Premature aging and immune senescence in HIV-infected children.

Objective: Several pieces of evidence indicate that HIV-infected adults undergo premature aging. The effect of HIV and antiretroviral therapy (ART) exposure on the aging process of HIV-infected children may be more deleterious since their immune system coevolves from birth with HIV. Design: Seventy-one HIV-infected (HIV+), 65 HIV-exposed-uninfected (HEU), and 56 HIV-unexposed-uninfected (HUU) children, all aged 0-5 years, were studied for biological aging and immune senescence. Methods: Telomere length and T-cell receptor rearrangement excision circle levels were quantified in peripheral blood cells by real-time PCR. CD4+ and CD8+ cells were analysed for differentiation, senescence, and activation/exhaustion markers by flow cytometry. Results: Telomere lengths were significantly shorter in HIV+ than in HEU and HUU children (overall, P < 0.001 adjusted for age); HIV+ ART-naive (42%) children had shorter telomere length compared with children on ART (P = 0.003 adjusted for age). T-cell receptor rearrangement excision circle levels and CD8+ recent thymic emigrant cells (CD45RA+CD31+) were significantly lower in the HIV+ than in control groups (overall, P = 0.025 and P = 0.005, respectively). Percentages of senescent (CD28−CD57+), activated (CD38+HLA-DR+), and exhausted (PD1+) CD8+ cells were significantly higher in HIV+ than in HEU and HUU children (P = 0.004, P < 0.001, and P < 0.001, respectively). Within the CD4+ cell subset, the percentage of senescent cells did not differ between HIV+ and controls, but programmed cell death receptor-1 expression was upregulated in the former. Conclusions: HIV-infected children exhibit premature biological aging with accelerated immune senescence, which particularly affects the CD8+ cell subset. HIV infection per se seems to influence the aging process, rather than exposure to ART for prophylaxis or treatment. Keywords: immune activation, immune senescence, microbial translocation, pediatric HIV/AIDS, premature aging, telomere length, T-cell receptor rearrangement excision circl

Chromosomal instability in aneuploid acute lymphoblastic leukemia associates with disease progression

Chromosomal instability (CIN) lies at the core of cancer development leading to aneuploidy, chromosomal copy-number heterogeneity (chr-CNH) and ultimately, unfavorable clinical outcomes. Despite its ubiquity in cancer, the presence of CIN in childhood B-cell acute lymphoblastic leukemia (cB-ALL), the most frequent pediatric cancer showing high frequencies of aneuploidy, remains unknown. Here, we elucidate the presence of CIN in aneuploid cB-ALL subtypes using single-cell whole-genome sequencing of primary cB-ALL samples and by generating and functionally characterizing patient-derived xenograft models (cB-ALL-PDX). We report higher rates of CIN across aneuploid than in euploid cB-ALL that strongly correlate with intraclonal chr-CNH and overall survival in mice. This association was further supported by in silico mathematical modeling. Moreover, mass-spectrometry analyses of cB-ALL-PDX revealed a "CIN signature" enriched in mitotic-spindle regulatory pathways, which was confirmed by RNA-sequencing of a large cohort of cB-ALL samples. The link between the presence of CIN in aneuploid cB-ALL and disease progression opens new possibilities for patient stratification and offers a promising new avenue as a therapeutic target in cB-ALL treatment.</p

The relapsed acute lymphoblastic leukemia network (ReALLNet): a multidisciplinary project from the spanish society of pediatric hematology and oncology (SEHOP)

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with survival rates exceeding 85%. However, 15% of patients will relapse; consequently, their survival rates decrease to below 50%. Therefore, several research and innovation studies are focusing on pediatric relapsed or refractory ALL (R/R ALL). Driven by this context and following the European strategic plan to implement precision medicine equitably, the Relapsed ALL Network (ReALLNet) was launched under the umbrella of SEHOP in 2021, aiming to connect bedside patient care with expert groups in R/R ALL in an interdisciplinary and multicentric network. To achieve this objective, a board consisting of experts in diagnosis, management, preclinical research, and clinical trials has been established. The requirements of treatment centers have been evaluated, and the available oncogenomic and functional study resources have been assessed and organized. A shipping platform has been developed to process samples requiring study derivation, and an integrated diagnostic committee has been established to report results. These biological data, as well as patient outcomes, are collected in a national registry. Additionally, samples from all patients are stored in a biobank. This comprehensive repository of data and samples is expected to foster an environment where preclinical researchers and data scientists can seek to meet the complex needs of this challenging population. This proof of concept aims to demonstrate that a network-based organization, such as that embodied by ReALLNet, provides the ideal niche for the equitable and efficient implementation of “what's next” in the management of children with R/R ALL

Clonal heterogeneity and rates of specific chromosome gains are risk predictors in childhood high-hyperdiploid B-cell acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is the commonest childhood cancer. High hyperdiploidy (HHD) identifies the most frequent cytogenetic subgroup in childhood B-ALL. Although hyperdiploidy represents an important prognostic factor in childhood B-ALL, the specific chromosome gains with prognostic value in HHD-B-ALL remain controversial, and the current knowledge about the hierarchy of chromosome gains, clonal heterogeneity and chromosomal instability in HHD-B-ALL remains very limited. We applied automated sequential-iFISH coupled with single-cell computational modeling to identify the specific chromosomal gains of the eight typically gained chromosomes in a large cohort of 72 primary diagnostic (DX, n = 62) and matched relapse (REL, n = 10) samples from HHD-B-ALL patients with either favorable or unfavorable clinical outcome in order to characterize the clonal heterogeneity, specific chromosome gains and clonal evolution. Our data show a high degree of clonal heterogeneity and a hierarchical order of chromosome gains in DX samples of HHD-B-ALL. The rates of specific chromosome gains and clonal heterogeneity found in DX samples differ between HHD-B-ALL patients with favorable or unfavorable clinical outcome. In fact, our comprehensive analyses at DX using a computationally defined risk predictor revealed low levels of trisomies +18+10 and low levels of clonal heterogeneity as robust relapse risk factors in minimal residual disease (MRD)-negative childhood HHD-B-ALL patients: relapse-free survival beyond 5 years: 22.1% versus 87.9%, P < 0.0001 and 33.3% versus 80%, P < 0.0001, respectively. Moreover, longitudinal analysis of matched DX-REL HHD-B-ALL samples revealed distinct patterns of clonal evolution at relapse. Our study offers a reliable prognostic sub-stratification of pediatric MRD-negative HHD-B-ALL patients

Caracterización biológica de la leucemia mieloide aguda con translocación t(8;16)(p11;p13) y reordenamiento MYST3-CREBBP

[spa] INTRODUCCIÓN. La leucemia mieloide aguda (LMA) es una enfermedad heterogénea desde el punto de vista clínico y biológico. En los últimos años se vienen reconociendo diversas alteraciones moleculares que definen entidades específicas. En este contexto, la LMA con translocación t(8;16)(p11;p13) y reordenamiento MYST3 (MOZ)/CREBBP (CBP) es una variedad infrecuente mal caracterizada desde el punto de vista biológico. HIPÓTESIS Y OBJETIVOS. La proteína quimérica MYST3-CREBBP, resultante de la translocación t(8;16)(p11;p13), podría conferir a este subtipo de LMA una individualidad biológica propia, con rasgos diferenciados respecto al resto de leucemias. Para confirmar esta hipótesis general los objetivos de la presente tesis doctoral fueron: 1) diseñar una técnica de PCR para el diagnóstico rápido y específico del reordenamiento MYST3-CREBBP; 2) caracterizar el punto de ruptura de los genes implicados en la translocación en una serie de pacientes y 3) estudiar el perfil de expresión génica de las LMA con reordenamiento MYST3-CREBBP y compararlo con el de otros subtipos bien definidos de LMA.PACIENTES Y MÉTODOS. Se estudió una serie de pacientes con LMA y reordenamiento MYST3-CREBBP (n=7) y se compararon sus características biológicas con otros casos de LMA. Para ello se diseñó una técnica de PCR nueva para la detección del reordenamiento MYST3-CREBBP, mientras que los puntos de ruptura de los genes implicados en la translocación se estudiaron mediante secuenciación directa. El estudio del perfil de expresión génica de la LMA con reordenamiento MYST3-CREBBP se abordó utilizando microarrays de oligonucleótidos (Affymetrix HU133A). La diferencia entre la expresión génica entre diferentes subtipos de leucemia se analizó con diversas técnicas estadísticas (ANOVA, t-test), utilizando diferentes programas informáticos. Los resultados de este análisis se validaron en una serie independiente de pacientes estudiados mediante RT-PCR cuantitativa utilizando arrays de baja densidad. RESULTADOS. Los pacientes afectos de LMA con reordenamiento MYST3-CREBBP presentaron un inmunofenotipo característico (CD34-, HLA-DR-, CD117-, CD56+, expresión de marcadores mielomonocíticos). Por otro lado, el análisis molecular reveló que el tránscrito tipo I del gen quimérico MYST3-CREBBP es el más común en estos pacientes. Por otra parte, el análisis sobre el perfil de expresión génica mostró una firma característica para las LMA con reordenamiento MYST3-CREBBP, consistente en la sobreexpresión de determinados genes HOX (HOXA9, HOXA10), de los oncogenes RET y PRL y la infraexpresión de genes como CCND2, STAT5 y WT1. Por otro lado, se observó una similitud en la expresión de algunos genes entre las leucemias MYST3-CREBBP y las LMA con reordenamiento de MLL, lo que sugiere un mecanismo de leucemogénesis parcialmente compartido por los dos tipos de leucemia.CONCLUSIONES. La técnica de RT-PCR implementada es útil para la detección rápida del reordenamiento MYST3-CREBBP. El denominado tránscrito tipo I del gen quimérico MYST3-CREBBP es el más común en la LMA con t(8;16). La LMA con reordenamiento MYST3-CREBBP posee un perfil de expresión característico, con sobreexpresión de diversos oncogenes como RET y PRL y la presencia de un patrón específico de expresión de los genes homeobox

Glial GLT-1 blockade in infralimbic cortex as a new strategy to evoke rapid antidepressant-like effects in rats

Ketamine and deep brain stimulation produce rapid antidepressant effects in humans and rodents. An increased AMPA receptor (AMPA-R) signaling in medial prefrontal cortex (mPFC) has been suggested to mediate these responses. However, little research has addressed the direct effects of enhancing glutamate tone or AMPA-R stimulation in mPFC subdivisions. The current study investigates the behavioral and neurochemical consequences of glutamate transporter-1 (GLT-1) blockade or s-AMPA microinfusion in the infralimbic (IL) and prelimbic (PrL) cortex. Owing to the connectivity between the mPFC and raphe nuclei, the role of serotonin is also explored. The bilateral microinfusion of the depolarizing agent veratridine into IL -but not PrL- of rats evoked immediate antidepressant-like responses. The same regional selectivity was observed after microinfusion of dihydrokainic acid (DHK), a selective inhibitor of GLT-1, present in astrocytes. The DHK-evoked antidepressant-like responses appear to be mediated by an AMPA-R-driven enhancement of serotonergic activity, as (i) they were prevented by NBQX 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt) and mimicked by s-AMPA; (ii) DHK and s-AMPA elevated similarly extracellular glutamate in IL and PrL, although extracellular 5-HT and c-fos expression in the midbrain dorsal raphe increased only when these agents were applied in IL; and (iii) DHK antidepressant-like responses were prevented by 5-HT synthesis inhibition and mimicked by citalopram microinfusion in IL. These results indicate that an acute increase of glutamatergic neurotransmission selectively in IL triggers immediate antidepressant-like responses in rats, likely mediated by the activation of IL–raphe pathways, which then results in a fast increase of serotonergic activity.This work was supported by the Spanish Ministry of Economy and Competitiveness (grant numbers SAF2012-35183; SAF2015-68346; and BES2013-063241 to JG-C), co-financed by European Regional Development Fund (ERDF); Generalitat de Catalunya (grant number 2014-SGR798 and 2016FI-B00285 to MT-G) and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM).Peer reviewe

Astrocyte control of glutamatergic activity: Downstream effects on serotonergic function and emotional behavior

Major depressive disorder (MDD) is a leading cause of disability worldwide, with a poorly known pathophysiology and sub-optimal treatment, based on serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors. We review existing theories on MDD, paying special attention to the role played by the ventral anterior cingulate cortex (vACC) or its rodent equivalent, infralimbic cortex (IL), which tightly control the activity of brainstem monoamine neurons (including raphe 5-HT neurons) via descending afferents. Further, astrocytes regulate excitatory synapse activity via glutamate reuptake through astrocytic transporters EAAT1 and EAAT2 (GLAST and GLT-1 in rodents), and alterations of astrocyte number/function have been reported in MDD patients and suicide victims. We recently assessed the impact of reducing GLAST/GLT-1 function in IL on emotional behavior and serotonergic function in rodents. The acute pharmacological blockade of GLT-1 with dihydrokainate (DHK) in rat IL evoked an antidepressant-like effect mediated by local AMPA-R activation and a subsequent enhancement of serotonergic function. No effects were produced by DHK microinfusion in prelimbic cortex (PrL). In the second model, a moderate small interfering RNAs (siRNA)-induced reduction of GLAST and GLT-1 expression in mouse IL markedly increased local glutamatergic neurotransmission and evoked a depressive-like phenotype (reversed by citalopram and ketamine), and reduced serotonergic function and BDNF expression in cortical/hippocampal areas. As for DHK, siRNA microinfusion in PrL did not evoke behavioral/neurochemical effects. Overall, both studies support a critical role of the astrocyte-neuron communication in the control of excitatory neurotransmission in IL, and subsequently, on emotional behavior, via the downstream associated changes on serotonergic function.This study was supported by the Spanish Ministry of Economy and Competitiveness, SAF2015-68346-P MINECO/FEDER, UE (F.A.) and SAF2016-75797-R AEI/FEDER, UE (A.B.) co-financed by the European Regional Development Fund “A way to build Europe”. Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) and CERCA Programme/Generalitat de Catalunya is also acknowledged. The authors would also like to thank the support of the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (2017SGR717). N.F. was a recipient of a fellowship from Spanish Ministry of Education, Culture, and Sport. J.G-C was a recipient of a fellowship from the Spanish Ministry of Economy and Competitiveness (BES-2013-063241). M.T-G was a recipient of a fellowship from Generalitat de Catalunya (2016FI_B 00285) co-financed by the European Social Fund.Peer reviewe

Competencies in Basic Life Support after a Course with or without Rescue Ventilation: Historical Cohort Study

Background: Simplifying the international guidelines to improve skills after training and their retention over time has been one of the top priorities in recent years. The objective of our study was to compare the results of the practical skills learned during training in basic life support with and without pulmonary ventilation. Methods: This was a comparative study of historical cohorts consisting of undergraduate students in health sciences. In one cohort, rescue breathing was performed, and in the other, it was not. The same data collection instruments were used for both cohorts: a test type examination of knowledge, data from a smart mannequin and an instructor observation grid. The means of knowledge and practical skills scores collected by the mannequin were compared using independent sample t-tests. Results: 497 students were recruited without significant differences between the two cohorts. The mean scores for knowledge and skills determined by the instructor and the mannequin were statistically higher in the cohort that did not perform rescue breathing. Conclusion: Students who participated in basic life support training that did not include rescue breathing scored better than those who participated in training that included this skill. Training with only compressions simplifies the guidelines and increases learning and content retention

Serotonergic mechanisms involved in antidepressant-like responses evoked by GLT-1 blockade in rat infralimbic cortex

Novel fast-acting antidepressant strategies, such as ketamine and deep brain stimulation, enhance glutamatergic neurotransmission in medial prefrontal cortex (mPFC) regions via AMPA receptor (AMPA-R) activation. We recently reported that the regionally-selective blockade of the glial glutamate transporter-1 (GLT-1) by dihydrokainic acid (DHK) microinfusion in rat infralimbic cortex (IL), the most ventral part of the mPFC, evoked immediate (10 min) antidepressant-like responses, which involved AMPA-R activation and were associated to increased serotonin (5-hydroxytryptamine, 5-HT) release. Given the reciprocal connectivity between the mPFC and the serotonergic dorsal raphe nucleus (DR), here we examined the serotoninergic mechanisms involved in the reported antidepressant-like responses of DHK microinfusion. First, we show that antidepressant-like responses evoked by IL application of DHK and citalopram are mediated by local 5-HT1A receptors (5-HT1A-R), since they are cancelled by previous IL WAY100635 microinfusion. Second, IL DHK microinfusion increases excitatory inputs onto DR, as shown by an increased glutamate and 5-HT release in DR and by a selective increase of c-Fos expression in DR 5-HT neurons, not occurring in putative GABAergic neurons. This view is also supported by an increased 5-HT release in ventral hippocampus following IL DHK microinfusion. Interestingly, antidepressant-like responses evoked by IL DHK lasted for 2¿h and could be prolonged for up to 24¿h by attenuating self-inhibitory effects via 5-HT1A autoreceptors. In contrast, the antidepressant-like effects of S-AMPA microinfusion in IL were short-lasting. Together, our results further support a prominent role of the IL-DR pathway and of ascending 5-HT pathways in mediating antidepressant-like responses evoked by glutamatergic mechanisms.This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2015-68346 and BES2013-063241 to J.G-C; BFU2015-68568-P to A.O.) co-financed by European Regional Development Fund; Generalitat de Catalunya (CERCA Programme; 2014-SGR798, 2016FI-B00285 to M.T-G, 2016 FI-B00531 to S.M-T and ICREA Acadèmia to A.O.); Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM)

Bone marrow MSC from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity

BACKGROUND: Although adoptive transfer of CD19-directed chimeric antigen receptor (CAR) T-cells (CD19-CAR T-cells) achieves high rates of complete response in patients with B-cell acute lymphoblastic leukemia (B-ALL), relapse is common. Bone marrow (BM) mesenchymal stem/stromal cells (BM-MSC) are key components of the hematopoietic niche and are implicated in B-ALL pathogenesis and therapy resistance. MSC exert an immunosuppressive effect on T-cells; however, their impact on CD19-CAR T-cell activity is understudied. METHODS: We performed a detailed characterization of BM-MSC from pediatric patients with B-ALL (B-ALL BM-MSC), evaluated their immunomodulatory properties and their impact on CD19-CAR T-cell activity in vitro using microscopy, qRT-PCR, ELISA, flow cytometry analysis and in vivo using a preclinical model of severe colitis and a B-ALL xenograft model. RESULTS: While B-ALL BM-MSC were less proliferative than those from age-matched healthy donors (HD), the morphology, immunophenotype, differentiation potential and chemoprotection was very similar. Likewise, both BM-MSC populations were equally immunosuppressive in vitro and anti-inflammatory in an in vivo model of severe colitis. Interestingly, BM-MSC failed to impair CD19-CAR T-cell cytotoxicity or cytokine production in vitro using B-ALL cell lines and primary B-ALL cells. Finally, the growth of NALM6 cells was controlled in vivo by CD19-CAR T-cells irrespective of the absence/presence of BM-MSC. CONCLUSIONS: Collectively, our data demonstrate that pediatric B-ALL and HD BM-MSC equally immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity.Financial support for this work was obtained from the European Research Council (CoG-2014-646903, PoC-2018-811220), the Spanish Ministry of Economy and Competitiveness (MINECO, SAF2016-80481R), the Fundación Uno entre Cienmil, the Obra Social La Caixa (LCF/PR/HR19/52160011), the Leo Messi Foundation, and the 'Heroes hasta la médula' initiative to PM. SRZ was supported by a Marie Sklodowska Curie Fellowship (GA 795833). MV is supported by a Juan de la Cierva fellowship from the MINECO. PM is an investigator of the Spanish Cell Therapy cooperative network (TERCEL)