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    The Role of Lymph Flow on MTLn3 induced Breast Cancer
    (2015-03) Castillo, Rudy A.; Carter, KiahRae J.; Hodge, Lisa
    Purpose: 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.
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    Independent of bacterial clearance, Interleukin-17A acts to exacerbate pulmonary inflammation in BALB/c mice infected with Mycoplasma pulmonis
    (2015-03) Mize, Maximillion T.; Simecka, Jerry
    Purpose: Accounting for >50,000 deaths per year, lung infections are a leading cause of mortality in the United States. Chronic lung diseases complicated by infectious disease result in an additional 140,000 deaths. As a leading cause of pneumonia in man and animals worldwide, mycoplasmas cause persistent infections that induce debilitating chronic lung inflammation. An incomplete understanding on the pathogenesis of disease has contributed to the absence of effective vaccines against these organisms. Using a naturally occurring murine model mirroring human and animal infections, interleukin-17A (IL-17A) was found to increase within the lungs of susceptible BALB/c and resistant C57BL/6 mice inoculated with M. pulmonis. In a previous study, C57BL/6 mice deficient in IL-17 receptor expression were shown to have impaired clearance of mycoplasma. We hypothesized that IL-17A may have a different role in susceptible BALB/c mice. Consistent with this idea, we report that injection of neutralizing antibodies against IL-17A reduced respiratory inflammation in BALB/c infected with M. pulmonis. This was seen through abrogated lymphoid and neutrophil recruitment when compared to littermate controls. In addition, both gross lung and alveolar lesions were significantly reduced upon IL-17A neutralization. Surprisingly, blocking IL-17A did not alter bacterial burden, thus solely influencing the inflammatory process during infection in BALB/c mice. Interestingly, anti-IL-17A antibody administration did not have an effect mycoplasma infection in resistant C57BL/6 mice, suggesting that the IL-17A pathways differ between susceptible and resistant strains of mice.
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    Osteopathic Lymphatic Pump Treatment Does Not Alter Antibiotic Distribution in a Rat Model
    (2015-03) Redman, Charlotte J.; Hodge, Lisa
    Purpose: 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.
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    Age-related Thymic Involution Perturbs Negative Selection Leading to Autoreactive T Cells That Induce Chronic Inflammation (Inflammaging)
    (2015-03) Coder, Brandon; Wang, Hongjun; Ruan, Linhui; Su, Dong-Ming
    The presence of chronic low-level pro-inflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality. Recently, inflammaging has been partially attributed to the persistent activation of immune cells thought to arise from latent viral infection, but the contribution of activated autoreactive T cells towards the development of inflammaging remains unclear. To address our hypothesis that age-related thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we performed experiments including: adoptive transfer, kidney capsule transplantation, and tetramer detection of autoreactive T cells on a FoxN1 conditional knock-out (FoxN1-cKO) mouse model that mimics natural thymic involution while maintaining a young periphery. We found that thymic involution leads to T cell activation shortly after thymic egress, which is accompanied by a chronic inflammatory phenotype consisting of cellular infiltration into non-lymphoid tissues and elevated serum IL-6 and TNFα levels. Autoreactive T cell clones were detected in the periphery of FoxN1-cKO mice. A failure of negative selection, facilitated by decreased expression of Aire rather than impaired regulatory T cell (Treg) generation, led to autoreactive T cell generation. Furthermore, the young environment can reverse age-related Treg accumulation but not inflammatory infiltration. Together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a source of chronic age-related inflammation (inflammaging).
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    Extracellular Superoxide Dismutase Promotes Immature Neutrophil Egress from the Bone Marrow
    (2015-03) Witter, Alexandra R.; Break, Timothy J.; Indramohan, Mohanalaxmi; Mummert, Mark E.; Berg, Rance E.
    Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using three groups of mice with varying levels of ecSOD activity, we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes (LM) leading to increased host susceptibility. However, it is unclear whether ecSOD activity affects neutrophil recruitment and function in a cell-intrinsic manner or by modulating the extracellular environment. Using adoptive transfer experiments, we observed that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we determined that ecSOD activity protects the extracellular matrix (ECM) and leads to an increase in phenotypically immature neutrophils in the bone marrow and liver. 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 LM. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.
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    HIV-1 Tat and gp120 Regulate Astrocyte Tissue Inhibitor of Matrix Metalloproteinases-1 (TIMP)-1
    (2015-03) Joshi, Chaitanya R.; Ghorpade, Anuja
    Purpose: While antiretroviral therapy (ART) has improved the quality of life and survival of HIV-1-infected patients, HIV-1-associated neurocognitive disorders (HAND) remain a major problem in over 30% of cases. All forms of HAND are associated with CNS inflammation. Astrocytes, the principal type of glial cells, are involved in signaling, homeostasis, and repair during CNS pathology. Some astrocytes become non-productively infected by HIV-1. The balance between matrix metalloproteinases (MMP) and their inhibitors must be tightly regulated during CNS inflammation. In the brain, tissue inhibitor of MMPs (TIMP)-1 protects human neurons from HIV-1-induced apoptosis and is mainly produced by astrocytes. Further, astrocyte TIMP-1 is differentially regulated during acute and chronic IL-1β-activation. However, the direct or indirect effects of astrocyte HIV-1 protein expression on TIMP-1 regulation are not well studied. Here, we investigated downstream effects of HIV-1 Tat and gp120 expression in astrocytes on the TIMP-1/MMP balance. Methods: Primary human astrocytes were transfected with HIV-1 protein-expressing plasmids (Tat72, Tat101, gp120JR-FL). First, HIV-1 Tatand gp120 expression levels were compared using RT2-PCR and western blot. Cell viability and proliferation were evaluated as measures of cytotoxicity using MTT and BrdU assays, respectively. Concurrently, time-dependent changes in TIMP-1 and CCL2 mRNA and protein levels were measured as indicators of astrocyte activation. Since C/EBPβ is a known TIMP-1 regulator, alterations in C/EBPβ mRNA and protein levels were analyzed. Results: HIV-1 Tat and gp120 expression in astrocytes significantly reduced cell viability. Transfected astrocytes showed higher cytokine and chemokine mRNA as well as protein levels. C/EBPβ mRNA expression was differentially altered in astrocytes expressing either HIV-1 Tat72, Tat101 and gp120 indicating potentially distinct underlying mechanisms. Conclusions: We propose that TIMP-1 is differentially regulated by HIV-1 protein expression in astrocytes, which mimic viral CNS reservoirs, and may have implications in HAND neuropathogenesis.