RESUMEN
The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial differentiation is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition, associated with differences in interkinetic nuclear migration and cell cycle length. Comparative RNA sequencing (RNA-seq) reveals differences in expression dynamics of cell morphogenesis factors, including ZEB2, a known epithelial-mesenchymal transition regulator. We show that ZEB2 promotes neuroepithelial transition, and its manipulation and downstream signaling leads to acquisition of nonhuman ape architecture in the human context and vice versa, establishing an important role for neuroepithelial cell shape in human brain expansion.
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Evolución Biológica , Encéfalo/citología , Forma de la Célula/fisiología , Animales , Encéfalo/metabolismo , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Transición Epitelial-Mesenquimal/genética , Expresión Génica , Gorilla gorilla , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Organoides/citología , Organoides/metabolismo , Pan troglodytes , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismoRESUMEN
Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.
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Epirregulina , Neocórtex , Animales , Humanos , Ratones , Proliferación Celular , Epirregulina/genética , Epirregulina/metabolismo , Gorilla gorilla/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neocórtex/citología , Neocórtex/metabolismo , Primates/fisiologíaRESUMEN
In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.
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Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Humanos , Transporte Activo de Núcleo Celular , Células Endoteliales/metabolismo , Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Carioferinas/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Ribonucleoproteínas/metabolismo , ARN Mensajero/metabolismo , Replicación ViralRESUMEN
Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential prophylactics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-mediated antiviral activity and its susceptibility to viral subversion remain incompletely understood. Pretreatment of cells with iBETs inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. The antiviral activity manifested itself by reduced reporter expression of recombinant viruses, and reduced viral RNA quantities and infectious titers in the culture supernatant. While we confirmed JQ-1-mediated downregulation of expression of angiotensin-converting enzyme 2 (ACE2) and interferon-stimulated genes (ISGs), multi-omics analysis addressing the chromatin accessibility, transcriptome and proteome uncovered induction of an antiviral nuclear factor erythroid 2-related factor 2 (NRF-2)-mediated cytoprotective response as an additional mechanism through which JQ-1 inhibits SARS-CoV-2 replication. Pharmacological inhibition of NRF-2, and knockdown of NRF-2 and its target genes reduced JQ-1-mediated inhibition of SARS-CoV-2 replication. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variant, which exhibited resistance to JQ-1 and increased sensitivity to exogenously administered type I interferon (IFN-I), suggesting a minimised need for SARS-CoV-2 ORF6-mediated repression of IFN signalling in the presence of JQ-1. Importantly, JQ-1 exhibited a transient antiviral activity when administered prophylactically in human airway bronchial epithelial cells (hBAECs), which was gradually subverted by SARS-CoV-2, and no antiviral activity when administered therapeutically following an established infection. We propose that JQ-1 exerts pleiotropic effects that collectively induce an antiviral state in the host, which is ultimately nullified by SARS-CoV-2 infection, raising questions about the clinical suitability of the iBETs in the context of COVID-19.
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COVID-19 , Interferón Tipo I , Humanos , SARS-CoV-2/metabolismo , Interferón Tipo I/farmacología , Proteínas Virales/metabolismo , Antivirales/farmacologíaRESUMEN
Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/genética , Esparcimiento de Virus , Anticuerpos BloqueadoresRESUMEN
PURPOSE: To compare transorbital point-of-care ultrasound techniques -optic nerve sheath diameter (US-ONSD) and optic disc elevation (US-ODE)- with fundoscopic papilledema to detect potentially raised intracranial pressure (ICP) with treatment indication in children. METHODS: In a prospective study, 72 symptomatic children were included, 50 with later proven disease associated with raised ICP (e.g. pseudotumour cerebri, brain tumour, hydrocephalus) and 22 with pathology excluded. Bilateral US-ONSD and US-ODE were quantified by US using a 12-MHz-linear-array transducer. This was compared to fundoscopic optic disc findings (existence of papilledema) and, in 28 cases, invasively measured ICP values. RESULTS: The sensitivity and specificity of a cut-off value of US-ONSD (5.73 mm) to detect treatment indication for diseases associated with increased ICP was 92% and 86.4%, respectively, compared to US-ODE (0.43 mm) with sensitivity: 72%, specificity: 77.3%. Fundoscopic papilledema had a sensitivity of 46% and a specificity of 100% in this context. Repeatability and observer-reliability of US-ODE examination was eminent (Cronbach's α = 0.978-0.989). Papilledema was detected fundoscopically only when US-ODE was > 0.67 mm; a US-ODE > 0.43 mm had a positive predictive value of 90% for potentially increased ICP. CONCLUSION: In our cohort, transorbital point-of-care US-ONSD and US-ODE detected potentially elevated ICP requiring treatment in children more reliably than fundoscopy. US-ONSD and US-ODE indicated the decrease in ICP after treatment earlier and more reliably than fundoscopy. The established cut-off values for US-ONSD and US-ODE and a newly developed US-based grading of ODE can be used as an ideal first-line screening tool to detect or exclude conditions with potentially elevated ICP in children.
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Hipertensión Intracraneal , Papiledema , Niño , Humanos , Papiledema/complicaciones , Papiledema/diagnóstico por imagen , Estudios Prospectivos , Sistemas de Atención de Punto , Reproducibilidad de los Resultados , Presión Intracraneal/fisiología , Nervio Óptico/diagnóstico por imagen , Nervio Óptico/patología , Hipertensión Intracraneal/complicaciones , Hipertensión Intracraneal/diagnóstico por imagen , Ultrasonografía/métodosRESUMEN
OBJECTIVE: Posterior fossa pediatric low-grade glioma involving the brainstem and cerebellar peduncles (BS-pLGG) are a subgroup with higher risks at surgery. We retrospectively analyzed the role of surgery in the interdisciplinary armamentarium of treatment options in our institutional series of BS-pLGG with various degrees of brainstem involvement. MATERIAL AND METHODS: We analyzed data of 52 children with BS-pLGG after surgical intervention for clinical/molecular characteristics, neurological outcome, factors influencing recurrence/progression pattern, and tumor volumetric analysis of exclusively surgically treated patients to calculate tumor growth velocity (TGV). Tumors were stratified according to primary tumor origin in four groups: (1) cerebellar peduncle, (2) 4th ventricle, (3) pons, (4) medulla oblongata. RESULTS: The mean FU was 6.44 years. Overall survival was 98%. The mean PFS was 34.07 months. Two patients had biopsies only. Fifty-two percent of patients underwent remission or remained in stable disease (SD) after initial surgery. Patients with progression underwent further 23 resections, 15 chemotherapies, 4 targeted treatments, and 2 proton radiations. TGV decreased after the 2nd surgery compared to TGV after the 1st surgery (p < 0.05). The resection rates were significantly higher in Groups 1 and 2 and lowest in medulla oblongata tumors (Group 4) (p < 0.05). More extended resections were achieved in tumors with KIAA1549::BRAF fusion (p = 0.021), which mostly occurred in favorable locations (Groups 1 and 2). Thirty-one patients showed postoperatively new neurological deficits. A total of 27/31 improved within 12 months. At the end of FU, 6% had moderate deficits, 52% had mild deficits not affecting activities, and 36% had none. Fifty percent of patients were free of disease or showed remission, 38% were in SD, and 10% showed progression. CONCLUSION: The first surgical intervention in BS-pLGG can control disease alone in overall 50% of cases, with rates differing greatly according to location (Groups 1 > 2 > 3 > 4), with acceptable low morbidity. The second look surgery is warranted except in medullary tumors. With multimodality treatments almost 90% of patients can obtain remission or stable disease after > 5 years of follow-up. An integrated multimodal and multidisciplinary approach aiming at minimal safe residual disease, combining surgery, chemo-, targeted therapy, and, as an exception, radiation therapy, is mandatory.
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PURPOSE: Pediatric low-grade gliomas (pLGG) are the most common brain tumors in children and achieving complete resection (CR) in pLGG is the most important prognostic factor. There are multiple intraoperative tools to optimize the extent of resection (EOR). This article investigates and discusses the role of intraoperative ultrasound (iUS) and intraoperative magnetic resonance imaging (iMRI) in the surgical treatment of pLGG. METHODS: The tumor registries at Tuebingen, Rome and Pretoria were searched for pLGG with the use of iUS and data on EOR. The tumor registries at Liverpool and Tuebingen were searched for pLGG with the use of iMRI where preoperative CR was the surgical intent. Different iUS and iMRI machines were used in the 4 centers. RESULTS: We included 111 operations which used iUS and 182 operations using iMRI. Both modalities facilitated intended CR in hemispheric supra- and infratentorial location in almost all cases. In more deep-seated tumor location like supratentorial midline tumors, iMRI has advantages over iUS to visualize residual tumor. Functional limitations limiting CR arising from eloquent involved or neighboring brain tissue apply to both modalities in the same way. In the long-term follow-up, both iUS and iMRI show that achieving a complete resection on intraoperative imaging significantly lowers recurrence of disease (chi-square test, p < 0.01). CONCLUSION: iUS and iMRI have specific pros and cons, but both have been proven to improve achieving CR in pLGG. Due to advances in image quality, cost- and time-efficiency, and efforts to improve the user interface, iUS has emerged as the most accessible surgical adjunct to date to aid and guide tumor resection. Since the EOR has the most important effect on long-term outcome and disease control of pLGG in most locations, we strongly recommend taking all possible efforts to use iUS in any surgery, independent of intended resection extent and iMRI if locally available.
RESUMEN
BACKGROUND: Idiopathic intracranial hypertension in children often presents with non-specific symptoms found in conditions such as hydrocephalus. For definite diagnosis, invasive intracranial pressure measurement is usually required. Ultrasound (US) of the optic nerve sheath diameter provides a non-invasive method to assess intracranial pressure. Transtemporal US allows imaging of the third ventricle and thus assessment for hydrocephalus. OBJECTIVE: To investigate whether the combination of US optic nerve sheath and third ventricle diameter can be used as a screening tool in pediatric idiopathic intracranial hypertension to indicate elevated intracranial pressure and exclude hydrocephalus as an underlying pathology. Further, to analyze whether both parameters can be used to monitor treatment outcome. MATERIALS AND METHODS: We prospectively included 36 children with idiopathic intracranial hypertension and 32 controls. Using a 12-Mhz linear transducer and a 1-4-Mhz phased-array transducer, respectively, optic nerve sheath and third ventricle diameters were determined initially and during the course of treatment. RESULTS: In patients, the mean optic nerve sheath diameter was significantly larger (6.45±0.65 mm, controls: 4.96±0.32 mm) and the mean third ventricle diameter (1.69±0.65 mm, controls: 2.99±1.31 mm) was significantly smaller compared to the control group, P<0.001. Optimal cut-off values were 5.55 mm for the optic nerve sheath and 1.83 mm for the third ventricle diameter. CONCLUSIONS: The combined use of US optic nerve sheath and third ventricle diameter is an ideal non-invasive screening tool in pediatric idiopathic intracranial hypertension to indicate elevated intracranial pressure while ruling out hydrocephalus. Treatment can effectively be monitored by repeated US, which also reliably indicates relapse.
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Nervio Óptico , Seudotumor Cerebral , Humanos , Femenino , Masculino , Niño , Seudotumor Cerebral/diagnóstico por imagen , Nervio Óptico/diagnóstico por imagen , Adolescente , Preescolar , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Estudios de Seguimiento , Tercer Ventrículo/diagnóstico por imagen , Estudios Prospectivos , Ultrasonografía Intervencional/métodos , LactanteRESUMEN
Recent findings in permanent cell lines suggested that SARS-CoV-2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air-liquid interface cultures of primary human bronchial epithelial cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active types I (α/ß) and III (λ) interferons and protected cells from super-infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza-like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron-induced interferon signaling was mediated via double-stranded RNA recognition by MDA5, as MDA5 knockout prevented it. The JAK/STAT inhibitor baricitinib inhibited the Omicron-mediated antiviral response, suggesting it is caused by MDA5-mediated interferon production, which activates interferon receptors that then trigger JAK/STAT signaling. In conclusion, our study (1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, (2) shows that Omicron infection protects cells from influenza A virus super-infection, and (3) indicates that BA.1 and BA.5 induce comparable antiviral states.
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COVID-19 , Subtipo H1N1 del Virus de la Influenza A , Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Inhibidores de las Cinasas Janus , Humanos , SARS-CoV-2 , Interferones , AntiviralesRESUMEN
Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.
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Células Clonales/citología , Secuenciación del Exoma/métodos , Células Madre Pluripotentes Inducidas/citología , Mutación , Neoplasias/genética , Anciano , Ciclo Celular , Muerte Celular , Diferenciación Celular , Línea Celular , Células Cultivadas , Reprogramación Celular , Células Clonales/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Pluripotentes Inducidas/química , Neoplasias/patologíaRESUMEN
We reviewed our experience in managing of NF2-associated vestibular schwannoma (VS) in children and young adults regarding the effect of surgery and postoperative bevacizumab treatment. A total of 579 volumetric and hearing data sets were analyzed. The effect of surgery on tumor volume and growth rate was investigated in 46 tumors and on hearing function in 39 tumors. Long-term hearing follow-up behavior was compared with 20 non-operated ears in additional 15 patients. Sixteen operated VS were treated with bevacizumab. Mutation analysis of the NF2 gene was performed in 25 patients. Surgery significantly slowed down VS growth rate. Factors associated with a higher growth rate were increasing patient age, tumor volume, and constitutional truncating mutations. Immediately after surgery, functional hearing was maintained in 82% of ears. Deterioration of hearing was associated with initial hearing quality, larger tumor volumes, and larger resection amounts. Average hearing scores were initially better in the group of non-operated VS. Over time, hearing scores in both groups worsened with a similar dynamic. During bevacizumab treatment of residual tumors, four different patterns of growth were observed. Decompression of the internal auditory canal with various degrees of tumor resection decreases the postoperative tumor growth rates. Carefully tailored BAEP-guided surgery does not cause additional hearing deterioration. Secondary bevacizumab treatment showed heterogenous effects both regarding tumor size and hearing preservation. It seems that postoperative tumor residuals, that grow slower, behave differently to bevacizumab than reported for not-operated faster growing VS.
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Neurofibromatosis 2 , Neuroma Acústico , Bevacizumab/uso terapéutico , Niño , Genes de la Neurofibromatosis 2 , Audición , Humanos , Neurofibromatosis 2/complicaciones , Neurofibromatosis 2/tratamiento farmacológico , Neurofibromina 2 , Neuroma Acústico/tratamiento farmacológico , Neuroma Acústico/cirugía , Resultado del Tratamiento , Carga Tumoral , Adulto JovenRESUMEN
PURPOSE: The hallmark of neurofibromatosis type 2 (NF2) is the presence of bilateral vestibular schwannomas (VS) which however have not yet developed or grown to large size in children and young adolescents. Therefore, early diagnosis in pediatric patients without family history of NF2 has to be made by signs and symptoms not related to VS which will be reviewed in this study. METHODS: A total of 70 children diagnosed for NF2 at an age of < 18 years were identified from our patient cohort. Age and symptoms, signs and pathology at symptom onset, age at NF2 diagnosis and symptoms leading to diagnosis as well as genetic findings were retrospectively reviewed. RESULTS: The average age at symptom/sign onset was 8 ± 6 (range 0-17) years and 11 ± 5 (range 1-17) years at time of diagnosis. Fifteen children had a positive family history and were diagnosed upon additional clinical symptoms. The most frequent first presenting symptom/signs were ophthalmological abnormalities (49%), followed by cutaneous features (40%), non-VS-related neurological deficits (33%), and symptoms attributable to VS (21%). VS were not only the most common symptomatic neoplasm but also the most frequent pathological evidence for the diagnosis (72%). In 42 patients with available genetic testing results, pathogenic mutations were most frequently identified (n = 27). CONCLUSION: The presenting symptoms in NF2 children appear "unspecific" or less specific for classical NF2 compared with adult NF2 patients, posing a challenge particularly for cases without family history. In children, ophthalmological and cutaneous features should raise clinical suspicion for NF2 and referral to an NF2 specialized center is recommended.
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Neurofibromatosis 2 , Adolescente , Adulto , Niño , Preescolar , Humanos , Lactante , Recién Nacido , Mutación , Neurofibromatosis 2/complicaciones , Neurofibromatosis 2/diagnóstico , Neurofibromatosis 2/genética , Estudios RetrospectivosRESUMEN
Myocardial interstitial fibrosis (MIF) is characterized by excessive extracellular matrix (ECM) deposition, increased myocardial stiffness, functional weakening, and compensatory cardiomyocyte (CM) hypertrophy. Fibroblasts (Fbs) are considered the principal source of ECM, but the contribution of perivascular cells, including pericytes (PCs), has gained attention, since MIF develops primarily around small vessels. The pathogenesis of MIF is difficult to study in humans because of the pleiotropy of mutually influencing pathomechanisms, unpredictable side effects, and the lack of available patient samples. Human pluripotent stem cells (hPSCs) offer the unique opportunity for the de novo formation of bioartificial cardiac tissue (BCT) using a variety of different cardiovascular cell types to model aspects of MIF pathogenesis in vitro. Here, we have optimized a protocol for the derivation of hPSC-derived PC-like cells (iPSC-PCs) and present a BCT in vitro model of MIF that shows their central influence on interstitial collagen deposition and myocardial tissue stiffening. This model was used to study the interplay of different cell types-i.e., hPSC-derived CMs, endothelial cells (ECs), and iPSC-PCs or primary Fbs, respectively. While iPSC-PCs improved the sarcomere structure and supported vascularization in a PC-like fashion, the functional and histological parameters of BCTs revealed EC- and PC-mediated effects on fibrosis-related cardiac tissue remodeling.
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Diferenciación Celular/genética , Fibrosis/terapia , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/metabolismo , Neovascularización Patológica/terapia , Órganos Bioartificiales , Células Endoteliales/citología , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fibrosis/genética , Fibrosis/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/patología , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Pericitos/citología , Pericitos/metabolismo , Sarcómeros/genética , Sarcómeros/metabolismo , Remodelación Ventricular/genéticaRESUMEN
Induced pluripotent stem cells (iPSCs) offer great promise for the field of regenerative medicine, and iPSC-derived cells have already been applied in clinical practice. However, potential contamination of effector cells with residual pluripotent cells (e.g., teratoma-initiating cells) or effector cell-associated side effects may limit this approach. This also holds true for iPSC-derived hematopoietic cells. Given the therapeutic benefit of macrophages in different disease entities and the feasibility to derive macrophages from human iPSCs, we established human iPSCs harboring the inducible Caspase-9 (iCasp9) suicide safety switch utilizing transcription activator-like effector nuclease (TALEN)-based designer nuclease technology. Mono- or bi-allelic integration of the iCasp9 gene cassette into the AAVS1 locus showed no effect on the pluripotency of human iPSCs and did not interfere with their differentiation towards macrophages. In both, iCasp9-mono and iCasp9-bi-allelic clones, concentrations of 0.1 nM AP20187 were sufficient to induce apoptosis in more than 98% of iPSCs and their progeny-macrophages. Thus, here we provide evidence that the introduction of the iCasp9 suicide gene into the AAVS1 locus enables the effective clearance of human iPSCs and thereof derived macrophages.
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Caspasa 9/genética , Células Madre Pluripotentes Inducidas/citología , Macrófagos/citología , Caspasa 9/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Genes Transgénicos Suicidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos/metabolismo , Medicina Regenerativa , Tacrolimus/análogos & derivados , Tacrolimus/farmacologíaRESUMEN
BACKGROUND: Medulloblastoma is the most common malignant brain tumor in childhood and adolescence. Although some patients present with distinct genetic alterations, such as mutated TP53 or MYC amplification, pediatric medulloblastoma is a tumor entity with minimal mutational load and low immunogenicity. METHODS: We identified tumor-specific mutations using next-generation sequencing of medulloblastoma DNA and RNA derived from primary tumor samples from pediatric patients. Tumor-specific mutations were confirmed using deep sequencing and in silico analyses predicted high binding affinity of the neoantigen-derived peptides to the patients' human leukocyte antigen molecules. Tumor-specific peptides were synthesized and used to induce a de novo T-cell response characterized by interferon gamma and tumor necrosis factor alpha release of CD8+ cytotoxic T cells in vitro. RESULTS: Despite low mutational tumor burden, at least two immunogenic tumor-specific peptides were identified in each patient. T cells showed a balanced CD4/CD8 ratio and mostly effector memory phenotype. Induction of a CD8-specific T-cell response was achieved for the neoepitopes derived from Histidine Ammonia-Lyase (HAL), Neuraminidase 2 (NEU2), Proprotein Convertase Subtilisin (PCSK9), Programmed Cell Death 10 (PDCD10), Supervillin (SVIL) and tRNA Splicing Endonuclease Subunit 54 (TSEN54) variants. CONCLUSION: Detection of patient-specific, tumor-derived neoantigens confirms that even in tumors with low mutational load a molecular design of targets for specific T-cell immunotherapy is possible. The identified neoantigens may guide future approaches of adoptive T-cell transfer, transgenic T-cell receptor transfer or tumor vaccination.
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Antígenos de Neoplasias/inmunología , Inmunoterapia , Meduloblastoma/genética , Meduloblastoma/terapia , Mutación/genética , Linfocitos T/inmunología , Adolescente , Secuencia de Aminoácidos , Niño , Epítopos/inmunología , Femenino , Humanos , Lactante , Masculino , Meduloblastoma/inmunología , Péptidos/químicaRESUMEN
PURPOSE: To demonstrate the clinical use of FDG-PET/MRI for monitoring enlargement and metabolism of plexiform neurofibromas (PNF) in patients with neurofibromatosis type 1 (NF1), in whom the development of a malignant peripheral nerve sheath tumor (MPNST) is often a life limiting event. METHODS: NF1 patients who underwent a simultaneous FDG-PET/MRI examination in our institution from September 2012 to February 2018 were included. Indication was suspicion of malignant transformation of a PNF to MPNST. A maximum of six peripheral nerve lesions per patient were defined as targets. Standardized uptake values (SUV) and apparent diffusion coefficients (ADC) were measured. The presence of target sign and contrast-medium enhancement was visually recorded. Growth rates were estimated comparing prior or follow-up examinations and correlated with FDG uptake and ADC values. The presence of CNS lesions in cerebral T2 weighted images was recorded. RESULTS: In 28 NF1 patients a total number of 83 peripheral nerve tumors, 75 benign PNFs and eight MPNSTs, were selected as target lesions. The SUVs of MPNSTs were significantly higher than the SUVs of PNF (3.84 ± 3.98 [SUVmean MPNSTs] vs. 1.85 ± 1.03 [SUVmean PNF], P < .01). Similarly, lesion SUVmean-to-liver SUVmean ratios significantly differed between MPNSTs and PNF (3.20 ± 2.70 [MPNSTs] vs. 1.23 ± 0.61 [PNF]; P < .01). For differentiation between still benign PNF and MPNSTs, we defined SUVmax ≥ 2.78 as a significant cut-off value. Growth rate of PNF correlated significantly positively with SUVmean (rs = .41; P = .003). MRI parameters like ADCmean (1.87 ± 0.24 × 10-3âmm2/s [PNF] vs. 1.76 ± 0.11 × 10-3âmm2/s [MPNSTs]; P > .05], contrast medium enhancement (P = .50) and target sign (P = .86) did not differ between groups. CONCLUSION: Simultaneous FDG-PET/MRI is a comprehensive imaging modality for monitoring PNF in NF1 patients. The combined acquisition of both morphologic information in MRI and metabolic information in PET enables the correlation of lesion growth rates with metabolic activity and to define SUV thresholds of significance to identify malignant transformation, which is of utmost clinical significance.
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Fluorodesoxiglucosa F18 , Imagen por Resonancia Magnética , Imagen Multimodal , Neurofibromatosis 1/patología , Neurofibromatosis 1/fisiopatología , Tomografía de Emisión de Positrones , Adolescente , Adulto , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Encéfalo/fisiopatología , Niño , Preescolar , Femenino , Humanos , Masculino , Neurofibromatosis 1/diagnóstico por imagen , Estudios Retrospectivos , Adulto JovenRESUMEN
Maladaptive cardiac remodeling after myocardial infarction (MI) is increasingly contributing to the prevalence of chronic heart failure. Women show less severe remodeling, a reduced mortality and a better systolic function after MI compared to men. Although sex hormones are being made responsible for these differences, it remains currently unknown how this could be translated into therapeutic strategies. Because we had recently demonstrated that inhibition of the conversion of testosterone to its highly active metabolite dihydrotestosterone (DHT) by finasteride effectively reduces cardiac hypertrophy and improves heart function during pressure overload, we asked here whether this strategy could be applied to post-MI remodeling. We found increased abundance of DHT and increased expression of androgen responsive genes in the mouse myocardium after experimental MI. Treatment of mice with finasteride for 21â¯days (starting 7â¯days after surgery), reduced myocardial DHT levels and markedly attenuated cardiac dysfunction as well as hypertrophic remodeling after MI. Histological and molecular analyses showed reduced MI triggered interstitial fibrosis, reduced cardiomyocyte hypertrophy and increased capillary density in the myocardium of finasteride treated mice. Mechanistically, this was associated with decreased activation of myocardial growth-signaling pathways, a comprehensive normalization of pathological myocardial gene-expression as revealed by RNA deep-sequencing and with direct effects of finasteride on cardiac fibroblasts and endothelial cells. In conclusion, we demonstrated a beneficial role of anti-androgenic treatment with finasteride in post-MI remodeling of mice. As finasteride is already approved for the treatment of benign prostate disease, it could potentially be evaluated as therapeutic strategy for heart failure after MI.
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Antagonistas de Andrógenos/uso terapéutico , Finasterida/uso terapéutico , Expresión Génica/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , Análisis de Varianza , Animales , Cardiomegalia/tratamiento farmacológico , Línea Celular , Dihidrotestosterona/metabolismo , Células Endoteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibrosis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Pluripotentes Inducidas , Masculino , Ratones , Contracción Muscular/efectos de los fármacos , Miocardio/patología , Neovascularización Fisiológica/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Although infants following major surgery frequently require RBC transfusions, there is still controversy concerning the best definition for requirement of transfusion in the individual patient. The aim of this study was to determine the impact of RBC transfusion on cerebral oxygen metabolism in noncardiac and cardiac postsurgical infants. DESIGN: Prospective observational cohort study. SETTING: Pediatric critical care unit of a tertiary referral center. PATIENTS: Fifty-eight infants (15 after pediatric surgery and 43 after cardiac surgery) with anemia requiring RBC transfusion were included. INTERVENTIONS: RBC transfusion. MEASUREMENTS AND MAIN RESULTS: We measured noninvasively regional cerebral oxygen saturation and microperfusion (relative cerebral blood flow) using tissue spectrometry and laser Doppler flowmetry before and after RBC transfusion. Cerebral fractional tissue oxygen extraction and approximated cerebral metabolic rate of oxygen were calculated. Fifty-eight RBC transfusions in 58 patients were monitored (15 after general surgery, 24 after cardiac surgery resulting in acyanotic biventricular physiology and 19 in functionally univentricular hearts including hypoplastic left heart following neonatal palliation). The posttransfusion hemoglobin concentrations increased significantly (9.7 g/dL vs 12.8 g/dL; 9.7 g/dL vs 13.8 g/dL; 13.1 g/dL vs 15.6 g/dL; p < 0.001, respectively). Posttransfusion cerebral oxygen saturation was significantly higher than pretransfusion (61% [51-78] vs 72% [59-89]; p < 0.001; 58% [35-77] vs 71% [57-88]; p < 0.001; 51% [37-61] vs 58% [42-73]; p = 0.007). Cerebral fractional tissue oxygen extraction decreased posttransfusion significantly 0.37 (0.16-0.47) and 0.27 (0.07-039), p = 0.002; 0.40 (0.2-0.62) vs 0.26 (0.11-0.57), p = 0.001; 0.42 (0.23-0.52) vs 0.32 (0.1-0.42), p = 0.017. Cerebral blood flow and approximated cerebral metabolic rate of oxygen showed no significant change during the observation period. The increase in cerebral oxygen saturation and the decrease in cerebral fractional tissue oxygen extraction were most pronounced in patients after cardiac surgery with a pretransfusion cerebral fractional tissue oxygen extraction greater than or equal to 0.4. CONCLUSION: Following RBC transfusion, cerebral oxygen saturation increases and cerebral fractional tissue oxygen extraction decreases. The data suggest that cerebral oxygenation in postoperative infants with cerebral fractional tissue oxygen extraction greater than or equal to 0.4 may be at risk in instable hemodynamic or respiratory situations.
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Anemia/terapia , Circulación Cerebrovascular/fisiología , Transfusión de Eritrocitos/métodos , Consumo de Oxígeno/fisiología , Procedimientos Quirúrgicos Operativos/efectos adversos , Anemia/etiología , Estudios de Cohortes , Femenino , Hemodinámica/fisiología , Humanos , Lactante , Recién Nacido , Unidades de Cuidado Intensivo Pediátrico , Flujometría por Láser-Doppler/métodos , Masculino , Estudios Prospectivos , Espectroscopía Infrarroja Corta/métodosRESUMEN
PURPOSE: Ventriculo-peritoneal (VP) shunts are effective for treatment of hydrocephalus in all age groups; however, they are associated with complications, a common one being ventricular catheter (VC) obstruction. VC position is likely to influence VC survival; however, most VCs are positioned freehand without guidance. This paper describes the accuracy of ultrasound guidance for VC placement and the impact of tip location on VC occlusion rate. METHODS: This is a retrospective cohort study of hydrocephalic children with first-time VP shunt and ultrasound-guided VC placement. Data recorded were age, sex, cause of hydrocephalus, side (left or right) and location (frontal or occipital) of VC, and exact postoperative position within the ventricle on first postoperative imaging: middle of ventricle (optimal position), near or touching the medial or lateral ventricle wall, within the third ventricle, and at the contralateral side. RESULTS: Of the 128 screened patients, 85 had a first postoperative imaging that clearly defined the VC position and were included. The follow-up was at least 12 months. Seventy-three percent of VCs were placed on the right and 71% via a frontal burhole. Eighty-three of 85 VC tips (95%) were in the intended ventricle, 61% at optimal position. Nine of 85 VCs (10%) obstructed within the first 12 months. Seven of nine (78%) obstructed VCs were located in a nonoptimal position (p = 0.016). Two of nine (22%) obstructed VCs entered through a frontal and seven of nine (78%) through an occipital burrhole (p = 0.016). CONCLUSION: Ultrasound-guided VC placement is as precise as frameless navigated placement. The optimal VC position was associated to a significant lower VC obstruction rate. The frontal position was superior to the occipital. Intraoperative US guidance is fast with almost no extra time and no extra cost. US-guided VC placement should become standard of care in VP shunt surgery.