Υποθέσαμε ότι το χρόνιο ιστικό στρες που προκαλείται από τις αλλοαντιδράσεις μετά από αλλογενή Μεταμόσχευση Αιμοποιητικών Κυττάρων (άλλο-ΜΑΚ) μπορεί να επάγει την εμφάνιση γονιδιωματικής αστάθειας (GI) στους επιθηλιακούς ιστούς. Για αυτό, 176 στοματικά επιχρίσματα από 71 ασθενείς μετά από άλλο-ΜΑΚ αναλύθηκαν για την ανίχνευση αστάθειας μικροδορυφορικών αλληλουχιών (microsatellite instability-MSI). Tα αποτελέσματα συσχετίσθηκαν με κλινικές παραμέτρους. Σε ένα in vitro σύστημα ανίχνευσης μεταλλάξεων ελέγξαμε την υπόθεση ότι οι αλλοαντιδράσεις μπορεί να επάγουν τη GI στην κυτταρική σειρά HaCaT. Ανιχνεύθηκε MSI στο 52% των αλλομεταμοσχευμένων ασθενών ενώ δεν ανιχνεύθηκε MSI σε ασθενείς μετά από αυτόλογη ΜΑΚ και υγιείς εθελοντές. Βρέθηκε σημαντική συσχέτιση της εμφάνισης MSI με την ηλικία του ασθενούς και του δότη, τη μεταμόσχευση από γυναίκα σε άνδρα και τον αριθμό CD34+ κυττάρων στο μόσχευμα. Οι ασθενείς με ιστορικό σοβαρού βαθμού αντίδρασης μοσχεύματος εναντίον ξενιστή είχαν μεγαλύτερο σχετικό κίνδυνο για εμφάνιση MSI. Δευτεροπαθής κακοήθεια διαγνώσθηκε σε 5 από τους MSI+ ενώ μόνο σε ένα από τους MSI- ασθενείς. Στο in-vitro σύστημα ανίχνευσης GI παρατηρήθηκε σημαντική επαγωγή μεταλλάξεων και βλάβης του DNA μετά από επώαση των HaCaT κυττάρων με Μεικτή Λεμφοκυτταρική Καλλιέργεια (MLC) ενώ η επίδραση με κυτταροκίνες, υπερκείμενο MLC ή ενεργοποιημένα με PHA περιφερικά μονοπύρηνα κύτταρα δεν προκάλεσε την επαγωγή GI. Τα αποτελέσματά μας υποδεικνύουν τη συμμετοχή των ενεργών ριζών οξυγόνου στον υποκείμενο μηχανισμό. Οι in vivo και in vitro μελέτες μας επιβεβαιώνουν ότι παράγοντες που ενέχονται στο αλλοαντιδραστικό μικροπεριβάλλον μετά από άλλο-ΜΑΚ μπορεί να προκαλέσουν GI στο επιθήλιο. Η κατανόηση του υποκείμενου μηχανισμού μπορεί να αναδείξει νέους βιοδείκτες και θεραπευτικούς στόχους. / We hypothesized that chronic tissue stress due to interaction of alloreactive donor cells with host epithelium after allogeneic hematopoietic cell transplantation (allo-HCT) may cause genomic alterations. We therefore analysed 176 buccal samples obtained from 71 unselected allo-transplanted patients for microsatellite instability (MSI). MSI was observed in 52% of allo-transplanted patients but never in 31 healthy or auto-transplanted controls. The patient age, the donor age, a female-to-male transplantation and a low number of CD34+ cells in the graft were significantly correlated with genomic instability. There was a trend for increasing risk of MSI for patients who experienced severe graft-versus-host-disease. Secondary malignancy was diagnosed in 5 (14%) of the MSI+ and only in 1 (3%) MSI- patient. In an in-vitro model of mutation analysis we found significant induction of frameshift mutations and DNA strand breaks in HaCaT keratinocytes co-cultured with Mixed Lymphocyte Cultures (MLC) but not after their exposure to IFN-γ, TNF-α, TGF-β, MLC-supernatant, peripheral blood mononuclear cells (PBMC) or phytohemagglutinin stimulated PBMC. A ROS mediated mechanism is implicated. Our in-vivo and in-vitro data show that alloreactions after allo-HCT may induce genomic alterations in epithelium. Progress in understanding DNA damage and repair after allo-HCT can potentially provide molecular biomarkers and therapeutic targets.

O pacu, Piaractus mesopotamicus, é um teleósteo da Família Characidae, intensivamente cultivado no Brasil por causa de sua rusticidade, crescimento rápido e fácil adaptação, além de seu excelente sabor. Para uma melhor produção de peixes, informações sobre seu sistema imunológico incluindo a histologia dos órgãos linfóides se faz necessária. Sendo assim, o objetivo deste estudo foi descrever histomorfologicamente os órgãos linfóides: rim cefálico, fígado, baço e timo do Piaractus mesopotamicus, analisando os perfis celulares de cada órgão. Foram utilizados 30 animais juvenis, com idade que variaram entre 5 meses a um ano, com peso médio de 588.1 g e comprimento total médio de 27,51 cm. Os órgãos foram pós-fixados em solução de paraformaldeído 4% e Karnovsky, depois desidratados, diafanizados em xilol e incluídos em parafina. Os cortes foram obtidos com uma espessura de 4µm e corados em hematoxilina-eosina. Os esfregaços sanguíneos foram corados em Giemsa-May Grünwald para microscopia de luz. Para microscopia eletrônica de transmissão as amostras fixadas em Karnovsky foram lavadas em sacarose e pósfixadas em tetróxido de ósmio 1%, desidratadas e emblocadas em resina Spurr. O resultado da análise macroscópica mostra que a distribuição do rim, rim cefálico, timo, fígado e baço são as mesmas encontradas na maioria dos teleósteos. Quanto à forma desses órgãos, o fígado apresentou uma variação anatômica na forma e números de lobos, sendo constituído por três lobos. O rim apresentou uma forma em \"H\", onde a região central deste se expandia sobre a bexiga natatória. O rim cefálico, em animais com idade mais avançada, se apresentou como uma dilatação na região cranial do rim, se mostrando bem visível. O timo e o baço apresentaram localização e forma similares aos de outros teleósteos. A microscopia mostrou similaridades entre os órgãos do Piaractus mesopotamicus e outros teleósteos. Na microscopia eletrônica de transmissão foram observadas similaridades ultraestruturais das células encontradas no rim cefálico, fígado, timo e baço e as já descritas em peixes teleósteos. Hemácias, trombócitos, linfócitos, eosinófilos e células granulocíticas especiais encontrados no sangue periférico, tanto de animais jovens quanto dos animais com idade mais avançada, foram os mesmos tipos celulares descritos na literatura de teleósteos. Observando nossos resultados concluímos que histologicamente os órgão linfóides de Piaractus mesopotamicus são similares aos de outros teleósteos e comparando os resultados de microscopia de transmissão concluímos que as estruturas encontradas nas nossas análises são as mesmas das descritas em outras espécies. Os resultados das nossas observações macroscópicas mostraram que o pacu é uma espécie que apresenta algumas características morfológicas singulares e que podem estar ligadas ao tipo de alimentação e a adaptações evolutivas. Esses achados demonstram que a anatomia dos órgãos de peixes e suas variações precisam ser analisados e correlacionados com suas funções e forma de vida de cada espécie. / Piaractus mesopotamicus (Pacu) is a fish from the Characidae Family, it is intensively culture in Brazil because of its rusticity, easy raising and adaptation, besides its excellent flavour. In order to produce a healthy fish, information on its immunological system, including the histology of the lymphoid organs is needed. Therefore, the objective of this study is to describe histomorphologicaly the lymphoid organs: head kidney, liver, spleen and thymus of the Piaractus mesopotamicus, analyzing the cellular profiles of each organ. Thirty young animals, with age varying between 5 months to a year, with average weight and total length of 588.1 g and a 27.51 cm, respectively, were used. The organs were fixed in 4% paraformaldehyde solution, and Karnovsky, afterwards dehydrated, diafanized in xilol and included in paraffin. Four µm sections were obtained and stained with hematoxilin-eosin. Blood smears were stained with Giemsa-May Grünwald for light microscopy. Samples for transmission electric microscopy were fixed in Karnovsky, washed in sacarose and post-fixed with osmium tetroxide 1%, dehydrated and embedded in resin Spurr. The macroscopic analysis show that the localization of the kidney, head kidney, thymus, liver and spleen are very similar to most of the teleosts. Some particularities were observed in the liver, which presented an anatomical variation in the shape and number of lobes, being constituted by three lobes. The kidney presented existe \"H\" shape, where the central region overlaps the swimming bladder. The head-kidney, in adult animals, presented an evident dilation in the cranial region of the kidney. The thymus and spleen presented a similar location and shape to that of the teleosts. The light microscopy studies showed similarities between the organs of the Piaractus mesopotamicus and other teleost fishes. The transmission electron microscopy studies showed ultrastructural similarities, of the cells from the head kidney, liver, thymus and spleen with the literature. Eritrocytes, trombocytes, lymphocytes, eosinophils and special granulocytic cells were found in peripheral blood of both juvenile and adult animals and were similar to the cellular profiles described in the literature to the other teleost fishes. Our studies were successful in describing the macro, micro and ultrastructure of Piaractus mesopotamicus organs and may be used as reference for further research aiming the improvement of fish health status in aquaculture.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23383号 / 医博第4752号 / 新制||医||1052(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 河本 宏, 教授 髙折 晃史, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / SIRT1 (Sirtuin 1) is a founding member of a family of seven proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1-/- mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1-/- mouse embryonic stem (mES) cells in vitro, and hematopoietic progenitors in SIRT1+/+, SIRT1+/-, and SIRT1-/- mice. SIRT1-/- ES cells exhibited markedly delayed/immature formation of blast colony-forming cells (BL-CFCs). When individual blast colonies were analyzed for hematopoietic and endothelial potential, replated SIRT1-/- BL-CFC possessed limited hematopoietic potential, whereas endothelial potential was essentially unaltered. The ability of SIRT1-/- ES cells to form primitive erythroid progenitors was not only delayed but greatly decreased. Moreover, after differentiation of SIRT1-/- mES cells, there were also significant decreases in granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5, decreased β-H1 globin, β-major globin, and Scl gene expression and reduced activation of the Erk1/2 pathway upon SIRT1-/- ES cell commitment. Reintroduction of WT SIRT1 into SIRT1-/- cells partially rescued the primitive erythroid progenitor formation of SIRT1-/- cells and the expression of hemoglobin genes, Hbb-bh1 and Hbb-b1, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1-/- cells. To confirm the requirement for SIRT1 for normal development of hematopoietic progenitor cells, we assessed embryonic and adult hematopoiesis in SIRT1+/+, SIRT1+/- and SIRT1-/- mice. Yolk sacs from SIRT1 mutant embryos generated fewer primitive erythroid precursors compared to wild-type (WT) and heterozygous mice. Moreover, knockout of SIRT1 decreased primary bone marrow hematopoietic progenitor cells (HPCs) in 5 week and 12 month old mice, which was especially notable at lower (5%) O2 tension. In addition these progenitors survived less well in vitro under conditions of delayed growth factor addition. Taken together, these results demonstrate that SIRT1 plays a role in ES cell hematopoietic differentiation and mouse hematopoiesis.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a common clinical problem. Tanshinone IIA is a compound purified from the Chinese herb Danshen ( Radix Salviae Miltiorrhiza Bge). Thrombopoietin (TPO) and Erythropoietin (Epo) are hematopoietic growth factors. The effects of tanshinone IIA, EPO and TPO on hypoxia-ischemia brain injury were investigated in this study, using in vitro model of neural cell culture and an in vivo model of hypoxic-ischemic brain damage. / Our observation provided the first evidence showing the expression of functional TPO receptor c-mpl in central nervous system. It revealed that novel agents TPO, EPO and tanshinone IIA have neuroprotection effects against brain injury induced by hypoxia-ischemia in neonatal rats, and these agents could be developed for clinical applications. / To investigate the effect of TPO, EPO and tanshinone IIA on in-vivo neural protection, a neonatal rat model of hypoxic-ischemic brain damage was established. Our results demonstrated significant and sustained brain injury in the hypoxic-ischemic and vehicle-treated group, measured by the reduction in relative weights of the ipsilateral (right) to the contralateral (left) brain at 1 and 3 weeks post-surgery, compared with those of sham-operated animals. At 3 weeks post-surgery, the hypoxic-ischemic animals had decreased cortical neuron density quantified by neuron-specific enolase (NSE) staining, and compromised sensorimotor functions in response to the postural reflex test. Treatment with TPO, EPO and tanshinone IIA significantly reduced the severity of brain injury, as indicated by the significantly increased ipsilateral brain weight and neuron density. Recoveries of sensorimotor functions (p < 0.05) and histopathology were also observed in animals that received TPO, EPO and tanshinone IIA. The plasma of tanshinone IIA-treated animals exhibited higher antioxidant activities (oxygen radical absorbance capacity assay) than those from vehicle-treated rats. / TPO and TPO receptor (c-mpl) mRNA was identified in human cerebral hemispheres, cerebellum, mouse neural progenitor cell line C17.2 and four neuroblastoma cell lines (SK-N-MC, MHH-NB-11, SK-N-AS and SH-SY-5Y) using RT-PCR methods. TPO proteins were detected in human cerebrospinal fluid (CSF) and plasma by ELISA. Furthermore, TPO receptor c-mpl was confirmed in human cerebral hemispheres, hippocampus, cerebellum, brainstem and spinal cord using immunohistostaining. TPO had a stimulating effect on the growth of neural progenitor cell C17.2 in culture via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway as demonstrated by Western blot. The anti-apoptotic effects of TPO, EPO on C17.2 cells were demonstrated by staining with Annexin-V and PI. EPO exerted a protective effect against SHSY-5Y cell damage induced by NMDA (N-methyl-d-aspartate), as demonstrated by the MTT and LDH assay. The anti-oxidative property of tanshinone IIA was studied in the C17.2 cell line. Tanshinone IIA increased the viability of these cells subjected to 2,2'-azobis (2-amidino propane hydrochloride) (AAPH)-induced oxidative stress. / by Xia Wen-Jie. / "May 2005." / Advisers: Kwok-Pui Fung, Tai-Fai Fok. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0126. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 126-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Rituximab (R) is increasingly incorporated in reduced intensity conditioning (RIC) regimens for allogeneic hematopoietic cell transplantation (alloHCT) in patients with B-cell malignancies, not only to improve disease control, but also to prevent graft-versus-host disease (GVHD). There are no randomized prospective data to validate this practice, although single center data and the CIBMTR analysis have shown promising results. We aimed at validation of these findings in a large registry study. We conducted a retrospective analysis using the EBMT registry of 3,803 adult patients with B-cell malignancies undergoing alloHCT (2001–2013) with either rituximab (R-RIC-9%) or nonrituximab (RIC-91%) reduced intensity regimens respectively. Median age and median follow up were 55 years (range 19.1–77.3) and 43.2 months (range 0.3–179.8), respectively. There was no difference in transplant outcomes (R-RIC vs RIC), including 1-year overall survival (69.9% vs 70.7%), 1-year disease-free survival (64.4% vs 62.2%), 1-year non-relapse mortality (21% vs 22%), and day-100 incidence of acute GVHD 2-4° (12% vs 12%). In summary, we found that addition of rituximab in RIC regimens for B-cell malignancies had no significant impact on major transplant outcome variables. Of note, data on chronic GVHD was not available, limiting the conclusions that can be drawn from the present study.

Células mononucleares de sangue de cordão umbilical (SCU) e sangue periférico mobilizado (SPM) com G-CSF, foram cultivadas in vitro com citocinas, na presença ou não de estroma de medula óssea. Os objetivos foram avaliar a capacidade proliferativa de células progenitoras, a ocorrência de apoptose e expressão de integrina. Nas culturas sem estroma, a celularidade aumentou 5 vezes (SCU) e não se alterou nas de SPM. O total de células CD34+ caiu em ambas culturas. Com estroma, o total de células nucleadas aumentou 7 vezes (SCU) e 2,3 vezes (SPM). O total de células CD34+ permaneceu o mesmo. A apoptose foi menor nas culturas de SCU. A expressão de integrina caiu, na população de células CD34+ e de CD45+ / Mononuclear cells from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (MPB), were cultured in vitro, in the presence of cytokines, with or without bone marrow stroma. The aims were to evaluate the proliferative response of progenitor cells, occurrence of apoptosis and expression of adhesion molecule. In cultures without stroma, cellularity increased 5-fold for UCB, but has not changed for MPB. The number of CD34+ cells has dropped in both culture. With stroma, total nucleated cells had a 7-fold increse (UCB) and a 2,3-fold (MBP), however, CD34+ cells number has not changed. Apoptosis was lower in UCB culture. The expression of integrin decreased, in the CD34+ and CD45+ population

Células mononucleares de sangue de cordão umbilical (SCU) e sangue periférico mobilizado (SPM) com G-CSF, foram cultivadas in vitro com citocinas, na presença ou não de estroma de medula óssea. Os objetivos foram avaliar a capacidade proliferativa de células progenitoras, a ocorrência de apoptose e expressão de integrina. Nas culturas sem estroma, a celularidade aumentou 5 vezes (SCU) e não se alterou nas de SPM. O total de células CD34+ caiu em ambas culturas. Com estroma, o total de células nucleadas aumentou 7 vezes (SCU) e 2,3 vezes (SPM). O total de células CD34+ permaneceu o mesmo. A apoptose foi menor nas culturas de SCU. A expressão de integrina caiu, na população de células CD34+ e de CD45+ / Mononuclear cells from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (MPB), were cultured in vitro, in the presence of cytokines, with or without bone marrow stroma. The aims were to evaluate the proliferative response of progenitor cells, occurrence of apoptosis and expression of adhesion molecule. In cultures without stroma, cellularity increased 5-fold for UCB, but has not changed for MPB. The number of CD34+ cells has dropped in both culture. With stroma, total nucleated cells had a 7-fold increse (UCB) and a 2,3-fold (MBP), however, CD34+ cells number has not changed. Apoptosis was lower in UCB culture. The expression of integrin decreased, in the CD34+ and CD45+ population