IRIS UniSR (’Università Vita-Salute San Raffaele)
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Targeting of the G9a, DNMT1 and UHRF1 epigenetic complex as an effective strategy against pancreatic ductal adenocarcinoma
No full textBackground: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with limited treatment options and a poor prognosis. The critical role of epigenetic alterations such as changes in DNA methylation, histones modifications, and chromatin remodeling, in pancreatic tumors progression is becoming increasingly recognized. Moreover, in PDAC these aberrant epigenetic mechanisms can also limit therapy efficacy. This study aimed to investigate the expression and prognostic significance of a key epigenetic complex encompassing DNA methyltransferase-1 (DNMT1), the histone methyltransferase G9a, and the scaffold protein UHRF1 in PDAC. We also evaluated the therapeutic potential of an innovative inhibitor targeting these epigenetic effectors. Methods: Immunohistochemical analysis of DNMT1, G9a, and UHRF1 expression was conducted in human PDAC tissue samples. Staining was semi-quantitatively scored, and overexpression was defined as moderate to strong positivity. The prognostic impact was assessed by correlating protein expression with patient survival. The antitumoral effects of the dual DNMT1-G9a inhibitor CM272 were tested in PDAC cell lines, followed by transcriptomic analyses to identify underlying mechanisms. The in vivo antitumoral efficacy of CM272 was evaluated in PDAC xenograft and syngeneic mouse models, both alone and in combination with anti-PD1 immunotherapy. Results: DNMT1, G9a, and UHRF1 were significantly overexpressed in PDAC cells and stroma compared to normal pancreatic tissues. Simultaneous overexpression of the three proteins was associated with significantly reduced survival in resected PDAC patients. CM272 exhibited potent antiproliferative activity in PDAC cell lines, inducing apoptosis and altering key metabolic and cell cycle-related genes. CM272 also enhanced chemotherapy sensitivity and significantly inhibited tumor growth in vivo without detectable toxicity. Combination of CM272 with anti-PD1 therapy further improved antitumor responses and immune cell infiltration, particularly CD4 + and CD8 + T cells. Conclusions: The combined overexpression of DNMT1, G9a, and UHRF1 in PDAC is a strong predictor of poor prognosis. CM272, by targeting this epigenetic complex, shows promising therapeutic potential by inducing apoptosis, reprogramming metabolic pathways, and enhancing immune responses. The combination of CM272 with immunotherapy offers a novel, effective treatment strategy for PDAC
LNA-ASO based therapy for the treatment of SARS-CoV-2 infection in pre-clinicals models
No full textThe COVID-19 crisis and the development of the first approved mRNA vaccine have highlighted the potential of RNA-based therapeutic strategies. Mutated variant strains of SARS-CoV-2 that evade immunity to previous infection or vaccination are continuously emerging, resulting in new outbreaks. Thus, there is a clear and urgent need for alternative approaches that are active against multiple variants of concern. Antisense oligonucleotides (ASOs) represent a new and promising class of RNA-targeted therapy. This study explored the design, evaluation, and optimization of ASOs targeting SARS-CoV-2 RNA, with the aim of providing a scalable therapeutic strategy capable of adapting to viral evolution. By integrating bioinformatics, in vitro screening, and delivery optimization, we report the key factors influencing ASO efficacy and highlight the challenges but also the potential of this approach.
Twenty-four ASOs were designed to target highly conserved, single-stranded regions of the SARS-CoV-2 genome, for maximal broad-spectrum efficacy against multiple variants. Extensive in vitro screening and optimization led to the identification of four LNA-modified ASOs that exhibited exceptional efficacy. These LNA-ASOs significantly reduced intracellular Spike RNA and virion Nucleoprotein RNA across 4 SARS-CoV-2 variants, highlighting them as potential therapeutic candidates. Initial experiments utilizing lipofectamine-based delivery methods revealed toxicity concerns. Therefore, optimization of the delivery method was carried out. Interestingly, we discovered that treatment with LNA-ASOs did not significantly alter the expression of interferon-stimulated genes when cells were either infected or not, demonstrating toxicity concerns were likely due to the delivery method alone. However, limited uptake of naked LNA-ASOs in the lung was observed in vivo, highlighting the challenge of ASO delivery to target cells. The exploration of a less toxic approach led us to the use of polymer-based delivery systems: P(SpAA-co-DAA) polymers (JH-26 and JH-27). These innovative polymers demonstrated improved LNA-ASO uptake and reduced toxicity compared to lipofectamine. The design of these polymers was based on a rational approach, considering factors such as charge density, biodegradability, and endosomal escape capabilities. Unexpectedly, the combination of JH-27 polymer with LNA-ASOs did not perform as effectively in reducing viral titers in A549-hACE2 cell.
Taken together, these findings highlight a meticulous methodological approach to the development of an RNA-based anti-viral therapy from design to in vivo experimentation.La crisi da COVID-19 e lo sviluppo del primo vaccino approvato a base di mRNA hanno messo in luce il potenziale delle strategie terapeutiche basate sull'RNA. Varianti mutate di SARS-CoV-2, capaci di sfuggire all'immunità acquisita derivante da precedenti infezioni o vaccinazioni, continuano ad emergere e a causare nuovi focolai. Vi e’ un bisogno urgente di approcci alternativi efficaci contro le nuove varianti di interesse. In questo contesto, gli oligonucleotidi antisenso (ASOs) rappresentano una classe promettente di agenti terapeutici. Il mio studio ha progettato, valutato e ottimizzato ASOs mirati all'RNA di SARS-CoV-2, con l'obiettivo di sviluppare una strategia terapeutica scalabile e capace di adattarsi all'evoluzione del virus. Integrando bioinformatica, screening in vitro e ottimizzazione della somministrazione sono stati identificati i principali fattori che influenzano l'efficacia degli ASOs, evidenziando le sfide ma anche il grande potenziale di questo approccio.
Ho progettato ventiquattro ASOs complementari a regioni altamente conservate e a singolo filamento del genoma del SARS-CoV-2, al fine di massimizzare l'efficacia ad ampio spettro contro più varianti. Lo screening e le ottimizzazioni in vitro hanno portato all'identificazione di quattro ASOs modificati con LNA che hanno dimostrato un'efficacia eccezionale. Questi LNA-ASOs hanno ridotto significativamente gli RNA intracellulari per la proteina Spike e per la Nucleoproteina virale in quattro varianti di SARS-CoV-2. Tuttavia, i primi esperimenti effettuati con somministrazione mediante lipofectamina hanno rivelato problemi di tossicità. È inoltre emerso che il trattamento con LNA-ASOs non altera significativamente l'espressione dei geni indotti dall'interferone, sia in cellule infette che non infette, suggerendo che i problemi di tossicità fosssero dovuti principalmente al metodo di somministrazione. Inoltre, è stato osservata una limitata capacita’ di transfezione degli LNA-ASOs "nudi" nei polmoni in vivo. Nel loro insieme questi risultati hanno evidenziato un problema da risolvere nella somministrazione agli organi bersaglio.
Ho quindi condotto un’ottimizzazione del metodo di somministrazione. Questa esplorazione ha portato all’uso di sistemi di somministrazione a base polimerica: i polimeri P(SpAA-co-DAA) H-26 e JH-27. Questi polimeri innovativi hanno migliorato la transfezione degli LNA-ASOs e hanno ridotto la tossicità rispetto alla lipofectamina. La progettazione di questi polimeri si è basata su un approccio razionale, tenendo conto di fattori come densità di carica, biodegradabilità e capacità entrare e uscire dagli endosomi. Tuttavia, in modo inaspettato, la combinazione del polimero JH-27 con LNA-ASOs non è risultata sufficientemente efficace nel ridurre i titoli virali nelle cellule A549-hACE2.
Complessivamente, questi risultati rappresentano un approccio metodologico rigoroso, dalla progettazione iniziale agli esperimenti in vivo, per lo sviluppo di una terapia antivirale basata sull'RNA
Dual concomitant CytoSorb hemoadsorption therapy in severe rhabdomyolysis: A novel approach to myoglobin clearance and organ preservation
No full textSevere rhabdomyolysis, characterized by extensive muscle breakdown and release of myoglobin and creatine kinase (CK), is a life-threatening condition often complicated by acute kidney injury (AKI) and multi-organ failure (MOF). Even when conventional treatments such as fluid resuscitation and renal replacement therapy (RRT) are timely applied, severe cases remain challenging to manage. Among therapies available in this setting, hemadsorption with CytoSorb has the potential not only to treat rhabdomyolysis through removal of circulating molecules but also to limit or even prevent rhabdomyolysis-related renal failure and MOF. In this case series we present a preliminary experience with a novel use CytoSorb hemoadsorption therapy, which encompassed the use of two CytoSorb cartridges running in parallel, to enhance myoglobin and cytokine clearance. Clinical data from the three patients with severe rhabdomyolysis treated with dual concomitant CytoSorb treatments highlighted marked improvements in CK, renal, hepatic, and inflammatory markers, with possible remarkable impact in containing rhabdomyolysis-related organ failure and death. Further investigation is warranted to establish the opportunity of tailored protocols for critically ill patients
Autonomous B-cell Receptor Signaling in Chronic Lymphocytic Leukemia
No full text: Chronic lymphocytic leukemia (CLL) is driven by both antigen-dependent and antigen-independent cell-autonomous B-cell receptor (BcR) signaling. The latter has been documented in CLL cases in different disease subgroups and correlates with disease progression. Autonomous signaling is also relevant in monoclonal B-cell lymphocytosis and other B-cell malignancies, including diffuse large B-cell lymphoma and splenic marginal zone lymphoma. This article examines its molecular basis, prognostic significance, and potential therapeutic relevance, highlighting the need for novel strategies to overcome resistance and improve outcomes
Early outcomes of the Third-generation of ClosureFast radiofrequency ablation for great saphenous vein reflux
No full textBackground: This study aimed to assess the safety of the third-generation ClosureFast catheter for radiofrequency ablation (RFA) in the treatment of great saphenous vein (GSV) reflux in patients presenting to a dedicated vein center. Materials and methods: All consecutive patients with incompetent GSV who underwent RFA between December 2023 and May 2024 were retrospectively analyzed. The primary study endpoints were technical success and postoperative complication rate at 30 days. Secondary study endpoints were freedom from GSV recanalization and recurrent varicose vein (RVV) rate over the follow-up. The improvement in symptoms (measured by the Venous Clinical Severity Score [VCSS]) was evaluated. Results: During the study period, 50 limbs were treated in 50 consecutive patients (mean age 55.8±13.4 years; 56% women; CEAP 2-4; VCSS >5). The technical success rate was achieved in 100% of cases. There was no significant incidence of 30-day complications. There were no instances of deep venous thrombosis or puncture site thermal injury. One patient (2%) had hyperpigmentation; two patients (4%) had ecchymosis; 4 patients (8%) had pain. At a mean follow-up of 2.9±1.4 months, GSV occlusion and freedom from reintervention rates were both 100% within 1 week and 30 days respectively. No patients had RVV over the follow-up. The VCSS score had decreased a median of 3.5 (IQR: 2.4-5) points from baseline (p<0.01). The mean CEAP class had decreased to 1.59 points from baseline, reflecting a shift towards milder disease categories (C0-C2). Conclusions: The third generation of RFA is safe and effective to ablate the GSV with a low complication rate in the perioperative period. However, durability over the follow-up and further studies with larger cohorts of patients are still needed to confirm these outcomes
External Validation of Nomograms for the Identification of Pelvic Nodal Dissection Candidates Among Prostate Cancer Patients with Negative Preoperative Prostate-specific Membrane Antigen Positron Emission Tomography
No full textBackground and objective: Extended pelvic lymph node dissection (ePLND) is recommended in selected radical prostatectomy (RP) prostate cancer (PCa) patients for staging purposes. We aim to externally validate available tools to predict lymph node invasion (LNI) in men with negative preoperative prostate-specific membrane antigen positron emission tomography (miN0). Methods: Overall, 282 intermediate- to high-risk PCa patients with miN0 disease undergoing RP and ePLND at ten centers between 2016 and 2023 were identified. The Memorial Sloan Kettering Cancer Center (MSKCC); Amsterdam-Brisbane-Sydney; and Briganti 2017, 2019, and 2023 tools predicting LNI were validated externally using calibration plots, C-indexes, and decision-curve analyses to assess calibration, discrimination, and net benefit. Key findings and limitations: Overall, 36 (13%) patients had LNI. The C-indexes of the MSKCC, Briganti 2017, Briganti 2019, Amsterdam-Brisbane-Sydney, and Briganti 2023 nomograms were 64%, 69%, 72%, 64%, and 77%, respectively. The Briganti 2023 nomogram exhibited higher net benefit than the other available nomograms, and the use of a 5% cutoff would have spared 47% ePLND procedures (vs 14% and 4.3% for the Briganti 2019 and Amsterdam-Brisbane-Sydney nomograms, respectively) at the cost of missing only five (3.8%) LNI cases. Heterogeneity in patient selection and imaging protocols represents the main limitations. Conclusions and clinical implications: The Briganti 2023 nomogram outperformed other available tools in predicting LNI in men with miN0 PCa. The use of this tool resulted in a considerable number of unnecessary ePLND procedures spared and optimization of ePLND recommendations in a contemporary clinical setting
The Influence of Repeated Abutment Changes on Peri-Implant Tissue Stability and Keratinised Tissue on Peri-Implant Health: 12-Year Post-Loading Results From a Multicentre Randomised Controlled Trial
No full textAimsTo evaluate the influence of at least three abutment disconnections in conventionally loaded implants against placement of a definitive abutment in immediately non-occlusal loaded implants on hard and soft tissue changes. A secondary aim was to evaluate whether the presence of less than 2 mm of keratinised mucosa is associated with increased peri-implant marginal bone loss and soft tissue recessions.MethodsSixty patients requiring one single crown or one fixed partial prosthesis supported by a maximum of three implants were randomised, after implants were placed with a torque superior to 35 Ncm, according to a parallel group design to receive definitive abutments which were loaded immediately (definitive abutment group) or transmucosal abutments which were delayed loaded after 3 months and were removed at least three times (repeated disconnection group). Patients were treated in three centres, and each patient contributed to the study with only one prosthesis followed for 12 years after initial loading. Outcome measures were: prosthesis failures, implant failures, complications, pink esthetic score (PES), buccal recessions, patient satisfaction, peri-implant marginal bone level changes, and height of the keratinised mucosa.ResultsThirty patients were randomly allocated to each group according to a parallel group design. Six patients dropped out or died from the definitive abutment group and seven from the repeated disconnection group. At 12 years post-loading, no patient in the definitive abutment group had implant failures versus three patients who lost five implants in the repeated disconnection group (difference = 13.04%; 95% CI: -6.7 to 26.8 to; p = 0.109; Fisher's exact test). No patient in the definitive abutment group had a prosthesis failure versus four patients of the repeated disconnection group. The difference was statistically significant (difference = 17.39%; 95% CI: 1.9 to 32.9; p = 0.049; Fisher's exact test). Four patients from the definitive abutment group versus seven patients from the repeated disconnection group were affected by complications (difference = 13.77%; 95% CI: -37.8 to 10.2; p = 0.318; Fisher's exact test), the difference not being statistically significant. PES scores did not show any statistically significant differences between the two groups: 10.84 +/- 1.95 for the definitive abutment group and 10.31 +/- 2.57 for the repeated abutment changes group (difference = -0.53; 95% CI: -1.14 to 2.20; p = 0.505). Buccal recessions amounted to 0.30 +/- 0.98 mm for the definitive abutment group and 0.15 +/- 0.54 mm for the repeated abutment changes group with no statistically significant differences between the two groups (difference = -0.14; 95% CI: -0.69 to 0.41; p = 0.592). All patients were declared to be very satisfied with the function and aesthetics of the prostheses and would undergo the same procedure again. Mean peri-implant marginal bone loss was 0.25 +/- 0.49 mm for the definitive abutment group and 0.70 +/- 1.04 mm for the repeated abutment changes group (difference = 0.45; 95% CI: -0.16 to 1.05; p = 0.135), the difference not being statistically significant. Height of keratinised mucosa was 2.56 +/- 1.75 for the definitive abutment group and 2.77 +/- 2.07 for the repeated abutment changes group (difference = 0.21 mm; 95% CI: -1.06 to 1.49; p = 0.746). There was no significantly increased marginal bone loss (difference = 0.20; 95% CI: -0.06 to 0.33; p = 0.268) or buccal recessions (difference = 0.10; 95% CI: -0.05 to 0.33; p = 0.203) at implants having less than 2 mm of keratinised mucosa at loading.ConclusionMore prosthesis failures were observed at 12 years after loading when comparing implants that underwent at least three repeated abutment disconnections to implants subjected to no disconnection. Immediate non-occlusal loading is a viable alternative to conventional loading. No increased bone loss or buccal recessions were noticed at implants with less than 2 mm of keratinised mucosa compared to those having more than 2 mm of keratinised mucosa
A comprehensive update on neuroimaging endpoints in amyotrophic lateral sclerosis
No full textIntroduction: There are currently few treatments approved for amyotrophic lateral sclerosis (ALS). Additionally, there remains a significant unmet need for reliable, standardized biomarkers to assess endpoints in clinical trials. Magnetic resonance imaging (MRI)- and positron emission tomography (PET)-derived metrics could help in patient selection and stratification, shortening trial duration and reducing costs. Areas covered: This review focuses on the potential use of neuroimaging endpoints in the context of ALS therapeutic trials, providing insights on structural and functional neuroimaging, plexus and muscle alterations, glial involvement and neuroinflammation, envisioning how these surrogates of disease progression could be implemented in clinical trials. A PubMed search covering the past 15 years was performed. Expert opinion: Neuroimaging is essential in understanding ALS pathophysiology, aiding in disease progression tracking and evaluating therapeutic interventions. High costs, limited accessibility, lack of standardization, and patient tolerability limit their use in routine ALS care. Addressing these obstacles is essential for fully harnessing neuroimaging potential in improving diagnostics and treatment in ALS
The awareness, characterization, and burden of Cognitive Impairment Associated with Schizophrenia (CIAS) in clinical practice: Results from a nationwide survey in Italy
No full textIntroduction: Cognitive Impairment (CIAS) is a core aspect of schizophrenia and one of the main obstacles to clinical and functional recovery in patients. People with CIAS have difficulties with learning and using information in real world. Despite its well-recognized role, it is not yet a diagnostic criterion in DSM-5 and ICD system. The effective management of CIAS represents a critical unmet need of schizophrenia treatment. Methods: To evaluate the awareness of CIAS in the Italian landscape, we conducted a quantitative survey on psychiatrists highly specialized in schizophrenia, focused on its awareness, assessment, burden, and treatment. Results: Of 152 participants, 139 (91.4 %) consider CIAS assessment important. The terminology most frequently used to describe CIAS is ‘cognitive impairment’. CIAS is assessed, clinically or with formal tools, in approximately 43 % of patients after stabilisation either during follow-up visits (N = 88, 67.7 %) or during hospital stay (N = 57, 43.8 %). 65 % of evaluated patients are considered affected by CIAS. Formal assessment tools (tests, questionnaires, interviews) are used in about 20 % of the centers. The Mini Mental State Examination (MMSE) (N = 75, 72.1 %) and the Wechsler Adult Intelligence Scale (WAIS) (N = 62, 59.6 %) are the most frequently used tools for CIAS evaluation. Conclusions: The clinical characteristics of the patient, structural barriers like the lack of trained personnel or inadequate economic resources, and organizational problems influence the assessment rate. Despite this awareness, greater effort must be made to overcome the barriers, especially economic and organizational ones, which prevent the assessment and treatment of CIAS from becoming established in routine clinical care