京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22737号 / 医博第4655号 / 新制||医||1046(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 伊佐 正, 教授 林 康紀, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Human palatine tonsil lymphocytes, when compared with peripheral blood lymphocytes (PBL), were in an activated state, even though there was no in vitro stimulation. When these tonsil lymphocytes were cultured in the absence of serum and polyclonal mitogens or antigens, the supernatant fluid often inhibited the proliferative response of target PBL to concanavalin A. The extent of this suppression ranged from 22% to 84%, and target cell viability was 90% or greater. There was no evidence for the presence of immunoglobulins or α2-macroglobulin in whole supernatant fluids. The suppressor was partially denatured at 80°C and was rendered completely inactive upon exposure to 100°C for 5 min. It was trypsin-sensitive, and had an apparent molecular weight of 100,000 or greater. The protein adhered strongly to DE-52, and the most active material was eluted with 0.4–0.6 M NaCl. Only one component was detected in this fraction by polyacrylamide gel electrophoresis. The suppressor had an isoelectric point of 5.0 ± 0.6.

A inflamação associada à obesidade é caracterizada por uma ativação crônica e de baixa intensidade do sistema imune. Diversos autores demonstraram mudanças em parâmetros inflamatórios após perda de peso. A cirurgia bariátrica é um método para tratar obesidade com alta eficiência e menor risco de recidiva. Os mononucleares de sangue periférico (MNSP) constituem um material biológico interessante para pesquisa, visto a capacidade de refletir alterações de expressão gênica de diferentes tecidos e o fácil acesso para análise. O presente estudo teve por objetivo avaliar os efeitos da obesidade e da perda de peso induzida por cirurgia bariátrica sobre a atividade imunológica, por meio de cultura primária de MNSP de pacientes com obesidade grau III (IMC >= 40 kg/m2). Foram coletadas amostras de sangue de veia periférica de 10 voluntários com peso normal (grupo controle) e antes e após a cirurgia de 20 voluntários com obesidade grau III. Após a separação dos mononucleares pelo gradiente de Ficoll-HyPaque, as células foram estimuladas por lipopolissacarídeo (LPS) ou concanavalina A (Con-A) e os sobrenadantes das culturas coletados para dosagem de IL-1?, IL-6, TNF-?, IFN-?, IL-10 e IL-17 por teste ELISA. As amostras de sangue também foram utilizadas para exames bioquímicos, dosagens de adiponectina, leptina e citocinas séricas. Os resultados evidenciaram maiores concentrações de IL-6, TNF-?, IL-1? e IL-10 nos sobrenadantes das culturas de MNSP do grupo com obesidade em relação ao grupo controle. Na comparação entre dosagens de citocinas do grupo com obesidade, observamos redução de TNF-?, IL-1? e IL-10 após 6 meses da cirurgia, a qual não foi observada após 1 ano, e aumento de IL-17 após 1 ano de tratamento. Não houve diferença significativa nas concentrações de citocinas séricas na comparação entre os grupos com obesidade e controle ou pré e pós-operatório. Observamos correlações das citocinas de sobrenadante das culturas de MNSP e séricas com resultados laboratoriais relacionados à homeostase glicêmica em pacientes com obesidade antes e após a cirurgia bariátrica, além da correlação entre citocinas do sobrenadante e o estado de adiposidade no pós-operatório. Concluímos que a obesidade grau III está associada a modificações da produção de citocinas por MNSP e a perda de peso induzida por cirurgia bariátrica influencia esta produção no primeiro ano de tratamento. / The obesity-associated inflammation is characterized by a chronic and low intensity activation of the immune system. Several authors have shown changes in inflammatory parameters after weight loss. Bariatric surgery is a method for treating obesity with high efficiency and less risk of recurrence. Peripheral blood mononuclear cells (PBMC) are an interesting biological material for research, due to the ability to reflect changes in gene expression in different tissues and easy access to analysis. This study aimed to evaluate the effects of obesity and weight loss induced by bariatric surgery on immune activity through PBMC culture of morbidly obese patients (BMI >= 40 kg/m2). Peripheral vein blood samples were collected from 10 volunteers with normal weight (control group) and before and after the surgery in 20 volunteers with morbid obesity. After separation of the mononuclear cells by Ficoll-Hypaque gradient centrifugation, cells were stimulated with lipopolysaccharide (LPS) and concanavalin A (Con-A) and culture supernatants collected for IL-1?, IL-6, TNF-?, IFN-?, IL-10 and IL-17 dosage by ELISA. Blood samples were also used for biochemical examinations and adiponectin, leptin and serum cytokine dosage. The results showed higher concentrations of IL-6, TNF-?, IL-1? and IL-10 in the supernatants of the MNSP cultures of the obesity group in relation to the control group. In the comparison between cytokine dosages of the obesity group, we observed reduction of TNF-?, IL-1? and IL-10 after 6 months of surgery, which was not observed after 1 year, and IL-17 increased after 1 year of treatment. There was no significant difference in serum cytokine concentrations in the comparison between obesity and control groups or operated group. We observed correlations of cytokines obtained in PBMC culture supernatant and serum with laboratory results related to glucose homeostasis in patients with obesity before and after bariatric surgery, as well as correlation between cytokines of the supernatant and the state of adiposity postoperatively. We conclude that morbid obesity is associated with changes in cytokine production by PMNC and weight loss induced by bariatric surgery influences cytokine production in the first year of treatment.

Myocardial infarction (MI) occurs following acute thrombotic occlusion of a coronary artery, and triggers a robust inflammatory response. Within hours, neutrophils are recruited to the infarcted myocardium followed by the infiltration of pro-inflammatory Ly6Chi monocytes. Transition from the pro-inflammatory macrophage phenotype (M1) to an anti-inflammatory, pro-resolution phenotype (M2-like) is critical to successful infarct healing. Interventions that polarize macrophages towards an anti-inflammatory 'M2-like' phenotype improve infarct healing in the experimental MI mouse model and reduce subsequent adverse remodelling of the myocardium, but the endogenous mechanisms that regulate repair are not well understood. Furthermore, differences in the resolution of inflammation in C57BL/6 and BALB/c mice, which are two of the commonly used wild-type mouse strains in experimental MI have not been characterised. We previously found that low peripheral blood eosinophil count is associated with increased short-term risk of mortality in low-intermediate risk patients with ischaemic heart disease. This suggests that eosinophils may have a role in the successful remodelling and repair of the heart following myocardial infarction. Eosinophils express a number of immuno-modulating cytokines and lipid mediators implicated in the resolution of inflammation. Increasingly prominent is interleukin-4 (IL-4), a cytokine that has been found to maintain the anti-inflammatory M2-like phenotype in macrophages. We therefore hypothesised that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are key in regulating the subsequent inflammatory response and scar tissue formation during infarct repair and cardiac remodelling. Experimental MI was induced by permanent left anterior descending artery ligation in isofluorane anaesthetized 12-15 week-old male wild-type (WT) BALB/c, WT C57BL/6, IL4Rα-/-, IL-4Rαflox/-, IL-4Rαflox/-LysMCre mice and eosinophil-deficient ΔdblGATA mice. Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of single cell infarct and remote zone tissue digests. Blood eosinophil count and 6-month all-cause mortality were assessed in 732 consecutive patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction (STEMI). The rate of mortality due to cardiac rupture was significantly higher in C57BL/6 mice in comparison with BALB/c mice at Day 7 post-MI. This was associated with a higher proportion of pro-inflammatory Ly-6Chi macrophages infiltrating the infarct zone tissue of C57BL/6 mice following MI. An accompanying reduction in the number of splenic Ly-6Chi monocytes post-MI, suggestive of splenic monocyte mobilisation, was seen in C57BL/6 mice but not found in BALB/c mice. Furthermore, C57BL/6 mice had a delayed transition in macrophage polarisation towards an anti-inflammatory phenotype. Disruption of IL4Rα signalling, in mice null for the IL4Rα gene, resulted in increased F4/80+ macrophage and pro-inflammatory Ly6Chi macrophage infiltration of the infarct zone and reduced expression of the anti-inflammatory macrophage marker CD206, compared to wild-type controls. Furthermore, expression of GATA3 and ST2, both associated with the immunosuppressive function of (CD4+ Foxp3+) regulatory T cells, was reduced in infarct zone regulatory T cells from IL4Rα-/- mice. These findings were associated with defective wound healing with impaired angiogenesis, increased scar size, disarrayed infarct zone collagen deposition, accompanied by modified expression of plod2 that encodes the collagen cross-linking enzyme lysyl hydroxylase 2. Resulting in greater left ventricular dilatation and loss of cardiac function, as well as a higher 7- day mortality due to cardiac rupture in IL4Rα-/- mice. This indicates that successful infarct repair requires the engagement of IL-4Rα signalling to facilitate the accumulation of anti-inflammatory macrophages and highly immunosuppressive ST2+ regulatory T cells in the heart following MI. Resident cardiac macrophages from naïve hearts of IL-4Rαflox/-LysMCre mice failed to undergo LysMCre-mediated deletion of the IL-4Rα gene, potentially because low or absent expression of Lyz2 (encoding lysozyme M). In both ST-elevation MI (STEMI) patients and mice after acute MI, there was a decline in peripheral blood eosinophil count, with activated eosinophils being recruited to the infarct zone and paracardial adipose tissue of mice. The transcription factors GATA-1 plays a role in the differentiation of eosinophils from eosinophil progenitor cells. Deletion of GATA-1 results in loss of the eosinophil lineage and has been exploited to develop the eosinophil-deficient ΔdblGATA mouse. ΔdblGATA mice were used to address the role of eosinophils in cardiac remodelling following MI. ΔdblGATA mice had increased left ventricular dilatation and reduced ejection fraction after induction of MI, relative to wild-type mice. ΔdblGATA mice had increased scar size with disarrayed infarct zone collagen deposition, accompanied by modified expression of the genes plod2 and lox, which are associated with collagen cross-linking. The proportion of CD206+ anti-inflammatory macrophages was less in the infarct zone of ΔdblGATA mice, but was restored by adoptive transfer of eosinophils from WT mice. Furthermore, adverse cardiac remodelling in eosinophil-deficient ΔdblGATA mice was rescued by provision of IL-4 complex following MI. In conclusion, an enhanced inflammatory response following MI underlies the increased risk of cardiac rupture seen with WT C57BL/6 mice in comparison to WT BALB/c mice. WT BALB/c mice are protected from cardiac rupture, which was associated with an absence of splenic monocyte mobilisation following ischaemic injury. The resolution of inflammation was found to be dependent on IL4Rα signalling which is crucial for cardiac repair and remodelling, through modulating inflammatory cell recruitment and phenotype, as well as scar formation. Eosinophils are recruited to the heart post-MI and are essential for regulating cardiac repair and remodelling, likely through provision of IL-4. Therefore, we were able to show that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are both key in regulating the subsequent inflammatory response and scar tissue formation during infarct healing and cardiac remodelling.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / A doenÃa de Chagas à causada pelo parasito intracelular Trypanosoma cruzi e afeta em torno de 17 milhÃes de pessoas na AmÃrica Latina. A fase crÃnica da doenÃa caracteriza-se por baixo nÃvel de parasitemia e alto nÃvel de anticorpos Anti-T.cruzi e o diagnÃstico à feito preferencialmente utilizando-se mÃtodos sorolÃgicos, incluindo imunofluorescÃncia indireta (IFI), hemaglutinaÃÃo indireta (HAI) e imunoensaio enzimÃtico (ELISA). SÃo testes que apresentam alta sensibilidade, mas sofrem de baixa especificidade, e um variÃvel nÃmero de indivÃduos apresenta testes sorolÃgicos inconclusivos. Nos Ãltimos anos, muitos estudos tÃm usado a tecnologia da ReaÃÃo em Cadeia da Polimerase (PCR) para detectar sequÃncias de DNA de T. cruzi em sangue de pacientes chagÃsicos crÃnicos. A alta especificidade da PCR tem apontado para sua aplicaÃÃo como mÃtodo confirmatÃrio no diagnÃstico de pacientes com provas sorolÃgicas inconclusivas. O objetivo desse trabalho foi realizar uma anÃlise comparativa entre a PCR e os mÃtodos convencionais mais utilizados para o diagnÃstico - sorologia e xenodiagnÃstico, na detecÃÃo da infecÃÃo por T. cruzi, em indivÃduos com doenÃa de Chagas crÃnica. Sangue de 67 pacientes chagÃsicos crÃnicos, ambos os sexos, provenientes do ambulatÃrio de doenÃa de Chagas do Hospital UniversitÃrio Walter CantÃdio, da Universidade Federal do CearÃ, foram avaliados para T. cruzi, utilizando testes sorolÃgicos (IFI, HAI e ELISA), PCR-Multiplex e xenodiagnÃstico. AlÃm disso, foi avaliado tambÃm a PCR-Multiplex em dejetos de triatomÃneos provenientes do xenodiagnÃstico destes pacientes. De acordo com o resultado dos testes sorolÃgicos, os pacientes foram classificados em 3 grupos: 1Â. Sorologia positiva (quando 2 ou 3 resultados dentre os 3 testes sorolÃgicos foram positivos), 2Â. Sorologia inconclusiva ou indeterminado (apenas 2 resultados foram negativos) e 3Â. Sorologia negativa (quando os 3 resultados foram negativos). Dentre as 59 amostras com sorologia positiva foram obtidos 18 (30,5%) resultados positivos de PCR-Multiplex em sangue perifÃrico e 41 (69,5%) resultados negativos. Nos PCR-Multiplex em dejetos de triatomÃneos foram obtidos 20 (33,9%) resultados positivos e 39 (66,1%) resultados negativos. As 2 amostras com sorologia inconclusiva foram negativas na PCR-Multiplex, tanto em sangue perifÃrico como em dejetos de triatomÃneos. As amostras com sorologia negativa (n=6) apresentaram 2 resultados positivos (33,3%) na PCR-Multiplex em sangue e 4 resultados negativos (66,7%). Nos dejetos de triatomÃneos, as 6 amostras com sorologia negativa foram negativas na PCR-Multiplex. Estes resultados mostraram que o ensaio da PCR-Multiplex em amostras de sangue perifÃrico e em dejetos de triatomÃneos, empregando iniciadores para regiÃes diferentes, no caso, DNA nuclear e DNA do cinetoplasto, podem ser aplicados para o diagnÃstico da doenÃa de Chagas crÃnica (mesmo com diferentes metodologias de extraÃÃo de DNA total). Os dados mostraram tambÃm que a PCR-Multiplex em dejetos de triatomÃneos à mais sensÃvel que o ensaio do xenodiagnÃstico.

Telomeres are protective structures at the end of our chromosomes, composed of multiple repeats of the DNA sequence TTAGGG. They are essential for maintaining chromosomal stability by preventing damage and degradation of the chromosome ends. Telomeres are normally shortened with each cell division until a critical length is reached, at which stage cell cycle arrest is induced. Telomere shortening can be prevented in the presence of the telomere-­‐elongating enzyme telomerase. Telomerase is expressed during embryogenesis and in certain normal cell types, but most somatic cells exhibit undetectable levels of telomerase activity. In contrast, most cancer cells express telomerase enabling them to proliferate indefinitely. There is a search for reliable molecular markers that can be used to help predict cancer risk and outcome. The interest of investigating telomere length as a potential biomarker in malignancy has grown rapidly, and both tumors and normal tissues have been in focus for telomere length measurements. In this thesis, telomere length was investigated in breast cancer patients and in patients with renal cell carcinoma (RCC). The breast cancer patients were found to have significantly longer mean telomere length in peripheral blood cells (i.e. immune cells) compared to a tumor-­‐free control group. Moreover, patients with the longest blood telomere length had a significantly worse outcome compared to patients with shorter blood telomeres. In a patient group with clear cell RCC, telomere length was investigated in peripheral blood cells, in tumors and in corresponding kidney cortex. Again, patients with the longest blood telomere length had a significantly worse prognosis compared to those with shorter blood telomeres. In contrast, telomere length in tumor and kidney cortex tissues did not predict outcome per se. Immunological components may play a role in telomere length dynamics as well as in cancer development. We aimed to investigate a possible association between telomere length and certain immunological parameters, including various cytokines and peripheral levels of a blood cell type with suppressor function [regulatory T cells (Tregs)]. In our patients with clear cell RCC, three cytokines correlated significantly with tumor telomere length, but not with telomere length in peripheral blood cells. In a separate patient group with various RCC tumors, blood telomere length correlated positively with the amount of Tregs. It might be speculated that a subset of patients with long blood telomeres has a less efficient immune response due to high Treg levels, contributing to a worse prognosis. Another aim of this thesis was to explore telomere length changes over time. Evaluation of blood samples collected at a 6-­‐month interval from 50 individuals, showed that half of the participants experienced a decline in mean telomere length during the time period. This group had longer telomere length at baseline compared to those who demonstrated increased/stable telomere length. In a separate group of five blood donors, a remarkable drop in telomere length was detected in one donor over a 6-­‐month period, whereas the other donors exhibited only small fluctuations in telomere length. In conclusion, the results of this thesis indicate that blood telomere length has potential to act as an independent prognostic marker in malignancy. Adding to the complexity is the fact that changes in blood telomere length might occur within relatively short time spans, indicating that telomere length is a dynamic character.

Telomeres are repetitive TTAGGG structures ending each chromosome and thereby protecting its integrity. Due to the end-replication problem, telomeres shorten with each cell division. When reaching a critical telomere length (TL), the cells stop dividing and enter replicative senescence. It has been speculated that telomeres might regulate lifespan at the organism level but this hypothesis is controversial. However, telomeres in human blood cells do shorten with increasing age. Telomerase is an enzyme capable of lengthen telomeres. It consists of a catalytic subunit, hTERT, and a RNA template, hTR. Telomerase is active in germ cells, stem cells, activated lymphocytes and highly proliferating epithelial cells while no activity is found in other somatic cells. One step in order to produce a tumour mass is that cancer cells need to have a limitless replicative potential and this can be achieved by activating telomerase. Most tumour cells express telomerase activity and hence, the enzyme is an interesting target for cancer therapy. Telomere length is in part inherited. Two separate family cohorts were investigated to elucidate the inheritance pattern and a strong paternal inheritance was observed. In the larger, multifamily cohort spanning up to four generations, a weak correlation between the TL of the mother and the child was also found, as well as a significant correlation between grandparent-grandchild pairs. Interestingly, the heritable impact diminished with increasing age, indicating than non-heritable factors might influence TL during life. A functional T to C transition polymorphism in the hTERT promoter was previously reported, showing that the -1327C/C genotype was correlated with shorter TL compared to the alternative genotypes in healthy individuals and in coronary artery disease patients. When investigating 226 myocardial infarction patients and 444 controls separately, no differences were observed regarding mean TL or increased attrition rate between the different genotypes. TL in blood cells is shown to be altered in patients with certain types of solid tumours. In our breast cancer cohort, TL was a strong prognostic marker. Short telomeres were associated with increased survival, especially in young patients and in those with advanced tumours. It has been speculated that cancer patients might have a faster telomere attrition rate than controls but this has not been experimentally proven. Two blood samples from the same individual taken with 9-11 years interval was investigated. Some were diagnosed with a malignancy after the second blood draw. When comparing patients with controls, telomere attrition rate was not correlated to future tumour development. About one third of the individuals elongated their telomeres over a decade and the individual telomere attrition rate was telomere length dependent, showing an inverse correlation to TL at a highly significant level. This strongly suggests that the TL maintenance mechanism shown to provide protection for short telomeres in vitro is important also in human cells in vivo.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

Dendritic cells (DCs) are potent antigen presenting cells (APCs) that link the innate and adaptive immune system by their unique ability to induce and direct immune responses towards various T helper (Th)-type of immune responses such as Th1-, Th2-, Th9-, Th17-, Th22- or T regulatory (TR). The type of Th response generated very much depends on the nature of the antigen encountered and allows for an effective and proficient immune response. For example, Th1 responses are used to clear intracellular pathogens while Th2 responses are needed to clear extracellular pathogens The ability to specifically modulate Th-responses is an area of intense research, as it allows for the development of more effective vaccines and immunotherapeutics. Immunomodulation of DCs is one strategy by which specific Th-type immune responses may be tailored. Current research is focused on identifying agents that have the capacity to immunomodulate DCs such as host defense peptides (HDPs). Apart from their anti-microbial activities, HDPs have a number of immune functions including recruitment and subsequent activation of DCs. The goal of this study was to examine the immunomodulatory effects of HDPs on porcine DC functions. This research was part of a larger multinational research project to develop a novel adjuvant platform for single-immunization vaccines against pertussis in neonates. The pig model was used for this research because of its physiological similarities to humans and the recently developed pertussis infection model in young piglets. A series of experiments was conducted to characterize and describe porcine DC functions. Two subsets of DCs were successfully characterized and tested for their response to stimulation with HDPs. Initial results demonstrated a minimal effect of HDPs on DC functions, therefore we expanded the number of HDPs used to include both synthetic derivatives of HDPs known as innate defense regulators (IDRs) and naturally- occurring HDPs. We examined these effects on peripheral blood mononuclear cells (PBMC) in vitro and found that HDPs induce expression of the chemokine interleukin (IL)-8, which resulted in PBMC recruitment in vitro. We then proceeded to evaluate the HDPs in vivo by intradermally administering them into the flank of pigs. Surprisingly, treatment with the HDPs did not result in recruitment of neutrophils in vivo. We also examined the effects of formulating IDR-1002 as an adjuvant with the academic antigen Keyhole Limpet Hemocyanin (KLH) on the development of KLH-specific immune responses in vaccinated pigs. While there was no difference in antibody titers between vaccinated and control animals, we found that co-formulation with IDR-1002 decreased both antigen-specific and mitogen-induced proliferation in KLH/IDR-1002 vaccinated animals as long as four weeks post-treatment. These results demonstrate that specific IDRs can suppress certain aspects of the pro-inflammatory immune response making them potentially highly versatile tools to modulate and tailor the immune response in disease states characterized by a pro-inflammatory component.

<p>My thesis research has centered on the concept of the peripheral blood cell (PBC) as an indicator of disease and environmental exposure. The PBC is not only easily accessible and constantly replenished, but it provides a snapshot of an individual's health. Doctors have long utilized PBCs as indicators of health based on count, morphology or the expression of particular cell surface markers. Using these methods, PBCs can serve as indicators of infection, inflammation or certain types of hematological malignancies. Now PBCs can be characterized as a function of their gene expression profiles in response to disease and toxicant exposure. Advances in cDNA microarray technology have made it possible to analyze global gene expression in small volumes of whole blood, or even in a sorted population of blood cells. The resulting gene expression data can serve as a molecular phenotype, or signature, of disease or toxicant exposure. These signatures serve a twofold purpose. First, they act as biological markers (biomarkers) that can indicate the presence of disease or aid monitoring the response to treatment. Second, they provide insight into the underlying biological mechanisms that are at work, by revealing genes, networks and pathways that are affected by the disease or toxin. This paradigm has been applied in a number of contexts, including infection, inflammation, leukemia, lymphoma, neurological disorders, cardiovascular disease, environmental exposures and solid tumors. </p><p>In the work presented here, we describe signatures of lead (Pb) exposure and breast cancer based on peripheral blood gene expression. Our objective in generating a blood-based signature of lead exposure was to develop a potential predictor of past and present exposure. This is particularly relevant because of continued widespread lead exposure through both environmental and occupational sources. Pb causes significant toxicities in a number of different organ systems including the hematological, endocrine, neurological and renal systems. Pb is considered a potential carcinogen due to evidence that it causes cancer in animal models and contributes to an elevated cancer risk in humans. Pb is thought to contribute to cancer risk indirectly through a variety of mechanisms, such as inhibition of DNA synthesis and repair, oxidative damage, interaction with DNA-binding proteins and tumor suppressor proteins, causing chromosomal aberrations and alterations to gene transcription. In addition, it has been shown to exacerbate the effects of other mutagens. Recent work also indicates that even low-level Pb exposure (defined here as levels below the threshold of detection of many common tests or below the level set by the CDC as an "elevated blood lead level" in children, or 10µg/dL) can impact health, especially in children, who are more susceptible to these negative health consequences. </p><p>We hypothesized that we could detect subtle and lasting changes in the PBC transcriptome that correlated to Pb exposure. We used a mouse model of per os Pb exposure to generate signatures corresponding to two different doses of Pb. One dose reflected a high-level exposure and the other a low-level exposure. We also analyzed the gene expression changes following removal of the Pb source. We were able to generate robust, dose-specific signatures of Pb exposure. This supports the growing body of evidence that even low levels of Pb exposure can have biological effects, and that there is likely no safe level of exposure. We also utilized a collection of pathway signatures to identify those pathways that were activated or repressed in response to Pb exposure compared to controls. We observed an increase in interferon-gamma pathway activity in response to low-level Pb exposure and an increase in E2F1 pathway activity in response to high-level Pb exposure. These results support previous findings that low-level Pb exposure can increase interferon-gamma production, whereas high-level Pb has been shown to increase DNA synthesis. The Pb signatures we report here were not predictive of a past lead exposure. These results suggest that the effect of Pb exposure on PBC gene expression is transient, perhaps due to the rapid turnover of blood cells and the absorption of Pb by the bones. We have proposed further studies to identify cells in the bone marrow that may serve as indicators of past Pb exposure based previous reports on the lasting effects of genotoxic stress on this tissue.</p><p>We also describe a predictor of human breast cancer based on peripheral blood gene expression. The objective of this study was to identify and characterize PBC gene expression patterns associated with the presence of a breast tumor. This work has the potential to make a significant impact on breast cancer screening and diagnosis. Despite the success of mammography in reducing mortality from breast cancer, many cancers go undetected due to factors such as breast density, age of the woman, or type of cancer. A blood-based breast tumor predictor would potentially offer an easy and noninvasive means of detecting primary breast cancer as well as monitoring patients for recurrences or metastases. In addition, the concept of using a blood-based biomarker for cancer detection would have positive implications for other types of cancer. For instance, patients with ovarian cancer are typically diagnosed at a late stage because of the absence of definitive symptoms and the lack of effective screenings methods. </p><p>We were able to successfully identify robust predictors of both mouse mammary tumors and human breast tumors based on PBC gene expression. The human breast tumor predictor exhibits a high level of sensitivity and specificity in distinguishing breast cancer patients and controls in an independent validation cohort. However, the true novelty in this study is that it integrates a factor modeling approach and a transgenic mouse model of breast cancer to identify biologically meaningful gene expression changes in the mouse PBC transcriptome. These genes were then used as the starting point for developing a human breast cancer predictor. This establishes an experimental system in which we can address questions that are inherently difficult to answer in human studies, such as whether this predictor is useful in detecting breast tumors early or in monitoring patients for recurrence or metastasis. In fact, our work suggests that tumor-associated gene expression changes in the PBCs can be detected in asymptomatic mice. Our results support those of previous studies, which identified blood gene expression profiles that are associated with a variety of solid tumors, including breast cancer. However, the sensitivity and specificity of our predictor are higher than that of the previously reported breast cancer signature. This may suggest that our strategy of using a mouse model to first identify informative genes allowed us to focus on those genes most relevant to the presence of a breast tumor and overcome the influence of the high degree of variation in blood gene expression in our human population. In order to be clinically useful, the predictor we report here would need to be tested in additional, large validation sets to establish its utility in an early detection setting and its specificity in distinguishing breast cancer from other cancer types as well as other potentially confounding conditions such as infection and inflammation. We describe some preliminary experiments in the mouse model intended to address these important questions.</p> / Dissertation