THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

The role of STAT3 in the emergency neutrophil response

Neutrophils are an essential component of innate immunity, serving to provide an immediate response to microbial invasion. In response to emergency situations such as an infection, serum levels of granulocyte colony-stimulating factor (G-CSF) are induced, causing a boost in neutrophil production and a rapid mobilization of bone marrow neutrophils to the blood, where they can circulate to clear foreign pathogens. Signal transducer and activator of transcription 3 (STAT3) is a principal downstream signaling intermediate of the G-CSF receptor. Mice null for STAT3 are embryonic lethal; therefore, to examine the role that STAT3 has in granulocytic development and function in vivo, we utilized a conditional knockout mouse that deletes functional STAT3 in the hematopoietic system (referred to herein as STAT3-deficient). Using this model, we show that STAT3 is required for G-CSF-induced expansion of granulocytic progenitor cells within the bone marrow and for acute G-CSF-dependent neutrophil mobilization into the blood. Thus, STAT3 has a critical role in the immediate G-CSF-response in vivo. Sustained G-CSF exposure causes skewed granulocytic production and mobilization in STAT3-deficient mice, suggesting an atypical granulocytic developmental pathway. To determine if STAT3-deficient neutrophils were functional, we examined neutrophil chemotaxis, since neutrophil function relies on proper chemoattractant-induced migration to infected tissue sites. STAT3-deficient neutrophils have impaired chemotaxis in response to the potent neutrophil chemoattractants MIP-2 and KC, both ligands for the chemokine receptor CXCR2. Additionally, STAT3-deficient mice have a defect in NIIP-2-induced acute neutrophil mobilization in vivo. Chemotaxis in response to fMLP and SDF-1, which utilize distinct seven-transmembrane chemokine receptors, was similar between wild type and STAT3-deficient neutrophils, suggesting that STAT3 specifically regulates CXCR2-mediated migration. MIP-2-induced activation of the Raf/MEK/ERK signaling cascade, which we show is required for MIP-2-dependent neutrophil chemotaxis, was impaired in STAT3-deficient neutrophils. Interestingly, acute G-CSF administration induced CXCR2 expression and Raf/MEK/ERK activation in neutrophils from wild type mice, whereas these responses were abrogated in neutrophils from STAT3-deficient mice. Thus, STAT3 regulation of CXCR2 functions may also contribute to STAT3's control of the acute G-CSF mobilization response. These combined results place STAT3 as a critical intermediate in neutrophil migration and G-CSF-induced neutrophil production responses required for emergency granulopoiesis. ^

Receptor tyrosine kinase HER2-mediated signaling pathways in metastasis and angiogenesis

Metastasis, the major cause of morbidity and mortality in most cancers, is a highly organized and organ-selective process. The receptor tyrosine kinase HER2 enhances tumor metastasis, however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the malignant cancer cells to specific organs. Here we show that HER2 enhances the expression of CXCR4 by increasing CXCR4 protein synthesis and inhibiting its degradation. We also observed significant correlation between HER2 and CXCR4 expression in human breast tumor tissues, and an association between CXCR4 expression and a poor overall survival rate in patients with breast cancer. Furthermore, we found that CXCR4 is required for HER2-induced invasion, migration, and adhesion activities in vitro . Finally we established stable transfectants using retroviral RNA interference to inhibit CXCR4 expression and showed that the CXCR4 is required for HER2-mediated lung metastasis in vivo. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and homing to metastatic organs, and establish a functional link between the receptor tyrosine kinase HER2 and the chemokine receptor CXCR4 signaling pathways. ^ The HER2 overexpression activates PI-3K/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia inducible factors (HIF) are the key regulator for angiogenesis and energy metabolism, and thereby enhance tumor growth and metastasis. HIF activation occurs in the majority of human cancers, including the HER2 overexpressing cancer cells. Previous reports suggested that increased PI-3K/Akt may activate HIF pathway in various tumors, but the detail mechanism is still not completely understood. Here we found that HER2/PI-3K/Akt pathway induces HIF-1α activation, which is independent of hypoxia, but relatively weaker than hypoxic stimulation. This phenomenon was further observed in Akt knock out mouse embryonic fibroblast cells. The PI-3K/Akt pathway does not affect HIF-1α binding with its E3 ligase VHL, but enhances the binding affinity between HIF-1α and β unit. Furthermore, we found Akt phosphorylates HIF-1β at serine 271 and further regulated HIF transcriptional activity. Our findings provided one mechanism that HER2 induce HIF activation via Akt to promote angiogenesis, and this process is independent on hypoxia, which may have implications in the oncogenic activity of HER2 and PI-3K/Akt pathway. ^

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^

EFFICACY AND MECHANISM OF â-DEFENSIN2 FUSED ANTIGEN PROTEIN VACCINES

Vaccines which use the strategy of fusing adjuvant murine â-defensin2 (mBD2) to an antigen in order to elicit stronger anti-antigen immune responses are referred to as murine â-defensin2 (mBD2) vaccines. Previous studies have validated the potential of mBD2 vaccines, thus in this study we focus on increasing vaccine efficacy as well as mechanism elucidation. Initially, we demonstrate superior IFN-ã release levels by antigen specific effector T cells when antigen is crosspresented by dendritic cells (DC) which absorbed mBD2 vaccine (mBD2 fused antigen protein) over antigen alone. We move unto an in vivo model and note significant increases in the expansion of antigen specific class I T cells but not class II T cells when receiving mBD2 vaccine over antigen alone. Further, knowing mBD2’s link with CC chemokine receptor 6 (CCR6) and Toll-like receptor 4 (TLR4) we note that this enhanced class I T cell expansion is CCR6 independent but TLR4 dependent. With anti-tumor responses desired, we demonstrate in tumor protection experiments with mice, compelling tumor protection when combining adoptive T cell therapy and mBD2 vaccine immunization. We further note that mBD2 vaccines are not limited by the antigen and characterize a viable strategy for enhancing tumor antigen immunogenicity.