Introdução: Doenças pulmonares ou infecções que ocorrem no início da vida podem ter permanente impacto na vida adulta. Pouco se sabe sobre o perfil de células do sistema imunológico em pulmões de crianças lactentes. Objetivo: Descrever o perfil de células do sistema imunológico no pulmão de lactentes sem doença pulmonar. Métodos: Amostras de pulmões histologicamente normais, obtidas através de autópsia de dez crianças que morreram de causas acidentais ou de doenças não pulmonares, foram marcadas por anticorpos contra linfócitos B e T, macrófagos, células NK (natural Killer), células citotóxicas, células dendríticas e mastócitos. As células foram quantificadas no epitélio, na camada interna, na camada externa das vias aéreas e nos septos alveolares. Membrana basal e septos alveolares foram medidos através de análise de imagem. Resultados expressos em células/mm de membrana basal epitelial brônquica ou alveolar. Resultados: A mediana das idades foi 2,5 meses (1-730 dias). Os resultados mostraram que a camada interna apresentou pequena densidade celular. No epitélio da via aérea e no parênquima houve predominância de células que estão relacionadas com a imunidade inata, tais como: CD56+, Granzyme + e CD68+. A camada externa e o parênquima alveolar apresentaram a maior densidade celular. Poucas células T CD4+ e células dendríticas foram encontradas na maioria dos compartimentos do pulmão. Conclusão: Há uma compartimentalização de células relacionadas com o sistema imunológico ao longo da via aérea e parênquima dos pulmões das crianças estudadas. Esta configuração pode estar relacionada com o desenvolvimento dos mecanismos de defesa da imunidade inata e da imunidade adquirida. Este conhecimento é importante para entender os mecanismos da imunocompetência pós-natal dos pulmões / Introduction: Pulmonary diseases or infections occurring early in life may have a permanent impact in adulthood. Little is known about the normal immune cell profile in the lungs of infants. Objective: To describe the immune cell profile of infants without lung disease. Methods: Histologically normal lung samples obtained at autopsy of ten infants that died either due to incidental or inflicted causes or non-pulmonary diseases were stained for antibodies against B and T lymphocytes, macrophages, NK cells, cytotoxic cells, dendritic cells and mast cells. Cells were quantified in the airway epithelial layer, inner layer, outer layer and alveolar septa. Basement membrane or alveolar septa lengths were assessed by image analysis. Results are expressed as cells/mm. Results: The median age of patients was 2.5 months and ranged from 1- 730 days. The inner layer of the airways was the region with the smallest density of cells. There was a predominance of cells related to the innate immunity such as CD56+, Granzyme B+ and CD68+ cells in the epithelial layer and alveolar parenchyma. The outer layer and the lung parenchyma presented the highest cellular density. There were very few CD4+ T cells or dendritic cells in most of the lung compartments. Conclusions: There was a compartmentalization of immune cells along airways and parenchyma in infants, which may be related to the development of innate and acquired lung defense mechanisms. This knowledge is important to understand mechanisms of postnatal immune competence of the lungs

The metabolic pathways required for adenosine triphosphate (ATP) production within the cell are well understood, however recent publications suggest that metabolic pathways are closely linked to immune cell activation and inflammatory diseases. There has been little examination of the metabolic pathways that modulate mast cell activation and the feedback regulator lactic acid. Here we examine metabolic pathways and regulation within mast cells in the context of lipopolysaccharide (LPS) and interleukin (IL-33) activation, for which there has been little to no reported studies. First, we examine the effects of lactic acid, previously considered only a by-product of glycolysis and now understood to act as a negative feedback regulator of inflammation in the context of LPS activation and sepsis. Lactic acid is elevated in septic patients and associated with mortality, potentially due to suppressive effects on LPS signaling and contribution to late phase immunosuppression. By attenuating glycolysis and reducing ATP availability for signaling and cytokine transcription, lactic acid impairs the function of immune cells to fight the initial or subsequent infections. We support this with in vitro and in vivo data. Additionally, our lab has published that lactic acid can suppress IL-33 activation, potentially by metabolic modulation as with LPS activation; however there has been no study of the metabolic requirements for IL-33 activation. We report here that glycolysis is required for ATP and reactive oxygen species (ROS) production to augment signaling and cytokine production downstream of the IL-33 receptor. Together, these studies examine the contribution of metabolism to mast cell activation and may provide potential targets for treatments of diseases that involve LPS- or IL-33-dependent mast cell activation.

Myeloid Derived Suppressor Cells (MDSC) represent a significant hurdle to cancer immunotherapy because they dampen anti-tumor cytotoxic T cell responses. Previous studies have reported on the myelo-depletive effects of certain chemotherapies. Using guadecitabine, a next-generation DNA methyltransferase inhibitor (DNMTi), we observed significantly reduced tumor burden in the 4T1 murine mammary carcinoma model. Guadecitabine treatment prevents excessive tumor-induced myeloid proliferation and systemic accumulation, and skews remaining MDSCs toward a beneficial antigen-presenting phenotype. Together, this alters the splenic environment to improve T cell activation and interferon-gamma (IFNg) production. Additionally, guadecitabine enhances the therapeutic effect of adoptively transferred antigen-experienced lymphocytes to diminish tumor growth and improve overall survival. Based on these findings, the immune-modulatory effects of guadecitabine can help rescue the anti-tumor immune response and could contribute to the overall effectiveness of current cancer immunotherapies. Allergies and asthma are common ailments that are on the rise around the world. Mast cells play a direct role in the signs and symptoms characteristic in allergic patients. The family of A Disintegrin And Metalloproteinases (ADAMs) are involved in regulating many cellular processes by cleaving surface receptors, ligands, and signaling molecules. We sought to determine the role of ADAM17 in mast cell activity. In studies using ADAM17-deficient mast cells, percent degranulation and cytokines released by IgE-mediated activation were significantly reduced. Interestingly, ionomycin-activation was unchanged, suggesting ADAM17 may be involved in IgE-mediated mast cell activation upstream of calcium release. Additionally, ADAM17MC-/- mice showed protection from IgE-, but not histamine-, mediated passive systemic anaphylaxis (PSA). The underlying mechanism behind the reduced degranulation occurs through signaling deficiencies downstream of Lyn phosphorylation. Together, the data suggest that ADAM17 is required for proper mast cell signaling through its interaction with the Src family kinase, Lyn.

Mast cells are tissue-resident immune cells known as effector cells for the innate and adaptive immune systems. Mast cells contribute to host defenses against parasites such as large roundworm parasites, bacterial pathogens, and toxins, and participate in wound healing, but they are mostly known for their role in allergic diseases. It has been well established that during allergic diseases, mast cells are stimulated by IgE cross-linkage to release proinflammatory mediators. However, a newly discovered cytokine, IL-33 has also been implicated in allergic disease. Recently, IL-33 has been implicated as a driver of several Type I sensitivities and previous studies have shown that IL-33 can stimulate mast cells in atopic inflammation. Although the importance of IL-33 has been established, there are still several things unknown about IL-33 signaling regulation or treatment. This dissertation will present two separate studies involving the modulation of IL-33-mediated mast cells function In the first study, the effects of fluvastatin are explored. In a previous study, fluvastatin was shown to inhibit proinflammatory functions of IgE crosslinked mast cells. Contrasting to IgE stimulation, fluvastatin augments IL-6 and TNF production in IL-33 stimulated mast cells, but suppressed MCP-1. This phenomenon was seen in mouse and human mast cells in vitro and replicated in a mast cell-dependent murine model of IL-33-induced inflammation in vivo. In the second study, IL-33 was found to induce miR-146a expression in mouse mast cells and mast cell-derived exosomes in vitro, and in plasma exosomes in vivo. IL-33 induced miR-146a was of interest because miR-146a is a known negative regulator of TLR signaling, which shares the MyD88 signaling pathway with IL-33. We found that miR-146a KO mast cells are hyperresponsive to IL-33 stimulation, data that were replicated by suppressing miR-146a-5p in WT mast cells. In an acute mast cell repopulation model, kitW-sh/W-sh mice containing miR-146a KO BMMC had increased IL-33 induced neutrophilia in comparison to their controls. Collectively, these data reveal new IL-33 signaling pathways and means of altering its inflammatory effects on mast cells. Because IL-33 has important roles in allergy and other Th2-mediated diseases, these results advance clinically relevant areas of immunology.

The development of microarray technology has provided an unprecedented wealth of data on gene expression in various tissue and cell types. Few studies have, however, taken full advantage of these data by selecting and then extensively characterising the functions of particular genes chosen from these microarray datasets. In this study, after analysing differentially-regulated genes revealed by microarray analysis of human mast cells activated via Fc??RI cross-linking, we chose two promising gene candidates for further research, A20 and Gem. Our group??s extensive gene expression database of major leukocytes showed that both A20 and Gem were up-regulated in other leukocyte types, and yet neither of these genes has been extensively explored in mast cells or in the immune system prior to our study. In order to investigate the first of these genes selected for further study, A20, we utilised both A20-deficient mast cells and mast cells in which A20 was over-expressed. Our findings establish for the first time that A20 is an important regulator of mast cell inflammatory responses to both LPS and Fc??RI cross-linking, and that it plays a novel role in mast cell proliferation. Our study of the second gene chosen for investigation, Gem, was conducted in a Gemdeficient mouse model developed by our group. In this study, we investigated the effect of Gem deficiency in two key immune cell types, macrophages and T-cells, complementing the work of a previous group member who investigated Gem deficiency in mast cells. Our results clearly exclude a role for Gem in macrophage and T-cell effector responses, and further establish that Gem is dispensable for in vivo inflammatory responses in models of delayed-type hypersensitivity and allergic airway inflammation. In addition to these findings, and given that the physiological role of Gem was not yet understood prior to our study, we extended our investigation to explore a potential function for Gem in the metabolic system. Using Gem-deficient mice, we found that Gem is necessary for insulin secretion from pancreatic islets. These findings confirm the potential for microarray expression data to reveal excellent gene candidates for further research and functional characterisation.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 199-227).

<p>När rumsligt omvälvande projekt skall genomföras är det ofta konsekvenserna för naturmiljö, kulturmiljö, estetiska miljöaspekter som ger upphov till diskussioner. Uppförandet av telemaster till mobiltelefonsystemet 3G är ett infrastrukturprojekt som kommer att vara visuellt påtagliga på många platser i landskapet. I Sverige uppförs två stycken parallella mastsystem och detta har givit upphov till diskussioner kring projektets påverkan på vår omgivning. Uppsatsen studerar hur miljöpåverkan från ett stort infrastrukturprojekt, uppförandet av telemaster för 3G-systemet, behandlats av länsstyrelsen i samrådsärenden enligt 12 kapitlet 6§ miljöbalken, samt diskuterar kring hur handläggare på länsstyrelsen hanterar och upplever dessa ärenden. </p><p>Studien visar att handläggare på de undersökta länsstyrelserna har begränsat mandat att minska antalet master genom samlokalisering. Önskemålen om att teleoperatörerna ska dela på själva masten är svåra att genomföra eftersom operatörerna inte har samverkansavtal. Laglig rätt att förplikta samlokalisering fanns inte under den studerade perioden. Det är vanskligt att säga om den sammantagna inverkan som masterna sägs ha kunnat begränsas på denna nivå. Det som identifierats som ett problem av aktörer som Boverket och PTS, d.v.s. antalet master, fastlades redan i ett tidigare skede. </p><p>Samråden är inriktade på att undvika ur naturmiljösynpunkt olämpliga mastplaceringar. Dock utgör den fastslagna utbyggnaden av nätet och kravet på geografisk täckning att det kan uttryckas som att det för länsstyrelsen är inte en fråga om i fall masterna ska uppföras, utan om var. Lokaliseringen av masterna blir ett samspel mellan var operatörerna föredrar att placera master samt var det är lämpligt ut naturmiljösynpunkt. Den på nationell nivå fastlagda geografiska täckningskravet gör att bedömningen av masters påverkan på vissa landskap, där intrycket i landskapsbilden blir stort, upplevs svår av de intervjuade handläggarna. Bedömning av masters påverkan på just landskapsbilden kan upplevas som subjektiv. </p><p>Det stora antalet anmälningar om samråd har resulterat i få förelägganden och förbud. MKB har endast använts i ett litet antal fall. Samråden synes mycket inriktade på att hitta alternativa lösningar innan förbud utfärdas. Landskapsbilden är den enskilda motivering till förbud som förekommer flest gånger.</p>

<p>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.</p><p>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.</p><p>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. </p><p>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.</p><p>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.</p>

<p>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. </p><p>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.</p><p>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.</p>

<p>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.</p><p>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 (<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. </p>