Thesis advisor: Thomas Chiles / Thesis advisor: James Kim / The purpose of this thesis is to describe some of the research I conducted in Dr. Markmann’s laboratory at Massachusetts General Hospital in the field of transplantation immunology. The first portion provides background information on the immune system and its different components, eventually providing an in-depth look at regulatory T cells, and their role in transplant immunology. The second portion of the thesis is dedicated to my experiments. This part presents the materials and methods used, the previous findings that led to my experiments, the data analysis and results, and a conclusive discussion. The research I did that is included in this thesis pertains to regulatory T cells. It is believed that a potential pathway to prolonging allograft survival is to drive antigen-specific CD4+Foxp3- T cells to convert to CD4+Foxp3+ T cells. I studied the effects of retinoic acid (RA) and anti-CD45RB treatment on Foxp3 expression in CD4+ T cells. These studies were done using different lines of transgenic mice that provided models of antigen-specificity. My in vitro data demonstrated that RA is capable of boosting TGF-β-mediated Foxp3 upregulation in a synergistic manner and that anti-CD45RB is also able to increase Foxp3 expression. In vivo RA experiments were inconclusive, and due to some misfortune and time constraints in vivo anti-CD45RB experiments were not conducted. Research funded by the following sources: 2R56AI048820, 5R01AI057851, and 5K01DK079207. / Thesis (BS) — Boston College, 2011. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

Clinicians lack appropriate non-invasive methods to be able to predict, diagnose, and reduce the risk of rejection in the years following kidney transplantation. Protocol biopsies and monitoring of serum creatinine levels are the most common methods of monitoring graft function after transplant; however, they have several negative aspects. Use of traditional factors regarding donors and recipients such as Human Leukocyte Antigen (HLA) DNA typing, pre-transplant anti-HLA antibody levels, and basic demographics (age, ethnicity/race, gender), has proved inadequate for post-transplant graft monitoring past the first few years. We propose that by utilizing immunologic factors available to clinicians across the United States, development of a non-invasive model for predicting renal graft outcome will provide a useful tool for post-transplant patient monitoring. We advocate an expanded model which incorporates both the traditional factors, as well as new factors, which have shown promise in predicting kidney outcome and are widely available for testing using commercial kits. These additional factors include major histocompatibility complex class I chain-related gene A (MICA) typing of donor and recipient, degree of matching for killer cell immunoglobulin-like receptors (KIRs) between donor and recipient, detection of MICA antibodies, and soluble CD30 level (sCD30). This proposed graft-function prediction model is the first to include all of these factors. Using multi-center data from adult recipients of standard-criteria deceased-donor (SCD) kidneys, we were able to construct models, containing the traditional factors only, for prediction of outcome at 1 year and 3 years post-transplant. Using single-center data from adult recipients of standard-criteria deceased-donor kidneys, we developed comparison models containing traditional factors only, as well as, expanded models containing the new suggested variables for prediction of outcome post-transplant. These additional variables, when incorporated into the expanded models provided greater positive predictive values, greater negative predictive values, and lower false negative rates for graft outcome at 1 year and at 3 years post-transplant than the models utilizing traditional factors only. Our results indicate that evaluation of sCD30, MICA and KIR as part of routine protocol testing, is helpful to clinicians for predicting risk of kidney graft rejection.

The location of the immunologic response to an allograft is not known with certainty. However, organized collections of T cells, B cells and antigen presenting cells have been found in peripheral tissue, in close proximity to organs undergoing rejection. It is hypothesized that this tertiary lymphoid tissue may be a location in which activation of lymphocytes can occur, leading to rejection of an allograft. We report here that in a splenectomized aly/aly mouse, which is devoid of secondary lymphoid organs and will normally fail to reject an allograft, the presence of tertiary lymphoid organs is associated with graft rejection. We additionally find that tertiary lymphoid organs can act as lymph nodes, and can support effector and memory allograft rejection responses. It is demonstrated that ectopic lymphoid tissue in aly/aly mice will support the multiplication and transformation of transferred naïve CD4 and CD8 T cells into cells that display phenotypic markers characteristic of effector and memory lymphocytes. These results demonstrate that ectopic lymphoid tissue is associated with the loss of immunologic ignorance and is sufficient to enable graft rejection. This suggests that allograft rejection may take place within ectopic lymphoid tissue, and suggests that techniques to interfere with the development of this tissue might offer a therapeutic approach to preserving organ allografts.

Cell transplantation therapy is a potentially powerful tool and can be used to replace defective cells with healthy cells. This offers the possibility of alleviating the destructive symptoms for many diseases such as Parkinson's disease, Alzheimer's disease, stroke, spinal cord trauma, Type I diabetes and many more. While there are many diseases that could be positively impacted from cell transplantation therapy, the focus of this research is insulin dependent, Type I Diabetes. The Islets of Langerhans are composed of various types of cells located in the pancreas and are responsible for a variety of biochemical functions. Specifically, the beta Islet cells are responsible for production of the hormone insulin that regulates and aids in biosynthesis of glucose. Transplantation of isolated allografted pancreatic islets, which contain insulin producing cells, into diabetic rats has proven to be highly successful. However, these transplantations involve using medications for long term immunosuppression to defend against an undesired host immune response. Immunosuppressive medications are both costly and illicit additional side effects that can be detrimental to the host. This research focuses on the use of testicular derived Sertoli cells that have been publicized to provide localized immunoprotection. Electrofusion is a process that can be used to fuse homogeneous and heterogeneous cell types by promoting the creation of micropores in the cell's lipid bilayer. This renders the cell temporarily fusogenic, or capable of facilitating fusion. Cells must then be brought into contact with one another via mechanical, chemical or viral means. This research study proposes to optimize electrofusion technology to create novel, secretory hybrids composed of Islet and Sertoli cells that are immunoprotected and produce insulin in response to a glucose challenge. The components of the electrofusion device include a Sterlitech 0.2 ìm microporous membrane, a woven cellulose absorbent pad, two aluminum electrodes and a chamber body and top injection molded using Delrin. Preliminary experiments using B16-F10 murine melanoma cells incorporated with centrifugation to increase cell to cell contact resulted in an average fusion yield of 18.9% ± 8.1 SD using a field strength of 2500 V/cm, 8 pulses and a 250 ìs pulse length. Additionally, lab synthesized electroporation buffers containing 8.5% sucrose (w/v) and 0.3% glucose increased total and viable fusion yields to 37.1% ± 9.3 SD and 13.8% ± 2.1 SD, respectively. These results showed promise and should be further validated with additional cell lines and tissues to corroborate reproducibility.

Patient expectations have been shown to be a major predictor of outcomes. Furthermore, fulfilled expectations have been linked to increased patient satisfaction and rehabilitation adherence. Expectations may be influenced by a variety of factors, including patient characteristics, pre-operative function, or disease characteristics. However, it is currently unknown what factors and to what degree they may influence patient expectations prior to knee surgery. Furthermore, understanding the importance and values of those expectations for recovery using qualitative methods has not previously been conducted in this patient population. A mixed methods design was used. Twenty-one participants scheduled to undergo cartilage repair of the knee, including autologous chondrocyte implantation, osteochondral allograft transplantation, or meniscal transplant were included. During their pre-operative visit, participants completed an expectations survey (Hospital for Special Surgery (HSS) Knee Surgery Expectations Survey) and the Knee Injury and Osteoarthritis Outcome Score (KOOS) as a measure of functional ability. At their first post-operative visit, patients completed the Self-Efficacy for Rehabilitation Scale (SER). Rehabilitation adherence was collected by the participant’s rehabilitation provider. A selected sample of 6 participants participated in a semi-structured interview 6 months following surgery to better understand their expectations for recovery. Pearson correlation coefficients were used to determine relationships between expectations and KOOS scores, SER scores, and measures of adherence. Results demonstrated that patients have moderate expectations for recovery and these expectations were positively associated with pre-operative pain, activities of daily living, and knee-related quality of life as measured by the KOOS. In addition, a negative relationship was found between patient expectations and adherence with home exercises, use of a brace, and weight-bearing restrictions. Four qualitative themes emerged as participants’ described how previous recovery experiences shaped their recovery following cartilage repair of the knee. Patient education, pre-habilitation, and the use of psychological skills during rehabilitation may help to manage patient expectations, improve rehabilitation adherence, and assist clinicians in providing more focused and individualized patient care.

Proposição: O presente trabalho propõe o estudo histológico descritivo da aplicação de um campo magnético sepultado, estático e permanente em área de enxerto ósseo alógeno liofilizado e dento-alveolar associada a defeito cirúrgico em mandíbulas de ratos. Metodologia: estudo experimental in vivo, randomizado. Amostra foi composta por 21 ratos, da espécie Rattus novergicus albinus, linhagem Wistar, machos, divididos em três grupos, correspondendo aos tempos experimentais de sete, 21 e 45 dias. Foram realizadas ostectomias associadas a odontossecções, na face lateral externa do corpo mandibular e no ramo ascendente, atingindo transversalmente a raiz do incisivo inferior e os ápices dos molares. Este defeito cirúrgico permitiu a adaptação de dispositivos metálicos magnetizados (grupo teste) e não magnetizados (grupo controle) associados às regiões apicais dos molares e ao coto distal do incisivo inferior, local este onde realizou-se enxertia óssea alógena liofilizada. A intensidade média do campo magnético sepultado e permanente foi de 250 gauss.Resultados: observou-se no grupo teste e controle, nos diferentes tempos experimentais a gradativa integração do enxerto ósseo alógeno liofilizado, a manutenção da vitalidade pulpar dos molares e coto proximal do incisivo inferior, além da contínua erupção do incisivo inferior do rato. Nos grupos teste, principalmente aos 45 dias, constatou-se uma diferenciada e exuberante neoformação óssea centrípeta em direção aos dispositivos metálicos imantados. Conclusão: o campo magnético sepultado in vivo foi capaz de favorecer o processo de cicatrização óssea no defeito cirúrgico criado. / Purpose: The present study provides a descriptive histological analysis of the use of a buried, static and permanent magnetic field in lyophilized bone allografts in a dentoalveolar area associated with surgical defects in rat mandibles. Method: a randomized in vivo experimental study was carried out with 21 male Wistar rats (Rattus norvegicus albinus), which were split into three groups, corresponding to 7, 21 and 45 experimental days. Ostectomies associated with odontotomies were performed on the outer lateral face of the mandibular body and in the ascending branch, cross-sectionally to the root of the lower incisor and to the molar apices. This surgical defect allowed for the use of magnetized (test group) and non-magnetized (control group) metal devices associated with molar apices and with the distal stump of the lower incisor, where the lyophilized bone allograft was performed. The intensity of the buried and permanent magnetic field amounted to 250 gauss. Results: In the test and control groups, there was gradual integration of the lyophilized bone allograft, maintenance of pulp vitality of the molars and of the proximal stump of the lower incisor, in addition to continuous eruption of the lower incisor. In the test groups, especially at 45 days, there was distinct and pronounced centripetal new bone formation in a direction toward the magnetized metal devices. Conclusion: the in vivo buried magnetic field favored bone healing at the site of the surgical defect.

Made available in DSpace on 2014-06-11T19:33:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2003-09-09Bitstream added on 2014-06-13T20:05:26Z : No. of bitstreams: 1 martins_mc_dr_arafo.pdf: 2338184 bytes, checksum: d19d1faa6e5b721d78be39854a2bfb47 (MD5) / O propósito do presente estudo foi o de avaliar histomorfometricamente a neoformação óssea da região posterior de maxila após os procedimentos cirúrgicos de elevação de seio maxilar e enxerto ósseo autógeno inlay associado ou não ao Plasma Rico em Plaquetas, em dois períodos de tempo: 6 e 8 meses. Foram selecionados 20 pacientes, com idade média de 42 anos, em boas condições de saúde geral. Os pacientes foram divididos, aleatoriamente em três grupos sendo: Grupo 1: enxerto ósseo autógeno e biópsia da área 8 meses após a cirurgia, 4 pacientes (grupo controle). Grupo 2: enxerto ósseo autógeno associado ao plasma rico em plaquetas e biópsia da área 6 meses após a cirurgia, 8 pacientes. Grupo 3: enxerto ósseo autógeno associado ao plasma rico em plaquetas e biópsia da área 8 meses após a cirurgia, 8 pacientes. Encontrou-se diferenças significantes nos períodos (G2 vs G3) em relação ao tecido ósseo (p=0,05) e em relação aos tecidos não calcificados (p=0,045), e nos tratamento (G1 vs G3) em relação a presença de material de enxerto (p=0,041). Baseado nos resultados obtidos, pôde-se concluir que em relação à quantidade e qualidade do tecido ósseo formado o uso do Plasma Rico em Plaquetas não apresentou benefícios adicionais à utilização do osso autógeno nos procedimentos cirúrgicos de elevação de seio maxilar. / The aim of the present study was to evaluated, histologic and histomorphometrically, bone formation after maxillary sinus floor augmentation by grafting with an association of PRP and autogenous bone from the symphyseal area. Out of 20 patients were used in this study, and they were distributed in three groups, G1: control - autogenous bone graft, 8 months healing period, G2 - autogenous bone graft and PRP, 6 months healing period, and G3: autogenous bone graft and PRP, 8 months healing period. Before surgery the blood was collected and proper stored, until PRP preparation. Subantral augmentation was performed and the graft was collected. The blood was centrifuged, the concentrate platelet-rich plasma layer was removed and associated to the autogenous graft. The mixture assumed a gel-like consistency, which was used to fill the sinus. After the period healing, implants were placed and core biopsies were taken from the area. Routine histological processing and staining were performed. There were no significant differences between the treatments (G1 vs G3). In regard to the healing period (G2 vs G3) we found significant differences due to bone (p=0,05) and non-calcified tissues (p=0,045), in regard to the type of treatment (G1 vs G3) we found significant differences due to the presence of grafted material (p=0,041). Within the limits of this study the association of PRP and autogenous bone did not bring any additional benefits in regard to bone formation.

Proposição: O presente trabalho propõe o estudo histológico descritivo da aplicação de um campo magnético sepultado, estático e permanente em área de enxerto ósseo alógeno liofilizado e dento-alveolar associada a defeito cirúrgico em mandíbulas de ratos. Metodologia: estudo experimental in vivo, randomizado. Amostra foi composta por 21 ratos, da espécie Rattus novergicus albinus, linhagem Wistar, machos, divididos em três grupos, correspondendo aos tempos experimentais de sete, 21 e 45 dias. Foram realizadas ostectomias associadas a odontossecções, na face lateral externa do corpo mandibular e no ramo ascendente, atingindo transversalmente a raiz do incisivo inferior e os ápices dos molares. Este defeito cirúrgico permitiu a adaptação de dispositivos metálicos magnetizados (grupo teste) e não magnetizados (grupo controle) associados às regiões apicais dos molares e ao coto distal do incisivo inferior, local este onde realizou-se enxertia óssea alógena liofilizada. A intensidade média do campo magnético sepultado e permanente foi de 250 gauss.Resultados: observou-se no grupo teste e controle, nos diferentes tempos experimentais a gradativa integração do enxerto ósseo alógeno liofilizado, a manutenção da vitalidade pulpar dos molares e coto proximal do incisivo inferior, além da contínua erupção do incisivo inferior do rato. Nos grupos teste, principalmente aos 45 dias, constatou-se uma diferenciada e exuberante neoformação óssea centrípeta em direção aos dispositivos metálicos imantados. Conclusão: o campo magnético sepultado in vivo foi capaz de favorecer o processo de cicatrização óssea no defeito cirúrgico criado. / Purpose: The present study provides a descriptive histological analysis of the use of a buried, static and permanent magnetic field in lyophilized bone allografts in a dentoalveolar area associated with surgical defects in rat mandibles. Method: a randomized in vivo experimental study was carried out with 21 male Wistar rats (Rattus norvegicus albinus), which were split into three groups, corresponding to 7, 21 and 45 experimental days. Ostectomies associated with odontotomies were performed on the outer lateral face of the mandibular body and in the ascending branch, cross-sectionally to the root of the lower incisor and to the molar apices. This surgical defect allowed for the use of magnetized (test group) and non-magnetized (control group) metal devices associated with molar apices and with the distal stump of the lower incisor, where the lyophilized bone allograft was performed. The intensity of the buried and permanent magnetic field amounted to 250 gauss. Results: In the test and control groups, there was gradual integration of the lyophilized bone allograft, maintenance of pulp vitality of the molars and of the proximal stump of the lower incisor, in addition to continuous eruption of the lower incisor. In the test groups, especially at 45 days, there was distinct and pronounced centripetal new bone formation in a direction toward the magnetized metal devices. Conclusion: the in vivo buried magnetic field favored bone healing at the site of the surgical defect.

Resumo: O propósito do presente estudo foi o de avaliar histomorfometricamente a neoformação óssea da região posterior de maxila após os procedimentos cirúrgicos de elevação de seio maxilar e enxerto ósseo autógeno inlay associado ou não ao Plasma Rico em Plaquetas, em dois períodos de tempo: 6 e 8 meses. Foram selecionados 20 pacientes, com idade média de 42 anos, em boas condições de saúde geral. Os pacientes foram divididos, aleatoriamente em três grupos sendo: Grupo 1: enxerto ósseo autógeno e biópsia da área 8 meses após a cirurgia, 4 pacientes (grupo controle). Grupo 2: enxerto ósseo autógeno associado ao plasma rico em plaquetas e biópsia da área 6 meses após a cirurgia, 8 pacientes. Grupo 3: enxerto ósseo autógeno associado ao plasma rico em plaquetas e biópsia da área 8 meses após a cirurgia, 8 pacientes. Encontrou-se diferenças significantes nos períodos (G2 vs G3) em relação ao tecido ósseo (p=0,05) e em relação aos tecidos não calcificados (p=0,045), e nos tratamento (G1 vs G3) em relação a presença de material de enxerto (p=0,041). Baseado nos resultados obtidos, pôde-se concluir que em relação à quantidade e qualidade do tecido ósseo formado o uso do Plasma Rico em Plaquetas não apresentou benefícios adicionais à utilização do osso autógeno nos procedimentos cirúrgicos de elevação de seio maxilar. / Abstract: The aim of the present study was to evaluated, histologic and histomorphometrically, bone formation after maxillary sinus floor augmentation by grafting with an association of PRP and autogenous bone from the symphyseal area. Out of 20 patients were used in this study, and they were distributed in three groups, G1: control - autogenous bone graft, 8 months healing period, G2 - autogenous bone graft and PRP, 6 months healing period, and G3: autogenous bone graft and PRP, 8 months healing period. Before surgery the blood was collected and proper stored, until PRP preparation. Subantral augmentation was performed and the graft was collected. The blood was centrifuged, the concentrate platelet-rich plasma layer was removed and associated to the autogenous graft. The mixture assumed a gel-like consistency, which was used to fill the sinus. After the period healing, implants were placed and core biopsies were taken from the area. Routine histological processing and staining were performed. There were no significant differences between the treatments (G1 vs G3). In regard to the healing period (G2 vs G3) we found significant differences due to bone (p=0,05) and non-calcified tissues (p=0,045), in regard to the type of treatment (G1 vs G3) we found significant differences due to the presence of grafted material (p=0,041). Within the limits of this study the association of PRP and autogenous bone did not bring any additional benefits in regard to bone formation. / Orientador: Ricardo Samih Georges Abi Rached / Coorientador: Elcio Marcantonio Junior / Banca: Luís Carlos Spolidorio / Banca: Joni Augusto Cirelli / Banca: Luís Antônio Violin Dias Pereira / Banca: Elton Gonçalves Zenóbio / Doutor

Proposição: O presente trabalho propõe o estudo histológico descritivo da aplicação de um campo magnético sepultado, estático e permanente em área de enxerto ósseo alógeno liofilizado e dento-alveolar associada a defeito cirúrgico em mandíbulas de ratos. Metodologia: estudo experimental in vivo, randomizado. Amostra foi composta por 21 ratos, da espécie Rattus novergicus albinus, linhagem Wistar, machos, divididos em três grupos, correspondendo aos tempos experimentais de sete, 21 e 45 dias. Foram realizadas ostectomias associadas a odontossecções, na face lateral externa do corpo mandibular e no ramo ascendente, atingindo transversalmente a raiz do incisivo inferior e os ápices dos molares. Este defeito cirúrgico permitiu a adaptação de dispositivos metálicos magnetizados (grupo teste) e não magnetizados (grupo controle) associados às regiões apicais dos molares e ao coto distal do incisivo inferior, local este onde realizou-se enxertia óssea alógena liofilizada. A intensidade média do campo magnético sepultado e permanente foi de 250 gauss.Resultados: observou-se no grupo teste e controle, nos diferentes tempos experimentais a gradativa integração do enxerto ósseo alógeno liofilizado, a manutenção da vitalidade pulpar dos molares e coto proximal do incisivo inferior, além da contínua erupção do incisivo inferior do rato. Nos grupos teste, principalmente aos 45 dias, constatou-se uma diferenciada e exuberante neoformação óssea centrípeta em direção aos dispositivos metálicos imantados. Conclusão: o campo magnético sepultado in vivo foi capaz de favorecer o processo de cicatrização óssea no defeito cirúrgico criado. / Purpose: The present study provides a descriptive histological analysis of the use of a buried, static and permanent magnetic field in lyophilized bone allografts in a dentoalveolar area associated with surgical defects in rat mandibles. Method: a randomized in vivo experimental study was carried out with 21 male Wistar rats (Rattus norvegicus albinus), which were split into three groups, corresponding to 7, 21 and 45 experimental days. Ostectomies associated with odontotomies were performed on the outer lateral face of the mandibular body and in the ascending branch, cross-sectionally to the root of the lower incisor and to the molar apices. This surgical defect allowed for the use of magnetized (test group) and non-magnetized (control group) metal devices associated with molar apices and with the distal stump of the lower incisor, where the lyophilized bone allograft was performed. The intensity of the buried and permanent magnetic field amounted to 250 gauss. Results: In the test and control groups, there was gradual integration of the lyophilized bone allograft, maintenance of pulp vitality of the molars and of the proximal stump of the lower incisor, in addition to continuous eruption of the lower incisor. In the test groups, especially at 45 days, there was distinct and pronounced centripetal new bone formation in a direction toward the magnetized metal devices. Conclusion: the in vivo buried magnetic field favored bone healing at the site of the surgical defect.