Lucia Stefanini

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing health concern given rising global prevalence, limited diagnostic and therapeutic tools, and strong association with cardiovascular disease. Platelets and oxidized phospholipids have been implicated in MASLD pathogenesis; however, the drivers and activities of platelets in MASLD remain overlooked. METHODS: We conducted a cross-sectional study to determine platelet metabolic and functional states in MASLD and to investigate whether oxidized phospholipids, which accumulate during MASLD, contribute to platelet dysfunction. The study included age-matched individuals with simple steatosis (n=42), steatohepatitis (n=28), metabolic dysfunction-associated cirrhosis (n=16), and controls without steatosis (n=23). Platelet function and metabolism were characterized by flow cytometry and Seahorse analysis. In vivo markers of platelet activation and plasma oxidized low-density lipoprotein were quantified by ELISA. The causal relationship between oxidized phospholipids and the platelet phenotype was investigated by comparing Ldlr ^−/− mice fed an Amylin Liver NASH diet to Amylin Liver NASH-fed E06-scFv/Ldlr ^−/− , transgenic mice that express a natural antibody (E06) that neutralizes oxidized phospholipids. RESULTS: We report that since early MASLD, in vivo markers of platelet secretion were elevated in plasma and platelets displayed heightened metabolic activity. In advanced MASLD, associated with MASH and cirrhosis, platelets were refractory to G-protein-coupled receptor stimulation, but more responsive to GPVI stimulation, promoting a secretory and procoagulant phenotype. Platelet dysfunction correlated with increased platelet mitochondrial activity and with blood levels of oxidized low-density lipoprotein, a major source of oxidized lipids. Ex vivo, the oxidized phospholipid oxPAPC recapitulated the increased mitochondrial activity and functional reprogramming observed in MASH platelets. In a mouse model of MASLD, neutralization of oxidized phospholipids partially mitigated diet-induced platelet dysfunction and binding to liver macrophages. CONCLUSIONS: In MASLD, we observed heightened platelet metabolic activity and changes in platelet reactivity, providing the basis to target platelet dysfunction for preventing hepatic and extra-hepatic complications of MASLD. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT05128253.

Background and Aims: Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are distinct autoimmune liver diseases (AILDs) that differ in pathogenesis and response to therapy. Moreover, some patients exhibit features of both, known as AIH/PBC overlap. Although AILDs are traditionally considered T cell‐driven, B cells have emerged as key contributors to disease progression. In this study, we investigated CD11c ⁺ T‐bet ⁺ B cells, implicated in different autoimmune diseases and never explored in AILDs. Methods: Circulating peripheral blood mononuclear cells (PBMC) were isolated from whole blood obtained from patients with AIH ( n = 15), PBC ( n = 8), AIH/PBC overlap syndrome ( n = 11), and healthy donors (HD, n = 12). B and T cell subsets were analysed by conventional and spectral flow‐cytometry. Clinical and laboratory parameters were recorded at the time of sample collection, and serum IL‐21, sCD40L, and BAFF levels were measured by ELISA and by flow cytometry. Results: Total CD19 ⁺ B‐cell frequencies were comparable across groups. However, the canonical atypical B‐cell subset defined as CD11c ⁺ T‐bet ⁺ CD21 ⁻ was significantly expanded in AIH and AIH/PBC patients but not in PBC. Phenotypic characterisation showed that these cells were enriched in double‐negative and naïve‐like B‐cell subsets and displayed the expected chemokine receptor profile of atypical B cells, with higher CXCR3 and lower CXCR5 expression compared with conventional B cells. Circulating follicular helper T cells (cTfh) were increased in AIH and AIH/PBC and correlated with the overall T‐bet ⁺ B‐cell compartment. Serum IL‐21 levels were higher in patients with AILDs, particularly in treatment non‐responders. Conclusions: Expansion of atypical CD11c ⁺ T‐bet ⁺ B cells, together with increased cTfh frequencies, hallmarks AIH and AIH/PBC. These findings support disease‐specific B‐cell alterations and distinct immune signatures in AIH and AIH/PBC compared with PBC, highlighting potential therapeutic implications for targeting pathogenic B cell subsets in AIH.

Background: Sex differences in cardiovascular diseases (CVD) are a matter of immediate concern, with platelets playing a pivotal role as major contributors to CVD. While the protective effects of estrogens on vascular health have been largely investigated, the impact of endogenous reproductive hormones on platelet function is less clear. In the SHOW (Sexual Hormones and Hemostasis: Observations for Women Health) study, we aimed to assess the association between the levels of endogenous reproductive hormones and in-vivo platelet activation among pre-menopausal healthy female subjects. Methods: Serum levels of estradiol, progesterone (PG), testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and in vivo platelet activation markers were quantified at four time-points across the menstrual cycle (day (d) 1, d5 ± 2, d14 ± 2, and d21 ± 2). Results: Among 21 healthy participants (mean age: 30 years), significant variations of thromboxane B2 (TxB2), soluble P-selectin (sP-selectin) and soluble NOX2-derived peptide (sNOX2-dp) are detected across the menstrual cycle. Linear mixed model analysis shows that sP-selection is associated with LH levels (F = 6.400, p = .016) in a time-dependent manner. TxB2 is associated with FSH across all time-points (F = 6.051, p = .019) and is significantly reduced on d1 in individuals with self-reported heavy menstrual bleeding. Soluble CD40L (sCD40L) changes most significantly in individuals with an ovulatory cycle (i.e. PG ≥ 3 ng/ml at d21). Conclusions: In healthy pre-menopausal female subjects, changes of in-vivo platelet activation markers are associated with changes of PG and gonadotropins. These findings suggest the need for caution in the interpretation of platelet biomarkers in clinical studies enrolling pre-menopausal female individuals.

The burden of metabolic dysfunction-associated steatotic liver disease (MASLD) is of immediate concern, as its prevalence is increasing worldwide. MASLD often progresses to liver fibrosis, posing significant health risks. Age-independent, noninvasive tools to evaluate fibrosis are needed to improve diagnostic accuracy across all age groups. Eighty-four inflammatory, hematological, and metabolic variables were quantified in the blood of 63 individuals with MASLD, with varying degrees of fibrosis, and 22 age-matched controls. Linear regression models were used to identify markers strongly correlated with liver fibrosis that were not influenced by age. Logistic regression models were used to evaluate the ability of various indices to discriminate between no/mild and severe liver fibrosis. Glutamine and propionate levels were identified as strongly correlated with fibrosis but not with age and were combined to form the GP index. The GP index demonstrated superior predictive power for liver fibrosis compared to existing scores, like FIB-4 and FIB-3. The study introduces the GP index, a novel metabolite-based score for diagnosing and monitoring liver fibrosis in patients with MASLD, which demonstrated robust performance irrespective of patient age in this cohort. By excluding age-dependent markers, the GP index can potentially reduce false positives and improve diagnostic accuracy, particularly in older populations. The combination of glutamine and propionate in this index represents a novel approach, capturing both intrinsic hepatic metabolic changes and extrinsic influences from the gut microbiota, providing a simple yet effective solution for liver fibrosis staging.

Background: Liver cirrhosis (LC) is a leading global cause of morbidity and mortality, with inflammation playing a key role in disease progression and clinical complications of LC. The Neutrophil/Lymphocyte Ratio (NLR), a readily available marker of systemic inflammation, has been linked to short-term adverse outcomes in LC, but data on long-term follow-up are limited. This study aimed to investigate the relationship between NLR and long-term all-cause mortality in an unselected cohort of LC patients. Methods: Data were gathered from the Italian multicenter observational study “PRO-LIVER”. Patients with available data to calculate NLR at baseline were included. Baseline clinical determinants of NLR and the association of NRL with all-cause mortality at 2-year follow-up were evaluated. Results: From the overall cohort (n = 753), 506 patients with LC (31% female, mean age 64.8 ± 11.9 years) were included in the analysis. Median value of NLR was 2.42 (Interquartile Range [IQR]: 1.61–3.52). At baseline, patients with NLR ≥ 2.42 were more likely to have Child–Pugh B or C, hepatocellular carcinoma (HCC), or portal vein thrombosis (PVT). After a median follow-up of 21 months, 129 patients died: 44 (17%) with NLR < 2.42 and 85 (34%) with NLR ≥ 2.42 (p < 0.001). At multiple-adjusted Cox regression analysis, NLR ≥ 2.42 was independently associated with all-cause mortality (HR: 1.65; 95% CI: 1.12–2.44; p = 0.012), along with age, Child–Pugh C class, HCC and PVT. Conclusions: NLR is associated with long-term all-cause mortality in LC. NLR may serve as a potentially easily available tool to aid risk refinement in LC. Trial registration number: ClinicalTrials.gov Identifier: NCT01470547.

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease (NAFLD), encompasses a spectrum of liver diseases characterized by hepatic steatosis, the presence of at least one cardiometabolic risk factor, and no other apparent cause. Metabolic syndrome (MetS) is a cluster of clinical conditions associated with increased risk of cardiovascular disease, type 2 diabetes, and overall morbidity and mortality. This narrative review summarizes the changes in the management of people with MetS and NAFLD/MASLD from screening to therapeutic strategies that have occurred in the last decades. Specifically, we underline the clinical importance of considering the different impacts of simple steatosis and advanced fibrosis and provide an up-to-date overview on non-invasive diagnostic tests (i.e., imaging and serum biomarkers), which now offer acceptable accuracy and are globally more accessible. Early detection of MetS and MASLD is a top priority as it allows for timely interventions, primarily through lifestyle modification. The liver and cardiovascular benefits of a global and multidimensional approach are not negligible. Therefore, a holistic approach to both conditions, MetS and related chronic liver disease, should be applied to improve overall health and longevity.

Background: Adherence to healthy dietary patterns, such as the Mediterranean diet (Med-diet), is recommended for the maintenance of cardiovascular health. The determinants for adherence to Med-diet and its importance in secondary cardiovascular disease prevention are still unclear. The aim of the study was to evaluate the influence of sex- and psycho-socio-cultural (i.e., gender-related) factors on Med-diet adherence and its role in preventing major cardiovascular events (MACEs) in patients with ischemic heart disease (IHD). Methods: Med-diet adherence was evaluated among 503 consecutive adults with IHD. MACEs were collected during a long-term follow-up. Results: Male Bem Sex-Role Inventory score (i.e., male personality traits) and physical functional capacity were associated with higher adherence, while cohabitation with a smoker and physical inactivity with poorer adherence. During a median follow-up of 22 months, 48 participants experienced MACEs (17.5%, 8.1%, and 3.9% of patients with low, medium, and high adherence, respectively; p = 0.016). At multivariate Cox--regression analysis, a greater adherence remained inversely associated with MACEs (HR: 0.49; 95% CI: 0.29–0.82; p = 0.006) after adjusting for confounding factors. Conclusion: The study suggests that gender-related factors have a role in maintaining a healthy dietary pattern. Improving Med-diet adherence may lower the risk of recurring cardiovascular events.

New-generation mRNA and adenovirus vectored vaccines against SARS-CoV-2 spike protein are endowed with immunogenic, inflammatory and immunomodulatory properties. Recently, BioNTech developed a noninflammatory tolerogenic mRNA vaccine (MOGm1Ψ) that induces in mice robust expansion of antigen-specific regulatory T (Treg) cells. The Pfizer/BioNTech BNT162b2 mRNA vaccine against SARS-CoV-2 is identical to MOGm1Ψ except for the lipid carrier, which differs for containing lipid nanoparticles rather than lipoplex. Here we report that vaccination with BNT162b2 led to an increase in the frequency and absolute count of CD4^posCD25^highCD127^low putative Treg cells; in sharp contrast, vaccination with the adenovirus-vectored ChAdOx1 nCoV-19 vaccine led to a significant decrease of CD4^posCD25^high cells. This pilot study is very preliminary, suffers from important limitations and, frustratingly, very hardly can be refined in Italy because of the >90% vaccination coverage. Thus, the provocative perspective that BNT162b2 and MOGm1Ψ may share the capacity to promote expansion of Treg cells deserves confirmatory studies in other settings.