<p>Cytokines play important roles in bone metabolism, participating in the complex interplay necessary for normal bone formation and turnover. The aim of the present thesis was to investigate the effects of two anti-inflammatory cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13) on bone. </p><p>Influence of pro- and anti-inflammatory cytokines on interleukin-6 (IL-6) formation in cultured human osteoblasts (hOBs) was investigated. IL-4 and IL-13 as well as interleukin-1 (IL-1) and tumour necrosis factor alpha and beta (TNF-α/β) stimulated IL-6 secretion in hOBs. Also, IL-4 and IL-13 synergistically potentiated the effect of IL-1 and TNFs on IL-6 secretion. </p><p>Effects of IL-4 and IL-13 on markers of osteoblastic activity in hOBs were investigated. IL-4 and IL-13 induced a dose-dependent increase in the formation of alkaline phosphatase (ALP) and pro-collagen type I carboxy-peptide (PICP) together with enhanced mineralization rate in hOBs. Formation of osteocalcin (OC) was unaffected. </p><p>The mechanism behind inhibited proliferation by IL-4 and IL-13 in hOBs was investigated. IL-4 and IL-13 caused a dose-dependent increase in DNA-fragmentation together with escalating Caspase-3 activity in hOBs, reflecting induced apoptosis. Osteoblast apoptosis was also confirmed by TNF-α, dexamethasone and by serum starvation.</p><p>The skeletal phenotype of IL-13^-/-, IL-4^-/-IL-13^-/- and WT mice was compared. An altered cortical bone mass was detected in adult male IL-4^-/-IL-13^-/- mice. They displayed a reduction in cortical bone mineral content (BMC) secondary to reduced cortical thickness. Mechanical strength of the cortical bone was reduced in level with the reduction detected in BMC. Trabecular bone mineral density (tvBMD) was unaffected. </p><p>Callus formation in IL-4^-/-IL-13^-/- and WT male mice was compared. No differences were found concerning radiological healing, biomechanical properties, callus parameters or histology. Heterotopic bone formation in IL-4^-/-IL-13^-/- and WT mice was compared using DXBM implants. No differences were found concerning mineralization of implants. Immuno-histology showed inhibition of autonomic nerves and lack of implant vascularization in IL-4^-/-IL-13^-/- mice. </p><p>In summery, the two anti-inflammatory cytokines IL-4 and IL-13 influence osteoblast activity and apoptosis <i>in vitro</i>. They also selectively influence cortical bone formation <i>in vivo</i>. These findings suggest a role for IL-4 and IL-13 in osteoblast differentiation, in bone metabolism and in bone formation. </p>

Cytokines play important roles in bone metabolism, participating in the complex interplay necessary for normal bone formation and turnover. The aim of the present thesis was to investigate the effects of two anti-inflammatory cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13) on bone. Influence of pro- and anti-inflammatory cytokines on interleukin-6 (IL-6) formation in cultured human osteoblasts (hOBs) was investigated. IL-4 and IL-13 as well as interleukin-1 (IL-1) and tumour necrosis factor alpha and beta (TNF-α/β) stimulated IL-6 secretion in hOBs. Also, IL-4 and IL-13 synergistically potentiated the effect of IL-1 and TNFs on IL-6 secretion. Effects of IL-4 and IL-13 on markers of osteoblastic activity in hOBs were investigated. IL-4 and IL-13 induced a dose-dependent increase in the formation of alkaline phosphatase (ALP) and pro-collagen type I carboxy-peptide (PICP) together with enhanced mineralization rate in hOBs. Formation of osteocalcin (OC) was unaffected. The mechanism behind inhibited proliferation by IL-4 and IL-13 in hOBs was investigated. IL-4 and IL-13 caused a dose-dependent increase in DNA-fragmentation together with escalating Caspase-3 activity in hOBs, reflecting induced apoptosis. Osteoblast apoptosis was also confirmed by TNF-α, dexamethasone and by serum starvation. The skeletal phenotype of IL-13-/-, IL-4-/-IL-13-/- and WT mice was compared. An altered cortical bone mass was detected in adult male IL-4-/-IL-13-/- mice. They displayed a reduction in cortical bone mineral content (BMC) secondary to reduced cortical thickness. Mechanical strength of the cortical bone was reduced in level with the reduction detected in BMC. Trabecular bone mineral density (tvBMD) was unaffected. Callus formation in IL-4-/-IL-13-/- and WT male mice was compared. No differences were found concerning radiological healing, biomechanical properties, callus parameters or histology. Heterotopic bone formation in IL-4-/-IL-13-/- and WT mice was compared using DXBM implants. No differences were found concerning mineralization of implants. Immuno-histology showed inhibition of autonomic nerves and lack of implant vascularization in IL-4-/-IL-13-/- mice. In summery, the two anti-inflammatory cytokines IL-4 and IL-13 influence osteoblast activity and apoptosis in vitro. They also selectively influence cortical bone formation in vivo. These findings suggest a role for IL-4 and IL-13 in osteoblast differentiation, in bone metabolism and in bone formation.

Anaplastic Lymphoma Kinase (ALK) was discovered in 1994, as a chromosomal translocation, t(2;5)(p23;q35), often seen in Anaplastic Large Cell Lymphomas (ALCL). Since then ALK has been extensively studied in this disease as well as in different model organisms. Due to its expression pattern within the central and peripheral nervous system ALK has been implicated in neuronal development. This hypothesis has been further strengthened by studies from Drosophila which have shown Alk to have an important role in optic lobe development. A recently described ALK mouse knockout model do not indicate an essential role for ALK in development, although a potential role within the central nervous system was strengthened. This since ALK-/- animals has an increased number of progenitor cells in the hippocampus and display altered behavior. The overall aim of the studies included in this thesis was to elucidate the function of ALK in the mouse. As a first step toward this goal we conducted an analysis of ALK mRNA and protein expression patterns during development. The strong expression of ALK in neuronal structures supports a role for ALK in neuronal development during embryogenesis. To further investigate the function of ALK in a physiological context we have developed two different ALK knockout strains, the ALK Kinase knockout (KO) and the ALK exon1 KO. The only visible phenotype in these strains is a reduction of total body weight which is apparent in the ALK-/- population when compared to wild type littermates. This size difference seems to take place after birth and is not due to an alteration in food consumption. We have also extensively studied the ALK Kinase KO with respect to gross development, the gastrointestinal canal and the olfactory system. ALK displays a very distinct expression pattern within the gastrointestinal canal being confined to enteric neuron precursors during embryogenesis and enteric nerves in the adult tissue. From these studies we conclude that ALK is not needed for development and viability in mice although it does play a role in regulation of body weight via a presently unknown mechanism. In addition, we have investigated the relationship between the Drosophila and mouse ALK receptor by examining the ability of the Drosophila Alk ligand Jelly-Belly, Jeb, to activate mouse ALK. Using different in vivo and in vitro techniques, we have shown that activation of mouse ALK cannot be accomplished by Drosophila Jeb. From this study we draw the conclusion that during development ligands for the Drosophila and mouse ALK has diverged to a level at which they can no longer substitute for each other.

Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis. The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice). Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels. Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates. Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.

mPIP is a mouse homologue of human PIP/GCDFP-15 which is an established marker of both malignant and benign pathological conditions of the mammary gland. mPIP gene expression has been identified in both lacrimal and salivary glands of healthy mice and the mPIP protein has been detected in saliva. The mPIP protein has been found to bind oral bacteria, showing the highest affinity for streptococci, suggesting a potential function of mPIP in the non-immune host defense in the mouse oral cavity. Since the exact functions of mPIP are still unknown, we examined the roles of mPIP through both in vitro and in vivo studies, specifically to address the possible role of this protein in non-immune host response through modulating the oral flora. The in vitro studies were primarily focused on elucidation of the consequences of interaction between mPIP and oral bacteria, in particular to examine whether mPIP plays a role in bacterial aggregation. The in vivo studies addressed the roles of mPIP through the analysis of an mPIP knockout mouse model generated in our laboratory. Following confirmation of the null mutation, the delineating the phenotype of this model was pursued through morphopathological analysis as well as examination of the impact of the lack of mPIP on the mouse oral flora. The null mutation in the mPIP knockout mice was confirmed by both the gene and protein analysis. Histological analysis revealed lymphocytic proliferation in both the submaxillary and prostate glands of the mPIP knockout mice. In addition, both quantitative and composition differences in the oral flora of mPIP knockout mice were identified when compared with wild-type controls. Specifically, a higher proportion of the oral bacteria of mPIP knockout mice were found to belong to genus Streptococcus and certain genera were found to be absent from the oral cavity of these mice. The effect of knockout mouse saliva, which lacks mPIP, on the aggregation of oral bacteria was compared to wild-type mouse saliva. Our data suggests that mPIP contributes to saliva-induced bacterial aggregation. While oral flora has multiple functions, including protection against infection, mPIP might play a role in the non-innate host defense through modulating the resident oral flora in the mouse. The identification of lymphocytic proliferation in submaxillary and prostate glands of mPIP knockout mice suggests that mPIP might also interfere with lymphocyte activity, playing a possible immunomodulatory role. / May 2008

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 261-288).

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Knockdown of the M5 acetylcholine muscarinic receptor in the ventral tegmental area (VTA) reduces brain-stimulation reward sensitivity in rats. Knockout (KO) of the M5 receptor in mice reduces morphine-induced dopamine efflux, locomotion, conditionedplace- preference, and mating-induced 30-110 kHz ultrasonic vocalizations (USVs). The GFP-labeled M5 receptor gene was transfected using a Herpes simplex virus either into the VTA or 0.2-0.7 mm posterior in the medial tegmentum (MT) of male M5 KO mice. HSV-M5-GFP transfection in VTA fully restored mating-induced USVs and augmented morphine-induced locomotion and stereotypy consistent with activation of DA neurons by M5 receptors. HSV-M5-GFP transfection sites in the MT inhibited USVs and morphine-induced locomotion presumably through inhibition of DA neurons. Putative transfection of M5 in GABA neurons of the rostromedial tegmental nucleus (RMTg) or 5HT neurons of the median raphe (mR) may explain this inhibition. Therefore, HSV-M5- GFP transfection in the VTA enhances DA-mediated behaviours while MT transfections inhibits these behaviours.