Τα μηνιγγιώματα αποτελούν ιδιαίτερα συχνούς όγκους του Kεντρικού Νευρικού Συστήματος, όχι όμως επαρκώς μελετημένοι. Στην πλειοψηφία τους είναι καλοήθεις όγκοι (WHO grade I), όμως το 10% όλων των μηνιγγιωμάτων εμφανίζουν κακοήθη χαρακτηριστικά, όπως διήθηση των παρακείμενων ιστών, αυξημένα ποσοστά υποτροπής μετά από εξαίρεση, κ.α. Το σηματοδοτικό μονοπάτι Hippo είναι ένα διατηρημένο εξελικτικά μονοπάτι που εμπλέκεται σε διαδικασίες ρύθμισης του μεγέθους των οργάνων, κυτταρικού πολλαπλασιασμού, διαφοροποίησης και ανάπτυξης καρκίνου. Λίγα είναι γνωστά όσον αφορά το ρόλο του Hippo μονοπατιού στα μηνιγγιώματα. Σκοπός της παρούσας εργασίας είναι η μελέτη του Hippo μονοπατιού στα χαμηλής και υψηλής κακοήθειας μηνιγγιώματα. Χρησιμοποιήθηκαν 53 δείγματα από εξαιρεθέντα μηνιγγιώματα, 34 χαμηλής κακοήθειας και 19 υψηλής κακοήθειας, τα οποία μελετήθηκαν ανοσοϊστοχημικά ως προς την έκφραση των παραγόντων του Hippo CD44, YAP και TAZ. Παρατηρήσαμε στατιστικά σημαντική συσχέτιση μεταξύ της έκφρασης των CD44, ΥΑΡ και ΤΑΖ. Υπήρχε στατιστικά σημαντική συσχέτιση μεταξύ του βαθμού κακοήθειας και της έκφρασης των παραπάνω μορίων. Παρατηρήθηκε στατιστικά σημαντική διαφορά στην πυρηνική εντόπιση των ΥΑΡ και ΤΑΖ μεταξύ των υψηλής και χαμηλής κακοήθειας μηνιγγιωμάτων. Βάσει των παραπάνω αποτελεσμάτων, συμπεραίνουμε ότι το σηματοδοτικό μονοπάτι Hippo είναι απενεργοποιημένο στα υψηλής κακοήθειας μηνιγγιώματα με αποτέλεσμα οι μεταγραφικοί συνενεργοποιητές ΥΑΡ και ΤΑΖ να δρουν στον πυρήνα προάγοντας τον κυτταρικό πολλαπλασιασμό και διαδικασίες αντιαπόπτωσης. Αυτό, είναι ίσως εφικτό μέσω της δράσης του CD44 που αλληλεπιδρά με την μερλίνη, upstream ρυθμιστής του μονοπατιού. Απαιτούνται περαιτέρω μελέτες για τη διευκρίνηση του ακριβούς μηχανισμού. / Meningiomas are among the most common primary tumors of the Central Nervous System, but relatively understudied. The majority of meningiomas are benign tumors (WHO grade I), but almost 10% of all diagnosed meningiomas exhibit malignant features, such as invasiveness to the surrounding brain tissue, high recurrence rate, etc. Hippo signaling pathway is an evolutionary highly conserved protein kinase cascade involved in organ size control, cell proliferation and apoptosis, differentiation and cancer development. Very few are known about the role of Hippo pathway and meningiomas. In this study we attempt to find out whether Hippo path functions differently between high and low grade meningiomas. For this purpose paraffin embedded tissues obtained from 53 patients who underwent surgical removal of meningiomas were examined histoimmunologicaly. 34 out of 53 cases were low grade meningiomas and 19 out of 53 were high grade meningiomas. The paraffin sections were immunostained with CD44, YAP and TAZ antibodies, components of the Hippo signaling pathway. We observed statistically significant association between the expression of CD44, YAP and TAZ. There was a significant correlation between high grade meningiomas and expression of the examined factors. Moreover there was a significant difference in nuclear accumulation of YAP and TAZ between high and low grade meningiomas. Our findings suggest that in high grade meningiomas, Hippo pathway is inactivated and YAP and TAZ transcriptional co activators are able to insert nucleus and promote proliferation and antiapoptosis. This may be due to CD44 function, which interacts with merlin, an upstream regulator of the Hippo path. More experiments are required to verify the exact mechanism.

SOUSA, Daniel Willian Lustosa de. Expressão da L-selectina e do CD44 nas leucemias linfóides agudas em crianças e adolescentes. 2009. 118 f. Dissertação (Mestrado em Patologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2009. / Submitted by denise santos (denise.santos@ufc.br) on 2013-12-03T12:41:12Z No. of bitstreams: 1 2009_dis_dwlsousa.pdf: 2111442 bytes, checksum: 1ddc01feade951f9f82cbb760935f58d (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2013-12-03T12:42:22Z (GMT) No. of bitstreams: 1 2009_dis_dwlsousa.pdf: 2111442 bytes, checksum: 1ddc01feade951f9f82cbb760935f58d (MD5) / Made available in DSpace on 2013-12-03T12:42:22Z (GMT). No. of bitstreams: 1 2009_dis_dwlsousa.pdf: 2111442 bytes, checksum: 1ddc01feade951f9f82cbb760935f58d (MD5) Previous issue date: 2009 / Introduction – Altered expression or function of adhesion molecules on leukemic blasts may contribute to the evolution of acute leukemia and its biological behavior. The elevated expression of adhesion molecules in ALL might be correlated with the extramedullary dissemination of blast cells, CNS involvement and leukemia tumor burden. Purpose – To analyze the expression of L-selectin and CD44 in ALL in children and adolescents. As well as to evaluate the prognostic factors (age, gender, initial leukocyte count, immunophenotype, FAB and EGIL classification, DNA index and early response to treatment) and the extramedullary presentation of ALL, to finally correlate the prognostic factors with these adhesion molecules. Patients and Methods – From November 2007 to November 2008, 76 patients with newly diagnosed ALL started on Brazilian GBTLI-ALL Protocol. The diagnosis was based on cytological, immunophenotypic, and cytogenetic methods. The mean fluorescence intensity (MFI) and the percentage of the adhesion molecules blasts cells was measured by flow cytometry using triple staining with McAb directly conjugated. CD45-PerCP positive cells were gated for blasts analysis. Anti-CD44-PE (Clone HP2/9 - Immunostep®) and CD62L-FITC (Clone HI62L - Immunostep®) were used to mark the adhesion molecules. The Cell Quest program was used for data acquisition and analysis. Statistical analysis was done by SPSS 16.0 Software. The association of features, prognosis and response to treatment was assessed by Chi-square, Fisher exact and Mann-Whitney tests. Overall survival curves were constructed by the Kaplan-Meier method and the log-rank test. Multivariate Cox regression analysis showed independent prognostic factors. Results – The mean age at diagnosis was 6.3±0.5 years (range 9mo to 17yr) and 65% of them were boys. Clinical findings were hepatomegaly (63%), splenomegaly (58%), lymphadenopathy (44%). CNS involvement was detected in 6.6% of cases and mediastinal mass appeared in 11.8% of them. Patients were classified into low risk (54%) and high risk (46%). FAB classification identified 83% as L1 and 17% as L2. Immunophenotypically, 89.5% of patients were classified as B-lineage ALL and 10.5% as T-lineage ALL. The most frequent EGIL subtype was B common and pre-B-ALL (51.5% and 45.5%, respectively). DNA index greater than 1.16 was found in 19% of the patients and was associated with favorable prognosis. On the D8 evaluation, 95% of the patients had blast count lower than 1.000/mm3 and leukocyte count lower than 5.000/mm3. The remission induction rate was 95% and there was a rate of 2.6% of death during induction therapy. CD44 had greater expression to the rate of 87.5% in T-cell ALL (MFI=150.44±20.29) with no statistical correlation. A significant positive correlation was demonstrated between 84% of CD44 expression and Leukemia burden tumor cases (p=0.01; OR=4.8). There was statistical correlation between L-selectin expression (87.5%/MFI=272.33±52.72) and T-cell ALL (p=0,004). No significant correlation was detected between L-selectin and CD44 expression and other prognostic factors. Multivariate statistical analysis (adjusted for overall survival) indicated that initial leukocyte count, gender, T immunophenotype and L-selectin were independent factors. Conclusion – L-selectin and CD44 expressions were elevated in ALL studied, mainly in T-cell ALL. The research demonstrated that there is an association between CD44 expression and leukemia tumor burden, which might be involved in the dissemination of leukemic cells and the progression of the disease. / Introdução – Alterações na expressão ou função das moléculas de adesão (MA) nas células leucêmicas podem contribuir para a evolução e no comportamento biológico das leucemias agudas. A expressão aumentada nas LLAs parece relacionar-se aos mecanismos de disseminação extramedular dos linfoblastos, infiltração do SNC e formação de massas tumorais. Objetivos – Analisar a expressão da L-selectina e do CD44 nas LLAs em crianças e adolescentes. Avaliar os fatores prognósticos (idade, sexo, leucometria ao diagnóstico, imunofenótipo, classificação FAB, EGIL, índice de DNA e resposta ao tratamento de indução) e as apresentações extramedulares das LLAs e correlacioná-los com essas MA. Pacientes e Métodos – Foram avaliados 76 pacientes com LLA, tratados com o Protocolo GBTLI-LLA. O diagnóstico foi baseado em critérios FAB, imunofenotípicos (EGIL) e citogenéticos. A expressão das MA foi avaliada por citometria de fluxo, utilizando tripla marcação. O anticorpo monoclonal CD45-PerCP (Immunotech®) foi utlizado como marcador dos linfoblastos. O CD44-PE (Clone HP2/9 - Immunostep®) e o CD62L-FITC (Clone HI62L - Immunostep®) foram utilizados para a marcação das MA. Para a análise das amostras e o cálculo da intensidade média de fluorescência foi utilizado o programa Cell Quest. Na análise estatística utilizou-se o software SPSS 16.0. A associação entre as variáveis, os fatores prognósticos e resposta foi realizada com os testes de Qui-quadrado, exato de Fisher e Mann-Whitney. Sobrevida global foi determinada por curvas Kaplan-Meier e teste log-rank. Análise multivariada por modelo proporcional de Cox foi utilizada para assegurar a independência dos fatores prognósticos. Resultados – A média de idade foi 6,3±0,5 anos (5m -17a) e predominou o sexo masculino (65%). Ao diagnóstico os achados clínicos foram: hepatomegalia (63%), esplenomegalia (58%) e linfadenomegalia (44%). A infiltração SNC ocorreu em 6,6% dos casos e o alargamento de mediastino em 11,8%. Quanto ao risco, 54% eram baixo risco e 46% alto risco. A classificação FAB determinou 83% como L1 e 17% L2. Diagnóstico de LLA-B foi mais frequente (89,5%) e o da LLA-T em 10,5% dos pacientes. O subtipo EGIL mais prevalente foi B II e B III, 51,5% e 45% respectivamente. O IDNA ≥ 1.16 foi encontrado em 19% dos pacientes e associou-se a bom prognóstico. Na avaliação do D8, 95% dos pacientes apresentaram contagem de blastos <1000/mm3 e leucócitos < 5.000/mm3. A taxa de remissão de indução foi de 95% e ocorreram 2,6% de óbitos na indução. Observou-se uma maior expressão do CD44 na LLA-T (87,5%/ IMF=150,44±20,29), porém sem significância estatística. LLAs com massa tumoral apresentaram 84% de expressão do CD44, quando comparada a 52% das LLAs sem massa tumoral (p=0.01; OR=4,8). Expressão aumentada da L-selectina na LLA-T (87,5%/IMF=272,33±52,72) foi estatisticamente significante (p=0,004), comparado a LLA-B (54,5%/ IMF= 115,90±12,75). Não houve correlação entre os outros fatores prognósticos e essas MA. Na análise multivariada as variáveis de maior impacto para a sobrevida foram: a leucometria ao diagnóstico, sexo, imunofenótipo T e a L-selectina. Conclusão – A expressão da L-selectina e do CD44 estão aumentadas nas LLAs estudadas, principalmente na LLA-T. O CD44 correlacionou-se com LLAs com massas tumorais e parece estar relacionado aos mecanismos de disseminação extramedular dos linfoblastos

Made available in DSpace on 2014-06-12T16:27:55Z (GMT). No. of bitstreams: 2 arquivo5637_1.pdf: 2299687 bytes, checksum: fbbb54dcc69a0fc5feca85fbb9bbbfe1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Circulação Extracorpórea (CEC) induz uma reação sistêmica inflamatória, que aumenta a morbimortalidade nas cirurgias cardíacas. Os neonatos e lactentes são especialmente suscetíveis a essa reação e ao desenvolvimento da Síndrome do Vazamento Capilar pós-operatória (SVC). A SVC resulta na perda da integridade endotelial, com extravasamento de grandes volumes de plasma e seus constituintes no espaço extracelular. Uma síndrome similar à SVC é desencadeada pela Interleucina-2, através de um possível mecanismo de apoptose, mediado pela molécula de adesão CD44. A ativação da CD44, em leucócitos na CEC, pode mediar a SVC em crianças de baixo peso. Na presente investigação, foi avaliada a expressão da CD44 em linfócitos T, células NK e demais leucócitos do sangue total, de um grupo consecutivo de 17 crianças submetidas à cirurgia cardíaca com CEC, nos dez primeiros meses de vida. Foi, também, avaliado o comportamento de células TCD4+ e TCD8+, no que se refere a freqüência de células no pós-operatório imediato. Duas crianças evoluíram para óbito precoce e foram excluídas do estudo. A pesquisa foi realizada em amostras de sangue arterial coletadas no início e no fim da cirurgia e, no pós-operatório, de quatro em quatro horas, em cerca de dez amostras por paciente. Os leucócitos isolados foram marcados com anticorpos monoclonais (antiCD44, antiCD3, antiCD56 e antiCD8) e as células separadas por citometria de fluxo. Foi observado aumento significante na freqüência de células CD44+ e CD3- até oito horas do final da cirurgia, e depleção significante de linfócitos T (CD3+ e CD44+) e células NK (CD56+ e CD44+), com pico na quarta hora, pósoperatória . Os resultados mostram também diminuição de linfócitos TCD4+ duas vezes maior do que de TCD8+. Pode-se concluir que existe um estado de imunossupressão com pico na quarta hora de pós-operatório, que envolve as células T e NK, ambas CD44+; enquanto as demais células brancas (CD44+ e CD3-) apresentam aumento na freqüência nas primeiras oito horas

CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored. The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development. The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading. The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified. Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

全文データ差替え待ち / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20268号 / 医博第4227号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 武藤 学, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DAM

Two important mechanisms of lymphocyte-mediated cytotoxicity, one perforin based and the other Fas ligand (FasL) based, have been characterized recently. It has also been shown that CD44, an adhesion molecule, can participate in signaling cytotoxic activity of cytotoxic T lymphocytes (CTLs). In the current study we tested the hypothesis that activation of natural killer (NK) or lymphokine activated killer (LAK) cells induces the expression of FasL, perforin, and CD44 which together contribute towards increased cytolytic activity. To this effect, we used wild-type mice, perforin-knockout mice, and mice lacking a functional FasL. We observed that both interleukin-2 (IL-2) and Poly I:C triggered NK/LAK cells to lyse targets through the perforin- and FasL- pathways. In addition, Fas+ tumor targets were more susceptible to lysis by poly I:C and IL-2 activated NK/LAK cells when compared to Fas- targets. Furthermore, Fas- tumor cells injected subcutaneously into syngeneic mice could grow and induce tumors, whereas, Fas+ tumors were rejected. IL-2 treatment increased the CD44 expression on NK cells, which was responsible for the lysis of endothelial cells through its ligand, hyaluronate. Upregulation of perforin and FasL in activated NK/LAK cells may explain why such cells can kill a wide variety of tumor cells efficiently. On the other hand, activated NK/LAK cells express increase increased levels of CD44 and use this molecule to mediate cytotoxicity of endothelial cells, which may account for the vascular leak seen during IL-2 therapy. / Master of Science

Sabemos hoje que os tumores sólidos apresentam uma composição celular heterogênia e que apenas uma pequena parcela dessas células apresenta capacidade de se proliferar e gerar novos tumores. Estudos prévios sobre a formação do câncer de mama têm sido realizados com base na combinação dos marcadores de superfície celular CD44 e CD24. Já foi demonstrado que uma subpopulação de células do câncer de mama com alta expressão de CD44 e baixa expressão de CD24 (CD44+/CD24-) tem maior capacidade de gerar tumores, quando comparadas com a subpopulação de células CD44-/CD24+. O objetivo do estudo foi identificar a taxa de células com fenótipo CD44+/CD24- presentes nos tumores mamários e relaciona-la com a taxa de comprometimento dos linfonodos axilares ipsilaterais por neoplasia, além de avaliar também sua relação com outros fatores sabidamente relacionados com mal prognóstico da paciente. Pacientes e métodos: avaliamos prospectivamente 53 amostras cirúrgicas provenientes de 42 pacientes com diagnóstico histopatológico de carcinoma de mama, quantificando as células CD44+/CD24- por citometria de fluxo. Relacionamos a porcentagem destas células encontrada em cada amostra com o comprometimento axilar, os receptores hormonais e Her-2, a idade da paciente, o grau histológico do tumor, o diâmetro patológico do tumor e o tipo histológico. Resultados: verificamos um significante aumento da população de células CD44+/CD24- no grupo de carcinomas ductais invasivos em pacientes que apresentavam metástase axilar [mediana 8,53% (3,6 71,2%)] em relação ao grupo de pacientes sem linfonodos comprometidos pela neoplasia [mediana 1,49% (0,3 17,1%)] (p=0,0002). Conclusão: concluímos então que quando estudamos vários tumores mamários invasivos de mesma classificação histológica, podemos notar que existe uma variação na quantidade de células CD44+/CD24- entre eles. Nosso estudo mostrou que essa variação está relacionada à agressividade tumoral e à sua capacidade de gerar metástases já que, tumores com maior quantidade de células CD44+/CD24- apresentam maior taxa de comprometimento dos linfonodos axilares. / It is known that solid tumors are composed by a heterogeneous combination of cells and only a small portion of these cells has the capacity to proliferate and generate new tumors. Previous studies about the breast cancer initiation have been based on a combination of CD44 and CD24cell surface markers. It has been shown that this subpopulation of breast cancer cells with high expression of CD44 and low expression of CD24 (CD44+/CD24-) has a greater capacity to generate tumors when compared with the subpopulation of cells CD44- /CD24+. The study objective was to identify whether the rate of cells with CD44+/CD24- phenotype present in breast tumors is related with the rate of ipsilateral lymph node metastasis, in addition to evaluate its relationship with other risk factors known to be related with worst prognosis. Patients and methods: we prospectively evaluated 53 surgical specimens from 42 patients with histological diagnosis of breast cancer, quantifying CD44+/CD24- cells through flow cytometry. We list the percentage of these cells found in each sample with axillary lymph node status, hormone receptors and Her-2, patient age, histological grade, pathological tumor diameter and histological tumorclassification. Results: we find a significant increase of CD44+/CD24- population in the invasive ductal carcinomas, in patients with axillary metastasis [median 8.53% (3.6 - 71.2%)] than in the group of patients without lymph nodes metastasis [median 1.49% (0.3 - 17.1%)] (p = 0.0002). Conclusion: when we studied several invasive breast tumors of same histological classification, we note that there is variation in the number of CD44+/CD24- cells. Our study showed that this variation is related to tumor aggressiveness and their ability to generate metastasis, because tumors with high rate of CD44+/CD24- cells have a higher rate of lymph node metastasis.

Thèse de doctorat : Bioingénierie. Ingénierie Cellulaire et Tissulaire : Nancy 1 : 2007. / Titre provenant de l'écran-titre.