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Cellular therapies rely on the delivery of therapeutic cells into patients, but their safety can be compromised by the manipulation of cells ex vivo or their placement outside of their natural context in vivo. Cell Fate Control Gene Therapy (CFCGT) offers the possibility of establishing pharmacological controls over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. In its simplest form, 'suicide' gene therapy (SGT), stable introduction of a 'suicide' gene that can activate a non-toxic prodrug establishes control over the survival of GMCs. Current SGT modalities are sub-optimal in clinical setting. To overcome the many limitation of current strategies, we have developed a next-generation CFCGT approach based on the active site-engineered variants of human deoxyCytidine Kinase (dCK), which enable robust activation of multiple Nucleoside Analogue (NA)-based prodrugs, act early in the pathway enabling rapid accumulation of activated NAs in target cells, and also provide the capabilities for the direct imaging of GMCs. Stable introduction of dCK variants into target cells by means of Lentiviral (LV) gene transfer significantly increases their sensitivity to multiple prodrugs. Our dCK variant with only two active site amino acid substitutions is expected to be non-immunogenic yet capable of specifically activating deoxythymidine- and deoxyuridine-based NAs that are not substrates for the wild-type enzyme, such as bromovinyldeoxyuridine (BVdU) and L-deoxythymidine (LdT). We show here that dCK can be used for controlling the survival of GMCs, in cell lines and primary cells in vitro and in a murine xenogeneic transplant models in vivo. To characterize dCK/prodrug-mediated killing mechanisms in GMCs, we have examined the levels of active metabolites in cells and the cellular pathways they antagonize. We describe here the experimental basis for the application of this novel CFCGT in bone marrow transplantation for management of Graft-versus-Host Disease (GvHD) and in enhancing chemotherapy in direct treatment of tumors. In summary, we have developed a novel and robust strategy for effective CFCGT that addresses the many shortcomings of existing modalities. Future studies will validate this novel system in a variety of primary cells and animal disease models, including models of hematopoietic transplantation and ES/iPS-based cell therapies.

Cellular therapies rely on the delivery of therapeutic cells into patients, but their safety can be compromised by the manipulation of cells ex vivo or their placement outside of their natural context in vivo. Cell Fate Control Gene Therapy (CFCGT) offers the possibility of establishing pharmacological controls over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. In its simplest form, 'suicide' gene therapy (SGT), stable introduction of a 'suicide' gene that can activate a non-toxic prodrug establishes control over the survival of GMCs. Current SGT modalities are sub-optimal in clinical setting. To overcome the many limitation of current strategies, we have developed a next-generation CFCGT approach based on the active site-engineered variants of human deoxyCytidine Kinase (dCK), which enable robust activation of multiple Nucleoside Analogue (NA)-based prodrugs, act early in the pathway enabling rapid accumulation of activated NAs in target cells, and also provide the capabilities for the direct imaging of GMCs. Stable introduction of dCK variants into target cells by means of Lentiviral (LV) gene transfer significantly increases their sensitivity to multiple prodrugs. Our dCK variant with only two active site amino acid substitutions is expected to be non-immunogenic yet capable of specifically activating deoxythymidine- and deoxyuridine-based NAs that are not substrates for the wild-type enzyme, such as bromovinyldeoxyuridine (BVdU) and L-deoxythymidine (LdT). We show here that dCK can be used for controlling the survival of GMCs, in cell lines and primary cells in vitro and in a murine xenogeneic transplant models in vivo. To characterize dCK/prodrug-mediated killing mechanisms in GMCs, we have examined the levels of active metabolites in cells and the cellular pathways they antagonize. We describe here the experimental basis for the application of this novel CFCGT in bone marrow transplantation for management of Graft-versus-Host Disease (GvHD) and in enhancing chemotherapy in direct treatment of tumors. In summary, we have developed a novel and robust strategy for effective CFCGT that addresses the many shortcomings of existing modalities. Future studies will validate this novel system in a variety of primary cells and animal disease models, including models of hematopoietic transplantation and ES/iPS-based cell therapies.

Από την δεκαετία του '70 είναι γνωστό ότι οι αλλογενείς μεταγγίσεις αίματος (ABT) προκαλούν ανοσοκαταστολή, ωστόσο, μετά το 1990 έγινε γνωστό ότι για το φαινόμενο αυτό ευθύνεται ο Τ λεμφοκυτταρικός πληθυσμός των ρυθμιστικών κυττάρων (Tregs). Στην παρούσα εργασία, μελετήθηκε η επίδραση της μετάγγισης σε ασθενείς που είχαν υποβληθεί σε ολική ή μερική αρθροπλαστική γόνατος ή ισχίου, στον πληθυσμό των φυσικών (nTreg) και επαγώγιμων (iTreg) Τ ρυθμιστικών κυττάρων. Στους ίδιους ασθενείς μελετήθηκε και η αλλαγή προτύπου στην έκκριση των κυτταροκινών. Συλλέχθηκαν δείγματα ολικού αίματος από 46 ασθενείς, 7 άντρες και 39 γυναίκες. Από αυτούς, οι 36 ασθενείς έλαβαν μετάγγιση (Group1) ενώ οι 10, δεν έλαβαν. Η συλλογή των δειγμάτων έγινε πριν την εγχείρηση (BS), αμέσως μετά το χειρουργείο (Day0), μια εβδομάδα μετά (Day7), ένα μήνα μετά (1month) και κατά τον επανέλεγχο των ασθενών (>3months). Στα δείγματα έγινε απομόνωση των PBMC και καθορίστηκε το ποσοστό των CD4+CD25+Foxp3+ και CD4+CD25high/+CD127low/- Tregs με την μέθοδο FACS ενώ στο πλάσμα καθορίστηκαν τα επίπεδα των κυτταροκινών με τις μεθόδους Cytometric Bead Array (CBA) και ELISA. Επιπρόσθετα, μελετήθηκε η λειτουργικότητας των Treg από δείγμα αίματος ασθενών μέσα στην πρώτη εβδομάδα μετά το χειρουργείο. Καλλιεργήθηκαν διάφοροι λόγοι Tregs: Teff για 72 ώρες, παρουσία PHA και CFSE. Με την μέθοδο Cytometric Bead Array (CBA) καθορίστηκαν τα επίπεδα των κυτταροκινών IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, IFN-γ ενώ με την μέθοδο της ELISA καθορίστηκαν τα επίπεδα των TNF-α και των υποδοχέων TNF-RI(p55/p60) και TNF-RII(p75/p80) καθώς επίσης και οι TGF-β1 και TGF-β2. Από τα πειράματα προέκυψε ότι οι πληθυσμοί τόσο των φυσικών CD4+CD25+Foxp3+ όσο και των επαγώγιμων CD4+CD25high/+CD127low/- Tregs, αυξάνονται μετά το χειρουργείο (day 0), μετά την μετάγγιση, ενώ μειώνονται την πρώτη εβδομάδα μετά το χειρουργείο. Αντίθετα αποτελέσματα παρατηρήθηκαν στους ασθενείς οι οποίοι υποβλήθηκαν σε χειρουργείο αλλά δεν μεταγγίστηκαν. Με τα πειράματα λειτουργικότητας, φάνηκε ότι τα Tregs ήταν λειτουργικά και ικανά να προκαλούν αναστολή του πολλαπλασιασμού των Teff. Σχεδόν όλες οι κυτταροκίνες που αναλύθηκαν, οι IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, IFN-γ, ο υποδοχέας TNF-RI(p55/p60) και TNF-RII(p75/p80) και TGF-β2 εμφάνισαν αύξηση μετά το χειρουργείο, μετά την μετάγγιση. Ωστόσο, μόνο η αύξηση της IL-6, και των υποδοχέων TNF-RI(p55/p60) και TNF-RII(p75/p80) ήταν στατιστικώς σημαντική μετά το χειρουργείο, μετά την μετάγγιση. Τα επίπεδα του TGF-β1 μειώθηκαν μετά το χειρουργείο, μετά την μετάγγιση (Th3 απόκριση). Συμπερασματικά, τα ποσοστά των Tregs αυξήθηκαν στους ασθενείς που υποβλήθηκαν σε αρθροπλαστική και μεταγγίστηκαν. Οι πληθυσμοί των Tregs παρέμειναν αυξημένοι μέχρι και τον πρώτο μήνα μετά το χειρουργείο. Τα Tregs είναι λειτουργικά και ικανά να καταστέλλουν τον πολλαπλασιασμό των Teff, παρουσία PHA. Μετά το χειρουργείο, ύστερα από μετάγγιση, τα επίπεδα των IL-6, TNF-RI και TNF-RII αυξάνονται ως αντίδραση κατά του μοσχεύματος. Μετά την εγχείρηση (day 0) και μετά από μετάγγιση, οι ασθενείς αναπτύσσουν Th1 απόκριση και πολλαπλασιασμό των Tregs. Σταδιακά, τα Tregs καταστέλλουν τις προφλεγμονώδης αποκρίσεις μέχρι να επανέλθει η ισορροπία των ληπτών μετά την εγχείρηση. / Clinical and experimental studies have established that allogeneic blood transfusion (ABT) can cause immunosuppression. In this work we determined whether and to which extend Tregs contribute to this effect. Material and methods: Heparinized peripheral blood samples were collected from 46 patients (7 male and 39 female, age 28-88 years) that underwent joint replacement surgery. The samples were collected immediately before surgery (BS) and after surgery (AS) on days 0, 7, 1 month, and 3 months to 1 year. Thirty six patients received ABT and 10 did not. PBMC were isolated and the numbers and % of CD4+CD25+Foxp3+ Tregs and CD4+CD25high/+CD127low/- Tregs were determined by FACS. Tregs and T effectors (Teff) were isolated from patients on days 0-7 and Treg functional assays were performed by culturing Tregs with PHA-stimulated Teff at different ratios for 72h with CFSE, and analyzed by FACS. Cytokine serum level determined with Cytometric Bead Array (CBA) for IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, IFN-γ and ELISA for TNF-α, the receptor TNF-RI(p55/p60) and II(p75/p80), TGF-β1 and β2. Results: Both, natural (CD4+CD25+Foxp3+) and inducible (CD4+CD25high/+CD127low/-) Tregs increased in day 0 and decreased in day 7 until BS levels, after ABT. Opposite results (small reduction) observed in patients without ABT. With functional assays proved that Tregs are functional and suppress the T-cell proliferation. IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, IFN-γ, the receptor TNF-RI(p55/p60) and II(p75/p80), TGF-β1 and β2 increased after the surgery, after ABT. IL-6, TNF-RI(p55/p60) and II(p75/p80) levels increased with SSD after the surgery, after ABT. TGF-β1 levels decreased in day 0 until BS levels (Th3 response). Th3 cells growth from CD4+CD25- FoxP3- Th0 peripheral cells. Th3 prevents maturation of DCs, Th2 T-cells. Induce the TGF-β secretion and inhibit IL-2 and antibodies secretion. Conclusion: In patients underwent scheduled joint replacement surgery, Tregs increased after ABT until 1 month and are functional and suppress Teff proliferation under PHA condition. IL-6, TNF-RI and TNF-RII are activation markers of immune system and suppressed after ABT. In day 0, IL-6, TNF-RI and II levels increased as a reaction graft against host’s antigen. After surgery (day 0), patients develop Th1 response and Tregs proliferation, after ABT. Gradually, Tregs suppress the proinflammatory responses until the balance in the recipient, after the surgery.

INTRODUÇÃO: O Transplante de Células-tronco Hematopoéticas (TCTH) alogênico é uma modalidade de tratamento com capacidade de curar várias doenças hematológicas benignas e malignas. Os progressos nesta área aumentaram o número e melhoraram os desfechos dos procedimentos realizados, porém sua morbimortalidade permanece elevada. Em 2009, o escore de risco do EBMT foi validado como um método simples para predizer, com dados pré-transplante, os desfechos de um paciente após o TCTH alogênico. OBJETIVOS: O objetivo deste estudo foi validar a aplicabilidade do escore de risco do EBMT em pacientes brasileiros do Hospital de Cínicas de Porto Alegre (HCPA), submetidos a TCTH alogênico. MÉTODOS: Foi realizado um estudo retrospectivo, observacional, com dados coletados de prontuários de 278 pacientes (156 homens (56%) com mediana de idade de 32 anos) submetidos ao TCTH alogênico no HCPA para doenças malignas e anemia aplástica severa, entre 1994 e 2010. Foi aplicado o escore de risco do EBMT e analisados os desfechos sobrevida global (OS), mortalidade não relacionada à recaída (NRM) e taxa de recaída (RR). RESULTADOS: OS, NRM e RR em cinco anos foram de 53,4%, 39% e 30,7%, respectivamente. A OS em pacientes com risco 0 foi significativamente maior (81,8%) do que os de risco 6 (20%) (p<0,001). Da mesma forma, pacientes com risco 0 tiveram menor NRM (13,6%) do que os com risco 6 (80%) (p=0,001). O estágio avançado da doença foi associado com aumento de RR em todas as patologias neoplásicas avaliadas. CONCLUSÃO: O escore de risco do EBMT pode ser utilizado como um dado adicional na avaliação de pacientes com doenças malignas e anemia aplástica severa com indicação de TCTH alogênico no nosso centro. / BACKGROUND: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is a treatment modality able to cure many hematological disorders. Although utilized with increasing frequency and success, it is still associated with a high transplant related mortality rate. In 2009, the EBMT risk score was validated as a simple tool to predict outcome after allogeneic HSCT for acquired hematological disorders. OBJECTIVES: The aim of this study was to validate the applicability of the EBMT risk score for allogeneic HSCT on south Brazilian patients, from a single center. METHODS: A retrospective observational study was performed based on patients’ records and data base of Hematology and Bone Marrow Transplantation Department at Hospital de Clínicas de Porto Alegre, including all allogeneic transplants for malignant and severe aplastic anemia from 1994 to 2010. Patients were categorized according to EBMT risk score and overall survival (OS), non-relapse mortality (NRM) and relapse rate (RR) were analyzed. RESULTS: There were 278 evaluable patients, of whom 56% were male, and the median age was 32 years. OS, NRM, and RR at five years median follow up, were 53,4%, 39%, and 30,7%, respectively. The OS was 81,8% for risk score 0 and 20% for score 6 (p<0,001), and NRM 13,6% and 80% for risk score 0 and 6, respectively (p=0,001). Advanced disease stage was associated with an increased RR in all evaluated neoplastic disorders. CONCLUSION: The EBMT risk score can be utilized as a tool for clinical decision-making before allogeneic HSTC for malignant hematological diseases and severe aplastic anemia at a single center, in Brazil.

Introdução: O transplante de células tronco hematopoiéticas é capaz de curar as doenças hematológicas. O papel da repopulação linfocitária precoce no período pós transplan te visa combater a células neoplásicas que resistiram ao regime de condicionamento e prevenir as infecções oportunistas graves. Sendo assim, uma contagem elevada de linfócitos no período pós transplante é capaz de reduzir a mortalidade relacionada ao transplante (TRM), melhorar a sobrevida livre de doença e reduzir a taxa de recidiva. Objetivos: Avaliar a recuperação linfocitária precoce no D+21 e D+30 pós transplante correlacionado com a taxa de recidiva da doença de base, mortalidade, sobrevida global e livre de doença. Analisar a freqüência das complicações infecciosas neste período. Métodos: Analisado o número absoluto de linfócitos no D+21 e D+30 pós transplante de células tronco hematopoiéticas. Conforme dados da literatura definiu-se no D+21 e no D+30 aqueles com número absoluto de linfócitos abaixo e acima de 300 e se correlacionou os dados obtidos com a taxa de óbito, taxa de recidiva, sobrevida global em 5 anos, sobrevida livre de doença, em 5 anos, TRM em 100 dias. e mortalidade não relacionada a recaída (NRM). Resultados: Neste estudo foram incluídos 100 pacientes portadores das seguintes neoplasias hematológicas: leucemia mielóide aguda, leucemia linfocítica aguda, leucemias secundárias e síndrome mielodisplásica. Destes, 55 pacientes eram do sexo masculino e 45 do sexo feminino. A média de idade foi de 27,9 anos (mínima 9 meses e máxima 55 anos). A mediana do tempo de seguimento foi de 601 dias (IC 95% 106-1845). A mediana de CD 34 infundidos foi de 4,0 (IC 95% 2,4-5,7) e quanto a origem destas células CD 34 infundidas 85% foram de medula óssea (MO), 12% periférica (PBSC) e 3% sangue de cordão umbilical (SCU). Quanto ao tipo de condicionamento realizado 22% foram não mieloablativos e 78% mieloablativos.A mediana de linfócitos no D+21 foi de 460 (IC 95% 0 - 6250) e no D+30 foi de 760 (IC 95% 40-6370). Com relação a taxa de infecções observou-se que 19% das infecções foram de etiologia viral, 65 % bacteriana e 17% fúngicas. A sobrevida global (OS) em 5 anos foi de 44 % , sobrevida livre de doença (DFS) foi de 37,7% , a mortalidade relacionada ao transplante (TRM) em 100 dias foi de 32,5%. E a mortalidade não relacionada a recidiva (NRM) em 5 anos foi de 40,2%. No desfecho óbito observamos que 69% dos pacientes que foram a óbito no D+21 tinham linfócitos abaixo de 300, e 43,9% tinham linfócitos acima de 300 (p<0,05). Pacientes com valores menores que 300 no dia 30 tem 2,20 vezes o risco de irem a óbito quando comparados com aqueles com valores acima de 300 (IC 95% 1,03-4,69) ajustado para DECH e CD34. Pacientes com valores menores que 300 no dia 30 tem 3,76 vezes o risco de irem a óbito em menos de 100 dias quando comparados com aqueles com valores acima de 300 (IC 95% 1,23-11,46) Conclusões: A reconstituição linfocitária precoce (> 300) no D+21 e no D+30 melhora a sobrevida global e livre de doença, bem como reduz a taxa de recidiva da doença de base e reduz a mortalidade. / Background: The role of repopulating lymphocyte after allogenic stem cell transplantation (SCT) includes the prevention of serious infections and attacking residual tumor cells in the early post transplant phase. Therefore, the current study analysed the role of the absolute lymphocyte count (ALC) on day 21 and 30 after SCT in predicting transplant outcomes of patients in terms of the risk of transplant related mortality (TRM) recurrence of original disease and risk of opportunistic infections. Objective: Evaluate early lymphocyte recovery on D +21 and D +30 posttransplant correlated with the rate of recurrence of the underlying disease, mortality, overall survival and disease free survival. Analyzed the frequency of infectious complications in this period. Methods: Analyzed the absolute lymphocyte count in the D +21 and D +30 after hematopoietic stem cell transplantation. According to literature data set the we correlate the absolute lymphocyte count in the D +21D +30 below and above 300 these data with the rate of death, relapse rate, overall survival in 5 years, disease-free survival in 5 years , TRM in 100 days and mortality unrelated to relapse (NRM). Results : Included in the study 100 patients with the following hematologic malignancies: acute myeloid leukemia, acute lymphocytic leukemia, secondary leukemia and myelodysplastic syndrome. Of these, 55 patients were male and 45 female. The average age was 27.9 years (minimum 9 months and maximum 55 years). The median follow-up was 601 days (95% CI 106-1845). The CD 34 median that was infused was 4.0 (95% CI 2.4 to 5.7).The source of stem cells infused was 85% of bone marrow (BM), peripheral 12% (PBSC) and 3 % of umbilical cord blood (UCB). Regarding the type of conditioning performed 22% were non myeloablative and 78% of lymphocytes were mieloablativos. The median of absolute lymphocyte count in the D +21 was 460 (95% CI 0 to 6250) and D +30 was 760 (95% CI 40- 6370 ). Regarding the rate of infections were observed 19% viral infections , bacterial in 65% and fungal in 17%. Overall survival (OS) at 5 years was 44%, disease-free survival (DFS) was 37.7%, transplant related mortality (TRM) in 100 days was 32.5%. Non relapsed mortality (NRM) at 5 years was 40.2%. The death rate found that 69% of patients who died at the D +21 had presented lymphocytes count below 300, and 43.9% were above 300 lymphocytes (p <0.05). Patients with counts less than 300 in D+30 presented 2.20 times risk of death when compared with those who presented values above 300 (95% CI 1.03 to 4.69) adjusted for GVHD and CD34. Patients presenting values less than 300 in 30 days have 3.76 times more risk of death in less than 100 days compared with those with values above 300 (95% CI 1.23 to 11.46). Conclusions: The early lymphocyte reconstitution (> 300) in D +21 D +30 improves overall survival and disease-free and reduces the relapse rate of the underlying disease and reduces mortality.

Orientador: Ana Liz Garcia Alves / Resumo: O tendão flexor digital superficial (TFDS) é uma estrutura importante para a espécie equina e para animais de alto desempenho atlético. Atualmente, a medicina regenerativa vem evoluindo de forma significativa no tratamento de diversas enfermidades, no entanto, o entendimento a respeito do comportamento celular quando do transplante de células tronco mesenquimais (CTMs) em tecido hígido ainda não é completamente conhecido. Sendo assim, este trabalho tem como objetivo a avaliação da resposta inflamatória local após aplicação de células tronco mesenquimais alogênicas derivadas do tecido adiposo (AdCTMs) em tendão equino comparado ao transplante autólogo e ao grupo controle. Para isso, 18 membros torácicos de 9 animais, divididos em três grupos, foram submetidos à aplicação de AdCTMs em tendão hígido, sendo que em Galog os animais receberam aplicação de AdCTMs alogênicas no membro torácico (MT); em Gauto, aplicação de células autólogas no MT e em Gcont aplicou-se o PBS como grupo controle. Esses animais foram avaliados por parâmetros físicos, exames ultrassonográficos e termográficos até o momento da biópsia, que foi realizada uma semana após aplicação das células autólogas e alogênicas para avaliar a possível reação inflamatória aguda, decorrente do implante de células no tecido tendíneo. O transplante alogênico de CTMs não resultou em reação adversa ou inflamatória que comprometesse o uso dessas células, elucidando a sua segurança neste trabalho. O seu comportamento mostrou-se ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The superficial digital flexor tendon (SDFT) is an important structure for the equine species and for high performance animals. Currently, regenerative medicine has evolved significantly in the treatment of several diseases, even though the cellular behavior during the implantation of mesenchymal stem cells in healthy tissue is not completely known, despite the fact that it has been tirelessly studied in the last few years. This study aims to evaluate and compare the local inflammatory response upon injection in equine tendon of either allogeneic and autologous mesenchymal stem cells derived from adipose tissue (AdMSC). For this purpose, 18 forelimbs, 9 animals were submitted to the application of AdMSC in healthy tendon, which were divided into 3 groups: in Galog the animals received application of allogeneic AdMSC in the thoracic member; in Gauto, autologous AdMSC were injected in the thoracic member; in Gcont, PBS was implanted as control group. These animals were evaluated through physical exams, ultrasound, and thermography up until the moment of the biopsy, which was performed a week after the application of AdMSC in order to evaluate the inflammatory response due to the implantation of the cells in the tendinous tissue. Biopsy samples were taken to histopathology and immunohistochemistry in order to evaluate the actions of allogeneic AdMSC. The allogeneic MSCs implantation did not result in adverse or inflammatory reaction which could compromise the use of these cells,... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-12-17T15:30:54Z (GMT). No. of bitstreams: 1 PedroHASL_Dissret.pdf: 4618668 bytes, checksum: 5bca73a0220258bdbd17a75573adb995 (MD5) Previous issue date: 2009-08-31 / Oral and facial bone defects can undertake appearance, psychosocial well-being and stomathognatic function of its patients. Over the yerars several strategies for bone defect regeneration have arised to treat these pathologies, among them the use of frozen and irradiated bone allograft. Manipulation of bone grafts it s not determined yet, and several osteotomy alternatives can be observed. The present work evaluated with a microscope the bone fragments obtained from different osteotomy methods and irrigation on rings and blocks allografts irradiated and frozen at 80? negative in a rabbit model. The study is experimental in vitro and it sample was an adult male New Zealand rabbit. The animal was sacrificed to obtain long bones, that were submitted to freezing at 80? negative and irradiated with Cobalt- 60. Then the long bones were sectioned into 24 bone pieces, divided into 4 groups: G1 (n=06) osteotomy was performed with bur No. 6 forming rings with 5 mm thickness with high-speed handpiece with manual irrigation; G2 (n=06) osteotomy was performed with bur No. 6 forming rings with 5 mm thick with surgical motor with a manual irrigation rotation 1500 rpm; GA (n=06), osteotomy with trephine using manual irrigation with saline; and GB (n=06), osteotomy with trephine using saline from peristaltic pumps of surgical motor. Five bone pieces of each group were prepared for analysis on light microscopy (LM) and one on electronic scan electronic microscopy (SEM). On the SEM analysis edges surface, presence of microcracks and Smear Layer were evaluated. Analyzing osteotomy technics on SEM was observed: increased presence of microcracks cutting with high speed; increased presence of areas covered by Smear Layer when cutting with motor implant. The irrigation analysis with SEM was observed: that the presence of microcracks does not depend on the type of irrigation; on manual irrigation, there was greater discrepancy between the cutting lines. The descriptive analysis of the osteotomy and irrigation process on LM showed: histological analysis showing the bony margins with clear tissue changed layer, composed of blackened tissue of charred appearance near to the cortical bone; on the edges of the bony part, bone fragments that were displaced during the bone cut and bone irregularities were observed. After analysis of results we can conclude: that there was greater regularity of the bone cut using high-speed handpiece than using motor implant; the cut with trephine using saline irrigated from peristaltic pumps of surgical motor showed greater homogeneity when compared with manual irrigation; charred tissue was found in all obtained bone samples, whit no significant statistically difference on the proportion of carbonization of the two analysed technics / Os defeitos ?sseos bucais e faciais comprometem a apar?ncia, o bem estar psicossocial e a fun??o estomatogn?tica dos seus portadores. Diante da necessidade de tratamento dessas patologias, surgiram, com o decorrer do tempo, diversas estrat?gias para a regenera??o de defeitos ?sseos, dentre elas o uso de aloenxerto ?sseo congelado e irradiado. A manipula??o dos enxertos ?sseos ainda n?o est? determinada, podendo-se observar v?rias alternativas de osteotomia. Este trabalho avaliou microscopicamente os fragmentos ?sseos obtidos por diferentes m?todos de osteotomia e de irriga??o sobre an?is e blocos de aloenxertos irradiados e congelados a 80?C negativos de coelho. O estudo ? do tipo Experimental in vitro e teve como amostra 01 coelho, adulto, da ra?a New Zealand. O animal foi sacrificado para obten??o de ossos longos, os quais foram congelados a 80? negativos e irradiados com Cobalto-60. Em seguida, os ossos foram seccionados em 24 pe?as ?sseas, divididos em quatros grupos: G1 (n=06) foi realizado a osteotomia com broca esf?rica n? 6 formando an?is com 5 mm de espessura com caneta de alta rota??o com irriga??o manual; G2 (n=06) foi realizado a osteotomia com broca esf?rica n? 6 formando an?is com 5 mm de espessura com motor cir?rgico com irriga??o manual a uma rota??o de 1500 rpm; GA (n=06), osteotomia com trefina usando irriga??o manual com soro fisiol?gico; e GB (n=06), osteotomia com trefina usando soro fisiol?gico proveniente de bombas perist?lticas do motor cir?rgico. De cada grupo, cinco pe?as ?sseas foram destinadas ? an?lise em Microscopia de Luz (ML) e uma analisada por Microscopia Eletr?nica de Varredura (MEV). A an?lise por ML levou em considera??o a presen?a de tecido carbonizado. Na an?lise por MEV levou em considera??o: superf?cie das bordas; presen?a de microfissuras e Smear Layer. Ao analisar a t?cnica de osteotomia utilizando MEV observa-se: maior presen?a de microfissuras ao corte com alta rota??o; maior presen?a de ?reas cobertas por uma camada de smear layer em cortes com motor de implante. Na analise da irriga??o com MEV observou-se que: a presen?a de microfissuras n?o depende do tipo de irriga??o; na irriga??o manual, verificou-se uma discrep?ncia maior entre as linhas de corte. Ao avaliar descritivamente o processo de osteotomia e irriga??o na ML, verificou-se que: na an?lise histol?gica as margens ?sseas apresentavam uma evidente camada alterada de tecido, composta por um tecido enegrecido de aspecto carbonizado, pr?ximo ao osso cortical; nas margens da pe?a ?ssea foram observados fragmentos ?sseos deslocados durante o corte ?sseo e irregularidades ?sseas. Ap?s an?lise dos resultados, pode-se concluir que: houve maior regularidade do corte ?sseo utilizando caneta de alta rota??o do que motor de implante; o corte com trefina usando irriga??o com bombas perist?lticas do motor de implante se mostrou mais homog?nea quando comparada ? t?cnica com irriga??o manual; tecido carbonizado foi encontrado em todos os esp?cimes ?sseos obtidos, sem diferen?a estatisticamente significativa na propor??o de carboniza??o nas duas t?cnicas de irriga??o estudadas

INTRODUÇÃO: O Transplante de Células-tronco Hematopoéticas (TCTH) alogênico é uma modalidade de tratamento com capacidade de curar várias doenças hematológicas benignas e malignas. Os progressos nesta área aumentaram o número e melhoraram os desfechos dos procedimentos realizados, porém sua morbimortalidade permanece elevada. Em 2009, o escore de risco do EBMT foi validado como um método simples para predizer, com dados pré-transplante, os desfechos de um paciente após o TCTH alogênico. OBJETIVOS: O objetivo deste estudo foi validar a aplicabilidade do escore de risco do EBMT em pacientes brasileiros do Hospital de Cínicas de Porto Alegre (HCPA), submetidos a TCTH alogênico. MÉTODOS: Foi realizado um estudo retrospectivo, observacional, com dados coletados de prontuários de 278 pacientes (156 homens (56%) com mediana de idade de 32 anos) submetidos ao TCTH alogênico no HCPA para doenças malignas e anemia aplástica severa, entre 1994 e 2010. Foi aplicado o escore de risco do EBMT e analisados os desfechos sobrevida global (OS), mortalidade não relacionada à recaída (NRM) e taxa de recaída (RR). RESULTADOS: OS, NRM e RR em cinco anos foram de 53,4%, 39% e 30,7%, respectivamente. A OS em pacientes com risco 0 foi significativamente maior (81,8%) do que os de risco 6 (20%) (p<0,001). Da mesma forma, pacientes com risco 0 tiveram menor NRM (13,6%) do que os com risco 6 (80%) (p=0,001). O estágio avançado da doença foi associado com aumento de RR em todas as patologias neoplásicas avaliadas. CONCLUSÃO: O escore de risco do EBMT pode ser utilizado como um dado adicional na avaliação de pacientes com doenças malignas e anemia aplástica severa com indicação de TCTH alogênico no nosso centro. / BACKGROUND: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is a treatment modality able to cure many hematological disorders. Although utilized with increasing frequency and success, it is still associated with a high transplant related mortality rate. In 2009, the EBMT risk score was validated as a simple tool to predict outcome after allogeneic HSCT for acquired hematological disorders. OBJECTIVES: The aim of this study was to validate the applicability of the EBMT risk score for allogeneic HSCT on south Brazilian patients, from a single center. METHODS: A retrospective observational study was performed based on patients’ records and data base of Hematology and Bone Marrow Transplantation Department at Hospital de Clínicas de Porto Alegre, including all allogeneic transplants for malignant and severe aplastic anemia from 1994 to 2010. Patients were categorized according to EBMT risk score and overall survival (OS), non-relapse mortality (NRM) and relapse rate (RR) were analyzed. RESULTS: There were 278 evaluable patients, of whom 56% were male, and the median age was 32 years. OS, NRM, and RR at five years median follow up, were 53,4%, 39%, and 30,7%, respectively. The OS was 81,8% for risk score 0 and 20% for score 6 (p<0,001), and NRM 13,6% and 80% for risk score 0 and 6, respectively (p=0,001). Advanced disease stage was associated with an increased RR in all evaluated neoplastic disorders. CONCLUSION: The EBMT risk score can be utilized as a tool for clinical decision-making before allogeneic HSTC for malignant hematological diseases and severe aplastic anemia at a single center, in Brazil.

Introdução: O transplante de células tronco hematopoiéticas é capaz de curar as doenças hematológicas. O papel da repopulação linfocitária precoce no período pós transplan te visa combater a células neoplásicas que resistiram ao regime de condicionamento e prevenir as infecções oportunistas graves. Sendo assim, uma contagem elevada de linfócitos no período pós transplante é capaz de reduzir a mortalidade relacionada ao transplante (TRM), melhorar a sobrevida livre de doença e reduzir a taxa de recidiva. Objetivos: Avaliar a recuperação linfocitária precoce no D+21 e D+30 pós transplante correlacionado com a taxa de recidiva da doença de base, mortalidade, sobrevida global e livre de doença. Analisar a freqüência das complicações infecciosas neste período. Métodos: Analisado o número absoluto de linfócitos no D+21 e D+30 pós transplante de células tronco hematopoiéticas. Conforme dados da literatura definiu-se no D+21 e no D+30 aqueles com número absoluto de linfócitos abaixo e acima de 300 e se correlacionou os dados obtidos com a taxa de óbito, taxa de recidiva, sobrevida global em 5 anos, sobrevida livre de doença, em 5 anos, TRM em 100 dias. e mortalidade não relacionada a recaída (NRM). Resultados: Neste estudo foram incluídos 100 pacientes portadores das seguintes neoplasias hematológicas: leucemia mielóide aguda, leucemia linfocítica aguda, leucemias secundárias e síndrome mielodisplásica. Destes, 55 pacientes eram do sexo masculino e 45 do sexo feminino. A média de idade foi de 27,9 anos (mínima 9 meses e máxima 55 anos). A mediana do tempo de seguimento foi de 601 dias (IC 95% 106-1845). A mediana de CD 34 infundidos foi de 4,0 (IC 95% 2,4-5,7) e quanto a origem destas células CD 34 infundidas 85% foram de medula óssea (MO), 12% periférica (PBSC) e 3% sangue de cordão umbilical (SCU). Quanto ao tipo de condicionamento realizado 22% foram não mieloablativos e 78% mieloablativos.A mediana de linfócitos no D+21 foi de 460 (IC 95% 0 - 6250) e no D+30 foi de 760 (IC 95% 40-6370). Com relação a taxa de infecções observou-se que 19% das infecções foram de etiologia viral, 65 % bacteriana e 17% fúngicas. A sobrevida global (OS) em 5 anos foi de 44 % , sobrevida livre de doença (DFS) foi de 37,7% , a mortalidade relacionada ao transplante (TRM) em 100 dias foi de 32,5%. E a mortalidade não relacionada a recidiva (NRM) em 5 anos foi de 40,2%. No desfecho óbito observamos que 69% dos pacientes que foram a óbito no D+21 tinham linfócitos abaixo de 300, e 43,9% tinham linfócitos acima de 300 (p<0,05). Pacientes com valores menores que 300 no dia 30 tem 2,20 vezes o risco de irem a óbito quando comparados com aqueles com valores acima de 300 (IC 95% 1,03-4,69) ajustado para DECH e CD34. Pacientes com valores menores que 300 no dia 30 tem 3,76 vezes o risco de irem a óbito em menos de 100 dias quando comparados com aqueles com valores acima de 300 (IC 95% 1,23-11,46) Conclusões: A reconstituição linfocitária precoce (> 300) no D+21 e no D+30 melhora a sobrevida global e livre de doença, bem como reduz a taxa de recidiva da doença de base e reduz a mortalidade. / Background: The role of repopulating lymphocyte after allogenic stem cell transplantation (SCT) includes the prevention of serious infections and attacking residual tumor cells in the early post transplant phase. Therefore, the current study analysed the role of the absolute lymphocyte count (ALC) on day 21 and 30 after SCT in predicting transplant outcomes of patients in terms of the risk of transplant related mortality (TRM) recurrence of original disease and risk of opportunistic infections. Objective: Evaluate early lymphocyte recovery on D +21 and D +30 posttransplant correlated with the rate of recurrence of the underlying disease, mortality, overall survival and disease free survival. Analyzed the frequency of infectious complications in this period. Methods: Analyzed the absolute lymphocyte count in the D +21 and D +30 after hematopoietic stem cell transplantation. According to literature data set the we correlate the absolute lymphocyte count in the D +21D +30 below and above 300 these data with the rate of death, relapse rate, overall survival in 5 years, disease-free survival in 5 years , TRM in 100 days and mortality unrelated to relapse (NRM). Results : Included in the study 100 patients with the following hematologic malignancies: acute myeloid leukemia, acute lymphocytic leukemia, secondary leukemia and myelodysplastic syndrome. Of these, 55 patients were male and 45 female. The average age was 27.9 years (minimum 9 months and maximum 55 years). The median follow-up was 601 days (95% CI 106-1845). The CD 34 median that was infused was 4.0 (95% CI 2.4 to 5.7).The source of stem cells infused was 85% of bone marrow (BM), peripheral 12% (PBSC) and 3 % of umbilical cord blood (UCB). Regarding the type of conditioning performed 22% were non myeloablative and 78% of lymphocytes were mieloablativos. The median of absolute lymphocyte count in the D +21 was 460 (95% CI 0 to 6250) and D +30 was 760 (95% CI 40- 6370 ). Regarding the rate of infections were observed 19% viral infections , bacterial in 65% and fungal in 17%. Overall survival (OS) at 5 years was 44%, disease-free survival (DFS) was 37.7%, transplant related mortality (TRM) in 100 days was 32.5%. Non relapsed mortality (NRM) at 5 years was 40.2%. The death rate found that 69% of patients who died at the D +21 had presented lymphocytes count below 300, and 43.9% were above 300 lymphocytes (p <0.05). Patients with counts less than 300 in D+30 presented 2.20 times risk of death when compared with those who presented values above 300 (95% CI 1.03 to 4.69) adjusted for GVHD and CD34. Patients presenting values less than 300 in 30 days have 3.76 times more risk of death in less than 100 days compared with those with values above 300 (95% CI 1.23 to 11.46). Conclusions: The early lymphocyte reconstitution (> 300) in D +21 D +30 improves overall survival and disease-free and reduces the relapse rate of the underlying disease and reduces mortality.