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.

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.

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.

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.

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.

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.

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.

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.

Chronic exposure to IL-6 induces a desensitized phenotype of the microglia.

BACKGROUND: When the homeostasis of the central nervous system (CNS) is altered, microglial cells become activated displaying a wide range of phenotypes that depend on the specific site, the nature of the activator, and particularly the microenvironment generated by the lesion. Cytokines are important signals involved in the modulation of the molecular microenvironment and hence play a pivotal role in orchestrating microglial activation. Among them, interleukin-6 (IL-6) is a pleiotropic cytokine described in a wide range of pathological conditions as a potent inducer and modulator of microglial activation, but with contradictory results regarding its detrimental or beneficial functions. The objective of the present study was to evaluate the effects of chronic IL-6 production on the immune response associated with CNS-axonal anterograde degeneration. METHODS: The perforant pathway transection (PPT) paradigm was used in transgenic mice with astrocyte-targeted IL6-production (GFAP-IL6Tg). At 2, 3, 7, 14, and 21 days post-lesion, the hippocampal areas were processed for immunohistochemistry, flow cytometry, and protein microarray. RESULTS: An increase in the microglia/macrophage density was observed in GFAP-IL6Tg animals in non-lesion conditions and at later time-points after PPT, associated with higher microglial proliferation and a major monocyte/macrophage cell infiltration. Besides, in homeostasis, GFAP-IL6Tg showed an environment usually linked with an innate immune response, with more perivascular CD11b+/CD45high/MHCII+/CD86+ macrophages, higher T cell infiltration, and higher IL-10, IL-13, IL-17, and IL-6 production. After PPT, WT animals show a change in microglia phenotype expressing MHCII and co-stimulatory molecules, whereas transgenic mice lack this shift. This lack of response in the GFAP-IL6Tg was associated with lower axonal sprouting. CONCLUSIONS: Chronic exposure to IL-6 induces a desensitized phenotype of the microglia.

Astrocyte-targeted IL-10 production decreases proliferation and induces a downregulation of activated microglia/macrophages after PPT.

When central nervous system (CNS) homeostasis is altered, microglial cells become rapidly activated, proliferate and release a broad range of molecules. Among the plethora of molecules involved in the regulation of microglial activation, cytokines are considered crucial. Although production of interleukin-10 (IL-10) has been demonstrated after different types of CNS injuries and associated with protective functions, the specific role played by IL-10 modulating microglial cells remains unclear. Hence, the objective of this study was to evaluate the effects of transgenic astrocyte IL-10 production on microglial activation associated with axonal anterograde degeneration. To address it, the hippocampal area subjected to perforant pathway transection (PPT) was analyzed by immunohistochemistry (IHC), flow cytometry and protein microarray in transgenic (GFAP-IL10Tg) mice and their corresponding wild types (WT) littermates. Our results demonstrated increased microglial/macrophages density in nonlesioned and PPT-lesioned GFAP-IL10Tg animals when compared with nonlesioned and lesioned WT, respectively. This increase was not due to proliferation, as GFAP-IL10Tg mice showed a reduced proliferation of microglial cells, but was related to an increased population of CD11b+/CD45high monocyte/macrophages. Despite this higher number, the microglia/macrophage population in transgenic animals displayed a downregulated phenotype characterized by lower MHCII, ICOSL, and CD11c. Moreover, a sustained T-cell infiltration was found in transgenic animals. We strongly suggest these modifications must be associated with indirect effects derived from the influence of IL-10 on astrocytes and/or neurons, which express IL-10R. We finally suggested that TGF-ß produced by astrocytes, along with IL-2 and CXCL10 might be crucial molecules mediating the effects of transgenic IL-10.

Reduced cuprizone-induced cerebellar demyelination in mice with astrocyte-targeted production of IL-6 is associated with chronically activated, but less responsive microglia.

BACKGROUND: Cerebellar pathology is a frequent feature of multiple sclerosis (MS), a demyelinating and neuroinflammatory disease of the central nervous system (CNS). Interleukin (IL)-6 is a multifunctional cytokine with a potential role in MS. Here we studied cuprizone-induced cerebellar pathology in transgenic mice with astrocyte-targeted production of IL-6 (GFAP-IL6), specifically focusing on demyelination, oligodendrocyte depletion and microglial cell response. RESULTS: Over the course of cuprizone treatment, when compared with WT mice, GFAP-IL6Tg showed a reduced demyelination in the deep lateral cerebellar nuclei (LCN). The oligodendrocyte numbers in the LCN were comparable between WT and GFAP-IL6Tg mice after 4-6weeks of cuprizone treatment, however after the chronic cuprizone treatment (12weeks) we detected higher numbers of oligodendrocytes in GFAP-IL6Tg mice. Contrary to strong cuprizone-induced microglial activation in the LCN of WT mice, GFAP-IL6Tg mice had minimal cuprizone-induced microglial changes, despite an already existing reactive microgliosis in control GFAP-IL6Tg not present in control WT mice. CONCLUSIONS: Our results show that chronic transgenic production of IL-6 reduced cuprizone-induced cerebellar demyelination and induced a specific activation state of the resident microglia population (Iba1+, CD11b+, MHCII+, CD68-), likely rendering them less responsive to subsequent injury signals.

Purine Signaling and Microglial Wrapping.

Microglial cells are highly dynamic cells with processes continuously moving to survey the surrounding territory. Microglia possess a broad variety of surface receptors and subtle changes in their microenvironment cause microglial cell processes to extend, retract, and interact with neuronal synaptic contacts. When the nervous system is disturbed, microglia activate, proliferate, and migrate to sites of injury in response to alert signals. Released nucleotides like ATP and UTP are among the wide range of molecules promoting microglial activation and guiding their migration and phagocytic function. The increased concentration of nucleotides in the extracellular space could be involved in the microglial wrapping found around injured neurons in various pathological conditions, especially after peripheral axotomy. Microglial wrappings isolate injured neurons from synaptic inputs and facilitate the molecular dialog between endangered or injured neurons and activated microglia. Astrocytes may also participate in neuronal ensheathment. Degradation of ATP by microglial ecto-nucleotidases and the expression of various purine receptors might be decisive in regulating the function of enwrapping glial cells and in determining the fate of damaged neurons, which may die or may regenerate their axons and survive.

Relación entre el nivel de sensibilidad periférica, obesidad y los parámetros de la marcha y de equilibrio en adultos mayores con diabetes tipo 2 / The relationship between level of peripheral sensitivity, obesity and gait and balance parameters in older adults with type-2 diabetes

RESUMEN Objetivo: Conocer la relación entre la sensibilidad de los pies, índice de masa corporal (IMC), y los parámetros de la marcha y de equilibrio en adultos mayores entre 65 y 75 años de edad. Material y método: Estudio des criptivo, correlacional y transversal, con una muestra de 119 adultos mayores con diagnóstico de diabetes tipo 2 (DT2). Se aplicaron las siguientes mediciones: el sistema GAITRite® Electronic Walkway para los parámetros de la marcha, la Batería Corta de Desempeño Físico para equilibrio y monofilamento de Semmes-Weinstein, North Coast Medical de 10 gramos para la sensibilidad y mediciones antropométricas para IMC. Se realizó un análisis estadístico con pruebas no paramétricas. Resultados: Se encontraron diferencias significativas en la velocidad, longitud del paso, de la zancada y el equilibrio entre los adultos mayores que tenían sensibilidad disminuida y normal. Conclusión: Una menor sensibilidad de los pies y mayor IMC se relacionan con mayor alteración de los parámetros de la marcha y el equilibrio en adultos mayores de 65 a 75 años de edad con DT2.

ABSTRACT Objective: To determine the relationship between feet sensitivity, body mass index (BMI), and gait and balance parameters in older adults between 65 and 75 years of age. Material and methods: A descriptive, correlational and cross-sectional study with a sample of 119 older adults diagnosed with type 2 diabetes (DT2). The following measurements were applied: the GAITRite® Electronic Walkway system for gait parameters, the Short Physical Performance Battery for balance, the Semmes-Weinstein monofilament examination, the North Coast Medical test using 10 grams for foot sensitivity and anthropometric measurements for BMI. A statistical analysis with non-parametric tests was performed. Results: Significant differences were found in speed, step length, stride length and balance between older adults who had decreased and normal sensitivity. Conclusion: Less foot sensitivity and a higher BMI are associated with a greater impairment reflected by the walking and balance parameters in older adults aged 65 to 75 with DT2.

Astrocyte-targeted production of interleukin-6 reduces astroglial and microglial activation in the cuprizone demyelination model: Implications for myelin clearance and oligodendrocyte maturation.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system. Interleukin (IL)-6 is a pleiotropic cytokine with a potential role in MS. Here we used transgenic mice with astrocyte-targeted production of IL-6 (GFAP-IL6Tg) to study the effect of IL-6 in the cuprizone-induced demyelination paradigm, which is an experimental model of de- and re-myelination, both hallmarks of MS. Our results demonstrated that cuprizone-treated GFAP-IL6Tg mice showed a significant reduction in astroglial and especially microglial activation/accumulation in the corpus callosum in comparison with the corresponding cuprizone-treated wild type (WT). Production of a key microglial attracting chemokine CXCL10, as well as CXCL1 and CCL4 was lower in cuprizone-treated GFAP-IL6Tg mice compared with cuprizone-treated WT. Reduced microglial cell accumulation was associated with inefficient removal of degraded myelin and axonal protection in cuprizone-treated GFAP-IL6Tg mice, compared with WT mice at the peak of demyelination. In addition, transgenic production of IL-6 did not alter initial oligodendrocyte (OL) apoptosis and oligodendrocyte precursor cell recruitment to the lesion site, but it impaired early OL differentiation, possibly due to impaired removal of degraded myelin. Indeed, a microglial receptor involved in myelin phagocytosis, TREM2, as well as the phagolysosomal protein CD68 were lower in cuprizone-treated GFAP-IL6Tg compared with WT mice. Our results show for the first time that astrocyte-targeted production of IL-6 may play a role in modulating experimental demyelination induced by cuprizone. Further understanding of the IL-6-mediated molecular mechanisms involved in the regulation of demyelination is needed, and may have implications for the development of future therapeutic strategies for the treatment of MS. GLIA 2016;64:2104-2119.

Alterations in microglial phenotype and hippocampal neuronal function in transgenic mice with astrocyte-targeted production of interleukin-10.

Interleukin-10 (IL-10) is a cytokine classically linked with anti-inflammatory and protective functions in the central nervous system (CNS) in different neurodegenerative and neuroinflammatory conditions. In order to study the specific role of local CNS produced IL-10, we have created a new transgenic mouse line with astrocyte-targeted production of IL-10 (GFAP-IL10Tg). In the present study, the effects of local CNS IL-10 production on microglia, astrocytes and neuronal connectivity under basal conditions were investigated using immunohistochemistry, molecular biology techniques, electrophysiology and behavioural studies. Our results showed that, in GFAP-IL10Tg animals, microglia displayed an increase in density and a specific activated phenotype characterised by morphological changes in specific areas of the brain including the hippocampus, cortex and cerebellum that correlated with the level of transgene expressed IL-10 mRNA. Distinctively, in the hippocampus, microglial cells adopted an elongated morphology following the same direction as the dendrites of pyramidal neurons. Moreover, this IL-10-induced microglial phenotype showed increased expression of certain molecules including Iba1, CD11b, CD16/32 and F4/80 markers, "de novo" expression of CD150 and no detectable levels of either CD206 or MHCII. To evaluate whether this specific activated microglial phenotype was associated with changes in neuronal activity, the electrophysiological properties of pyramidal neurons of the hippocampus (CA3-CA1) were analysed in vivo. We found a lower excitability of the CA3-CA1 synapses and absence of long-term potentiation (LTP) in GFAP-IL10Tg mice. This study is the first description of a transgenic mouse with astrocyte-targeted production of the cytokine IL-10. The findings indicate that IL-10 induces a specific activated microglial phenotype concomitant with changes in hippocampal LTP responses. This transgenic animal will be a very useful tool to study IL-10 functions in the CNS, not only under basal conditions, but also after different experimental lesions or induced diseases.

Effects of astrocyte-targeted production of interleukin-6 in the mouse on the host response to nerve injury.

Interleukin-6 (IL-6) is a pleiotropic cytokine with a key role in the control of inflammatory/immune responses. In the central nervous system (CNS), an increase in IL-6 occurs in a wide range of pathological conditions such as excitotoxicity and traumatic brain injury. We evaluated the effects of astrocyte-targeted production of IL-6 in the CNS in the sterile-nerve injury model of facial nerve axotomy. To accomplish this, facial nerve transection was performed in transgenic mice (glial fibrillary acidic protein [GFAP]-IL6Tg) with IL-6 production under the GFAP promoter. Neuronal death, glial activation, lymphocyte recruitment, and integrin expression were evaluated by immunohistochemistry and flow cytometry from 3 to 28 days postinjury. Our findings revealed an increase in motor neuron cell death in GFAP-IL6Tg mice correlating with changes in the microglial activation pattern, characterized principally by less attachment to neurons and reduced expression of both CD11b and CD18. We also found a higher CD4(+) T-lymphocyte recruitment in GFAP-IL6Tg mice. In addition, changes in the expression pattern of different integrins and their receptors were observed in transgenic animals. Specifically, alterations in osteopontin expression in motor neurons and its receptors CD44 and CD49e in lymphocytes and microglia, respectively, which may account for the variations related to glial reactivity and lymphocyte infiltration. In conclusion, our results indicated that forced local production of IL-6 has a direct impact on the outcome of nerve injury in the CNS inducing an increase in neurodegeneration, changes in glial response, and lymphocyte recruitment as well as in the expression of different integrins and their receptors.

Overexpression of the nuclear factor kappaB inhibitor A20 is neurotoxic after an excitotoxic injury to the immature rat brain.

BACKGROUND: The zinc finger protein A20 is an ubiquitinating/deubiquitinating enzyme essential for the termination of inflammatory reactions through the inhibition of nuclear factor kappaB (NF-kappaB) signaling. Moreover, it also shows anti-apoptotic activities in some cell types and proapoptotic/pronecrotic effects in others. Although it is known that the regulation of inflammatory and cell death processes are critical in proper brain functioning and that A20 mRNA is expressed in the CNS, its role in the brain under physiological and pathological conditions is still unknown. METHODS: In the present study, we have evaluated the effects of A20 overexpression in mixed cortical cultures in basal conditions: the in vivo pattern of endogenous A20 expression in the control and N-methyl-d-aspartate (NMDA) excitotoxically damaged postnatal day 9 immature rat brain, and the post-injury effects of A20 overexpression in the same lesion model. RESULTS: Our results show that overexpression of A20 in mixed cortical cultures induced significant neuronal death by decreasing neuronal cell counts by 45 ± 9%. in vivo analysis of endogenous A20 expression showed widespread expression in gray matter, mainly in neuronal cells. However, after NMDA-induced excitotoxicity, neuronal A20 was downregulated in the neurodegenerating cortex and striatum at 10-24 hours post-lesion, and it was re-expressed at longer survival times in reactive astrocytes located mainly in the lesion border. When A20 was overexpressed in vivo 2 hours after the excitotoxic damage, the lesion volume at 3 days post-lesion showed a significant increase (20.8 ± 7.0%). No A20-induced changes were observed in the astroglial response to injury. CONCLUSIONS: A20 is found in neuronal cells in normal conditions and is also expressed in astrocytes after brain damage, and its overexpression is neurotoxic for cortical neurons in basal mixed neuron-glia culture conditions and exacerbates postnatal brain excitotoxic damage.