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Browsing by Author "Gates, Kayla L"
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Item Award Recognition for "Developing a Minimally Invasive Cell-Based Model to Predict Response to Major Trauma"(2021-08) Gates, Kayla L; Nunge, Rebecca A; Adom, Jamila; Kaplan, Mark; McKinley, ToddBackground: The physiologic response to injury is heavily influenced by the immune system. The complexities of the immunologic response to injury are becoming increasingly understood as researchers have leveraged computational methods that allow temporal and spatial coordination of immune mediator orchestration to be quantified. Recently, early differences in immunologic orchestration have been shown to stratify individual tolerance to injury. Specifically, there are subsets of trauma patients that are either sensitive or tolerant to hemorrhage that demonstrated notably different early immunologic orchestration of mediators from clusters of cytokines that are primarily tissue protective or pro/anti-inflammatory. These differentiating networks of mediators formed and dissipated over the initial 72 hours after injury clearly demonstrating that the immunologic response to injury is an acute dynamic event that has pathomechanistic relevance to outcomes after injury. Additionally, it is distinctly possible that individualized differences in immune response may determine tolerance/sensitivity to injury. The differential immunologic response to trauma represents an opportunity to discover specific factors that may be predictive of a patients’ response to traumatic injury and subsequent hemorrhagic shock. Accordingly, we have embarked on a line of experimentation to explore potential precision approaches based on individual immunologic response to injury. Here we report our initial experimental findings in conceptual model development with the ultimate goal of developing minimally invasive/non-injurious testing that will accurately identify individual tolerance to hemorrhage and injury. In this experiment, an in-vitro cell-based assay was designed to mimic traumatic injury. Specifically, we tested the immunologic response in murine splenocytes to a simulated hypoxic injury, a simulated mechanical injury and a simulated open wounding injury. The development of a reliable cell-based model will allow investigation to determine correspondence and relevance between cell-based responses to non-traumatic injury and in vivo immunologic response to trauma, with the overall goal of developing a reliable test to predict response to traumatic injury in humans. Methods: In-vitro cellular responses of murine splenocytes are reflective of peripheral blood cell responses and were used for pilot experiments. Splenocytes from C57BL/6 (B6) BALB/c or CH3/ HeJ strains mice were used with stimuli that mimic traumatic injury using chemical (hypoxia or sepsis) or mechanical (shear stress) stimuli that might from an open wounding type of injury. Hypoxia was simulated by subjecting cell cultures to hydrogen peroxide. Sepsis was simulated by subjecting cells to lipopolysaccharide (LPS). Some culture conditions included several individual cytokines associated with acute inflammation and external pathogens (interleukin (IL)-6, IL-1β, IL- 33), the damage molecule high mobility group box protein (HMGB)-1, or combinations of LPS and the cytokines. Following treatment, cDNA was prepared and used for qPCR amplification of TNFα, HIF1α, and BAX to assess inflammation, hypoxia, and apoptosis, respectively. Multiplex analysis of IL-21, IL-4, IL-22, IL-5, and IL-10 expression was performed from culture supernatant collected at 24 hours after stimulation. Flow cytometry was performed to assess proliferation of immune cells following treatment. ELISA was conducted to quantify production of the cytokine IL-9 that occurred following splenocyte stimulation. Results: Analysis of C57BL/6 splenocyte viability show that any combination of cytokines or LPS did not impact cell survival, while hydrogen peroxide reduced survival significantly in each treatment group. From the qPCR data, LPS generated a 4x increase in TNFα expression relative to control, while cytokine treatment yielded no expression changes. Treatment with LPS + cytokines closely resembled the LPS treatment group. LPS treatment reduced expression of HIF1α, while hydrogen peroxide increased expression of HIF1α. The addition of cytokines reduced expression of HIF1α in groups that were treated with both hydrogen peroxide and cytokines. ELISA analysis of the proinflammatory cytokine IL-9 indicated increased production of IL-9 following treatment with LPS + cytokines. In the second experiment, the model was applied to three different strains of mice in order to gauge differences in the immune response to the same cellular stress. Multiplex analysis showed no significant changes in IL-4 or IL-21 expression in any of the strains. C3H mice showed no response to LPS, which was expected due to LPS resistance in these strains. In the B6 and BALB mice, IL-10 was induced by LPS treatment. BALB mice also showed increased expression of IL-5 and IL-22 in response to mechanical stress, while the other strains showed no response. IL-10 expression was not induced by mechanical injury in any strain. Flow cytometry analysis was used to assess immune cell response to stimuli. Both B6 and C3H mice showed increased percentages of CD4 and CD8 cells in response to mechanical stimulus, LPS, and LPS + cytokine treatment relative to control. Macrophage levels were more elevated in B6 mice in response to mechanical stimulus, whereas levels decreased in the C3H mice. Discussion: The overall goal of this line of investigation is to develop minimally invasive and non-injurious testing that can be used to determine individual tolerance/sensitivity to trauma and hemorrhage. These pilot studies were used to determine how immune cells can be isolated and stimulated to mimic injury. Splenocytes were used as they encompass a broad cross-section of white blood cells. Clear inter-strain differences were evident between the B6, BALB and C3H mice. Hypoxia stimuli consistently resulted in roughly a 50% loss of cell viability and accordingly may not be a viable strategy. The greatest effects were encountered with LPS +/- addition of stimulating cytokines. We measured changes in five of six cytokines in B6 mice and four of six BALB mice involving reparative cytokines (IL-21 and 22), anti-inflammatory cytokines (IL-10) and in type 2 cytokines (IL-4 and IL-5). Accordingly, these strains and stimulation methods will be expanded to determine effects on production on a broader panel of cytokines. In addition, computational methods will be leveraged on the next experiment to determine in-vitro effects on immunologic mediator orchestration to account for time-dependent mediator networks and spatial networks of mediators. Moving forward, these experiments will be repeated to reproduce our findings and improve our ability to distinguish between varying immune responses. Results will then be paired with studies examining the responses to traumatic injury among these and other strains. The overall goal of this project is to accurately predict the response to an in-vivo injury using an in-vitro non traumatic stimulus. Findings from this project will enable the development of a clinical test that accurately predicts immunologic response to trauma and stratify individual tolerance to hemorrhage and injury.Item Developing a Minimally Invasive Cell-Based Model to Predict Response to Major Trauma(2020-07-31) Nunge, Rebecca A; Gates, Kayla L; Adom, Jamila; Kaplan, Mark; McKinley, ToddBackground. Physical trauma results in a systemic inflammatory response. Preliminary research in orthopedic trauma patients suggests that patients with similar demographics and severity of injury vary in their response to traumatic injury. Analysis of the immunological response post-injury showed a sustained pro-inflammatory response with delayed reparative cytokine expression in trauma sensitive patients, while the trauma tolerant patients had an early inflammatory expression with resolution by 72 hours post-injury. Thus, we hypothesize that differential response to non-traumatic injury might serve as a predictive tool for the identification of trauma tolerant and sensitive patients prior to injury. The goal of this research is to test whether immunological changes to inflammatory stimuli can predict tolerance or sensitivity to trauma using an-vitro cell-based assay. Methods. Splenocytes were isolated from naive C57BL/6 mice and subjected to biological trauma in vitro using LPS (100 ng/mL) or hypoxic trauma using hydrogen peroxide (50 µM, 100 µM, and 200 µM) with or without proinflammatory cytokines, IL-1β (1 ng/mL) , IL-6 (200 ng/mL), and IL-33 (150 ng/mL). Inflammation and hypoxia were assessed using IL-6 and HIF-1ɑ expression respectively via qPCR 24 hours post-treatment. Cell death and pro-inflammatory cytokine production using multiplex analysis were used to measure outcomes. Results. Both types of treatments showed increased cell death compared to the control group. qPCR data is pending. Conclusion. With these studies as a core of the experimental approach, this in vitro cell-based assay will be used to assess immunologic response to inflammatory stimuli across the genetic variation of mouse strains. Findings from this project could enable the development of a clinical test that accurately predicts immunologic response to trauma and related-complications based on patients’ sensitivity to pre-traumatic injury.Item Developing a Minimally Invasive Cell-Based Model to Predict Response to Major Trauma(2020-07-31) Nunge, Rebecca A; Gates, Kayla L; Adom, Jamila; McKinley, ToddBackground. Physical trauma results in a systemic inflammatory response. Preliminary research in orthopedic trauma patients suggests that patients with similar demographics and severity of injury vary in their response to traumatic injury. Analysis of the immunological response post-injury showed a sustained pro-inflammatory response with delayed reparative cytokine expression in trauma sensitive patients, while the trauma tolerant patients had an early inflammatory expression with resolution by 72 hours post-injury. Thus, we hypothesize that differential response to non-traumatic injury might serve as a predictive tool for the identification of trauma tolerant and sensitive patients prior to injury. The goal of this research is to test whether immunological changes to inflammatory stimuli can predict tolerance or sensitivity to trauma using an-vitro cell-based assay. Methods. Splenocytes were isolated from naive C57BL/6 mice and subjected to biological trauma in vitro using LPS (100 ng/mL) or hypoxic trauma using hydrogen peroxide (50 µM, 100 µM, and 200 µM) with or without proinflammatory cytokines, IL-1β (1 ng/mL) , IL-6 (200 ng/mL), and IL-33 (150 ng/mL). Inflammation and hypoxia were assessed using IL-6 and HIF-1ɑ expression respectively via qPCR 24 hours post-treatment. Cell death and pro-inflammatory cytokine production using multiplex analysis were used to measure outcomes. Results. Both types of treatments showed increased cell death compared to the control group. qPCR data is pending. Conclusion. With these studies as a core of the experimental approach, this in vitro cell-based assay will be used to assess immunologic response to inflammatory stimuli across the genetic variation of mouse strains. Findings from this project could enable the development of a clinical test that accurately predicts immunologic response to trauma and related-complications based on patients’ sensitivity to pre-traumatic injury.Item IU School of Medicine Correctional Medicine Student Outreach Project(2022-04-28) Nunge, Rebecca A; Gates, Kayla L; Messmore, Niki MBackground: This project was founded on the basis that correctional medicine is an important component frequently missing from medical school curriculums. Opportunities to participate in medical care within correctional facilities, while concurrently engaging in discussions about the institutions that have contributed to disproportionate incarceration of certain populations and mass incarceration as a whole, will cultivate empathetic, socially-engaged, and passionate young physicians. This student organization was formed to facilitate clinical opportunities within correctional facilities and host events that focus on the broader socioeconomic and political context and forms of structural and cultural violence that have contributed to mass incarceration in the United States. Methods: The overall goal of this project is to send medical students into Indiana Department of Corrections (IDOC) facilities, or county jails, to work closely with the medical providers as they provide care to incarcerated individuals. In order to facilitate this goal, a relationship was fostered between IUSM and Dr. Kristen Dauss, the Chief Medical Officer of the IDOC. Upon completion and approval of the contract between Centurion, the IDOC medical provider, and IUSM, students will have opportunities to shadow providers at the Indiana Women’s Prison. This pilot location was chosen due to the unique opportunity it presents to learn about the medical needs of the incarcerated women and young children, as it is the only facility in the state with a mother-baby unit. Since its creation, IUCM has also hosted numerous virtual educational lectures, panels, and journal clubs in collaboration with other student organizations and scholars in the field of correctional health. Lastly, the organization encourages engagement with original research through the IMPRS program, in coordination with Niki Messmore, our faculty advisor. Conclusions: As physicians who will practice medicine in the country with the highest incarceration rate in the world, having a fundamental understanding of topics related to correctional health, adverse health experiences while incarcerated, and longstanding traumatic effects of incarceration is imperative. IUCM’s goal is to create introductory materials and share resources with medical students relating to the create introductory materials and share resources with medical students relating to the Powered by Qualtrics A socioeconomic and political context which has led to mass incarceration, the lived experiences of people who are incarcerated, and the deficits in care for currently and formerly incarcerated people. Developing a better understanding of the justice system as well as the emotional, mental, and physical impact incarceration has on patients, and will stimulate interest in engaging with these concepts through research, volunteer work, educational events, and in patient care. In the future, each campus will partner with local correctional facilities and community organizations. After accruing a critical mass of student involvement at educational events and within facilities, we will utilize surveys to study the impact of the project. Additionally, we hope to expand the project to include other healthcare professional programs, and involve other facilities statewide. Lastly, due to our belief that correctional health should be a component of the core curriculum, we are currently working with IUSM administration to create material for the FCP course related to these topics.Item IU School of Medicine Correctional Medicine Student Outreach Project(2022-10-22) Nunge, Rebecca A; Gates, Kayla L; Fazle, Trilliah; Garcia, Jennifer; Messmore, Nicole M; Agarwal, NeetaBackground: This project was founded on the basis that correctional medicine is an important component frequently missing from medical education. Opportunities to participate in medical care within correctional facilities, while concurrently engaging in discussions about disproportionate incarceration of certain populations and mass incarceration as a whole, will cultivate empathetic, socially-engaged, and passionate young physicians. This student organization was formed to facilitate clinical opportunities within correctional facilities and host events that focus on the broader socioeconomic and political context and forms of structural and cultural violence that have contributed to mass incarceration in the United States. Methods: In order to facilitate organizational goals, a relationship was fostered between IUSM and Dr. Kristen Dauss, the Chief Medical Officer of the IDOC. Following affiliation agreements, students may now gain clinical exposure at any facility in the state. Since its creation, IUCM has hosted virtual educational lectures, panels, and journal clubs, in collaboration with other student organizations and scholars in the field. The organization encourages engagement with original research in coordination with faculty advisors. We have also worked with administration to incorporate correctional health topics officially into the curriculum. Conclusions: As physicians who will practice medicine in the country with the highest incarceration rate in the world, having a fundamental understanding of topics related to correctional health, adverse health experiences while incarcerated, and longstanding traumatic effects of incarceration is imperative. IUCM’s goal is to create introductory materials and share resources relating to the socioeconomic and political context which has led to mass incarceration and the deficits in care for currently and formerly incarcerated people. Developing a better understanding of the justice system as well as the emotional, mental, and physical impact incarceration has on patients, and will stimulate interest in engaging with these concepts through research, volunteer work, educational events, and in patient care.