NF-κB Signaling is Required for X-Chromosome Inactivation Maintenance Following T cell Activation.

X Chromosome Inactivation (XCI) is a female-specific process which balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of Xist RNA and heterochromatic modifications on the inactive X chromosome (Xi), and these modifications become enriched at the Xi after cell stimulation. Here, we examined allele-specific gene expression and the epigenomic profiles of the Xi following T cell stimulation. We found that the Xi in unstimulated T cells is largely dosage compensated and is enriched with the repressive H3K27me3 modification, but not the H2AK119-ubiquitin (Ub) mark, even at promoters of XCI escape genes. Upon CD3/CD28-mediated T cell stimulation, the Xi accumulates H2AK119-Ub and H3K27me3 across the Xi. Next, we examined the T cell signaling pathways responsible for Xist RNA localization to the Xi and found that T cell receptor (TCR) engagement, specifically NF-κB signaling downstream of TCR, is required. Disruption of NF-κB signaling, using inhibitors or genetic deletions, in mice and patients with immunodeficiencies prevents Xist/XIST RNA accumulation at the Xi and alters expression of some X-linked genes. Our findings reveal a novel connection between NF-κB signaling pathways which impact XCI maintenance in female T cells.

The conneXion between sex and immune responses.

There are notable sex-based differences in immune responses to pathogens and self-antigens, with female individuals exhibiting increased susceptibility to various autoimmune diseases, and male individuals displaying preferential susceptibility to some viral, bacterial, parasitic and fungal infections. Although sex hormones clearly contribute to sex differences in immune cell composition and function, the presence of two X chromosomes in female individuals suggests that differential gene expression of numerous X chromosome-linked immune-related genes may also influence sex-biased innate and adaptive immune cell function in health and disease. Here, we review the sex differences in immune system composition and function, examining how hormones and genetics influence the immune system. We focus on the genetic and epigenetic contributions responsible for altered X chromosome-linked gene expression, and how this impacts sex-biased immune responses in the context of pathogen infection and systemic autoimmunity.

c-Myc uses Cul4b to preserve genome integrity and promote antiviral CD8+ T cell immunity.

During infection, virus-specific CD8+ T cells undergo rapid bursts of proliferation and differentiate into effector cells that kill virus-infected cells and reduce viral load. This rapid clonal expansion can put T cells at significant risk for replication-induced DNA damage. Here, we find that c-Myc links CD8+ T cell expansion to DNA damage response pathways though the E3 ubiquitin ligase, Cullin 4b (Cul4b). Following activation, c-Myc increases the levels of Cul4b and other members of the Cullin RING Ligase 4 (CRL4) complex. Despite expressing c-Myc at high levels, Cul4b-deficient CD8+ T cells do not expand and clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) in vivo. Cul4b-deficient CD8+ T cells accrue DNA damage and succumb to proliferative catastrophe early after antigen encounter. Mechanistically, Cul4b knockout induces an accumulation of p21 and Cyclin E2, resulting in replication stress. Our data show that c-Myc supports cell proliferation by maintaining genome stability via Cul4b, thereby directly coupling these two interdependent pathways. These data clarify how CD8+ T cells use c-Myc and Cul4b to sustain their potential for extraordinary population expansion, longevity and antiviral responses.

Impaired dynamic X-chromosome inactivation maintenance in T cells is a feature of spontaneous murine SLE that is exacerbated in female-biased models.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a highly female-biased systemic autoimmune disease, but the molecular basis for this female bias remains incompletely elucidated. B and T lymphocytes from patients with SLE and female-biased mouse models of SLE exhibit features of epigenetic dysregulation on the X chromosome which may contribute to this strong female bias. We therefore examined the fidelity of dynamic X-chromosome inactivation maintenance (dXCIm) in the pathogenesis of two murine models of spontaneous lupus-NZM2328 and MRL/lpr-with disparate levels of female-bias to determine whether impaired dXCIm contributes to the female bias of disease. METHODS: CD23+ B cells and CD3+ T cells were purified from age-matched C57BL/6 (B6), MRL/lpr, and NZM2328 male and female mice, activated in vitro, and processed for Xist RNA fluorescence in situ hybridization, H3K27me3 immunofluorescence imaging, qPCR, and RNA sequencing analyses. RESULTS: The dynamic relocalization of Xist RNA and the canonical heterochromatin mark, H3K27me3, to the inactive X chromosome was preserved in CD23+ B cells, but impaired in activated CD3+ T cells from the MRL/lpr model (p < 0.01 vs. B6), and even more impaired in the heavily female-biased NZM2328 model (p < 0.001 vs. B6; p < 0.05 vs. MRL/lpr). RNAseq of activated T cells from NZM2328 mice revealed the female-biased upregulation of 32 X-linked genes distributed broadly across the X chromosome, many of which have roles in immune function. Many genes encoding Xist RNA-interacting proteins were also differentially expressed and predominantly downregulated, which may account for the observed mislocalization of Xist RNA to the inactive X chromosome. CONCLUSIONS: Although evident in T cells from both the MRL/lpr and NZM2328 models of spontaneous SLE, impaired dXCIm is more severe in the heavily female-biased NZM2328 model. The aberrant X-linked gene dosage in female NZM2328 mice may contribute towards the development of female-biased immune responses in SLE-prone hosts. These findings provide important insights into the epigenetic mechanisms contributing to female-biased autoimmunity.

Time to get ill: the intersection of viral infections, sex, and the X chromosome.

Females have more robust immune responses than males, and viral infections are more severe for males. Hormones and genetic sex, namely the X chromosome, influence sex differences with immune responses. Here, we review recent findings underlying sexual dimorphism of disease susceptibility for two prevalent viral infections, influenza and SARS-CoV-2, which exhibit male-biased disease severity. Viral infections are proposed to be an initiating event for autoimmunity, which exhibits a female bias. We also review recent work elucidating the epigenetic and genetic contribution of X-Chromosome Inactivation maintenance, and X-linked gene expression, for the autoimmune disorder Systemic Lupus Erythematosus, and highlight the complex considerations required for identifying underlying hormonal and genetic contributions responsible for sex differences in immune responses.

Ectromelia-encoded virulence factor C15 specifically inhibits antigen presentation to CD4+ T cells post peptide loading.

Smallpox and monkeypox pose severe threats to human health. Other orthopoxviruses are comparably virulent in their natural hosts, including ectromelia, the cause of mousepox. Disease severity is linked to an array of immunomodulatory proteins including the B22 family, which has homologs in all pathogenic orthopoxviruses but not attenuated vaccine strains. We demonstrate that the ectromelia B22 member, C15, is necessary and sufficient for selective inhibition of CD4+ but not CD8+ T cell activation by immunogenic peptide and superantigen. Inhibition is achieved not by down-regulation of surface MHC- II or co-stimulatory protein surface expression but rather by interference with antigen presentation. The appreciable outcome is interference with CD4+ T cell synapse formation as determined by imaging studies and lipid raft disruption. Consequently, CD4+ T cell activating stimulus shifts to uninfected antigen-presenting cells that have received antigen from infected cells. This work provides insight into the immunomodulatory strategies of orthopoxviruses by elucidating a mechanism for specific targeting of CD4+ T cell activation, reflecting the importance of this cell type in control of the virus.

NASA-TLX Assessment of Surgeon Workload Variation Across Specialties.

OBJECTIVE: With advancements in surgical equipment and procedures, human-system interactions in operating rooms affect surgeon workload and performance. Workload was measured across surgical specialties using surveys to identify potential predictors of high workload for future performance improvement. SUMMARY BACKGROUND DATA: Surgical instrumentation and technique advancements have implications for surgeon workload and human-systems interactions. To understand and improve the interaction of components in the work system, NASA-Task Load Index can measure workload across various fields. Baseline workload measurements provide a broad overview of the field and identify areas most in need of improvement. METHODS: Surgeons were administered a modified NASA-Task Load Index survey (0 = low, 20 = high) following each procedure. Patient and procedural factors were retrieved retrospectively. RESULTS: Thirty-four surgeons (41% female) completed 662 surgery surveys (M = 14.85, SD = 7.94), of which 506 (76%) have associated patient and procedural data. Mental demand (M = 7.7, SD = 5.56), physical demand (M = 7.0, SD = 5.66), and effort (M = 7.8, SD = 5.77) were the highest rated workload subscales. Surgeons reported difficulty levels higher than expected for 22% of procedures, during which workload was significantly higher (P < 0.05) and procedural durations were significantly longer (P > 0.001). Surgeons reported poorer perceived performance during cases with unexpectedly high difficulty (P < 0.001). CONCLUSIONS: When procedural difficulty is greater than expected, there are negative implications for mental and physical demand that result in poorer perceived performance. Investigations are underway to identify patient and surgical variables associated with unexpected difficulty and high workload. Future efforts will focus on re-engineering the surgical planning process and procedural environment to optimize workload and performance for improved surgical care.

Recombinant Poxviruses: Versatile Tools for Immunological Assays.

The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, Vaccinia virus, a poxvirus closely related to Cowpox virus, has enjoyed widespread use for this purpose. Recombinant poxvirus-based expression has a number of advantages over other systems. Poxviruses accommodate the insertion of large pieces of recombinant DNA into their genome, and recombination and selection are relatively efficient. Poxviruses readily infect a variety of cell types, and they drive rapid and high levels of antigen expression. Additionally, they can be utilized in a variety of assays to study both MHC class I restricted and MHC class II restricted antigen processing and presentation. Ultimately, the numerous advantages of poxvirus recombinants have made the Vaccinia expression system a mainstay in the study of processing and presentation over the past two decades. In an attempt to address one shortcoming of Vaccinia virus while simultaneously retaining the benefits inherent to poxviruses, our laboratory has begun to engineer recombinant Ectromelia viruses. Ectromelia virus, or mousepox, is a natural pathogen of murine cells and performing experiments in the context of a natural host-pathogen relationship may elucidate unknown factors that influence epitope generation and host response. This chapter will describe several recombinant poxvirus system protocols used to study both MHC class I and class II antigen processing and presentation, as well as provide insight and troubleshooting techniques to improve the reproducibility and fidelity of these experiments.

Poor Antigen Processing of Poxvirus Particles Limits CD4+ T Cell Recognition and Impacts Immunogenicity of the Inactivated Vaccine.

CD4+ T cells play critical roles in defending against poxviruses, both by potentiating cellular and humoral responses and by directly killing infected cells. Despite this central role, the basis for pox-specific CD4+ T cell activation, specifically the origin of the poxvirus-derived peptides (epitopes) that activate CD4+ T cells, remains poorly understood. In addition, because the current licensed poxvirus vaccines can cause serious adverse events and even death, elucidating the requirements for MHC class II (MHC-II) processing and presentation of poxviral Ags could be of great use. To address these questions, we explored the CD4+ T cell immunogenicity of ectromelia, the causative agent of mousepox. Having identified a large panel of novel epitopes via a screen of algorithm-selected synthetic peptides, we observed that immunization of mice with inactivated poxvirus primes a virtually undetectable CD4+ T cell response, even when adjuvanted, and is unable to provide protection against disease after a secondary challenge. We postulated that an important contributor to this outcome is the poor processability of whole virions for MHC-II-restricted presentation. In line with this hypothesis, we observed that whole poxvirions are very inefficiently converted into MHC-II-binding peptides by the APC as compared with subviral material. Thus, stability of the virion structure is a critical consideration in the rational design of a safe alternative to the existing live smallpox vaccine.

Characteristics of team briefings in gynecological surgery.

Preoperative briefings have been proven beneficial for improving team performance in the operating room. However, there has been minimal research regarding team briefings in specific surgical domains. As part of a larger project to develop a briefing structure for gynecological surgery, the study aimed to better understand the current state of pre-operative team briefings in one department of an academic hospital. Twenty-four team briefings were observed and video recorded. Communication was analyzed and social network metrics were created based on the team member verbal interactions. Introductions occurred in only 25% of the briefings. Network analysis revealed that average team briefings exhibited a hierarchical structure of communication, with the surgeon speaking the most frequently. The average network for resident-led briefings displayed a non-hierarchical structure with all team members communicating with the resident. Briefings conducted without a standardized protocol can produce variable communication between the role leading and the team members present.

Shortcut assessment: Can residents' operative performance be determined in the first five minutes of an operative task?

BACKGROUND: The aim was to validate the potential use of a single, early procedure, operative task as a predictive metric for overall performance. The authors hypothesized that a shortcut psychomotor assessment would be as informative as a total procedural psychomotor assessment when evaluating laparoscopic ventral hernia repair performance on a simulator. METHODS: Using electromagnetic sensors, hand motion data were collected from 38 surgery residents during a simulated laparoscopic ventral hernia repair procedure. Three time-based phases of the procedure were defined: Early Phase (start time through completion of first anchoring suture), Mid Phase (start time through completion of second anchoring suture), and Total Operative Time. Correlations were calculated comparing time and motion metrics for each phase with the final laparoscopic ventral hernia repair score. RESULTS: Analyses revealed that execution time and motion, for the first anchoring suture, predicted procedural outcomes. Greater execution times and path lengths correlated to lesser laparoscopic ventral hernia repair scores (r = -0.56, P = .0008 and r = -0.51, P = .0025, respectively). Greater bimanual dexterity measures correlated to Greater LVH repair scores (r = + 0.47, P = .0058). CONCLUSIONS: This study provides validity evidence for use of a single, early operative task as a shortcut assessment to predict resident performance during a simulated laparoscopic ventral hernia repair procedure. With the continued development and decreasing costs of motion technology, faculty should be well-versed in the use of motion metrics for performance measurements. The results strongly support the use of dexterity and economy of motion (path length + execution time) metrics as early predictors of operative performance.

Interruptions Experienced by Emergency Nurses: Implications for Subjective and Objective Measures of Workload.

INTRODUCTION: This study aimed to describe interruptions experienced by emergency nurses and establish convergence validity of 1 objective workload measure by linking interruption characteristics to objective and subjective measures of workload. METHODS: Interruptions were captured in real time across 8- or 12-hour shifts using a previously validated Workflow Interruptions Tool (WIT). Data collected on each interruption included type, priority, and location where the interruption occurred. At mid- and end-shift, the Surgery Task Load Index (SURG-TLX) and the Rapid Cognitive Assessment Tool (RCAT) were administered to participating nurses to measure workload subjectively and objectively. RESULTS: Thirty-eight emergency nurse shifts were observed. A total of 3,229 interruptions were recorded across 372.5 clinical hours and 38 shifts (means [M] = 85.0 interruptions per shift, standard deviation [SD] = 34.9; M = 8.7 interruptions per hour, SD = 3.36). The median duration per interruption was 13.0 seconds. A moderate positive association was identified between the number of interruptions experienced during a shift and the increased overall SURG-TLX workload reported at end-shift, r(36) = 0.323, P = 0.048. Also, a moderate positive association was identified between increased reaction times during the RCAT task and increased mental demand experienced at end of shift, r(36) = 0.460, P < 0.001. DISCUSSION: This study observed interruptions throughout the entirety of a nursing shift and found that the majority of interruptions caused by the environment were low priority. Targeting interventions to reduce low-priority and environmental interruptions may aid in alleviating the impact of interruptions on clinical staff and patient care. Furthermore, results demonstrate that the frequency of interruptions was perceived to increase the nursing staff workload overall.

Ectromelia virus lacking the E3L ortholog is replication-defective and nonpathogenic but does induce protective immunity in a mouse strain susceptible to lethal mousepox.

All known orthopoxviruses, including ectromelia virus (ECTV), contain a gene in the E3L family. The protein product of this gene, E3, is a double-stranded RNA-binding protein. It can impact host range and is used by orthopoxviruses to combat cellular defense pathways, such as PKR and RNase L. In this work, we constructed an ECTV mutant with a targeted disruption of the E3L open reading frame (ECTVΔE3L). Infection with this virus resulted in an abortive replication cycle in all cell lines tested. We detected limited transcription of late genes but no significant translation of these mRNAs. Notably, the replication defects of ECTVΔE3L were rescued in human and mouse cells lacking PKR. ECTVΔE3L was nonpathogenic in BALB/c mice, a strain susceptible to lethal mousepox disease. However, infection with ECTVΔE3L induced protective immunity upon subsequent challenge with wild-type virus. In summary, E3L is an essential gene for ECTV.

Physician, Interrupted: Workflow Interruptions and Patient Care in the Emergency Department.

BACKGROUND: It is unclear how workflow interruptions impact emergency physicians at the point of care. OBJECTIVES: Our study aimed to evaluate interruption characteristics experienced by academic emergency physicians. METHODS: This prospective, observational study collected interruptions during attending physician shifts. An interruption is defined as any break in performance of a human activity that briefly requires attention. One observer captured interruptions using a validated tablet PC-based tool that time stamped and categorized the data. Data collected included: 1) type, 2) priority of interruption to original task, and 3) physical location of the interruption. A Kruskal-Wallis H test compared interruption priority and duration. A chi-squared analysis examined the priority of interruptions in and outside of the patient rooms. RESULTS: A total of 2355 interruptions were identified across 210 clinical hours and 28 shifts (means = 84.1 interruptions per shift, standard deviation = 14.5; means = 11.21 interruptions per hour, standard deviation = 4.45). Physicians experienced face-to-face physician interruptions most frequently (26.0%), followed by face-to-face nurse communication (21.7%), and environment (20.8%). There was a statistically significant difference in interruption duration based on the interruption priority, χ2(2) = 643.98, p < 0.001, where durations increased as priority increased. Whereas medium/normal interruptions accounted for 53.6% of the total interruptions, 53% of the interruptions that occurred in the patient room (n = 162/308) were considered low priority (χ2 [2, n = 2355] = 78.43, p < 0.001). CONCLUSIONS: Our study examined interruptions over entire provider shifts and identified patient rooms as high risk for low-priority interruptions. Targeting provider-centered interventions to patient rooms may aid in mitigating the impacts of interruptions on patient safety and enhancing clinical care.

Research Residents' perceptions of skill decay: Effects of repeated skills assessments and scenario difficulty.

INTRODUCTION: Skills decay is a known risk for surgical residents who have dedicated research time. We hypothesize that simulation-based assessments will reveal significant differences in perceived skill decay when assessing a variety of clinical scenarios in a longitudinal fashion. METHODS: Residents (N = 46; Returning: n = 16, New: n = 30) completed four simulated procedures: urinary catheterization, central line, bowel anastomosis, and laparoscopic ventral hernia repair. Perception surveys were administered pre- and post-simulation. RESULTS: Perceptions of skill decay and task difficulty were similar for both groups across three procedures pre- and post-simulation. Due to a simulation modification, new residents were more confident in urinary catheterization than returning residents (F(1,4) = 11.44, p = 0.002). In addition, when assessing expectations for skill reduction, returning residents perceived greater skill reduction upon reassessment when compared to first time residents (t(35) = 2.37, p = 0.023). CONCLUSION: Research residents may benefit from longitudinal skills assessments and a wider variety of simulation scenarios during their research years. TABLE OF CONTENTS SUMMARY: As part of a longitudinal study, we assessed research residents' confidence, perceptions of task difficulty and surgical skill reduction. Residents completed surveys pre- and post-experience with four simulated procedures: urinary catheterization, subclavian central line insertion, bowel anastomosis, and laparoscopic ventral hernia repair. Returning residents perceived greater skill reduction upon reassessment when compared to residents participating for the first time. In addition, modification of the clinical scenarios affected perceptions of skills decay.

Do errors and critical events relate to hernia repair outcomes?

BACKGROUND: The study aimed to validate an error checklist for simulated laparoscopic ventral hernia (LVH) repair procedures. We hypothesize that residents' errors can be assessed with a structured checklist and the results will correlate significantly with procedural outcomes. METHODS: Senior residents' (N = 7) performance on a LVH simulator were video-recorded and analyzed using a human error checklist. Junior residents (N = 38) performed two steps of the same simulated LVH procedure. Performance was evaluated using the error checklist and repair quality scores. RESULTS: There were no significant differences between senior and junior residents' checklist errors (p > 0.1). Junior residents' errors correlated with hernia repair quality (p = 0.05). CONCLUSIONS: The newly developed assessment tool showed significant correlations between performance errors, critical events, and hernia repair quality. These results provide validity evidence for the use of errors in performance assessments. SUMMARY: This study validated an error checklist for simulated laparoscopic ventral hernia (LVH) repair procedures. The checklist was designed based on errors committed by chief surgery residents during LVH repairs. In a separate data collection, junior residents were evaluated using the checklist. Hernia repair quality was also assessed. Errors significantly correlated with hernia repair quality (p = 0.05).

Giving CD4+ T cells the slip: viral interference with MHC class II-restricted antigen processing and presentation.

Activation of CD4+ T cells through interactions with peptides bound to Major Histocompatibility Complex Class II (MHC-II) molecules is a crucial step in clearance of most pathogens. Consequently, many viruses have evolved ways of blocking this aspect of adaptive immunity, from specific targeting of processing and presentation components to modulation of signaling pathways that regulate peptide presentation in addition to many other host defense mechanisms. Such cases of interference are far less common compared to what has been elucidated in MHC-I processing and presentation. This may be attributable in part to the complexity of MHC-II antigen processing, the scope of which is only now coming to light.

Long-range enhancer activity determines Myc sensitivity to Notch inhibitors in T cell leukemia.

Notch is needed for T-cell development and is a common oncogenic driver in T-cell acute lymphoblastic leukemia. The protooncogene c-Myc (Myc) is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates Myc is unknown. Here, we identify a distal enhancer located >1 Mb 3' of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter. The Notch1 binding element in this region activates reporter genes in a Notch-dependent, cell-context-specific fashion that requires a conserved Notch complex binding site. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Leukemia cells selected for resistance to Notch inhibitors express Myc despite epigenetic silencing of enhancer domains near the Notch transcription complex binding sites. Notch-independent expression of Myc in resistant cells is highly sensitive to inhibitors of bromodomain containing 4 (Brd4), a change in drug sensitivity that is accompanied by preferential association of the Myc promoter with more 3' enhancer domains that are strongly dependent on Brd4 for function. These findings indicate that altered long-range enhancer activity can mediate resistance to targeted therapies and provide a mechanistic rationale for combined targeting of Notch and Brd4 in leukemia.