A self-sustaining layer of early-life-origin B cells drives steady-state IgA responses in the adult gut.

The adult immune system consists of cells that emerged at various times during ontogeny. We aimed to define the relationship between developmental origin and composition of the adult B cell pool during unperturbed hematopoiesis. Lineage tracing stratified murine adult B cells based on the timing of output, revealing that a substantial portion originated within a restricted neonatal window. In addition to B-1a cells, early-life time-stamped B cells included clonally interrelated IgA plasma cells in the gut and bone marrow. These were actively maintained by B cell memory within gut chronic germinal centers and contained commensal microbiota reactivity. Neonatal rotavirus infection recruited recurrent IgA clones that were distinct from those arising by infection with the same antigen in adults. Finally, gut IgA plasma cells arose from the same hematopoietic progenitors as B-1a cells during ontogeny. Thus, a complex layer of neonatally imprinted B cells confer unique antibody responses later in life.

Effects of psychiatric comorbidities in persons with epilepsy on recurrent emergency department visits.

BACKGROUND: Coexisting mental health disorders in persons with epilepsy present as substantial burdens to patients and healthcare systems. STUDY DESIGN AND METHODS: In this retrospective study, we reviewed 160 patients presenting to a safety net hospital Emergency Department (ED) with seizures to investigate whether differences in clinical workup, follow-ups, and ED visit recurrence existed between epilepsy patients with epilepsy with and without a coexisting psychiatric disorder. RESULTS: Patients with epilepsy with a psychiatric comorbidity had more subsequent ED visits (45 % vs 26 %, p = 0.01) and fewer outpatient follow-up opportunities (74 % vs 87 %, p = 0.042) compared to patients with epilepsy without psychiatric comorbidities, highlighting a healthcare gap that needs to be addressed. INTERPRETATION: Our findings suggest a need for ED providers to shift their clinical practice in favor of offering more outpatient follow-up opportunities, to ensure long-term management of seizures in patients with epilepsy with comorbid psychiatric disorders.

Human Primary Airway Basal Cells Display a Continuum of Molecular Phases from Health to Disease in Chronic Obstructive Pulmonary Disease.

Airway basal cells are crucial for regeneration of the human lung airway epithelium and are believed to be important contributors to chronic obstructive pulmonary disease (COPD) and other lung disorders. To reveal how basal cells contribute to disease and to discover novel therapeutic targets, these basal cells need to be further characterized. In this study, we optimized a flow cytometry-based cell sorting protocol for primary human airway basal cells dependent on cell size and NGFR (nerve-growth factor receptor) expression. The basal cell population was found to be molecularly and functionally heterogeneous, in contrast to cultured basal cells. In addition, significant differences were found, such as KRT14 expression exclusively existing in cultured cells. Also, colony-forming capacity was significantly increased in cultured cells showing a clonal enrichment in vitro. Next, by single-cell RNA sequencing on primary basal cells from healthy donors and patients with Global Initiative for Chronic Obstructive Lung Disease stage IV COPD, the gene expression revealed a continuum ranging from healthy basal cell signatures to diseased basal cell phenotypes. We identified several upregulated genes that may indicate COPD, such as stress response-related genes GADD45B and AHSA1, together with with genes involved in the response to hypoxia, such as CITED2 and SOD1. Taken together, the presence of healthy basal cells in stage IV COPD demonstrates the potential for regeneration through the discovery of novel therapeutic targets. In addition, we show the importance of studying primary basal cells when investigating disease mechanisms as well as for developing future cell-based therapies in the human lung.

Emergency department visits and readmissions in patients with psychogenic nonepileptic seizures (PNES) at a safety net hospital.

INTRODUCTION: Readmissions and emergency department (ED) visits after an index admission have been become a quality measure due to associations with poor outcomes and increased healthcare costs. Readmissions and ED encounters have been studied in a variety of conditions including epilepsy but have not been examined exclusively in psychogenic nonepileptic seizures (PNES). In this study we examined the rate of readmissions and ED visits after a discharge from an Epilepsy Monitoring Unit (EMU) in a safety net hospital. We also determined patient phenotypes that are associated with readmissions. MATERIAL AND METHODS: This was a retrospective chart review study with index admission being a discharge from an EMU between January 1 and December 31 2016 with follow-up until August 31 2020. We obtained data regarding demographics, medical and psychiatric history, and social history and treatment interventions. Our outcome variables were both all-cause and seizure-related hospital readmissions and ED visits 30 days following the index discharge and readmissions and ED visits 30 days thereafter. RESULTS: Eleven of 122 patients (9%) had a non-seizure-related ED visit and/or hospitalization within 30 days of index discharge while 45 (37%) had re-contact with the health system thereafter for non-seizure-related issues. Seven of 122 patients (6%) had a seizure-related ED visit or hospital readmission within 30 days of discharge. Twenty-eight (23%) had a seizure-related readmission or ED visit after 30 days. Of these 28, 4 patients had been to an ER within 7 days of EMU discharge. The majority of subsequent encounters with the healthcare system were through the ED (n = 38) as compared to hospital (n = 10) and EMU readmissions (n = 9). On bivariate statistical analysis, charity or self-pay insurance status (p < 0.01), homelessness (p < 0.01), emergent EMU admission on index admission (p < 0.01), history of a psychiatric diagnosis (p < 0.02), and ED encounters 12 months prior to admission (p < 0.01) were significantly associated with readmission; however, on multivariate analysis only charity insurance status was a significant predictor. CONCLUSIONS: In this study of readmissions and ED visits after discharge with a diagnosis of PNES at a safety net hospital, we found a seizure-related readmission rate of approximately 6% in 30 days and 23% thereafter with the majority of re-contact with the hospital being in the ED. On multi-variate analysis insurance status was a significant factor associated with readmission and ED visits. Our future research directions include examining referrals and treatment completion at the hospital's PNES clinic as well as creating a risk score to better identify patients with PNES at risk of readmission.

Comparison of psychiatric comorbidities and impact on quality of life in patients with epilepsy or psychogenic nonepileptic spells.

OBJECTIVES: Psychiatric comorbidity is common in people with epilepsy (PWE) and psychogenic nonepileptic spells (PNES). These comorbidities can be detrimental to quality of life (QOL) and are often underdiagnosed and undertreated. Some types of epilepsy, such as focal temporal lobe epilepsy (TLE), have been associated with higher rates of psychiatric comorbidity. This study examined the impact of psychiatric comorbidity on QOL in patients admitted to two level 4 epilepsy monitoring units (EMUs). METHODS: In this prospective observational study, 200 patients admitted to two level 4 EMUs completed standardized surveys including the Quality of Life in Epilepsy (QOLIE-31-P), Generalized Anxiety Disorder 7-item (GAD-7), Patient Health Questionnaire (PHQ-9), and Beck Depression Inventory-II (BDI-II). Hierarchal multiple regression was performed to assess impact on QOL. RESULTS: Of the 200 participants, 113 had a diagnosis of epilepsy, 36 had a diagnosis of PNES, and 51 were excluded for nondiagnostic evaluation or dual diagnosis. Of those with epilepsy, 65 had TLE, 28 had focal extratemporal lobe epilepsy (ETLE), and 20 had nonfocal epilepsy. Patients with PNES had higher self-reported anxiety and depression levels (GAD-7: p = 0.04, PHQ-9: p < 0.01; BDI-II: p < 0.01) but similar QOL to PWE (p = 0.78). Using hierarchal multiple regression, symptoms of anxiety and depression were significant predictors of lower QOL in PWE but not in patients with PNES. There was no difference in QOL in those with ETLE and TLE. CONCLUSIONS: Our findings suggest that self-reported anxiety and depression symptoms are common in patients admitted to level 4 EMUs regardless of diagnosis and play an important role in predicting QOL in PWE. Our findings emphasize the importance of routinely screening all EMU patients for psychiatric comorbidity.

Cannabis and Epilepsy.

Objective: In recent years, the use of cannabidiol in the treatment of refractory epilepsy has been increasingly investigated and has been gaining public support as a novel way to treat these disorders. Marijuana has been used for medical purposes for thousands of years, and a lot of research has been conducted over the last several decades into the chemistry and pharmacology of marijuana and its many compounds, including cannabidiol. Methods: Using PubMed, we performed a review of the literature regarding the history of cannabinoid use in treating epilepsy. Results and conclusions: There are historical and recent scientific developments that support the use of cannabidiol in rare severe epilepsy syndromes.

Lentiviral gene therapy using cellular promoters cures type 1 Gaucher disease in mice.

Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase ß-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease.

Coxsackie B virus myositis in a healthy young man with mumps co-infection.

Infection is an established but uncommon etiology of myositis, and Coxsackie B virus has only been rarely described as a causative agent. We present a case of a 38-year-old male who presented with weakness, myalgias, and testicular pain following two weeks of upper respiratory infection. Laboratory tests revealed an elevated creatine kinase and positive serology for Coxsackie B4 and mumps. This unusual presentation of Coxsackie B myositis and mumps co-infection in a previously healthy young patient illustrates the importance of including infectious etiologies in the differential diagnosis and the potential life-threatening consequences of biased clinical reasoning.

Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways.

Cachectic muscle wasting is a frequent complication of many inflammatory conditions, due primarily to excessive muscle catabolism. However, the pathogenesis and intervention strategies against it remain to be established. Here, we tested the hypothesis that Toll-like receptor 4 (TLR4) is a master regulator of inflammatory muscle catabolism. We demonstrate that TLR4 activation by lipopolysaccharide (LPS) induces C2C12 myotube atrophy via up-regulating autophagosome formation and the expression of ubiquitin ligase atrogin-1/MAFbx and MuRF1. TLR4-mediated activation of p38 MAPK is necessary and sufficient for the up-regulation of atrogin1/MAFbx and autophagosomes, resulting in myotube atrophy. Similarly, LPS up-regulates muscle autophagosome formation and ubiquitin ligase expression in mice. Importantly, autophagy inhibitor 3-methyladenine completely abolishes LPS-induced muscle proteolysis, while proteasome inhibitor lactacystin partially blocks it. Furthermore, TLR4 knockout or p38 MAPK inhibition abolishes LPS-induced muscle proteolysis. Thus, TLR4 mediates LPS-induced muscle catabolism via coordinate activation of the ubiquitin-proteasome and the autophagy-lysosomal pathways.

Physical activity status and quality of life in patients with epilepsy - Survey from level four epilepsy monitoring units.

PURPOSE: People with epilepsy (PWE) tend to have sedentary lifestyles which may predispose them to a lower perceived quality of life (QOL). Moreover, the relationship between physical activity (PA) and QOL in populations of PWE with high disease burden has been under-studied. The goal of this study was to evaluate PA level and its impact on health-related QOL in PWE who were admitted to Level-4 epilepsy monitoring units (EMU). METHODS: In this prospective observational study, 200 patients from two EMUs in Dallas, Texas completed the following standard surveys: Rapid Assessment of Physical Activity (RAPA), the Quality of Life in Epilepsy (QOLIE-31), Patient Health Questionnaire-9 (PHQ-9), and Generalized Anxiety Disorder 7-item (GAD-7) questionnaire. Information on self-reported epilepsy history, severity of disease, and socioeconomic status were also collected. The diagnosis of epilepsy was confirmed by video-EEG monitoring. RESULTS: Among the 200 who completed the survey, 113 had a diagnosis of epilepsy and 109 of them completed the RAPA. Ninety-two (84 %) of these PWE reported a sedentary level of physical activity (RAPA < 6) and 16 % reported an active level (RAPA ≥ 6). Self-reported QOL was slightly higher in PWE with an active level of PA compared to PWE with a sedentary level of PA (63.8 ± 15.0 vs 53.7 ± 17.9, p = 0.07), even though there was no difference in the severity of self-reported mood symptoms. After controlling for employment and seizure frequency, physical activity level measured by RAPA score was also positively related to QOL (r = 0.39, p = 0.01) and negatively correlated with anxiety symptoms (r = -0.28, p = 0.02) and depression symptoms (r = -0.25, p = 0.04). CONCLUSION: The majority of PWE in this survey reported sedentary lifestyles despite most of them being young to middle-aged adults. Higher PA level was associated with fewer self-reported mood symptoms and higher QOL. In conjunction with the literature, these results suggest that PWE with a wide range of disease burden should be encouraged to participate in regular exercise to potentially improve QOL.

Emergent Admissions to the Epilepsy Monitoring Unit in the Setting of COVID-19 Pandemic-related, State-mandated Restrictions: Clinical Decision Making and Outcomes.

Due to the coronavirus disease 2019 (COVID-19) pandemic, the state of Texas-limited elective procedures to conserve beds and personal protective equipment (PPE); therefore, between March 22 and May 18, 2020, admission to the epilepsy monitoring unit (EMU) was limited only to urgent and emergent cases. We evaluated clinical characteristics and outcomes of these patients who were admitted to the EMU. Nineteen patients were admitted (one patient twice) with average age of 36.26 years (11 female) and average length of stay 3 days (range: 2-9 days). At least one event was captured on continuous EEG (cEEG) and video monitoring in all 20 admissions (atypical in one). One patient had both epileptic (ES) and psychogenic non-epileptic seizures (PNES) while 10 had PNES and 9 had ES. In 8 of 9 patients with ES, medications were changed, while in 5 patients with PNES, anti-epileptic drugs (AED) were stopped; the remaining 5 were not on medications. Of the 14 patients who had seen an epileptologist pre-admission, 13 (or 93%) had their diagnosis confirmed by EMU stay; a statistically significant finding. While typically an elective admission, in the setting of the COVID-19 pandemic, urgent and emergent EMU admissions were required for increased seizure or event frequency. In the vast majority of patients (13 of 19), admission lead to medication changes to either better control seizures or to change therapeutics as appropriate when PNES was identified.

Non-lesional mesial temporal lobe epilepsy requires bilateral invasive evaluation.

PURPOSE: Mesial temporal lobe epilepsy (MTLE) usually responds well to surgical treatment, although in non-lesional cases up to 50% of patients experience seizure relapse. The possibility of bilateral independent seizure onset should be considered as a reason for epilepsy surgery failure. METHODS: In a cohort of 177 patients who underwent invasive presurgical evaluation with stereo-tactically placed electrodes in two level four epilepsy centers, 29 had non-lesional MTLE. Invasive evaluation results are described. RESULTS: Among 29 patients with non-lesional MRI and mesial temporal lobe seizure onset recorded during stereo-EEG (SEEG) evaluation, four patients with unilateral preimplantation hypothesis had independent bilateral mesial temporal seizures on SEEG despite of unilateral non-invasive evaluation data. Three of these patients were treated with bitemporal responsive neurostimulator system (RNS). Independent bilateral mesial temporal seizures have been confirmed on RNS ECoG (electrocorticography). The fourth patient underwent right anterior temporal lobectomy. CONCLUSION: We propose that patients with non-lesional mesial temporal lobe epilepsy would benefit from bilateral invasive evaluation of mesial temporal structures to predict those patients who would be at most risk for surgical failure. Neurostimulaiton could be an initial treatment option for patients with independent bitemporal seizure onset.

Lin28b controls a neonatal to adult switch in B cell positive selection.

The ability of B-1 cells to become positively selected into the mature B cell pool, despite being weakly self-reactive, has puzzled the field since its initial discovery. Here, we explore changes in B cell positive selection as a function of developmental time by exploiting a link between CD5 surface levels and the natural occurrence of self-reactive B cell receptors (BCRs) in BCR wild-type mice. We show that the heterochronic RNA binding protein Lin28b potentiates a neonatal mode of B cell selection characterized by enhanced overall positive selection in general and the developmental progression of CD5+ immature B cells in particular. Lin28b achieves this by amplifying the CD19/PI3K/c-Myc positive feedback loop, and ectopic Lin28b expression restores both positive selection and mature B cell numbers in CD19-/- adult mice. Thus, the temporally restricted expression of Lin28b relaxes the rules for B cell selection during ontogeny by modulating tonic signaling. We propose that this neonatal mode of B cell selection represents a cell-intrinsic cue to accelerate the de novo establishment of the adaptive immune system and incorporate a layer of natural antibody-mediated immunity throughout life.

Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells.

Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at the single cell level, through subsequent transplantation and high-throughput sequencing. Furthermore, cellular barcoding allows for bona fide hematopoietic stem cells (HSCs) to be defined based on functional rather than immunophenotypic parameters. This protocol describes the work flow of lentiviral cellular barcoding, tracking 14.5 days post coitum (d.p.c.) fetal liver (FL) Lineage-Sca+cKit+ (LSK) HSPCs following long-term reconstitution (Figure 1) ( Kristiansen et al., 2016 ), but can be adapted to the cell type or time frame of choice. Figure 1.Summary of experimental workflow ( Naik et al., 2013 ).

Hepatic Leukemia Factor Maintains Quiescence of Hematopoietic Stem Cells and Protects the Stem Cell Pool during Regeneration.

The transcription factor hepatic leukemia factor (HLF) is strongly expressed in hematopoietic stem cells (HSCs) and is thought to influence both HSC self-renewal and leukemogenesis. However, the physiological role of HLF in hematopoiesis and HSC function is unclear. Here, we report that mice lacking Hlf are viable with essentially normal hematopoietic parameters, including an intact HSC pool during steady-state hematopoiesis. In contrast, when challenged through transplantation, Hlf-deficient HSCs showed an impaired ability to reconstitute hematopoiesis and became gradually exhausted upon serial transplantation. Transcriptional profiling of Hlf-deficient HSCs revealed changes associated with enhanced cellular activation, and cell-cycle analysis demonstrated a significant reduction of quiescent HSCs. Accordingly, toxic insults targeting dividing cells completely eradicated the HSC pool in Hlf-deficient mice. In summary, our findings point to HLF as a critical regulator of HSC quiescence and as an essential factor for maintaining the HSC pool during regeneration.

Preoxygenation and apneic oxygenation using Transnasal Humidified Rapid-Insufflation Ventilatory Exchange for emergency intubation.

PURPOSE: Hypoxia is one of the leading causes of anesthesia-related injury. In response to the limitations of conventional preoxygenation, Transnasal Humidified Rapid-Insufflation Ventilatory Exchange (THRIVE) has been used as a method of providing both preoxygenation and apneic oxygenation during intubation. MATERIALS AND METHODS: In this prospective, observational study, THRIVE was introduced in a critical care unit (CCU), operating room (OR), and emergency department (ED) during emergency intubation of patients at high risk of hypoxia. Linear regression analysis tested for correlation between apnea time or body mass index and hemoglobin saturation (Spo2). RESULTS: Across 71 sequential patients, the interquartile range for apnea time and decrease in Spo2 were 60 to 125 seconds and 0% to 3%, respectively. Significant desaturation occurred in 5 (7%) patients. There was no evidence of correlation between apnea time or body mass index and Spo2 (R2=0.04 and 0.08 for CCU/ED and OR and 0.01 and 0.04 CCU/ED and OR, respectively). There were no complications reported from using THRIVE. CONCLUSIONS: This study demonstrated that preoxygenation and apneic oxygenation using THRIVE were associated with a low incidence of desaturation during emergency intubation of patients at high risk of hypoxia in the CCU, OR, and ED. THRIVE has the potential to minimize the risk of hypoxia in these patient groups.

The simultaneous use of a heat and moisture exchanger and a heated humidifier causes critical airway occlusion in less than 24 hours.

PURPOSE: Heat and moisture exchangers (HMEs) and heated humidifiers (HHs) may be used together inadvertently. Such an incident occurred at our institution resulting in airway occlusion. MATERIAL AND METHODS: A bench-top study was conducted to compare the incidence of airway occlusion when using (1) no airway humidification, (2) HME alone, (3) HH alone, and (4) both HME and HH in combination as part of a standard breathing circuit. RESULTS: The simultaneous use of a HME and a HH was associated with a reduction in tidal volume (no airway humidification, P ≤ .05; HME alone, P ≤ .01; and HH alone, P ≤ .01) and an increased incidence of airway occlusion (no airway humidification, 0/7; HME alone, 0/7; HH alone, 0/7; and HME and HH in combination, 7/7; P < .0001). CONCLUSIONS: The use of a HME and a HH in combination is likely to result in airway occlusion. Precautions should be taken to ensure that both systems are not used together in clinical practice.

A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome.

Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome for prophylaxis against aortic aneurysm progression, despite limited evidence for their efficacy and safety in the disorder. Unexpectedly, Marfan mice treated with CCBs show accelerated aneurysm expansion, rupture, and premature lethality. This effect is both extracellular signal-regulated kinase (ERK1/2) dependent and angiotensin-II type 1 receptor (AT1R) dependent. We have identified protein kinase C beta (PKCß) as a critical mediator of this pathway and demonstrate that the PKCß inhibitor enzastaurin, and the clinically available anti-hypertensive agent hydralazine, both normalize aortic growth in Marfan mice, in association with reduced PKCß and ERK1/2 activation. Furthermore, patients with Marfan syndrome and other forms of inherited thoracic aortic aneurysm taking CCBs display increased risk of aortic dissection and need for aortic surgery, compared to patients on other antihypertensive agents.

The SMAD-binding domain of SKI: a hotspot for de novo mutations causing Shprintzen-Goldberg syndrome.

Shprintzen-Goldberg syndrome (SGS) is a rare, systemic connective tissue disorder characterized by craniofacial, skeletal, and cardiovascular manifestations that show a significant overlap with the features observed in the Marfan (MFS) and Loeys-Dietz syndrome (LDS). A distinguishing observation in SGS patients is the presence of intellectual disability, although not all patients in this series present this finding. Recently, SGS was shown to be due to mutations in the SKI gene, encoding the oncoprotein SKI, a repressor of TGFß activity. Here, we report eight recurrent and three novel SKI mutations in eleven SGS patients. All were heterozygous missense mutations located in the R-SMAD binding domain, except for one novel in-frame deletion affecting the DHD domain. Adding our new findings to the existing data clearly reveals a mutational hotspot, with 73% (24 out of 33) of the hitherto described unrelated patients having mutations in a stretch of five SKI residues (from p.(Ser31) to p.(Pro35)). This implicates that the initial molecular testing could be focused on mutation analysis of the first half of exon 1 of SKI. As the majority of the known mutations are located in the R-SMAD binding domain of SKI, our study further emphasizes the importance of TGFß signaling in the pathogenesis of SGS.

Genome Sequences of Mycobacteriophages AlanGrant, Baee, Corofin, OrangeOswald, and Vincenzo, New Members of Cluster B.

AlanGrant, Baee, Corofin, OrangeOswald, and Vincenzo are newly isolated phages of Mycobacterium smegmatis mc(2)155 discovered in Pittsburgh, Pennsylvania, USA. All five phages share nucleotide similarity with cluster B mycobacteriophages but span considerable diversity with Corofin and OrangeOswald in subcluster B3, AlanGrant and Vincenzo in subcluster B4, and Baee in subcluster B5.