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Objective Previous reports have indicated that the short term prognosis for patients with malignant diseases and serious adverse events requiring mechanical ventilation (SAEV) is improving. The purpose of this study was to determine whether these patients can be cured of malignant disease or whether they survive SAEV only to subsequently relapse. Patients and methods The authors report the outcome of children with SAEV treated in the multicentre studies ALL-BFM 95 and AML-BFM 98. Data from 1182 patients with acute lymphoblastic leukaemia (ALL) and 334 patients with acute myeloid leukaemia (AML) were analysed. 88 patients (51 ALL and 37 AML) developed SAEV. Results The prognosis was almost identical in ALL and AML patients (survival of SAEV patients: 48%, 95% CI 38% to 58%; overall survival after 5 years: 31%, 95% CI 21% to 41%). Prognosis was independent of the time between leukaemia diagnosis and SAEV. Approximately 20% of children who required haemodialysis (n=14) or cardiac resuscitation (n=16) achieved long term survival, but no patient who fulfilled more than three of six identified risk factors (age >=10 years, high risk leukaemia, C reactive protein >=150 mg/l, administration of inotropic infusion, cardiac resuscitation and haemodialysis) survived (n=16; 0%, 95% CI 0% to 20%). Conclusions Intensive care improves the short and long term survival of children with leukaemia. 64% (95% CI 50% to 78%) of children with acute leukaemia who survived SAEV achieved long term survival. Prognosis mainly depends on age and leukaemia risk group. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play dual roles in acute lymphoblastic leukemia (ALL) as both tumor suppressors and oncogenes, and miRNA expression profiles can be used for patient risk stratification. Precise assessment of miRNA levels is crucial for understanding their role and function in gene regulation. Quantitative real-time polymerase chain reaction (qPCR) is a reliable, rapid, and cost-effective method for analyzing miRNA expression, assuming that appropriate normalization to stable references is performed to ensure valid data. In this study, we evaluated the stability of six commonly used miRNA references (5sRNA, SNORD44, RNU6, RNU1A1, miR-103a-3p, and miR-532-5p) across nine B-cell precursor (BCP) ALL cell lines, 22 patient-derived xenograft (PDX) BCP ALL samples from different organ compartments of leukemia bearing mice, and peripheral blood mononuclear cells (PBMCs) from six healthy donors. We used four different algorithms (Normfinder, ∆CT, geNorm, and BestKeeper) to assess the most stably expressed reference across all samples. Moreover, we validated our data in an additional set of 13 PDX ALL samples and six healthy controls, identifying miR-103a-3p and miR-532-5p as the most stable references for miRNA normalization in BCP ALL studies. Additionally, we demonstrated the critical importance of using a stable reference to accurately interpret miRNA data., (© 2024. The Author(s).)

Purpose: Genetic hypomorphic defects in X chromosomal IKBKG coding for the NF-κB essential modulator (NEMO) lead to ectodermal dysplasia and immunodeficiency in males and the skin disorder incontinentia pigmenti (IP) in females, respectively. NF-κB essential modulator (NEMO) Δ-exon 5-autoinflammatory syndrome (NEMO-NDAS) is a systemic autoinflammatory disease caused by alternative splicing and increased proportion of NEMO-Δex5. We investigated a female carrier presenting with IP and NEMO-NDAS due to non-skewed X-inactivation., Methods: IKBKG transcripts were quantified in peripheral blood mononuclear cells isolated from the patient, her mother, and healthy controls using RT-PCR and nanopore sequencing. Corresponding proteins were analyzed by western blotting and flow cytometry. Besides toll-like receptor (TLR) and tumor necrosis factor (TNF) signaling, the interferon signature, cytokine production and X-inactivation status were investigated., Results: IP and autoinflammation with recurrent fever, oral ulcers, hepatitis, and neutropenia, but no immunodeficiency was observed in a female patient. Besides moderately reduced NEMO signaling function, type I interferonopathy, and elevated IL-18 and CXCL10 were found. She and her mother both carried the heterozygous variant c.613 C > T p.(Gln205*) in exon 5 of IKBKG previously reported in NEMO-deficient patients. However, X-inactivation was skewed in the mother, but not in the patient. Alternative splicing led to increased ratios of NEMO-Dex5 over full-length protein in peripheral blood cell subsets causing autoinflammation. Clinical symptoms partially resolved under treatment with TNF inhibitors., Conclusion: Non-skewed X-inactivation can lead to NEMO-NDAS in females with IP carrying hypomorphic IKBKG variants due to alternative splicing and increased proportions of NEMO-∆ex5., (© 2024. The Author(s).)

Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Accordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using Oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL., (© 2024. The Author(s).)

Background: Neuroblastoma is the most common extracranial solid tumour in children. Relapsed or refractory neuroblastoma is associated with a poor outcome. We assessed the combination of irinotecan-temozolomide and dasatinib-rapamycin (RIST) in patients with relapsed or refractory neuroblastoma., Methods: The multicentre, open-label, randomised, controlled, phase 2, RIST-rNB-2011 trial recruited from 40 paediatric oncology centres in Germany and Austria. Patients aged 1-25 years with high-risk relapsed (defined as recurrence of all stage IV and MYCN amplification stages, after response to treatment) or refractory (progressive disease during primary treatment) neuroblastoma, with Lansky and Karnofsky performance status at least 50%, were assigned (1:1) to RIST (RIST group) or irinotecan-temozolomide (control group) by block randomisation, stratified by MYCN status. We compared RIST (oral rapamycin [loading 3 mg/m 2 on day 1, maintenance 1 mg/m 2 on days 2-4] and oral dasatinib [2 mg/kg per day] for 4 days with 3 days off, followed by intravenous irinotecan [50 mg/m 2 per day] and oral temozolomide [150 mg/m 2 per day] for 5 days with 2 days off; one course each of rapamycin-dasatinib and irinotecan-temozolomide for four cycles over 8 weeks, then two courses of rapamycin-dasatinib followed by one course of irinotecan-temozolomide for 12 weeks) with irinotecan-temozolomide alone (with identical dosing as experimental group). The primary endpoint of progression-free survival was analysed in all eligible patients who received at least one course of therapy. The safety population consisted of all patients who received at least one course of therapy and had at least one post-baseline safety assessment. This trial is registered at ClinicalTrials.gov, NCT01467986, and is closed to accrual., Findings: Between Aug 26, 2013, and Sept 21, 2020, 129 patients were randomly assigned to the RIST group (n=63) or control group (n=66). Median age was 5·4 years (IQR 3·7-8·1). 124 patients (78 [63%] male and 46 [37%] female) were included in the efficacy analysis. At a median follow-up of 72 months (IQR 31-88), the median progression-free survival was 11 months (95% CI 7-17) in the RIST group and 5 months (2-8) in the control group (hazard ratio 0·62, one-sided 90% CI 0·81; p=0·019). Median progression-free survival in patients with amplified MYCN (n=48) was 6 months (95% CI 4-24) in the RIST group versus 2 months (2-5) in the control group (HR 0·45 [95% CI 0·24-0·84], p=0·012); median progression-free survival in patients without amplified MYCN (n=76) was 14 months (95% CI 9-7) in the RIST group versus 8 months (4-15) in the control group (HR 0·84 [95% CI 0·51-1·38], p=0·49). The most common grade 3 or worse adverse events were neutropenia (54 [81%] of 67 patients given RIST vs 49 [82%] of 60 patients given control), thrombocytopenia (45 [67%] vs 41 [68%]), and anaemia (39 [58%] vs 38 [63%]). Nine serious treatment-related adverse events were reported (five patients given control and four patients given RIST). There were no treatment-related deaths in the control group and one in the RIST group (multiorgan failure)., Interpretation: RIST-rNB-2011 demonstrated that targeting of MYCN-amplified relapsed or refractory neuroblastoma with a pathway-directed metronomic combination of a multkinase inhibitor and an mTOR inhibitor can improve progression-free survival and overall survival. This exclusive efficacy in MYCN-amplified, relapsed neuroblastoma warrants further investigation in the first-line setting., Funding: Deutsche Krebshilfe., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.)

Despite advances in the treatment and care of severe physical injuries, trauma remains one of the main reasons for disability-adjusted life years worldwide. Trauma patients often suffer from disturbances in energy utilization and metabolic dysfunction, including hyperglycemia and increased insulin resistance. White adipose tissue plays an essential role in the regulation of energy homeostasis and is frequently implicated in traumatic injury due to its ubiquitous body distribution but remains poorly studied. Initial triggers of the trauma response are mainly damage-associated molecular patterns (DAMPs) such as histones. We hypothesized that DAMP-induced adipose tissue inflammation contributes to metabolic dysfunction in trauma patients. Therefore, we investigated whether histone release during traumatic injury affects adipose tissue. Making use of a murine polytrauma model with hemorrhagic shock, we found increased serum levels of histones accompanied by an inflammatory response in white adipose tissue. In vitro, extracellular histones induced an inflammatory response in human adipocytes. On the molecular level, this inflammatory response was mediated via a MYD88-IRAK1-ERK signaling axis as demonstrated by pharmacological and genetic inhibition. Histones also induced lytic cell death executed independently of caspases and RIPK1 activity. Importantly, we detected increased histone levels in the bloodstream of patients after polytrauma. Such patients might benefit from a therapy consisting of activated protein C and the FDA-approved ERK inhibitor trametinib, as this combination effectively prevented histone-mediated effects on both, inflammatory gene activation and cell death in adipocytes. Preventing adipose tissue inflammation and adipocyte death in patients with polytrauma could help minimize posttraumatic metabolic dysfunction., (© 2024. The Author(s).)

In recent years, the discovery of functional and communicative cellular tumour networks has led to a new understanding of malignant primary brain tumours. In this review, the authors shed light on the diverse nature of cell-to-cell connections in brain tumours and propose an innovative treatment approach to address the detrimental connectivity of these networks. The proposed therapeutic outlook revolves around three main strategies: (a) supramarginal resection removing a substantial portion of the communicating tumour cell front far beyond the gadolinium-enhancing tumour mass, (b) morphological isolation at the single cell level disrupting structural cell-to-cell contacts facilitated by elongated cellular membrane protrusions known as tumour microtubes (TMs), and (c) functional isolation at the single cell level blocking TM-mediated intercellular cytosolic exchange and inhibiting neuronal excitatory input into the malignant network. We draw an analogy between the proposed therapeutic outlook and the Alcatraz Federal Penitentiary, where inmates faced an impassable sea barrier and experienced both spatial and functional isolation within individual cells. Based on current translational efforts and ongoing clinical trials, we propose the Alcatraz-Strategy as a promising framework to tackle the harmful effects of cellular brain tumour networks., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Glioblastoma, the most common and lethal primary adult brain tumor, cannot be successfully removed surgically due to its highly invasive nature. Therapeutically, approaches must be aimed at a systemic brain disease and not merely at a tumor located within the brain, unless a successful containment strategy can be found. Reelin, an extracellular matrix glycoprotein, plays an important role in neuronal migration and serves here as a natural stop signal. Interestingly, the expression of reelin is negatively associated with tumor grade and, within glioblastoma, correlates with increased overall survival. To further elucidate a potential biological reason for these findings, we looked at the cellular behavior of glioblastoma cell lines grown on a pure fibronectin matrix or a matrix with reelin inserts. While reelin had no significant effects on cellular metabolism, proliferation, or resistance to chemotherapeutic agents, it did significantly affect the cells' interaction with fibronectin. Both matrix attachment and detachment were modulated by reelin, and thus, the invasion and motility of cells interacting with a reelin-containing matrix were altered. The data presented in this work strongly suggest that reelin might be a potential modulator of underlying molecular mechanisms that contribute to glioblastoma invasion.

For patients with inborn errors of immunity (IEI) and other inborn diseases, mixed donor chimerism is a well-accepted outcome of hematopoietic stem cell transplantation (HSCT). Cytoreductive chemotherapy for a secondary malignancy is a potential challenge for the stability of the graft function after HSCT. We report on a boy with X-SCID who developed Ewing sarcoma ten years after HSCT which was successfully treated with cytoreductive chemotherapy, surgery and local radiation. Surprisingly, this treatment had a positive impact on mixed chimerism with an increase of donor-cell proportions from 40% for neutrophils and 75% for non-T-mononuclear cells (MNCs) to >90% for both. T-cell counts remained stable with 100% of donor origin. This is -to our knowledge- the first report on the impact of cytoreductive chemotherapy on post-HSCT mixed chimerism and provides an important first impression for future patients., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation., (© 2023 Lui et al.)

Increased expression of BIRC5/survivin, a crucial regulator of the mitotic spindle checkpoint, is associated with poor prognosis in neuroblastoma (NB), the most common extracranial tumor of childhood. Transcriptional inhibitors of survivin have been tested in adult cancers and inhibitors of survivin homodimerization are emerging. We compared genetic inhibition of survivin transcription with the inhibition of survivin homodimerization by S12 and LQZ-7I, chosen from a larger panel of survivin dimerization inhibitors with activity against NB cells. Mice hemizygous for Birc5 were crossed with NB-prone TH-MYCN mice to generate Birc5 +/-/ MYCNtg /+ mice. The marked decrease of survivin transcription in these mice did not suffice to attenuate the aggressiveness of NB, even when tumors were transplanted into wild-type mice to assure that immune cell function was not compromised by the lack of survivin. In contrast, viability, clonogenicity and anchorage-independent growth of NB cells were markedly decreased by S12. S12 administered systemically to mice with subcutaneous NB xenotransplants decreased intratumoral hemorrhage, albeit not tumor growth. LQZ-7I, which directly targets the survivin dimerization interface, was efficacious in controlling NB cell growth in vitro at markedly lower concentrations compared to S12. LQZ-7I abrogated viability, clonogenicity and anchorage-independent growth, associated with massively distorted mitotic spindle formation. In vivo, LQZ-7I effectively reduced tumor size and cell proliferation of NB cells in CAM assays without apparent toxicity to the developing chick embryo. Collectively, these findings show that inhibiting survivin homodimerization with LQZ-7I holds promise for the treatment of NB and merits further investigation.

The risk of developing severe COVID-19 rises dramatically with age. Schoolchildren are significantly less likely than older people to die from SARS-CoV-2 infection, but the molecular mechanisms underlying this age-dependence are unknown. In primary infections, innate immunity is critical due to the lack of immune memory. Children, in particular, have a significantly stronger interferon response due to a primed state of their airway epithelium. In single-cell transcriptomes of nasal turbinates, we find increased frequencies of immune cells and stronger cytokine-mediated interactions with epithelial cells, resulting in increased epithelial expression of viral sensors (RIG-I, MDA5) via IRF1. In vitro, adolescent peripheral blood mononuclear cells produce more cytokines, priming A549 cells for stronger interferon responses to SARS-CoV-2. Taken together, our findings suggest that increased numbers of immune cells in the airways of children and enhanced cytokine-based interactions with epithelial cells tune the setpoint of the epithelial antiviral system. Our findings shed light on the molecular basis of children's remarkable resistance to COVID-19 and may suggest a novel concept for immunoprophylactic treatments., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Background: Neuroblastoma (NB), the most common extracranial solid malignancy in children, carries a poor prognosis in high-risk disease, thus requiring novel therapeutic approaches. Survivin is overexpressed in NB, has pro-mitotic and anti-apoptotic functions, and impacts on oxidative phosphorylation (OXPHOS) and aerobic glycolysis. The subcellular localization and hence function of survivin is directed by the GTPase Ran., Aim: To determine efficacy and modes of action of the survivin-Ran inhibitor LLP-3 as a potential novel therapy of NB., Methods: Survivin and Ran mRNA expression in NB tumors was correlated to patient survival. Response to LLP-3 in NB cell lines was determined by assays for viability, proliferation, apoptosis, clonogenicity and anchorage-independent growth. Interaction of survivin and Ran was assessed by proximity-linked ligation assay and their subcellular distribution by confocal immunofluorescence microscopy. Expression of survivin, Ran and proteins important for OXPHOS and glycolysis was determined by Western blot, hexokinase activity by enzymatic assay, interaction of survivin with HIF-1α by co-IP, and OXPHOS and glycolysis by extracellular flux analyzer., Results: High mRNA expression of survivin and Ran is correlated with poor patient survival. LLP-3 decreases viability, induces apoptosis, and inhibits clonogenic and anchorage-independent growth in NB cell lines, including those with MYCN amplification, and mutations of p53 and ALK. LLP-3 inhibits interaction of survivin with Ran, decreasing their concentration both in the cytoplasm and the nucleus. LLP-3 impairs flexibility of energy metabolism by inhibiting both OXPHOS and glycolysis. Metabolic inhibition is associated with mitochondrial dysfunction and attenuated hexokinase activity but is independent of HIF-1α., Conclusion: LLP-3 attenuates interaction and concentration of survivin and Ran in NB cells. It controls NB cells with diverse genetic alterations, associated with inhibition of OXPHOS, aerobic glycolysis, mitochondrial function and HK activity. Thus, LLP-3 warrants further studies as a novel drug against NB., (© 2023. The Author(s).)

Therapy-resistant viral reactivations contribute significantly to mortality after hematopoietic stem cell transplantation. Adoptive cellular therapy with virus-specific T cells (VST) has shown efficacy in various single-center trials. However, the scalability of this therapy is hampered by laborious production methods. In this study we describe the in-house production of VST in a closed system (CliniMACS Prodigy® system, Miltenyi Biotec). In addition, we report the efficacy in 26 patients with viral disease following hematopoietic stem cell transplantation in a retrospective analysis (adenovirus, n=7; cytomegalovirus, n=8; Epstein-Barr virus, n=4; multi-viral, n=7). The production of VST was successful in 100% of cases. The safety profile of VST therapy was favorable (n=2 grade 3 and n=1 grade 4 adverse events; all three were reversible). A response was seen in 20 of 26 patients (77%). Responding patients had a significantly better overall survival than patients who did not respond (P<0.001). Virus-specific symptoms were reduced or resolved in 47% of patients. The overall survival of the whole cohort was 28% after 6 months. This study shows the feasibility of automated VST production and safety of application. The scalability of the CliniMACS Prodigy® device increases the accessibility of VST treatment.

Hormone absence or inactivity is common in congenital disease, but hormone antagonism remains controversial. Here, we characterize two novel homozygous leptin variants that yielded antagonistic proteins in two unrelated children with intense hyperphagia, severe obesity, and high circulating levels of leptin. Both variants bind to the leptin receptor but trigger marginal, if any, signaling. In the presence of nonvariant leptin, the variants act as competitive antagonists. Thus, treatment with recombinant leptin was initiated at high doses, which were gradually lowered. Both patients eventually attained near-normal weight. Antidrug antibodies developed in the patients, although they had no apparent effect on efficacy. No severe adverse events were observed. (Funded by the German Research Foundation and others.)., (Copyright © 2023 Massachusetts Medical Society.)

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Introduction: Durability of immune protection against reinfection with SARS-CoV-2 remains enigmatic, especially in the pediatric population and in the context of immune-evading variants of concern. Obviously, this knowledge is required for measures to contain the spread of infection and in selecting rational preventive measures., Methods: Here, we investigated the serum neutralization capacity of 36 seropositive adults and 34 children approximately one year after infection with the ancestral Wuhan strain of SARS-CoV-2 by using a pseudovirus neutralization assay., Results: We found that 88.9% of seropositive adult (32/36) and 94.1% of seropositive children (32/34) convalescents retained the neutralizing activity against the SARS-CoV-2 Wuhan strain (WT). Although, the neutralization effect against Omicron BA.1 (B.1.1.529.1) was significantly lower, 70.6% (24/34) of children and 41.7% (15/36) of adults possessed BA.1 cross-neutralizing antibodies. The spike 1 (S1)-specific T cell recall capacity using an activation-induced marker assay was analyzed in 18 adults and 16 children. All participants had detectable S1-specific CD4 T cells against WT, and 72.2% (13/18) adults and 81,3% (13/16) children had detectable S1 WT-specific CD8 T cells. CD4 cross-reactivity against BA.1 was demonstrated in all investigated adults (18/18), and 66.7% (12/18) adult participants had also detectable specific CD8 BA.1 T cells while we detected BA.1 S1 reactive CD4 and CD8 T cells in 81.3% (13/16) children., Discussion: Together, our findings demonstrate that infection with the ancestral strain of SARS-CoV-2 in children as well as in adults induces robust serological as well as T cell memory responses that persist over at least 12 months. This suggests persistent immunological memory and partial cross-reactivity against Omicron BA.1., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Seidel, Jacobsen, Fabricius, Class, Zernickel, Blum, Conzelmann, Weil, Groß, Bode, Renk, Elling, Stich, Kirchhoff, Debatin, Müench and Janda.)

Vaccines are doubtlessly one of the most crucial life-saving medical interventions to date. However, perplexingly, they court more public controversy than their objectively excellent safety profile warrants. While doubts about the safety of vaccines, as well as opposition to vaccine policies, can be traced back at least to the mid-19th century, the modern anti-vaccine movement has come in 3 distinct waves, or generations, each precipitating around distinct key events. Here, we describe the first 2 generations and trace the origins of an emerging third generation anti-vaccine movement. Currently, this third generation is an integral part of the larger anti-COVID movement and in this more libertarian environment propagates the idea of individualism superseding the responsibility for community health. We highlight the need for a better science education of the young, as well as the general public to further enhance overall science literacy and suggests strategies to achieve these goals.

Background: The utility for liquid biopsy of tumor-associated circulating single-nucleotide variants, as opposed to mutations, of the mitochondrial (mt) and nuclear genomes in neuroblastoma (NB) is unknown., Procedure: Variants of the mt and nuclear genomes from tumor, blood cells, and consecutive plasma samples of five patients with metastatic NB that relapsed or progressed were analyzed. Targeted parallel sequencing results of the mt genome, and of the coding region of 139 nuclear genes and 22 miRNAs implicated in NB, were correlated with clinical imaging and laboratory data., Results: All tumors harbored multiple somatic mt and nuclear single nucleotide variants with low allelic frequency, most of them not detected in the circulation. In one patient a tumor-associated mt somatic variant was detected in the plasma before and during progressive disease. In a second patient a circulating nuclear tumor-associated DNA variant heralded clinical relapse. In all patients somatic mt and nuclear variants not evident in the tumor biopsy at time of diagnosis were found circulating at varying timepoints. This suggests either tumor heterogeneity, evolution of tumor variants or a confounding contribution of normal tissues to somatic variants in patient plasma. The number and allelic frequency of the circulating variants did not reflect the clinical course of the tumors. Mutational signatures of mt and nuclear somatic variants differed. They varied between patients and were detected in the circulation without mirroring the patients' course., Conclusions: In this limited cohort of NB patients clinically informative tumor-associated mt and nuclear circulating variants were detected by targeted parallel sequencing in a minority of patients., (© 2022 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

Immune reactions after trauma are characterized by immediate activation of innate immunity and simultaneously downregulation of adaptive immunity leading to a misbalanced immunohomeostasis and immunosuppression of the injured host. Therefore, the susceptibility to secondary infections is strongly increased after trauma. Immune responses are regulated by a network of immune cells influencing each other and at the same time modifying their functions dependent on the inflammatory environment. Although myeloid-derived suppressor cells (MDSCs) are initially described as T-cell suppressors, their immunomodulatory capacity after trauma is mostly undefined. Therefore, in vitro-generated MDSCs were adoptively transferred into mice after blunt chest trauma (TxT). A single MDSC treatment-induced splenic T-cell expansion decreased apoptosis sensitivity and improved proliferation in the absence of T-cell exhaustion until 2 weeks after trauma. MDSC treatment had a long-lasting effect on the genomic landscape of CD4+ T cells by upregulating primarily Th2-associated genes. Remarkably, immune-activating functions of MDSCs supported the ability of TxT mice to respond to post-traumatic secondary antigen challenge. Secondary insults were mimicked by immunizing MDSC-treated TxT mice with ovalbumin (OVA), followed by OVA restimulation in vitro. MDSC treatment significantly increased the frequency of OVA-specific T cells, enhanced their Th1/Th2 cytokine expression, and induced upregulation of cytolytic molecules finally improving OVA-specific cytotoxicity. Overall, we could show that therapeutic MDSC treatment after TxT improves post-traumatic T-cell functions, which might enable the traumatic host to counterbalance trauma-induced immunoparalysis., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

The COVID-19 course and immunity differ in children and adults. We analyzed immune response dynamics in 28 families up to 12 months after mild or asymptomatic infection. Unlike adults, the initial response is plasmablast-driven in children. Four months after infection, children show an enhanced specific antibody response and lower but detectable spike 1 protein (S1)-specific B and T cell responses than their parents. While specific antibodies decline, neutralizing antibody activity and breadth increase in both groups. The frequencies of S1-specific B and T cell responses remain stable. However, in children, one year after infection, an increase in the S1-specific IgA class switch and the expression of CD27 on S1-specific B cells and T cell maturation are observed. These results, together with the enhanced neutralizing potential and breadth of the specific antibodies, suggest a progressive maturation of the S1-specific immune response. Hence, the immune response in children persists over 12 months but dynamically changes in quality, with progressive neutralizing, breadth, and memory maturation. This implies a benefit for booster vaccination in children to consolidate memory formation., (© 2022. The Author(s).)

20 years ago, we described a human cell strain derived from subcutaneous adipose tissue of an infant supposed to have Simpson-Golabi-Behmel Syndrome (SGBS), thus called "SGBS cells". Since then, these cells have emerged as the most commonly used cell model for human adipogenesis and human adipocyte biology. Although these adipocyte derived stem cells have not been genetically manipulated for transformation or immortalization, SGBS cells retain their capacity to proliferate and to differentiate into adipocytes for more than 50 population doublings, providing an almost unlimited source of human adipocyte progenitor cells. Original data obtained with SGBS cells led to more than 200 peer reviewed publications comprising investigations on adipogenesis and browning, insulin sensitivity, inflammatory response, adipokine production, as well as co-culture models and cell-cell communication. In this article, we provide an update on the characterization of SGBS cells, present basic methods for their application and summarize results of a systematic literature search on original data obtained with this cell strain., (© 2022. The Author(s).)

Introduction: Death due to cancer is mostly associated with therapy ineffectiveness, i.e. tumor cells no longer responding to treatment. The underlying dynamics that facilitate this mutational escape from selective pressure are well studied in several other fields and several interesting approaches exist to combat this phenomenon, for example in the context of antibiotic-resistance in bacteria., Areas Covered: Ninety percent of all cancer-related deaths are associated with treatment failure. Here, we discuss the common treatment modalities and prior attempts to overcome acquired resistance to therapy. The underlying molecular mechanisms are discussed and the implications of emerging resistance in other systems, such as bacteria, are discussed in the context of cancer., Expert Opinion: Reevaluating emerging therapy resistance in tumors as an evolutionary mechanism to survive in a rapidly and drastically altering fitness landscape leads to novel treatment strategies and distinct requirements for new drugs. Here, we propose a scheme of considerations that need to be applied prior to the discovery of novel therapeutic drugs.

Background: Long COVID in children and adolescents remains poorly understood due to a lack of well-controlled studies with long-term follow-up. In particular, the impact of the family context on persistent symptoms following SARS-CoV-2 infection remains unknown. We examined long COVID symptoms in a cohort of infected children, adolescents, and adults and their exposed but non-infected household members approximately 1 year after infection and investigated clustering of persistent symptoms within households., Methods: 1267 members of 341 households (404 children aged <14 years, 140 adolescents aged 14-18 years and 723 adults) were categorized as having had either a SARS-CoV-2 infection or household exposure to SARS-CoV-2 without infection, based on three serological assays and history of laboratory-confirmed infection. Participants completed questionnaires assessing the presence of long COVID symptoms 11-12 months after infection in the household using online questionnaires., Findings: The prevalence of moderate or severe persistent symptoms was statistically significantly higher in infected than in exposed women (36.4% [95% CI: 30.7-42.4%] vs 14.2% [95% CI: 8.7-21.5%]), infected men (22.9% [95% CI: 17.9-28.5%] vs 10.3% [95% CI: 5.8-16.9%]) and infected adolescent girls (32.1% 95% CI: 17.2-50.5%] vs 8.9% [95%CI: 3.1-19.8%]). However, moderate or severe persistent symptoms were not statistically more common in infected adolescent boys aged 14-18 (9.7% [95% CI: 2.8-23.6%] or in infected children <14 years (girls: 4.3% [95% CI: 1.2-11.0%]; boys: 3.7% [95% CI: 1.1-9.6%]) than in their exposed counterparts (adolescent boys: 0.0% [95% CI: 0.0-6.7%]; girls < 14 years: 2.3% [95% CI: 0·7-6·1%]; boys < 14 years: 0.0% [95% CI: 0.0-2.0%]). The number of persistent symptoms reported by individuals was associated with the number of persistent symptoms reported by their household members (IRR=1·11, p=·005, 95% CI [1.03-1.20])., Interpretation: In this controlled, multi-centre study, infected men, women and adolescent girls were at increased risk of negative outcomes 11-12 months after SARS-CoV-2 infection. Amongst non-infected adults, prevalence of negative outcomes was also high. Prolonged symptoms tended to cluster within families, suggesting family-level interventions for long COVID could prove useful., Funding: Ministry of Science, Research and the Arts, Baden-Württemberg, Germany., Competing Interests: Declaration of interests The authors declare that they have no relevant conflicts of interest., (Copyright © 2022. Published by Elsevier B.V.)

Background: Pulmonary involvement is the leading cause of morbidity and mortality after severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Long-term impairment has been reported in adults with severe infection. However, most infections cause only mild symptoms or are even asymptomatic, especially in children. There is insufficient evidence regarding pulmonary outcome measures in mild SARS-CoV-2. The objectives of this study were to determine spirometry parameters after SARS-CoV-2 infection and correlate those with reported persisting symptoms in children, adolescents, and adults., Methods: Data on clinical symptoms during acute infection as well as SARS-CoV-2 serology results were recorded. Twelve months after infection, spirometry was performed and information on persisting symptoms was collected using a structured questionnaire. 182 participants (108 SARS-CoV-2 positive) from 48 families were included; 53 children (< 14 years), 34 adolescents and young adults (14-25 years), and 95 adults., Results: Spirometry values did not significantly differ between the particular subgroups of the cohort (adults, adolescents, children; infected and non-infected individuals). Adults reported more symptoms during acute infection as well more persisting fatigue (29.7% of participants), reduced physical resilience (34.4%), and dyspnea (25.0%) 12 months after infection than adolescents (fatigue 26.7%, reduced physical resilience 20%, and 0% dyspnea) and children (4%, 0%, 0%, respectively). There was no correlation between persistent subjective symptoms and spirometry results., Discussion: Children and adolescents are less affected than adults by acute SARS-CoV-2 as well as by post-infection persistent symptoms. Spirometry was not able to demonstrate any differences between healthy individuals and participants who had suffered from mild SARS-CoV-2 12 months after the infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bode, Haendly, Fabricius, Mayer, Zernickel, Haddad, Frieh, Elling, Renk, Stich, Jacobsen, Debatin and Janda.)

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and has a poor prognosis in high-risk cases, requiring novel therapies. Pathways that depend on phospho-signaling maintain the aggressiveness of NB. Protein phosphatase 2 (PP2A) with its catalytic subunit PPP2CA is a major phosphatase in cancer cells, including NB. We show that reduction of PPP2CA by knock-down decreased growth of NB cells and that complete ablation of PPP2CA by knock-out was not tolerated. Thus, NB cells are addicted to PPP2CA, an addiction augmented by MYCN activation. SET, a crucial endogenous inhibitor of PP2A, was overexpressed in poor-prognosis NB. The SET inhibitor OP449 effectively decreased the viability of NB cells, independent of their molecular alterations and in line with a tumor suppressor function of PPP2CA. The contrasting concentration-dependent functions of PPP2CA as an essential survival gene at low expression levels and a tumor suppressor at high levels are reminiscent of other genes showing this so-called Goldilocks phenomenon. PP2A reactivated by OP449 decreased activating phosphorylation of serine/threonine residues in the AKT pathway. Conversely, induced activation of AKT led to partial rescue of OP449-mediated viability inhibition. Dasatinib, a kinase inhibitor used in relapsed/refractory NB, and OP449 synergized, decreasing activating AKT phosphorylations. In summary, concomitantly reactivating phosphatases and inhibiting kinases with a combination of OP449 and dasatinib are promising novel therapeutic approaches to NB., Competing Interests: Author MV was employed by Cognosci, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Galiger, Dahlhaus, Vitek, Debatin and Beltinger.)

An association between certain ABO/Rh blood groups and susceptibility to SARS-CoV-2 infection has been proposed for adults, although this remains controversial. In children and adolescents, the relationship is unclear due to a lack of robust data. Here, we investigated the association of ABO/Rh blood groups and SARS-CoV-2 in a multi-center study comprising 163 households with 281 children and 355 adults and at least one SARS-CoV-2 seropositive individual as determined by three independent assays as a proxy for previous infection. In line with previous findings, we found a higher frequency of blood group A (+ 6%) and a lower frequency of blood group O (-6%) among the SARS-CoV-2 seropositive adults compared to the seronegative ones. This trend was not seen in children. In contrast, SARS-CoV-2 seropositive children had a significantly lower frequency of Rh-positive blood groups. ABO compatibility did not seem to play a role in SARS-CoV-2 transmission within the families. A correction for family clusters was performed and estimated fixed effects of the blood group on the risk of SARS-CoV-2 seropositivity and symptomatic infection were determined. Although we found a different distribution of blood groups in seropositive individuals compared to the reference population, the risk of SARS-CoV-2 seropositivity or symptomatic infection was not increased in children or in adults with blood group A or AB versus O or B. Increasing age was the only parameter positively correlating with the risk of SARS-CoV-2 infection. In conclusion, specific ABO/Rh blood groups and ABO compatibility appear not to predispose for SARS-CoV-2 susceptibility in children., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Janda, Engel, Remppis, Enkel, Peter, Hörber, Ganzenmueller, Schober, Weinstock, Jacobsen, Fabricius, Zernickel, Stamminger, Dietz, Groß, Bode, Haddad, Elling, Stich, Tönshoff, Henneke, Debatin, Franz and Renk.)

Targeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies., (© 2022. The Author(s).)

Autoinflammatory diseases are a heterogenous group of disorders defined by fever and systemic inflammation suggesting involvement of genes regulating innate immune responses. Patients with homozygous loss-of-function variants in the OTU-deubiquitinase OTULIN suffer from neonatal-onset OTULIN-related autoinflammatory syndrome (ORAS) characterized by fever, panniculitis, diarrhea, and arthritis. Here, we describe an atypical form of ORAS with distinct clinical manifestation of the disease caused by two new compound heterozygous variants (c.258G>A (p.M86I)/c.500G>C (p.W167S)) in the OTULIN gene in a 7-year-old affected by a life-threatening autoinflammatory episode with sterile abscess formation. On the molecular level, we find binding of OTULIN to linear ubiquitin to be compromised by both variants; however, protein stability and catalytic activity is most affected by OTULIN variant p.W167S. These molecular changes together lead to increased levels of linear ubiquitin linkages in patient-derived cells triggering the disease. Our data indicate that the spectrum of ORAS patients is more diverse than previously thought and, thus, supposedly asymptomatic individuals might also be affected. Based on our results, we propose to subdivide the ORAS into classical and atypical entities., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Covalent Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop as the result of a mutation in cysteine 481 of BTK (C481S), which prevents irreversible binding of the drugs. In the present study we performed preclinical characterization of vecabrutinib, a next-generation noncovalent BTK inhibitor that has ITK-inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wild-type BTK. In the murine Eμ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, whereas the naive populations were increased. Of importance, vecabrutinib treatment significantly reduced the frequency of regulatory CD4+ T cells in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on the activation and proliferation of isolated T cells. Lastly, combination treatment with vecabrutinib and venetoclax augmented treatment efficacy, significantly improved survival, and led to favorable reprogramming of the microenvironment in the murine Eμ-TCL1 model. Thus, noncovalent BTK/ITK inhibitors, such as vecabrutinib, may be efficacious in C481S BTK mutant CLL while preserving the T-cell immunomodulatory function of ibrutinib., (© 2022 by The American Society of Hematology.)

Glioblastoma represents the most aggressive tumor of the central nervous system. Due to invasion of glioblastoma stem cells into the healthy tissue, chemoresistance, and recurrence of the tumor, it is difficult to successfully treat glioblastoma patients, which is demonstrated by the low life expectancy of patients after standard therapy treatment. Recently, we found that diisothiocyanate-derived mercapturic acids, which are isothiocyanate derivatives from plants of the Cruciferae family, provoked a decrease in glioblastoma cell viability. These findings were extended by combining diisothiocyanate-derived mercapturic acids with dinaciclib (a small-molecule inhibitor of cyclin-dependent kinases with anti-proliferative capacity) or temozolomide (TMZ, standard chemotherapeutic agent) to test whether the components have a cytotoxic effect on glioblastoma cells when the dosage is low. Here, we demonstrate that the combination of diisothiocyanate-derived mercapturic acids with dinaciclib or TMZ had an additive or even synergistic effect in the restriction of cell growth dependent on the combination of the components and the glioblastoma cell source. This strategy could be applied to inhibit glioblastoma cell growth as a therapeutic interference of glioblastoma., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)