Analisi morfologica, funzionale e fisiopatologica delle inclusioni ottenute in cellule PC12 mediante trattamento con metanfetamina

La presenza di inclusioni neuronali è una caratteristica anatomopatologica di un ampio numero di malattie neurodegenerative come la Malattia di Parkinson (MdP). Questi inclusi, noti anche come corpi di Lewy, sono stati descritti come aggregati citoplasmatici neuronali eosinofili che si possono distinguere in base alla sede di localizzazione ed alle caratteristiche strutturali in una forma classica ed una corticale. I classici corpi di Lewy sono localizzati a livello del tronco encefalico e del proencefalo e presentano un core denso circondato da un alone pallido. Gli inclusi corticali sono presenti nel telencefalo, presentano un aspetto uniforme senza differenza tra le regioni esterne e quelle centrali e sono debolmente eosinofili. Attualmente alcune ricerche propongono che le due forme siano in realtà stadi di diversa maturazione e che i corticali siano la forma più immatura che naturalmente evolve verso la forma classica con il progredire del danno cellulare. Queste ipotesi sono fondate sullo studio dei meccanismi di formazione delle inclusioni neuronali che chiamano in causa il sistema ubiquitina-proteasoma (UP), una delle principali vie per la degradazione di proteine anomale, mutate od eccessivamente espresse. Il mancato funzionamento del sistema UP anche in maniera parziale è in grado di determinare un accumulo di proteine nel citosol cellulare che, aggregandosi, formano le inclusioni o corpi di Lewy. La presenza di proteine appartenenti al sistema UP ed enzimi associati allo stesso, all’interno degli inclusi neuronali conferma il ruolo chiave di questo sistema, come evidenziato anche in studi post-mortem. Gli studi condotti dal nostro gruppo su modelli animali trattati con metanfetamina (MA) hanno permesso di osservare la formazione di inclusioni neuronali citoplasmatiche a livello della substantia nigra. Al fine di indagare questo fenomeno in maniera più approfondita abbiamo condotto una serie di ricerche su modelli in vitro. La prima parte del progetto ha previsto una serie di esperimenti pilota che hanno permesso di individuare la dose di MA e il tempo d’esposizione ottimali ottenendo la maggior quantità di cellule con inclusi e il maggior numero di inclusi per cellula. Gli studi condotti successivamente hanno confermato una correlazione tempo dipendente nel numero degli inclusi e nella loro ultrastruttura. Questo dato ha permesso di stabilire una progressione nell’evoluzione della formazione dell’incluso che passa da un aspetto multimembranale concentrico ad una fase intermedia, in cui si osserva una progressiva fusione delle membrane in senso centrifugo, fino a perdere la conformazione membranosa del core, ottenendo un aspetto analogo ai corpi di Lewy.Questo dato conferma l’evoluzione dinamica degli inclusi che sono soggetti a modificazioni sia morfologiche strutturali.La seconda parte ha permesso di confermare, anche in vitro, la positività per antigeni specifici dei corpi di Lewy, come α-sinucleina e ubiquitina. Questo è stato effettuato su diversi stadi di maturazione dell’incluso, dopo frazionamento per mezzo di centrifugazione differenziale. Infine sono stati effettuati studi di immunoblotting per valutare la presenza ed una eventuale variazione di espressione dell’ubiquitina e dell’α-sinucleina nelle varie frazioni. Questo ampio progetto di ricerca ha confermato quindi che la MA induce la formazione di inclusioni citoplasmatiche in cellule PC12 in maniera dose e tempodipendente. Inoltre le formazioni ottenute presentano una immunopositività per i markers osservati negli inclusi presenti nelle malattie neurodegenerative e ne indicano una evoluzione morfologica e strutturale, indicando che sono prodotte da un meccanismo patogenetico comune in cui è coinvolta l’attività del sistema UP

Decompressive surgery in cerebral venous sinus thrombosis due to vaccine-induced immune thrombotic thrombocytopenia

Background and purpose: Cerebral venous sinus thrombosis due to vaccine-induced im-mune thrombotic thrombocytopenia (CVST-VITT) is an adverse drug reaction occurring after severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) vaccination. CVST-VITT patients often present with large intracerebral haemorrhages and a high proportion undergoes decompressive surgery. Clinical characteristics, therapeutic management and outcomes of CVST-VITT patients who underwent decompressive surgery are described and predictors of in- hospital mortality in these patients are explored.Methods: Data from an ongoing international registry of patients who developed CVST within 28 days of SARS-CoV- 2 vaccination, reported between 29 March 2021 and 10 May 2022, were used. Definite, probable and possible VITT cases, as defined by Pavord et al. (N Engl J Med 2021; 385: 1680–1689), were included. Results: Decompressive surgery was performed in 34/128 (27%) patients with CVST- VITT. In- hospital mortality was 22/34 (65%) in the surgical and 27/94 (29%) in the non- surgical group (p< 0.001). In all surgical cases, the cause of death was brain herniation. The highest mortality rates were found amongst patients with preoperative coma (17/18, 94% vs. 4/14, 29% in the non-comatose; p< 0.001) and bilaterally absent pupillary re-flexes (7/7, 100% vs. 6/9, 67% with unilaterally reactive pupil, and 4/11, 36% with bi-laterally reactive pupils; p= 0.023). Postoperative imaging revealed worsening of index haemorrhagic lesion in 19 (70%) patients and new haemorrhagic lesions in 16 (59%) pa-tients. At a median follow-up of 6 months, 8/10 of surgical CVST-VITT who survived ad-mission were functionally independent.Conclusions: Almost two-thirds of surgical CVST-VITT patients died during hospital ad-mission. Preoperative coma and bilateral absence of pupillary responses were associated with higher mortality rates. Survivors often achieved functional independence.Peer reviewe

Management of Cerebral Venous Thrombosis Due to Adenoviral COVID-19 Vaccination

Objective Cerebral venous thrombosis (CVT) caused by vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare adverse effect of adenovirus-based severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccines. In March 2021, after autoimmune pathogenesis of VITT was discovered, treatment recommendations were developed. These comprised immunomodulation, non-heparin anticoagulants, and avoidance of platelet transfusion. The aim of this study was to evaluate adherence to these recommendations and its association with mortality. Methods We used data from an international prospective registry of patients with CVT after the adenovirus-based SARS-CoV-2 vaccination. We analyzed possible, probable, or definite VITT-CVT cases included until January 18, 2022. Immunomodulation entailed administration of intravenous immunoglobulins and/or plasmapheresis. Results Ninety-nine patients with VITT-CVT from 71 hospitals in 17 countries were analyzed. Five of 38 (13%), 11 of 24 (46%), and 28 of 37 (76%) of the patients diagnosed in March, April, and from May onward, respectively, were treated in-line with VITT recommendations (p < 0.001). Overall, treatment according to recommendations had no statistically significant influence on mortality (14/44 [32%] vs 29/55 [52%], adjusted odds ratio [OR] = 0.43, 95% confidence interval [CI] = 0.16-1.19). However, patients who received immunomodulation had lower mortality (19/65 [29%] vs 24/34 [70%], adjusted OR = 0.19, 95% CI = 0.06-0.58). Treatment with non-heparin anticoagulants instead of heparins was not associated with lower mortality (17/51 [33%] vs 13/35 [37%], adjusted OR = 0.70, 95% CI = 0.24-2.04). Mortality was also not significantly influenced by platelet transfusion (17/27 [63%] vs 26/72 [36%], adjusted OR = 2.19, 95% CI = 0.74-6.54). Conclusions In patients with VITT-CVT, adherence to VITT treatment recommendations improved over time. Immunomodulation seems crucial for reducing mortality of VITT-CVT. ANN NEUROL 2022Peer reviewe

Characteristics and Outcomes of Patients With Cerebral Venous Sinus Thrombosis in SARS-CoV-2 Vaccine–Induced Immune Thrombotic Thrombocytopenia

Importance: Thrombosis with thrombocytopenia syndrome (TTS) has been reported after vaccination with the SARS-CoV-2 vaccines ChAdOx1 nCov-19 (Oxford-AstraZeneca) and Ad26.COV2.S (Janssen/Johnson & Johnson). Objective: To describe the clinical characteristics and outcome of patients with cerebral venous sinus thrombosis (CVST) after SARS-CoV-2 vaccination with and without TTS. Design, setting, and participants: This cohort study used data from an international registry of consecutive patients with CVST within 28 days of SARS-CoV-2 vaccination included between March 29 and June 18, 2021, from 81 hospitals in 19 countries. For reference, data from patients with CVST between 2015 and 2018 were derived from an existing international registry. Clinical characteristics and mortality rate were described for adults with (1) CVST in the setting of SARS-CoV-2 vaccine-induced immune thrombotic thrombocytopenia, (2) CVST after SARS-CoV-2 vaccination not fulling criteria for TTS, and (3) CVST unrelated to SARS-CoV-2 vaccination. Exposures: Patients were classified as having TTS if they had new-onset thrombocytopenia without recent exposure to heparin, in accordance with the Brighton Collaboration interim criteria. Main outcomes and measures: Clinical characteristics and mortality rate. Results: Of 116 patients with postvaccination CVST, 78 (67.2%) had TTS, of whom 76 had been vaccinated with ChAdOx1 nCov-19; 38 (32.8%) had no indication of TTS. The control group included 207 patients with CVST before the COVID-19 pandemic. A total of 63 of 78 (81%), 30 of 38 (79%), and 145 of 207 (70.0%) patients, respectively, were female, and the mean (SD) age was 45 (14), 55 (20), and 42 (16) years, respectively. Concomitant thromboembolism occurred in 25 of 70 patients (36%) in the TTS group, 2 of 35 (6%) in the no TTS group, and 10 of 206 (4.9%) in the control group, and in-hospital mortality rates were 47% (36 of 76; 95% CI, 37-58), 5% (2 of 37; 95% CI, 1-18), and 3.9% (8 of 207; 95% CI, 2.0-7.4), respectively. The mortality rate was 61% (14 of 23) among patients in the TTS group diagnosed before the condition garnered attention in the scientific community and 42% (22 of 53) among patients diagnosed later. Conclusions and relevance: In this cohort study of patients with CVST, a distinct clinical profile and high mortality rate was observed in patients meeting criteria for TTS after SARS-CoV-2 vaccination.info:eu-repo/semantics/publishedVersio

Cerebral venous sinus thrombosis due to vaccine-induced immune thrombotic thrombocytopenia in middle-income countries

Background: Adenovirus-based COVID-19 vaccines are extensively used in low- and middle-income countries (LMICs). Remarkably, cases of cerebral venous sinus thrombosis due to vaccine-induced immune thrombotic thrombocytopenia (CVST-VITT) have rarely been reported from LMICs. Aims: We studied the frequency, manifestations, treatment, and outcomes of CVST-VITT in LMICs. Methods: We report data from an international registry on CVST after COVID-19 vaccination. VITT was classified according to the Pavord criteria. We compared CVST-VITT cases from LMICs to cases from high-income countries (HICs). Results: Until August 2022, 228 CVST cases were reported, of which 63 were from LMICs (all middle-income countries [MICs]: Brazil, China, India, Iran, Mexico, Pakistan, Turkey). Of these 63, 32 (51%) met the VITT criteria, compared to 103 of 165 (62%) from HICs. Only 5 of the 32 (16%) CVST-VITT cases from MICs had definite VITT, mostly because anti-platelet factor 4 antibodies were often not tested. The median age was 26 (interquartile range [IQR] 20–37) versus 47 (IQR 32–58) years, and the proportion of women was 25 of 32 (78%) versus 77 of 103 (75%) in MICs versus HICs, respectively. Patients from MICs were diagnosed later than patients from HICs (1/32 [3%] vs. 65/103 [63%] diagnosed before May 2021). Clinical manifestations, including intracranial hemorrhage, were largely similar as was intravenous immunoglobulin use. In-hospital mortality was lower in MICs (7/31 [23%, 95% confidence interval (CI) 11–40]) than in HICs (44/102 [43%, 95% CI 34–53], p = 0.039). Conclusions: The number of CVST-VITT cases reported from LMICs was small despite the widespread use of adenoviral vaccines. Clinical manifestations and treatment of CVST-VITT cases were largely similar in MICs and HICs, while mortality was lower in patients from MICs.</p

Sex differences in cerebral venous sinus thrombosis after adenoviral vaccination against COVID-19

Introduction: Cerebral venous sinus thrombosis associated with vaccine-induced immune thrombotic thrombocytopenia (CVST-VITT) is a severe disease with high mortality. There are few data on sex differences in CVST-VITT. The aim of our study was to investigate the differences in presentation, treatment, clinical course, complications, and outcome of CVST-VITT between women and men. Patients and methods: We used data from an ongoing international registry on CVST-VITT. VITT was diagnosed according to the Pavord criteria. We compared the characteristics of CVST-VITT in women and men. Results: Of 133 patients with possible, probable, or definite CVST-VITT, 102 (77%) were women. Women were slightly younger [median age 42 (IQR 28–54) vs 45 (28–56)], presented more often with coma (26% vs 10%) and had a lower platelet count at presentation [median (IQR) 50x109/L (28–79) vs 68 (30–125)] than men. The nadir platelet count was lower in women [median (IQR) 34 (19–62) vs 53 (20–92)]. More women received endovascular treatment than men (15% vs 6%). Rates of treatment with intravenous immunoglobulins were similar (63% vs 66%), as were new venous thromboembolic events (14% vs 14%) and major bleeding complications (30% vs 20%). Rates of good functional outcome (modified Rankin Scale 0-2, 42% vs 45%) and in-hospital death (39% vs 41%) did not differ. Discussion and conclusions: Three quarters of CVST-VITT patients in this study were women. Women were more severely affected at presentation, but clinical course and outcome did not differ between women and men. VITT-specific treatments were overall similar, but more women received endovascular treatment.</p

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation