Indiana University-Purdue University Indianapolis (IUPUI) / Graft-versus-host disease (GVHD) remains the major complication after allogeneic hematopoietic stem cell transplantation (HSCT), resulting from immunological attack on target organs such as gastrointestinal (GI) tract, liver and skin from donor allogeneic T cells. The most common treatment for GVHD is immunosuppressive drugs such as corticosteroids, which may result in many side effects including the loss of the beneficial graft-versus-leukemia (GVL) effect and increased infection rates. However, GVHD-specific drugs have yet to be implemented. Here we show that by targeting on a novel pathogenic CD4+ T cell subpopulation that our lab previously found in patients with GI GVHD, we can develop new avenues to treat GVHD. This novel population is characterized as CD146+CCR5+ T cells, co-expressing IL-17A and IFN-γ. We found that the inducible T-cell costimulator (ICOS), which has been reported to be important for human Th17 differentiation in vitro, is critical for the development of this nonconventional T Helper 1 (Th1*)-polarized CD146+CCR5+ conventional T cells (Tconvs) population. Furthermore, we found that ICOS can induce the generation of Th1*-polarized CD146+CCR5+ regulatory T cells (Tregs) population, lowering the frequencies of phenotypic markers of functional Tregs. Our data also showed that inhibiting the major transcriptional factor of Th17, RAR-related orphan receptor gamma t (RORγt), could prevent the development of CD146+CCR5+ Tconvs in vitro. Our results demonstrate how pathogenic CD146+CCR5+ T cells are induced through ICOS or RORγt, suggesting new targets for GVHD treatment. We anticipate our assay to be a starting point for the development of novel GVHD-specific drugs. For example, the treatments that focus on inhibiting RORγ would have fewer side effects than general immunosuppressive drugs that GVHD patients use today and inhibit GVHD while sparing the GVL effect. Furthermore, we expect the CD146+CCR5+ Tconvs and/or Tregs can be used as GVHD biomarkers. These biomarkers may guide preemptive treatments such as RORγt inhibitor.

Allogeneic haematopoietic stem cell transplantation (alloHSCT) is an established therapy for many haematological disorders. Unfortunately, the new donor-derived immune system may damage host cells (graft-versus-host disease (GvHD)), causing significant morbidity and mortality. Since regulatory T cells (Tregs) can modulate immune responses, it was hypothesised that Treg numbers in the haematopoietic stem cell grafts and/or peripheral blood may influence the development of GvHD and other transplant-related complications. In this project, a prospective observational clinical study of putative Tregs in human alloHSCT was performed in Oxford. Flow cytometry and methylation-specific qPCR assays were developed to quantify putative Tregs and lymphocyte populations within the grafts and post-transplant blood samples. Although low CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell numbers were not associated with increased incidence of GvHD, low proportions of CD25(+)FOXP3(+)CD127(-/dim) cells in the graft (as a percentage of total CD4(+) T cells) were independently associated with poor engraftment, increased non-relapse mortality and inferior overall survival. Similarly, falling CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell counts over the first three months post-transplant were associated with higher non-relapse mortality and inferior overall survival. In view of these novel findings, strategies that increase CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T cells in alloHSCT may improve clinical outcomes. One possible route for increasing Tregs is through cellular therapy. This project therefore tested the hypothesis that CD4(+)CD25(+)FOXP3(+) Tregs can be produced in vitro from conventional CD4(+) T cells. In the presence of TGFβ and Azacitidine, FOXP3 was expressed in the majority of activated CD4(+) T cells. These cells also had a demethylated FOXP3 TSDR enhancer which is specific to natural Tregs. However, most of these cells produced pro-inflammatory cytokines, for example, TNFα. Therefore, under these conditions, FOXP3 expression was not sufficient to produce a Treg phenotype. It is proposed that current focus for generating Tregs for human clinical trials should be directed towards improving isolation and expansion of ex vivo isolated Tregs.

Introduction: Chronic graft-versus-host disease (cGVHD) is a serious complication following allo-HSCT characterized by immune dysregulation, organ dysfunction, risk for infection, and distressing symptoms. Complications may include scleroderma, hepatic dysfunction and bronchiolitis obliterans. Advances in allo-HSCT for many hematologic dyscrasias (e.g. acute and chronic leukemias, aplastic anemia, and myelodysplastic syndrome) have improved survival which has generated a renewed focus on survivorship issues. Distressing symptoms are noted as negatively impacting quality of life (QoL). The relationship between inflammation and behavioral responses may impact symptoms. Examining patterns and levels of inflammation with symptoms is relevant. Objective: The aims of this study were to examine: 1) levels of symptoms (cGVHD specific, general symptoms, and cluster symptoms [pain, depression and fatigue]), inflammation (cytokines [Interleukin {IL}-1β, IL-6, IL-10, TNF, and INF-γ] and C-reactive protein [CRP]) and QoL in patients diagnosed with cGVHD and 2) relationships between and among symptoms, inflammation and QoL in individuals with cGVHD. Methods: A cross-sectional study design examined 24 individuals (ages 29-79) with cGVHD enrolled from an NCI-designated cancer center after obtaining informed consent. Data were collected using medical record and validated questionnaires. Plasma cytokine levels were measured using BioRad® multiplex assay. C-reactive protein levels were measures using an enzyme-linked immunosorbant assay. Statistical analyses included descriptive statistics and pairwise correlations. Results: A total of 24 participants (58.3% female) with cGVHD enrolled in this study. Multiple, concurrent symptoms were noted. Several pro-inflammatory cytokines were higher in participants with symptoms versus those without symptoms. IL-6 correlated with lack of energy (r= .42; p= .04) and dry mouth (r= .42; p= .04). IL-10 was correlated with difficulty sleeping (r= .43; p= .03). Sexual dysfunction correlated with social well-being (r= -.44; p=.03). Many symptoms negatively correlated with QoL. Conclusion: Findings from this study, one of the first to examine levels of symptoms and inflammatory markers in individuals with cGVHD, demonstrate significant relationships among symptoms, inflammation, and quality of life. The relationship of inflammatory biomarkers with symptoms emphasizes the need for further interdisciplinary research. Better understanding mechanisms associated with symptoms is necessary for the development and testing of targeted interventions to improve QoL for individuals with cGVHD.

Doctor of Philosophy / Department of Anatomy and Physiology / Mark L. Weiss / Graft Versus Host Disease (GVHD) is the major complication following hematopoietic stem cell transplantation. GVHD is activated by immunocompetent T cells presented in the donor grafted tissue. Due to the increased use of bone marrow transplantation to treat diverse malignancies, the incidence of GVHD has shown a notable increase. Depending of the degree of immunological mismatch between donor and host, 50-70% of patients develop GVHD after allogeneic Bone Marrow Transplantation (BMT). Once GVHD develops, mortality reaches up to 50% in humans. Several studies using Mesenchymal Stromal Cells (MSCs) to prevent and treat GVHD have produced controversial results. It is thought that distinct MSCs sources used in those studies might be an important factor that produces different outcomes. For cellular therapy, the most attractive characteristics of MSCs are their reduced immunogenic potential, and their abilities to modulate immune responses. This dissertation addressed the hypothesis that Wharton’s jelly cells (WJCs) would prevent the pathology and death associated with GVHD after BMT. To accomplish this, I created a clinically relevant model of GVHD by transplanting allogeneic bone marrow across minor histocompatibility antigen (HA) barriers in the rat. To enhance alloreactive T-cell stimulation, bone marrow (BM) was co-administered with a fraction of CD8[superscript]+ cells magnetically selected from spleen to induce GVHD. Bone marrow tissue was isolated from a donor rat Fischer 344 (F344, RT1lv) and transplanted into lethally irradiated (10 Gray) Lewis rat (LEW, RT1l). Once GVHD was induced, MSCs derived from umbilical cord WJCs were either co-transplanted at day 0 with bone marrow, or given on day 2 post-BMT intravenously. The prophylactic potential of WJCs in an in vivo GVHD model was assessed as survival time, clinical symptomatology occurrence, and histopathology injuries in target tissues. Results indicate that while co-administration of WJCs with hematopoietic cells on day 0 failed to alleviate GVHD associated symptomatology and mortality. WJCs administered on day 2 post-induction ameliorated GVHD-associated symptomatology, improved engraftment and survival.

Orientador: Ana Gabriela Sálvio / Resumo: A Doença Enxerto Contra o Hospedeiro (DECH) é uma das principais complicações em pacientes pós transplante de células tronco hematopoiéticas (TCTH), onde as células T do doador agridem os tecidos do receptor resultando em uma das principais causas de mortalidade e morbidade. Ocorre em cerca de metade dos pacientes que realizaram transplante de células tronco hematopoiéticas (TCTH). A pele é um dos órgãos mais acometidos. A DECH é dividida da forma aguda (DECH-a) e crônica (DECH-c), de acordo com o tempo de aparecimento dos achados clínicos e histopatológicos. A DECH de pele é classificada em graus variados, de I a IV, de acordo com o comprometimento. O presente estudo teve como objetivo avaliar a incidência de DECH-a cutânea entre os pacientes submetidos ao TCTH alogênico, aparentado, no Serviço de Transplante de Medula Óssea do Hospital Amaral Carvalho em Jaú/SP. Também foi objetivo do estudo a análise do perfil destes pacientes, a incidência e o impacto da doença na mortalidade precoce nos 100 primeiros dias pós TCTH. Os procedimentos metodológicos envolveram análise retrospectiva de dados retirados de planilhas do setor administrativo do Serviço de Transplante de Medula Óssea do Hospital Amaral Carvalho em Jaú/SP, contendo informações de 1113 pacientes que estiveram internados para a realização de transplante de medula óssea alogênico, aparentado, no período de agosto de 1996 a dezembro de 2013. Foram incluídos 582 pacientes cujas as doenças eram de linhagem mielóide - as ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Submitted by ANA LETICIA SGAVIOLLI null (ana.lsgaviolli@sp.senac.br) on 2016-10-25T21:37:48Z No. of bitstreams: 1 Submissão - 25.10.16.pdf: 954213 bytes, checksum: f27af9c710378005f768acd829e6aae3 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-11-01T19:55:43Z (GMT) No. of bitstreams: 1 serignolli_als_me_bot.pdf: 954213 bytes, checksum: f27af9c710378005f768acd829e6aae3 (MD5) / Made available in DSpace on 2016-11-01T19:55:43Z (GMT). No. of bitstreams: 1 serignolli_als_me_bot.pdf: 954213 bytes, checksum: f27af9c710378005f768acd829e6aae3 (MD5) Previous issue date: 2016-09-02 / A Doença Enxerto Contra o Hospedeiro (DECH) é uma das principais complicações em pacientes pós transplante de células tronco hematopoiéticas (TCTH), onde as células T do doador agridem os tecidos do receptor resultando em uma das principais causas de mortalidade e morbidade. Ocorre em cerca de metade dos pacientes que realizaram transplante de células tronco hematopoiéticas (TCTH). A pele é um dos órgãos mais acometidos. A DECH é dividida da forma aguda (DECH-a) e crônica (DECH-c), de acordo com o tempo de aparecimento dos achados clínicos e histopatológicos. A DECH de pele é classificada em graus variados, de I a IV, de acordo com o comprometimento. O presente estudo teve como objetivo avaliar a incidência de DECH-a cutânea entre os pacientes submetidos ao TCTH alogênico, aparentado, no Serviço de Transplante de Medula Óssea do Hospital Amaral Carvalho em Jaú/SP. Também foi objetivo do estudo a análise do perfil destes pacientes, a incidência e o impacto da doença na mortalidade precoce nos 100 primeiros dias pós TCTH. Os procedimentos metodológicos envolveram análise retrospectiva de dados retirados de planilhas do setor administrativo do Serviço de Transplante de Medula Óssea do Hospital Amaral Carvalho em Jaú/SP, contendo informações de 1113 pacientes que estiveram internados para a realização de transplante de medula óssea alogênico, aparentado, no período de agosto de 1996 a dezembro de 2013. Foram incluídos 582 pacientes cujas as doenças eram de linhagem mielóide - as mielopatias malignas (Leucemia Mieloide Aguda, Leucemia Mieloide Crônica, Síndrome Mielodisplásica e Doença Mieloproliferativa Crônica) para que o grupo pesquisado se tornasse homogêneo em termos de drogas e condicionamento. Esses 582 pacientes englobaram 2 grupos: pacientes que apresentaram DECH-a exclusiva de pele e pacientes que não apresentaram nenhum tipo de DECH. Todos os dados foram revisados e submetidos a análise estatística univariada e multivariada. Na análise univariada, observa-se maior sobrevida em 10 anos para os indivíduos com DECH-a de pele grau I e II (p=0.039) e para os transplantados com células de Medula Óssea (0.018). Essa significância estatística foi mantida na análise multivariada. A principal causa da mortalidade nos primeiros 100 primeiros dias do transplante foi a recidiva da doença de base, sendo a DECH aguda responsável por 6% dos óbitos nesse período. Não houve diferença significativa para os outros dados pesquisados. Conclui-se então que uma escolha adequada de doadores, além do uso de fonte de medula óssea como principal escolha para realização de transplante de medula óssea, podem diminuir a incidência de DECH-a de pele, além de aumentar a sobrevida dos pacientes transplantados. / Graft versus host disease (GVHD) is a major complication occurred in patients posttransplantation hematopoietic stem cell transplantation (HSCT), where the donor's T cells attack the recipient's tissues resulting in a major cause of mortality and morbidity.This is a recurrent and severe event, which occurs in about half of patients who underwent hematopoietic stem cell transplantation (HSCT), the skin is one of the most affected organs. GVHD is divided as acute (GVHD) and chronic (chronic GVHD), according to the time, clinical and histopathological findings. This study aimed to evaluate the incidence of GVHDskin among patients undergoing allogeneic HSCT, akin in Marrow Transplant Service Bone Amaral Carvalho Hospital, Jaú / SP, the profile of this patient and the impact of the disease in early mortality in the first 100 days after HSCT. The methodological procedures involved retrospective analysis of data from spreadsheets in the administrative sector of the Bone Marrow Transplant Service, Hospital Amaral Carvalho - Jau, containing information of 1,113 patients who were hospitalized for performing allogeneic bone marrow transplantation, related in the period August 1996 to December 2013. to equalize the analysis were included only 582 patients whose diseases were of myeloid lineage - malignant myelopathy (Acute myeloid Leukemia, Chronic myeloid Leukemia, Myelodysplastic Syndrome and Chronic myeloproliferative disease) so that the study group became homogeneous in terms of drugs and conditioning. These 582 patients were divided into 2 groups: patients with GVHDexclusive skin and patients without any GVHD. All data were reviewed and subjected to statistical analysis. In univariate analysis, there is greater survival at 10 years for patients with GVHD to skin grade I and II (p = 0.039) and the transplanted cells Bone Marrow (0018). This significance was maintained in multivariate analysis. The main cause of mortality in the first 100 early days of transplantation was the recurrence of the underlying disease, and acute GVHD responsible for 6% of deaths in this period. There was no significant difference for the other surveyed data. It was concluded that a suitable choice of donors, and the use of bone marrow source as the primary choice for bone marrow transplantation, can reduce the incidence of GVHD, the skin, and increase the survival rate of transplant patients.

Mesenchymal stem cells and their regenerative and immunomodulatory potential Abstract Mesenchymal stem cells (MSCs) possess multidirectional regenerative ability, which, together with their immunomodulatory potential, makes them promising cell type for therapy of wide variety of diseases. Despite ongoing research, which proved MSCs application to be safe, reported effect of MSCs administration on patients is not convincingly beneficial yet. In our work we focused on elucidation of MSCs role in regeneration of vital organs, heart and liver, where a large damage is life threatening for patients and any improvement in therapy would save many lives. Similar situation is in Graft versus host disease (GVHD), where MSCs immunomodulatory properties could be beneficial. Role of MSCs in heart regeneration was examined in vitro. Primary adult swine cardiomyocytes (CMCs) were co-cultured with or without swine MSCs for 3 days and morphological and functional parameters (contractions, current, respiration) of CMCs were measured. MSCs showed supportive effect on CMCs survival, especially at day 3 of the experiment, where in co-culture was significantly higher number of viable CMCs with physiological morphology and maintained function. Effect of MSCs on liver regeneration was observed in swine model of chronic liver...

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21018号 / 医博第4364号 / 新制||医||1028(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 小川 誠司, 教授 柳田 素子, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Mesenchymal stem cells and their regenerative and immunomodulatory potential Abstract Mesenchymal stem cells (MSCs) possess multidirectional regenerative ability, which, together with their immunomodulatory potential, makes them promising cell type for therapy of wide variety of diseases. Despite ongoing research, which proved MSCs application to be safe, reported effect of MSCs administration on patients is not convincingly beneficial yet. In our work we focused on elucidation of MSCs role in regeneration of vital organs, heart and liver, where a large damage is life threatening for patients and any improvement in therapy would save many lives. Similar situation is in Graft versus host disease (GVHD), where MSCs immunomodulatory properties could be beneficial. Role of MSCs in heart regeneration was examined in vitro. Primary adult swine cardiomyocytes (CMCs) were co-cultured with or without swine MSCs for 3 days and morphological and functional parameters (contractions, current, respiration) of CMCs were measured. MSCs showed supportive effect on CMCs survival, especially at day 3 of the experiment, where in co-culture was significantly higher number of viable CMCs with physiological morphology and maintained function. Effect of MSCs on liver regeneration was observed in swine model of chronic liver...