A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses.

Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17ß-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyer's patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-ß, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.

Role of CAMK2D in neurodevelopment and associated conditions.

The calcium/calmodulin-dependent protein kinase type 2 (CAMK2) family consists of four different isozymes, encoded by four different genes-CAMK2A, CAMK2B, CAMK2G, and CAMK2D-of which the first three have been associated recently with neurodevelopmental disorders. CAMK2D is one of the major CAMK2 proteins expressed in the heart and has been associated with cardiac anomalies. Although this CAMK2 isoform is also known to be one of the major CAMK2 subtypes expressed during early brain development, it has never been linked with neurodevelopmental disorders until now. Here we show that CAMK2D plays an important role in neurodevelopment not only in mice but also in humans. We identified eight individuals harboring heterozygous variants in CAMK2D who display symptoms of intellectual disability, delayed speech, behavioral problems, and dilated cardiomyopathy. The majority of the variants tested lead to a gain of function (GoF), which appears to cause both neurological problems and dilated cardiomyopathy. In contrast, loss-of-function (LoF) variants appear to induce only neurological symptoms. Together, we describe a cohort of individuals with neurodevelopmental disorders and cardiac anomalies, harboring pathogenic variants in CAMK2D, confirming an important role for the CAMK2D isozyme in both heart and brain function.

Localized Plasmonic Heating for Single-Molecule DNA Rupture Measurements in Optical Tweezers.

To date, studies on the thermodynamic and kinetic processes that underlie biological function and nanomachine actuation in biological- and biology-inspired molecular constructs have primarily focused on photothermal heating of ensemble systems, highlighting the need for probes that are localized within the molecular construct and capable of resolving single-molecule response. Here we present an experimental demonstration of wavelength-selective, localized heating at the single-molecule level using the surface plasmon resonance of a 15 nm gold nanoparticle (AuNP). Our approach is compatible with force-spectroscopy measurements and can be applied to studies of the single-molecule thermodynamic properties of DNA origami nanomachines as well as biomolecular complexes. We further demonstrate wavelength selectivity and establish the temperature dependence of the reaction coordinate for base-pair disruption in the shear-rupture geometry, demonstrating the utility and flexibility of this approach for both fundamental studies of local (nanometer-scale) temperature gradients and rapid and multiplexed nanomachine actuation.

The protective effect of 1400W against ischaemia and reperfusion injury is countered by transient medullary kidney endothelial dysregulation.

Renal ischaemia and reperfusion (I/R) is caused by a sudden temporary impairment of the blood flow. I/R is a prevalent cause of acute kidney injury. As nitric oxide generated by inducible nitric oxide synthase (iNOS) has detrimental effects during I/R, the pharmacological blockade of iNOS has been proposed as a potential strategy to prevent I/R injury. The aim of this study was to improve the understanding of 1400W (an iNOS inhibitor) on renal I/R as a pharmacological strategy against kidney disease. BALB/c mice received 30 min of bilateral ischaemia, followed by 48 h or 28 days of reperfusion. Vehicle or 1400W (10 mg/kg) was administered 30 min before inducing ischaemia. We found that after 48 h of reperfusion 1400W decreased the serum creatinine, blood urea nitrogen, neutrophil gelatinase-associated lipocalin and proliferating cell nuclear antigen 3 in the I/R animals. Unexpectedly, we observed mRNA upregulation of genes involved in kidney injury, cell-cycle arrest, inflammation, mesenchymal transition and endothelial activation in the renal medulla of sham animals treated with 1400W. We also explored if 1400W promoted chronic kidney dysfunction 28 days after I/R and did not find significant alterations in renal function, fibrosis, blood pressure or mortality. The results provide evidence that 1400W may have adverse effects in the renal medulla. Importantly, our data point to 1400W-induced endothelial dysfunction, establishing therapeutic limitations for its use. KEY POINTS: Acute kidney injury is a global health problem associated with high morbidity and mortality. The pharmacological blockade of inducible nitric oxide synthase (iNOS) has been proposed as a potential strategy to prevent AKI induced by ischaemia and reperfusion (I/R). Our main finding is that 1400W, a selective and irreversible iNOS inhibitor with low toxicity that is proposed as a therapeutic strategy to prevent kidney I/R injury, produces aberrant gene expression in the medulla associated to tissue injury, cell cycle arrest, inflammation, mesenchymal transition and endothelial activation. The negative effect of 1400W observed in the renal medulla at 48 h from drug administration, is transient as it did not translate into a chronic kidney disease condition.

Outbreak of Intermediate Species Leptospira venezuelensis Spread by Rodents to Cows and Humans in L. interrogans-Endemic Region, Venezuela.

Leptospirosis is a common but underdiagnosed zoonosis. We conducted a 1-year prospective study in La Guaira State, Venezuela, analyzing 71 hospitalized patients who had possible leptospirosis and sampling local rodents and dairy cows. Leptospira rrs gene PCR test results were positive in blood or urine samples from 37/71 patients. Leptospira spp. were isolated from cultured blood or urine samples of 36/71 patients; 29 had L. interrogans, 3 L. noguchii, and 4 L. venezuelensis. Conjunctival suffusion was the most distinguishing clinical sign, many patients had liver involvement, and 8/30 patients with L. interrogans infections died. The Leptospira spp. found in humans were also isolated from local rodents; L. interrogans and L. venezuelensis were isolated from cows on a nearby, rodent-infested farm. Phylogenetic clustering of L. venezuelensis isolates suggested a recently expanded outbreak strain spread by rodents. Increased awareness of leptospirosis prevalence and rapid diagnostic tests are needed to improve patient outcomes.

Lower Microscopy Sensitivity with Decreasing Malaria Prevalence in the Urban Amazon Region, Brazil, 2018-2021.

Malaria is increasingly diagnosed in urban centers across the Amazon Basin. In this study, we combined repeated prevalence surveys over a 4-year period of a household-based random sample of 2,774 persons with parasite genotyping to investigate the epidemiology of malaria in Mâncio Lima, the main urban transmission hotspot in Amazonian Brazil. We found that most malarial infections were asymptomatic and undetected by point-of-care microscopy. Our findings indicate that as malaria transmission decreases, the detection threshold of microscopy rises, resulting in more missed infections despite similar parasite densities estimated by molecular methods. We identified genetically highly diverse populations of Plasmodium vivax and P. falciparum in the region; occasional shared lineages between urban and rural residents suggest cross-boundary propagation. The prevalence of low-density and asymptomatic infections poses a significant challenge for routine surveillance and the effectiveness of malaria control and elimination strategies in urbanized areas with readily accessible laboratory facilities.

Serum Extracellular Vesicles Cargo Approach in Bitches with Mammary Tumors.

This study investigated serum extracellular vesicles (EVs) in bitches with mammary neoplasms, in order to understand their size, shape, and concentration, as well as their association with tumor malignancy. Thirty bitches were categorized into control (n = 10), mammary tumor grades I and II (GI, n = 13), and grade III (GII, n = 7). Serum was separated from blood collected during mastectomy, and EVs were isolated using size exclusion chromatography. The analysis revealed no significant differences in EV concentrations among groups, with similar concentrations for control, GI, and GII. Ninety-one proteins were identified in EV-enriched samples, with six showing varied abundance across groups. Notably, keratin 18 was highly abundant in GI, while sushi domain-containing protein, EvC ciliary subunit 2, and the joining chain of multimeric IgM and IgA were increased in GII. Additionally, protocadherin 17 and albumin were upregulated in both GI and GII. ROC curves identified potential biomarkers for differentiating tumor grades. Enrichment pathway analysis revealed AFP gene upregulation in the GI. Mass spectrometry proteomics data were deposited in Mendeley Data. The study provides valuable insights into serum EV characterization in bitches, suggesting keratin 18 and protocadherin 17 as potential biomarkers for canine mammary neoplasia, with implications for future diagnostic and therapeutic strategies.

Applying two approaches to detect unmeasured confounding due to time-varying variables in a self-controlled risk interval design evaluating COVID-19 vaccine safety signals, using myocarditis as a case example.

We test the robustness of the self-controlled risk interval (SCRI) design in a setting where time between doses may introduce time-varying confounding, using both negative control outcomes (NCOs) and quantitative bias analysis (QBA). All vaccinated cases identified from 5 European databases between 1 September 2020 and end of data availability were included. Exposures were doses 1-3 of the Pfizer, Moderna, AstraZeneca, and Janssen COVID-19 vaccines; outcomes were myocarditis and otitis externa (NCO). The SCRI used a 60-day control window and dose-specific 28-day risk windows, stratified by vaccine brand and adjusted for calendar time. The QBA included two scenarios: (i) baseline probability of the confounder was higher in the control window and (ii) vice versa. The NCO was not associated with any of the COVID-19 vaccine types or doses except Moderna dose 1 (IRR = 1.09, 95%CI 1.01-1.09). The QBA suggested even the strongest literature-reported confounder (COVID-19; RRmyocarditis = 18.3) could only explain away part of the observed effect from IRR = 3 to IRR = 1.40. The SCRI seems robust to unmeasured confounding in the COVID-19 setting, although a strong unmeasured confounder could bias the observed effect upward. Replication of our findings for other safety signals would strengthen this conclusion.

Expanding the phenotype of neurofibromatosis type 1 microdeletion syndrome.

Neurofibromatosis type 1 (NF-1) microdeletion syndrome accounts for 5 to 11% of individuals with NF-1. The aim of our study was to characterize a large cohort of individuals with NF-1 microdeletion syndrome and expand its natural history. We conducted a retrospective chart review from 1994 to 2024 of individuals with NF-1 microdeletion syndrome followed at two large Neurofibromatosis Clinics. This cohort consists of 57 individuals with NF-1 microdeletion syndrome (28 type-1, 4 type-2, 2 type-3, 9 atypical deletions, and 14 indeterminate). We note 38/56 (67.9%) with describable facial features, 25/57 (43.8%) with plexiform neurofibromas, and 3/57 (5.2%) with malignant peripheral nerve sheath tumors within the observed period. The most reported neurodevelopmental manifestations from school-age or older individuals included 39/49 (79.6%) with developmental delays, 35/49 (71.4%) with expressive and/or receptive speech delays, 33/41 (80.5%) with learning difficulties, and 23/42 (54.8%) with attention-deficit/hyperactivity disorder. Full-scale IQ testing data was available for 22 individuals (range: 50-96). Of the 21 adults in this cohort, 14/21 (66.7%) graduated from high school, and 4/21 (19.0%) had some college experience. Many individuals received academic support (i.e., special education, individual education plan). In this cohort, neurocognitive outcomes in adults varied more than typically reported in the literature.

Aerobic exercise training combined with local strength exercise restores muscle blood flow and maximal aerobic capacity in long-term Hodgkin lymphoma survivors.

It is unclear whether muscle blood flow (MBF) is altered in long-term Hodgkin lymphoma (HL) survivors. We tested the hypothesis that 1) MBF response during mental stress (MS) is impaired in long-term HL survivors and 2) aerobic exercise training combined with local strength exercise (ET) restores MBF responses during MS in these survivors. Eighteen 5-year HL survivors and 10 aged-paired healthy subjects (HC) were studied. Twenty HL survivors were randomly divided into two groups: exercise-trained (HLT, n = 10) and untrained (HLUT, n = 10). Maximal aerobic capacity was evaluated by a cardiopulmonary exercise test and forearm blood flow (FBF) by venous occlusion plethysmography. MS was elicited by Stroop color and word test. ET was conducted for 4 mo, 3/wk for 60 min each session. The aerobic exercise intensity corresponded to anaerobic threshold up to 10% below the respiratory compensation point. The strength exercises consisted of two to three sets of chest press, pulley and squat exercises, 12-15 repetitions each exercise at 30-50% of the maximal voluntary contraction. Baseline was similar in HL survivors and HC, except peak oxygen consumption (peak V̇o2, P = 0.013) and FBF (P = 0.006) that were lower in the HL survivors. FBF responses during MS were lower in HL survivors (P < 0.001). ET increased peak V̇o2 (11.59 ± 3.07%, P = 0.002) and FBF at rest (33.74 ± 5.13%, P < 0.001) and during MS (24 ± 5.31%, P = 0.001). Further analysis showed correlation between the changes in peak V̇o2 and the changes in FBF during MS (r = 0.711, P = 0.001). In conclusion, long-term HL survivors have impaired MBF responses during MS. ET restores MBF responses during MS.NEW & NOTEWORTHY Long-term Hodgkin lymphoma (HL) survivors have impaired muscle blood flow responses during mental stress and decreased maximal aerobic capacity. Supervised aerobic exercise training combined with local strength exercises restores muscle blood flow responses during mental stress and maximal aerobic capacity in these survivors. These findings provide evidence of safety and effectiveness of exercise training in HL survivors. Moreover, they highlight the importance of exercise training in the treatment of this set of patients.

Morphological heterogeneity of neurons in the human central amygdaloid nucleus.

The central amygdaloid nucleus (CeA) has an ancient phylogenetic development and functions relevant for animal survival. Local cells receive intrinsic amygdaloidal information that codes emotional stimuli of fear, integrate them, and send cortical and subcortical output projections that prompt rapid visceral and social behavior responses. We aimed to describe the morphology of the neurons that compose the human CeA (N = 8 adult men). Cells within CeA coronal borders were identified using the thionine staining and were further analyzed using the "single-section" Golgi method followed by open-source software procedures for two-dimensional and three-dimensional image reconstructions. Our results evidenced varied neuronal cell body features, number and thickness of primary shafts, dendritic branching patterns, and density and shape of dendritic spines. Based on these criteria, we propose the existence of 12 morphologically different spiny neurons in the human CeA and discuss the variability in the dendritic architecture within cellular types, including likely interneurons. Some dendritic shafts were long and straight, displayed few collaterals, and had planar radiation within the coronal neuropil volume. Most of the sampled neurons showed a few to moderate density of small stubby/wide spines. Long spines (thin and mushroom) were observed occasionally. These novel data address the synaptic processing and plasticity in the human CeA. Our morphological description can be combined with further transcriptomic, immunohistochemical, and electrophysiological/connectional approaches. It serves also to investigate how neurons are altered in neurological and psychiatric disorders with hindered emotional perception, in anxiety, following atrophy in schizophrenia, and along different stages of Alzheimer's disease.

Evaluation of long-term immune response in cattle to botulism using a recombinant E. coli bacterin formulated with Montanide™ ISA 50 and aluminum hydroxide adjuvants.

Botulism is a severe disease caused by potent botulinum neurotoxins (BoNTs) produced by Clostridium botulinum. This disease is associated with high-lethality outbreaks in cattle, which have been linked to the ingestion of preformed BoNT serotypes C and D, emphasizing the need for effective vaccines. The potency of current commercial toxoids (formaldehyde-inactivated BoNTs) is assured through tests in guinea pigs according to government regulatory guidelines, but their short-term immunity raises concerns. Recombinant vaccines containing the receptor-binding domain have demonstrated potential for eliciting robust protective immunity. Previous studies have demonstrated the safety and effectiveness of recombinant E. coli bacterin, eliciting high titers of neutralizing antibodies against C. botulinum and C. perfringens in target animal species. In this study, neutralizing antibody titers in cattle and the long-term immune response against BoNT/C and D were used to assess the efficacy of the oil-based adjuvant compared with that of the aluminum hydroxide adjuvant in cattle. The vaccine formulation containing Montanide™ ISA 50 yielded significantly higher titers of neutralizing antibody against BoNT/C and D (8.64 IU/mL and 9.6 IU/mL, respectively) and induced an immune response that lasted longer than the response induced by aluminum, extending between 30 and 60 days. This approach represents a straightforward, cost-effective strategy for recombinant E. coli bacterin, enhancing both the magnitude and duration of the immune response to botulism.

Correlation-Pattern-Based Continuous Variable Entanglement Detection through Neural Networks.

Entanglement in continuous-variable non-Gaussian states provides irreplaceable advantages in many quantum information tasks. However, the sheer amount of information in such states grows exponentially and makes a full characterization impossible. Here, we develop a neural network that allows us to use correlation patterns to effectively detect continuous-variable entanglement through homodyne detection. Using a recently defined stellar hierarchy to rank the states used for training, our algorithm works not only on any kind of Gaussian state but also on a whole class of experimentally achievable non-Gaussian states, including photon-subtracted states. With the same limited amount of data, our method provides higher accuracy than usual methods to detect entanglement based on maximum-likelihood tomography. Moreover, in order to visualize the effect of the neural network, we employ a dimension reduction algorithm on the patterns. This shows that a clear boundary appears between the entangled states and others after the neural network processing. In addition, these techniques allow us to compare different entanglement witnesses and understand their working. Our findings provide a new approach for experimental detection of continuous-variable quantum correlations without resorting to a full tomography of the state and confirm the exciting potential of neural networks in quantum information processing.

T-cell immune profile in blood of systemic mastocytosis: Association with disease features.

BACKGROUND: Systemic mastocytosis (SM) is a heterogeneous disease characterized by an expansion of KIT-mutated mast cells (MC). KIT-mutated MC display activated features and release MC mediators that might act on the tumour microenvironment and other immune cells. Here, we investigated the distribution of lymphocyte subsets in blood of patients with distinct subtypes of SM and determined its association with other disease features. METHODS: We studied the distribution of TCD4+ and TCD4- cytotoxic cells and their subsets, as well as total NK- and B cells, in blood of 115 SM patients-38 bone marrow mastocytosis (BMM), 67 indolent SM (ISM), 10 aggressive SM (ASM)- and 83 age-matched healthy donors (HD), using spectral flow cytometry and the EuroFlow Immunomonitoring panel, and correlated it with multilineage KITD816V, the alpha-tryptasemia genotype (HαT) and the clinical manifestations of the disease. RESULTS: SM patients showed decreased counts (vs. HD) of TCD4- cytotoxic cells, NK cells and several functional subsets of TCD4+ cells (total Th1, Th2-effector memory, Th22-terminal effector and Th1-like Tregs), together with increased T-follicular-helper and Th1/Th17-like Treg counts, associated with different immune profiles per diagnostic subtype of SM, in multilineal versus MC-restricted KITD816V and in cases with a HαT+ versus HαT- genotype. Unique immune profiles were found among BMM and ISM patients with MC-restricted KITD816V who displayed HαT, anaphylaxis, hymenoptera venom allergy, bone disease, pruritus, flushing and GI symptoms. CONCLUSION: Our results reveal altered T- and NK-cell immune profiles in blood of SM, which vary per disease subtype, the pattern of involvement of haematopoiesis by KITD816V, the HαT genotype and specific clinical manifestations of the disease.

Retrospective multicenter study of elderly patients with platinum-sensitive relapsed ovarian cancer treated with trabectedin and pegylated liposomal doxorubicin (pld) in a real-world setting: a geico study.

BACKGROUND: Trabectedin in combination with pegylated liposomal doxorubicin (PLD) is approved for the treatment of patients with platinum-sensitive relapsed ovarian cancer. Nevertheless, there is currently limited information regarding this treatment in elderly patients with ovarian cancer in a real-world setting. METHODS: This observational and multicentric study retrospectively evaluated trabectedin plus PLD in a real-world setting treatment of elderly patients diagnosed with platinum-sensitive relapsed ovarian cancer, treated according to the Summary of Product Characteristics (SmPC) from 15 GEICO-associated hospitals. Patients ≥ 70 years old at the time of treatment initiation and platinum-free intervals ≥ 6 months were considered eligible. RESULTS: Forty-three patients with a median age of 74.0 years were treated between January 1st, 2015, and December 31st, 2019 in 15 Spanish centers. Four patients achieved complete response (9.3%), 14 (32.6%) partial response, and 13 (30.2%) stable disease as the best radiological response. In the analysis of biological overall response according to CA125 serum levels (i.e., Rustin criteria), 14 responded to the treatment (32.6%), 11 responded and normalized (25.6%), three patients stabilized (7.0%) and three progressed (7.0%). Median progression-free survival (PFS) and overall survival (OS) in the study population were 7.7 and 19.5 months, respectively. The most common grade 3/4 adverse events were neutropenia (n = 8, 18.7%) and asthenia (n = 5, 11.6%). CONCLUSIONS: This analysis demonstrated that trabectedin combined with PLD is a feasible and effective treatment in elderly patients with platinum-sensitive relapsed ovarian cancer, showing an acceptable safety profile, which is crucial in the palliative treatment of these patients.

Dandy-Walker malformation in an individual with ABL1 variant.

Dandy-Walker malformation (DWM) is often sporadic, but there are a growing number of genetic disorders that have been associated with this condition. We present a female individual with a de novo variant in ABL1, c.734A>G (p.Y245), who was diagnosed prenatally with DWM. ABL1-related neurodevelopmental disorder was recently identified but brain malformations have not been well characterized to date. We reviewed the published literature and identified one additional individual with DWM and ABL1-related disorder, which suggests a possible association with this malformation.

Homozygosity for disease-causing variants in AMT and GLDC in a patient with severe nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is a relatively well-characterized inborn error of metabolism that results in a combination of lethargy, hypotonia, seizures, developmental arrest, and, in severe cases, death early in life. Three genes encoding components of the glycine cleavage enzyme system-GLDC, AMT, and GCSH-are independently associated with NKH. We report on a patient with severe NKH in whom the homozygous pathogenic variant in AMT (NM_000481.3):c.602_603del (p.Lys201Thrfs*75) and the homozygous likely pathogenic variant in GLDC(NM_000170.2):c.2852C>A (p.Ser951Tyr) were both identified. Our patient demonstrates a novel combination of two homozygous disease-causing variants impacting the glycine cleavage pathway at two different components, and elicits management- and genetic counseling-related challenges for the family.

Aminotransferase trends in propionic acidemia.

Propionic acidemia is a metabolic condition with multiple serious acute and chronic presentations that require strict monitoring. Literature on liver function abnormalities in propionic acidemia is scarce, and the mechanism of liver impairment in this condition remains unclear. Currently, there is no indication for liver-function tests during follow-up and their clinical or prognostic utility is unknown. This study aimed to determine aminotransferase trends in individuals with propionic acidemia at a single institution. We retrospectively evaluated and classified the aminotransferases of 12 patients with propionic acidemia during hospital admissions and routine office visits. The present findings suggest that aminotransferase elevations are very common in this population and can persist beyond acute illness. During hospitalization events, aminotransferases were not a predictor of severity, duration of stay, and readmission within 1 month. Understanding aminotransferase trends in these patients will help clinicians make decisions in the acute setting and potentially in the follow-up of new therapies.

Presentation of ichthyosis after substrate reduction therapy in Gaucher type 1.

We describe a case in which a type 1 Gaucher patient developed ichthyosis weeks after starting substrate reduction therapy (SRT) with eliglustat. There are no reports of ichthyosis in the literature in enzyme replacement or SRT for Gaucher disease. Ichthyosis is seen with type 2 and 3 Gaucher disease, but not type 1. This raises the question: Why would a patient develop ichthyosis after starting SRT?

The value of additional electrodes when stereo-electroencephalography is inconclusive.

OBJECTIVE: Stereo-electroencephalography (SEEG) is the preferred method for intracranial localization of the seizure-onset zone (SOZ) in drug-resistant focal epilepsy. Occasionally SEEG evaluation fails to confirm the pre-implantation hypothesis. This leads to a decision tree regarding whether the addition of SEEG electrodes (two-step SEEG - 2sSEEG) or placement of subdural electrodes (SDEs) after SEEG (SEEG2SDE) would help. There is a dearth of literature encompassing this scenario, and here we aimed to characterize outcomes following unplanned two-step intracranial EEG (iEEG). METHODS: All 225 adult SEEG cases over 8 years at our institution were reviewed to extract patient data and outcomes following a two-step evaluation. Three raters independently quantified benefits of additional intracranial electrodes. The relationship between two-step iEEG benefit and clinical outcome was then analyzed. RESULTS: Fourteen patients underwent 2sSEEG and nine underwent SEEG2SDE. In the former cohort, the second SEEG procedure was performed for these reasons-precise localization of the SOZ (36%); defining margins of eloquent cortex (21%); and broadening coverage in the setting of non-localizable seizure onsets (43% of cases). Sixty-four percent of 2sSEEG cases were consistently deemed beneficial (Light's κ = 0.80). 2sSEEG performed for the first two indications was much more beneficial than when onsets were not localizable (100% vs 17%, p = .02). In the SEEG2SDE cohort, SDEs identified the SOZ and enabled delineation of margins relative to eloquent cortex in all cases. SIGNIFICANCE: The two-step iEEG is useful if the initial evaluation is broadly concordant with the original electroclinical hypothesis, where it can clarify onset zones or delineate safe surgical margins; however, it provides minimal benefit when the implantation hypothesis is erroneous, and we recommend that 2sSEEG not be generally utilized in such cases. SDE implantation after SEEG minimizes the need for SDEs and is helpful in delineating surgical boundaries relative to ictal-onset zones and eloquent cortex.