Infecções fúngicas invasivas têm emergido como causa de alta morbimortalidade entre pacientes com neoplasia hematológicas, principalmente os submetidos a transplante de células-tronco hematopoéticas (TCTH). Fungos estão onipresentes na natureza, logo, medidas com o objetivo de reduzir a carga fúngica em ambientes hospitalares têm sido preconizadas. No presente estudo, foi realizada coleta de fungos no ar em dois centros de referência em TCTH do sul do Brasil, que possuem instalações distintas, assim como diferentes formas de controle de ar. Todos os quartos do hospital 2 são equipados com filtro de partículas de ar de alta eficiência (HEPA), enquanto no hospital 1 não há sistema específico de filtragem do ar; além disso, os pacientes internados no hospital 2 são de maior risco para doença fúngica invasiva, em função de fatores relacionados ao hospedeiro. Foram realizadas 130 coletas de ar no período de dezembro de 2009 a janeiro de 2011, sendo as amostras provenientes de quartos, banheiros e corredor. Para fins de análise, os fungos pertencentes ao gênero Aspergillus, Rhizopus e Fusarium foram considerados como fungos filamentosos potencialmente patogênicos, enquanto os demais fungos foram classificados como ambientais. A comparação entre os corredores dos hospitais 1 e 2 não mostrou diferença quanto a quantidade de fungos isolados (p=0,114 para fungos ambientais e p=0,622 para fungos filamentosos potencialmente patogênicos). Já os quartos de ambos os hospitais apresentaram redução significativa na quantidade de fungos filamentosos potencialmente patogênicos, quando comparados com os corredores (p<0,0001). Comparando-se os quartos dos hospitais 1 e 2, observou-se menor quantidade de fungos ambientais no hospital 2 (p<0,0001); contudo, para fungos filamentosos potencialmente patogênicos não se encontrou diferença (p=0,7145). Durante o período de estudo, a incidência de doença fúngica invasiva por fungos filamentosos foi de 2,1% no hospital 1 e 7,6% no hospital 2. A baixa carga fúngica nos quartos do hospital 1 poderia ser explicadas pelo uso de medidas protetoras adicionais, incluindo janelas e portas fechadas, reforçando-se a importância de tais medidas no cuidados em ambientes protegidos. / Invasive fungal infections have emerged as a cause of high morbidity and mortality among patients with hematologic malignancies, especially among those undergoing hematopoietic stem cell transplantation. Fungi are ubiquitous in nature, therefore measures aimed at reducing fungal burden in hospitals have been emphasized. In this study air samples were collected in two HSCT centers in Southern Brazil, which have distinct facilities, as well diferent air control systems. All rooms of hospital 2 are equipped with HEPA filters. In addition, patients hospitalized in this unit are at a higher risk for invasive fungal diseases. A total of 130 samples were obtained during December 2009 to January 2011 from rooms, restrooms and corridors. For analysis, all fungi belonging to the genus Aspergillus, Rhizopus and Fusarium were considered filamentous fungi potentially pathogenic, while others were considered environmental fungi. The comparison between corridors of hospital 1 and 2 showed no difference in fungal concentration (p=0.114 for environmental fungi and p=0.622 for potentially pathogenic). The rooms of both hospitals showed a significant lower concentration in PPF, as compared to corridors (p<0.0001). Comparing rooms of hospital 1 e 2 there was a lower amount of environmental fungi in hospital 2 (p<0.0001) – however no difference was observed for potentially pathogenic (p=0.714). During the period of study, the incidence of invasive mold infection was 2.1% in hospital 1 and 7.6% in hospital 2. The low fungal burden in rooms in hospital 1 may be explained by the implementation of additional protective measures, emphasizing the importance of such measures in protected environments.

O bussulfano (BU) e a ciclofosfamida (CY) são fármacos utilizados nos regimes de condicionamento pré-transplante de células tronco hematopoéticas (TCTH). O BU apresenta estreito intervalo terapêutico, alta variabilidade interindividual na farmacocinética e graves reações adversas. O presente estudo avaliou a administração de uma dose teste de BU oral para a individualização do regime de dosagem, definiu o melhor tempo de coletas esparsas para o monitoramento terapêutico do BU e validou um algoritmo baseado em modelo compartimental e farmacocinética populacional em pacientes submetidos ao TCTH. Trinta pacientes portadores de doenças hematológicas tiveram o tratamento com BU individualizado baseado em uma dose teste oral de 0,25 mg/Kg de BU. As doses foram baseadas no clearance aparente calculado na dose teste e as concentrações plasmáticas foram confirmadas após a quinta dose de tratamento. Os coeficientes de variação obtidos entre os valores de clearance avaliados na dose teste e na quinta dose foram <= 30%, exceto para 5 pacientes. Não foram observadas associação entre os parâmetros farmacocinéticos do BU e a evolução clínica dos pacientes. Com a finalidade de estimar os melhores tempos de coletas ideais para aplicação no monitoramento terapêutico do BU, um modelo farmacocinética populacional foi utilizado e um esquema de coletas esparsas com não mais de cinco amostras por paciente (t = 0,5; 2,25; 3; 4 e 5 horas após a dose) demonstrou ser suficiente para a caracterização da farmacocinética do BU. O presente estudo avaliou também a farmacocinética dos enantiômeros da ciclofosfamida (CY) e seus metabólitos (4-hidroxiciclofosfamida e carboxiciclofosfamida), em pacientes submetidos ao TCTH. Foram investigados pacientes portadores de esclerose sistêmica (n=10) e esclerose múltipla (n=10) em regime de condicionamento com 50 mg CY /kg/dia durante 4 dias. Dois ensaios específicos baseados na análise por LC-MS/MS foram desenvolvidos e validados para analisar os enantiômeros da CY e seus metabólito 4- hidroxiciclofosfamida (HCY) e carboxiciclofosfamida (CEPM) em plasma humano. Os parâmetros farmacocinéticos dos enantiômeros da CY e seus metabólitos foram calculados empregando o programa WinNonlin e mostraram acúmulo plasmático dos enantiômeros (S)- (-)-CY (AUC 215, 0 vs 186,2 ?g.h/mL para os paciente EM e 219,1 vs 179,2 ?g.h/mL para os paciente ES) e HCY (1), provavelmente o (R)-(+)-HCY (AUC 5,6 vs 3,7 ?g.h/mL para os paciente EM e 6,3 vs 5,6 ?g.h/mL para os paciente ES) em ambos os grupos de pacientes investigados. A disposição cinética do metabólito CEPM não mostrou enantiosseletividade. A farmacocinética da CY e seus metabólitos HCY e CEPM não diferiu entre os pacientes portadores de EM ou ES. Não foi observado correlação entre o metabolismo da CY e os genótipos avaliados (CYP2B6 e CYP2C9). Não foi possível correlacionar os valores de AUC0-? dos enantiômeros da CY e/ou dos metabólitos HCY e CEPM com a toxicidade ao uso de CY em virtude do pequeno número de pacientes investigados. / Busulfan (BU) and cyclophosphamide (CY) are drugs used during conditioning treatment for hematopoietic stem cell transplantation (HSCT). BU presents narrow therapeutic window, high interindividual variability in the pharmacokinetics and serious adverse effects. The present study evaluated the administration of a BU test dose for dose individualization, set the best sparse sampling scheme for BU therapeutic monitoring and validated an algorithm based on compartmental and population pharmacokinetics model in HSCT patients. Thirty patients received BU individualized treatment based on an oral test dose of 0.25 mg/kg. Doses were based on apparent clearance calculated with BU test dose. Plasma concentrations were confirmed after the fifth treatment dose. Coefficients of variation obtained between the clearance values evaluated in the test dose and fifth dose were <= 30%, except for 5 patients. No association between BU pharmacokinetic parameters and clinical outcome was observed. To estimate the ideal sampling scheme for BU therapeutic drug monitoring, a population pharmacokinetic model was used. Sparse sampling scheme with no more than five samples per patient (t = 0.5, 2.25, 3, 4 and 5 hours after dosing) was shown to be sufficient to characterize the BU pharmacokinetics. This study also evaluated the pharmacokinetics of the cyclophosphamide enantiomers and its metabolites (4-hydroxycyclophosphamide and carboxicyclophosphamide) in HSCT patients. We investigated patients with systemic sclerosis (SS) (n = 10) and multiple sclerosis (MS) (n = 10) in the conditioning regimen with CY 50 mg/kg/day for 4 days. Two specific tests based on LC-MS/MS analysis were developed and validated to analyze the CY enantiomers and its metabolite 4-hydroxycyclophosphamide (HCY) and carboxicyclophosphamide (CEPM) in human plasma. Pharmacokinetics of CY enantiomers and its metabolites were calculated using WinNonlin software and showed plasma accumulation of (S)-(-)-CY (AUC 215.0 vs 186.2 ?g.h/mL for the MS patient and 219.1 vs. 179.2 ?g.h/mL for the SS patient) and HCY (1), probably the (R)-(+)-HCY (AUC 5.6 vs 3.7 ?g.h /mL for MS patients and 6.3 vs 5.6 ?g.h/ mL for the SS patients) enantiomers in both groups of investigated patients. CEPM kinetics disposition showed lack of enantioselectivity. The pharmacokinetics of CY and its metabolites (HCY and CEPM) did not differ between patients with MS or SS. There was no correlation between the metabolism of CY, CYP2B6 and CYP2C9 genotypes. It was not possible to correlate the AUC0-? of CY enantiomers and/or its metabolites (HCY and CEPM) with CY toxicity due to the small number of patients investigated.

Orientador: Maria Filomena Ceolim / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Enfermagem / Made available in DSpace on 2018-08-23T23:37:16Z (GMT). No. of bitstreams: 1 FurlaniCotrim_Renata_D.pdf: 1606137 bytes, checksum: c07e87834fa65c007065f0a1f8e21512 (MD5) Previous issue date: 2013 / Resumo: Os distúrbios de sono estão presentes nos seguimentos de transplante de células-tronco hematopoiéticas (TCTH) e fazem parte de um conjunto de sintomas que vêm associados ao decréscimo da qualidade de vida. Permanecer em um ambiente hospitalar pode precipitar o surgimento destes distúrbios. Este estudo destinou-se à avaliação da qualidade de sono de pacientes com câncer hematológico submetidos ao TCTH, no intuito de identificar fatores preditivos de qualidade de sono antes da realização do transplante (primeira etapa), durante a hospitalização (segunda etapa) e após a alta hospitalar (terceira etapa). O estudo foi realizado em um hospital público do interior do estado de São Paulo. Participaram 47 portadores de câncer hematológico que atenderam aos critérios do estudo. Os dados foram coletados por meio dos seguintes instrumentos: Questionário de Caracterização - Aspectos Sócio-Econômicos, Demográficos e Clínicos; Índice de Qualidade de Sono de Pittsburgh (PSQI-BR); instrumento de qualidade de vida Short Form-12 (SF-12), Diário de Sono (DS) e escala de fadiga. Dosagens de interleucina-6 e proteína-C reativa foram obtidas durante a hospitalização. Os dados foram submetidos a testes estatísticos não-paramétricos e a análises de regressão logística e linear. O sono de má qualidade predominou nas três etapas do estudo atingindo pior pontuação durante a hospitalização. Verificou-se associação do sono de má qualidade à presença de fadiga na primeira e na segunda etapa do estudo e melhor avaliação do componente físico (PCS) do SF-12 em sujeitos com sono de boa qualidade nas três etapas do estudo. Na primeira etapa, o sono de má qualidade foi predominante em indivíduos do sexo feminino e, na terceira etapa, observou-se uma tendência à associação do sono de má qualidade à idade igual ou superior a 40 anos. Nestas duas etapas, o sono de má qualidade associou-se, ainda, ao diagnóstico de mieloma e ao TCTH autólogo, achado contrário ao verificado em outros estudos. Na hospitalização, a prestação de assistência pela equipe de saúde e a necessidade de usar o banheiro foram as principais causas de interrupção do sono noturno. Verificou-se melhor padrão de sono depois da enxertia de neutrófilos em relação ao período anterior à enxertia, no qual as dosagens de PCR e do número de sinais e sintomas foram mais elevados. Não foi verificada variação significativa na dosagem de interleucina-6. Em T5, momento anterior à enxertia, o número de sinais e sintomas explicou 28% da variação da qualidade de sono. viii Os sujeitos com maior número de cochilos demonstraram maior necessidade de sono noturno e pior avaliação da qualidade de sono durante à hospitalização. Pertencer ao sexo feminino aumentou em quase 20% a chance de sono de má qualidade na primeira etapa do estudo e, na terceira etapa, a cada um ponto (1,0) de aumento no componente físico (PCS) e mental (MCS) de qualidade de vida, a chance de má qualidade de sono diminuiu, respectivamente, em 15% e 18%. Este estudo destaca a importância de adotar medidas que garantam um sono de boa qualidade na fase aguda do transplante, sobretudo durante a hospitalização, no intuito de minimizar o impacto provocado pelo TCTH na vida dos sujeitos. / Abstract: Sleep disturbances are present in hematopoietic stem cell transplantation (HSCT) and are part of a set of symptoms have been associated with decreased quality of life. Staying in a hospital setting may precipitate the onset of these disorders. This study aims to evaluate the quality of sleep in patients with hematological cancer undergoing HSCT, in order to identify predictors of sleep quality prior to transplantation (first stage), during hospitalization (second stage) and after discharge (third stage). The study was conducted in a public hospital in the state of Sao Paulo. Participants 47 patients with hematological cancer who met the study criteria. Data were collected using: Characterization Questionnaire - Socio-Economic, Demographic and Clinical; Index Pittsburgh Sleep Quality (PSQI-BR); quality of life Short Form-12 (SF-12), Sleep Diary (SD) and fatigue scale. Interleukin-6 and C-reactive protein doses were obtained during hospitalization. The data were subjected to non-parametric statistical tests and logistic and linear regression analyzes. The poor sleep predominated in the three stages of the study reaching worst score during hospitalization. An association of poor sleep and presence of fatigue was verified in the first and second stage of the study, and evaluation of the best physical component (PCS) of the SF-12 between subjects with good sleep quality in the three stages of the study. In the first stage, the poor quality of sleep was predominant in females and, in the third stage, there was a tendency for the association of poor sleep and age (greater than 40 years). In these two steps, the poor sleep was associated also to the diagnosis of myeloma and autologous HSCT, a opposed finding to that observed in other studies. During hospitalization, the care provision and the need to use bathroom were the main causes of nocturnal sleep disruption. Better sleeping pattern was verified after engraftment. Before engraftment, CRP dosages and the signs and symptoms number were higher. Variation in the interleukin-6 dose was not significant. At T5, prior to engraftment, the number of signs and symptoms explained 28% of the variation in quality of sleep. The subjects with the highest number of naps showed greater need for nocturnal sleep and worse sleep quality assessment during hospitalization. Being female increased by almost 20% chance of poor sleep in the first stage of the study and, in the third step, each point (1.0) increase in physical component (PCS) and mental (MCS) of quality of life, the chance of poor sleep quality decreased, respectively, 15% and 18%. This study highlights the importance of adopting measures that guarantee a good quality sleep in the acute phase of transplantation, particularly during hospitalization, in order to minimize the impact caused by HSCT in subjects' lives. / Doutorado / Enfermagem e Trabalho / Doutora em Enfermagem

Nous avons montré que l'enzyme de conversion de l'angiotensine (ACE) est un nouveau marqueur de la cellule souche hématopoïétique et identifie l’émergence de l'hématopoïèse dans tous les sites hématogènes de l’embryon humain. L'ACE fait partie du système rénine-angiotensine (SRA) dont la fonction principale est d'agir sur l'angiotensine I pour former l'angiotensine II (AngII), un puissant vasoconstricteur.De plus, nous montrons que les principaux composants du SRA (les récepteurs AT1 et AT2, l’angiotensinogène et la rénine) sont exprimés dans la même région de l'embryon qui exprime l'ACE, suggérant ainsi l’existence d’un SRA local dans l'embryon précoce. Des tests fonctionnels, conduits in vitro chez l'embryon de la souris, montrent que l’Ang II stimule dans la culture l'émergence des progéniteurs hématopoïétiques, effet qui peut être bloqué par un antagoniste spécifique de l’AT1. Ces observations suggèrent pour la première fois, le rôle direct du SRA dans l’émergence hématopoïétique au cours de l’ontogenèse. De plus, nous mettons en évidence l'existence d'un SRA local dans la moelle osseuse (MO) adulte et nous montrons que les principaux éléments de ce système sont surexprimés dans la MO de patients atteints de leucémie aiguë myéloïde, aussi bien dans les blastes que dans les cellules stromales. Ces observations suggèrent une contribution du SRA à la dérégulation de la niche observée dans les hémopathies.Ainsi, la présence d’un SRA local dans la niche hématopoïétique intra-embryonnaire et dans la MO chez l’adulte place ce système dans une position stratégique comme acteur important de l’émergence et de la régulation du système sanguin définitif. / We have shown that the angiotensin-converting enzyme (ACE) is a new marker of human hematopoietic stem cells and also identifies emerging hematopoiesis in all hemogenic sites inside the human embryo. ACE is a key component of renin-angiotensin system (RAS) as it catalyses the production of angiotensin II (Ang II) well known for its effect in the control of blood pressure, through AT1 and AT2 receptors.Furthermore, we observe the presence of the main elements of the RAS (AT1, AT2 receptors, angiotensinogen and renin) in the same region of the embryo expressing ACE, meaning that a local RAS exists in the embryo. Functional in vitro analyses, carried out in mouse model, show a stimulatory effect of AngII in the hematopoietic precursors emergence, an effect inhibited by a specific AT1 antagonist. These observations suggest for the first time a direct role of RAS in the emergence of hematopoiesis during ontogeny. In addition, our data indicate the presence of a local RAS inside the adult bone marrow (BM). This system is overexpressed in the BM of acute myeloid leukemia (AML) patients, both in hematopoietic cells and in stromal cells suggesting a RAS contribution to the bone marrow niche deregulation, always observed in these hemopathies.Therefore, the existence of a local RAS in the intraembryonic niche and in the adult bone marrow suggests that this system is an important actor in the emergence and regulation of the definitive blood system.

Class of 2011 Abstract / OBJECTIVES: The purpose of this study was to analyze the impact of patient and hospital characteristics as well as selected comorbidities on inpatient mortality and charges in pediatric HSCT. We have determined the top 25 comorbidities reported during all inpatient stays for HSCT as well as for those stays ending in mortality. METHODS: All data was extracted from the AHRQ KID databases for the years 1997, 2000, 2003, and 2006. Two regression analyses were performed to determine the contribution of various independent variables on mortality and charges. Subjects of this study included all cases of HSCT reported in the Healthcare Cost and Utilization Project (HCUP) KID as ICD-9 41.XX. RESULTS: Factors accounting for larger increases in cost included death during hospital stay, the development of disseminated intravascular coagulation (DIC), pneumonia, and length of stay (LOS). The largest decreases in charges were seen for patients coming from a small or “micropolitan” location, patients cared for in teaching hospitals, and in hospitals with large bedsizes. Variables associated with increased risk of mortality on linear regression included development of DIC, sepsis, or pneumonia. CONCLUSION: Further study relating to HSCT is necessary to determine the contribution of specific comorbidities to mortality and charges. Importantly, DIC is associated with both greater risk of mortality and greater charges. It would be prudent to recommend increased monitoring and early treatment for DIC based on these results.

Orientador: Sandra Cecilia Botelho Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T10:34:08Z (GMT). No. of bitstreams: 1 Costa_ClaudiaRaquelCantarelli.pdf: 4518268 bytes, checksum: fb54bd9384d947b72d5d29ed906ae70f (MD5) Previous issue date: 2009 / Resumo: O cytomegalovirus humano (HCMV) e o herpesvirus humano 6 (HHV-6) são ß-herpesvirus com homologia superior a 67% e alta soroprevalência na população adulta. A infecção primaria por estes herpesvirus ocorre comumente na infância e é normalmente subclinica, ou pode causar mononucleose (HCMV) ou exantema súbito (HHV-6) sendo resolvidos na maioria dos casos sem complicações. Após a infecção primária os vírus permanecem no hospedeiro por toda vida podendo ser reativado de seu estado de latência em indivíduos adultos imunocomprometidos como os receptores de células tronco hematopoiéticas (TCTH). A reativação ou reinfecção por estes vírus causam serias complicações em pacientes submetidos ao transplante de células tronco hematopoiéticas como pneumonia intersticial, febre, gastroenterite, mielossupressão, encefalite e doença do enxerto contra o hospedeiro (GVHD). A reativação do HHV-6 após o transplante é associada com o desenvolvimento de infecções oportunistas, doença causada pelo citomegalovírus humano e possíveis episódios de rejeição aguda. Com efetivos tratamentos antivirais disponíveis, um monitoramento adequado destes vírus distinguindo entre latência e reativação é critico para estes pacientes. Monitoramos 30 pacientes submetidos à TCTH quanto a infecção ativa por HCMV e HHV-6 pelas técnicas de nested-PCR em soro e células, PCR- em tempo real em soro e células e transcrição reversa acoplada a nestedPCR (RT-nPCR). 29 pacientes (96,66%) apresentaram infecção ativa por HCMV sendo 21 pacientes (70%) pela nested-PCR em células, 17 pacientes(56,66%) pela neste-PCR em soro ,23 pacientes(76,67%) pela PCR em tempo real em células,19 pacientes (63,33%) pela PCR em tempo real em soro e 15 pacientes (53,3%) pela RT-nPCR. 25pacientes (83,33%) apresentaram infecção ativa por HHV-6, sendo 14 pacientes (46,7%) pela nested-PCR em células, 2 pacientes(6,6%) pela PCR em tempo real em células,23 pacientes (76,67%) %) pela PCR em tempo real em soro e 9 pacientes (30%) pela RT-nPCR. Todos os pacientes que apresentaram infecção ativa por HCMV apresentaram também presença do HHV-6, e 25 pacientes (83,33%) apresentaram co-infecção HCMV/HHV-6, sendo a infecção por HHV-6 precoce em relação ao HCMV. O presente estudo encontrou também associação entre infecção ativa por HCMV e doença do enxerto contra o hospedeiro. / Abstract: Human cytomegalovirus (HCMV) and human herpesvirus type 6 (HHV-6) are ß-herpesvirinae extremely closely related with a homology > 67% with a high seroprevalence in the adult population. Primary infection commonly appears in early childhood and is usually subclinical, or may cause mononucleosis (HCMV) or febrile illness, including exanthema subitum (HHV-6), solving, in the majority of cases, without complications. After primary infection, the viruses persist in the infected individual through life and can be reactivated from their state of latency in immunocompromised hosts. Reactivation or reinfection causes severe clinical diseases in patients who underwent hematopoietic stem cell transplantation, like interstitial pneumonia, fever, gastroenteritis, myelossupression, encephalitis and graft-versus-host-disease (GVHD). A potential increase in virulence of HHV-6 in the course of a simultaneous CMV reactivation, leading to a great risk of CMV-associated disease. In this present study, 30 patients who received HSCT were monitoring for active HCMV and HHV-6 infection by Nested PCR in serum and peripheral blood leukocytes (PBL) samples, real time PCR in serum and PBL and RT-nPCR. In 29 patients (96,66%) active HCMV infection was detected: 21 patients (70%) by PBL nested-PCR, 17 patients (56,66%) by serum neste-PCR, 23 patients(76,67%) by PBL real-time-PCR,19 patients (63,33%) by serum real-time-PCR and 15 patients (53,3%) by RT-nPCR. In 25 patients (83,33%) active HHV-6 infection was detected : 14 patients (46,7%) by PBL nested-PCR, 2 patients(6,6%) by PBL real time -PCR,23 patients (76,67%) by serum real time-PCR and 9 patients (30%) by RT-nPCR. In all patients who had active HCMV infection, HHV-6 DNA was detected. 25 patients (83,33) had HCMV/HHV-6 co-infection, and the active HHV-6 infection was detected earlier in the majority of the cases. Our results showed a correlation between GVHD and active HCMV infection and detection of active HCMV infection by serum nested-PCR and PBL and serum real time-PCR. / Universidade Estadual de Campi / Ciencias Basicas / Doutor em Clínica Médica

Orientador: Maria Elvira Pizzigatti Corrêa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T08:02:08Z (GMT). No. of bitstreams: 1 Torregrossa_ViniciusRabelo_M.pdf: 2074120 bytes, checksum: 7ac320774930da79cb9aea0a6c37ec73 (MD5) Previous issue date: 2015 / Resumo: INTRODUÇÃO: Alterações salivares quantitativas e qualitativas são complicações comuns ao Transplante de Células-Tronco Hematopoiéticas alogênico (TCTHa). Essas alterações salivares são frequentemente relacionas à fase tardia do TCTHa, principalmente na presença da Doença do Enxerto-Contra-Hospedeiro crônica (DECHc). Poucos estudos abordaram a influência da toxicidade aguda dos regimes de condicionamento sobre as alterações precoces do fluxo salivar. O objetivo deste trabalho foi avaliar a influência do TCTHa nas alterações precoces do fluxo salivar e validar critérios clínicos utilizados para o diagnóstico de hipossalivação nesta população de pacientes. MÉTODOS: O estudo prospectivo das alterações quantitativas da saliva envolveu 69 pacientes adultos submetidos ao primeiro TCTHa. A saliva não estimulada foi coletada e os pacientes foram submetidos à avaliação da saúde oral, do grau de mucosite oral, e de critérios clínicos de hipossalivação previamente ao início do regime de condicionamento, e entre os dias D+8-10 pós-TCTH. A avaliação da condição de saúde oral incluiu a obtenção do índice de Dentes Cariados, Perdidos e Obturados (CPOD), Índice Gengival (IG), e do Índice de Placa (IP). Para a avaliação da hipossalivação foram utilizados quatro critérios clínicos objetivos e quatro critérios subjetivos, considerando-se hipossalivação quando o Fluxo Salivar Não Estimulado (FSNE) ?0,2 mL/min. A avaliação da mucosite oral foi realizada entre os dias D+8-10 pós-TCTH, conforme os critérios da OMS. O estudo de validação dos critérios de hipossalivação envolveu 120 pacientes não consecutivos submetidos ao primeiro TCTH alogênico. As avaliações orais e as coletas de saliva foram realizadas simultaneamente e em diferentes períodos pós-TCTH. Para o estudo das alterações precoces do fluxo salivar, as variáveis categóricas foram analisadas pelo teste de associação Qui-quadrado, ou pelos testes de Fisher ou de Mann-Whitney. O teste t pareado foi utilizado na comparação das variáveis contínuas nos diferentes períodos. No estudo de validação dos critérios de hipossalivação, o teste alfa de Cronbach foi aplicado para medir a consistência interna e confiabilidade dos critérios clínicos utilizados. Foram então selecionados 5/8 critérios clínicos de hipossalivação, e um Sistema de Pontuação para Boca Seca (SPBS) foi elaborado. O teste de Mann-Whitney e a correlação de Pearson foram utilizados na análise da distribuição do FSNE em relação às pontuações obtidas, a partir dos critérios clínicos de hipossalivação selecionados. RESULTADOS: Foi observado um aumento do fluxo salivar (p=0.03) e do grau de inflamação gengival (p=0.03) entre os dias D+8-10 pós-TCTH. O aumento do fluxo salivar neste período foi correlacionado à gravidade da mucosite oral (p=0.02), à presença de vômito (p=0.03), ou ao uso de nutrição parenteral total (p=0.03) nos dias das coletas de saliva. Apesar da hipossalivação não ter sido um achado frequente no período estudado, mulheres e pacientes com doenças de alto risco apresentaram um menor fluxo salivar (p=0.01 e p=0.03, respectivamente). O SPBS incluiu quatro critérios clínicos validados e uma questão subjetiva de hipossalivação: 1.Alta aderência da espátula de madeira na mucosa jugal; 2.Ausência de lago sublingual; 3.Saliva espessa e viscosa; 4.Ausência de secreção salivar após ordenha do ducto parotídeo; 5. Você sente sua boca seca?. O SPBS foi dicotomizado entre as pontuações 0-1 e 2-5, de acordo com os valores obtidos do FSNE. Após a dicotomização, 66 (55%) pacientes apresentaram pontuações de 0-1, com uma média do FSNE de 0.65 mL/min (0.1-9.0 mL/min), e 54 (45%) pacientes apresentaram pontuações de 2-5 com uma média do FSNE de 0.34 mL/min (0.01-6.7 mL/min). Maiores pontuações obtidas no SPBS foram correlacionadas a um FSNE reduzido (p=0.006, r=-25%), confirmado pelo teste de Mann-Whitney (p<0.0001). CONCLUSÕES: O aumento do fluxo salivar nos dias D+8-10 pós-TCTH pode estar relacionado à intensa reação inflamatória e dano tecidual e induzidos pela toxicidade dos regimes de condicionamento, que se traduz pela gravidade da mucosite oral observada clinicamente. O SPBS provou ser uma ferramenta confiável para o diagnóstico de hipossalivação em pacientes submetidos ao TCTH alogênico / Abstract: INTRODUCTION: Qualitative and quantitative salivary changes are common complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). These salivary changes are related to late effects of HSCT, mostly in the presence of chronic graft-versus-host disease (cGVHD). Little is known about the influence of the conditioning regimens-related toxicity on the very early salivary flow changes. Thus, the aim of the present study was to understand the influence of allo-HSCT on the very early salivary flow changes. A secondary aim was to validate clinical criteria for the diagnosis of hyposalivation in allo-HSCT patients. METHODS: The prospective study of quantitative salivary changes enrolled 69 adult patients undergoing their first allo-HSCT. The unstimulated whole saliva was collected and patients were assessed for their oral health status, the degree of oral mucositis (OM), and for clinical criteria used for the diagnosis of hyposalivation before the start of the pretransplant conditioning regimens and between the days D+8-10 posttransplantation. The oral health exam included the evaluation of Decayed-Missing-Filled teeth (DMFT) index, Gingival Index (GI), and Plaque Index (PI). The clinical assessment of hyposalivation was composed by four objective clinical criteria and by four subjective questions. Hyposalivation was considered when the unstimulated whole saliva flow rate (UWSFR) was ? 0.2 mL/min. OM severity was evaluated at days D+8-10 according to the World Health Organization (WHO) criteria. The validation study of the clinical criteria for the diagnosis of hyposalivation enrolled 120 non-consecutive patients undergoing their first allo-HSCT. The oral health exams and saliva collection were taken simultaneously and in different periods of HSCT. For the study of very early salivary flow changes, chi-square or Fischer¿s test, besides of the Mann-Whitney U Test were applied according to the variable type. Pared T-test was used to compare continuous variables in different periods. For the validation study of the clinical criteria for the diagnosis of hyposalivation, a Cronbach¿s alpha test was applied in order to measure the internal consistency and satisfactory reliability of all criteria used. Five of eight clinical criteria of hyposalivation were selected, and a scoring system called Oral Dryness Score (ODS) was developed. Mann-Whitney U test was applied to analyze the UWSFR distribution among the dichotomized ODS scores, in addition to the Pearson¿s correlation coefficient. RESULTS: An increase of the UWSFR (p=0.03) and the worsening of the gingival index (p=0.03) were observed at days D+8-10 posttransplantation. A positive correlation was found between an increase of the UWSFR and a greater severity of OM (p=0.02), the use of total parenteral nutrition (TPN) (p=0.03), and with vomiting episodes (p=0.03). Although hyposalivation was not a frequent finding among the studied population, a reduced UWSFR was observed in women (p=0.01), and in the group of patients with a high risk underlying disease (p=0.03). The ODS included four validated objective clinical criteria and a subjective question of hyposalivation: 1.Higher adherence of the wood spatula to the jugal mucosa; 2.No saliva pooling in the anterior floor of mouth; 3.Increased viscosity and thickness of saliva; 4.Absence of salivary secretion of the parotid duct under manual pressure; 5.Does your mouth feels dry?. The ODI was dichotomized between 0-1 scores and 2-5 scores, respecting its behavior according to the UWSFR. After dichotomization, 66 (55%) patients presented 0¿1 scores, with a UWSFR median of 0.65 mL/min (0.1¿9.0 mL/min), and 54 (45%) patients presented 2¿5 scores with a UWSFR median of 0.34 mL/min (0.01¿6.7 mL/min). A higher ODS was correlated with a decreased UWSFR (p=0.006, r=-25%), confirmed by a Mann-Whitney U test (p<0.0001). CONCLUSIONS: The clinical impact of the conditioning regimens toxicity showed through the OM severity seemed to have influenced the very early salivary flow changes. If the influence of HSCT on the very early salivary changes could be better characterized, as well as its correlation with short and long-term oral outcomes of HSCT patients, the proper clinical management could be improved. The ODS was correlated with a decreased UWSFR, proving to be a reliable tool for the diagnosis of hyposalivation in this population / Mestrado / Estomatologia / Mestre em Estomatopatologia

Orientador: Maria Elvira Pizzigatti Corrêa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-28T03:50:50Z (GMT). No. of bitstreams: 1 Vieira_RaizaMeira_M.pdf: 2654950 bytes, checksum: 493dac1ecf0ca2acb92f0ff8829107aa (MD5) Previous issue date: 2015 / Resumo: Complicações orais estão presentes em cerca de 80% dos pacientes durante o Transplante de Células Tronco Hematopoéticas (TCTH), sendo a mucosite oral (MO) e as alterações salivares umas da que possuem maior impacto para a qualidade de vida do paciente. A utilização do perfil proteico salivar (PPS) na abordagem diagnóstica em diversas doenças tem sido frequente. A identificação de um PPS que auxilie o entendimento das manifestações agudas orais do TCTH poderia sobremaneira, influenciar nas decisões terapêuticas visando melhora no manejo do paciente. O objetivo deste trabalho foi identificar alterações do PPS do início do regime do condicionamento pré TCTH até a recuperação medula e correlacioná- las com dados clínicos orais. Para tanto, foi utilizado o banco de dados de proteomica salivar do grupo de Odontologia do Hemocentro, encontrada por Feio et AL (2013). Nesta avaliação foram incluídos 16 pacientes submetidos ao primeiro TCTH alogênico na Unidade de Transplante de Medula Óssea do Hospital de Clínicas da UNICAMP. As amostras de saliva total não estimulada (STNE) foram coletadas em dois momentos: previamente ao condicionamento (coleta A) e a segunda (coleta B), entre os dias D+8 e D+10 pós-TCTH. Dados sobre saúde oral, grau de MO foram coletados, além da avaliação de hiposalivação. O estudo do PPS foi realizado por espectometria de massas no LNBio. Os PPS das duas coletas, A e B foram tabelados no programa Excel® 2007 (Microsoft, WA, EUA), juntamente com os dados clínicos. O teste T pareado foi utilizado para a comparação entre os PPSs com os tempos das coletas. Os critérios clínicos de hiposalivação foram comparados com as divergências proteicas durante as duas coletas por ANOVA. A comparação entre os dados clínicos de cada coleta e seu respectivo PPS, foi feito pelo teste T pareado, sendo considerado significativo p<0,005. Sete proteínas apresentaram intensidades divergentes entre as coletas A e B: Prolactin-inducible protein, Alpha-Amylase 1, Cystatin-SN, Submaxillary gland androgen-regulated protein 3B, Sthaterin, cDNA FLJ60163, highly similar to Carbonic anhydrase 6 apresentaram-se em decréscimo na coleta B comparada a coleta A e a vitamin D-binding protein isoform 1 precursor se apresentou aumentada na coleta B quando comparada a coleta A. A mucosite oral foi correlacionada com as proteinas, Immunoglobulin J chain, Pulative uncharacterized protein DKFZp686N02209 que se demonstraram com intensidade diminuída naqueles pacientes que apresentaram graus III e IV em comparação àqueles com grau 0¿II de MO; a proteína Statherin esteve presente em maior intensidade naqueles pacientes que apresentaram MO com grau III e IV. Esses resultados podem estar relacionados principalmente à toxicidade do regime de condicionamento mieloablativo nas glândulas salivares e sugerem que as proteinas Statherin, Immunoglobulin J chain e Pulative uncharacterized protein DKFZp686N02209 podem ser candidatas a um painel de PPS para a MO no TCTH / Abstract: Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for various hematological diseases. Oral Complications are present in about 80% of patients who undergo to HSCT. Oral mucositis (OM), oral infections and salivary changes, among others are the most common oral clinical founds in the acute phase of the HSCT. The use of salivary profile as a tool for diagnosis and management of treatment responses has been shown a promising future in different clinical settings. Thus the discovery of a salivary protein profile (SPP) of patients submitted to the allogeneic HSCT during the acute phase of the transplantation may be helpful on the management of the oral effects of the HSCT. The aim of this research was to identify the SPP changes during the acute phase of allogeneic HSCT and correlate them with the oral clinical manifestation of the acute phase of HSCT. This study enrrolled 16 patients with hematological diseases who, underwent to their first allogenic HSCT at the Bone Marrow Transplantation Unit ¿ UNICAMP. Unstimulated salivary samples were collected in two periods: first (collection A), it was performed prior the initiation of the conditioning regimen for the HSCT and collection B; performed between day D+8-10 days after HSCT. Oral health indices were obtained in both moments of the saliva collection. The severity of oral mucositis was collected on the collection B. The PPS analysis was performed by mass spectrometry on a previous study performed by Feio et al (2013). Student T test paired was used in order to between the 39 identified proteins equally between the collections A and B. Clinical correlations were also compared to the different PPS using one-way ANOVA analysis. For both comparisons, considered significant p < 0.05. Among the collections A and B, 7 proteins were presented with differing intensities: Prolactin-inducible protein, Alpha-Amylase 1, Cystatin SN, Submaxillary gland androgen-regulated protein 3B, Sthaterin, cDNA FLJ60163 Similar to highly Carbonic anhydrase 6 showed a decrease intensity in the collection B and the vitamin D-binding protein isoform 1 precursor quantity showed a increased in the collection B. Three proteins were shown altered intensity when correlated with the severity of OM: Immunoglobulin J chain, Pulative uncharacterized protein DKFZp686N02209v. These proteins showed a decreased of intensity in those patients grade III-IV OM when compared to patients with grade 0-II OM. The protein Statherin showed in increased intensity in patients with OM grade III-IV. These results indicate the influence of the toxicity of the conditioning regimens on the salivary protein profiles in the acute phase of the HSCT. The proteins: Immunoglobulin J chain, Pulative uncharacterized protein DKFZp686N02209v and Statherin might be a potential candidates for a panel of salivary biomarkers for OM / Mestrado / Estomatologia / Mestra em Estomatopatologia

Network biology has proven to be powerful tool for representing and analyzing complex molecular networks. It has also been successfully applied to biological field helping understand various biological processes. However, our current knowledge about the dynamics of gene networks during disease progression is rather limited. On the other hand, network construction is a prerequisite of network analysis. When the number of samples is limited, state-of-art computational methods for network construction are not robust in terms of low statistical power. In addition, molecular networks have been used extensively to improve the inference accuracy of causal coding variants, but this potential has not been investigated to the same extent for noncoding variants. To address those limitations, I first developed inference of multiple differential modules (iMDM) algorithm to study network dynamics. This method is able to identify both unique and shared modules from multiple gene networks, each of which denoting a different perturbation condition. Using iMDM algorithm, I identified different types of modules to understand heart failure progression and disease dynamics. Next, I developed a computational framework to construct condition specific transcriptional regulatory network. I also developed a computational method to rank transcription factors in the transcriptional regulatory network. Applying this framework to RNA-seq data for hematopoietic stem cell development, I successfully constructed corresponding transcriptional regulatory network and identified key transcriptional factors that play important roles. Finally, I developed Annotation of Regulatory Variants using Integrated Networks (ARVIN), a network-based algorithm, to identify causal genetic variants for diseases. By applying ARVIN to various diseases, we obtained a systems understanding of the gene circuitry that is affected by all enhancer mutations in a given disease.

Hematopoietic cells exploit the multistep paradigm of cell migration to ultimately enable them to perform their function. This process is dictated by the ability of adhesion molecules on the circulating hematopoietic cells to find their counter-receptors on endothelial cells. Of those molecules, the selectin family and their respective ligands induce the initial transient interactions between circulating cells and the opposing endothelium. In this thesis, I focused on studying E-selectin mediated cellular migration in two hematopoietic cell types, namely human hematopoietic stem and progenitor cells (HSPCs) and human T-lymphocytes. HSPCs derived from pluripotent sources theoretically offers a novel, unlimited source for hematopoietic stem cell transplantation therapy. In vitro pluripotent stem cell derived- hematopoietic stem/progenitor cells (ES/iPS-HSPCs) behave much like somatic HSPCs in that they exhibit clonal expansion and multilineage hematopoietic capacity. However, unlike somatic sources, ES/iPS-HSPCs do not give rise to effective hematopoietic repopulation, which may be due to insufficient HSPCs homing to the bone marrow. HSPCs exploit E- and P-selectin to home and engraft into bone marrow niches. Thus, one of my objectives in this thesis was to study the expression of E-selectin ligands associated with ES/iPS-HSPCs. I showed that ES/iPS-HSPCs lack functional E-selectin ligand(s). In an effort to enhance the interaction between Eselectin and ES/iPS-HSPCs, we decorated the cell surface with sialyl-Lewis x (sLex) using the ex-vivo glycan engineering technology. However, this decoration did not improve the engraftment capacity of ES/iPS-HSPCs, in vivo. Induction of E-selectin expression during inflammation is key to recruitment of immune cells and therefore I also focused on analyzing the expression of E-selectin ligands on activated human T-cells. I identified several novel glycoproteins that may function as E-selectin ligands. Specifically, I compared the role of the known E-selectin ligands, namely PSGL-1 and CD43, to CD44. I showed that CD44 purified from in vitro human activated T-cells or from psoriasis patients acts as a functional E-selectin ligand. Furthermore, our knock-down studies demonstrated that CD44, and not CD43, cooperates with P-selectin glycoprotein ligand-1 (PSGL-1) as a major E-selectin ligand.