Infectious inflammation and injury in oral mucosa(35%)? Chronic periodontal diseases, which are complex, inflammatory oral diseases involving gram-negative microorganisms such as P. gingivalis, are characterized by destruction of supporting tissues surrounding the dentition. However, it is becoming increasingly clear that while the presence of these organisms is central to pathogenesis, the destructive process is largely host-derived. To define underlying parameters involved in this disfiguring pathogenesis, gene expression profiles of human gingival biopsy samples from patients with adult periodontitis were compared to healthy gingiva. Based on our initial data, genes involved in tissue turnover/inflammatory processes were over-represented in disease relative to non-disease samples. Of significant interest was the fact that many of the identified genes were those associated with activated mononuclear phagocytic cell populations, consistent with in situ analysis of inflamed gingival tissues revealing infiltrates of CD68+ macrophages and DC-SIGN+ dendritic cells in association with lymphocytes. Given the seminal role of antigen presenting cells in innate and adaptive immunity, and the common lineage of monocytes, macrophages and dendritic cells, we hypothesized that these cells would express both convergent and divergent functional genomic profiles when exposed to periodontopathogens, and that these transcriptional signatures may provide insight into the complex, initial inflammatory response to microbial challenge. Because P. gingivalis (Pg) is considered a primary etiologic agent in oral mucosal disease, we focused on defining the response of myeloid cells to lipopolysaccharide derived from this pathogen and the immediate early transcriptional response monitored using the Affymetrix system. We identify a Pg LPS-inducible convergent, transcriptional core response of >500 annotated genes/ESTs among these populations, reflected by a shared, but quantitatively distinct proteomic response. Nonetheless, despite these similarities, clear differences emerged between the monocytes, DC and macrophages. The finding that long-lived myeloid inflammatory cells, particularly DC, rapidly and aggressively respond to Pg LPS with generation of chemokines, proteases and cytokines capable of driving T helper cell lineage (Th17) polarization without evidence of corresponding immunosuppressive pathways highlights their prominent role in host defense and progressive tissue pathogenesis. The shared, unique and/or complementary transcriptional and proteomic profiles may frame the context of the host response to P. gingivalis, contributing to the destructive nature of periodontal inflammation, including osteoclastogenesis. These studies are of additional importance since P. gingivalis, once considered to be primarily an oral pathogen, has more recently been linked to several systemic conditions, such as coronary artery disease and preterm delivery of low birth weight infants.? ? SLPI regulation of proteolytic cascade in inflammation and cancer (35%)? Enhanced inflammation, delayed healing and susceptibility to infection have suggested a link between SLPI deficiency and regulation of inflammatory responses. How SLPI mediates these disparate activities, particularly those related to cell regulation, has remained a mystery. In our search for a cell surface molecule, we identified annexin II as a SLPI-binding protein. Among other activities, annexin II serves as a docking station for tissue-type plasminogen activator and plasminogen, facilitating plasminogen activation and generation of the protease, plasmin, prompting us to hypothesize that SLPI might influence plasmin generation. Based on a series of experiments, we demonstrated that the SLPI-annexin II interaction interrupts the proteolytic cascade responsible for plasmin generation. Moreover, we have shown that in SLPI null mice, macrophages process unregulated levels of plasmin, which contributes to excess tissue degradation, increased inflammation and failed healing following tissue injury. By inhibiting plasmin, which has a myriad of downstream consequences relative to complement activation, fibrinolysis, cytokine regulation, TGF-beta activation and other inflammatory sequelae, SLPI can exert control over proteolytic and inflammatory activation networks. Because fibrinolytic processes are fundamental not only to inflammation, coagulation and tissue repair, but growing evidence highlights their dysregulation in the pathogenesis of multiple chronic inflammatory disorders, cardiovascular disease and malignancy, further defining and regulating these pathways is of major significance.? ? Relevant to these findings, we have been exploring the role of SLPI as a protective agent in metastic head and neck squamous cell carcinoma (HNSCC), which remains one of the 10 most frequently occurring cancers worldwide with a very poor prognosis, in collaboration with investigators at UMD. Building on newly identified genetic links in the biochemical signals that influence neoplasia, we surmised that SLPI might influence tumor progression by multiple mechanisms. Based on immunohistochemical analysis of human oral squamous carcinoma tissues, SLPI expression was higher in noninvolved than in malignant tissue, and levels significantly inversely correlated with oral squamous cancer stage, pattern of invasion and NFkB activation. Thus, our data imply that SLPI may have anti-tumor activities, suggesting a strategy to predict (biomarker) and prevent (therapy) tumor invasive potential in oral cancer. Our continuing studies include exploring these pathways in a model of squamous cell carcinoma in SLPI null and WT mice and we have obtained initial data that the absence of SLPI is associated with more rapid and extensive development of tumors.? ? Autoimmunity and Sjogrens syndrome (30%)? An important objective of our studies is to explore underlying mechanisms by which inflammatory and immune responses go awry, resulting in autoimmune pathogenesis, as exemplified by Sjogrens syndrome (SS). The autoimmune hallmarks of SS are attested by the focal lymphoid cell infiltration of the exocrine glands (focus score) and the production of autoantibodies. The etiology of SS remains largely unknown and a conundrum is presented by the failure of many drugs that are effective in other autoimmune disorders, particularly TNF antagonists, such as etanercept. Importantly, plasma type 1 IFN was elevated in SS patients at baseline and not diminished by etanercept and paradoxically, TNF increased in the treated primary (p)SS patient group. Significantly increased levels of IFN and IFN-inducible genes, including B cell activating factor (BAFF), are consistent with failure of etanercept to suppress disease, and may explain some anti-TNF related adverse events. Another likely clue to the lack of efficacy of TNF inhibitors is the elevated IL-17 in Sjogrens patients peripheral circulation, which is also unmodulated by anti-TNF therapy. Collectively, these data indicated that TNF is not a pivotal cytokine in the pathogenesis of SS, driving our studies to define the underlying immunopathogenic mechanisms responsible for exocrinopathy and thereby, to define potential new target molecules for treatment. In salivary gland biopsies, we are performing gene profiling to correlate specific gene expression with T cell subsets and/or their products within the inflamed tissues. We will continue to explore the clinical aspects of SS along with our animal model studies, including the TGF- beta null mice in which the salivary glands are a target of autoimmune lymphocytic infiltration, sharing histopathologic features with those typical of SS in humans.
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