Characterisation of choroid plexus-infiltrating T cells reveals novel therapeutic targets in murine neuropsychiatric lupus.

OBJECTIVE: Diffuse central nervous system manifestations, referred to as neuropsychiatric lupus (NPSLE), are observed in 20-40% of lupus patients and involve complex mechanisms that have not yet been adequately elucidated. In murine NPSLE models, choroid plexus (ChP)-infiltrating T cells have not been fully evaluated as drivers of neuropsychiatric disease. METHOD: Droplet-based single-cell transcriptomic analysis (single-cell RNA sequencing) and immune T-cell receptor profiling were performed on ChP tissue from MRL/lpr mice, an NPSLE mouse model, at an 'early' and 'late' disease state, to investigate the infiltrating immune cells that accumulate with NPSLE disease progression. RESULTS: We found 19 unique clusters of stromal and infiltrating cells present in the ChP of NPSLE mice. Higher resolution of the T-cell clusters uncovered multiple T-cell subsets, with increased exhaustion and hypoxia expression profiles. Clonal analysis revealed that the clonal CD8+T cell CDR3 sequence, ASGDALGGYEQY, matched that of a published T-cell receptor sequence with specificity for myelin basic protein. Stromal fibroblasts are likely drivers of T-cell recruitment by upregulating the VCAM signalling pathway. Systemic blockade of VLA-4, the cognate ligand of VCAM, resulted in significant resolution of the ChP immune cell infiltration and attenuation of the depressive phenotype. CONCLUSION: Our analysis details the dynamic transcriptomic changes associated with murine NPSLE disease progression, and highlights its potential use in identifying prospective lupus brain therapeutic targets.

Voices Unveiled: Personal Reflections of Faculty of Color in Nursing Academia.

ABSTRACT: The presence of faculty of color (FOC) provides critical role modeling and mentorship for diverse students and thereby contributes to increasing diversity in the nursing workforce. Personal reflections of FOC within a predominantly White institution involved feelings of isolation, not belonging, and lacking support and mentorship. Addressing negative experiences of FOC supports retention and advances diversity, equity, and inclusion.

Advances in the diagnosis, pathogenesis and treatment of neuropsychiatric systemic lupus erythematosus.

PURPOSE OF REVIEW: Diagnosing and treating neuropsychiatric systemic lupus erythematosus (NPSLE) remains challenging as the pathogenesis is still being debated. In this review, we discuss studies evaluating recent advances in diagnostic methods, pathogenic mediators and potential treatments. RECENT FINDINGS: Screening tools used for neurodegenerative diseases were found to be both sensitive and moderately specific for cognitive dysfunction in NPSLE. Neuroimaging can be used to distinguish systemic lupus erythematosus (SLE) patients from healthy controls, but further refinement is needed to differentiate between lupus patients with and without neuropsychiatric manifestations. Elevated levels of specific molecules in the cerebrospinal fluid and/or serum, as well as the presence of certain autoantibodies, have been identified as potential biomarkers in attempts to facilitate a more accurate and objective diagnosis. Among such autoantibodies, anti-NR2 and anti-ribosomal P autoantibodies also have a pathogenic role, although newer studies demonstrate that blood-brain barrier damage may not always be required as previously believed. These and other observations, together with new evidence for disease attenuation after microglial modulation, suggest direct involvement of the central nervous system in NPSLE pathogenesis. SUMMARY: Neuropsychiatric involvement of SLE includes a variety of symptoms that impact quality of life and patient prognosis. There have been recent advances in improving the diagnosis of NPSLE as well as in dissecting the underlying pathogenesis. The attenuation of neuropsychiatric disease in mouse models demonstrates the potential for targeted therapies, which are based on a clearer understanding of the pathogenesis of NPSLE. Further assessment of these treatments is required in NPSLE patients, as well as the potential use of neuroimaging to distinguish between SLE patients with or without neuropsychiatric manifestations.

Are lupus animal models useful for understanding and developing new therapies for human SLE?

Systemic lupus erythematosus is a systemic autoimmune disease driven by a complex combination of genetic, environmental, and other immunoregulatory factors. The development of targeted therapies is complicated by heterogeneous clinical manifestations, varying organ involvement, and toxicity. Despite advances in understanding the mechanisms contributing to SLE, only one biologic drug, belimumab, is FDA-approved. The identification and development of potential therapies have largely been driven by studies in lupus animal models. Therefore, direct comparison of both the therapeutic and immunological findings in human and murine SLE studies is critical and can reveal important insights into indeed how useful and relevant are murine studies in SLE drug development. Studies involving belimumab, mycophenolate mofetil, abatacept, rituximab, and anti-interferon strategies generally demonstrated analogous findings in the attenuation of SLE manifestations and modulation of select immune cell populations in human and murine SLE. While further basic and translational studies are needed to identify SLE patient subsets likely to respond to particular therapeutic modalities and in dissecting complex mechanisms, we believe that despite some inherent weaknesses SLE mouse models will continue to be integral in developing targeted SLE therapies.

Patellofemoral kinematics in dogs with cranial cruciate ligament insufficiency: an in-vivo fluoroscopic analysis during walking.

BACKGROUND: Complete rupture of the cranial cruciate ligament (CrCL) in dogs causes profound disturbance to stifle joint biomechanics. The objective of this study was to characterize the effects of cranial cruciate ligament (CrCL) insufficiency on patellofemoral (PF) kinematics in dogs during walking. Ten client-owned dogs (20-40 kg) with natural unilateral complete CrCL rupture were included. Dogs underwent computed tomographic scans to create digital bone-models of the patella and femur. Lateral projection fluoroscopy of the stifles was performed during treadmill walking. Sagittal plane PF kinematics were calculated throughout the gait cycle by overlaying digital bone models on fluoroscopic images using a previously described 2D-3D registration technique. For acquisition of kinematics in the contralateral (control) stifle, fluoroscopy was repeated 6-months after stabilizing surgery of the affected side. Results were compared between the pre-operative CrCL-deficient and 6-month post-operative control stifles. RESULTS: Craniocaudal PF translation was similar between CrCL-deficient and control stifles throughout the gait cycle. The patella was more distal and positioned in greater flexion throughout the gait cycle in CrCL-deficient stifles when compared to the control stifle at equivalent time points. There was no significant difference in PF poses between CrCL-deficient and control stifles at equivalent femorotibial flexion angles; however, common femorotibial flexion angles were only found over a small range during the swing phase of gait. CONCLUSIONS: CrCL insufficiency altered PF kinematics during walking, where the changes were predominately attributable to the femorotibial joint being held in more flexion. Abnormal PF kinematics may play a role in the development of osteoarthritis that is commonly observed in the PF joint CrCL-deficient stifles.

Normal patellofemoral kinematic patterns during daily activities in dogs.

BACKGROUND: Patellar abnormalities are a common cause of pain and lameness in dogs; however, in vivo the relative motion between the femur and patella in dogs is not well described. The objective of this study was to define normal in vivo sagittal plane patellofemoral kinematics in three axes of motion using non-invasive methods. We hypothesized patellofemoral alignment in the sagittal plane would tightly correlate with the femorotibial flexion angle. Six healthy dogs without orthopedic disease underwent computed tomography (CT) of their hind limbs to create 3-D models of the patella and femur. Normal stifle joint motion was captured via flat-panel imaging while each dog performed a series of routine activities, including sitting, walking, and trotting. The 3-D models of the patella and femur were digitally superimposed over the radiographic images with shape-matching software and the precise movement of the patella relative to the femur was calculated. RESULTS: As the femorotibial joint flexed, the patellofemoral joint also flexed and the patella moved caudally and distally within the femoral trochlea during each activity. Patellar flexion and distal translation during walk and sit were linearly coupled with the femorotibial flexion angle. Offset was evident while trotting, where patella poses were significantly different between early and late swing phase (p ≤ 0.003). Patellar flexion ranged from 51 to 6° while trotting. The largest flexion angle (92°) occurred during sit. The patella traversed the entire proximodistal length of the femoral trochlea during these daily activities. CONCLUSIONS: Using single-plane flat-panel imaging, we demonstrated normal in vivo patellofemoral kinematics is tightly coupled with femorotibial kinematics; however, trot kinematic patterns did not follow the path defined by walking and stand-to-sit motions. Our normal data can be used in future studies to help define patellofemoral joint kinematics in dogs with stifle abnormalities.

Analysis of a Needs Assessment Survey to Develop an Online Resource Repository Supporting Nurse Anesthesia Educators.

BACKGROUND: Without highly qualified nurse anesthesia educators and administrators, the health care system will be threatened by the inadequate supply of certified registered nurse anesthetists (CRNAs). PURPOSE: American Association of Nurse Anesthesiologists' Faculty Stabilization Task Force (FSTF) analyzed reasons for high faculty turnover and developed recommendations to support nurse anesthesia faculty and administrators. METHODS: A survey evaluated participants' current role, leadership development opportunities, mentorship experiences, and resource needs. RESULTS: Of 109 respondents, 87 (80%) were program administrators or assistant administrators with less than 5 years of experience in their role. Despite academic experience, 51% felt adequately prepared for their role. CONCLUSIONS: The FSTF provided 2 recommendations: to create a robust faculty development program for all faculty at all levels of CRNA education and a repository of information needed for program administrators and faculty to oversee and educate students in a high-quality CRNA program.

Defining Ultra-Massive Transfusion through a Systematic Review.

Background: Despite the widespread use of ultra-massive transfusion (UMT) as an intervention for trauma patients in hemorrhagic shock, no standard definition exists. We performed a systematic review to determine a consensus definition for UMT. Methods: A search was performed from 1979-2022. The authors screened studies defining UMT and associated outcomes as defined by our prespecified PICO questions. The PRISMA guidelines were used. Results: 1662 articles met criteria for eligibility assessment, 17 for full-text review and eight for data extraction. Only two studies demonstrated a consensus definition of UMT, which used ≥20 units of red blood cell product within 24hrs. Parameters associated with increased mortality included lower blood pressure, lower pulse and lower Glasgow Coma Score at the time of presentation and a higher injury severity score and undergoing a resuscitative thoracotomy. Conclusions: The absence of a consensus definition for UMT raises challenges from clinical, research and ethical perspectives. Based on our findings, the authors advocate for the feasibility of standardizing the definition of UMT as ≥20 units of red blood cell product within 24hrs.

The nurse-led equitable learning framework for training programs: A framework to grow, bolster and diversify the nursing and public health workforce.

BACKGROUND: Addressing threats to the nursing and public health workforce, while also strengthening the skills of current and future workers, requires programmatic solutions. Training programs should be guided by frameworks, which leverage nursing expertise and leadership, partnerships, and integrate ongoing evaluation. PURPOSE STATEMENT: This article provides a replicable framework to grow, bolster, and diversify the nursing and public health workforces, known as the Nurse-led Equitable Learning (NEL) Framework for Training Programs. The framework has been applied by several multipronged, federally funded training programs led by investigators embedded in an academic nursing institution. METHODS: The NEL framework focuses on: (1) increasing equitable access to the knowledge, skills, and competencies needed to prepare a diverse workforce to deliver effective interventions; (2) fostering academic-practice linkages and community partnerships to facilitate the deployment of newly gained knowledge and skills to address ongoing and emerging challenges in care delivery; and (3) continuously evaluating and disseminating findings to inform expansion and replication of programs. RESULTS: Ten programs using this framework have successfully leveraged $18.3 million in extramural funding to support over 1000 public health professionals and trainees. Longitudinal evaluation efforts indicate that public health workers, including nurses, are benefiting from the programs' workplace trainings, future clinicians are being rigorously trained to identify and address determinants of health to improve patient and community well-being, and educators are engaging in novel pedagogical opportunities to enhance their ability to deliver high quality public health education. CONCLUSIONS: Training programs may apply the NEL framework to ensure that the nursing and public health workforces achieve equitable, sustainable growth and deliver high quality evidence-based care.

Choroid Plexus-Infiltrating T Cells as Drivers of Murine Neuropsychiatric Lupus.

OBJECTIVE: T cells are critical in the pathogenesis of systemic lupus erythematosus (SLE) in that they secrete inflammatory cytokines, help autoantibody production, and form autoreactive memory T cells. Although the contribution of T cells to several forms of organ-mediated damage in SLE has been previously demonstrated, the role of T cells in neuropsychiatric SLE (NPSLE), which involves diffuse central nervous system manifestations and is observed in 20-40% of SLE patients, is not known. Therefore, we conducted this study to evaluate how behavioral deficits are altered after depletion or transfer of T cells, to directly assess the role of T cells in NPSLE. METHODS: MRL/lpr mice, an NPSLE mouse model, were either systemically depleted of CD4+ T cells or intracerebroventricularly injected with choroid plexus (CP)-infiltrating T cells and subsequently evaluated for alterations in neuropsychiatric manifestations. Our study end points included evaluation of systemic disease and assessment of central nervous system changes. RESULTS: Systemic depletion of CD4+ T cells ameliorated systemic disease and cognitive deficits. Intracerebroventricular injection of CP-infiltrating T cells exacerbated depressive-like behavior and worsened cognition in recipient mice compared with mice who received injection of splenic lupus T cells or phosphate buffered saline. Moreover, we observed enhanced activation in CP-infiltrating T cells when cocultured with brain lysate-pulsed dendritic cells in comparison to the activation levels observed in cocultures with splenic T cells. CONCLUSION: T cells, and more specifically CP-infiltrating antigen-specific T cells, contributed to the pathogenesis of NPSLE in mice, indicating that, in the development of more targeted treatments for NPSLE, modulation of T cells may represent a potential therapeutic strategy.

Supporting the Foundation of Nurse Anesthesia Education: A Report of the Faculty Stabilization Taskforce.

Without highly qualified educators and educational program administrators, the ability to provide anesthesia and pain management services throughout the healthcare system will be threatened by the inadequate supply of Certified Registered Nurse Anesthetists (CRNAs). The average turnover rate between the years 2016-2020, for CRNA program administrators, was 15% with some programs changing leadership as often as every two to four years. In response, the American Association of Nurse Anesthesiology (AANA) and the Council on Accreditation of Nurse Anesthesia Educational Programs (COA) created the Faculty Stabilization Taskforce (FSTF) to review reasons for the high turnover rate and to develop methods and tools to support program faculty and administrators in their role of educating the future CRNA workforce. The FSTF provided two recommendations: 1) to create a robust faculty development program for all faculty at all levels of CRNA education, and 2) to create a repository of information needed to administrate, and educate students, in a high-quality CRNA program. This article summarizes the report made to the AANA Board of Directors (BOD) which was unanimously accepted and is already being implemented by AANA and COA staff. The full text of the report can be found on the AANA's website at www.aana.com/FSTF.

Second Victim Syndrome in Trauma Practitioners and Other Ancillary Staff.

In health care, second victims are traumatized clinicians involved in unanticipated or untoward patient events. Programs that address second victim syndrome are sparse and its diagnosis often goes unrecognized. Consistently, literature has identified gaps in support resources, leading to compromised patient care and provider health. This project evaluates the need for second victim resources in trauma care providers at a tertiary public level 1 trauma hospital by electronically implementing a validated second victim survey over 5 weeks. Our results illustrate that second victim syndrome is prevalent among 57.1% of trauma care providers, of which 22.9% agree that second victim syndrome results in some form of undesirable work intentions.

Microbial Communities in Retail Draft Beers and the Biofilms They Produce.

In the beer brewing industry, microbial spoilage presents a consistent threat that must be monitored and controlled to ensure the palatability of a finished product. Many of the predominant beer spoilage microbes have been identified and characterized, but the mechanisms of contamination and persistence remain an open area of study. Postproduction, many beers are distributed as kegs that are attached to draft delivery systems in retail settings where ample opportunities for microbial spoilage are present. As such, restaurants and bars can experience substantial costs and downtime for cleaning when beer draft lines become heavily contaminated. Spoilage monitoring on the retail side of the beer industry is often overlooked, yet this arena may represent one of the largest threats to the profitability of a beer if its flavor profile becomes substantially distorted by contaminating microbes. In this study, we sampled and cultured microbial communities found in beers dispensed from a retail draft system to identify the contaminating bacteria and yeasts. We also evaluated their capability to establish new biofilms in a controlled setting. Among four tested beer types, we identified over a hundred different contaminant bacteria and nearly 20 wild yeasts. The culturing experiments demonstrated that most of these microbes were viable and capable of joining new biofilm communities. These data provide an important reference for monitoring specific beer spoilage microbes in draft systems and we provide suggestions for cleaning protocol improvements. IMPORTANCE Beer production, packaging, and service are each vulnerable to contamination by microbes that metabolize beer chemicals and impart undesirable flavors, which can result in the disposal of entire batches. Therefore, great effort is taken by brewmasters to reduce and monitor contamination during production and packaging. A commonly overlooked quality control stage of a beer supply chain is at the retail service end, where beer kegs supply draft lines in bars and restaurants under nonsterile conditions. We found that retail draft line contamination is rampant and that routine line cleaning methods are insufficient to efficiently suppress beer spoilage. Thus, many customers unknowingly consume spoiled versions of the beers they consume. This study identified the bacteria and yeast that were resident in retail draft beer samples and also investigated their abilities to colonize tubing material as members of biofilm communities.

The T Cell Receptor Repertoire in Neuropsychiatric Systemic Lupus Erythematosus.

Objective: In systemic lupus erythematosus (SLE), widespread T cell infiltration into target organs contributes to inflammation and organ damage. Autoreactive T cells become aberrantly activated in this disease due to dysfunctional T cell receptor signaling that lowers the activation threshold. Characterizing the T cell repertoire can provide further insight into the specific homing and proliferation of these T cells into lupus target organs. In the spontaneous lupus model, MRL/lpr, the TCR repertoire has not been fully elucidated, especially for T cells infiltrating the brain. Our aim was to investigate and compare the TCR repertoire between MRL/lpr mice and its congenic controls, MRL/MpJ, and within MRL/lpr tissues. Methods: Spleen, salivary gland, and brain choroid plexus were isolated from female MRL/lpr mice and MRL/MpJ mice. The TCRß CDR3 region was analyzed by multiplex PCRs and sequencing. Results: Significant differences were seen not only between the MRL/lpr and MRL/MpJ spleens, but also between MRL/lpr tissues. The TCR repertoire in MRL/lpr choroid plexus tissues had significantly increased clonality and sequence homology compared to MRL/lpr spleen and salivary gland. The consensus sequence, CASSQDWGGYEQYFF, was identified in the MRL/lpr choroid plexus repertoire. Conclusions: The TCR repertoire in lupus prone mice is not uniform between target organs, and suggests that T cells are specifically recruited into the choroid plexus of MRL/lpr mice. Further studies are needed to determine the antigen specificities for these infiltrating T cells in target organs of lupus mice, and their possible contribution to the pathogenesis of neuropsychiatric disease and other lupus manifestations.

Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis.

Increasing evidence suggests that neutrophil extracellular traps (NETs) may play a role in promoting atherosclerotic plaque lesions in humans and in murine models. The exact pathways involved in NET-driven atherogenesis remain to be systematically characterized. To assess the extent to which myeloid-specific peptidylarginine deiminase 4 (PAD4) and PAD4-dependent NET formation contribute to atherosclerosis, mice with myeloid-specific deletion of PAD4 were generated and backcrossed to Apoe-/- mice. The kinetics of atherosclerosis development were determined. NETs, but not macrophage extracellular traps, were present in atherosclerotic lesions as early as 3 weeks after initiating high-fat chow. The presence of NETs was associated with the development of atherosclerosis and with inflammatory responses in the aorta. Specific deletion of PAD4 in the myeloid lineage significantly reduced atherosclerosis burden in association with diminished NET formation and reduced inflammatory responses in the aorta. NETs stimulated macrophages to synthesize inflammatory mediators, including IL-1ß, CCL2, CXCL1, and CXCL2. Our data support the notion that NETs promote atherosclerosis and that the use of specific PAD4 inhibitors may have therapeutic benefits in this potentially devastating condition.

Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7-dependent lupus.

The peptidylarginine deiminases PAD2 and PAD4 are implicated in the pathogenesis of several autoimmune diseases. PAD4 may be pathogenic in systemic lupus erythematosus (SLE) through its role in neutrophil extracellular trap (NET) formation that promotes autoantigen externalization, immune dysregulation, and organ damage. The role of this enzyme in mouse models of autoimmunity remains unclear, as pan-PAD chemical inhibitors improve clinical phenotype, whereas PAD4-KO models have given conflicting results. The role of PAD2 in SLE has not been investigated. The differential roles of PAD2 and PAD4 in TLR-7-dependent lupus autoimmunity were examined. Padi4-/- displayed decreased autoantibodies, type I IFN responses, immune cell activation, vascular dysfunction, and NET immunogenicity. Padi2-/- mice showed abrogation of Th subset polarization, with some disease manifestations reduced compared with WT but to a lesser extent than Padi4-/- mice. RNA sequencing analysis revealed distinct modulation of immune-related pathways in PAD-KO lymphoid organs. Human T cells express both PADs and, when exposed to either PAD2 or PAD4 inhibitors, displayed abrogation of Th1 polarization. These results suggest that targeting PAD2 and/or PAD4 activity modulates dysregulated TLR-7-dependent immune responses in lupus through differential effects of innate and adaptive immunity. Compounds that target PADs may have potential therapeutic roles in T cell-mediated diseases.

A role for muscarinic receptors in neutrophil extracellular trap formation and levamisole-induced autoimmunity.

Levamisole, an anthelmintic drug with cholinergic properties, has been implicated in cases of drug-induced vasculitis when added to cocaine for profit purposes. Neutrophil extracellular trap (NET) formation is a cell death mechanism characterized by extrusion of chromatin decorated with granule proteins. Aberrant NET formation and degradation have been implicated in idiopathic autoimmune diseases that share features with levamisole-induced autoimmunity as well as in drug-induced autoimmunity. This study's objective was to determine how levamisole modulates neutrophil biology and its putative effects on the vasculature. Murine and human neutrophils exposed to levamisole demonstrated enhanced NET formation through engagement of muscarinic subtype 3 receptor. Levamisole-induced NETosis required activation of Akt and the RAF/MEK/ERK pathway, ROS induction through the nicotinamide adenine dinucleotide phosphate oxidase, and peptidylarginine deiminase activation. Sera from two cohorts of patients actively using levamisole-adulterated cocaine displayed autoantibodies against NET components. Cutaneous biopsy material obtained from individuals exposed to levamisole suggests that neutrophils produce NETs in areas of vasculitic inflammation and thrombosis. NETs generated by levamisole were toxic to endothelial cells and impaired endothelium-dependent vasorelaxation. Stimulation of muscarinic receptors on neutrophils by cholinergic agonists may contribute to the pathophysiology observed in drug-induced autoimmunity through the induction of inflammatory responses and neutrophil-induced vascular damage.

Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by synovial joint inflammation and by development of pathogenic humoral and cellular autoimmunity to citrullinated proteins. Neutrophil extracellular traps (NETs) are a source of citrullinated autoantigens and activate RA synovial fibroblasts (FLS), cells crucial in joint damage. We investigated the molecular mechanisms by which NETs promote proinflammatory phenotypes in FLS, and whether these interactions generate pathogenic anti-citrulline adaptive immune responses. NETs containing citrullinated peptides are internalized by FLS through a RAGE-TLR9 pathway promoting FLS inflammatory phenotype and their upregulation of MHC class II. Once internalized, arthritogenic NET-peptides are loaded into FLS MHC class II and presented to Ag-specific T cells. HLADRB1*0401 transgenic mice immunized with mouse FLS loaded with NETs develop antibodies specific to citrullinated forms of relevant RA autoantigens implicated in RA pathogenesis as well as cartilage damage. These results implicate FLS as mediators in RA pathogenesis, through the internalization and presentation of NET citrullinated peptides to the adaptive immune system leading to pathogenic autoimmunity and cartilage damage.