimostrare la reale prevalenza delle infezioni asintomatiche. Gli anticorpi anti-Leishmania sono stati rilevati in 27/260 campioni analizzati mediante Western blot (10,4%), mentre il DNA di Leishmania è stato ritrovato in 4/260 campioni sottoposti a real-time PCR (prevalenza totale=11.5%). Da questo studio è emerso che la Valsamoggia è una zona ad alta prevalenza di infezioni asintomatiche, dato rilevante soprattutto perché la leishmaniosi è solo la punta di un iceberg in cui bisogna considerare il rischio di riattivazione in soggetti immunocompromessi, come i pazienti HIV positivi, che necessitano di una diagnosi precoce.Among proteins encoded by HIV-1, Tat appears to be responsible for the regulation of B cells of the germinal center by constitutive activation of the NF-kB factor involved in lymphomagenesis. To try to define this interaction, in the first part of the project the presence of a possible correlation between Tat and NF-kB in cultures of germinal center cells (GCs), isolated from lymphoid organs, was sought. The expression of NF-kB was evaluated by Western blot from cultures of activated GCs and treated with Tat. The data obtained indicate that the expression of NF-kB in GCs cells is finely regulated over time and its modulation is related to the presence of Tat. In the future, the micro-array analysis of gene expression profiles in Tat presence/absence is necessary to determine the signal transduction pathways and the transcription factors involved. Since many infections occur during HIV infection, the Leishmania/HIV coinfection was analyzed in the second part of the study. The early diagnosis of Leishmania infection is essential to introduce targeted therapy, but is rarely considered in diagnostic algorithms with consequent under-diagnosis. Recently, in Emilia Romagna there has been an increase in cases of leishmaniasis, therefore, a study was conducted on healthy donors in Valsamoggia (BO) to demonstrate the real prevalence of asymptomatic infections. Anti-Leishmania antibodies were detected in 27/260 samples analyzed by Western blot (10.4%), while Leishmania DNA was found in 4/260 samples by real-time PCR (total prevalence = 11.5%) . From this study it has emerged that Valsamoggia is an area with a high prevalence of asymptomatic infections, especially since it is only the tip of an iceberg in which the risk of reactivation must be considered in immunocompromised subjects, such as HIV-positive patients, who need an early diagnosis

COMMUNITY ENGAGEMENT AND GREENING STRATEGIES AS ENABLING PRACTICES FOR INCLUSIVE AND RESILIENT CITIES

The climate change challenges call for innovative and sustainable policies and governance models, capable of achieving adaptation and mitigation goals working on a necessary behavioural societal change, both at individual and collective levels. Cities and their public spaces represent an ideal ground for the implementation of innovative strategies, which combine participatory and engagement practices to physical transformations of urban areas in a regenerative perspective. Co-design and participatory paths can trigger reactivation and re-appropriation of underused spaces, generate new dynamics in the public space use and provide effective solutions to tackle climate change, improving outdoor microclimatic comfort conditions. The implementation of demonstrative and temporary interventions – based on greening actions co-created with local administrations, stakeholders and citizens and supported by technologies – represents a viable and effective practice in order to experiment, test, monitor and evaluate shared pathways to more liveable, resilient and sustainable cities. This combined approach was experimented in the Bologna University area by the EU Horizon 2020 project ROCK – Regeneration and Optimisation of Cultural Heritage in Creative and Knowledge Cities (GA 730280) – through a series of pilot actions aimed at public open space utilization and potential enhancement in particular in the historical city centres, generating new resilient processes in terms of environmental sustainability and social inclusion

Changes in membrane lipids drive increased endocytosis following Fas ligation

Once activated, some surface receptors promote membrane movements that open new portals of endocytosis, in part to facilitate the internalization of their activated complexes. The prototypic death receptor Fas (CD95/Apo1) promotes a wave of enhanced endocytosis that induces a transient intermixing of endosomes with mitochondria in cells that require mitochondria to amplify death signaling. This initiates a global alteration in membrane traffic that originates from changes in key membrane lipids occurring in the endoplasmic reticulum (ER). We have focused the current study on specific lipid changes occurring early after Fas ligation. We analyzed the interaction between endosomes and mitochondria in Jurkat T cells by nanospray-Time-of-flight (ToF) Mass Spectrometry. Immediately after Fas ligation, we found a transient wave of lipid changes that drives a subpopulation of early endosomes to merge with mitochondria. The earliest event appears to be a decrease of phosphatidylcholine (PC), linked to a metabolic switch enhancing phosphatidylinositol (PI) and phosphoinositides, which are crucial for the formation of vacuolar membranes and endocytosis. Lipid changes occur independently of caspase activation and appear to be exacerbated by caspase inhibition. Conversely, inhibition or compensation of PC deficiency attenuates endocytosis, endosome-mitochondria mixing and the induction of cell death. Deficiency of receptor interacting protein, RIP, also limits the specific changes in membrane lipids that are induced by Fas activation, with parallel reduction of endocytosis. Thus, Fas activation rapidly changes the interconversion of PC and PI, which then drives enhanced endocytosis, thus likely propagating death signaling from the cell surface to mitochondria and other organelles

Comparison of the Aptima HIV-1 Quant Dx Assay with the COBAS\uae AmpliPrep/COBAS\uae TaqMan\uae HIV-1 v2.0 Test for HIV-1 Viral Load Quantification in Plasma Samples from HIV-1-Infected Patients.

Background and aims: HIV\u20101 RNA viral load (VL) in plasma samples of HIV\u20101\u2013positive patients is used to assess the level of viral replication, the risk of disease progression, and the response and efficacy to antiretroviral treatment. Knowing the performance of different tests for HIV\u20101 RNA detection is, therefore, important for clinical care. This study compared the performance of the recently introduced Aptima HIV\u20101 Quant Dx assay (Hologic, Inc) and the standard COBAS AmpliPrep/COBAS TaqMan HIV\u20101 v2.0 Test (CAP/CTM2) (Roche Molecular System, Inc) for HIV\u20101 RNA quantitation. Methods: Assay performance was assessed using 335 clinical samples, a standard HIV\u20101 low VL panel, and 2 diluted samples from well\u2010characterized patients infected with different HIV\u20101 subtypes tested in 5 replicates over 3 days. All samples were tested on both assays to evaluate inter\u2010assay agreement, both qualitatively and quantitively. Altogether, we evaluated assay sensitivity, linearity, accuracy, precision, repeatability, and reproducibility. Results: Assay agreement for qualitative results in 335 clinical samples was fair (80.6%). Correlation of quantitative assay results (n = 164) was excellent (R2 = 0.97), with 96.3% of the results within the 95% limit of assay agreement ( 120.42 to +0.86 log), and 98.8% within 1 log of each other. Aptima\u2010HIV\u20101 yielded results, on average, 0.22 log higher than CAP/CTM2. Both assays accurately quantitated the HIV\u20101 standard at low VL (R2 65 0.94), with all samples within 0.5 log of the target. Conclusion: Aptima\u2010HIV\u20101 assay demonstrated sensitivity, accuracy, reproducibility, and precision for the detection and quantitation of HIV\u20101 RNA across a wide dynamic range of VLs. Its performance, together with full automation and high throughput, suggests that Aptima\u2010HIV\u20101 could be a suitable assay for reliable monitoring of HIV\u20101 VL in patients undergoing treatment

Perhexiline maleate enhances antitumor efficacy of cisplatin in neuroblastoma by inducing over-expression of NDM29 ncRNA

High Risk Neuroblastoma (HR-NB) is a pediatric cancer characterized by high malignancy and remarkable cell heterogeneity within the tumour nodules. In a recent study, we demonstrated that in vitro and in vivo over-expression of the non-coding RNA NDM29 (neuroblastoma differentiation marker 29) induces NB cell differentiation, dramatically reducing their malignancy. Among gene expression changes, differentiated phenotype induced by NDM29 is characterized by decrease of the expression of ABC transporters responsible for anticancer drug resistance. Thus, the pharmacological induction of NDM29, in principle, might represent a possible novel strategy to increase cytotoxic drug responses. In this work, we identify a small molecule able to induce the expression of NDM29 in NB cells, conferring to malignant cells increased susceptibility to cisplatin cytotoxic effects. We demonstrate that the pharmacological induction of NDM29 expression in vivo enhances the antitumoral effects of chemotherapy specifically on tumour initiating/cancer stem cells sub-population, usually refractory to therapies and responsible for tumour relapse. In summary, we suggest a novel therapeutical approach possibly useful to treat very aggressive NB cases with poor prognosis. This novel pharmacological strategy aims to promote differentiation of "stem-like" cells to render them more susceptible to the killing action of cytotoxic anticancer drugs

Relationship between gender differences and clinical outcome in patients with the antiphospholipid syndrome

Antiphospholipid syndrome (APS), characterized by artherial and/or venous thrombosis, pregnancy morbidity and "antiphospholipid" antibodies (aPLs), is more common in women than in men, with a female to male ratio of about 3.5:1. Only few studies have investigated the clinical differences between male and female patients with APS. Therefore, this study was aimed to analyze the differences of clinical manifestations and laboratory tests, at diagnosis, between female and male APS patients and the clinical outcome. We enrolled 191 consecutive APS patients (125 with primary APS, PAPS, and 66 with secondary APS, SAPS) with a female predominant ratio of approximately 3:1 (142 vs 49). The prevalence of PAPS was higher in males than females (p<0.001). The analysis of aPL profile revealed that high IgM anti-cardiolipin (aCL) and high-medium IgG aCL titers were more frequent in males. In thrombotic APS peripheral arterial thrombosis was more common in male than female patients (p=0.049), as well as myocardial infarction (p=0.031). Multivariate analysis to correct for cardiovascular risk factors, high titer of aPLs and triple positivity for aPLs, revealed that the odds ratio for myocardial infarction in male was 3.77. Thus, APS may be considered as a disease in which serological (IgM titer) and clinical profiles are influenced by gender

An altered lipid metabolism characterizes Charcot-Marie-Tooth type 2B peripheral neuropathy.

Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-14C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations

Impact of the flame retardant 2,2’4,4’-tetrabromodiphenyl ether (PBDE-47) in THP-1 macrophage-like cell function via small extracellular vesicles

2,2’4,4’-tetrabromodiphenyl ether (PBDE-47) is one of the most widespread environmental brominated flame-retardant congeners which has also been detected in animal and human tissues. Several studies have reported the effects of PBDEs on different health issues, including neurobehavioral and developmental disorders, reproductive health, and alterations of thyroid function. Much less is known about its immunotoxicity. The aim of our study was to investigate the effects that treatment of THP-1 macrophage-like cells with PBDE-47 could have on the content of small extracellular vesicles’ (sEVs) microRNA (miRNA) cargo and their downstream effects on bystander macrophages. To achieve this, we purified sEVs from PBDE-47 treated M(LPS) THP-1 macrophage-like cells (sEVsPBDE+LPS) by means of ultra-centrifugation and characterized their miRNA cargo by microarray analysis detecting the modulation of 18 miRNAs. Furthermore, resting THP-1 derived M(0) macrophage-like cells were cultured with sEVsPBDE+LPS, showing that the treatment reshaped the miRNA profiles of 12 intracellular miRNAs. This dataset was studied in silico, identifying the biological pathways affected by these target genes. This analysis identified 12 pathways all involved in the maturation and polarization of macrophages. Therefore, to evaluate whether sEVsPBDE+LPS can have some immunomodulatory activity, naïve M(0) THP-1 macrophage-like cells cultured with purified sEVsPBDE+LPS were studied for IL-6, TNF-α and TGF-β mRNAs expression and immune stained with the HLA-DR, CD80, CCR7, CD38 and CD209 antigens and analyzed by flow cytometry. This analysis showed that the PBDE-47 treatment does not induce the expression of specific M1 and M2 cytokine markers of differentiation and may have impaired the ability to make immunological synapses and present antigens, down-regulating the expression of HLA-DR and CD209 antigens. Overall, our study supports the model that perturbation of miRNA cargo by PBDE-47 treatment contributes to the rewiring of cellular regulatory pathways capable of inducing perturbation of differentiation markers on naïve resting M(0) THP-1 macrophage-like cells

MRI index lesion radiomics and machine learning for detection of extraprostatic extension of disease: a multicenter study

Objectives: To build a machine learning (ML) model to detect extraprostatic extension (EPE) of prostate cancer (PCa), based on radiomics features extracted from prostate MRI index lesions. Methods: Consecutive MRI exams of patients undergoing radical prostatectomy for PCa were retrospectively collected from three institutions. Axial T2-weighted and apparent diffusion coefficient map images were annotated to obtain index lesion volumes of interest for radiomics feature extraction. Data from one institution was used for training, feature selection (using reproducibility, variance and pairwise correlation analyses, and a correlation-based subset evaluator), and tuning of a support vector machine (SVM) algorithm, with stratified 10-fold cross-validation. The model was tested on the two remaining institutions' data and compared with a baseline reference and expert radiologist assessment of EPE. Results: In total, 193 patients were included. From an initial dataset of 2436 features, 2287 were excluded due to either poor stability, low variance, or high collinearity. Among the remaining, 14 features were used to train the ML model, which reached an overall accuracy of 83% in the training set. In the two external test sets, the SVM achieved an accuracy of 79% and 74% respectively, not statistically different from that of the radiologist (81-83%, p = 0.39-1) and outperforming the baseline reference (p = 0.001-0.02). Conclusions: A ML model solely based on radiomics features demonstrated high accuracy for EPE detection and good generalizability in a multicenter setting. Paired to qualitative EPE assessment, this approach could aid radiologists in this challenging task

An aqueous olive leaf extract (OLE) ameliorates parameters of oxidative stress associated with lipid accumulation and induces lipophagy in human hepatic cells

Fatty liver is a disease characterized by a buildup of lipids in the liver, often resulting from excessive consumption of high-fat-containing foods. Fatty liver can degenerate, over time, into more severe forms of liver diseases, especially when oxidative stress occurs. Olive leaf extract (OLE) is a reliable source of polyphenols with antioxidant and hypolipidemic properties that have been successfully used in medicine, cosmetics, and pharmaceutical products. Using "green" solvents with minimal impact on the environment and human health, which simultaneously preserves the extract's beneficial properties, represents one of the major challenges of biomedical research. In the present study, we assayed the potential antioxidant and lipid-lowering effect of a "green" OLE obtained by a water ultrasound-assisted extraction procedure, on the human hepatic HuH7 cell line, treated with a high concentration of free fatty acids (FFA). We found that high FFA concentration induced lipid accumulation and oxidative stress, as measured by increased hydrogen peroxide levels. Moreover, the activity of antioxidant enzymes, catalase, superoxide dismutase, and glutathione peroxidase, was reduced upon FFA treatment. Coincubation of high FFA with OLE reduced lipid and H2O2 accumulation and increased the activity of peroxide-detoxifying enzymes. OLE ameliorated mitochondrial membrane potential, and hepatic parameters by restoring the expression of enzymes involved in insulin signaling and lipid metabolism. Electron microscopy revealed an increased autophagosome formation in both FFA- and FFA + OLE-treated cells. The study of the autophagic pathway indicated OLE's probable role in activating lipophagy