TNF is a proinflammatory cytokine whose pleiotropic biological properties are signaled through the receptor TNFR1. Activation of this signaling pathway has been implicated in a broad range of biological functions, including host defense, inflammation, apoptosis, autoimmunity, and cancer. TRADD is an adaptor protein that is recruited to TNFR1 upon receptor engagement. Using a Tradd-deficient murine model, we demonstrated that TRADD is essential for both TNF-mediated apoptosis and inflammatory responses. In addition to refining the role of TRADD in TNFR1 signaling, we have also identified a novel function of TRADD in TLR3 and TLR4 pathways, which are key drivers of the innate immune response. We showed that TRADD is involved in NF-κB activation upon TLR3 and TLR4 stimulation, and Tradd-deficient macrophages showed impaired inflammatory cytokine production in response to TLR ligands in vitro. These data reveal the multifaceted functions of TRADD in immune signaling pathways. Beyond its role in the immune response, TNF has also been shown to play a crucial, cell-non-autonomous role in driving tumor growth in various models of cancer. We initially sought to determine whether TRADD is essential for this aspect of TNF function by employing the use of a chemical induced skin carcinogenesis model in which the tumor-promoting role of TNF is very well established. In this model, H-Ras is the major driving oncogene. We found that Tradd deficiency accelerated tumor formation in mouse skin, in strong contrast to what was observed in Tnfr1-deficient mice. Further in vitro analyses revealed that upon expression of oncogenic H-Ras, Tradd-deficient murine fibroblasts displayed both reduced cell cycle arrest and repression of Ras induced cellular senescence. Importantly, the level of p19Arf induced by H-Ras expression was reduced in Tradd-deficient fibroblasts in a post-translational manner. Our biochemical evidence suggests that TRADD can shuttle dynamically between the cytoplasm and the nucleus; in doing so, nuclear TRADD interacts with ULF, a newly identified E3 ubiquitin ligase for p19Arf. Interaction between nuclear TRADD and ULF sequesters ULF away from p19Arf, leading to p19Arf stabilization and tumor suppression. Together, these data demonstrate the functional diversity of TRADD in different compartments of the cell.

This project established an in vivo method to identify and manipulate expression of markers of osteoarthritis (OA). Specifically, strategies that predictably induce joint inflammation to evaluate dietary methods of OA prevention in young horses have yet to be accomplished. Therefore, the 3 studies described herein were conducted to determine effectiveness of an intra-articular lipopolysaccharide (LPS) challenge on markers of inflammation and cartilage metabolism in young horses and potential of dietary glucosamine hydrochloride (HCl) to mitigate these alterations. In the first study, horses were challenged with 0.25 ng or 0.50 ng of intra-articular LPS solution or lactated ringer’s solution (control). Injection of LPS increased inflammation based on synovial prostaglandin E2 (PGE2) concentrations. Carboxypeptide of type II collagen (CPII), a maker of type II collagen synthesis, also increased in a dose-dependent manner. However, clinical parameters of health were not influenced and remained within normal ranges. Carpal circumference increased in response to repeated arthrocentesis. Lameness scores increased with LPS injection when compared to controls. This model of joint inflammation (0.5 ng LPS) was used in the second study to evaluate potential chondroprotective effects of oral glucosamine HCl supplementation in yearling horses. Specifically, the oral absorption of glucosamine HCl versus saline was determined by nasogastric dosing and incorporation of dietary glucosamine HCl into plasma and synovial fluid over time. Plasma and synovial fluid concentrations of glucosamine tended to increase over the 98-d period. In the third study, yearlings were challenged with intra-articular LPS to determine the potential of glucosamine HCl to mitigate inflammation when compared to contralateral joints. Injection of LPS increased synovial PGE2 and cartilage biomarkers CPII and collagenase cleavage neopeptide (C2C), a marker of type II collagen degradation. Oral glucosamine HCl decreased PGE2 and C2C concentrations, but increased levels of CPII. Results of these 3 studies provide a clearer understanding of joint inflammation and cartilage turnover in young horses and demonstrated a potential role of oral glucosamine to mitigate these effects and possibly prevent OA in horses.

Leukocyte exit from peripheral tissues is fundamental to host defense, yet little is known about the role of adhesive molecules in this process. In my thesis I ask the question “can an integrin regulate leukocyte exit from inflamed peripheral tissues” and specifically investigate the leukocyte integrin α1β1. This is an important question because leukocyte exit, or persistence, at an inflammatory lesion can have a profound effect on the immune response. In addition, I present special in situ staining techniques which had to be developed in order to assay endogenous leukocyte migration in a murine model. The introductory sections review functional differences between myeloid and lymphoid leukocyte subsets, the leukocyte adhesion cascade, integrins, chemokine receptors and the essential concepts of signaling and the relationship between chemokines and integrin activation. I also discuss the pro-migratory paradigm of leukocyte integrins, in other words that integrin adhesion is equated with leukocyte migration. The current literature regarding what is known about integrin function in peripheral tissues and leukocyte migration is also discussed. Chapter 2 characterizes my inflammatory model and implicates α1β1integrin and macrophages as important molecular and cellular entities respectively, involved in sustaining the inflammatory response. Chapter 3 develops endogenous in-situ labeling in the blood compartment, establishing the fundamentals of my in-situ approach. Chapter 4 extends this and establishes in-situ pulse labeling (ISPL) to label endogenous leukocytes in peripheral tissues. Chapter 4 then goes on to combine the technological advances and conceptual framework established in the previous chapters to elucidate a role for α1β1integrin in the exit of macrophages from inflamed peripheral tissues. Finally, in Chapter 5 I discuss the implications of my results in the context of the host defense, how it might impact the immune response and future directions for this research.

The present research is focused on the topic of inflammation-drug interaction. Inflammation complicates many human diseases and conditions ranging from obesity to cancer. Therefore, the study of the effect of inflammation on drug pharmacokinetics and pharmacodynamics is pivotal. First, we tested the hypothesis that controlling inflammation using valsartan can restore the previously reported altered verapamil pharmacokinetics and pharmacodynamics. Such an effect is expected due to the anti-inflammatory properties of angiotensin II inhibition. Inflammation resulted in L-type calcium channel target protein (Cav1.2) downregulation and reduced verapamil potency in pre-adjuvant arthritis rat model. Valsartan treatment reversed the observed downregulation of L-type calcium channels thereby enhancing verapamil potency. This beneficial interaction, once proven in humans, may be of value in cardiac patients with superimposing inflammatory diseases. Second, we investigated whether the response to verapamil is reduced in experimentally induced acute myocardial injury (AMI) in rats. AMI caused a 75% reduction in verapamil potency and Cav1.2 target protein downregulation. If extrapolated to humans, our observations may suggest that L-type calcium channel downregulation can contribute, at least in part, to the poor outcome in myocardial infarction patients treated with calcium channel blockers (CCBs). Third, we studied the effect of obesity on the pharmacological response of CCBs in children with renal disease. Our data indicated that obese children are less responsive to CCBs than non-obese ones. Therefore, obesity should be considered when initiating antihypertensive drug therapy in children. Last, we were interested in finding out if the expression of other target genes is also altered by inflammation. We used real time polymerase chain reaction, after determination of the best housekeeping gene to be used as an internal control. Inflammation resulted in significant alterations of several molecular targets and transporters affecting the pharmacokinetics and pharmacodynamics of drugs. These findings may provide an insight into the effect of inflammation on drug targets and modulators of disease pathogenesis. In conclusion, inflammation is a missed ring in the chain of therapy. The research presented in this thesis will add to the inflammation-drug interaction field important findings that will help understanding the role of inflammation in pharmacotherapy outcomes. / pharmaceutical sciences

<p>Includes Bibliographical references (leaves 83- 94).Forty-three male athymic nude mice were implanted with human vaginal mucosal cysts under general anaesthesia with Ketamine [25mg/kg] and Medetomidine [0.5mg/kg]. Cysts in 37 mice were recovered after 9 weeks of growth. twenty three cyst linings had retained the original structure of the vaginal epithelium. No marked deifference was present between the thickness of 9 week old linings and donor vaginal epithelium. The contaminants isolated from the skin of mice before implantation were mainly normal commercals of healthy experimental animals. There was no distinct difference in the number of cases with intact cyst formation between the terramycin/vitamin cocktaik group. The frequency of poor wound healing and/ or murine epidermis ingrowth was three times higher in animals stitched with silk sutures that in those cases where nylon sutures were used.</p>

The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

Alzheimer??s disease (AD) is the most common form of dementia affecting the elderly. Extracellular deposition of beta amyloid (Abeta plaques), intraneuronal tau accumulation, inflammation (activated astrocytes and microglia), and neuronal loss are all consistent pathological features of the disease. Unlike Abeta plaques, inflammation correlates with neuronal loss and cognitive decline in AD, suggesting it plays an important role in disease progression. Recent research has also identified soluble oligomeric Abeta species in the AD brain, which correlate with disease progression and are proposed to be more neurotoxic than their fibrillar counterpart. The main aims of this thesis were to determine whether oligomeric Abeta is a more powerful stimulator of the inflammatory response compared to fibrillar forms of the protein, to identify potential mediators of this response and to determine downstream neuronal changes. Isolated human primary astrocytic, microglial and neuronal cell cultures were used to assess which Abeta alloform and conformation was more neurotoxic and neuroinflammatory. A total of 17 inflammatory cytokines and chemokines were measured simultaneously using a Multiplex approach and neurotoxicity was assessed by lactate dehydrogenase release. Cell cultures involving all three cell types were also used to examine these inflammatory mediators in a more complex system, and direct changes in tau levels and/or phosphorylation determined by western immunoblotting. Further immunohistochemical analysis determined the localisation of oligomeric Abeta within post-mortem human AD brain tissue. The data clearly shows that oligomeric Abeta was more neurotoxic and neuroinflammatory than fibrillar Abeta, with oligomeric Abeta40 favoured in toxic plaque deposits in AD brain tissue. Cell culture experiments showed that glia clearly mediate neuroprotection against oligomeric Abeta. Soluble TNF-alpha, IL-2, IL-4 and IL-12 mediate this response early in disease, with a decline in their secretion and a sustained increase in IL-1alpha, IFN-gamma, GM-CSF, MIP-1beta, and IL-8 provoking increased tau protein expression. Overall, the data clearly demonstrates oligomeric Abeta more powerfully stimulates a suite of inflammatory mediators affecting neuronal survival and tau pathology, although the contribution of additional glial-derived factors must also be determined. The identification of these inflammatory mediators, in combination with other potential factors, may lead to the development of mechanistic therapeutic interventions that could target these early pathological changes.

Alzheimer??s disease (AD) is the most common form of dementia affecting the elderly. Extracellular deposition of beta amyloid (Abeta plaques), intraneuronal tau accumulation, inflammation (activated astrocytes and microglia), and neuronal loss are all consistent pathological features of the disease. Unlike Abeta plaques, inflammation correlates with neuronal loss and cognitive decline in AD, suggesting it plays an important role in disease progression. Recent research has also identified soluble oligomeric Abeta species in the AD brain, which correlate with disease progression and are proposed to be more neurotoxic than their fibrillar counterpart. The main aims of this thesis were to determine whether oligomeric Abeta is a more powerful stimulator of the inflammatory response compared to fibrillar forms of the protein, to identify potential mediators of this response and to determine downstream neuronal changes. Isolated human primary astrocytic, microglial and neuronal cell cultures were used to assess which Abeta alloform and conformation was more neurotoxic and neuroinflammatory. A total of 17 inflammatory cytokines and chemokines were measured simultaneously using a Multiplex approach and neurotoxicity was assessed by lactate dehydrogenase release. Cell cultures involving all three cell types were also used to examine these inflammatory mediators in a more complex system, and direct changes in tau levels and/or phosphorylation determined by western immunoblotting. Further immunohistochemical analysis determined the localisation of oligomeric Abeta within post-mortem human AD brain tissue. The data clearly shows that oligomeric Abeta was more neurotoxic and neuroinflammatory than fibrillar Abeta, with oligomeric Abeta40 favoured in toxic plaque deposits in AD brain tissue. Cell culture experiments showed that glia clearly mediate neuroprotection against oligomeric Abeta. Soluble TNF-alpha, IL-2, IL-4 and IL-12 mediate this response early in disease, with a decline in their secretion and a sustained increase in IL-1alpha, IFN-gamma, GM-CSF, MIP-1beta, and IL-8 provoking increased tau protein expression. Overall, the data clearly demonstrates oligomeric Abeta more powerfully stimulates a suite of inflammatory mediators affecting neuronal survival and tau pathology, although the contribution of additional glial-derived factors must also be determined. The identification of these inflammatory mediators, in combination with other potential factors, may lead to the development of mechanistic therapeutic interventions that could target these early pathological changes.

Thesis (M.S.)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Mary P. Miles. Includes bibliographical references (leaves 69-79).

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.