RESUMEN
Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.
Asunto(s)
Encefalopatías Metabólicas Innatas/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virología , ARN/química , ARN/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Encefalopatías Metabólicas Innatas/patología , Tronco Encefálico/patología , Encefalitis Viral/genética , Escherichia coli/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Herpesvirus Humano 1 , Humanos , Interferones/metabolismo , Intrones/genética , Masculino , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Sistemas de Lectura Abierta/genética , Linaje , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/deficiencia , ARN Nucleotidiltransferasas/genética , Receptor Toll-Like 3/metabolismo , Replicación ViralRESUMEN
Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Some HSE children carry inborn errors of the Toll-like receptor 3 (TLR3)-dependent IFN-α/ß- and -λ-inducing pathway. Induced pluripotent stem cell (iPSC)-derived cortical neurons with TLR3 pathway mutations are highly susceptible to HSV-1, due to impairment of cell-intrinsic TLR3-IFN immunity. In contrast, the contribution of cell-intrinsic immunity of human trigeminal ganglion (TG) neurons remains unclear. Here, we describe efficient in vitro derivation and purification of TG neurons from human iPSCs via a cranial placode intermediate. The resulting TG neurons are of sensory identity and exhibit robust responses to heat (capsaicin), cold (icilin), and inflammatory pain (ATP). Unlike control cortical neurons, both control and TLR3-deficient TG neurons were highly susceptible to HSV-1. However, pretreatment of control TG neurons with poly(I:C) induced the cells into an anti-HSV-1 state. Moreover, both control and TLR3-deficient TG neurons developed resistance to HSV-1 following pretreatment with IFN-ß but not IFN-λ. These data indicate that TG neurons are vulnerable to HSV-1 because they require preemptive stimulation of the TLR3 or IFN-α/ß receptors to induce antiviral immunity, whereas cortical neurons possess a TLR3-dependent constitutive resistance that is sufficient to block incoming HSV-1 in the absence of prior antiviral signals. The lack of constitutive resistance in TG neurons in vitro is consistent with their exploitation as a latent virus reservoir in vivo. Our results incriminate deficiencies in the constitutive TLR3-dependent response of cortical neurons in the pathogenesis of HSE.
Asunto(s)
Inmunidad/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Receptor Toll-Like 3/metabolismo , Antivirales/farmacología , Diferenciación Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Niño , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Humanos , Inmunidad/genética , Células Madre Pluripotentes Inducidas/citología , Interferón beta/farmacología , Mutación , Neuronas/efectos de los fármacos , Neuronas/virología , Poli I-C/farmacología , Receptor Toll-Like 3/genética , Ganglio del Trigémino/citologíaRESUMEN
In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-ß (IFN-ß) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-ß and IFN-λ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-ß ( IFN-α/ß) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/ß intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies.
Asunto(s)
Sistema Nervioso Central/patología , Herpesvirus Humano 1/inmunología , Células Madre Pluripotentes Inducidas/citología , Receptor Toll-Like 3/deficiencia , Astrocitos/inmunología , Astrocitos/virología , Biomarcadores , Diferenciación Celular , Linaje de la Célula , Separación Celular , Células Cultivadas , Sistema Nervioso Central/citología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/virología , Niño , Susceptibilidad a Enfermedades , Encefalitis por Herpes Simple/inmunología , Encefalitis por Herpes Simple/metabolismo , Encefalitis por Herpes Simple/patología , Encefalitis por Herpes Simple/virología , Herpesvirus Humano 1/patogenicidad , Humanos , Inmunidad Innata , Células Madre Pluripotentes Inducidas/virología , Interferones/inmunología , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/genética , Células-Madre Neurales/inmunología , Células-Madre Neurales/virología , Neuronas/inmunología , Neuronas/patología , Neuronas/virología , Oligodendroglía/inmunología , Oligodendroglía/patología , Oligodendroglía/virología , Receptor Toll-Like 3/genéticaRESUMEN
The protein-coding exome of a patient with a monogenic disease contains about 20,000 variants, only one or two of which are disease causing. We found that 58% of rare variants in the protein-coding exome of the general population are located in only 2% of the genes. Prompted by this observation, we aimed to develop a gene-level approach for predicting whether a given human protein-coding gene is likely to harbor disease-causing mutations. To this end, we derived the gene damage index (GDI): a genome-wide, gene-level metric of the mutational damage that has accumulated in the general population. We found that the GDI was correlated with selective evolutionary pressure, protein complexity, coding sequence length, and the number of paralogs. We compared GDI with the leading gene-level approaches, genic intolerance, and de novo excess, and demonstrated that GDI performed best for the detection of false positives (i.e., removing exome variants in genes irrelevant to disease), whereas genic intolerance and de novo excess performed better for the detection of true positives (i.e., assessing de novo mutations in genes likely to be disease causing). The GDI server, data, and software are freely available to noncommercial users from lab.rockefeller.edu/casanova/GDI.
Asunto(s)
Exoma , Enfermedades Genéticas Congénitas/genética , Humanos , Curva ROCRESUMEN
Human embryonic stem cells (hESCs) have enormous potential for applications in basic biology and regenerative medicine. However, harnessing the potential of hESCs toward generating homogeneous populations of specialized cells remains challenging. Here we describe a novel technology for the genetic identification of defined hESC-derived neural cell types using bacterial artificial chromosome (BAC) transgenesis. We generated hESC lines stably expressing Hes5::GFP, Dll1::GFP, and HB9::GFP BACs that yield green fluorescent protein (GFP)(+) neural stem cells, neuroblasts, and motor neurons, respectively. Faithful reporter expression was confirmed by cell fate analysis and appropriate transgene regulation. Prospective isolation of HB9::GFP(+) cells yielded purified human motor neurons with proper marker expression and electrophysiological activity. Global mRNA and microRNA analyses of Hes5::GFP(+) and HB9::GFP(+) populations revealed highly specific expression signatures, suggesting that BAC transgenesis will be a powerful tool for establishing expression libraries that define the human neural lineage and for accessing defined cell types in applications of human disease.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Electrofisiología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Receptores Notch/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare TLR3 variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-ß and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients' iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN-mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.
Asunto(s)
Gripe Humana/genética , Patrón de Herencia/genética , Neumonía/genética , Receptor Toll-Like 3/deficiencia , Alelos , Niño , Preescolar , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Resultado Fatal , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Recién Nacido , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/fisiología , Interferones/metabolismo , Mutación con Pérdida de Función/genética , Pulmón/patología , Masculino , Mutación Missense/genética , Poli I-C/farmacología , Transporte de ProteínasRESUMEN
Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.
Asunto(s)
Encefalitis por Herpes Simple/genética , Herpesvirus Humano 1/genética , Neuronas/inmunología , ARN Nucleolar Pequeño/genética , Adulto , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/virología , Preescolar , Encefalitis por Herpes Simple/inmunología , Encefalitis por Herpes Simple/patología , Encefalitis por Herpes Simple/virología , Femenino , Predisposición Genética a la Enfermedad , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Humanos , Inmunidad/genética , Lactante , Masculino , Metagenoma/genética , Metagenoma/inmunología , Persona de Mediana Edad , Neuronas/virología , ARN Nucleolar Pequeño/inmunologíaRESUMEN
Familial dysautonomia (FD) is a debilitating disorder that affects derivatives of the neural crest (NC). For unknown reasons, people with FD show marked differences in disease severity despite carrying an identical, homozygous point mutation in IKBKAP, encoding IκB kinase complex-associated protein. Here we present disease-related phenotypes in human pluripotent stem cells (PSCs) that capture FD severity. Cells from individuals with severe but not mild disease show impaired specification of NC derivatives, including autonomic and sensory neurons. In contrast, cells from individuals with severe and mild FD show defects in peripheral neuron survival, indicating that neurodegeneration is the main culprit for cases of mild FD. Although genetic repair of the FD-associated mutation reversed early developmental NC defects, sensory neuron specification was not restored, indicating that other factors may contribute to disease severity. Whole-exome sequencing identified candidate modifier genes for individuals with severe FD. Our study demonstrates that PSC-based modeling is sensitive in recapitulating disease severity, which presents an important step toward personalized medicine.
Asunto(s)
Sistema Nervioso Autónomo/fisiopatología , Disautonomía Familiar/fisiopatología , Células Madre Pluripotentes Inducidas , Células Receptoras Sensoriales/citología , Adolescente , Adulto , Sistema Nervioso Autónomo/citología , Sistema Nervioso Autónomo/crecimiento & desarrollo , Proteínas Portadoras/genética , Estudios de Casos y Controles , Supervivencia Celular/genética , Niño , Disautonomía Familiar/genética , Femenino , Genotipo , Humanos , Masculino , Modelos Neurológicos , Mutación , Cresta Neural/citología , Neuronas/citología , Fenotipo , Análisis de Secuencia de ADN , Índice de Severidad de la Enfermedad , Factores de Elongación Transcripcional , Adulto JovenRESUMEN
Herpes simplex virus 1 (HSV-1) is a common virus that can rarely invade the human central nervous system (CNS), causing devastating encephalitis. The permissiveness to HSV-1 of the various relevant cell types of the CNS, neurons, astrocytes, oligodendrocytes, and microglia cells, as well as their response to viral infection, has been extensively studied in humans and other animals. Nevertheless, human CNS cell-based models of anti-HSV-1 immunity are of particular importance, as responses to any given neurotropic virus may differ between humans and other animals. Human CNS neuron cell lines as well as primary human CNS neurons, astrocytes, and microglia cells cultured/isolated from embryos or cadavers, have enabled the study of cell-autonomous anti-HSV-1 immunity in vitro. However, the paucity of biological samples and their lack of purity have hindered progress in the field, which furthermore suffers from the absence of testable primary human oligodendrocytes. Recently, the authors have established a human induced pluripotent stem cells (hiPSCs)-based model of anti-HSV-1 immunity in neurons, oligodendrocyte precursor cells, astrocytes, and neural stem cells, which has widened the scope of possible in vitro studies while permitting in-depth explorations. This mini-review summarizes the available data on human primary and iPSC-derived CNS cells for anti-HSV-1 immunity. The hiPSC-mediated study of anti-viral immunity in both healthy individuals and patients with viral encephalitis will be a powerful tool in dissecting the disease pathogenesis of CNS infections with HSV-1 and other neurotropic viruses.
RESUMEN
Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient's leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient's dermal fibroblasts and induced pluripotent stem cell (iPSC)-derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.
Asunto(s)
Heterocigoto , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/inmunología , Factor 7 Regulador del Interferón/genética , Interferón Tipo I/biosíntesis , Síndrome de Dificultad Respiratoria/inmunología , Niño , Células Dendríticas/inmunología , Femenino , Fibroblastos/inmunología , Genes Recesivos , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Gripe Humana/complicaciones , Gripe Humana/genética , Interferón Tipo I/genética , Leucocitos/inmunología , Pulmón/inmunología , Mutación , Síndrome de Dificultad Respiratoria/genética , Síndrome de Dificultad Respiratoria/virología , Mucosa Respiratoria/inmunologíaRESUMEN
Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.
Asunto(s)
Técnicas Citológicas/métodos , Células Madre Pluripotentes Inducidas/citología , Cresta Neural/citología , Células Madre/citología , Animales , Diferenciación Celular/fisiología , Humanos , RatonesRESUMEN
The long-term risk of malignancy associated with stem cell therapies is a significant concern in the clinical application of this exciting technology. We report a cancer-selective strategy to enhance the safety of stem cell therapies. Briefly, using a cell engineering approach, we show that aggressive cancers derived from human or murine induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are strikingly sensitive to temporary MYC blockade. On the other hand, differentiated tissues derived from human or mouse iPSCs can readily tolerate temporary MYC inactivation. In cancer cells, endogenous MYC is required to maintain the metabolic and epigenetic functions of the embryonic and cancer-specific pyruvate kinase M2 isoform (PKM2). In summary, our results implicate PKM2 in cancer's increased MYC dependence and indicate dominant MYC inhibition as a cancer-selective fail-safe for stem cell therapies.
Asunto(s)
Ingeniería Celular , Tratamiento Basado en Trasplante de Células y Tejidos/normas , Células Madre Pluripotentes Inducidas/citología , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/diagnóstico por imagen , Femenino , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Imagen por Resonancia Magnética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/terapia , Neurogénesis , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Radiografía , Hormonas Tiroideas/metabolismo , Proteínas de Unión a Hormona TiroideRESUMEN
OBJECTIVE: To determine the proportion of children with herpes simplex encephalitis (HSE) displaying TLR3 deficiency, the extent of TLR3 allelic heterogeneity, and the specific clinical features of TLR3 deficiency. METHODS: We determined the sequence of all exons of TLR3 in 110 of the 120 patients with HSE enrolled in our study who do not carry any of the previously described HSE-predisposing mutations of TLR3 pathway genes (TLR3, UNC93B1, TRIF, TRAF3, and TBK1). All the new mutant TLR3 alleles detected were characterized experimentally in-depth to establish the causal relationship between the genotype and phenotype. RESULTS: In addition to the 3 previously reported TLR3-deficient patients from the same cohort, 6 other children or young adults with HSE carry 1 of 5 unique or extremely rare (minor allele frequency <0.001) missense TLR3 alleles. Two alleles (M374T, D592N) heterozygous in 3 patients are not deleterious in vitro. The other 3 are deleterious via different mechanisms: G743D+R811I and L360P heterozygous in 2 patients are loss-of-function due to low levels of expression and lack of cleavage, respectively, and R867Q homozygous in 1 patient is hypomorphic. The 3 patients' fibroblasts display impaired TLR3 responses and enhanced herpes simplex virus 1 susceptibility. Overall, TLR3 deficiency is therefore found in 6 (5%) of the 120 patients studied. There is high allelic heterogeneity, with 3 forms of autosomal dominant partial defect by negative dominance or haploinsufficiency, and 2 forms of autosomal recessive defect with complete or partial deficiency. Finally, 4 (66%) of the 6 TLR3-deficient patients had at least 1 late relapse of HSE, whereas relapse occurred in only 12 (10%) of the total cohort of 120 patients. CONCLUSIONS: Childhood-onset HSE is due to TLR3 deficiency in a traceable fraction of patients, in particular the ones with HSE recurrence. Mutations in TLR3 and TLR3 pathway genes should be searched and experimentally studied in children with HSE, and patients with proven TLR3 deficiency should be followed carefully.
Asunto(s)
Encefalitis por Herpes Simple/diagnóstico , Encefalitis por Herpes Simple/genética , Frecuencia de los Genes/genética , Mutación/genética , Receptor Toll-Like 3/deficiencia , Receptor Toll-Like 3/genética , Adolescente , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Linaje , Recurrencia , Factores de RiesgoRESUMEN
OBJECTIVE: Medically refractory epilepsy is amenable to neurosurgical intervention if the epileptogenic focus is accurately localized. If the scalp video-electroencephalography (EEG) and magnetic resonance imaging are nonlateralizing, yet a single focus is suspected, video-EEG monitoring with bilateral intracranial electrode placement is helpful to lateralize the ictal onset zone. We describe the indications, risks, and utility of such bilateral surveys at our institution. METHODS: We retrospectively reviewed 26 patients with medically refractory seizures who were treated over a 5-year period and underwent bilateral placement of intracranial electrodes. Subdural strips were used in all cases, and additional stereotactic implantation of depth electrodes into mesial temporal lobes occurred in 50%. The mean patient age was 37.7 years, and 65.4% of patients were male. RESULTS: The most common indication for bilateral invasive monitoring was bilateral ictal onsets on surface video-EEG (76.9%), followed by frequent interictal spikes contralateral to a single ictal focus (7.7%). Intracranial monitoring lasted an average of 8.2 days, with ictal events recorded in all cases. Ten patients (38.5%) subsequently underwent more extensive unilateral monitoring via implantation of subdural and depth electrodes through a craniotomy. A therapeutic procedure was performed in 17 patients (65.4%), whereas 1 patient underwent a palliative corpus callosotomy (3.8%). Nine patients underwent a resection without unilateral invasive mapping. Reasons for no therapeutic surgery (n = 8) included multifocal onsets, failing the Wada test, refusal of further treatment, and negative intraoperative electrocorticogram. There was 1 surgical complication, involving a retained electrode fragment that was removed in a separate minor procedure. Of the 26 patients, 15 (57.7%) are now seizure-free or have seizure disorders that have substantially improved (modified Engel classes I and II). Of the 17 patients who underwent a potentially curative surgery, 13 (76.5%) were Engel classes I and II. CONCLUSION: Bilateral placement of subdural strip and depth electrodes for epilepsy monitoring in patients with nonlateralizing scalp EEG and/or discordant imaging studies but clinical suspicion for focal seizure origin is both safe and effective. Given the safety and efficacy of this procedure, epileptologists should have a low threshold to consider bilateral implants for suitable patients.