Wear particle-induced inflammation leading to periprosthetic osteolysis remains a major cause of hip implant failure. As polyethylene particles from conventional metal-on-polyethylene implants have been associated with these failures, an interest in lower wear metal-on-metal (MM) bearings has emerged. However, the biological effects of nanometer-size chromium oxide particles, predominant type of wear particles produced by MM implants, remain mostly unknown. Therefore, this study aimed to determine the cytotoxicity of nanometer-size Cr2O3 particles on macrophages in vitro, by analyzing their effects on cell mortality and cytokine release and comparing them with those of similarly-sized alumina (Al2O3) particles (known to be relatively bioinert). Results showed that at high concentrations, nanometer-size Cr2O3 particles can be cytotoxic to macrophages, inducing significant decreases in total cell numbers and increases in necrosis. Results also showed that, at high concentrations, the cytotoxicity of Cr2O3 particles was overall higher than that of Al2O3 particles, even though Cr2O3 and Al2O3 are both stable forms of ceramic materials. However, it appeared to be lower than that of previously reported conventional polyethylene and CoCrMo particles. Therefore, chromium oxide particles may not be the main culprit in initiating the inflammatory reaction in MM periprosthetic tissues.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

Le facteur d'inhibition de la migration des macrophages (MIF) joue un rôle important dans la régulation du système immunitaire inné et adaptatif. Le MIF est impliqué dans plusieurs maladies inflammatoires chroniques dû à sa capacité à induire la sécrétion du facteur nécrosant tumoral (TNF-a) et de l'interleukine-12 (IL-12), en plus de favoriser l'expression des récepteurs de type Toll (TLR) 4 chez les macrophages. Paradoxalement, la neutralisation du MIF augmente l'activation des lymphocytes T cytotoxiques et diminue la réponse humorale in vivo qui sont des résultats inattendus considérant le rôle du MIF comme pro-inflammatoire et inducteur d'IL-12. Étant donné que la sécrétion de MIF est induite chez les souris BALB/c infectées avec Plasmodium chabaudi adami (DK) et que la résolution de cette infection nécessite une réponse Th1 couplée à une production d'IFN-y, nous avons évalué l'effet de la déficience en MIF sur la cinétique d'infection ainsi que sur l'activation des lymphocytes CD4+. Nos résultats montrent un meilleur contrôle de l'infection avec Plasmodium chabaudi adami (DK) chez des souris déficientes en MIF (MIF/KO), caractérisé par un pic et une parasitémie cumulative significativement plus faible. D'autre part, une anémie plus modérée, déterminée par une plus faible expression des précurseurs érythroïdes et des réticulocytes a aussi été mesurée chez des souris MIF/KO lors de l'infection. Au jour 4 post-infection, le pourcentage et le nombre absolu de cellules CD4+ spléniques activées était plus important chez les souris MIF/KO, de plus, ces cellules sécrétaient plus d'IFN-y, moins d'IL-10 et d'IL-4. De manière intéressante, la production précoce d'IL-4 par une population des cellules spléniques non B et non T (possiblement des basophiles) a été fortement inhibée chez les souris MIF/KO lors de l'infection. Une observation collatérale dans notre étude a été la surexpression du facteur de transcription T-bet chez des cellules T CD4 + naïves déficientes en MIF. Étant donné que les lymphocytes CD4+ déficients en MIf sécrètent plus d'IFN-y et moins d'IL-4 suite à leur stimulation in vitro, nos résultats suggèrent que le MIF module la capacité des lymphocytes CD4+ à sécréter des cytokines de type Th1. De plus, elle suggère aussi la présence d'une corrélation entre l'expression plus forte de T-bet et le phénotype Th1 accentué chez les lymphocytes CD4+ déficients en MIF. Par contre, cette régulation n'impliquerait pas la voie des récepteurs TLR étant donné qu'aucune modulation de ces récepteurs par le MIF n'a été observée. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : MIF, Plasmodium, IFN-y, IL-4, Lymphocyte

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 204-240).

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 47-49).

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 96-108).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 109-113).

About one in eight women in the United States will develop breast cancer over the course of her lifetime. Moreover, patient-to-patient variability in disease progression continues to complicate clinical decisions in diagnosis and treatment for breast cancer patients. Early detection of tumors is a key factor influencing patient survival, and advancements in diagnostic and imaging techniques has allowed clinicians to spot smaller sized lesions. There has also been an increase in premature treatments of non-malignant lesions because there is no clear way to predict whether these lesions will become invasive over time. Patient variability due to genetic polymorphisms has been investigated, but studies on variability at the level of cellular activity have been extremely limited. An individual’s biochemical milieu of cytokines, growth factors, and other stimuli contain a myriad of cues that pre-condition cells and induce patient variability in response to tumor progression or treatment. Circulating white blood cells called monocytes respond to these cues and enter tissues to differentiate into monocyte-derived macrophages (MDMs) and osteoclasts that produce cysteine cathepsins, powerful extracellular matrix proteases. Cathepsins have been mechanistically linked to accelerated tumor growth and metastasis. This study aims to elucidate the variability in disease progression among patients by examining the variability of protease production from tissue-remodeling macrophages and osteoclasts. Since most extracellular cues initiate multiple signaling cascades that are interconnected and dynamic, this current study uses a systems biology approach known as cue-signal-response (CSR) paradigm to capture this complexity comprehensively. The novel and significant finding of this study is that we have identified and predicted donor-to-donor variability in disease modifying cysteine cathepsin activities in macrophages and osteoclasts. This study applied this novel finding to the context of tumor invasion and showed that variability in tumor associated macrophage cathepsin activity and their inhibitor cystatin C level mediates variability in cancer cell invasion. These findings help to provide a minimally invasive way to identify individuals with particularly high remodeling capabilities. This could be used to give insight into the risk for tumor invasion and develop a personalized therapeutic regime to maximize efficacy and chance of disease free survival.

B cell development is a highly organized process, which commences in the fetal liver during embryogenesis and in the bone marrow (BM) after birth. Surface IgM+ immature B cells emigrate from the BM via the blood stream to the spleen and finally differentiate into conventional mature follicular B (FoB) cells and marginal zone (MZ) B cells. Conversely, some sIgM+ immature B cells can also mature into IgD+ FoB cells in the BM. The ubiquitously expressed cell surface glycoprotein CD47 and its receptor signal regulatory protein α (SIRPα) are members of the immunoglobulin superfamily. Both individually and upon their interaction, CD47 and SIRPα have been found to play important role in the homeostasis of T lymphocytes or CD8­ conventional dendritic cells (cDCs) in secondary lymphoid organs. However, their role in regulating B cell homeostasis has remained unknown. The present study describes important roles of CD47 and SIRPα in B cell homeostasis. Lack of SIRPα signaling in adult SIRPα mutant (MT - cytoplasmic domain deletion) mice resulted in an impaired B cell maturation in the BM and spleen, which was also reflected in the blood. In the BM and spleen of SIRPα MT mice, reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells were observed, while earlier BM B cell progenitors or splenic transitional B cells remained unaltered. In SIRPα MT mice, maturing B cells in BM and spleen were found to express higher levels of the pro-apoptotic protein BIM and contained an increased level of apoptotic cells. In contrast to that for FoB cells, the splenic MZ B cell population was increased with age in SIRPα MT mice without showing an increased level of activation markers. Immunohistochemical analysis revealed an increased follicular localization of MZ B cells in the spleens of SIRPα MT mice. In addition, MZ macrophages and marginal metallophilic macrophages were not restricted to their normal position in SIRPα MT spleens. Interestingly, CD47-deficient (CD47-/-) mice mimicked the FoB cell phenotype observed in SIRPα MT mice and had a reduced number of  FoB cells in the BM, blood and the spleen at 5­6 months of age, but not in younger mice. Similar to SIRPα MT mice, CD47-/- mice also displayed an increased number of splenic MZ B cells. Sera form both mouse strains did not show any signs of an increased production of autoantibodies or antinuclear antigens. BM reconstitution experiments identified a requirement for non-hematopoietic SIRPα signaling for normal B cell maturation in the BM and to maintain normal numbers and retention of MZ B cells in the splenic MZ. On the contrary, hematopoietic SIRPα signaling appeared to be important for FoB cell maturation in the spleen. Interestingly, hematopoietic SIRPα was required for normal MZ retention of MZ macrophages while normal distribution of metallophilic macrophages required non­hematopoietic SIRPα signaling.  Collectively, these findings revealed an important role of CD47 and of SIRPα signaling in B cell homeostasis in different lymphoid organs.