Diversity, equity, and inclusion (DEI) in medical education: DEI at the bedside.

BACKGROUND: The ability to recognize and address bias is an important communication skill not typically addressed during training. We describe the design of an educational curriculum that aims to identify and change behavior related to diversity, equity, and inclusion (DEI). "DEI at the Bedside" uses the existing infrastructure of bedside teaching and provides a tool to normalize DEI discussions and develop skills to address bias during a neurology inpatient rotation. METHODS: As part of traditional clinical rounds, team members on an inpatient service shared experiences with DEI topics, including bias. The team developed potential responses should they encounter a similar situation in the future. We report the results of our needs assessment and curriculum development to evaluate the feasibility of incorporating a DEI educational curriculum in the neurology inpatient setting. RESULTS: Forty-two DEI experiences were recorded. Medical students were the most frequent discussants (44%). Direction of bias occurred between healthcare team members (33%), against patients (31%), and patients against healthcare team members (28%). Experiences ranged from microaggressions to explicit comments of racism, sexism, and homophobia. CONCLUSIONS: Based on needs assessment data, we developed a DEI educational curriculum for the inpatient neurology setting aimed to improve knowledge and skills related to DEI topics as well as to normalize conversation of DEI in the clinical setting. Additional study will demonstrate whether this initiative translates into measurable and sustained improvement in knowledge of how bias and disparity show up in the clinical setting and behavioral intent to discuss and address them.

Neuroprotection mediated by inhibition of calpain during acute viral encephalitis.

Neurologic complications associated with viral encephalitis, including seizures and cognitive impairment, are a global health issue, especially in children. We previously showed that hippocampal injury during acute picornavirus infection in mice is associated with calpain activation and is the result of neuronal death triggered by brain-infiltrating inflammatory monocytes. We therefore hypothesized that treatment with a calpain inhibitor would protect neurons from immune-mediated bystander injury. C57BL/6J mice infected with the Daniel's strain of Theiler's murine encephalomyelitis virus were treated with the FDA-approved drug ritonavir using a dosing regimen that resulted in plasma concentrations within the therapeutic range for calpain inhibition. Ritonavir treatment significantly reduced calpain activity in the hippocampus, protected hippocampal neurons from death, preserved cognitive performance, and suppressed seizure escalation, even when therapy was initiated 36 hours after disease onset. Calpain inhibition by ritonavir may be a powerful tool for preserving neurons and cognitive function and preventing neural circuit dysregulation in humans with neuroinflammatory disorders.

Axons are injured by antigen-specific CD8(+) T cells through a MHC class I- and granzyme B-dependent mechanism.

Axon injury is a central determinant of irreversible neurological deficit and disease progression in patients with multiple sclerosis (MS). CD8(+) lymphocytes (CTLs) within inflammatory demyelinated MS lesions correlate with acute axon injury and neurological deficits. The mechanisms of these correlations are unknown. We interrogated CTL-mediated axon injury using the transgenic OT-I antigen-specific CTL model system in conjunction with a chambered cortical neuron culture platform that permitted the isolated manipulation of axons independent of neuron cell bodies and glia. Interferon gamma upregulated, through a dose dependent mechanism, the axonal expression of functional major histocompatibility complex class I (MHC I) molecules competent to present immunologically-relevant antigens derived from endogenously expressed proteins. Antigen-specific CTLs formed cytotoxic immune synapses with and directly injured axons expressing antigen-loaded MHC I molecules. CTL-mediated axon injury was mechanistically dependent upon axonal MHC I antigen presentation, T cell receptor specificity and axoplasmic granzyme B activity. Despite extensive distal CTL-mediated axon injury, acute neuron cell body apoptosis was not observed. These findings present a novel model of immune-mediated axon injury and offer anti-axonal CTLs and granzyme B as targets for the therapeutic protection of axons and prevention of neurological deficits in MS patients.

Hippocampal protection in mice with an attenuated inflammatory monocyte response to acute CNS picornavirus infection.

Neuronal injury during acute viral infection of the brain is associated with the development of persistent cognitive deficits and seizures in humans. In C57BL/6 mice acutely infected with the Theiler's murine encephalomyelitis virus, hippocampal CA1 neurons are injured by a rapid innate immune response, resulting in profound memory deficits. In contrast, infected SJL and B6xSJL F1 hybrid mice exhibit essentially complete hippocampal and memory preservation. Analysis of brain-infiltrating leukocytes revealed that SJL mice mount a sharply attenuated inflammatory monocyte response as compared to B6 mice. Bone marrow transplantation experiments isolated the attenuation to the SJL immune system. Adoptive transfer of B6 inflammatory monocytes into acutely infected B6xSJL hosts converted these mice to a hippocampal damage phenotype and induced a cognitive deficit marked by failure to recognize a novel object. These findings show that inflammatory monocytes are the critical cellular mediator of hippocampal injury during acute picornavirus infection of the brain.

The STAT3 beacon: IL-6 recurrently activates STAT 3 from endosomal structures.

Endocytic trafficking plays an important role in signal transduction. Signal transducer and activator of transcription 3 (STAT3) and mitogen-activate protein kinase (MAPK) have both been localized to endosomal structures and are dependent upon endocytosis for downstream function. While the dependence of MAPK signaling upon endosomes has been well characterized, the involvement of endosomes in regulating STAT3 signaling has not been defined. Consequently, this study evaluated the role of endosomes in the initiation, modulation, amplification and persistence of interleukin-6(IL-6)-induced STAT3 signal transduction and transcription, and utilized IL-6-induced MAPK signaling as a comparator. Using pharmacologic treatment and temperature control of endocytic trafficking, pulse-chase treatments and in vitro kinase assays, STAT3 was found to interact with endosomes in a markedly different fashion than MAPK. STAT3 was activated by direct interaction with internal structures upstream of the late endosome following IL-6 exposure and persistent STAT3 signaling depended upon recurrent activation from endocytic structures. Further, STAT3 subcellular localization was not dependent upon endocytic trafficking. Instead, STAT3 transiently interacted with endosomes and relocated to the nucleus by an endosome-independent mechanism. Finally, endocytic trafficking played a central role in regulating STAT3 serine 727 phosphorylation through crosstalk with the MAPK signaling system. Together, these data reveal endosomes as central to the genesis, course and outcome of STAT3 signal transduction and transcription.

CD8+ T cells cause disability and axon loss in a mouse model of multiple sclerosis.

BACKGROUND: The objective of this study was to test the hypothesis that CD8+ T cells directly mediate motor disability and axon injury in the demyelinated central nervous system. We have previously observed that genetic deletion of the CD8+ T cell effector molecule perforin leads to preservation of motor function and preservation of spinal axons in chronically demyelinated mice. METHODOLOGY/PRINCIPAL FINDINGS: To determine if CD8+ T cells are necessary and sufficient to directly injure demyelinated axons, we adoptively transferred purified perforin-competent CD8+ spinal cord-infiltrating T cells into profoundly demyelinated but functionally preserved perforin-deficient host mice. Transfer of CD8+ spinal cord-infiltrating T cells rapidly and irreversibly impaired motor function, disrupted spinal cord motor conduction, and reduced the number of medium- and large-caliber spinal axons. Likewise, immunodepletion of CD8+ T cells from chronically demyelinated wildtype mice preserved motor function and limited axon loss without altering other disease parameters. CONCLUSIONS/SIGNIFICANCE: In multiple sclerosis patients, CD8+ T cells outnumber CD4+ T cells in active lesions and the number of CD8+ T cells correlates with the extent of ongoing axon injury and functional disability. Our findings suggest that CD8+ T cells may directly injure demyelinated axons and are therefore a viable therapeutic target to protect axons and motor function in patients with multiple sclerosis.

Apoptosis of hippocampal pyramidal neurons is virus independent in a mouse model of acute neurovirulent picornavirus infection.

Many viruses, including picornaviruses, have the potential to infect the central nervous system (CNS) and stimulate a neuroinflammatory immune response, especially in infants and young children. Cognitive deficits associated with CNS picornavirus infection result from injury and death of neurons that may occur due to direct viral infection or during the immune responses to virus in the brain. Previous studies have concluded that apoptosis of hippocampal neurons during picornavirus infection is a cell-autonomous event triggered by direct neuronal infection. However, these studies assessed neuron death at time points late in infection and during infections that lead to either death of the host or persistent viral infection. In contrast, many neurovirulent picornavirus infections are acute and transient, with rapid clearance of virus from the host. We provide evidence of hippocampal pathology in mice acutely infected with the Theiler's murine encephalomyelitis picornavirus. We found that CA1 pyramidal neurons exhibited several hallmarks of apoptotic death, including caspase-3 activation, DNA fragmentation, and chromatin condensation within 72 hours of infection. Critically, we also found that many of the CA1 pyramidal neurons undergoing apoptosis were not infected with virus, indicating that neuronal cell death during acute picornavirus infection of the CNS occurs in a non-cell-autonomous manner. These observations suggest that therapeutic strategies other than antiviral interventions may be useful for neuroprotection during acute CNS picornavirus infection.

Up-regulation of heat shock protein 27 induces resistance to 17-allylamino-demethoxygeldanamycin through a glutathione-mediated mechanism.

17-Allylamino-demethoxygeldanamycin (17-AAG), currently in phase I and II clinical trials as an anticancer agent, binds to the ATP pocket of heat shock protein (Hsp90). This binding induces a cellular stress response that up-regulates many proteins including Hsp27, a member of the small heat shock protein family that has cytoprotective roles, including chaperoning of cellular proteins, regulation of apoptotic signaling, and modulation of oxidative stress. Therefore, we hypothesized that Hsp27 expression may affect cancer cell sensitivity to 17-AAG. In colony-forming assays, overexpression of Hsp27 increased cell resistance to 17-AAG whereas down-regulation of Hsp27 by siRNA increased sensitivity. Because Hsp27 is known to modulate levels of glutathione (GSH), we examined cellular levels of GSH and found that it was decreased in cells transfected with Hsp27 siRNA when compared with control siRNA. Treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, also sensitized cells to 17-AAG. Conversely, treatment of Hsp27 siRNA-transfected cells with N-acetylcysteine, an antioxidant and GSH precursor, reversed their sensitivity to 17-AAG. A cell line selected for stable resistance to geldanamycin relative to parent cells showed increased Hsp27 expression. When these geldanamycin- and 17-AAG-resistant cells were transfected with Hsp27 siRNA, 17-AAG resistance was dramatically diminished. Our results suggest that Hsp27 up-regulation has a significant role in 17-AAG resistance, which may be mediated in part through GSH regulation. Clinical modulation of GSH may therefore enhance the efficacy of Hsp90-directed therapy.