Non-redundant and critical roles for leukotriene B4 receptors BLT1 and BLT2 in mouse models of inflammatory arthritis.

Inflammation is now recognized as an important factor in several age-related diseases such as arthritis, atherosclerosis, multiple sclerosis and diabetes. In each case, sub-clinical chronic inflammation occurs over years and leads to progressive destruction of the tissue until the symptoms become clinically apparent. Eicosanoids such as prostaglandins and leukotrienes play an important role in inflammation. Among these, leukotriene B 4 (LTB 4 ) has had a long history of being associated with numerous inflammatory diseases. A high affinity receptor for LTB 4 , LTB 4 receptor 1 (BLT 1) has been well-characterized in human and murine tissues. While the molecular mechanisms remain unclear, its importance in diverse inflammatory diseases was demonstrated in mice lacking BLT1. More recently, a second highly conserved low affinity LTB 4 receptor, LTB 4 receptor 2 (BLT2) was identified, but its functional significance remains completely unknown. Both BLT1 and BLT2 are seven-transmembrane G protein coupled receptors transducing signals through heterotrimeric G-proteins. Chapter II of this thesis describes the generation and characterization of monoclonal antibodies to BLT1. Immunization of BLT1-deficient mice with 300.19, a murine pre-B cell line expressing high levels of either human or murine BLT1 allowed isolation of highly species specific anti-BLT1 antibodies. Using an approach involving a series of human/murine BLT1 chimeric receptors the monoclonal antibody binding sites were mapped to extracellular loop-2. Extensive characterization of BLT1 in murine primary cells revealed high levels of BLT1 in neutrophils and eosinophils and a previously unsuspected regulation of BLT1 in macrophages. While bone marrow derived macrophages expressed high levels of BLT1 its expression in peripheral tissue macrophages was significantly reduced suggesting a potential novel mechanism, and a role for BLT1 in inflammation-induced egress of bone marrow derived cells. The antibodies also allowed demonstration of normal BLT1 expression in the recently generated BLT2 deficient mice. Future studies could explore the therapeutic potential of these antibodies. In Chapter III, we investigated the role of BLT1 and BLT2 in inflammatory arthritis in mouse models. Collagen induced arthritis (CIA) is a model where mice are immunized with chicken collagen type II in complete freund\u27s adjuvant and leads to a polyarthritis in the distal joints. BLT1 deficient mice on the DBA/1 background were completely protected from the development of CIA which confirms the previous observations with BLT1 antagonists and BLT1 deficient mice on the C57B1/6 background. Arthritis was also induced by transfer of K/BxN serum to naÏve mice. K/BxN is a T cell receptor transgenic mouse (KRN) crossed with a NOD mouse. These mice spontaneously develop arthritis due to auto-antibody production against glucose-6-phosphate isomerase, a ubiquitous enzyme. In this model, we report for the first time that BLT2 deficient mice are completely protected from inflammatory arthritis. Histopathologic examination revealed a massive inflammatory cell influx in wild-type mice that was completely absent in BLT2 deficient mice. Further analysis of these mice using bone marrow transplantation studies demonstrated development of arthritis requires BLT2 expression on bone marrow-derived cells. Wild-type mice which received bone marrow from BLT2 -/- mice showed little sign of disease on histological analysis, while BLT2-/- mice receiving wild-type bone marrow showed intense inflammation leading to severe destruction in cartilage and bone. When BLT1/BLT2 double deficient mice were transplanted with a mixture of BLT1 deficient and BLT2 deficient bone marrow, a gain of clinical disease was observed. All these data demonstrate that BLT2 has a critical role in arthritis and it is non-redundant with the role played by BLT1. Further study is needed to determine the function of BLT2 and in which cell types it is import

Thermal Recovery of Multi-Limbed Robots with Electric Actuators

The problem of finding thermally minimizing configurations of a humanoid robot to recover its actuators from unsafe thermal states is addressed. A first-order, data-driven, effort based, thermal model of the robots actuators is devised, which is used to predict future thermal states. Given this predictive capability, a map between configurations and future temperatures is formulated to find what configurations, subject to valid contact constraints, can be taken now to minimize future thermal states. Effectively, this approach is a realization of a contact-constrained thermal inverse-kinematics (IK) process. Experimental validation of the proposed approach is performed on the NASA Valkyrie robot hardware

Role of Leukotriene B4 Receptors in the Development of Atherosclerosis: Potential Mechanisms

Objective-Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB 4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-/- mice. Methods and Results-Analysis of global changes in gene expression induced by LTB 4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2, which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E-/- mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4. Conclusions-The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation

PPARγ is a Major Driver of the Accumulation and Phenotype of Adipose-Tissue TregT_{reg} Cells

Obesity and type-2 diabetes have increased markedly over the past few decades, in parallel. One of the major links between these two disorders is chronic, low-grade inflammation. Prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue (VAT) and ultimately other tissues, leading to metabolic abnormalities such as insulin resistance, type-2 diabetes and fatty-liver disease. Although invasion of VAT by pro-inflammatory macrophages is considered to be a key event driving adipose-tissue inflammation and insulin resistance, little is known about the roles of other immune system cell types in these processes. A unique population of VAT-resident regulatory T $(T_{reg})$ cells was recently implicated in control of the inflammatory state of adipose tissue and, thereby, insulin sensitivity. Here we identify peroxisome proliferator-activated receptor (PPAR)-γ, the ‘master regulator’ of adipocyte differentiation, as a crucial molecular orchestrator of VAT $T_{reg}$ cell accumulation, phenotype and function. Unexpectedly, PPAR-γ expression by VAT $T_{reg}$ cells was necessary for complete restoration of insulin sensitivity in obese mice by the thiazolidinedione drug pioglitazone. These findings suggest a previously unknown cellular mechanism for this important class of thiazolidinedione drugs, and provide proof-of-principle that discrete populations of $T_{reg}$ cells with unique functions can be precisely targeted to therapeutic ends

Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector

Genetic deficiency in the expression of interleukin-10 (IL-10) is associated with the onset and progression of experimental inflammatory bowel disease (IBD). The clinical significance of IL-10 expression is supported by studies showing that immune-augmentation of IL-10 prevents inflammation and mucosal damage in animal models of colitis and in human colitis. Interleukin-10 (IL-10), an endogenous anti-inflammatory and immunomodulating cytokine, has been shown to prevent some inflammation and injury in animal and clinical studies, but the efficacy of IL-10 treatment remains unsatisfactory. We found that intra-peritoneal administration of adenoviral IL-10 to mice significantly reversed colitis induced by administration of 3% DSS (dextran sulfate), a common model of colitis. Adenoviral IL-10 (Ad-IL10) transfected mice developed high levels of IL-10 (394 +/- 136 pg/ml) within the peritoneal cavity where the adenovirus was expressed. Importantly, when given on day 4 (after the induction of colitis w/DSS), Ad-IL10 significantly reduced disease activity and weight loss and completely prevented histopathologic injury to the colon at day 10. Mechanistically, compared to Ad-null and DSS treated mice, Ad-IL10 and DSS-treated mice were able to suppress the expression of MAdCAM-1, an endothelial adhesion molecule associated with IBD. Our results suggest that Ad-IL10 (adenoviral IL-10) gene therapy of the intestine or peritoneum may be useful in the clinical treatment of IBD, since we demonstrated that this vector can reverse the course of an existing gut inflammation and markers of inflammation

Upper Lip Asymmetry During Smiling: An Analysis Using Three-Dimensional Images

Objective:The aim of this study was to use three-dimensional images to determine the presence of upper lip asymmetry at rest and during smiling in a group of individuals with no history of orthodontics or facial cosmetic surgery.MethodsStandardized three-dimensional frontal resting and smiling images of 54 volunteers were analyzed using the 3dMDvultus software (3dMD, Atlanta, GA). Measurements were made from the soft tissue nasion, ipsilateral ala, subnasale, and menton to the right and left commissures of the lip. A 2.5 mm or greater difference between the right and left sides was defined as an asymmetry. The agreement on the presence or absence of asymmetry between the subjects’ states of rest and smiling was determined by the McNemar’s chi-squared test. Statistical significance was defined as p<0.05.Results:Menton was the most stable facial landmark to evaluate the upper lip symmetry at rest and during smiling (p=0.002). Using menton as a landmark, only one of the 54 subjects showed asymmetry while resting, but 12 (22%) showed asymmetry when smiling.Conclusion:As part of treatment planning for orthodontics or orthognathic surgery, patients should be evaluated for the upper lip symmetry during resting and smiling. The presence of asymmetry during smiling is a significant clinical problem that needs to be recognized so that patients can be informed about the effect it can have on the final esthetic result

Development and Screening of Contrast Agents for In Vivo Imaging of Parkinson’s Disease

Purpose: The goal was to identify molecular imaging probes that would enter the brain, selectively bind to Parkinson’s disease (PD) pathology, and be detectable with one or more imaging modalities. Procedure: A library of organic compounds was screened for the ability to bind hallmark pathology in human Parkinson’s and Alzheimer’s disease tissue, alpha-synuclein oligomers and inclusions in two cell culture models, and alpha-synuclein aggregates in cortical neurons of a transgenic mouse model. Finally, compounds were tested for blood–brain barrier permeability using intravital microscopy. Results: Several lead compounds were identified that bound the human PD pathology, and some showed selectivity over Alzheimer’s pathology. The cell culture models and transgenic mouse models that exhibit alpha-synuclein aggregation did not prove predictive for ligand binding. The compounds had favorable physicochemical properties, and several were brain permeable. Conclusions: Future experiments will focus on more extensive evaluation of the lead compounds as PET ligands for clinical imaging of PD pathology