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Established in 1807, The School of Medicine is the first public and the fifth oldest medical school in the United States. Current programs include MD, MD/PhD, graduate, physical therapy & rehabilitation science, and medical research technology.
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Mast Cell Modulation of the Developing HippocampusBrain development requires orchestration of overlapping critical periods defined by responsiveness to endogenous or exogenous stimuli (i.e. hormones, light, touch). Simultaneously, sensitive periods represent heightened risk to perturbations by abnormal stimuli (i.e. inflammation). Though neuro-immune cooperation between resident microglia and neurons is essential during critical periods of brain development, the contribution of infiltrating immune cells to neurodevelopmental processes is largely elusive. We discovered a critical and sensitive period created by a population of peri-hippocampal mast cells (phMCs) in the lateral ventricles abundant from birth through 2-weeks-old in the neonatal rat but absent thereafter. This epoch of phMCs is maintained by self-replication inside the brain, creating a mix of mature and immature mast cells which constitutively piecemeal degranulate. phMCs harbor a unique transcriptomic identity to skin, bone marrow and other brain mast cells including transcripts encoding colony-stimulating factors (Csf1, Csf2) essential for microglial development. Pharmacological inhibition of phMC degranulation and secretion stunts microglial maturation, illuminating the role of phMC-derived factors in driving hippocampal microglial development. In contrast, activation of phMCs releases proinflammatory chemokines, compromises the blood-brain-barrier (BBB), and recruits peripheral immune cells across the CNS for at least two days. Together, these findings indicate that a transient population of peri-hippocampal mast cells creates a critical period for microglial maturation via piecemeal degranulation in the healthy brain and a sensitive period to inflammatory stimuli. The dual role of phMCs in homeostasis and inflammation highlight an essential need to understand how the immune system can adapt its function to serve neuronal populations and prevent inflammatory disturbances that lead to neurologic disease.
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Unraveling Cochlear Otic Mesenchyme Cells: The Role of POU3F4 During Cochlear DevelopmentThe cochlea consists of diverse cellular populations working in harmony to convert mechanical stimuli into electrical signals for the perception of sound. One such cell type are otic mesenchyme cells (OMCs), which are a specialized type of neural crest and cranial paraxial mesoderm that express multiple unique transcription factors (e.g., POU3F4), all of which are known deafness genes, highlighting the importance of OMCs in auditory function. OMCs are known to terminally differentiate into spatially and functionally distinct cell types, including fibrocytes of the lateral wall and spiral limbus, modiolar osteoblasts, and specialized tympanic border cells of the basilar membrane. Interestingly, consequences of Pou3f4 mutations are diverse and include a complete loss of endocochlear potential, shortening of the cochlear duct, and defective pathfinding and survival of spiral ganglion neurons (SGNs), indicating diverse roles of POU3F4 in each OMC-derived cell type. Here, we aim to illuminate the molecular distinctness and functionality of OMCs and show how loss of Pou3f4 impacts cochlear development. By utilizing scRNA sequencing, we elucidated that OMCs divide into four transcriptionally distinct subpopulations well before the onset of hearing, each of which corresponding to one of the OMC-derived cochlear structures. Furthermore, we show OMC subpopulations display distinct functional roles corresponding to their spatial localization. We also unravel the cochlear cellular communication pathways showcasing OMCs are the main contributors of outgoing signaling during cochlear development, including both global and subpopulation specifying signaling pathways. Finally, we indicate how Pou3f4 expression regulates gene expression in each OMC subpopulation and which signaling pathways are lost in Pou3f4 mutants which may be the cause of the defects in surrounding cell types. Our data suggest that OMC diversification occurs not long after the formation of the otocyst with further refinement until the onset of hearing, well before terminal differentiation. Our data also suggests OMCs are the main contributors of paracrine signaling during cochlear development, showcasing their importance in influencing surrounding cochlear cell types. Finally, we show how loss of Pou3f4 affects each OMC subpopulation differently, leading to diverse phenotypes in Pou3f4 mutants. Without cochlear OMCs and their later terminally differentiated cell types, normal auditory function would not be feasible highlighting the importance of tissue specific mesenchymal cells in cochlear development.
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Bacterial enzymatic combinatorial chemistry (BECC) enabled targeted lipid A modification of Shigella vaccine strains to reduce endotoxicity without compromising immunogenicity or invasivenessShigella spp. are Gram-negative bacteria that cause severe diarrheal disease, contributing significantly to morbidity and mortality worldwide. High transmissibility and increased antibiotic resistance have propelled the development of Shigella vaccine candidates; however, no FDA-approved vaccine exists to date. We have collaborated with Walter Reed Army Institute of Research (WRAIR) to improve their live-attenuated vaccine candidates, whose only drawback during clinical trials was febrile symptoms experienced in a few individuals. In this study, we sought to resolve those unwanted side effects by detoxifying the lipid A moiety (also known as endotoxin) that is thought to contribute to the febrile symptoms observed. To do so, we employed bacterial enzymatic combinatorial chemistry (BECC), whereby lipid A modification enzymes were ectopically expressed to induce structural alterations known to dampen lipid A signaling capacity. The enzymes LpxE (phosphatase) and PagL (deacylase) were expressed in both virulent and attenuated vaccine strains of Shigella to modify the bis-phosphorylated hexa-acylated lipid A structure ordinarily present in the Shigella outer membrane. The expected modifications were confirmed using mass spectrometric and gas chromatographic analyses when the enzymes were expressed individually or in combination (both LpxE and PagL) using a construct we refer to as “Dual”. These enzyme/enzyme combinations were subsequently integrated into the chromosome using Tn7 transposition to avoid the possibility of plasmid loss during production. The impact of the induced lipid A structural alterations on innate immune signaling was assessed by stimulation of NF-?B reporter cell lines and human PBMCs with the extracted lipopolysaccharide (LPS). Additionally, in vivo reactogenicity of the LPS in a murine acute endotoxemia model was assessed. We found that dephosphorylation, but not deacylation, of lipid A, was a powerful tool to reduce LPS-mediated signaling in live-attenuated Shigella vaccines, resulting in reduced toxicity of Shigella LPS in vivo. Additionally, we found that dephosphorylation of the lipid A moiety did not impair invasion of colonic epithelia or immunogenicity in a mouse pulmonary model. Overall, this study generated Shigella vaccine candidates with reduced endotoxicity, which upon oral ingestion in humans, will ultimately have reduced reactogenicity.
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The predictive accuracy of Shock Index in trauma outcomes in older injured patientsThe elderly is an increasing proportion of all cases treated at trauma centers. Shock index (SI) calculated as heart rate (HR) divided by systolic blood pressure (SBP), has been shown to be a good predictor of mortality and transfusion in injured patients. One limitation of SI is that its accuracy in different age groups, especially the elderly has not been fully evaluated. We studied the accuracy of admission SI in predicting early, 48-hour and in-hospital mortality, and major interventions (massive transfusion, ICU admission and surgery in 24 hours) in trauma patients admitted to a major trauma center. We examined whether age, injury severity, injury type, blood alcohol and comorbidities affected the predictive accuracy of SI. Of particular interest is the accuracy of SI in the elderly. We also compared the predictive accuracy of SI, HR and SBP. Optimal cut-points for SI were determined. SI had acceptable accuracy in predicting mortality outcomes, and ICU admission overall. Accuracy was good in the prediction of massive transfusion, and poor in the prediction of surgery in 24 hours. SI was better than HR or SBP in predicting mortality outcomes (all ages, elderly, and younger patients). However, in older patients, accuracy of SI in predicting major interventions was not different from that of SBP. Accuracy of SI in predicting 48-hour and all in-hospital mortality, and ICU admission was better in younger patients. Accuracy was also better among those with lower injury severity than in those who were more severely injured. Accuracy of SI in predicting massive transfusion was similar in older and younger trauma patients. Optimal cut-offs for predicting outcomes were lower for older patients (0.5-0.7 for mortality and major interventions) than in younger patients (0.6-0.9 for mortality and 0.6-0.8 for major interventions). Accuracy of SI in predicting all in-hospital death and massive transfusion was less among patients with elevated blood alcohol while comorbidities did not affect accuracy. In conclusion, SI is less accurate in in predicting mortality among older patients and is less accurate in predicting mortality and massive transfusion among blood alcohol-positive patients, potentially affecting its utility in triage and clinical management.
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TLR4-SNP Mice Reveal the Role of M2a Macrophages in Resolution of Chemically-Induced ColitisToll-like receptor 4 (TLR4) is an innate immune receptor responsive to Gram negative lipopolysaccharide (LPS). Single nucleotide polymorphisms (SNPs) in human TLR4 that encode an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I) render individuals hyporesponsive to LPS. In humans, these SNPs have also been associated with increased susceptibility to inflammatory bowel disease (IBD). IBD, including Crohn’s Disease (CD) and Ulcerative Colitis (UC), impacts millions of individuals worldwide and severely impairs quality of life for these patients. While multiple treatments are available for IBD, there are several problems: (1) not all patients respond, (2) responses may diminish over time, and (3) treatments often have undesirable side effects. Some patients with IBD express these SNPs and, using knock-in mice engineered to express the murine homologues of these human TLR4 mutations (“TLR4-SNP” mice), we have shown that TLR4-SNP mice develop significantly more severe colitis induced by dextran sodium sulfate (DSS) than wild-type (WT) mice. Previous studies have provided indirect evidence for a role of “tissue repair” M2 macrophages (Mφ) in the resolution of colitis. Signaling through the shared IL-4/IL-13 receptor, IL-4Rα, leading to activation of the transcription factor peroxisome proliferator-activated receptor (PPARγ) has been shown to be required for induction of M2a Mφ and our data provide direct evidence for the involvement of both in repair of DSS-induced colonic damage. In response to DSS, colons of TLR4-SNP mice produced reduced levels of M2a Mφ marker mRNA and protein. Additionally, PPARγ protein levels were reduced in colons from DSS-treated TLR4-SNP mice and therapeutic administration of the PPARγ agonist ligand, rosiglitazone, ameliorated colitis in TLR4-SNP mice. Together, these data indicate that the failure of TLR4-SNP mice to resolve DSS-induced colitis may be secondary to their failure to induce “tissue repair” M2a Mφ. Our findings provide insight into the potential development of novel therapies targeting Mφ signaling pathways that aim to alleviate the debilitating symptoms experienced by individuals with IBD.
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From What to How: An Exploration of How Genetic Counseling Program Directors Learn to TeachThe pathway from medical practitioner to academic teacher is not-well defined, specifically, there is a lack of clarity in how clinicians learn to teach, particularly within the field of genetic counseling. While there is a defined curricular pathway for entry into clinical practice there is not a defined pathway for instruction of health professionals who want to teach. While healthcare professionals enter an academic role with a defined set of clinical skills, they often lack formal training in how to teach. This qualitative phenomenological study, using a community of practice (CoP) theoretical model, explored the pathway to teaching for genetic counseling education program directors. Thirteen study participants shared their professional journey of learning to teach. Data from this study support the three phases of CoP in genetic counseling education program director development: engagement, imagination, and alignment. Participants reported content, instructional, and pedagogical reflection as they progressed in their understanding and development of their own teaching practice. Engagement with genetic counseling education programs was reported in three different ways: planned, sequential, and unplanned. All respondents acknowledged content reflection as a part of their early practice of learning to teach. The imagination phase is demonstrated as individuals expanded their understanding of teaching, program leadership, and responsibilities required to serve as a program director – in other words they began a practice of instructional reflection. Those who reported alignment did so based on mentorship and contribution to the community of genetic counseling education program directors and shared instances of pedagogical reflection. The findings of this study support the premise that genetic counseling education program directors, although not formal scholars in education prior to entry into their educator role, practice the scholarship of teaching and learning through a CoP. They are committed to the practice of evaluating how students learn to improve their own teaching. Further, they are committed to modeling professional development and learning as a member of a CoP. What they lack is the formal understanding of educational theory as it relates to genetic counseling instruction – without this knowledge it is difficult to conduct theoretically grounded educational research and advance the profession.
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Evaluation of Salmonella enterica serovar Typhimurium Vaccines in the Context of ImmunosenescenceNon-typhoidal Salmonella (NTS) is responsible for a high burden of foodborne infections and deaths worldwide. In the United States, NTS infections are the leading cause of hospitalizations and deaths due to foodborne illnesses, and older adults (≥65 years) are disproportionately affected by Salmonella infections. Due to this public health concern, we have developed a live attenuated vaccine, CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA), against Salmonella enterica serovar Typhimurium, a common serovar of NTS. The effects of age on parenteral vaccination are well documented, however, how advanced age impacts oral vaccine responses is less understood. In this dissertation, systemic and mucosal immune responses to CVD 1926 were evaluated in adult (six-to-eight-week-old) and aged (18-month-old) mice. We demonstrated that aged mice immunized with CVD 1926 failed to reduce bacterial burden upon challenge with wild-type S. Typhimurium, which was associated with lower vaccine-induced antibody titers and weaker T cell responses. Notably, characteristics of a successful mucosal vaccine response were weak in aged mice, suggesting that mucosal responses to oral vaccines decrease with advanced age. In efforts to develop a NTS vaccine that is effective for older adults, two vaccine approaches were evaluated: (i) a novel live-attenuated vaccine strain, CVD 1926 ΔsteD and (ii) heterologous prime boost strategy involving a mucosal prime with CVD 1926 that was followed by a parenteral boost with a conjugate S. Typhimurium vaccine. SteD is a Salmonella effector that suppresses CD4+ T cell responses during infection with wild-type S. Typhimurium. Using in vitro and in vivo assays, we demonstrated that CVD 1926 ΔsteD immunization elicits enhanced MHC-II expression, increased flagellin-specific CD4+ T cells, robust serum IgG and fecal IgA responses, and protection against S. Typhimurium colonization of the spleen, cecum, and small intestine upon challenge in aged mice. While the heterologous prime boost strategy induced robust Salmonella-specific antibody responses in aged mice, only modest protection against S. Typhimurium colonization was observed, suggesting that this vaccination approach cannot overcome immunosenescence. Taken together, these studies identify the age-associated deficits in mucosal vaccine responses and presents a promising prototype vaccine strain that may be effective for older adults.
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Predictors of Traumatic Stress in Hospital Nurses in the Third Year of COVID-19 and Their Lived Experiences: A Mixed Methods StudyBackground: Research on nurses early in the COVID-19 pandemic shows elevated severity of trauma-related stress, depression, anxiety, and poorer well-being than before the pandemic. Fewer studies examined nurses’ experiences three years into the pandemic and the relationships of the experiences that predict increased post-traumatic stress severity. Objectives: This study evaluates the relationships among peritraumatic distress, moral distress, resilience, and post-traumatic stress severity as its primary objective. The study also examined the relationships of depression, anxiety, sleep quality, and nurses’ perceived work environment to post-traumatic stress as its secondary objective. The overall goal is to understand targets for intervention on the path to the development of post-traumatic stress that could potentially reduce the impact of crises on nurses. Methods: This study evaluates the interrelationships of these variables using a concurrent triangulation mixed methods framework. Nurses participated in multiple surveys, and a subset of these nurses participated in semi-structured interviews. A structural equation model (SEM) examined the relationships of the primary outcomes, and multiple regression analyses investigate the independent predictive ability of the variables on post-traumatic stress. The interviews utilized a descriptive phenomenological methodology to describe the lived experiences of traumatic stress during the pandemic for these nurses. Results: In the SEM, moral distress partially complementarily mediated the direct positive effect of peritraumatic distress on post-traumatic stress and negatively moderated the direct negative effect of resilience on post-traumatic stress. A multiple regression with all variables excluding resilience and sleep quality (not significant at p < .20) accounted for 62.6% of the variability in post-traumatic stress symptom severity. The interviews revealed that nurses exist in three interrelated worlds: their “Internal World” (emotions and personal well-being), their ‘Hospital World” (coworkers, leadership, environment, etc.) and the “Outside World” (the public, social media, current events, etc.). Conclusion: Nurses require support for their mental health at work and outside the hospital during a crisis, and hospital policies must consider all three. Reduction in peritraumatic stress and moral distress and support for nurse resilience are some of the most critical areas to focus on to reduce the post-traumatic stress severity in nurses during a long-term crisis.
