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    (2013-04-12) Indramohan, Mohanalaxmi
    Purpose: Listeria monocytogenes (LM) is a Gram-positive intracellular pathogen that causes meningitis and septicemia in immunocompromised individuals, and spontaneous abortion in pregnant women. IL-23 is a pro-inflammatory cytokine that promotes the recruitment of immune cells during multiple infectious and autoimmune diseases. Using IL-23p19 knockout (KO) mice we have demonstrated, that IL-23 is required for resistance against LM, and for the efficient recruitment of neutrophils to the liver, and monocytes to the spleen. However, it is not known whether IL-23 can impact the function of phagocytic cells including monocytes, neutrophils, and macrophages during LM infection. Methods: Mice lacking IL-23p19 (IL-23p19 KO) and C57BL/6 (B6) mice were infected with LM. For the isolation of thioglycollate-elicited peritoneal macrophages, mice were intraperitoneally injected with 1 ml of 2% thioglycollate, three days prior to harvest. Flow cytometry based assays were performed to measure phagocytosis and production of reactive oxygen species (ROS) using the dyes hydroethidine (HE) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). ELISAs were performed to measure the amounts of TNF-ɑ and IL-6. Results: The phagocytic ability of neutrophils and monocytes in the spleen was equivalent between IL-23p19 KO and B6 mice. IL-23p19 KO mice had reduced monocyte-specific ROS production in spleen in comparison to B6 mice. However, the production of monocyte-specific or neutrophil-specific ROS was not induced or enhanced by the addition of recombinant IL-23 to B6 splenocytes. Interestingly, there was a reduction in the production of TNF-ɑ and IL-6 from thioglycollate-elicited peritoneal macrophages from IL-23p19 KO mice compared to B6 mice. Conclusions: IL-23 does not mediate phagocytosis by neutrophils and monocytes. Optimal production of monocyte-derived ROS requires IL-23 during LM infection. However, IL-23 does not directly mediate the production of ROS. Lastly, IL-23 is required for enhancing the production of TNF-ɑ and IL-6 from thioglycollate-elicited peritoneal macrophages.
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    (2013-04-12) Hall, Jessica
    Purpose: Our objective is to determine the role of chronic psychological stress on the cutaneous immune response during contact hypersensitivity (CHS) reactions. We hypothesize that chronic psychological stress induces suppression of the cutaneous immune response mediated by a disruption of leukocyte trafficking. Methods: Restraint stress was applied to female BALB/c mice for 2 hours for 30 days. The sensitizing agent, oxazalone, was then applied on the abdomen, and the mice were then challenged on the ear 7 days later. Weight measurements and corticosterone levels were evaluated. Ear swelling was measured by caliper and histology to assess inflammation. In vitro and in vivo assays were performed to analyze leukocyte proliferation. Cytokine and chemokine ELISAs were performed on the lymph node culture supernatant and ear homogenate. Flow cytometry was performed on draining lymph nodes. Circulating leukocytes were analyzed by Hemavet before and after the stressing period. Results: Decreased weight gain and elevated serum corticosterone in the Stressed group confirmed differential stress levels. Ear swelling in response to oxazalone challenge was reduced in the Stressed group. In vitro and in vivo analysis showed no difference in T cell proliferation or overall cell proliferation in auricular lymph nodes, respectively. Common inflammatory cytokine and chemokine concentration in ear homogenates at the site of inflammation did not significantly differ between the two groups. Flow cytometric analysis of auricular draining lymph nodes showed no difference in T cell activation, however CD4+ and CD8+ T cell percentages were elevated in auricular lymph nodes of the Stressed group. Hemavet analysis showed a reduction in circulating neutrophils, lymphocytes, and monocytes in the Stressed group in addition to overall reduced white blood cell number. Conclusions: Our results show that chronic restraint stress suppresses the cutaneous immune response in CHS reactions. The mechanism of this suppression is not due to reduced T cell proliferation, T cell activation, or total cell proliferation in draining lymph nodes. Our results indicate reduced lymphocyte, neutrophil, and monocyte trafficking to the site of inflammation and an accumulation of CD4+ and CD8+ T cells in draining lymph nodes may contribute to the suppression. Future studies will be performed to analyze CD4+ T cell, CD8+ T cell, neutrophil, and macrophage population at the site of inflammation using immunofluorescence staining.
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    (2013-04-12) Shaw, Jennifer
    Purpose: Aging induced thymic involution is proposed to be related to an increased incidence of autoimmune disorders because one of roles of a functional thymus is to generate central immune tolerance to prevent autoimmunity. Th17 cells have been shown to play a role in autoimmunity and can be generated in the thymus. Therefore, it can be questioned whether self-reactive Th17 clones can be generated in the aged atrophied thymus and become involved in the process of autoimmune development in the elderly once being induced under certain circumstances. Methods: We used conditional FoxN1 knockout (FC) mice, which possess an atrophied thymus, mimicing aged mice when injected with tamoxifen. The control mice with the normal thymus are termed FF. We used a cell sorting approach to isolate CD4 single positive thymocytes and naive CD4 positive spleen T cells. Sorted cells were cultured in conditions to induce Th17 differentiation. Flow cytometry was used to analyze cell populations. We used Rag gene knockout mice as hosts to receive Th17 cell infusion for observation of autoimmune phenotypes by histochemistry. Results: 1. The percentage of induced Th17 thymocytes is higher in FC mice compared to FF mice, but without induction there is no difference in FC and FF two groups. This indicates that when given a stimulus such as infection the atrophied thymus may produce a higher percentage of auto-reactive Th17 cells compared to a normal thymus. 2. The percentage of induced Th17 splenocytes and non-induced Th17 splenocytes shows no difference between FC and FF mice. However, there is greater inflammation and cell infiltration in the organs (such as the colon and lung) in the Rag-/- mice infused with FC Th17 compared to that infused with FF Th17. The result shows that although the percentage of Th17 cells from an atrophied thymus is not affected in the periphery, they may have higher auto-reactive activity compared to a normal thymus. Conclusions: The atrophied thymus may be defective in the generation of central immune tolerance and therefore allow auto-reactive Th17 cells to survive and leave the thymus.
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    (2013-04-12) Break, Timothy
    Purpose: Listeria monocytogenes (LM) causes spontaneous abortions in pregnant females and septicemia and meningitis in immunocompromised individuals, and results in ~25% mortality rate in infected individuals. Extracellular superoxide dismutase (ecSOD) converts superoxide into hydrogen peroxide in the extracellular milieu and protects against oxidative stress. The use of mice with varying levels of ecSOD serves as a novel approach to determine how an extracellular enzyme can impact immunity against an intracellular pathogen. Methods: Congenic mice with high ecSOD activity (ecSOD HI), wild type ecSOD activity (ecSOD WT), or lacking ecSOD (ecSOD KO), on the C57Bl/6 background, were infected with LM. Colony forming units (CFUs) were counted to determine bacterial load. Percentages of different cell types, cell-surface markers, and intracellular molecules (including ROS and proteins) were determined by flow cytometry. Neutrophil depletions were performed by i.p. injection of anti-Ly6G or isotype control antibody. Cytokine and chemokine concentrations were measured by ELISA. Results: EcSOD HI mice were more susceptible to LM infection than ecSOD WT and ecSOD KO mice. Interestingly, ecSOD HI mice have higher percentages of neutrophils in the liver compared to ecSOD KO mice, which was at least partially mediated by increased ecSOD-induced neutrophil-attracting chemokine production. Neutrophil depletions were performed, and ecSOD WT and KO livers had increased CFUs, while ecSOD HI mice showed slightly decreased CFUs, compared to isotype-treated mice. Furthermore, TNF-ɑ, which is important for clearance of LM, was highest in ecSOD KO mice. Interestingly, ecSOD activity decreased the ability of neutrophils to undergo oxidative burst, a mechanism to kill LM, and increased the amount of LM inside neutrophils. Lastly, ecSOD activity increased the percentage of myeloid-derived suppressor cells, which suppress immune responses. Conclusions: Our data show that ecSOD is detrimental during the early response to LM infection. An increased percentage of neutrophils in ecSOD HI livers, but no concurrent decrease in CFUs, suggests that ecSOD can impair the function of neutrophils. One way this may occur is through the internalization of ecSOD during phagocytosis, which decreases oxidative burst, allowing for LM to survive inside neutrophils. Furthermore, an increase in myeloid-derived suppressor cells in the ecSOD HI livers helps explain why these mice are more susceptible to infection.
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    (2013-04-12) Racine, Ronny
    Purpose: Our objective was to determine the impact of CD44 expression on T-ALL tumorigenesis and metastasis using Jurkat cells engineered to express surface CD44. Methods: The E6.1 Jurkat cells were engineered to express the CD44 standard isoform. DNA content and aneuploidy was assessed by propidium iodide staining and karyotyping. Myristoylated-Akt and LY294002 were used to increase or decrease Akt activation in an attempt to rescue cells from aneuploidy or to induce aneuploidy. Propidium iodide and BrdU incorporation was used to investigate the restriction checkpoint regulation. RNA was isolated from CD44 expressing cells and the vector control and assessed using a Cell Cycle RT2-qPCR array and a Human Signal Transduction Pathway Finder RT2-qPCR array. Results: Flow cytometry confirmed the expression of CD44 on the transfected cell line but not the open vector control. DNA content analysis showed that CD44 expressing cells were aneuploid and had a higher number of chromosomes when compared to the open vector control. Myr-Akt did not rescue CD44 expressing cells from aneuploidy. LY294002, an inhibitor of Akt activation, was used to treat the open vector control, and there was no change in aneuploidy when Akt was inhibited in control cells. The restriction point of the cell cycle was determined to be defective in CD44 expressing cells, preventing the cells from undergoing apoptosis due to the detection of aneuploidy. The two PCR arrays contained significant downregulation in gene areas that focused on aneuploidy, proliferation, NOTCH signaling, and immune function. Detailed array analysis using IPA software provided Akt, EGR-1, NOTCH, and p53 as potential upstream effectors of the observed aneuploidy and potential decreased invasiveness of CD44 expressing cells. Conclusions: Our results show that expression of CD44 induces hypophosphorylation of Akt and aneuploidy. CD44 expression alters mRNA levels of several key proteins involved in invasion such as Serpin E1 and NOTCH. These results point us towards future experiments to investigate the invasiveness and metastatic potential of our cells in vivo. CD44 expressing tumor cells in patients show decreased NOTCH signaling and responsiveness to therapy, and our results are in agreement with clinical findings. Future work will focus on determining the link between aneuploidy, proliferation, and signaling pathway modifications due to CD44 expression within T-ALL cells and will pave the way towards using CD44 expression as a prognostic marker for T-ALL.
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    (2013-04-12) Smith, Ashley
    Purpose: The purpose of this study is to determine if Toll-like receptors (TLRs) on corneal epithelial cells are responsible for recognition of Acanthamoeba trophozoites. We hypothesize that TLR-4 recognizes the parasite and activates an initiate inflammatory responses. Methods: Human corneal epithelial (HCE), Chinese hamster corneal epithelial (HCORN) cells, or Human embryonic kidney (HEK 293) cells were cultured until confluent. The cells were then stimulated with either 1X 105/mL Acanthamoeba castellanii trophozoites, ultrapure lipopolysaccharides (LPS) at 10µg/mL as a positive control, or left un-stimulated for 24 hours. Once the incubation period was complete, the supernatant was removed and used for ELISA analysis for the production of IL-8 or CXCL2. RNA was collected from each experimental group and RT-PCR was performed using TLR-4 and chemokine primers. A TLR-4 neutralizing antibody was pre-incubated with the corneal cells 1 hr. prior to stimulation, the supernatant was collected for quantification of IL-8 production. Immunocytochemistry was used to visualize the TLR-4 protein on the cell membrane. The pathogenic Acanthamoeba castellanii and non-pathogenic Acanthamoeba neff strains were also compared utilizing this methodology. Results: Up-regulation of TLR-4 mRNA expression was seen in HCE cells and HCORN cells 24 hrs. after treatment. Increased production of cytokine/chemokine mRNA and IL-8 or CXCL2 protein was observed in the cells that were stimulated with Acanthamoeba castellanii trophozoites compared to un-stimulated control cells. This increase, was mitigated when a TLR-4 neutralizing antibody was added to the culture 1 hr. prior to treatment. We found that only the pathogenic Acanthamoeba castellanii strain up-regulates TLR-4 mRNA and chemokine production. Conclusions: These findings suggest that TLR-4 is responsible for the signaling cascade involved in the inflammatory response to Acanthamoeba infection. This cascade begins with the recognition of Acanthamoeba and leads to the production of cytokines and chemokines which attract neutrophils to the site of infection to combat the parasite.
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    (2013-04-12) Burnley, Preston I.
    Purpose: Current knowledge about regulating proliferation and differentiation of thymic epithelial cells (TECs) by the genes p63 and FoxN1, respectively, in the prenatal thymus has led our lab to determine whether these genes also regulate TEC homeostasis during aging in the postnatal thymus. Methods: We used murine models to verify the role of a p63-FoxN1 regulatory axis in TEC homeostasis during thymic aging. Results: Our studies show that a proportion of pan-p63+ TECs was increased with age, among which the isoform, TAp63, as oppose to ΔNp63, was increased and accompanied by increased senescent cell clusters with age. Furthermore, in the postnatal thymus blockade of the TEC differentiation via a conditional FoxN1 knockout demonstrated an increase in TAp63 and senescent cell clusters as well. This proportion of TAp63+ TECs, however, was decreased when FoxN1 cDNA was exogenously administered into the aged thymus intrathymically. In addition, we found that increased TAp63+ populations contained high proportions of phosphorylated-p53+ and apoptotic TECs but showed no changes in BrdU-labeled proliferation. Lastly, TAp63 cDNA intrathymically injected into the young thymus showed an increase in senescent cell clusters but little change in FoxN1. Conclusions: We conclude that the expression of TAp63 has a reverse correlation with expression of FoxN1. During the natural thymic aging, decrease in FoxN1 causes an accumulation of undifferentiated TECs. This most likely leads to an increase in TAp63, thus resulting in an increase in cellular senescence and exhaustion of epithelial stem cells. This finally will cause age-related thymic involution and deteriorate thymic function.
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    (2013-04-12) Sulak, Kyle
    Purpose: This report will discuss the manifestations and treatment of the rare complication of Macrophage Activation Syndrome (MAS) and superimposed life-threatening Cytomegalovirus (CMV) infection in Juvenile Idiopathic Arthritis (JIA). We will also discuss the potential causative factors of MAS. Methods: This report will discuss the manifestations and treatment of the rare complication of Macrophage Activation Syndrome (MAS) and superimposed life-threatening Cytomegalovirus (CMV) infection in Juvenile Idiopathic Arthritis (JIA). We will also discuss the potential causative factors of MAS. Results: Current treatment recommendations indicate management of JIA with Anakinra, low dose Prednisolone and Methotrexate. Indications that point to the diagnosis of MAS include an elevated PT, thrombocytopenia, hypofibrinogenemia, elevated D-dimers, elevated aspartate aminotransferase and clinical presentation. Clinical recommendations for the treatment of MAS include fresh frozen plasma, corticosteroids and supportive care as well as Cyclosporine when patients do not respond to those treatments. Initial treatment for CMV is intravenous ganciclovir. Additionally, immune globulin may be administered as well as foscarnet and vanganciclovir. Previous studies point to inciting agents of MAS as well as lab criteria for distinguishing the onset of MAS from an increase in systemic symptoms of JIA. Conclusions: In this patient's case, the onset of MAS is likely multifactorial and may include both CMV infection and use of methotrexate. This case showed that management of autoimmune disorders with life-threatening infections is challenging as treatment for one condition may worsen the other. Also, when evaluating laboratory data, it is important to remember that JIA causes an increase in many inflammatory markers, so a relative decrease may be more indicative of MAS than absolute low values. This may warrant for routine monitoring of inflammatory markers in order to make comparisons if a patient's symptoms worsen and MAS is suspected.
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    (2013-04-12) Coder, Brandon
    Purpose: Immunotolerance generates protection against autoimmunity by deleting self reactive T cells in the thymus through negative selection as well as the generation of natural regulatory T cells (nTregs) that will help suppress autoimmunity in the periphery. Natural aging is associated with a progressive loss of FoxN1 and thymic atrophy, and is also believed to be associated with increased autoimmunity and an increase in suppressive FoxP3+ regulatory T cells (Tregs). Deletion of self-reactive T cells requires self-antigen presentation by medullary thymic epithelial cells (mTECs). We set out to determine if thymic aging, characterized by the progressive loss of FoxN1 and mTEC disruption, alters immunotolerance by influencing negative selection or impacting the generation of suppressive Treg cells. Methods: For thymus population experiments, we induce thymic atrophy in our FoxN1 conditional knockout mice using tamoxifen injections. We then determine expression of CD4+, CD8+, and CD4+8+ double positive, and CD25+FoxP3+ regulatory thymocytes using flow cytometry. The peripheral Treg data is assessed by adoptive transfer of total spleenocytes from young and aged wild type mice into young Rag2-/- mice. Cell populations are determined using flow cytometry. Results: We found that the loss of FoxN1 induced thymic atrophy is associated with an impairment of negative selection, where CD4+ and CD8+ are increased in the thymus while CD4+CD8+ double positive thymocytes are decreased. This indicates that the age atrophied thymus is not able to delete additional single positive thymocytes, and these may be self-reactive thymocytes. Additionally, we found that nTregs in the aged and atrophied thymus are increased in proportion and their suppressor function remains intact. Furthermore, we found that when we transfer aged spleenocytes, in which there are increased Treg and decreased pro-apoptotic Bim protein, and young spleenocytes separately into young Rag2 knockout mice, that the young periphery is able to restore both Bim levels and Treg levels to that of the young mice. Conclusions: We conclude that loss of FoxN1 disrupts thymic mTEC structure and impairs negative selection, which may lead to an increase in self-reactive T cells. However, thymic atrophy does not compromise peripheral Tregs. The function and number of peripheral Tregs is dependent on the micro-environment in which they stay.
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    (2013-04-12) Witter, Alexandra
    Purpose: Listeria monocytogenes (LM) is an intracellular foodborne pathogen that causes severe disease in immunocompromised individuals, spontaneous abortion in pregnant women, and results in ~25% mortality rate in infected individuals, making it one of the leading causes of death from foodborne infection. Extracellular superoxide dismutase (ecSOD) converts superoxide into hydrogen peroxide in the extracellular milieu and protects against oxidative stress. While it has been shown that ecSOD decreases innate immune responses during LM infection, its role in a secondary infection model has not been explored. Methods: Congenic mice with high ecSOD activity (ecSOD HI), wild type ecSOD activity (ecSOD WT), or lacking ecSOD (ecSOD KO), on the C57Bl/6 background, were infected with LM/OVA ΔActA and after 40 days challenged with LM/OVA. Colony forming units (CFUs) were counted to determine bacterial burden in the spleen and liver. Percentages of different cell types were determined by flow cytometry. IFN-𝛾 production and concentrations were determined by flow cytometry and ELISA respectively. Results: Our results indicate that ecSOD is protective in a secondary infection since ecSOD HI mice are better able to control bacterial burden than ecSOD KO mice with the ecSOD WT mice showing intermediate CFUs. The ecSOD KO mice exhibit a significantly lower percentage of dendritic cells and corresponding decreases in percentages of both overall CD8 T cells as well as memory CD8 T cells. There was also a decrease in percentages of overall CD4 T cells and memory CD4 T cells, primarily in the spleen. Additionally, there was a significant decrease in CD8 T cell specific IFN-𝛾 production in the spleen after overnight culture with both specific and non-specific stimulation. Conclusions: Our data indicate that ecSOD plays an important role in modulating cell-mediated adaptive immune responses during secondary intracellular bacterial infection. The combination of decreased dendritic cell and CD8 T cell populations, as well as decreased ability of CD8 T cells to produce IFN-𝛾, in ecSOD KO mice suggest that ecSOD may play a role in facilitating the activation of CD8 T cells and their ability to effectively respond during secondary LM infection.
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    (2013-04-12) McDougal, April
    Purpose: Staphylococcus aureus is a pathogen often seen in the hospital setting and the cause of various infections in humans. Multiple antibiotic resistant S. aureus strains (MRSA) have been clinically isolated, which becomes problematic when S. aureus forms biofilms or slime layers on synthetic materials like catheters. Biofilm infections are difficult to treat, and if a patient has compromised immunity due to underlying medical conditions, the infection can be life threatening. To understand the impact of host immunity on the treatment of S. aureus biofilm infections, we subcutaneously (SC) implanted S. aureus infected catheter pieces in mice that have severe combined immunodeficiency (SCID), treated them for 7 days with rifampin, and measured the number of rifampin-resistant and rifampin-sensitive cells associated with the catheter pieces. Methods: Using broth media supplemented with glucose and sodium chloride, 1 cm Teflon catheters were inoculated with a non-resistant strain of S. aureus for one hour. Infected catheters were surgically placed subcutaneously in four mouse strains, BALB/c, BALB/c-SCID, C57BL/6J and C57BL/6J-SCID. Seven days after implantation, an antibiotic regimen was started using either rifampicin or vehicle, 2 times a day for 7 days. The catheters were aseptically removed within 20 hours of the final dose. The catheters were processed and plated on non-antibiotic agar media and media containing 1 mg/mL of rifampin. Results: Catheter-associated CFU stabilized at 6.3 log CFU in C57BL/6J mice and 7.4 log CFU in BALB/c mice 14 days after implantation. Rifampin 7-day treatment at 25mg/kg yielded mean log CFU reductions of 1.8 and 2.6 in BALB/c wild-type and SCID mice, respectively. The same treatment resulted in mean log CFU reductions of 2.3 and 2.7 in C57BL/6J wild-type and SCID mice, respectively. Rifampin resistant isolates (1mg/mL) were recovered at a mean log CFU of 3.8 and 2.6 in C57BL/6J wild-type and SCID mice, respectively. Conclusions: SCID mice are lymphocyte deficient and have an impaired adaptive immune response. Our data shows that the reduced immunity enhances rifampin's ability to treat the S. aureus biofilm infection, while reducing the frequency of rifampin-resistance in the model. Therefore, our data suggests that the host's immune response can influence an antibiotic's ability to treat S. aureus biofilm infections.
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    (2013-04-12) Carter, KiahRae J.
    Purpose: The lymphatic pump treatment (LPT), designed by the osteopathic medical profession, targets the musculoskeletal system and enhances the flow of lymph through the lymphatic system. Clinical studies showed that LPT plus antibiotic treatment enhances the recovery of pneumonia infections. Animal studies from our lab demonstrate that LPT significantly enhances thoracic and mesenteric lymph flow, mobilizes leukocytes from the gastrointestinal lymphoid tissue (GALT) into lymph circulation, and significantly increases the lymphatic flux of leukocytes, cytokines, chemokines, superoxide dismutase and reactive nitrogen species in lymph. We hypothesize that by increasing lymph flow, LPT can enhance immunity and drug delivery during pneumonia. Methods: To test this hypothesis rats were nasally infected with 1x108 Streptococcus pneumoniae colony forming units (CFU's) on day zero. Twenty-four hours post-infection, rats were randomized into control, sham, LPT, control plus 40 mg/kg (subcutaneous) levofloxacin, sham plus 40 mg/kg levofloxacin or LPT plus 40 mg/kg levofloxacin. Rats received treatment once daily for three consecutive days. Sham and LPT were applied as previously described. On day four post-infection, lungs were removed and bacteria were enumerated. Results: Three daily treatments of LPT significantly reduced (p<0.05) S. pneumoniae CFU's in the lung compared to control and sham. All Levofloxacin treated groups significantly reduced (p<0.05) S. pneumoniae CFU's compared to control, sham, and LPT. LPT plus Levofloxacin significantly reduced (p<0.01) S. pneumoniae CFU's compared to all treatment groups. These results suggest that LPT protects against pneumonia. Conclusions: Our study suggests that as a complementary therapy to antibiotics, LPT can enhance the clearance of bacteria from the lungs. In future studies, we will determine if LPT enhances the delivery of levofloxacin to the lung by measuring the concentration of levofloxacin in the serum and bronchiolar alveolar lung fluid (BALF). The results provide scientific support for the clinical use of LPT as a complementary therapy for the treatment of pneumonia.