Stefania Braidotti

Introduction: The adverse effect of metals and metalloids on neural development is a significant health concern, especially in vulnerable populations, such as pediatric patients. Excessive accumulation of these elements in the developing central nervous system (CNS) can induce oxidative stress, trigger cell death, and promote neuroinflammation. However, few studies have quantified metal and metalloid concentrations in cerebrospinal fluid (CSF) in pediatric patients, and, to our knowledge, none have compared these levels between children with and without neurological disorders. Methods: In this exploratory study, we compared pediatric patients with neurological diseases or CNS infections to control pediatric patients to assess differences in metal and metalloid concentrations and in CSF inflammatory cytokine profiles. Results: We observed higher levels of most metals and metalloids in neurological and infection patients, while group-specific differences were observed in cytokines. Notably, the cytokines Pentraxin-3 and IL-8 showed positive correlations with calcium, copper, iron, antimony, and chromium, suggesting a possible functional relationship in neuroinflammation. Discussion: In conclusion, this study presents a comparative analysis of CSF metal and metalloid levels in conjunction with inflammatory cytokine profiles in a pediatric population, providing a basis for further research into their roles in neuroinflammation and neurological disease.

Background/Objectives: Pediatric allogeneic hematopoietic stem cell transplantation (allo-HSCT) is complicated by iron overload and hypomagnesemia, both contributing to immune dysfunction and post-transplant morbidity. The combined impact of these metabolic disturbances on pediatric allo-HSCT outcomes remains unexplored. This study aims to determine whether hypomagnesemia can serve as a prognostic biomarker for delayed immune reconstitution and explores its interplay with iron overload in predicting post-transplant complications and survival outcomes. Methods: A retrospective analysis was conducted on 163 pediatric allo-HSCT recipients. Serum magnesium levels were measured at defined intervals post-transplant, and outcomes were correlated with CD4+ T cell recovery, time to engraftment, incidence of graft-versus-host disease (GVHD), and survival within 12 months. Iron status, including siderosis severity, was evaluated using imaging and laboratory parameters obtained from clinical records. Results: Patients who died within 12 months post-transplant exhibited significantly lower magnesium levels. Hypomagnesemia was associated with delayed CD4+ T cell recovery, prolonged engraftment, and an increased risk of acute GVHD. A strong inverse correlation was observed between magnesium levels and the severity of siderosis. Iron overload appeared to exacerbate magnesium deficiency. Additionally, the coexistence of hypomagnesemia and siderosis significantly increased the risk of immune dysfunction and early mortality. No significant association was found with chronic GVHD. Conclusions: Hypomagnesemia is a significant, early predictor of poor outcomes in pediatric allo-HSCT, particularly in the context of iron overload, underscoring the need for early intervention, including iron chelation and MRI, to improve outcomes.

High‐dose methotrexate (HD‐MTX) infusions are commonly used to consolidate remission in children with acute lymphoblastic leukemia (ALL). We investigate the potential role of candidate polymorphisms in SLCO1B1 (rs4149056 and rs2306283), ABCB1 (rs1045642), ABCC2 (rs717620), ABCC3 (rs9895420), and ABCC4 (rs7317112) drug transporters genes on HD‐MTX pharmacokinetics and patients' outcome (meant both as relapse and drug‐related toxicities) in an Italian cohort of 204 ALL pediatric patients treated according to the AIEOP‐BFM ALL 2009 protocol. TaqMan SNP genotyping assays determined patient's genotypes. Measurements of HD‐MTX plasma concentration were available for 814 HD‐MTX courses in 204 patients; MTX clearance was estimated by a two‐compartmental linear pharmacokinetic model with first‐order elimination and a Bayesian approach, via ADAPT. Independent contributions of age and ABCC4 SNP rs7317112 (A>G, intronic) on MTX clearance were detected in a multivariate analysis (p = 1.57 × 10⁻⁸ and p = 2.06 × 10⁻⁵, respectively), suggesting a delayed elimination of the drug in older patients and an accelerated one in carriers of the variant GG genotype. After multiple corrections, the association between ABCC2 SNP rs717620 (−24 C>T) and severe hematological toxicity was found (p < 0.005). Moreover, SLCO1B1 SNP rs4149056 (c.521T>C, p.V174A) affected patients' outcomes: carriers of the variant C allele presented a reduced risk of relapse compared to wild‐type TT (hazard risk: 0.27, 95% confidence interval [CI]: 0.08–0.90, p = 0.037). Taken together, these data highlighted the importance of variants in drug transporters genes on HD‐MTX disposition in the AIEOP‐BFM ALL 2009 protocol consolidation phase, and their putative role as predictive markers of outcome.

Background: Pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) face several risk factors influencing transplantation success, including nutritional status as measured by body mass index (BMI). Methods: This study analyzed BMI data collected from patients transplanted between 2003 and 2023, and aimed to evaluate whether deviations from normal BMI are associated with poorer clinical outcomes. BMI levels assessed before and after first-line treatment and pre-transplantation were analyzed retrospectively to determine a correlation with survival and post-transplant complications. Results: Underweight patients had significantly lower 12- and 36-month overall survival rates compared to normal-weight and overweight patients (p = 1.22 × 10⁻⁸ and p = 8.88 × 10⁻⁸, respectively). Event-free survival was also lower for underweight patients at all time points. A higher pre-transplant BMI increases the risk of acute graft-versus-host disease (GVHD, p = 0.00068). Otherwise, pre-transplant BMI was not significantly correlated with early TRCs and cGVHD. As secondary objectives, this study identified differences in BMI across primary disease groups, with solid tumor patients having the highest BMI and myelodysplastic syndrome patients having the lowest. BMI cut-offs were identified to predict or protect against serious outcomes, including delayed engraftment, TRCs, and acute and chronic GVHD. Conclusions: This study highlights the importance of nutritional assessment and management in pediatric patients undergoing allo-HSCT to optimize post-transplant outcomes, as deviations from a normal BMI can significantly impact post-transplant health. These findings underscore the importance of integrating BMI assessment throughout the entire pre-HSCT therapeutic course to identify patients at higher risk for complications and to define more effective nutritional management strategies.

Optimized use of prophylactic or therapeutic donor lymphocyte infusions (DLI) is aimed at improving clinical outcomes in patients with malignant and non-malignant hematological diseases who have undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT). Memory T-lymphocytes (CD45RA−/CD45RO+) play a crucial role in immune reconstitution post-HSCT. The infusion of memory T cells is proven to be safe and effective in improving outcomes due to the enhanced reconstitution of immunity and increased protection against viremia, without exacerbating graft-versus-host disease (GVHD) risks. Studies indicate their persistence and efficacy in combating viral pathogens, suggesting a viable therapeutic avenue for patients. Conversely, using virus-specific T cells for viremia control presents challenges, such as regulatory hurdles, cost, and production time compared to CD45RA-memory T lymphocytes. Additionally, the modulation of regulatory T cells (Tregs) for therapeutic use has become an important area of investigation in GVHD, playing a pivotal role in immune tolerance modulation, potentially mitigating GVHD and reducing pharmacological immunosuppression requirements. Finally, donor T cell-mediated graft-versus-leukemia immune responses hold promise in curbing relapse rates post-HSCT, providing a multifaceted approach to therapeutic intervention in high-risk disease scenarios. This comprehensive review underscores the multifaceted roles of T lymphocytes in HSCT outcomes and identifies avenues for further research and clinical application.

Oligodendroglioma (OG) is a brain tumor that contributes to <1% of brain tumor diagnoses in the pediatric population. Unfortunately, pediatric OG remains without definitive molecular characteristics to aid in diagnosis, and little is known about the tumor microenvironment. Tumor cells’ metabolism and proliferation rate are generally higher than those of healthy cells, so their iron demand is also significantly higher. This consideration underlines the great importance of iron for tumor development and progression. In this context, this study aims to evaluate the effect of iron in a cellular in vitro model of human oligodendroglioma brain tumor. Cell morphology, the effect of siderotic medium on cell growth, iron uptake, and the expression of iron-metabolism-related genes were evaluated via optic microscopy, ICP-MS, confocal microscopy, and real-time PCR, respectively. This study underlines the great importance of iron for tumor development and progression and also the possibility of reducing the available iron concentration to determine an antiproliferative effect on OG. Therefore, every attempt can be promising to defeat OG for which there are currently no long-term curative therapies.

Oligodendroglioma (OG) is a brain tumor that contributes to < 1% of brain tumor diagnoses in the pediatric population. Unfortunately, pediatric OG remains without definitive molecular characteristics to aid in diagnosis, and little is known about the tumor microenvironment. Tumor cells' metabolism and proliferation rate are generally higher than healthy cells, so their iron demand is also significantly increased. This consideration underlines the great importance of iron for tumor development and progression. In this context, this study aims to evaluate the effect of iron in a cellular in vitro model of human oligodendroglioma brain tumor. Cell morphology, the effect of siderotic medium on cell growth, iron uptake, and the expression of iron metabolism-related genes were evaluated by optic microscopy, ICP-MS, confocal microscopy, and RealTime PCR, respectively. This study underlines the great importance of iron for tumor development and progression, but also the possibility of reducing the available iron concentration to determine an antiproliferative effect on OGs. Therefore, every attempt can be promising to defeat the OGs for which there are currently no long-term curative therapies.

The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of BCR-ABL1 like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (P_ABL)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The P_ABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (P_ABL-_F) and fully-phosphorylated (P_PHOSPHO-_ABL) biosensors) were included in the assay. After incubation with whole cell lysates, average P_ABL phosphorylation was significantly increased (basal vs. P_ABL phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, p-value < 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to P_PHOSPHO-_ABL in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on P_ABL; a lower signal was instead observed for NALM6 and REH (p < 0.001 and p < 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib (p < 0.001 for K562, NALM6, ALL-SIL and p < 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by P_ABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel P_ABL-ELISA assay on leukemic cells isolated from patient’s bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia (BCR-ABL1 positive) and a child affected by ALL (BCR-ABL1 negative) were performed. Phosphorylation of P_ABL was specifically inhibited after the incubation of BCR-ABL1 positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the P_ABL-based ELISA assay provides a novel in vitro tool for screening both the aberrant ABL1 activity in BCR-ABL1 like ALL leukemic cells and their potential response to TK inhibitors.