Browsing by Author "Hodge, Lisa M."
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Item AMP-Activated Protein Kinase (AMPK) signaling regulates the age-related decline of hippocampal neurogenesis(2018-05) Wang, Brian S.; Jin, Kunlin; Hodge, Lisa M.; Singh, Meharvan; Sumien, Nathalie; Yang, ShaohuaAging is the progressive decline of physiological function and increased vulnerability to disease and death. By the year 2050, 2 billion people will be over the age of 60. Accompanying this, the incidence of age-associated neurological diseases is expected to rise. Thus, there is an urgent need to find therapies to promote healthy brain aging. The finding that neurogenesis continues into adulthood allows us to target endogenous neurogenesis as a potential therapeutic. However, the number of stem cells can decrease by about 80% in the aged brain and is a main cause for the decrease in brain function. The reasons for the age-related decline in neurogenesis can be due to intrinsic factors such as cell metabolism, which have been studied but its role in neurogenesis remains largely unexplored. Interestingly, neural stem cells (NSCs) possess metabolically different characteristics from their differentiated progeny, suggesting the need for a shift in cellular metabolism to accommodate the requirements for neurogenesis. In the process of the metabolic shift, the AMP-activated protein kinase (AMPK) plays a pivotal role for controlling stem cell proliferation and differentiation as a cell's master metabolic regulator. Additionally, AMPK has been reported to control the functions of signaling pathways that regulate the aging process, which suggests its potential involvement in the age-related decline of neurogenesis. Therefore, we hypothesize that inhibition of AMPK signaling activation (phosphorylation) in the old brain will cause a concomitant increase in hippocampal neurogenesis. Our specific aim is to establish whether AMPK signaling plays a critical role in the age-related decline of hippocampal neurogenesis. Our objectives for this aim are to (i) determine the expression pattern of AMPK in the subgranular and subventricular zones of young-adult and old mice using immunohistochemistry and Western blotting; and (ii) examine the impact of loss or gain of AMPK activation on hippocampal neurogenesis in young-adult and old mice using pharmacological agents Compound C (AMPK inhibitor) and 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, AMPK activator). Our results show that (i) AMPK subunit isoforms are differentially expressed in the neurogenic regions – most are localized to the cytoplasm in the subgranular zone (SGZ) with the exception of α2 and β1, while most isoforms are found in the nucleus in the subventricular zone (SVZ) except α1; (ii) AMPK signaling activation was significantly increased in the SGZ and SVZ; and (iii) short-term but not long-term pharmacological inhibition of AMPK signaling could partially rescue hippocampal neurogenesis in the old brain. Taken together, these results indicate that AMPK is a critical mediator in the regulation of downstream processes for the age-related decline in hippocampal neurogenesis.Item Anti-Tumor Immune Responses Against MTLn3 Mammary Adenocarcinoma(2014-05-01) Carter, KiahRae J.; Hodge, Lisa M.Lymphatic pump treatment (LPT) is used as a lymph enhancing therapy to treat edema. In animals, LPT enhanced lymphatic flow, released leukocytes and inflammatory mediators into lymph, and inhibited pulmonary tumor formation. Therefore, we hypothesized the administration of LPT would enhance immunity and inhibit primary breast tumor growth. Rats were subcutaneously injected with MTLn3 and divided into MTLn3, MTLn3+Sham-LPT and MTLn3+LTP group. Sham group received light touch under anesthesia and LPT group received treatment under anesthesia. There were no changes in tumor growth between groups. Administration of Sham-LPT resulted in an increase in tumor-adjacent lymph node weight. Collectively, our data suggests LPT did not enhance primary tumor growth and may also protect against the pathogenesis exhibited by sham-LPT.Item Cell-Free mtDNA Quantification in Alzheimer's Patients from the Mexican American Population(2020-05) House, Sara R.; Phillips, Nicole R.; Hodge, Lisa M.; Zascavage, Roxanne R.Abstract Background AD is a continuous problem in the 65+ population but it is especially challenging in the Hispanic population where not only is it more prevalent but more severe than Caucasian populations. This study explores the efficacy of using peripheral blood plasma as an alternative tissue for testing as well as the usefulness for future research assisting in identifying the population structure most at risk for developing AD based upon CF-mtDNA quantity results. Materials and Methods Samples tested included a total cohort (Mexican American and Caucasian) of 177 individuals (AD=45, MCI=74, NC=58). The Mexican American subset contained 92 individuals (AD=21, MCI=53, and NC=18). Peripheral blood plasma was collected from the TARCC biobank and quantified. CF-mtDNA was then tested for significance using correlation analyses, logistic and linear regression models. Results CF-mtDNA was significantly negatively correlated with education, age, sex, and hypertensive samples in the total and Mexican American populations. The greatest difference was expected to be in CF-mtDNA quantity from NC to AD samples. Instead, the most significant difference was between MCI and NC samples. As CF-mtDNA quantity increased, the MMSE and CDRSOB scores were less impaired. Conclusion In conclusion, CF-mtDNA is an easily accessible and easily tested molecular marker of diseases that are relevant to studies for cognitive decline. Although our findings were inconsistent with current literature, they bring to light the weight of confounding factors within limited sample studies. With the completion of the full sample set associated with this study, more power is needed to overcome these issues.Item Comparison of Deparaffinization Methods with DNA Quantification(2016-08-01) Leonard, Raechal P.; Warren, Joseph E. ; LaRue, Bobby L. ; Hodge, Lisa M.Formalin-fixed, paraffin embedded (FFPE) tissue samples are often the only sample type available for testing in pathological and clinical fields. Obtaining usable DNA from FFPE samples is difficult due to the formalin fixation method. Formaldehyde, a large component of formalin, causes DNA-protein cross-linking as well as other issues that must be overcome to obtain useable DNA. Many methods for deparaffinization exist to reduce the issues that arise when working with FFPE samples. This project focuses on comparing two methods of deparaffinization: a method described by Bosso and Al-Mulla and a method used by the University of North Texas Center For Human Identification (UNTCHI), to determine if there is a significant difference in DNA obtained from FFPE samples.Item Converting from Paper-Based to Electronic Data Capture and Record Keeping in Clinical Trial Management: Benefits, Challenges and Practical Considerations(2015-12-01) Castle, Colton; Gwirtz, Patricia A.; Bell, April M.; Hodge, Lisa M.Clinical research has lagged behind the technological advance of other healthcare fields. Most investigational sites depend on a paper‐based data capture and record retention system. This practicum project examined the various benefits and challenges of electronic data capture and electronic record keeping systems. Electronic systems can improve data integrity, reduce trial cost and increase efficiency in the course of a clinical trial. However, electronic systems can also pose some challenges, including implementation and training cost, decreased productivity, and issues with data security and health record privacy. This project discussed some practical considerations for investigational sites transitioning to electronic systems. These aims were accomplished by review of the literature and consulting investigational sites through an email questionnaire.Item Discovering the Optimal Hair Sections for Mitochondrial DNA Quantification via a Multiplex Real-Time PCR Assay(2015-05-01) Nakhla, Meriam I.; Warren, Joseph E.; Planz, John V.; Hodge, Lisa M.Hair is among the frequently encountered evidence found in crime scenes. The average person loses approximately 100 hairs a day. Because these hairs are telogen strands, or at the end of their life-phase, there is very little tissue present to obtain nuclear DNA. Hair shafts, however, contain mitochondrial DNA that can be used for identification purposes. There are two areas of concern involving mtDNA analysis of hair shafts: 1) will there be enough mtDNA present to obtain a full profile, and 2) and has the integrity of mtDNA been compromised due to oxidative properties, and/or the keratinization of the hair. The purpose of this project is to elucidate whether the amount of mitochondrial DNA changes from the proximal to the distal end of the hair shaft. Five hair samples were obtained from five subjects and the hairs were dissected at every fourth centimeter. DNA was extracted from each hair section, and subjected to mitochondrial DNA quantification (via the control region of the genome), as well as assessed for any deletions seen within the coding region as a sign of damage that may have occurred, using an assay validated by the University of North Texas- Health Science Center (UNTHSC, Fort Worth, Texas). It was found that there was generally a gradual decrease in mitochondria copy number throughout the hair strands from the proximal to the distal end. Also, it was found that mitochondrial DNA is more susceptible to damage towards the distal end. Mitochondrial DNA sequencing was performed on specific samples to observe any relationship between the concentration of mitochondria and the stability of the sequence.Item Evaluation of NK Cell – Astrocyte Interactions: Potential Role in HIV-Associated Neurocognitive Disorders and HIV- Associated Dementia(2015-05-01) Bowen, Kelly E.; Mathew, Porunelloor A.; Mathew, Stephen O.; Hodge, Lisa M.NK cells play important roles in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. During pathological conditions, NK cells were shown to be recruited to the CNS and could impact CNS physiology by killing glial cells and by secreting IFN-g. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders (HAND). There is little data on NK cell-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. This study aimed to identify NK-associated ligands expressed by human astrocytes that confer this NK-directed cytotoxicity of astrocytes and assay the cytotoxicity differences in presence and absence of HIV 3S peptide. Using a fusion protein consisting of the extracellular domain of NKp44 fused to Fc portion of human IgG, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with 3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study demonstrated that NKp44 has a protective effect on astrocytes from NK cell mediated killing during HIV infection. Astrocytes could also secrete cytokines that affect the expression of NK receptors on NK cells. We evaluated the expression of receptors on NK cells after co-culture with astrocytes. CD38 expression was increased on primary NK cells after incubation with astrocytes. CD38 is expressed on both NK cells and astrocytes and has an important implication in HIV-1 infection. Blocking CD38 signaling in our studies decreased astrocyte lysis, suggesting CD38 signaling has important implications in NK-astrocyte interactions. Future studies providing novel insights into the role of NK cells in the pathogenesis of HAND and other brain disorders might result in the development of NK cell based therapies for brain pathologies.Item Examination of Ventilation Before and After Advanced Airway Placement During Continuous Chest Compressions Cardiopulmonary Resuscitation(2020-05) Gordon, Teresa R.; Hodge, Lisa M.; Sumien, Nathalie; Mathew, Stephen O.Purpose: The aim of this project was to identify a method to measure ventilation during continuous chest compressions CPR that will improve outcomes for resuscitation attempts on out of hospital cardiac arrest patients Hypothesis: It is possible to identify ventilation waveforms using defibrillator bioimpedance and adequate ventilation prior to placement of an advanced airway during continuous chest compressions is associated with better outcomes. Design: Defibrillator files from 4 Resuscitation Outcomes Consortium sites were examined for evidence of ventilations. The number of ventilations, placement of an advanced airway, return of spontaneous circulation, initial heart rhythm, and ventilation rates were recorded. Results and Conclusion: It is feasible to identify ventilations during continuous chest compressions CPR using bioimpedance. There was no significant difference between ventilation before and after an advanced airway was placed. The association between ventilation and outcome is undetermined.Item Extracellular Superoxide Dismutase Indirectly Enhances the Release of Immature Neutrophils from the Murine Bone Marrow(2016-08-01) Witter, Alexandra R.; Berg, Rance E.; Hodge, Lisa M.; Mummert, Mark E.Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using ecSOD HI, ecSOD WT, and ecSOD KO mice, we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes leading to increased host susceptibility. Using adoptive transfer experiments, we observed that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we noted that ecSOD activity results in decreased retention of immature neutrophils in the bone marrow without altering granulopoiesis. Furthermore, we determined that ecSOD activity protects the extracellular matrix (ECM) and increases concentrations of neutrophil-attracting chemokines leading to an increase in immature neutrophils in the liver. Since ecSOD can be produced by cells from the hematopoietic lineage as well as non-hematopoietic cells, we used bone marrow chimeric mice to investigate the relative contribution of ecSOD produced by cells from each lineage. Ultimately, it was determined that ecSOD from both hematopoietic and non-hematopoietic cells contributes to the overall phenotype observed in ecSOD congenic mice. Collectively, our data suggest that ecSOD activity inhibits degradation of the ECM and promotes egress of immature neutrophils out of the bone marrow and into the liver where they provide inadequate protection against L. monocytogenes. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.Item Feasibility and Effectiveness of the GLB-AIM (Group Lifestyle Balance Adapted for Individuals With Impaired Mobility) Intervention for People Living with Spinal Cord Injury(2017-12-01) Mazurek, Zachary; Reeves, Rustin E.; Hodge, Lisa M.; Mathew, Stephen O.Purpose: The prevalence of obesity in the population living with spinal cord injury (SCI) is greater than the general population. Obesity linked coronary heart disease is a leading cause of morbidity and mortality in the population living with SCI. Behavioral interventions to promote weight loss are limited for the SCI population. GLB-AIM looks to address the lack of behavioral interventions by providing a feasible and effective program to promote weight loss for people living with SCI. Methods: The GLB-AIM was delivered to participants over the course of 12 months. The sample was assessed for feasibility as measured by attendance over 12-month program and compliance with dietary self-monitoring for the first 13 weeks. Effectiveness was evaluated by measuring weight change over 12 months. The data were analyzed using a mixed models analysis controlling for time living with injury, group assignment, and starting weight. Results: The 12-month retention rate was 62.5% (20/32), Session attendance for the core sessions averaged 74.6% and dropped to 48.9% during the support sessions. Dietary self-monitoring for group 1 averaged 33% over the first 13 sessions and increased to 77% among group 2. Analysis of the combined SCI groups indicated significant weight loss (p = 0.017) that averaged 5.03 +8.58 kg over the 12-month program. Discussion: The GLB-AIM was a feasible and effective approach for promoting weight loss over 12 months for a sample with SCI. Additional adaptations may increase attendance during the subsequent support sessions and reduce program attrition by addressing barriers related to health events and transportation issues. The GLB-AIM program promoted weight loss in people living with SCI, which highlights the program’s effectiveness. Future adaptations of the GLBAIM should seek to enhance weight loss through increased weight feedback and the providing individualized calorie targets.Item Genetic Diversity of Easter Island (Rapanui) Population from Identifiler® Plus autosomal, Y-filer®, and Y-Plex™ 6 Y-STR Loci(2015-05-01) Guadian, Laura; Chakraborty, Ranajit; Budowle, Bruce; Hodge, Lisa M.This study investigated the genetic diversity of the Easter Island (Rapanui) population using data on 15 autosomal Short Tandem Repeats (STRs) typed with the commercial STR kits Identifiler® Plus and 23 Y-chromosome STRs typed using Y-filer (17 loci) and Y- PLEX™ 6 (6 loci). The analysis was conducted using genotype and haplotype data of 122 presumably unrelated individuals that included 48 males and 74 females. This study: (i) examined if Easter Island population had reduced genetic diversity in comparison with cosmopolitan populations such as Mainland Chilean, Polynesian, European, and African; (ii) compared genetic affinity of the Easter Island population with historically related cosmopolitan populations; and (iii) investigated the forensic utility of autosomal STRs and Y-STRs in the Easter Island population.Item Immune and Inflammatory Responses Differ Between the Upper and Lower Respiratory Tract(2001-05-01) Hodge, Lisa M.; Simecka, Jerry; Goldfarb, Ronald H.; Mathew, Porunelloor A.The purpose of these studies was to evaluate the role of upper and lower respiratory immune responses during immunization against respiratory disease antigens, and to characterize which immune responses during immunization against respiratory disease antigens, and to characterize which immune responses contribute to protection in the respiratory tract during infection. After nasal immunization, antigen-specific IgA antibody forming cells dominated throughout the respiratory tract. However, IgG responses were significant in lungs, but not in nasal passages. Furthermore, parental immunization did not enhance humoral immunity in the upper respiratory tract even after a nasal challenge, whereas extrapulmonary lymphoid responses enhanced responses in the lung. After nasal immunization, inflammatory reactions developed within the lungs of mice, but not in nasal passages. Lowering dosages of CT reduced, but did not eliminate, these adverse reactions without compromising immunogenicity. Serum IgE responses were also enhanced in a dose dependent manner by inclusion of CT. During infection, mRNA expression for IL-4 was greater in the nasal passages, while both mRNAs for IL-4 and IFN-y were increased in the lungs. As well, we found increased mycoplasma organisms in the lungs of IFN-y-/- mice, suggesting a protective role for cell-mediated immunity in the lung. In contrast, IL-4-/- mice had greater mycoplasma organisms in the nasal passages, indicating IL-4 responses are crucial for upper respiratory tract protection. Consistent with antigen deposition, nasal inoculation with 10 μl volume of antigen plus CT resulted in significant IgA responses in the nasal passages compared to mice given 24 μl immunizations; however, lower respiratory tract immunizations generated antibody responses in both nasal passages and lungs. In addition, both immunizations resulted in equivalent serum antibody responses. Upper and total respiratory tract immunizations provided protection in the nasal passages when CT was added. However, in the lung, all immunizations resulted in protection against mycoplasma infection, regardless of the inclusion of CT, suggesting a different role for CT as an adjuvant in upper and lower respiratory tract immune protection. In conclusion, we found immune responses generated during immunization and infection are different between the upper and lower respiratory tracts, and the contribution of these responses to clearance of respiratory infection differs.Item Increased resolution screening of the pharmacogenetic gene CYP2D6 with microarray technology(2019-08) Davis, Carey P.; Budowle, Bruce; Luedtke, Robert R.; Hodge, Lisa M.; Phillips, Nicole R.; Woerner, August E.Autopsy is a primary methodology used for assessing cause and/or manner of death in medicolegal investigations. Some autopsies, however, do not resolve the cause of death unambiguously or there is no evident pathology to determine the cause of death. In addition, in some cases toxicology screens are negative or difficult to interpret because it is challenging to determine if a high concentration of a drug in the body derived from one large dose or has built up over time. Determining the genetic constitution of victims at specific target genes may clarify the cause of some of these unexplained deaths or at least indicate susceptibility to triggering effects. Cytochrome P450 (CYP450) is a super family of enzymes that detoxify foreign chemicals and are involved in the metabolism of drugs. One gene in this family that encodes CYP450 enzymes, CYP2D6, accounts for the metabolism of 25% of all drugs currently on the market. By examining the variability in the CYP2D6 gene, a SNP panel was developed and used to aid in personalized medicine with a long-term outcome of reducing risk in patients who partake in drug therapy. However, paralogs of the CYP2D6 gene can interfere with obtaining accurate typing results. The hypothesis of this dissertation is that it is possible to develop a targeted panel for clinically relevant variants in the CYP2D6 gene using array-based technology that can provide accurate and reliable genotyping results. The goal was first to demonstrate that high throughput sequencing, also known as next generation or massively parallel sequencing, could reliably sequence a complex target using a model system, i.e. the hypervariable regions of the human mitochondrial genome. Then, using this advanced sequencing capability define the baseline genetic variation of the CYP2D6 gene in a selected population and identify those genetic markers associated with metabolism capacity that would be verified against a database of actionable variants that cause reaction to drug exposure. The entire CYP2D6 gene was sequenced to identify SNPs at a population level. These SNPs were compared against a known database of clinically relevant samples with known metabolic responses of the same ethnic background to verify actionable variants. Once these variants were identified, a PCR assay workflow leveraging microarray technology was developed to quickly and efficiently screen individuals of interest by overcoming paralog interference. This assay can be used prior to administering drugs or post mortem to gain information about potential adverse drug reactions.Item Lymphatic Pump Treatment Enhances Pulmonary Immunity and Inhibits Solid Tumor Formation in the Lung(2009-12-01) Pedrueza, Mayela; Hodge, Lisa M.Item Lymphatic Pump Treatment Enhances the Lymphatic and Immune System and Ameliorates Disease Severity in a Rat Model of Respiratory Infection(2014-05-01) Schander, Artur; Hodge, Lisa M.The purpose of these studies was to explore the benefits, effects, and mechanisms of LPT in both a healthy and a diseased animal model, and hence provide scientific rationale for the clinical application of LPT. Novel findings in this dissertation demonstrate that in anesthetized canines: 1) LPT mobilizes leukocytes from the GALT into lymphatic circulation; 2) LPT mobilizes inflammatory mediators into lymphatic circulation; and 3) repeated application of LPT increases lymph flow, concentration of leukocytes, and flux of inflammatory mediators into lymphatic circulation. In addition, this dissertation for the first time demonstrates: 1) the development of a novel lymph enhancing rodent model, in which LPT increases leukocyte flux in the cisterna chili, predominantly from the GALT; and 2) that LPT facilitates the clearance of pneumococcal respiratory infection and suggests a mechanism by which LPT might facilitate the clearance of pneumococcal pneumonia. Our studies demonstrated that LPT transiently mobilized leukocytes from the mesenteric lymph nodes. We found a significant increase in the concentrations of MCP-1 and flux of IL-6 flux in TDL and MDL in anesthetized dogs. Interestingly, both IL-6 and MCP-1 were present in BALF of rats infected with pneumococcus, and LPT significantly increased IL-6 and moderately increased MCP-1 concentrations compared to Sham and Control animals, which supports our notion that LPT may increase cytokine/chemokine redistribution from the mesentery to the lung. We demonstrated that LPT enhanced the clearance of S. pneumoniae after 3 consecutive daily treatments and found that LPT increased the concentrations of SP-D, IL-6, IL-12p70, and IL-17 in BALF and enhanced the release of NO2- and IL-6 by AM 4 days post-infection. Collectively these studies suggest, that LPT re-distributes inflammatory mediators to the lung, enhances the recruitment of macrophages and neutrophils to the lung and skews alveolar macrophages towards a M1 phenotype, all of which may be responsible for and promote the clearance of S. pneumoniae.Item Modulation of Astrocyte Phenotype in Response to T-cell Interaction(2021-05) Hersh, Jessica M.; Yang, Shaohua; Smith, Michael L.; Jin, Kunlin; Hodge, Lisa M.We determined that T-cell astrocyte interaction modulates interleukin-10 (IL-10) production from both cell types. The impact of IL-10 on astrocytes was compared to IL-10 generated from T-cell-astrocyte interactions in vitro. We demonstrated that T-cells directly interact with astrocytes to upregulate gene expression and secretion of IL-10, confirmed by elevated STAT3p/STAT3 expression in astrocytes. IL-10 increased astrocytes proliferation. In addition, IL-10 treatment and CD4+ co-culture shifts primary astrocytes toward a more energetic phenotype. These findings indicate that direct interaction of CD4+ T-cells with astrocytes, activated the IL-10 anti-inflammatory pathway, altering astrocyte phenotype, metabolism, and proliferation.Item Obstacles Associated with Physician Referral of Patients into Clinical Trials(2017-05-01) Torrez, Nick; Hodge, Lisa M.; Mathew, Stephen O.; Jung, Marianna E.Understanding the safety and efficacy of potential new medications relies on evidence gained through the participation of subjects in clinical drug trials. Many clinical trial sites struggle with recruitment of suitable participants which can delay the progress of drug development. Physicians can play a significant role in influencing patients to enter into a clinical trial, however many physicians due not utilize their unique position to facilitate the recruitment of patients into clinical trials, which may help to advance medical science and improve future treatment options. The lack of participation by physicians in the referral of patients into clinical trials (Crosson et al. 2001; Daugherty C, 1995; Jenkins and Fallowfield, 2000; Lara et al., 2001) can potentially be explained by various obstacles. We propose that these obstacles may be issues such as time, lack of knowledge about clinical trials, lack of clinical trials suitable for patients, language barriers, conflict of interest, communication with local investigators, and trust in medical researchers.Item Osteopathic Lymphatic Pump Treatment as an Adjunctive Therapy to Protect Against Infection and Inflammation(2018-08) Castillo, Rudy A.; Hodge, Lisa M.; Simecka, Jerry W.; Mathew, Porunelloor A.; Goulopoulou, Styliani; Budowle, BruceThe lymphatic system transports interstitial fluid from the tissue into circulation. Osteopathic physicians recognize the importance of the lymphatic system and have designed osteopathic manipulative techniques, including lymphatic pump treatment (LPT), to enhance the flow of lymph. LPT is used clinically by osteopathic physicians as an adjunctive therapy for the treatment of pneumonia, although its mechanism of protection is unknown. Recent studies have demonstrated LPT increased lymphatic flow and reduced the concentration of bacteria in the lungs of rats infected with Streptococcus pneumoniae. The combination of LPT and antibiotic further reduced the concentration of S. pneumoniae compared to antibiotics alone. The goal of these studies was to identify the mechanism(s) by which LPT protects the lung during infection. Enhancing lymph output using therapies such as LPT might redistribute protective lymph-borne factors and pharmaceuticals to the lung, providing additional protection against pneumonia. In the first aim we hypothesized that LPT would enhance the delivery of levofloxacin from the blood to the lung, which might accelerate the clearance of pulmonary bacteria. We discovered that LPT increased the concentration of antibiotic in the epithelial lining fluid over time. These results suggest LPT may aid during treatment of pneumonia by facilitating the delivery of antibiotics the lung. In previous studies, LPT protected rats against pneumonia without the need for antibiotics, suggesting there is another mechanism of protection offered by LPT. LPT mobilizes lymph-borne factors into circulation that may modulate pulmonary inflammation. Therefore, the second aim of these studies was to determine the biological effect of thoracic duct lymph mobilized during LPT on macrophage activity in vitro. Thoracic duct lymph suppressed the inflammatory response of macrophages and alveolar macrophages to lipopolysaccharide, lipoteichoic acid, and interferon-gamma. Importantly, lung immunopathology is associated with morbidity and mortality during pneumonia caused by S. pneumonia. Therefore, by mobilizing lymph into circulation, LPT may protect the lung by suppressing this immunopathologic response. In conclusion, these studies have elucidated mechanisms of protection offered by LPT and support its use as an adjunctive therapy for the treatment of pneumonia. Once these mechanisms are fully understood, LPT can be optimally applied to patients with pneumonia, which may substantially reduce morbidity, mortality and the cost of hospitalization.Item Oxazolone as a Model to Induce Edema in the Lower Limb of Rats(2018-03-14) Castillo, Rudy; Hodge, Lisa M.; Pastrana, ChristopherPurpose: Peripheral edema is a condition characterized by the accumulation of excess interstitial fluid in distal tissues and commonly manifests in the arms or legs. Untreated complex peripheral edema can progress into chronic lymphedema as impaired fluid drainage and chronic inflammation cause irreversible damage to the surrounding tissue and local lymphatics. The overall goal of our research is to study the effectiveness of osteopathic manipulative medicine treatments on edema, infection and inflammation. The aim of this research was to evaluate to the oxazolone (OXA)-induced acute skin inflammation model to induce lower limb edema in the rat. Specifically, we hypothesized that the application of OXA would induce create a local inflammatory response, induce edema in the lower limbs decrease lymphatic vessel function. Methods: Female Sprague Dawley rats, weighing 200-250 g, were used for this study. To induce edema, on day zero the right lower limb was shaved and 750 µl of 5% OXA-acetone solution or 5% PBS-acetone solution vehicle (VEH) was applied to the exposed skin. Lower limb measurements were made at days zero and six using a Vernier caliper. Measurements were taken at the midpoints of the hind paw, ankle, tibia and femur of their right lower limbs to establish paw thickness; distance between paw-to-ankle and ankle-to-tibia were taken to calculate lower limb volume by truncated cone formula. At day six, the rats were euthanized, and the bilateral hind paws were removed above the calcaneus and weighed. The spleen and bilateral inguinal lymph nodes were removed, homogenized, centrifuged and cells were washed. Cell pellets were stained with PE mouse anti-rat granulocytes, FITC anti-rat CD3, and APC anti-rat CD161 antibodies. The percentage of granulocytes, T cells and dendritic cells were measured by flow cytometry. Data were analyzed by ANOVA followed a Tukey post-test. Comparisons were made between OXA and VEH groups at day six post-induction. Results: OXA did not induce significant (p [greater than] 0.05) changes in either hind paw thickness or lower limb volume. OXA significantly (P Conclusions: OXA induced an acute inflammatory response in the draining inguinal lymph nodes. However, as used in this approach, OXA did not induce peripheral edema. In future studies we investigate alternate strategies to induce lower limb edema in the rat.Item Oxazolone as a Model to Induce Edema in the Lower Limb of Rats(2018-05) Pastrana, Christopher; Hodge, Lisa M.; Simecka, Jerry W.; Mathis, Keisa W.; Sumien, NathaliePurpose. Peripheral edema is a condition characterized by the accumulation of excess interstitial fluid in distal tissues and commonly manifests in the arms or legs. Untreated complex peripheral edema can progress into chronic lymphedema as impaired fluid drainage and chronic inflammation cause irreversible damage to the surrounding tissue and local lymphatics. The overall goal of our research is to study the effectiveness of osteopathic manipulative medicine techniques (OMT) for the treatment of edema, infection and inflammation. The aim of this study was to evaluate oxazolone (OXA) as a model to induce acute inflammation and lower limb edema in the rat. Specifically, we hypothesized that a single application of OXA would induce a local inflammatory response and induce edema in the lower limb. Methods. Female Sprague Dawley rats, weighing 200-250 g, were used for this study. On day 0 the right lower limb was shaved and 750 µl of 5% OXA-acetone solution or 750 µl of a 5% phosphate buffered saline (PBS)-acetone solution vehicle (VEH) was applied to the exposed skin. Lower limb measurements were made at days 0 and 6 using a Vernier caliper. At day 6, the rats were euthanized, and the bilateral hind paws were removed above the calcaneus and weighed. The spleen and bilateral inguinal lymph nodes were removed, homogenized, centrifuged and cells were stained with phycoerythrin (PE) mouse anti-rat granulocytes, fluorescein (FITC) anti-rat CD3, and allophycocyanin (APC) anti-rat CD161 antibodies. The percentage of granulocytes, T cells and dendritic cells were measured by flow cytometry. Data were analyzed by analysis of variance (ANOVA) followed by Tukey-Kramer post-test or by Student's t-test. Comparisons were made between OXA and VEH groups at day 6 post-induction. Results. OXA did not induce significant (p[greater than]0.05) changes in either hind paw thickness or hind paw volume. OXA significantly (P[less than]0.05) increased the concentration of macrophages, neutrophils, and dendritic cells, and T cells within inguinal lymph nodes. Conclusions. OXA induced an acute local inflammatory response in the draining inguinal lymph nodes. However, as used in this approach, OXA did not induce peripheral edema. In future studies we will investigate alternate strategies to induce lower limb edema in the rat.