Roadmap for Postnatal Brain Maturation: Changes in Gray and White Matter Composition during Development Measured by Fourier Transformed Infrared Microspectroscopy.

Key events in postnatal brain development, such as neuronal migration, synaptogenesis, and myelination, shape the adult brain. These events are reflected in changes in gray and white matter (GM and WM) occurring during this period. Therefore, precise knowledge of GM and WM composition in perinatal brain development is crucial to characterizing brain formation as well as the neurodevelopmental disruption observed in diseases such as autism and schizophrenia. In this study, we combined histochemical and immunohistochemical staining with biochemical and biophysical analyses using Fourier transform infrared (IR) microspectroscopy (µFTIR) to better understand the chemical changes during postnatal developmental myelination. For this purpose, we analyzed the GM and WM in the mouse brain and cerebellum (strain C57BL/6) from postnatal day 0 (P0) to day P28 and established presumed correlations between staining and IR data. IR spectra allowed the (i) quantification of lipid and protein content through the CH2/amide I ratio, (ii) determination of chemical characteristics of lipids, such as the presence of unsaturated bonds in the carbonate chain or carbonyls from ester groups in the polar head, and (iii) determination of the protein secondary structure (α-helix and intramolecular ß-sheets). The results indicate that the increase in the CH2/amide I ratio calculated from the µFTIR data correlates well with lipid histochemical staining. IR data indicated a change in the lipid composition in WM since carbonyl and unsaturated olefinic groups do not increase when lipids accumulate during myelination. Our correlation analysis between IR data and immunohistochemical staining of myelin-associated proteins revealed that myelin oligodendrocyte protein correlated well with lipid accumulation, while myelin basic protein appeared before lipid modifications, which indicated that myelin-associated proteins and lipid deposition were not synchronic. These events were related to a decrease in the intramolecular ß/α protein ratio. Our results indicate that lipids and proteins in WM substantially change their composition due to primary myelination, and according to results obtained from staining, these modifications are better described by lipid histochemical staining than by immunohistochemistry against myelin-related proteins. In conclusion, µFTIR can be a useful technique to study WM during perinatal development and provide detailed information about alterations in the chemical composition related to neurodevelopmental diseases.

Younger age and induction failure predict outcomes in infant leukemia: 30 years of experience in a tertiary center.

Objectives: This study aimed to evaluate the characteristics and outcomes of infant patients with leukemia. Methods: A retrospective analysis was conducted in a cohort of 39 patients diagnosed with infant leukemia from 1990 to 2020 who underwent treatment at the pediatric hemato-oncology department of a tertiary hospital in Madrid, Spain. Results: Of the 588 diagnosed cases of childhood leukemia, 39 (6.6%) cases were infant leukemia. The 5-year event-free survival and the 5-year overall survival were 43.6% (SE 4.1) and 46.5% (SD 24.08), respectively. In a univariate analysis, a younger age at diagnosis was associated with poorer outcomes (p = 0.027), as was induction failure (p = 0.0024). Patients treated with hematopoietic stem cell transplantation had better outcomes than non-transplanted patients (p = 0.001); however, the group comparisons that exclude patients who were unable to undergo transplantation due to refractoriness/relapse or death during treatment showed no significant differences. Conclusions: The main risk factors that affected survival in our study were an age younger than 6 months and a poor response to induction therapy. It is important to identify poor prognostic factors in this population in order to seek different approaches that could improve outcomes.

Decoding the molecular heterogeneity of pediatric monomorphic post-solid organ transplant lymphoproliferative disorders.

Posttransplant lymphoproliferative disorders (PTLDs) represent a broad spectrum of lymphoid proliferations, frequently associated with Epstein-Barr virus (EBV) infection. The molecular profile of pediatric monomorphic PTLDs (mPTLDs) has not been elucidated, and it is unknown whether they display similar genetic features as their counterpart in adult and immunocompetent (IMC) pediatric patients. In this study, we investigated 31 cases of pediatric mPTLD after solid organ transplantation, including 24 diffuse large B-cell lymphomas (DLBCLs), mostly classified as activated B cell, and 7 cases of Burkitt lymphoma (BL), 93% of which were EBV positive. We performed an integrated molecular approach, including fluorescence in situ hybridization, targeted gene sequencing, and copy number (CN) arrays. Overall, PTLD-BL carried mutations in MYC, ID3, DDX3X, ARID1A, or CCND3 resembling IMC-BL, higher mutational burden than PTLD-DLBCL, and lesser CN alterations than IMC-BL. PTLD-DLBCL showed a very heterogeneous genomic profile with fewer mutations and CN alterations than IMC-DLBCL. Epigenetic modifiers and genes of the Notch pathway were the most recurrently mutated in PTLD-DLBCL (both 28%). Mutations in cell cycle and Notch pathways correlated with a worse outcome. All 7 patients with PTLD-BL were alive after treatment with pediatric B-cell non-Hodgkin lymphoma protocols, whereas 54% of patients with DLBCL were cured with immunosuppression reduction, rituximab, and/or low-dose chemotherapy. These findings highlight the low complexity of pediatric PTLD-DLBCL, their good response to low-intensity treatment, and the shared pathogenesis between PTLD-BL and EBV-positive IMC-BL. We also suggest new potential parameters that could help in the diagnosis and the design of better therapeutic strategies for these patients.

Microglia states and nomenclature: A field at its crossroads.

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

Tisagenlecleucel therapy for relapsed or refractory B-cell acute lymphoblastic leukaemia in infants and children younger than 3 years of age at screening: an international, multicentre, retrospective cohort study.

BACKGROUND: Children aged younger than 3 years were excluded from the ELIANA phase 2 trial of tisagenlecleucel in children with acute lymphoblastic leukaemia. The feasibility, safety, and activity of tisagenlecleucel have not been defined in this group, the majority of whom have high-risk (KMT2A-rearranged) infant acute lymphoblastic leukaemia and historically poor outcomes despite intensification of chemotherapy, and for whom novel therapies are urgently needed. We aimed to provide real-world outcome analysis of the feasibility, activity, and safety of tisagenlecleucel in younger children and infants with acute lymphoblastic leukaemia. METHODS: We did an international, multicentre, retrospective cohort study at 15 hospitals across ten countries in Europe. Eligible patients were children aged younger than 3 years at screening between Sept 1, 2018, and Sept 1, 2021, who were screened for tisagenlecleucel therapy for relapsed or refractory B-cell precursor acute lymphoblastic leukaemia according to licensed indications. Patients received a single intravenous infusion of tisagenlecleucel. We tracked chimeric antigen receptor T-cell therapy outcomes using a standardised data reporting form. Overall survival, event-free survival, stringent event-free survival, B-cell aplasia, and toxicity were assessed in all patients who received a tisagenlecleucel infusion. FINDINGS: 38 eligible patients were screened, of whom 35 (92%) received a tisagenlecleucel infusion. 29 (76%) of 38 patients had KMT2A-rearranged acute lymphoblastic leukaemia, and 25 (66%) had relapsed after previous allogeneic haematopoietic stem-cell transplantation (HSCT). Patients had previously received a median of 2 lines (IQR 2-3) of (non-HSCT) therapy. Seven (18%) of 38 patients had received inotuzumab and 14 (37%) had received blinatumomab. After a median of 14 months (IQR 9-21) of follow-up, overall survival at 12 months after tisagenlecleucel infusion was 84% (64-93; five patients had died), event-free survival was 69% (47-83; nine events), and stringent event-free survival was 41% (23-58; 18 events). The probability of ongoing B-cell aplasia was 70% (95% CI 46-84; seven events) at 12 months. Adverse events included cytokine release syndrome, which occurred at any grade in 21 (60%) of 35 patients and at grade 3 or worse in five (14%), and neurotoxicity at any grade in nine (26%), none of which were severe. Measurable residual disease-negative complete response with or without haematological recovery occurred in 24 (86%) of 28 patients who had measurable disease. INTERPRETATION: These data suggest that tisagenlecleucel has antitumour activity and has an acceptable safety profile for young children and infants with B-cell precursor acute lymphoblastic leukaemia. FUNDING: None.

Resultados preliminares tras la instauración del programa con medicamentos CAR-T en leucemia aguda linfoblástica en un centro pediátrico / Building a CAR-T cell therapy program in a pediatric hemato-oncology department. Preliminary data

No disponible

Astrocyte-targeted Overproduction of IL-10 Reduces Neurodegeneration after TBI.

Traumatic brain injury is the greatest cause of disability and death in young adults in the developed world. The outcome for a TBI patient is determined by the severity of the injury, not only from the initial insult but, especially, as a product of the secondary injury. It is proposed that this secondary injury is directly linked to neuro-inflammation, with the production of pro-inflammatory mediators, activation of resident glial cells and infiltration of peripheral immune cells. In this context, anti-inflammatory treatments are one of the most promising therapies to dampen the inflammatory response associated with TBI and to reduce secondary injury. In this sense, the main objective of the present study is to elucidate the effect of local production of IL-10 in the neurological outcome after TBI. For this purpose, a cryogenic lesion was caused in transgenic animals overproducing IL-10 under the GFAP promoter on astrocytes (GFAP-IL10Tg mice) and the neuro-protection, microglial activation and leukocyte recruitment were evaluated. Our results showed a protective effect of IL-10 on neurons at early time-points after TBI, in correlation with a shift in the microglial activation profile towards a down-regulating phenotype and lower production of pro-inflammatory cytokines. Concomitantly, we observed a reduction in the BBB leakage together with modifications in leukocyte infiltration into the affected area. In conclusion, local IL-10 production modifies the neuro-inflammatory response after TBI, shifting it to anti-inflammatory and neuro-protective conditions. These results point to IL-10 as a promising candidate to improve neuro-inflammation associated with TBI.

Advanced Molecular Characterisation in Relapsed and Refractory Paediatric Acute Leukaemia, the Key for Personalised Medicine.

Relapsed and refractory (R/r) disease in paediatric acute leukaemia remains the first reason for treatment failure. Advances in molecular characterisation can ameliorate the identification of genetic biomarkers treatment strategies for this disease, especially in high-risk patients. The purpose of this study was to analyse a cohort of R/r children diagnosed with acute lymphoblastic (ALL) or myeloid (AML) leukaemia in order to offer them a targeted treatment if available. Advanced molecular characterisation of 26 patients diagnosed with R/r disease was performed using NGS, MLPA, and RT-qPCR. The clinical relevance of the identified alterations was discussed in a multidisciplinary molecular tumour board (MTB). A total of 18 (69.2%) patients were diagnosed with B-ALL, 4 (15.4%) with T-ALL, 3 (11.5%) with AML and 1 patient (3.8%) with a mixed-phenotype acute leukaemia (MPL). Most of the patients had relapsed disease (88%) at the time of sample collection. A total of 17 patients (65.4%) were found to be carriers of a druggable molecular alteration, 8 of whom (47%) received targeted therapy, 7 (87.5%) of them in addition to hematopoietic stem cell transplantation (HSCT). Treatment response and disease control were achieved in 4 patients (50%). In conclusion, advanced molecular characterisation and MTB can improve treatment and outcome in paediatric R/r acute leukaemias.

Chronic Oral Anticoagulation Therapy and Prognosis of Patients Admitted to Hospital for COVID-19: Insights from the HOPE COVID-19 Registry.

Background: Most evidence regarding anticoagulation and COVID-19 refers to the hospitalization setting, but the role of oral anticoagulation (OAC) before hospital admission has not been well explored. We compared clinical outcomes and short-term prognosis between patients with and without prior OAC therapy who were hospitalized for COVID-19. Methods: Analysis of the whole cohort of the HOPE COVID-19 Registry which included patients discharged (deceased or alive) after hospital admission for COVID-19 in 9 countries. All-cause mortality was the primary endpoint. Study outcomes were compared after adjusting variables using propensity score matching (PSM) analyses. Results: 7698 patients were suitable for the present analysis (675 (8.8%) on OAC at admission: 427 (5.6%) on VKAs and 248 (3.2%) on DOACs). After PSM, 1276 patients were analyzed (638 with OAC; 638 without OAC), without significant differences regarding the risk of thromboembolic events (OR 1.11, 95% CI 0.59-2.08). The risk of clinically relevant bleeding (OR 3.04, 95% CI 1.92-4.83), as well as the risk of mortality (HR 1.22, 95% CI 1.01-1.47; log-rank p value = 0.041), was significantly increased in previous OAC users. Amongst patients on prior OAC only, there were no differences in the risk of clinically relevant bleeding, thromboembolic events, or mortality when comparing previous VKA or DOAC users, after PSM. Conclusion: Hospitalized COVID-19 patients on prior OAC therapy had a higher risk of mortality and worse clinical outcomes compared to patients without prior OAC therapy, even after adjusting for comorbidities using a PSM. There were no differences in clinical outcomes in patients previously taking VKAs or DOACs. This trial is registered with NCT04334291/EUPAS34399.

TRPV2: A Key Player in Myelination Disorders of the Central Nervous System.

Transient potential receptor vanilloid 2 (TRPV2) is widely expressed through the nervous system and specifically found in neuronal subpopulations and some glial cells. TRPV2 is known to be sensitized by methionine oxidation, which results from inflammation. Here we aim to characterize the expression and regulation of TRPV2 in myelination pathologies, such as hypomyelination and demyelination. We validated the interaction between TRPV2 and its putative interactor Opalin, an oligodendrocyte marker, in mixed glial cultures under pro- and anti-inflammatory conditions. Then, we characterized TRPV2 time-course expression in experimental animal models of hypomyelination (jimpy mice) and de-/remyelination (cuprizone intoxication and experimental autoimmune encephalomyelitis (EAE)). TRPV2 showed upregulation associated with remyelination, inflammation in cuprizone and EAE models, and downregulation in hypomyelinated jimpy mice. TRPV2 expression was altered in human samples of multiple sclerosis (MS) patients. Additionally, we analyzed the expression of methionine sulfoxide reductase A (MSRA), an enzyme that reduces oxidated methionines in TRPV2, which we found increased in inflammatory conditions. These results suggest that TRPV2 may be a key player in myelination in accordance with the recapitulation hypothesis, and that it may become an interesting clinical target in the treatment of demyelination disorders.

Venous thromboembolism in pediatric patients with acute lymphoblastic leukemia under chemotherapy treatment. Risk factors and usefulness of thromboprophylaxis. Results of LAL-SEHOP-PETHEMA-2013.

INTRODUCTION: Symptomatic venous thromboembolism (VTE) is diagnosed in 3%-14% of patients during pediatric acute lymphoblastic leukemia (ALL) therapy. There are well-known risk factors, but the role of others as inherited thrombophilia is still controversial. Prophylaxis with low molecular weight heparin (LMWH) has been described, but its use is not globally accepted. METHODS: A retrospective multicentric study in ALL patients 1-18 years old following SEHOP-PETHEMA-2013 treatment guideline was performed to evaluate VTE rate, anticoagulant treatment, outcome, risk factors, and safety and usefulness of LMWH administration as primary thromboprophylaxis in children with inherited thrombophilia. RESULTS: A total of 652 patients were included in the study. VTE incidence was 8.7%. Most of the cases occurred during induction therapy associated with central venous catheter. Univariant analysis showed that family history of thrombosis, presence of mediastinal mass, high-risk treatment group, and inherited thrombophilia were statistically significant risk factors. LMWH administration seemed to decrease VTE rate in patients with inherited thrombophilia and those with T-cell ALL phenotype. CONCLUSION: Most of the VTE cases occurred in patients without inherited thrombophilia, but when it is present, the VTE risk is higher. LMWH administration was useful to decrease VTE in these patients.

Chronic IL-10 overproduction disrupts microglia-neuron dialogue similar to aging, resulting in impaired hippocampal neurogenesis and spatial memory.

The subgranular zone of the dentate gyrus is an adult neurogenic niche where new neurons are continuously generated. A dramatic hippocampal neurogenesis decline occurs with increasing age, contributing to cognitive deficits. The process of neurogenesis is intimately regulated by the microenvironment, with inflammation being considered a strong negative factor for this process. Thus, we hypothesize that the reduction of new neurons in the aged brain could be attributed to the age-related microenvironmental changes towards a pro-inflammatory status. In this work, we evaluated whether an anti-inflammatory microenvironment could counteract the negative effect of age on promoting new hippocampal neurons. Surprisingly, our results show that transgenic animals chronically overexpressing IL-10 by astrocytes present a decreased hippocampal neurogenesis in adulthood. This results from an impairment in the survival of neural newborn cells without differences in cell proliferation. In parallel, hippocampal-dependent spatial learning and memory processes were affected by IL-10 overproduction as assessed by the Morris water maze test. Microglial cells, which are key players in the neurogenesis process, presented a different phenotype in transgenic animals characterized by high activation together with alterations in receptors involved in neuronal communication, such as CD200R and CX3CR1. Interestingly, the changes described in adult transgenic animals were similar to those observed by the effect of normal aging. Thus, our data suggest that chronic IL-10 overproduction mimics the physiological age-related disruption of the microglia-neuron dialogue, resulting in hippocampal neurogenesis decrease and spatial memory impairment.

Specific microglial phagocytic phenotype and decrease of lipid oxidation in white matter areas during aging: Implications of different microenvironments.

Physiological aging is characterized by an imbalance of pro-inflammatory and anti-inflammatory mediators leading to neuroinflammation. Microglial cells, which are highly regulated by the local microenvironment, undergo specific changes depending upon the brain area during aging. The aim of this study was to evaluate the influence of age over microglial cells along different brain areas and microenvironments. For this purpose, transgenic mice with overproduction of either the anti-inflammatory IL-10 cytokine or the pro-inflammatory IL-6 cytokine were used. Our results show that, during aging, microglial cells located in white matter (WM) areas maintain their phagocytic capacity but present a specific phagocytic phenotype with receptors involved in myelin recognition, arguing for aging-derived myelin damage. Whereas IL-10 overproduction anticipates the age-related microglial phagocytic phenotype, maintaining it over time, IL-6 overproduction exacerbates this phenotype in aging. These modifications were linked with a higher efficiency of myelin engulfment by microglia in aged transgenic animals. Moreover, we show, in a novel way, lower lipid oxidation during aging in WM areas, regardless of the genotype. The novelty of the insights presented in this study open a window to deeply investigate myelin lipid oxidation and the role of microglial cells in its regulation during physiological aging.

Evaluation of Myelin Phagocytosis by Microglia/Macrophages in Nervous Tissue Using Flow Cytometry.

Determination of microglial phagocytosis of myelin has acquired importance in the study of demyelinating diseases. One strategy to determine microglial phagocytosis capacity consists of assaying microglia with fluorescently labeled myelin; however, most approaches are performed in cell culture, where microglia usually show important phenotypic differences compared with in vivo conditions. In this article we describe an adapted flow cytometry protocol to assay myelin phagocytosis by microglia obtained directly from in vivo tissue of the central nervous system. Key steps for a first analysis of phagocytic microglia are provided. Additionally, we describe how to fluorescently label myelin using a pH-sensitive tag, pHrodo™ Green STP Ester. © 2021 Wiley Periodicals LLC. Basic Protocol: Assay for determination of myelin phagocytosis by microglia/macrophages using flow cytometry Support Protocol 1: Conjugation of isolated and purified myelin with pHrodo Green STP Ester Support Protocol 2: Quantification of phagocytic cell number by flow cytometry.

The role of early natural killer cell adoptive infusion before engraftment in protecting against human herpesvirus-6B encephalitis after naïve T-cell-depleted allogeneic stem cell transplantation.

BACKGROUND: Naïve T-cell-depleted grafts have been employed as an ex vivo T-cell depletion (TCD) platform to prevent graft-versus-host disease (GvHD) and improve immune reconstitution by providing rapid donor memory T-cell reconstitution after allogenic hematopoietic stem cell transplantation (allo-HSCT). CD45RA- memory T cells confer protection against viruses such as cytomegalovirus, Epstein-Barr virus, and adenovirus; however, reports have shown an unexpectedly high incidence of human herpesvirus (HHV)-6B encephalitis among pediatric allo-HSCT patients. METHODS: We report the first 18 consecutive allo-HSCT, 16 haplo-HSCT, and two human leukocyte antigen-matched related donors implanted with naïve TCD grafts. All donors were administered three cell products: first, a CD34+ stem cell product; second, a CD45RA+ TCD graft, followed by an adoptive natural killer (NK) cell infusion within 10 days after HSCT. The study's primary endpoint was the incidence of HHV-6B encephalitis. RESULTS: Engraftment was achieved in 94.5% of cases; 2-year overall survival, event-free survival, and GvHD/relapse-free survival were 87.2% (95% CI 78.6-95.8), 67.3% (95% CI 53.1-81.5), and 64% (95% CI 50.5-78.1), respectively. HHV-6B reactivation occurred in 7 of the haplo-HSCT patients, six of who received a cell infusion with an NK/CD4 ratio <2. None of the patients developed encephalitis. CONCLUSIONS: In this clinical study, we show that early adoptive NK cell infusion after a 45RA+ TCD allo-HSCT graft is safe and can prevent HHV-6B encephalitis. We recommend infusing adoptive NK cells after allo-HSCT using CD45RA+ TCD grafts.

Phase 2 Clinical Trial of Infusing Haploidentical K562-mb15-41BBL-Activated and Expanded Natural Killer Cells as Consolidation Therapy for Pediatric Acute Myeloblastic Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) accounts for approximately 20% of pediatric leukemia cases; 30% of these patients experience relapse. The antileukemia properties of natural killer (NK) cells and their safety profile have been reported in AML therapy. We proposed a phase 2, open, prospective, multicenter, nonrandomized clinical trial for the adoptive infusion of haploidentical K562-mb15-41BBL-activated and expanded NK (NKAE) cells as a consolidation strategy for children with favorable and intermediate risk AML in first complete remission after chemotherapy (NCT02763475). PATIENTS AND METHODS: Before the NKAE cell infusion, patients underwent a lymphodepleting regimen. After the NKAE cell infusion, patients were administered low doses (1 × 106/IU/m2) of subcutaneous interleukin-2. The primary study endpoint was AML relapse-free survival. We needed to include 35 patients to demonstrate a 50% reduction in relapses. RESULTS: Seven patients (median age, 7.4 years; range, 0.78-15.98 years) were administered 13 infusions of NKAE cells, with a median of 36.44 × 106 cells/kg (range, 6.92 × 106 to 193.2 × 106 cells/kg). We observed chimerism in 4 patients (median chimerism, 0.065%; range, 0.05-0.27%). After a median follow-up of 33 months, the disease of 6 patients (85.7%) remained in complete remission. The 3-year overall survival was 83.3% (95% confidence interval, 68.1-98.5), and the cumulative 3-year relapse rate was 28.6% (95% confidence interval, 11.5-45.7). The study was terminated early because of low patient recruitment. CONCLUSION: This study emphasizes the difficulties in recruiting patients for cell therapy trials, though NKAE cell infusion is safe and feasible. However, we cannot draw any conclusions regarding efficacy because of the small number of included patients and insufficient biological markers.