<p>Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of <u>gr</u>anule <u>s</u>erine <u>p</u>rote<u>ases</u>, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. </p><p>In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an <i>in vivo</i> substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.</p>

Understanding energetic potential of habitat patches is important for management designed to provide adequate habitat for wildlife species. Great Smoky Mountains National Park (GSMNP) has a high density of black bears that have been studied intensively from 1968-1997; habitats within the Park are relatively undisturbed, and similar vegetative cover types can be found throughout the southern Appalachian mountains. Black bear reproduction in the Park has been correlated to hard mast production, however little work has been done to assess the importance of soft mast. Geographic Information System (GIS) based habitat use models have been developed for bears in the Park, yet the importance of foods in determining habitat selection, and the possibility of sexual habitat segregation due to food availability have not yet been determined. The primary objectives of the study included estimation of the location, timing, and amount of caloric production by 19 important black bear foods and determination of the significance of caloric production by mast type, season, overstory vegetation type, and plant species. Secondary objectives were to test for correlation of bear habitat use with estimated caloric production from mast, and to test for sexual segregation of habitats based on caloric production. This study was limited to the northwest quadrant of GSMNP during 1995.

The airway epithelium plays a crucial role in protecting the underlying connective tissue (CT) from noxious agents. Damage and shedding of the epithelium are observed in the airways of asthma, cystic fibrosis and rhinitis patients. The aim of this study was to investigate the mechanisms by which epithelial damage occurs, and the consequences of such damage for the inflammatory process in the airways. In this study, cultured normal human bronchial epithelial cells, excised rat tracheae, and cultured murine mast cells were used as model systems. Metabolic alterations, morphological changes and cell-cell contact stabilities were investigated. The T-helper (Th)-1 cytokines, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin (IL)-1β were found to be pro-inflammatory, leading to major morphological changes, inhibitions in desmosome formation, and accelerated cell death. The Th2 cytokines, IL-4, IL-5, and IL-13 were found to cause no changes in cell death, nitric oxide levels and desmosome formation but instead an increase in proliferation, therefore were anti-inflammatory in this respect. Increasing the osmolarity of the airway surface liquid (ASL) altered the integrity of the tight junction (TJ) and allowed a 4-kDa compound to penetrate the epithelial layer and access the CT. This effect was reversible if the ASL was returned to 295 mOsm. Intentionally breaking the TJ with EGTA and subsequent osmolar changes in ASL demonstrated the importance of TJ and the fragility of the CT under hyperosmolar stress, leading to a disrupted CT with larger capillaries and altered elemental ion content and epithelial denudation. Hydrocortisone was shown to downregulate IL-4-induced IL-6 upregulation in murine mast cells. Furthermore, incubating mast cells with hydrocortisone lead to a new subpopulation that was morphologically unique, that displayed new cell surface markers (CD44 and CD61) and that lacked CD54. These changes modify the interactions of mast cells with surrounding cells in the CT and epithelium. In conclusion, the balance between pro- and anti-inflammatory cytokines and ASL osmolarity may influence the role of the airway epithelium as a barrier. The pharmacological use of hyperosmolarity to disrupt TJ reversibly may help facilitate the delivery of drugs through the airway epithelium.

Mast cells are very potent multifunctional effector cells of the immune system normally distributed throughout connective tissues. An accumulation of mast cells has been described in several pathological conditions such as allergic- and autoimmune inflammations and in certain tumours. This necessitates two different processes: 1) Recruitment of mast cell progenitors from peripheral blood; 2) Accretion of mature mast cells at sites of inflammation and tumour areas. Both processes are depending on the local production of chemotactic factors. The aim of this study was to investigate the role of chemokines and their corresponding receptors in mast cell chemotaxis. By cloning and mRNA-screening of cord blood derived mast cells several chemokine receptors were found to be expressed. Functional expression was confirmed of chemokine receptors CXCR4, CCR1 and CCR4. CXCL12, the only known ligand for CXCR4, acted as a mast cell chemotaxin and induced migration of progenitor cells with capacity to differentiate into mast cells. Of several ligands known to bind CCR1 and CCR4, only CCL5 induced migration of mast cells. The migration to CCL5 was mediated through both CCR1 and CCR4. In contrast, the ligands to CCR4, CCL17 and CCL22, could inhibit CCL5-induced migration. Expression of CCR1 and CCR4 could also be confirmed on mast cells in lung from asthmatic patients. Furthermore, we could demonstrate that mast cells were attracted by CCL5 produced by tumour cells in Hodgkin´s lymphoma. In conclusion, the work in this thesis has identified two chemokines that regulates mast cell migration. This knowledge helps us understand the mechanisms behind homing of mast cell progenitors from the blood into the tissue and the accumulation of mature mast cells at sites of inflammation and tumourigenesis.

It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease. The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (&lt;6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma.

Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of <u>gr</u>anule <u>s</u>erine <u>p</u>rote<u>ases</u>, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an in vivo substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.

Mast cells (MC) are potent inflammatory cells that are known primarily for their prominent role in IgE mediated allergies. However, they also provide beneficial functions to the host, e.g. in bacterial and parasitic defence. MCs react rapidly upon stimulation by releasing potent granule-stored mediators, and serine proteases of the chymase or tryptase families are such major granule constituents. As a first step towards a better understanding of the biological function of these proteases, we have determined the extended cleavage specificities of four mammalian mast cell chymases, by utilizing a substrate phage display approach. The specificities of these enzymes have then been used to compare their functional characteristics. The major mucosal MC chymase in mice, mMCP-1, was found to possess a strict preference in four amino acid positions of the peptide substrate. Using this sequence to search the mouse proteome for potential in vivo substrates led to the identification of several very interesting potential novel substrates. Some of them may explain the increased epithelial permeability provided by this enzyme. Human MCs, express only one single α-chymase, and the rodent α-chymases have secondarily gained elastase-like primary cleavage specificity. However, rodents express additional chymases, the β-chymases, and rodent β-chymases may have adopted the function of the α-chymases. The cleavage specificities of the human chymase and two rodent β-chymases were therefore determined (rat rMCP-1 and mouse mMCP-4). N-terminal of the cleaved bond the three chymases showed similar preferences, but C-terminal the human chymase and mMCP-4 shared a high preference for acidic amino acids in the P2´ position and therefore seem to be functional homologues. The molecular interactions mediating the preference for acidic amino acids in position P2´ were further investigated. By site-directed mutagenesis of the human chymase, amino acids Arg143 and Lys192 were concluded to synergistically mediate this preference. Our data show that chymases, of different MC subpopulations, display quite different extended cleavage specificities. However mouse do possess a MC chymase with almost identical cleavage specificity as the human MC chymase indicating a strong evolutionary pressure to maintain this enzyme specificity.

Anandamide (AEA) is an endogenous fatty acid which activates the same cannabinoid receptors as ∆9-tetrahydrocannabinol, the psychoactive substance in marijuana. In vivo, anandamide exerts a number of actions including effects upon pain and inflammation. However, AEA has a short duration of action since it is rapidly metabolised, primarily by the intracellular enzyme fatty acid amide hydrolase (FAAH). The general aim of this thesis has been to identify and characterize compounds capable of preventing the metabolism of anandamide. The chemical approach was based on the endogenous anti-inflammatory compound palmitoylethanolamide (PEA), a compound related to anandamide with the ability to inhibit anandamide degradation by substrate competition, but without the ability to directly activate cannabinoid receptors. A number of compounds were identified as inhibitors of rat brain FAAH in the initial in vitro studies, without having major affinity for the cannabinoid receptors. In particular, palmitoylisopropylamide (PIA) was found to reduce the metabolism of AEA in intact C6 glioma cells with potency similar to the prototypical AEA reuptake inhibitor AM404. This compound was in addition found to exert less effect upon C6 glioma cell proliferation than either AM404 or the closely related uptake inhibitor VDM11. To evaluate if PIA was effective in vivo, a model of mast cell dependent inflammation, oedema of the ear following local injection of compound 48/80, was set up using anaesthetised mice. Initially, a CB2 cannabinoid receptor selective agonist was used to probe the model and demonstrated to produce an anti-oedema effect. In contrast, the compound was inactive in vitro in skin slice preparations. PIA showed a similar pattern, although there was a large variation in responses which affected the significance of the result obtained, as did the vehicle used to dissolve the compound. Taken together, the present data would suggest that PIA can inhibit the degradation of AEA without having deleterious effects upon cell proliferation or affinity for the cannabinoid receptors. Further experimentation is necessary to elucidate the usefulness of this compound in vivo.

Background. Recent studies in cancer research have focused on the reciprocal interaction between cancer cells and their microenvironment. Tumour growth is angiogenesis dependent and the rate of angiogenesis correlates with a poor prognosis in many different cancers. We have shown that the rate of angiogenesis correlates with prognosis in Prostate Cancer (PC). We have also observed that the vasculature is involved during the involution of the prostate in rodents subsequent to hormonal ablation. Patients with metastatic PC are subjected to hormonal ablation therapy – a therapy unfortunately not curative. Our ambition is therefore to find means to enhance the effects of castration therapy of prostate tumours, possibly by a simultaneous inhibition of angiogenesis and of growth factors populating the tumour stroma. The angiopoietins are a family of growth factors that regulate angiogenesis by direct effects on endothelial cells in a context dependent manner. The purpose of this thesis was therefore to examine the role of the angiopoietins and the stroma in general in PC and to explore their potential as novel targets. Materials and Methods. We have had at our disposal access to clinical materials in the form of paraffin embedded samples from untreated PC patients with a long follow up. We have also used animal tumour models and in vitro cell culture systems followed by immunohistochemistry, in situ hybridization, western blotting, laser micro dissection, and quantitative real-time PCR for evaluation of the experiments. Results. In paper I, we found a significant correlation between high levels of angiopoietin 2 (Ang 2) and high vascular density, histological grade, metastases and poor prognosis in PC patients. In the second paper we found that the receptor for the angiopoietins, Tie 2, and the ligand Ang 1 mediated the decrease in vascular stability observed after castration treatment. This was not observed in prostate tumours subsequent to hormonal ablation (paper III), nor was there a decrease of other growth factor receptors. In summary (paper III), we found that a combined inhibition of the tumour stroma in terms of an inhibition of the PDGF-Rs by the use of Imatinib, and the vasculature in terms of a perturbed Tie 2 signalling, inhibited tumour growth. Finally, in paper IV, we found that Imatinib inhibited the castration induced influx of mast cells after castration therapy. The mast cells expressed high levels of FGF 2 and epiregulin, and inhibition of mast cell function inhibited tumour growth, by inhibiting angiogenesis. Conclusions. We have observed that the tumour stroma is of particular importance for tumour growth in PC. Targeting the tumour microenvironment, and in particular by a simultaneous inhibition of the vasculature and stroma, could prove beneficial for patients with advanced PC.

Flexor tendon injuries in zone II of the hand (i.e. between the distal volar crease and the distal interphalangeal joint) can be costly for both the afflicted individual and society because of the high cost of a long rehabilitation period, complicated by tendon ruptures or scarring with adhesion formation, causing impaired range of motion. The aim of the present thesis was to characterize more fully the deep flexor tendon, the tendon sheath and their response to injury in a rabbit model in order to find potential targets to improve the outcome of repair. The intrasynovial rabbit deep flexor tendon differed from the extrasynovial peroneus tendon in the expression of collagens and transforming growth factor-β1 gene expression. Differences were also found in collagen III and proteoglycans between regions of the flexor tendon subjected to either compressive or tensile load. After laceration and subsequent repair of the flexor tendon, a shift in collagen gene expression from type I to type III occurred. Proteoglycans were generally increased with the notable exception of decorin, a potential inhibitor of the profibrotic transforming growth factor-β1 which was markedly increased during the first two weeks after repair in tendon tissue but remained unaltered in the sheaths. Both vascular endothelial growth factor and basic fibroblast growth factor mRNA levels remained essentially unaltered, whereas insulin-like growth factor-1 increased later in the healing process, suggesting potential beneficial effects of exogenous addition, increasing tendon strength through stimulating tenocyte proliferation and collagen synthesis. Matrix metalloproteinase-13 mRNA levels increased and remained high in both tendon and sheath, whereas there was only a transient increase of matrix metalloproteinase-3 mRNA in tendon. We could also demonstrate a significant increase of the proportion of myofibroblasts, mast cells and neuropeptide containing nerve fibers in the healing tendon tissue, all components of the profibrotic myofibroblast-mast cell-neuropeptide pathway. / Biomolecular aspects of flexor tendon healing