Orientador: Sandra Helena Penha de Oliveira / Banca: Lúcia Helena Faccioli / Banca: Carla Máximo Prado / Resumo: O objetivo deste estudo foi avaliar o mecanismo envolvido na produção de TGF-β, FGF-2 e CCL3/MIP-1α induzida por Stem cell factor (SCF) em células da musculatura lisa de traqueia (CMLT) e as vias de transdução de sinalização ativadas. Traqueias de camundongos normais foram coletadas, fragmentadas e colocadas em garrafas contendo meio de cultura DMEM com 10% de Soro Fetal Bovino. CMLT foram estimuladas com SCF (1, 10 and 100 ng/mL) e avaliadas após 1, 6 e 24 horas. Características fenotípicas das CMLT foram analisadas por imunofluorescência para α-actina de músculo liso (α-AML), α-citoqueratina e α-proteína de ativação de fibroblastos (α-FAP). Ativação de c-kit em CMLT estimuladas por SCF foi avaliada por citometria de fluxo. A expressão de RNAm para TGF-β foi observada pela reação de polimerase em cadeia-transcriptase reversa (RT-PCR) e a produção da proteína, por ensaio de imunoabsorção enzimática (ELISA). A produção de FGF-2 foi avaliada por immunoblot e a produção de CCL3/MIP-1α por ELISA. Em outro conjunto de experimentos, CMLT foram pré-tratadas com inibidores de MAPK p42/44(PD 98059 [PD]), p38(SB 202190[SB]), e JNK (SP 600125 [SP]) por 30 minutos seguidos de estimulação com SCF (10 ng/mL) por 24 horas. Células pré-tratadas com anticorpos específicos não revelaram qualquer marcação para citoqueratina nem para α-FAP, contudo, ocorrendo a marcação para α-AML, indicando a pureza da linhagem celular primária. SCF induziu a expressão de receptores c-kit em CMLT. CMLT estimuladas por 10 ng/mL de SCF expressaram TGF-β mRNA e produziram TGF- β proteína, FGF-2 e CCL3/MIP-1α após 24 horas. O pré-tratamento com SB, PD e SP inibiu estas produções. Estas produções foram mediadas pela ativação das vias p42/44, p38, e JNK. CMLT parecem ser importantes células residentes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the mechanism involved in SCF-induced TGF-β, FGF-2 and CCL3/MIP-1α production in tracheal smooth muscle cells (tSMC) and the activated signaling transduction pathway. Normal mouse tracheas were collected, fragmented and placed in bottles containing the culture medium DMEM with 10% Fetal Bovine Serum. tSMC primary cultures were stimulated with SCF (1, 10 and 100 ng/mL) and evaluated at 1, 6 and 24 hours. The phenotypic characteristic of tSMC in primary culture was analyzed using immunofluorescence staining for α-smooth muscle actin (α-SMA), α-cytokeratin and α-Fibroblast Activation Protein (α-FAP). c-Kit activation in SCF-stimulated tSMC was evaluated by flow cytometric. The TGF-β mRNA expression was observed by reverse-transcriptase polymerase chain reaction (RT-PCR) and protein production by enzyme-linked immunosorbent assay (ELISA). FGF-2 production was evaluated by immunoblot and CCL3/MIP-1α production by ELISA. In other set of experiment, tSMC were pretreated p42/44 inhibitor (PD 98059 [PD]), p38 inhibitor (SB 202190[SB]), or JNK inhibitor (SP 600125 [SP]) for 30 minutes followed by stimulation with SCF (10 ng/mL) for 24 hour. Cells treated with specific antibodies, showing neither labeling for cytokeratin nor either FAP, however, labeling for α-SMA indicating purity of the primary cell line. SCF induces c-Kit expression on tSMC. SCF-stimulated tSMC express TGF-β mRNA expression and FGF-2 and CCL3/MIP-1α production at 10 ng/mL after 24 hours. SB, PD and SP pre-treatment inhibited these productions. These productions were mediated by activation pathways p42/44, p38, and JNK. tSMC seems to be important resident cells involved in the cell activation and tissue repair, since they are capable of producing growth factors and chemokines and added new... (Complete abstract click electronic access below) / Mestre

Mastzellen sind ubiquitär im Bindegewebe vorkommende Zellen, welche eine Vielzahl von Mediatoren physiologischer und pathologischer Prozesse exprimieren. Ihre Zahl ist in gesunden Geweben gering, am höchsten jedoch in der Haut und Schleimhäuten von Nase, Auge und Gastrointestinaltrakt sowie in der Lunge. Die Ergebnisse unserer Arbeitsgruppe zeigen, dass unter pathologischen Bedingungen, insbesondere bei entzündlichen Reaktionen und Erkrankungen des atopischen Formenkreises, die Mastzellzahlen um ein Vielfaches steigen. Dabei werden die Vorläuferzellen des Blutes, welche aus einer CD34+/c-Kit+ hämatopoetischen Stammzelle des Knochenmarkes rekrutiert werden, aktiviert und durch chemotaktisch wirkende Faktoren zur Einwanderung in die Gewebe stimuliert. Unter Verwendung von in vitro Kulturmodellen konnte gezeigt werden, dass diese Vorläuferzellen im Blut die Expression von c-Kit und CD34 herunterregulieren und als Zellpool im peripheren Blut zirkulieren. Nach Aktivierung der Zellen wurde c-Kit wieder nachgewiesen. Die Zellen wandern ins Gewebe ein und differenzieren dort unter Einfluss von Zytokinen, welche durch andere Gewebszellen freigesetzt werden, aus. Es wurden Wechselwirkungen in der Haut zwischen Mastzellen und Fibroblasten, Melanozyten, Keratinozyten und Nervenzellen gezeigt. Als Mittler dieser Wechselwirkungen konnten, neben den in der Literatur beschriebenen Faktoren, von uns SCF, GM-CSF, NGF und andere Neurotrophine zum Beispiel BDNF, NT-3 und NT-4 gezeigt werden. Die Regulation und Freisetzung dieser Faktoren ist bei pathologisch veränderter Haut, wie bei Atopischer Dermatitis, Psoriasis, Haarwachstumsstörungen, Tumoren der Haut und in der Wundheilung verändert. Die Modulation der Expression dieser Faktoren und ihrer Rezeptoren durch verschiedene Therapeutika, wie Glukokortikoide, Antihistaminika, Retinoide und UV-Bestrahlung konnte in verschiedenen Kulturmodellen gezeigt werden. Diese Erkenntnisse können in Zukunft bei der Entwicklung neuer Therapeutika zur Behandlung von verschiedenen Erkrankungen der Haut, Lunge sowie Darm und, da in zunehmendem Maße auch von Mastzellfunktionen in anderen Organen, wie Hirn, Herz, Leber und Niere berichtet wird, auch hier weiterführend beitragen. / Mast cells are ubiquitary connective tissue cells derived from bone marrow CD34+/c-Kit+ stem cells. They are able to produce various regulators of physiological and pathological processes. Normally, they are present in low numbers with highest density in skin and nasal, ophthalmic, gastrointestinal and pulmonary mucosa. The number is increased up to 100-fold in various pathological processes as inflammatory reactions and atopic diseases. During this processes mast cell precursors from peripheral blood are activated, migrate in the tissues by the effects of chemotactically acting factors. In order to elucidate the mechanisms involved in this processes, we established different in vitro cell culture models. Our results suggest that the precursor cells circulating in the peripheral blood do not express c-Kit. When the cells are activated, c-Kit expression is upregulated and the cells are able to migrate in the tissue, where they differentiate influenced by cytokines released from tissue cells. The interaction between mast cells and fibroblasts, melanocytes, keratinocytes and nerve cells were studied. Stem cell factor, GM-CSF, nerve growth factor and other neurotrophins as BDNF, NT-3 and NT-4 could be demonstrated as mediators of this interactions. The regulation and release of these factors are modified in pathological skin diseases as atopic dermatitis, psoriasis, changes in the hair cycle and skin tumors and in wound healing. Modulation of expression of these factors and its receptors by therapeutics as glucocorticoids, antihistamines, retinoids and UV-radiation was shown in different culture models. Our results may contribute to develop new therapeutics for skin, pulmonary and intestinal diseases and give new insights in mast cell functions in brain, liver and kidney.

Die Mechanismen der Mastzell(MZ)-Homöostase in peripheren Geweben sind weitgehend unbekannt. Die Regulation der MZ-Zahlen schließt wahrscheinlich die lokale Proliferation der Gewebe-MZ sowie die Rekrutierung und Differenzierung von MZ-Vorläuferzellen ein, wobei das Gewebe den MZ-Phänotyp beeinflusst. Fibroblasten (Fb) induzieren die Proliferation und Differenzierung unreifer Knochenmark-generierter MZ (BMCMC) zu Bindegewebs-MZ (CTMC). Da BMCMC an Fb adhärieren, wurde untersucht ob dieser direkte Zellkontakt für die Fb-induzierte Proliferation und die Differenzierung zu CTMC entscheidend ist. Cokultur-Experimente mit BMCMC und Fb zeigten, dass die verstärkte Proliferation, der erhöhte Histamingehalt und die Induktion der Expression der MZ Protease 4 (MCPT-4) mRNA (beides Marker der CTMC) vom direkten Zellkontakt abhängen. Adhäsionsversuche mit blockierenden Antikörpern demonstrierten, dass die Interaktion von alpha4beta7 Integrin auf den BMCMC mit Vascular Cell Adhesion Molecule 1 (VCAM-1), auf den Fb, an der Adhäsion beteiligt ist, die Proliferation und Differenzierung der MZ selbst jedoch nicht auslöst. Interessanterweise zeigten BMCMC die Kit, den Rezeptor für den wichtigen MZ-Wachstumsfaktor Stammzellfaktor (SCF), nicht exprimieren, ebenfalls die kontaktabhängigen Fb-induzierte Veränderungen, wenngleich im geringeren Ausmaß. Eine genomweite Expressionsanalyse wies schließlich die kontaktabhängige Hochregulation der Expression weiterer Gene, die mit dem Phänotyp der CTMC assoziiert sind, nach. Des Weiteren konnte die Verringerung der Expression bestimmter Gene gezeigt werden, die von unreifen MZ oder MZ-Vorläufern exprimiert werden. Insgesamt zeigen die Daten der hier vorliegenden Arbeit, dass für die durch Fb ausgelöste umfassenden Differenzierung und Ausreifung von unreifen MZ zu CTMC, eine teilweise durch VCAM-1/alpha4beta7 vermittelte direkte Zell-Zell-Interaktion notwendig ist, bei der sowohl Kit-abhängige, als auch Kit-unabhängige Signalwege involviert sind. / The precise mechanisms of mast cell (MC) homeostasis in peripheral tissues are largely unknown. Regulation of MC numbers is assumed to involve the proliferation of local MCs and the recruitment and subsequent differentiation of MC precursors, whereas the tissues determine the MC phenotype. Fibroblasts (Fb) induce proliferation and differentiation of bone marrow-derived cultured MCs (BMCMCs) towards connective tissue type MCs (CTMCs). Since BMCMCs exhibit adhesion to Fbs, it had been analyzed, whether this direct cell-to-cell crosstalk is mandatory for the differentiation towards CTMCs. It was found that Fb-cocultured immature MCs exhibit increased proliferation and histamine content and the induced expression of mast cell protease 4 (mcpt4) mRNA, both markers for mature CTMC, and that these changes required a directed cell-to-cell interaction. Adhesion Assays using blocking mAbs revealed that the interaction of alpha4beta7 integrin on BMCMCs with Fb-expressed Vascular Cell Adhesion Molecule 1 (VCAM-1) is largely responsible for the adhesion, which itself didn’t induce proliferation and differentiation of MCs. Most notably, MCs deficient for Kit, the receptor for the MC growth factor stem cell factor (SCF), also showed a significant Fb-induced increase in histamine content, mcpt4 expression, and proliferation, albeit to a lesser extent than wildtype BMCMCs. Whole genome expression analysis showed contact-dependently upregulated expression of several genes associated with CTMC phenotype and functions. Furthermore, downregulation of genes associated with MC progenitors had been shown. Collectively, the data show that the Fb-induced substantial differentiation of immature MCs towards CTMCs requires a partly VCAM-1/alpha4beta7-mediated cell-to-cell interaction and involves both Kit-dependent and -independent signaling pathways.

No description available.

Chronic Myelogenous Leukemia (CML) is a blood cancer that arises when hematopoietic cells acquire an abnormal protein known as BCR-ABL. Current therapies for CML include drugs that inhibit BCR-ABL. However, these drugs only suppress the disease and do not cure it. One reason is that BCR-ABL drugs fail to kill the primitive population of CML cells, referred to as leukemia stem cells (LSCs), which are responsible for initiating and propagating CML. Since LSCs are not killed, the cancer is not cured and many affected patients eventually relapse. Recent studies suggest that LSCs are protected from current therapies by the bone marrow micro-environment where they reside. There, cytokine signaling molecules are present, which mediate processes that protect LSCs from BCR-ABL drugs. The stem cell factor (SCF) is one of these signaling molecules. It activates the receptor c-Kit located on the surface of LSCs, and this activation in turn allows proliferating LSCs to resist BCR-ABL drugs, even without prior exposure to these drugs, i.e., innate resistance is observed. In this thesis, the mechanism of this innate resistance is investigated, so that a suitable treatment strategy can be developed. To this end, a co-agent approach based on synthetic antibodies (sABs) is proposed to inhibit the receptor c-Kit, with the goal of disrupting its activation by the ligand SCF. This disruption should in turn block the SCF-mediated innate resistance, thus potentially restoring BCR-ABL drug apoptotic activity. The method for this disruption involves targeting the c-Kit structural susceptibility. Specifically, the sABs are designed via antibody phage display technology to target the D1–D2–D3 domains representing the SCF binding sites, hence preventing downstream pathway activation. The hypothesis is that, by blocking the SCF-mediated innate resistance, a suitable combination of such an sAB co-agent and a BCR-ABL drug should be conducive to suppressing LSCs, thereby providing a potential means to improve CML treatment. In addition, to assess the performance of the proposed treatment strategy, a set of in vitro tests is conducted, focusing on performance behaviors such as cell binding, cell death, and the progenitor inhibition. The experimental results support the hypothesis that the proposed combinatorial strategy is indeed a promising approach to mitigate the innate resistance, thus restoring BCR-ABL drug apoptotic activity.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

No / Androgens stimulate many hair follicles to alter hair colour and size via the hair growth cycle; in androgenetic alopecia tiny, pale hairs gradually replace large, pigmented ones. Since stem cell factor (SCF) is important in embryonic melanocyte migration and maintaining adult rodent pigmentation, we investigated SCF/c-Kit signalling in human hair follicles to determine whether this was altered in androgenetic alopecia. Quantitative immunohistochemistry detected three melanocyte-lineage markers and c-Kit in four focus areas: the epidermis, infundibulum, hair bulb (where pigment is formed) and mid-follicle outer root sheath (ORS). Colocalisation confirmed melanocyte c-Kit expression; cultured follicular melanocytes also exhibited c-Kit. Few ORS cells expressed differentiated melanocyte markers or c-Kit, but NKI/beteb antibody, which also recognises early melanocyte-lineage antigens, identified fourfold more cells, confirmed by colocalisation. Occasional similar bulbar cells were seen. Melanocyte distribution, concentration and c-Kit expression were unaltered in balding follicles. Androgenetic alopecia cultured dermal papilla cells secreted less SCF, measured by ELISA, than normal cells. This identifies three types of melanocyte-lineage cells in human follicles. The c-Kit expression by dendritic, pigmenting, bulbar melanocytes and rounded, differentiated, non-pigmenting ORS melanocytes implicate SCF in maintaining pigmentation and migration into regenerating hair bulbs. Less differentiated, c-Kit-independent cells in the mid-follicle ORS stem cell niche and occasionally in the bulb, presumably a local reserve for long scalp hair growth, implicate other factors in activating stem cells. Androgens appear to reduce alopecia hair colour by inhibiting dermal papilla SCF production, impeding bulbar melanocyte pigmentation. These results may facilitate new treatments for hair colour changes in hirsutism, alopecia or greying.