Browsing by Author "Hodge, Lisa"
Now showing 1 - 14 of 14
- Results Per Page
- Sort Options
Item ANTI-TUMOR IMMUNE RESPONSES AGAINST MTLn3 MAMMARY ADENOCARCINOMA(2014-03) Carter, KiahRae J.; Orlowski, Ashley; Hodge, LisaBreast cancer is the leading cause of cancer-related deaths. More research needs to be done to examine the role of the lymphatic system during metastasis and therapies directed at the lymphatic system. Our rat model was used because it closely mimics human breast cancer. These results will allow for the future studies of therapies targeting the lymphatic system and if they will prevent metastasis. Purpose (a): Breast cancer is the leading cause of cancer-related morbidity and mortality. New research suggests the lymphatic vessels play a key role during the metastasis of breast cancer and therapies directed at the lymph system may aid in the treatment of breast cancer. MTLn3 is a mammary adenocarcinoma that is commonly used to study the effects of tumor metastasis in Fischer 344 rats. MTLn3 closely mimics human breast cancer pathogenesis, making it ideal for the study of breast cancer disease; however, little is known about the role of the lymphatic and immune systems in this disease model. The purpose of this study was to identify the type of immune response generated during MTLn3 disease. Specifically, we proposed that natural killer cells (NK), T cells, B cells and macrophages (MO) would increase in response to disease. Methods (b): To test our hypothesis, rats were randomized into control group or were sub-cutaneously injected in the right mammary fat pad with 1x106 MTLn3 tumor cells/mL on day 0. At days 0, 7, 14, 21 and 25 post-injection, lungs, tumor-adjacent lymph nodes (ALN), tumor–opposite lymph nodes (OLN) and spleens were removed and the concentration of leukocytes was determined. Primary tumors were excised and measured to calculate tumor volume. Blood was analyzed for the complete blood count and serum was measured for cancer-specific biomarkers. Results (c): All animals gained weight until day 14 post-injection. However, rats injected with MTLn3 suffered weight loss between days 14-25 post-injection. Furthermore, primary tumor size significantly (p < 0.05) increased during this time, suggesting weight loss may be related to disease. CD4+ T cells, B cells and MO in the spleen at day 21 decreased by day 25. Tumor adjacent lymph nodes experienced an increase in all cell populations, T cells, B Cells, MO, dendritic cells and NK. There were no differences in cell populations between ALN and OLN, except MO were significantly (p < 0.05) increased in ALN at Day 25. There was no change in pulmonary leukocytes by day 25. Neutrophils, monocytes and lymphocytes in the blood were significantly (p < 0.05) increased between control and 25 days post-injection rats, suggesting there is an immune response against MTLn3 tumor cells. Conclusions (d): Collectively, our results suggest MTLn3 initiates an immune response mediated by T cells, B cells, macrophages and NK cells between days 14-25 of disease. Of interest, these cells increase in the ALN at day 25 post-injection, suggesting they migrate into the lymph nodes in response to disease. In future studies, we will determine if MTLn3 metastasizes to the sentinel lymph nodes and the lung and determine if therapies targeting the lymphatic system inhibit this process.Item Cardiovascular Response to Endotoxin-Mediated Sepsis: A Dose-Response Study(2022) Aguirre, David Salinas; Martinez, Richard; Warne, Cooper; Mallet, Robert T.; Dick, Gregory; Tune, Johnathan; Hodge, LisaPurpose: Our long-term goal is to advance our understanding and treatment of sepsis, a potentially life-threatening condition that occurs when the response to infection causes tissue and organ damage. Sepsis can be caused by lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria. We used a swine model of LPS-induced sepsis to study the impact of the lymphatic and immune systems on the disease progression. Our first experiments were aimed at determining the optimal dose of Escherichia coli LPS in order to study the effect of sepsis on the cardiovascular system. We hypothesized that 2-hour intravenous infusions of 1- 50 µg/kg LPS would reveal dose- and time-dependent changes in hemodynamic parameters that are consistent with sepsis. Methods: Yorkshire pigs (61 ± 4 kg, n = 4, 2 male) were sedated, intubated, and ventilated. Femoral artery and venous lines were placed to allow measurement of blood pressure, infusion of LPS, and blood gas sampling. A thoracotomy was performed in order to secure a Transonic flow probe around the left anterior descending coronary artery and to insert a sampling catheter in the anterior cardiac vein. A dose of LPS (1, 5, 25, and 50 µg/kg) was given to each pig over 2 hours. Blood samples were collected immediately before LPS infusion and for every 30 minutes during and after LPS infusion for blood gas measurements. Vital signs were recorded as the animals developed sepsis. Results: Only the pig given the lowest dose of LPS (1 µg/kg) survived the full 6 hours (mean survival time in remaining 3 pigs was 180 ± 30 min). At 150 min, a ≈55% decrease in mean arterial pressure was observed (107 ± 4 to 48 ± 13 mmHg), resulting in a ≈60% increase in heart rate (91 ± 9 to 146 ± 14 beats/min). Coronary blood flow and myocardial oxygen consumption decreased ≈28% (0.53 ± 0.06 to 0.38 ± 0.02 ml/min/g) and ≈33% (60 ± 6 to 44 ± 2 µl/min/g), respectively. Ventricular fibrillation was the cause of death in the 3 non-surviving pigs. Conclusion: A dose of 1 µg/kg appears to be an optimal dose for future studies, as this dose was survivable in the desired time frame, while causing hypotension and tachycardia. In future studies, this model will allow us to study the effect of novel therapeutics during acute sepsis.Item Culture with Dextrose Reduced Macrophage Viability in A Dose Dependent Manner: Implications for Prolotherapy(2017-03-14) Castillo, Rudy; Conway, Allison; Rabago, David; Nourani, Booby; Hodge, Lisa; Marvin, HannahPurpose: Prolotherapy, coined from proliferant therapy, is an alternative injection-based therapy that has been used in clinical practice for over 80 years to treat various chronic musculoskeletal conditions. Modern hypotheses suggest prolotherapy promotes growth of normal cells and tissues to improve ligament mechanics, and decrease pain through inflammatory mechanisms. The most common injectant contains dextrose (D-glucose), a natural form of glucose found in the body. Recent literature indicates adults with symptomatic knee pain received the most relief from intra-articular injection of dextrose, compared to saline injections, or exercise. This study aims to begin to explain the mechanism of action of dextrose in the inflammatory response. Specifically, we hypothesized that dextrose would suppress the release of inflammatory mediators from LPS-activated macrophages. Methods: To test this hypothesis, murine RAW 264.7 macrophages were cultured in vitro with phosphate-buffered saline (PBS) or dextrose solution at 2.5%, 5%, and 10% of total volume per well, with or without 500ng lipopolysaccharide (LPS). Twenty-four hours after incubation at 37°C with 5% CO2, culture supernatants were stored and assayed for nitrite (NO2-) using Griess reagent. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide. Results: Dextrose did not significantly alter the production of NO2- in macrophages cultured without LPS. However, in LPS-activated macrophages, dextrose significantly (p2- compared to respective PBS controls. Specifically, 2.5% dextrose suppressed NO2-release by 78.31%, while 5% and 10% dextrose completely inhibited production of NO2- . Cell viability was also significantly (p Conclusions: Dextrose reduced viability and suppressed the production of NO2- by macrophages in vitro. Prolotherapy may protect against inflammation by reducing the inflammatory activities of macrophages. Future studies will examine the effect of dextrose on macrophage function in vivo using animal models.Item Cytokine Response in an Endotoxin-Mediated Sepsis Model(2022) Martinez, Richard; Aguirre, David Salinas; Warne, Cooper; Dick, Gregory; Mallet, Robert T.; Tune, Johnathan; Hodge, LisaPurpose: Sepsis is a life-threatening condition that develops secondary to infection and can manifest acute organ dysfunction due to the body's overactive systemic response. Sepsis affects approximately 1.7 million US adults and claims 270,000 lives as a result. The long-term goal of our project is to gain a better understanding of the roles of the lymphatic and immune systems in the progression of sepsis. The purpose of this study is to collect pilot data using a translational swine model of endotoxin-mediated sepsis. We chose a swine model because it closely mimics how sepsis progresses in humans. Sepsis was induced by infusion of lipopolysaccharide (LPS) from Escherichia coli. LPS was chosen because it is a key mediator in the activation of the immune system and the development of sepsis. We hypothesized that the administration of LPS would increase the concertation of the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-?) in a dose-dependent manner over time. Methods: Yorkshire pigs (61 � 4 kg, n = 4, 2 male) were sedated, intubated, and ventilated. Thoracotomy was performed under anesthesia to record flow data and sample cardiac blood for use in another study. Femoral artery and venous lines were placed to allow measurement of blood pressure and infusion of LPS. Specifically, LPS was prepared at 1, 5, 25, or 50 ?g/kg (pig body weight) in sterile saline. LPS was infused into anesthetized pigs over a 2-hour period. Blood samples were collected immediately prior to LPS administration and at 30 min intervals during 2 hours of LPS infusion up to 4 hours following LPS infusion. The plasma was analyzed via enzyme-linked immunosorbent immunoassay (ELISA) for the concentrations of IL-6 and TNF-? using commercially available kits. Results: As hypothesized, the infusion of LPS increased the concentration of the inflammatory mediators IL-6 and TNF-? over time compared to pre-LPS infusion. Specifically, the greatest increase in IL-6 was seen at 180 minutes in both the 50 and 25 ?g/kg LPS infused pigs. TNF-? concentration peaked between 30 to 90 minutes during LPS infusion in both the 50 and 25 ?g/kg LPS infused pigs. The lower doses of 1 and 5 ?g/kg LPS produced little to no IL-6 or TNF-?. Furthermore, we discovered that the pigs who received 50 or 25 ?g/kg of LPS died from septic shock within 180 minutes of LPS infusion, whereas the pigs that received 1or 5 ?g/kg of LPS survived longer. Conclusion: In this study, we identified the impact of increasing the doses of LPS on the production of IL-6 and TNF-? in swine. Our preliminary results suggest a dose range of 10-20 ?g/kg of LPS may be ideal to study the inflammatory response in this model. The acquisition of these data are essential to pursue our long-term research objective, which is to identify the role of the lymphatic and immune systems during sepsis.Item Differential Effects of Thoracic Duct Lymph on Pulmonary Macrophages(2016-03-23) Conway, Allison; Hodge, Lisa; Castillo, Rudy A.Purpose: The gut-lung axis remains a poorly defined mechanism that could impact etiology and treatment of gut and respiratory diseases. Early literature first described this gut-lung crosstalk in inflammatory bowel disease patients with chronic bronchopulmonary disease. Recent literature describes the role of intestinal lymph during acute respiratory distress syndrome (ARDS). In these studies, intestinal ischemia and reperfusion injury in animal models induces lung injury suggesting factors, such as cytokines and lipids, released from the gut during gastrointestinal shock can contribute to ARDS. The gut lymphatics provide a large pool of lymph rich in immune cells, inflammatory mediators, and lipids. Our lab has previously demonstrated lymph-enhancing techniques enhanced the flux of cytokines, chemokines and reactive oxygen and nitrogen species in thoracic and mesenteric lymph. We propose factors released from the gut travel through the lymphatics to the lung and suppress the immune response in the lung. Methods: To test this hypothesis, alveolar macrophages from bronchoalveolar lavage fluid (BALF), lung tissue macrophages (after BALF collected) and intraperitoneal (IP) macrophages from IP lavage were isolated from healthy F344 rats. In addition, a rat alveolar macrophage cell line (NR8383) was used for in vitro studies. Macrophages were cultured for 12 hours and canine thoracic duct lymph (TDL) at 10% total volume per well and/or LPS (500ng per well) added for 24 hours. Supernatants were stored and used to measure nitric oxide (NO) and tumor necrosis factor alpha (TNFa) using Griess assay and ELISA. Results: AM were the most sensitive to LPS activation compared to LM and IP macrophages. TDL suppressed LPS-induced NO (52% decrease) and TNFa (25% decrease) production. However, TDL did not significantly suppress LPS activation in LM and IP macrophages. In NR8383, TDL suppressed LPS-induced NO (86% decrease) and TNFa (66% decrease) production. TDL alone did not activate NR8383, AM, LM or IP macrophages and did not affect cell viability after culture. Conclusions: Our results suggest a biological factor in lymph selectively suppresses LPS activation in alveolar macrophages. The mobilization of healthy lymph may protect the lung from chronic inflammation caused by pathogens and pulmonary disease. Future studies will focus on identifying factors in lymph that may modulate the immune response that may improve disease outcome.Item The effects of esmolol on the control of coronary blood flow and myocardial oxygen supply-demand balance in sepsis(2024-03-21) Digilio, Michaela; Warne, Cooper; Heard, Michael; Tucker, Selina; Essajee, Sal; Bradford, Ni' Ja D.; Hodge, Lisa; Tune, Johnathan; Dick, GregoryPurpose: Sepsis is an acute organ dysfunction secondary to infection that results in tachycardia, tachypnea, fever, decreased blood pressure, and lactic acidosis. This results in an overall myocardial oxygen supply-demand imbalance leading to cardiac dysfunction and ultimately death. The current treatment for sepsis is antibiotic therapy, vasopressors, and fluid therapy. However, this regimen does not address the tachycardia that leads to cardiovascular decompensation. Beta-blocker therapy addresses this myocardial oxygen supply-demand imbalance and is expected to promote survival in sepsis. We hypothesize that treatment with beta-blocker therapy during acute sepsis will address the myocardial oxygen supply-demand imbalance to maintain coronary perfusion pressure, improve myocardial oxygen delivery, and promote survival. Methods: Female and male Yorkshire pigs were used as the animal model for this project. Pigs were anesthetized, intubated, and a rectal thermometer and oximeter were placed. Catheters placed in ear vein, great cardiac vein, femoral artery, and bilateral femoral veins. Pressure transducer placed in the femoral artery. A transonic flow transducer placed around the left anterior descending artery. After instrumentation, baseline values were collected. Then, infusion with Escherichia coli lipopolysaccharide (LPS) at 10 µg/kg over the course of 2 hours was used to induce sepsis. LPS was infused via the femoral vein at a rate of 0.5mL/min. After 2 hours, intervention began depending on the treatment group. Intervention lasted 4 hours. Experiment groups included Sham (without LPS, fluids, norepinephrine (NE), or esmolol), Control (with LPS, no fluids, NE, or esmolol), Standard (with LPS, fluids, and NE), and Experimental (with LPS, fluids, NE and esmolol). Doses: LPS 10 µg/kg, esmolol escalating from 100mg/hr, and NE escalating from 0.4 µg/kg/min. Goals during the intervention included keeping the mean arterial pressure (MAP) above 65mmHg and heart rate below 100. Results: All control pigs died during the 4-hour follow-up. 1 out of 3 standard treatment pigs survived. All esmolol-treated pigs survived. The esmolol group had better MAP, coronary blood flow, myocardial oxygen delivery, and oxygen extraction than the standard treatment group. Conclusion: Esmolol improves survival, coronary perfusion pressure, and myocardial oxygen delivery. This data provides support for our hypothesis and the clinical use of esmolol in sepsis.Item Lymphatic pump technique mobilizes lymph that inhibits the inflammatory response of macrophages in vitro(2018-03-14) Hodge, Lisa; Castillo, Rudy A.Purpose: The lymphatic system maintains tissue fluid homeostasis by returning excess fluid, known as lymph, into circulation. Osteopathic physicians recognize the importance of the lymphatic system and have designed a set of lymphatic pump techniques (LPT) that enhance the flow of lymph. LPT has been used clinically to treatment respiratory tract disease, infection, and edema. LPT has also been reported to enhance mesenteric and thoracic lymphatic flow, the concentration of leukocytes, and the flux of inflammatory mediators in lymph of dogs. We propose LPT may act as an adjunctive therapy by mobilizing lymph-borne factors into circulation that protect tissues during inflammation. In this study, we propose that lymph mobilized by LPT suppresses macrophage activation in vitro. Methods: To test this hypothesis, under anesthesia, the thoracic ducts of six mongrel dogs were cannulated and thoracic duct lymph (TDL) was collected before (baseline), during, and after (recovery) LPT. TDL supernatant was collected by centrifugation and frozen. Total protein was measured in TDL using the Bradford method. Murine RAW 264.7 macrophages were cultured with baseline, LPT, or recovery TDL at 5% total volume per well with or without lipopolysaccharide (LPS) for 24 hours at 37°C and 5% CO2. After culture, cell-free supernatants were assayed for nitrite (NO2-), tumor necrosis factor-alpha (TNF-alpha), and interleukin-10 (IL-10). Macrophage viability was measured using flow cytometry and markers, annexin V and propidium iodide. Results: LPT transiently increased TDL flow and protein flux (10-fold). Baseline, LPT, or recovery TDL did not increase the production of NO2-, TNF-alpha, IL-10 or alter macrophage viability. When macrophages were activated with LPS, the addition of baseline, LPT, or recovery TDL decreased the production of NO2- (2-fold), TNF-alpha (5-fold) and IL-10 (3-fold) compared to LPS. There were no significant (p [greater than] 0.05) differences in the production of NO2-, TNF-alpha, and IL-10 by macrophages cultured with baseline, LPT, or recovery TDL and LPS. Conclusions: Our data suggests that lymph contains biological factor(s) that suppress macrophage activation without altering cell viability. The redistribution of protective lymph during LPT may provide scientific rationale for the clinical use of LPT to treat inflammation and edema. Future studies will focus on the mechanism(s) responsible for these novel findings.Item Osteopathic Lymphatic Pump Treatment Does Not Alter Antibiotic Distribution in a Rat Model(2015-03) Redman, Charlotte J.; Hodge, LisaPurpose: Osteopathic doctors perform manual medicine treatments to promote the body to self-heal. The lymphatic pump technique (LPT) is one of these treatments and it has been shown to improve lymph circulation and delivery of immune factors. In 2010 we developed a rat model to study the effect of LPT on the lymphatic system. We found that 4 minutes of LPT significantly increased the lymph flow and the concentration of lymphocytes in the lymph. We also found that the combination of LPT and levofloxacin protected against Streptococcus pneumoniae-mediated pneumonia by reducing the concentration of bacteria in the lungs. Furthermore, LPT has been shown to increase the uptake of antigen from the interstitium; therefore, LPT may act as an adjunctive therapy during the treatment of pneumonia by enhancing the uptake and delivery of antibiotics. Specifically, we hypothesized that LPT would increase the concentration of levofloxacin in the lungs. Materials and Methods: Male, Fisher 344 rats with jugular vein catheters weighing between 200-300 grams were used. Rats were injected subcutaneously with 50 mg/kg levofloxacin and randomized into control, sham, or LPT groups. Control rats received no treatment or anesthesia, sham rats received anesthesia for 4 minutes, and LPT rats received LPT under anesthesia for 4 minutes. Ten minutes post-treatment, serum and bronchoalveolar lavage fluid (BALF) were collected and the concentration of levofloxacin was determined using a levofloxacin bioassay. Results: In serum, there was no significant difference (p value= 0.74) in the levofloxacin concentration between control (3.8±0.4 μg/ml), sham (3.9±0.6 μg/ml), or LPT (4.3±0.5 μg/ml). Similarly in the BALF, there was no significant difference (p value= 0.69) in the levofloxacin concentration between control (0.14±0.03 μg/ml), sham (0.10±0.03 μg/ml), LPT (0.12 ±0.03 μg/ml). Conclusion: In conclusion, the results from this experiment suggest that LPT does not enhance the delivery of levofloxacin to the lungs. Alternatively, LPT may act as an adjunctive therapy during the treatment of pneumonia by enhancing immune-mediated protection. Future studies are necessary to test this hypothesis.Item Pyruvate-Enriched Anti-inflammatory Preservation of Explanted, Machine-Perfused Porcine Kidneys(2020) Olivencia-Yurvati, Albert; Morales, Jessica; Hodge, Lisa; Mallet, Robert T.; Konty, Logan; Williams, Arthur; Ryou, Myoung-Gwi; Omar,SalmaPurpose: The gold-standard treatment for end-stage renal disease (ESRD) is kidney transplantation. Although the incidence of ESRD continues to mount, the supply of transplantable kidneys is woefully inadequate. Kidneys obtained from cardiac death victims are a potential source, but oxidative stress and inflammation predispose the organs to acute failure or delayed graft function after transplant. The intermediary metabolite pyruvate possesses energy-yielding, antioxidant and anti-inflammatory properties, and has been found to induce anti-inflammatory erythropoietin production in brain and heart. We postulate pyruvate-enriched preservation of machine-perfused kidneys will preserve organ integrity by increasing renal erythropoietin formation and dampening pro-inflammatory cytokine production. Methods: Kidneys are obtained from juvenile, Yorkshire pigs mechanically ventilated with 2and3% isoflurane. To model donation after cardiac death, the kidneys are harvested after induction of cardiac arrest and 60 minutes of ischemia in situ. After cold saline flush, the right kidney is explanted and perfused via the renal artery with standard organ preservation solution +/- 20 mM pyruvate in a LifePort® organ preservation system at 2-5°C for 72 h. Perfusate is sampled periodically for measurements of pro-inflammatory cytokines TNF-alpha and IL-6 and anti-inflammatory cytokines erythropoietin and IL-1beta. Results: The results are pending, as we are currently acquiring data. Conclusions: The conclusions are pending, but we expect hypothermic machine perfusion with pyruvate-enriched versus standard perfusate will suppress renal release of pro-inflammatory cytokines and increase anti-inflammatory cytokine formation, thereby blunting renal inflammation before transplant.Item Quality Assurance Training: Will a New Training Intervention Improve Data Collection of the Texas Emergency Medicine Research Associate Program (TEMRAP)?(2018-12) Saldana, Miguel Antonio; Hodge, Lisa; Pierce, Ava; Krishnamoorthy, RaghuIntroduction: Data collection is vital for the success of a clinical research project. The purpose of this practicum was to address the inadequate data collection by the Texas Emergency Medicine Research Associate Program (TEMRAP) research associates (RAs). The primary goal was to incorporate a more efficient training method to reduce the RAs' error rate in the documentation. The secondary aim of this experiment was to determine if RAs' knowledge of clinical research studies and/or their self-confidence when enrolling a patient had an effect on quality of data collection and if these variables could be improved by a new training method. Methods: A randomized clinical trial was used to evaluate the efficacy of simulated clinical research enrollment training as a teaching and/or learning method to reduce the error rate in submitted research packets by RAs. The returning RAs were randomized into an intervention group with new training (simulations) and a control group with current training (didactic presentations). A self-confidence survey and a knowledge questionnaire were completed by RAs pre/post-training and one-month follow-up. Quality of data collection was measured by comparing the error rates of data collection in completed clinical research enrollment packets submitted by the RAs in the intervention group versus the control group. Results: Results showed no statistically significant difference in the level of knowledge, confidence or error rates between the patient enrollment simulation (intervention) group and the didactic presentations (control) group after their respective training (p [greater than] .05). However, there was a statistically significant increase in knowledge and confidence post-training in patient simulations group. A significant association was present between confidence and error rate but not between knowledge and error rate for research associates in either training group. Conclusion: Clinical simulation training was not a significantly more effecting training method compared to current TEMRAP didactic presentation training. Even though knowledge and confidence did increase post-training there was no significant difference between the two types of training. Future experiments should explore the possibility of combining the two types of training and observing other potential variables affecting the quality of data, such as research associates' motivation. Additionally, the need for a larger sample size and enrolling participants with no prior research experience should be explored for significant results.Item The Effect of Osteopathic Manipulation Techniques on the Lymphatic System(2021) Parker, Madison; Williams, Arthur; Mallet, Robert T.; Tune, Johnathan; Hodge, LisaPurpose: Osteopathic manipulation techniques (OMT) have been utilized by osteopathic physicians to treat a variety of conditions. However, the mechanisms by which OMT aides the body in healing are not well understood. The long-term goal of our research is to advance our understanding of the impact of OMT during disease, such as sepsis. The purpose of this study is to develop a large animal model and to establish surgical techniques that will be used in these future studies. We hypothesized that the abdominal lymphatic pump technique (LPT) will enhance thoracic duct lymph (TDL) flow. Methods: The thoracic duct was exposed via thoracotomy then cannulated using an angio-catheter. TDL was collected from 2 swine (1 male, 1 female) during 10-minutes of baseline, 4-minutes of LPT, and 10-minutes post-LPT. TDL flow was measured by timed collection during each condition. TDL was centrifuged to remove the cellular components, and the supernatant was stored for biomarker analysis. Results: TDL flow increased from an average of 1.68 mL/min to 3.25 mL/min during LPT administration. Immunological assays will be performed to identify changes in lymph composition during OMT. Conclusion: In our pilot study, LPT increased TDL flow in both our swine subjects and demonstrated surgical feasibility. In future studies we will study the physiological effects of OMT during sepsis. This knowledge would provide an evidence-based foundation for the use, or contraindication, of OMT during sepsis and aid osteopathic physicians during their therapeutic decision making.Item The Role of Lymph Flow on MTLn3 induced Breast Cancer(2015-03) Castillo, Rudy A.; Carter, KiahRae J.; Hodge, LisaPurpose: Secondary lymphedema is the chronic accumulation of lymph with no definitive cure. At least 22 % of axillary lymph node dissection and biopsies in breast cancer (BC) patients can lead to secondary lymphedema. There are no pharmaceuticals approved for lymphedema however manual medicine techniques have been designed to enhance lymph flow. Many clinicians fear manual medicine techniques, such as osteopathic lymphatic pump treatment (LPT), could promote metastasis in BC patients although there is a lack of literature supporting this notion. Our previous studies have shown LPT increases lymph flow and leukocyte concentrations in the lymph of rats. Physical activity increases lymph flow and has been associated with improved quality of life in BC patients and proposed as a modulator of the immune system. Therefore we proposed increasing lymph flow does not promote primary tumor growth. Methods: To determine the effect of LPT on BC, rats were randomized on day 0 into control, sham and LPT groups and injected with MTLn3. The LPT group received LPT under anesthesia, the sham group received anesthesia and the control group did not receive LPT or anesthesia. Treatment was administered once daily at days 14-24 post-injection. At days 0, 7, 14, 21 and 25, primary tumors were excised, measured, weighed and prepared for histological examination. Axillary sentinel lymph nodes (SLN) were excised, weighed and leukocyte populations were measured. Results: In the control rats, tumor weight increased significantly between days 14 (0.06 grams) and 25 (2.86 grams) post-injection. Tumor volume in situ increased significantly between days 14 (1.17 cm3) and 25 (2.75 cm3) post injection. Consistent with tumor growth, immunofluorescent staining revealed angiogenesis between days 14 and 25. Furthermore, SLN weight increased significantly (three-fold increase) and pathology confirmed metastasis by day 25. The number of T cells, B cells, NK cells, dendritic cells and macrophages were significantly higher in the SLN by day 25. LPT did not increase primary tumor size compared to control and sham groups. Interestingly, sham significantly increased SLN size (five-fold increase) when compared to control and LPT decreased SLN size when compared to sham. Conclusions: Our results suggest LPT does not increase primary tumor growth and negated the effect of sham treatment on the sentinel lymph node. Therefore, future studies will focus on how LPT reduces sham induced enlargement and determine if LPT reduces tumor load or fluid in the SLN.Item Thoracic Duct Lymph Reduces the Production of TNF-alpha IFN-gamma by Pulmonary Leukocytes in vitro(2020) House, Sara; Morales, Jessica; Hodge, Lisa; Vo, RussellPurpose: Streptococcus pneumoniae, a cause of community acquired pneumonia, accounts for nearly one million hospitalizations in the U.S., annually. The lymphatic pump technique (LPT) is a manipulative medicine technique used by osteopathic physicians to mobilize lymph and treat pneumonia. Our objective was to identify the biological effect of thoracic duct lymph (TDL) mobilized with LPT on the immune response against S. pneumoniae. We hypothesized that lymph mobilized during LPT would suppress the in vitro activity of lung leukocytes in mice infected with S. pneumoniae. Methods: TDL was collected from dogs during 4min of baseline, 4min of LPT, and 10min post-LPT. Mice were intranasally infected with 5x10^5 CFU of S. pneumoniae. Lung leukocytes were isolated from healthy and infected mice (24hr post-infection.) Leukocytes were cultured with media plus 5% saline, 5% baseline TDL, 5% LPT TDL, or 5% post-LPT TDL, and co-cultured with/without LPS. The TNFa and TNFg were measured in supernatants after 24hrs. Results: When cultured with LPS, the addition of baseline LPT, LPT, or post-LPT lymph decreased TNFa and TNFg production by leukocytes from healthy mice. Leukocytes from infected mice did not produce cytokines even when stimulated with LPS, suggesting expended biological activity in vivo. There were no differences in TNFa and TNFg production by leukocytes cultured with baseline LPT, LPT, or post-LPT lymph. Conclusion: TDL reduced inflammatory cytokine production by lung leukocytes. Mobilization of lymph during LPT may release protective factors that limit inflammation and protect the lungs from pulmonary disease.Item Thoracic Duct Lymph Suppresses the Inflammatory Response of Macrophages in vitro(2017-03-14) Hodge, Lisa; Castillo, Rudy A.Purpose: The gastrointestinal lymphatic vessels redistribute a large pool of lymph rich in immune cells, inflammatory mediators, and lipids. Recent literature suggests that during gastrointestinal injury, soluble factors released from the mesentery redistribute duct to the lung via the thoracic duct where they initiate inflammation and contribute to multiple organ dysfunction syndrome (MODS). Alternatively, normal mesenteric lymph has been shown to suppress inflammation in vivo and in vitro. Specifically, under inflammatory conditions, normal mesenteric lymph reduced the expression of cell adhesion molecules and myeloperoxidase in pulmonary tissue and reduced expression of cell adhesion molecules on pulmonary endothelial cells. However, the role of normal lymph on phagocyte function remains unknown. Importantly, macrophages have been to shown to contribute to MODS. The aim of this study was to investigate the effect of normal lymph on macrophage function. Specifically, we hypothesized that normal thoracic duct lymph (TDL) would suppress the release of inflammatory mediators by LPS-activated macrophages. Methods: To test this hypothesis, under anesthesia the thoracic ducts of eight mongrel dogs were cannulated and lymph was collected. The TDL was centrifuged to remove cells and the TDL supernatant was frozen and stored at -80oC. Murine RAW 264.7 macrophages were cultured in vitro with TDL at 0.5, 1, 2, 5 or 10% total volume per well or phosphate-buffered saline at 5 or 10% total volume per well with or without lipopolysaccharide (LPS) for 24 hours at 37°C with 5% CO2. After incubation, cell cultures were centrifuged to remove cells and the supernatants were assayed for nitric oxide (NO) and tumor necrosis factor-alpha (TNF-a) production. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide to distinguish live cells from apoptotic cells. Results: TDL did not augment the production of NO2-, TNF-a or alter cell viability by macrophages cultured in media alone. However, when macrophages were activated with LPS, TDL suppressed the release of NO and TNF-a. Specifically, the addition of TDL at 5% total volume per well suppressed NO2- production (15±0.6 uM) and TNF-a production (5016±425 pg/mL) compared to LPS. Culture with LPS and/or TDL did not alter cell viability. Conclusions: Our data suggests that during stimulation with LPS, a biological factor in lymph suppressed the release of inflammatory mediators by macrophages. Furthermore, cell viability was unaltered, suggesting that that TDL altered macrophage function. Future studies will focus on the ability of lymph to suppress the inflammatory response in disease models.