published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

In two studies we tested the effect of vitamin B-6 (B-6) intake on in vitro lymphocyte, proliferation and IL-2 production in healthy young women. In Study I, 6 women were fed a constant diet containing 0.84 mg (4.96 μmols) of B-6 for 12 d, and 1.24 mg (7.33 μmols) and 2.44 mg (14.42 μmols) of B-6 during two subsequent 10-d periods. Lymphocyte proliferation in response to the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM) was significantly higher (p < 0.05) at 2.44 mg than at 0.84 mg intake and the pre-study value. In Study II, 10 women who consumed their self-selected diets were randomly divided into a PN and a placebo group of five each. Following a 5-d baseline period, the PN group received a daily supplement of 1.5 mg (7.29 μmols) and 50 mg (243 μmols) pyridoxine.HC1 (PN.HC1) for 7 and 6 d, respectively. This was followed by a 28-d washout period during which no supplement or placebo was administered. After daily 1.5 mg supplementary PN.HC1 for seven days, lymphocyte proliferation in response to phytohemagglutinin (PHA), Con A and PWM, and IL-2 production were significantly higher than the baseline (p < 0.05) and that of the placebo group (p < 0.05). The 50 mg PN.HC1 supplement increased IL-2 production, but not lymphocyte proliferation as compared with the 1.5 mg PN.HC1 supplement period. Lymphocyte proliferation and IL-2 production were significantly correlated with lymphocyte pyridoxal 5'- phosphate concentrations (p < 0.01). We conclude that a B-6 intake of 1.5 to 2 times the RDA improves lymphocyte proliferation and IL-2 production in healthy young women and this effect of B-6 on immunocompetence is transitory. To explore the basis for the effect of B-6 on immune function, putrescine was added to the culture medium (2 - 200 μmol/L). In vitro lymphocyte proliferation and IL-2 production were not changed by the addition of putrescine under our experimental conditions. / Graduation date: 1995

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Pain from bone metastases is multifaceted with clinical descriptors including ongoing pain, hypersensitivity to external stimuli and intermittent episodes of breakthrough pain characterized as a sudden and abrupt onset of severe pain on a background of well-controlled pain. Moreover, cancer-induced bone pain remains inadequately managed due to a myriad of side effects associated with the current pain relieving regimens, which primarily rely on administration of opiates. Despite advances made in cancer therapeutics, these patients experience an inferior quality of life with incapacitating pain with limited daily activities. Development of long-term novel, non-opiate mechanism-based therapeutics with limited side effects is considered beneficial in elevating the patients' quality of life. First part of this dissertation encompasses the role of p38 MAPK in a mouse model of cancer-induced bone pain in which breast cancer cells were injected and sealed into the femur. Our data demonstrated that both acute and prolonged inhibition of p38 MAPK blocked cancer-induced spontaneous pain but had no effect on the evoked pain indicating important differences in mechanisms mediating ongoing pain as opposed to evoked pain. Undermanaged control of breakthrough pain is attributed to poor understanding of underlying mechanisms and how they may differ from ongoing pain due, in part, to lack of a pre-clinical model in which these mechanisms can be studied. We have established a rat model of cancer-induced bone pain to examine ongoing pain and pain relief using conditioned place preference paradigm as well as breakthrough pain using palpation-induced conditioned place aversion. We have shown that while peripheral afferent input from the tumor-bearing tibia mediates cancer-induced ongoing pain and initiation of breakthrough pain, it does not contribute to the maintenance of breakthrough pain. These data suggest that molecular targets mediating these two mechanisms may be different. This hypothesis was confirmed by our findings in this model that acute blockade of interleukin-6 blocked movement-evoked breakthrough pain in tumor-bearing rats, but failed to block tumor-induced ongoing pain. Hence, we provide a platform to manipulate treatments that can be given alone or in combination with opiates in such a way that patients receive adequate control of breakthrough pain.

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Asthma is a multi-faceted chronic inflammatory disease accompanied by loss of airway epithelial integrity leading to remodeling of the airways. Perturbations to the lung redox environment, including alterations in glutathione (GSH) content, have been reported in asthma. GSH can be conjugated to protein cysteines, controlling protein function in an oxidant-dependent process known as protein S-glutathionylation (PSSG). The thioltransferase, glutaredoxin-1 (Glrx1), deglutathionylates proteins under physiological conditions, restoring sulfhydryl groups of target proteins. Glrx1 is emerging as a critical player in settings of allergic airway disease, but its function in regulating epithelial cell responses to asthma-relevant cytokines has not been examined. Furthermore, the role of Glrx1 in controlling the extent of airway remodeling in response to house dust mite (HDM) in vivo is still not well understood. Interleukin-17A (IL-17A) is a potent cytokine that stimulates epithelial cells to produce pro-inflammatory mediators, in part by activating the nuclear factor kappaB (NF-κB) pathway, a key regulator of inflammation. We demonstrate that interleukin-17A (IL-17A) induces rapid activation of both classical and alternative NF-κB, while simultaneously resulting in protein oxidation and PSSG. In particular, we show IL 17A induces S-glutathionylation of RelA (RelA-SSG) and IKKα (IKKα-SSG), which is enhanced following siRNA-mediated knockdown of Glrx1. We also demonstrate that absence of Glrx1 leads to increased nuclear content of RelA and RelB and enhanced production of NF-κB-driven pro-inflammatory genes, KC and CCL20 while decreasing IL-6 expression. Finally, we show that siRNA-mediated knockdown of IKKα attenuates nuclear RelA and RelB and dampens pro-inflammatory gene production. Together, these data indicate a crucial role for the Glrx1/PSSG axis in controlling RelA-SSG, IKKα-SSG and epithelial cell responsiveness to IL-17A. Mice lacking Glrx1 were previously shown to display enhanced resolution of allergic airway disease induced by ovalbumin (Ova) challenge. In this study, we determined the role of Glrx1 in a HDM model of allergic airway disease. Wild type (WT) mice and Glrx1 deficient (Glrx1-/-) mice demonstrated similar total lung cell counts, but Glrx1-/- mice displayed fewer neutrophils than WT mice. Conversely, mice overexpressing Glrx1 specifically in CCSP positive cells in the lung (Epi-Glrx1) showed attenuated total lung cell counts and lung eosinophils compared to control mice. Immunohistological analysis of remodeling markers revealed that Glrx1-/- mice displayed increased HDM-induced mucus metaplasia, α smooth muscle actin (αSMA) positivity and collagen staining compared to WT mice. Evaluation of total lung collagen showed that Glrx1-/- mice had significantly higher collagen content compared to WT mice. In Epi-Glrx1 mice, attenuation of mucus metaplasia, αSMA content and collagen staining was observed compared to control mice. Furthermore, Epi-Glrx1 mice also demonstrated significantly impaired collagen production compared to control mice. We also demonstrate that Glrx1 absence results in decreased expression of the epithelial cell marker, E-cadherin, and increased expression of αSMA, a mesenchymal marker. Together, these studies demonstrate a critical role for Glrx1 in controlling epithelial cell responses to IL-17A and in mediating in vivo collagen production in response to chronic allergen exposure.

Renal cell carcinoma accounted for an estimated 57,760 new cases and estimated 12,980 deaths in the United States in 2009. Current treatment options for systemic renal cell carcinoma yield markedly low response percentages; however, recent cytokine therapy experiments have produced promising results. A novel adenovirus, Ad.5/3-mda-7, has been synthesized to efficiently infect renal cancer cells with the mda-7 gene. This gene encodes for the cytokine MDA-7/IL-24 that has the ability to specifically target transformed cells. Assays performed with this adenovirus resulted in an increased percentage of cell death in renal cancer cells when compared to infection with Ad.5/3-cmv empty vector. Further assays that combined Ad.5/3-mda-7 infection with treatments of small molecule inhibitors increased the percentages of cell death by upregulating JNK and p38 MAPK pathways, downregulating the ERK1/2 MAPK pathway, and downregulating the PI3K pathway. Western blots confirmed upregulation and downregulation of these pathways by probing for key proteins. Renal cancer cells responded best to infection with Ad.5/3-mda-7 and treatment with PD184352, PX866, and Rapamycin. This combinatorial treatment caused a greater percentage of cell death than the sum of the two individual treatments, suggesting a synergistic inhibition of cell growth pathways. These findings suggest that the combination of Ad.5/3-mda-7 and specific small molecule inhibitors has developmental potential as a novel and more efficient treatment option for systemic renal cell carcinoma.

AbstractINTERLEUKIN-6 LEVELS IN GENERALIZED AND LOCALIZED AGGRESSIVE PERIODONTITIS PATIENTSBy Bindu Reddy, D.D.S.A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at the Virginia Commonwealth UniversityVirginia Commonwealth University, 2004Major Director: Joseph V. Califano, D.D.S., Ph.D.Associate Professor, Department of Periodontics Periodontitis is an inflammatory disease involving the supporting structures of the dentition. Many studies have shown that there is a relationship between periodontal disease, the presence of pro-inflammatory cytokines, and systemic disease such as cardiovascular disease and diabetes mellitus. The purpose of this study was to measure serum Interleukin-6 levels in generalized and localized aggressive periodontitis and non-periodontitis patients and look for relationships with measures of disease severity. We also examined variables known to have a relationship with IL-6. A total of 172 subjects, comprising three periodontal subgroups, non-periodontitis (NP=61), generalized aggressive periodontitis (GAP=77), and localized aggressive periodontitis (LAP= 34), had serum samples evaluated for IL-6 levels using a highly sensitive ELISA test. The IL-6 levels were compared with clinical and demographic data including age, race, gender, number of teeth, probing depth, attachment loss, bleeding index, plaque index, gingival index, cotinine levels, smoking status, and CRP levels. Using multiple regression analysis, smoking status (p=0.0015) was the only variable found to have a significant relationship with IL-6 levels for all three groups.

Systemic scleroderma (SSc) is a systemic connective tissue disease affecting skin and internal organs. The pathogenesis of SSc is characterized by inflammation, vasculopathy and fibrosis. No agent has been proven effective in the treatment of SSc. There is a lack of suitable biomarkers for monitoring the disease activity or the response to the treatment of SSc. Therefore our aim was to analyse the extracellular levels of S100A4, Hsp90 (Heat shock protein 90) and IL-35 (interleukin-35) in SSc. S100A4 and Hsp90 have been initially studied in tumours; in some of them considered as suitable prognostic markers and candidates for future therapies. We have recently described the profibrotic role of S100A4 and Hsp90 in the pathogenesis of SSc. Our results showed that inactivation of S100A4 and Hsp90 effectively prevented the development of experimental skin fibrosis. This was consequently confirmed by the analysis of S100A4 and Hsp90 in the peripheral blood of patients with SSc, where significant associations with disease activity and organ involvement were detected. IL-35 may become another potential biomarker of SSc. We detected increased expression of IL-35 in the affected skin, dermal fibroblasts and in serum of patients with SSc. Moreover, the main profibrotic mediator transforming growth factor...

Celiac disease is an autoimmune disease which occurs in susceptible individuals after ingestion of food containing gluten. Gluten and its monomeric fraction gliadin induce inflammatory damage of the small intestine by activating the immune cells that react strongly to gluten peptides. Gluten peptides have the ability to activate cells of adaptive as well as innate immune system. This work is focused on the production of interleukin (IL)-1 in antigen presenting cells stimulated with peptic gliadin digest. We found that monocytes and peripheral blood mononuclear cells (PBMC) isolated from blood of celiac patients secrete significantly more IL-1α and IL-1β than cells of healthy donors after stimulation with gliadin digest. The gliadin-induced IL-1β expression is controlled by a signaling cascade that includes MAPK kinase family molecules and transcription factor NF-κB. Moreover, we found that the adaptor proteins MyD88 and TRIF as well as Toll-like receptor (TLR) 2 and 4 play a role in the signaling cascade underlying gliadin-induced IL-1β expression by using murine bone marrow derived dendritic cells (BMDC). The precursor form of IL-1β in gliadin- stimulated PBMC and murine BMDC is maturated by caspase-1. In celiac PBMC the gliadin- induced maturation and secretion of IL-1β depends on the potassium...