Abstract
Oxidative Stress and Muscle Dysfunction Following Anterior Cruciate Ligament Surgery
Tyler Barker, PhD
Oregon State University, 2009
Despite the advances in surgery, physical therapy, and pharmaceutical agents, muscle dysfunction (ie, atrophy and weakness) continues to impair recovery from an anterior cruciate ligament (ACL) injury and surgery. Ischemia-reperfusion injury during surgery and the subsequent limb disuse are 2 events experienced by patients having ACL surgery. Oxidative stress and inflammation mediate muscle dysfunction; both of which can be modulated by antioxidants. The purpose of this project was to test the hypothesis that vitamin E and C supplementation would ameliorate muscle dysfunction following ACL surgery by attenuating the increase in mediators of muscle dysfunction.
A randomized, double-blind, placebo-controlled study was conducted in men who received one of two supplements: (1) antioxidant (AO; 400 IU of vitamin E and 1,000 mg vitamin C per day), or (2) matching placebo (PL) starting ∼2-weeks prior to (baseline) and concluding 3-months postsurgery. Lower limb strength and skeletal muscle fiber cross-sectional area measurements were used to assess muscle dysfunction; markers of oxidative stress and inflammation were evaluated in the circulation and muscle.
Plasma a-tocopherol (a-T) and ascorbic acid (AA) increased, while g-T concentrations decreased significantly with AO supplementation. Following surgery, oxidative stress (F2-isoprostanes), inflammation and muscle damage increased significantly in both groups. Compared to the PL group, AO supplementation ameliorated the increase in an anti-inflammatory cytokine (interleukin (IL)-10) and the depression of a pro- to anti-inflammatory cytokine ratio (IL-6:IL-10) immediately following surgery. Elevated AA decreased in the AO group and inversely correlated with a neutrophil chemoattractant cytokine immediately following surgery.
In contrast to our expectations, a significant atrophy response from pre- to post-surgery was not observed in muscle biopsies, using histologic techniques. In fact, AO supplementation increased inducible nitric oxide synthase and myeloperoxidase expression in the muscle following surgery. Furthermore, and unlike the PL group, the recovery in peak isometric force of the injured limb within the AO group did not significantly increase from baseline to 3-months postsurgery.
In summary, AO supplementation protected against immuno-suppression (increase in anti-inflammatory cytokines), but was ineffective in lowering oxidative stress induced by surgery. Moreover, AO supplementation did not minimize, and potentially contributed to muscle dysfunction following ACL reconstructive surgery.
The Functional and Biochemical Studies of the Epstein-Barr Virus, Bcl-2 Homolog, BHRF1
Anthony Lloyd Desbien, PhD
University of Colorado at Denver, 2009
Current knowledge suggests that the balance between life and death within a cell can be controlled by the stable engagement of Bc1–2-related pro-apoptotic proteins such as Bak, Box and Bim by survival proteins such as Bc1–2. BHRF1 is a pro-survival molecule from Epstein-Barr virus that has a high degree of homology to Bd-2. To understand how BHRF1 blocks apoptosis, BHRF1 and mutants of BHRF1 were expressed in primary cells and an IL-2 dependent T cell line. BHRF1 protected cells from apoptosis induced by cytokine withdrawal as well as blockade of protein synthesis. Biochemical analysis revealed BHRF1 bound the Executioner Bak and, when cells were cultured without cytokines, BHRF1 associated with Bim. A point mutation of BHRF1 that lost the ability to bind Bak, also lost the ability to protect cells from protein synthesis blockade. Interestingly, this mutant retained its ability to bind Bim and to protect cells from culture without cytokines. This demonstrated that it was the capacity of BHRF1 to bind Bak that facilitated its protection against protein synthesis blockade, and that BHRF1 bound Bim to provide protection against apoptosis induced by culture without cytokines. Intriguingly, the amount of Bim bound by BHRF1 was minimal when compared to the amount of Bim induced by apoptosis. Thus BHRF1 does not act by absorbing the excess Bim produced while cells prepare for death.
The Modulation of the Mesolimbic and Nigrostriatal Dopamine Pathways by CXCL12 and CXCR4
Jordan Trecki, PhD
Temple University, Pennsylvania, 2009
The role of chemokines in immune function is clearly established. Recent evidence suggests that these molecules also play an important role in the CNS as modulators of neuronal activity. The chemokine CXCL12 has been identified in several regions of the adult rat brain including the substantia nigra, ventral tegmental area, and caudate putamen. CXCR4, a receptor activated by CXCL12, is expressed by dopaminergic neurons in the substantia nigra. The research presented herein explored the behavioral modulation of CXCL12, expression of the CXCR4 receptor in the forebrain of the adult rat and the effect of CXCL12 administration on extracellular dopamine and glutamate release in the medial shell of the nucleus accumbens. This research furthered our understanding of how CXCL12 can affect behavior and suggested that the modulation of cocaine-induced behavior by CXCL12 is due to an interaction with CXCR4 receptors in the mesolimbic and nigrostriatal dopamine pathways.
The data presented tested the effects of intracranial injections of CXCL12 on cocaine-induced locomotion and stereotypic activity in adult male Sprague Dawley rats. Results demonstrate that intracerebroventricular administration of CXCL12 (25 ng/4 mL) 15 min prior to cocaine (20 mg/kg IP) produced a significant potentiation of both ambulatory and stereotypic activity as compared to cocaine alone. The effects of CXCL12 were blocked by administration of the selective CXCR4 antagonist, AMD 3100. Administration of CXCL12 into specific brain regions was performed to further understand the site of action of CXCL12. Bilateral administration of CXCL12 (25 ng/0.5 mL) into the ventral tegmental area 15 min prior to cocaine (20 mg/kg IP) significantly potentiated cocaine-induced ambulatory activity, whereas microinjections of CXCL12 into the caudate putamen selectively increased stereotypy. Conversely, administration of CXCL12 into the lateral shell of the nucleus accumbens resulted in an inhibition of cocaine-stimulated ambulatory activity. No alterations in ambulatory or stereotypic activity were observed following CXCL12 administration into the core of the nucleus accumbens.
Immunohistochemistry results showed evidence of CXCR4 within the caudate putamen and lateral shell of the nucleus accumbens. Dual labeling immunofluorescence demonstrated that CXCR4 is co-expressed on cholinergic and GABAergic neurons, including co-localization with the D1 dopamine receptor in both the caudate putamen and lateral shell of the nucleus accumbens. Results demonstrated that CXCR4 is co-expressed with choline acetyl transferase, a marker for cholinergic neurons, with GAD C38, a marker for GABAergic neurons, and with the D1 dopamine receptor, also a marker for GABAergic medium spiny neurons.
High pressure liquid chromatography studies were conducted using brain dialysate collected from microdialysis probes surgically implanted in the medial shell of the nucleus accumbens. Results demonstrated no significant change in extracellular dopamine or glutamate following an acute administration of CXCL12.
The research presented herein sought to determine the behavioral modifications of CXCL12 as well as the localization of CXCR4 in the forebrain of the adult rat. This research also examined changes in extracellular dopamine and glutamate levels following CXCL12 administration. Results demonstrated that CXCL12 does alter the behavioral activity of cocaine. Results also showed that CXCR4 is localized on cholinergic and GABAergic neurons that could be contributing to the behavioral modification. These results have extended our understanding of the complex mechanisms of CXCL12 and CXCR4 in the mesolimbic and nigrostriatal dopamine pathways of the adult rat brain.
Activation of the Innate Immune Response by the Alzheimer’s Amyloid Beta Protein via Toll-Like Receptors
Maria Udan, PhD
University of Missouri, Saint Louis, 2009
Alzheimer’s disease (AD) is the most common form of neurodegenerative disease characterized by the generation and deposition of amyloid beta plaques and the formation of neurofibrillary tangles. A wealth of data now demonstrate that inflammation is a prominent feature in AD pathology and a potential therapeutic target for the treatment and prevention of the disease. The emergence of evidence linking amyloid beta protein (Ab), the primary component of senile plaques, to inflammation has led to new insights into understanding AD pathology. Ab, a protein fragment resulting from cleavage of human amyloid precursor protein (APP), primarily exists in 2 forms: a slower-aggregating 40-amino acid long peptide (Ab(1–40)) and a faster-aggregating 42-residue peptide Ab(1–42). This investigation focused on elucidating the mechanism by which Ab provokes an inflammatory response in AD. For this study, we utilized THP-1 human monocytes/macrophages as an inflammatory model system due to their sensitivity to Ab. We hypothesized that fibrillar A(b(1–42) may utilize Toll-like receptors (TLRs), a family of transmembrane receptors that mediate recognition of certain conserved structural motifs in pathogens, for production of proinflammatory products and activation of the innate immune response. Biophysical characterization of the bioactive species of Ab(1–42) revealed that a soluble yet fibrillar species of Ab(1–42) invokes tumor necrosis factor alpha (TNF-a) production in THP-1 monocytes/macrophages. Moreover, using a TLR antibody neutralization assay, whereby receptor blockade inhibits cell responsiveness to TLR ligands, we showed that both TLR-2 and TLR-4 were highly involved in Ab(1–42)-induced TNF-a production. The role of TLR-2 in Ab-induced innate immune response was further substantiated by the production of proinflammatory interleukin-8 (IL-8) in transfected HEK293 cells, a mammalian cell line that does not express TLR-2, after stimulation with Ab(1–42). Furthermore, our results suggest the possible involvement of TLR-2/TLR-1 or TLR-2/TLR-6 for the Ab-induced activation of TLR downstream signaling. Taken together, our findings provide strong correlation between Ab and innate immune response activation via TLR-2 and TLR-4. The identification of TLRs that recognize Ab has opened new venues for understanding the mechanism of Ab-induced inflammatory response and may thus be a new therapeutic target for AD.
Constitutive Noncanonical NFκB Signaling in Pancreatic Cancer
Catherine Wharry, PhD
University of Pennsylvania, 2009
The NFκB signaling pathway regulates the expression of genes critical to the immune response, inflammation, and cell survival, and constitutive activation of this pathway has been associated with dozens of human cancers. Correspondingly, suppression of NFκB signaling is under intense scrutiny for potential clinical applications. However, the majority of studies regarding the contribution of NFκB signaling to tumorigenesis examine only classical NFκB signaling. The more recently described noncanonical NFκB pathway has not been specifically investigated for its role in cancer development. The noncanonical pathway is IKKa-dependent, and is characterized by stabilization of the protein NIK and processing of NFκB2/p100 to p52. Target genes of this pathway include the cytokine BAFF and the chemokines CXCL12, CXCL13, CCL19, and CCL21. These proteins function in lymphoid development, immune cell trafficking, and angiogenesis. Several of them have been observed in association with both lymphoid malignancies and solid tumors. We investigated the activation state of the noncanonical NFκB pathway and its target genes in pancreatic ductal adenocarcinoma (PDAC), and found this pathway to be constitutively activated. The NC NFκB proteins p52 and RelB were expressed in the nuclei of human pancreatic tumor tissues and PDAC cell lines. Additionally, the panel of PDAC cell lines exhibited NC activation in terms of modification and processing of p100 and stabilization of NIK. Strikingly, these cells also dramatically overexpressed NC NFκB target genes. We confirmed the role of NC activation in the up-regulation of these genes by inhibiting NC signaling and suppressing the expression of these chemokines and cytokines. We also found that PDAC cells release soluble factors that stimulate expression of NC target genes in endothelium, indicating a critical role for NC NFκB signaling in the tumor microenvironment. These observations demonstrate that the NC NFκB pathway is constitutively activated in PDAC cells, and these findings strongly suggest a functional role for this pathway in tumorigenesis.
Leptin and Inflammation in the Brain: Characterization of Cellular Targets
Véronique Lafrance, MSc
McGill University, Canada, 2009
The primary role of the adipocyte-derived hormone leptin is to regulate energy balance via direct action on hypothalamic neurons. Recently, a role in mediating the inflammatory response in the brain has been attributed to this hormone, although its cellular targets in mediating this function are still unknown. Microglia, the resident immune cells in the brain, have been suggested as one possible target. The aim of the present study was to explore this by using primary microglial cultures. Addition of leptin to the culture medium resulted in the induction and release of the inflammatory cytokine IL-1b, indicating that leptin can activate these cells directly. In addition, leptin was found to modulate cytokine release induced by other inflammatory stimuli such as lipopolysaccharide (LPS), an effect that is most likely due to the influence of leptin on the activation state of the cells as indicated by morphological changes. Collectively, these data suggest that leptin acts on microglia to induce an inflammatory response and to change their cellular conformation, modifying their responsiveness to secondary inflammatory stimuli.
Microfluidic Studies of Pulsatile CXCL12 Stimulation on Prostate Cancer Cells
Yao-Kuang Chung, PhD
University of Michigan, 2009
This project investigates effects of flow and CXC chemokine ligand-12, CXCL12 stimuli on prostate cancer PC3 cell adhesion and migration by using microfluidics. Prostate carcinoma (PCa) is the most frequently diagnosed cancer in men and the second leading cause of cancer death in American males. Bone metastasis, known to be exacerbated by CXC chemokine receptor 4 (CXCR4) signaling pathways, is a major cause of high morbidity and mortality rates.
Although inhibition of CXCR4 is known to modulate cancer metastasis in vivo, the detailed mechanisms are still ambiguous. In vitro studies are useful but lack many physiological features and may not reveal the full range of cancer cell behaviors. For example, the temporal patterns of CXCR4 stimulation by CXCL12 in vivo may be pulsatile rather than continuous as is the case in many in vitro studies. The pulsatile exposure to CXCL12 is expected due to pulsatile release, active degradation by proteases, scavenging by CXCR7 expressing cells, binding to extracellular matrix, and by presence of interstitial flows. Active scavenging by CXCR7 has been shown to be critical for cell directed sensing and polarizing toward CXCL12 stimuli in vivo further reinforcing the potentially important role of temporal patterns of stimulation. Pulsatile stimulation makes mechanistic sense also since CXCR4 is a G-protein-coupled receptor (GPCR) and continuous stimulation would simply lead to receptor desensitization.
Experiments by microfluidics demonstrate that pulses of CXCL12 rather than continuous stimulation induce significantly enhanced directed migration of PC3 cells. And as expected, CXCR4 knockdown PC3 cells migrated with significantly lower speed and directionality under CXCL12 stimulation compared with normal PC3 cells. During the course of studying the effect of temporal patterns of CXCL12 stimulation, it was unexpectedly discovered that PC3 cells showed significantly better adhesion and migration behavior under pulsatile flow than under steady flow even in the absence of chemical stimulation. The technology helps clarify some of the biophysical effect of CXCR4 that may be important for physiological function of malignant prostate cancer cells.
Modulation of the Immune Response at Systemic and Mucosal Sites by DNA Vaccine Adjuvants in HIV and Influenza Models
Kimberly Ann Kraynyak, PhD
University of Pennsylvania, 2009
Since the discovery of the human immunodeficiency virus type 1 (HIV-1) as the cause of acquired immunodeficiency syndrome (AIDS) almost 30 years, nearly 25 million people have died from AIDS worldwide. HIV-1/AIDS is a pandemic and there currently is no vaccine or cure for the devastating disease. Regardless of the route of infection, the mucosa is the primary site of viral replication and amplification. Therefore, the generation of immune responses at mucosal sites by and HIV-1 vaccine is likely important. We aimed to modulate the immune response generated through systemic vaccination by incorporating plasmid DNA expressing cytokine or mucosal chemokine adjuvants. First, we examined the potential of IL-15 in combination with its high-affinity receptor alpha chain (IL-15Ra) to augment immune responses when co-delivered with IL-15 through immunizations in mice. We found that IL-15Ra is a novel adjuvant in the absence of IL-15, increasing the potency of the CD8+ T-cell responses, even in IL-15 knockout mice. However, little impact on mucosal immunity was observed. Therefore, we chose 3 novel chemokines to examine for putative effects of mucosal immunity: CCL25 (thymus-expressed chemokine, TECK), CCL27 (cutaneous T-cell-attracting chemokine, CTACK), and CCL28 (mucosa-associated chemokine, MEC). Mice co-immunized with these mucosal chemokines and an HIV-1 gag antigenic plasmid exhibited increased levels of both systemic and mucosal immune responses. In addition, these responses were physiologically relevant in that they protected from a lethal influenza challenge. To further test the efficacy in the standard model for HIV-1 vaccine studies, immunization were carried out in the rhesus macaque model. Immunizations with CCL27 and simian (SIV) constructs delivered with electroporation elicited potent, but similar cellular and humoral immune responses in the periphery. The effects of CCL27 as an adjuvant were observed at mucosal sites, as shown by increased cytokine expression in CD4+ T and CD8+ T cells from the lung as well as antigen-specific IgA in the lung and fecal samples. We show for the first time that the incorporation of plasmid-encoded chemokines is capable of eliciting responses at mucosal sites in the rhesus macaque model.
Regulation of Cytokines and Chemokines During the Progression of Acute Inflammation in the Human Whole Blood Model
Devin L. Horton, PhD
University of Michigan, 2009
Traumatic injuries or infectious challenges activate the innate immune response, initiating inflammation and cytokines and chemokines that are released into the circulation in distinct kinetic patterns. In the clinic, patients often present after the onset of inflammation, however, few studies have investigated if the difference in cytokine and chemokine kinetics affects their ability to be regulated by anti-inflammatory reagents. To address this, the whole blood model was used to (1) characterize 24 h acute inflammation, (2) determine if postponing the addition of the anti-inflammatory glucocorticoid dexamethasone (DEX) until after an inflammatory stimulus had any consequences on its ability to regulate cytokines and chemokines, and (3) determine if the effects of post-stimulus DEX were cell-type specific. The Toll-like receptor 4 (TLR-4) agonist lipopolysaccharide (LPS) or the TLR-2 agonist Pam 3-cysteine-serine-lysine 4 (Pam) was used to activate the inflammatory response in whole blood. The levels of proinflammatory cytokines tumor necrosis factor, interleukin-1 beta, IL-6, IL-8, and growth related oncogene alpha (GROa) chemokines were indicators of inflammation. LPS stimulation of whole blood induced rapid TNF, IL-b, and IL-6 protein and messenger ribonucleic acid (mRNA) over 24 h. Pam stimulation caused slower induction of IL-10 and IL-6 protein. Both stimuli induced a continuous increase in IL-8 and GROa protein and mRNA levels. Concomitant addition of LPS or Pam and DEX to whole blood significantly suppressed cytokine and chemokine protein levels compared with either stimulus alone. Six-hour DEX significantly suppressed IL-8 and GROa mRNA at 24 h compared with LPS alone. Pam-induced cytokine and chemokine protein was also suppressed by 6-h DEX. Additional experiments designed to determine if these effects were cell-type specific indicated that 24 h after LPS stimulation, isolated neutrophils produced a substantial amount of IL-8 mRNA. Administration of DEX 6 h after LPS stimulation suppressed IL-8 mRNA levels in neutrophils and monocytes. These data indicate that the administration of anti-inflammatory reagents to a patient presenting in the clinic as little as 6 h after the onset of inflammation more likely improves inflammation by suppressing the message and thus protein of inflammatory mediators, such as chemokines, which persist beyond 6.
Role of Amyloid Precursor Protein (APP) and Amyloid Beta in Alzheimer’s Disease (AD): Modulation of APP Processing Detection of Abeta Oligomers, and Their Role in the Pathogenesis and Treatment of AD
Minkyu Leo Kim, PhD
University of Pennsylvania, 2009
Amyloid b (Ab) is an amyloidogenic peptide that forms senile plaques, a pathological hallmark found in the brains of Alzheimer’s disease (AD). Ab is generated from proteolytic cleavages of amyloid precursor protein (APP) by b- and g-secretase. Alternatively, APP can be cleaved within Ab domain by a-secretase, precluding the formation of the neurotoxic protein. One of the enzymes that consist of a-secretase pool for APP, tumor necrosis factor-a (TNF-a)-converting enzyme (TACE), is also responsible for the secretion of TNF-a, a proinflammatory cytokine that is implicated in the pathogenesis of AD. The dual role of TACE as an APP and a TNF-a-processing enzyme may confound the use of TACE inhibitors for AD in suppressing excessive TNF-a-mediated inflammatory reactions, should it result in an unwanted increase in the amyloidogenic cleavage of APP. The first half of this dissertation is driven by the hypothesis that a specific inhibitor of TACE can effectively block TACE activity without affecting Ab production. In this study, we tested effects of a novel TACE inhibitor, BMS-561392, on Ab production and APP processing in vitro and in vivo, and examined our theory that expression level of APP is not a rate-limiting factor in a- and b-cleavage reactions. Ab peptides can also aggregate into small oligomers, acutely synaptotoxic species responsible for synaptic malfunction in AD. Despite the important role Ab oligomers may play in the disease, developing a sensitive and reliable assay that can quantify Ab oligomers has remained elusive until recently. In the second study of this dissertation, we developed a highly specific oligomeric Ab enzyme-linked immunosorbent assay (ELISA) with picomolar sensitivity. Because Ab oligomers have the same sequence with its monomeric form, we relied on the previously characterized Nab61 antibody that recognizes a conformational epitopes of oligomeric Ab structures. We utilized the novel assay to detect and quantify Ab oligomers in brain extracts and cerebrospinal fluid (CSF) from AD patients and animal models. These studies have yielded novel insights into the dynamics between APP processing, inflammation, and Ab oligomers in AD brains and lay the groundwork for developing effective and specific treatments against AD.
The Cellular and Immunological Consequences of Listeriolysin O-Mediated Vacuolar Escape
Nicole P. Meyer-Morse, PhD
University of California, Berkeley, 2009
Listeria monocytogenes is a Gram-positive facultative intracellular pathogen capable of causing severe food born illness. Escape from the phagosome is essential for the intracellular life cycle of L. monocytogenes and has tremendous consequences on many biological processes including protective immunity and autophagy.
that results in the production of the cholesterol-dependent cytolysin listeriolysin O (LLO). Expression of LLO is required for phagosomal escape and for the induction of a cell-mediated immune response to L. monocytogenes. A mutant of L. monocytogenes that does not express LLO is avirulent, remains trapped in the phagosome, and is unable to induce cell-mediated immunity. The reason that L. monocytogenes mutants, lacking LLO, do not induce a cell-mediated immune response is not understood.
Using a mixed inoculum of cytosolic and phagosome-trapped L. monocytogenes, we found that when L. monocytogenes was confined to the vacuole, it suppressed the immunizing capability of L. monocytogenes in the cytosol. Further, as we increased the concentration of the bacteria trapped in the phagosome with an inoculum containing an immunizing dose of cytosolic L. monocytogenes, a concomitant decrease in the expression of proinflammatory cytokines and L. monocytogenes-specific T cells was observed. Additionally, immunosuppressive cytokines, including IL-10, were made in response to bacteria trapped in the phagosome, and blockage of the IL-10R restored the immunizing capability of cytosolic L. monocytogenes even in the presence of phagosome-trapped bacteria.
L. monocytogenes escape from the phagosome also induced autophagy. Autophagy is cellular bulk degradation pathway used to degrade long-lived proteins and organelles. We found L. monocytogenes mutants lacking LLO did not induce autophagy, whereas wild-type and other mutants induced relatively equal amounts of autophagy early in an infection of bone marrow-derived macrophages. Bacillus subtilis expressing LLO also induced autophagy, indicating that the induction was not specific to L. monocytogenes. Lastly, we found that liposomes containing LLO were sufficient to induce autophagy in the absence of infection. Thus, LLO was necessary and sufficient to induce autophagy in an L. monocytogenes infection.
In summary, we found that LLO expression by L. monocytogenes was required for both the proper induction of the cell-mediated immune response to L. monocytogenes and was also required for the induction of autophagy in response to infection with L. monocytogenes.