Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-14T12:30:34Z No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:05:05Z (GMT) No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Made available in DSpace on 2016-10-22T13:05:05Z (GMT). No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) Previous issue date: 2008-02-15 / Nos países tropicais, as perdas causadas pela infestação de carrapatos em bovinos acarretam um grande impacto no sistema de produção animal. Recentes estudos têm mostrado a importância de fatores genéticos ligados a resistência a carrapato em Bos taurus indicus e Bos taurus taurus e que as citocinas têm um papel crítico na prevenção ou progressão de doenças. O objetivo desse trabalho foi avaliar os níveis de expressão dos genes IL-10 e IL-4 relacionados ao perfil imunológico Th2 associado à susceptibilidade ao carrapato e os genes IL-2 e IFN- relacionados ao perfil imunológico Th1 associado à resistência ao parasito. Além destes genes, analisou-se o perfil de expressão do gene TLR-2, importante no processo de reconhecimento de patógenos e os genes IL-8 e TNF-α importantes no processo inflamatório inicial. Seis animais mais resistentes e seis animais mais susceptíveis de uma população F2 de 332 animais, originária do cruzamento de animais F1(½ Holandês: ½ Gir), foram selecionados baseado na contagem de carrapatos e valor genético. Amostras de tecido foram coletadas de pele no 5° e 12° dias após a infestação para extração de RNA total. As PCRs em tempo real foram realizadas usando o gene GAPDH como controle endógeno. Os animais resistentes e susceptíveis apresentaram aumento de expressão do gene IL-10 no 5° (p<0,01) e 12 ° dias após a infestação (p<0,05). O gene IL-2, nos animais resistentes e susceptíveis, no 5° dia após a infestação não apresentou alteração da expressão sendo que 12° dia, em ambos os grupos de animais, este gene passou a ser mais expresso em relação ao animal controle sugerindo um perfil de resposta imunológica do tipo de Th2 nos animais resistentes e susceptíveis nos primeiros dias após a infestação. O gene IL-4 apresentou uma tendência ao aumento de expressão nos animais resistentes e susceptíveis em relação ao controle, sendo o perfil Th2 sugerido atribuído a IL-10 produzida por linfócitos T regulatórios (p>0,05). O gene TNF- apresentou aumento de expressão nos animais susceptíveis no 5° dia após a infestação com posterior diminuição no 12° dia após a infestação (p<0,05). Nos animais resistentes não foi observada alteração da expressão deste gene, isto sugere que ele possa estar mais atuante no início do processo inflamatório, logo após a fixação do carrapato. A mesma observação estende-se para o gene IL-8, em que não foi verificada alteração de expressão nos animais resistentes, embora nos animais susceptíveis este gene apresentou diminuição da expressão no 12° dia após a infestação (p<0,05). Quanto ao gene IFN-, não houve diferença de expressão entre os animais resistentes e susceptíveis, sendo que este gene parece não estar relacionado ao mecanismo de resistência. O gene TLR-2 apresentou diminuição da expressão em ambos os grupos de animais. Estes resultados sugerem que a resposta imune adquirida avaliada neste trabalho não apresenta papel preponderante no mecanismo de resistência e que resposta imune inata poderia está envolvida no mecanismo de resistência ao carrapato. Portanto, avaliação da resposta imunológica horas após a fixação do carrapato poderia nos fornecer resultados mais conclusivos. / In tropical countries losses caused by tick infestation in cattle lead to a major impact on animal production systems. Recent studies have shown the importance of genetic factors linked to tick resistance in Bos indicus and Bos taurus as well as the critical role in the prevention or progression of diseases mediated by cytokines. The aim of this work was to evaluate gene expression of IL-10 and IL-4 in relation to tick susceptibility associated with the Th2 profile and gene expression of IL-2 and IFN- in relation to tick resistance associated with the Th1 profile. In addition, the expression of TLR-2, important in the process the recognition of pathogens, and TNF-α and IL-8 genes, important in the initial inflammatory process, were evaluated. Six tick-resistant and six tick-susceptible animals from a F2 population of 332 animals, originated from the cross of F1 animals (½ Holstein: ½ Gir), were selected based on tick count and breeding value for tick resistance. Skin biopsies were collected in the 5th and 12th days after tick infestation. The GAPDH was used as endogenous control to normalize the amount of starting cDNA target in the real-time PCR assay. Both resistant and susceptible animals showed increased gene expression of IL-10 in the 5th and 12th days after infestation in relation to control animal (p<0.05). The IL-2 gene showed no change of expression in the 5th day after infestation for the resistant and susceptible animals. In the 12th post infestation, both resistant and susceptible animals showed increased gene expression in relation to control animal. These results suggest an enhancement of Th2 profile through the increase of IL-10 mRNA levels and a possible inhibition of the Th1 pattern in both groups (resistant and susceptible) starting 5 days after infestation and return to normal by day 12. Despite our results suggest the occurrence of the Th2 profile, the susceptible and resistant animals did not show variation on gene expression for IL-4 in relation to control animal. The susceptible animals showed increased expression of TNF-α in the 5th day after infestation. However, in the 12th day post infestation it was noted a decrease in the gene expression level. The resistant animals showed no change in the expression of this gene in relation to control animals suggesting that TNF-α could be more actively expressed in the early steps of the inflammatory process. Similarly, the resistant animals showed no variation in the expression of IL-8 while the susceptible animals showed increased expression in the 12th day post infestation. There were no differences of expression between resistant and susceptible animals in relation to IFN-γ what suggests that this gene might not be involved in the resistance mechanism. The TLR-2 gene showed decreased expression in both resistant and susceptible animals (p<0.05). Finally, there was no difference in expression between susceptible and resistant animals in relation to all selected genes in the 5th and 12th days after infestation. These results suggest that the acquired immunity evaluated in this work might not have preponderant role in the resistance mechanism. The innate immunity might be playing a major role in the bovine tick resistance/susceptibility mechanism in early hours after infestation.

L’épuisement des lymphocytes T CD8+ (LT CD8) est une voie de différentiation unique survenant lors de contextes pathologiques particuliers ayant en commun la persistance d’antigènes dans l’hôte, tel que les infections virales chroniques (expl : VIH, hépatites B et C) et différents types de cancers. Il apparait aujourd’hui très clairement que ce mécanisme est à l’origine de l’échec de l’immunité adaptative face à ces pathologies particulièrement néfastes pour l’homme. L’étude de ce processus a mené à la découverte de cible thérapeutiques d’un grand intérêt (« immune checkpoints ») pouvant être ciblées pour corriger et/ou reverser l’épuisement. Les essais thérapeutiques ayant découlés de ces découvertes ont donné des résultats extrêmement prometteurs dans le traitement de plusieurs cancers. Cependant, bien que ces thérapies ciblées permettent un regain temporaire de la fonction des LT CD8+, elles ne permettent pas d’inverser le processus d’épuisement. Il est donc crucial aujourd’hui de se tourner vers les agents causateurs de cet état d’épuisement qui restent très méconnues à ce jour. La famille de cytokines partageant la chaine commune gamma (cytokines gamma c) comprenant l’IL-2 -4 -7 -9 -15 et -21 sont des acteurs solubles clés de l’immunité adaptative. Ces cytokines sont intimement liées aux processus de développement, d’homéostasie, de différenciation et de maintenance des lymphocytes T. Parmi elles, l’IL-2 et l’IL-15 ont un rôle majeur dans le processus de différenciation des LT CD8+ au cours d’une infection virale aigue. Malgré cela, l’implication de ces cytokines dans l’épuisement des LT CD8+ dans un contexte d’infection virale chronique n’a jamais été investiguée. En se basant sur les connaissances actuelles des rôles de l’IL-2 et de l’IL-15 sur la différenciation des LT CD8+ au cours d’une infection virale aigue, nous avons émis l’hypothèse que ces cytokines pourraient promouvoir l’épuisement dans un contexte d’infection virale chronique. Dans un premier temps, nous avons démontré chez l’homme (patients atteints d’hépatite C chronique) et la souris (modèle LCMV Clone 13) que la chaîne beta du récepteur à l’IL-2 (IL2R beta[CD122]) qui se lie à l’IL-2 et l’IL-15 reste sélectivement exprimée à la surface des LT CD8+ épuisés au cours d’une infection virale chronique. De plus, une expression élevée de cette chaîne de récepteur corrèle avec un épuisement plus sévère des LT CD8+ chez l’homme et la souris. En développant un modèle murin dans lequel les LT CD8+ sont déficients pour cette chaîne, nous avons démontré que l’IL-2 et IL-15 contrôlent plusieurs aspects clés du processus d’épuisement. Ces cytokines augmentent l’expression de plusieurs récepteurs inhibiteurs (caractéristiques de l’épuisement) et contrôlent même directement l’expression de certains d’entre eux (notamment 2B4 et TIM-3). L’IL-2 et l’IL-15 dirigent également la différenciation terminale des LT CD8+ vers un état d’épuisement extrême et abrogent de manière irréversible leur potentiel de différenciation en cellules mémoires. Nous montrons donc pour la première fois un rôle clé de l’IL-2 et l’IL-15 dans l’épuisement des LT CD8+ au cours d’une infection virale chronique. Dans un deuxième temps nous avons investigué les fonctions individuelles et redondantes de l’IL-2 et l’IL-15 dans l’épuisement des LT CD8+. Nous avons également déterminé les fenêtres d’actions déterminantes de ces cytokines et les mécanismes intracellulaires clés par lesquels elles contrôlent le processus d’épuisement. L’IL-2 et l’IL-15 coopèrent pour promouvoir l’expression de 2B4 et TIM-3 à la surface des LT CD8+ et ces cytokines semblent collaborer pour diriger leur différenciation terminale. En revanche, les signaux médiés par l’IL-2 pendant la phase de « priming » abrogent sélectivement leur potentiel de différenciation en cellules T centrales mémoires (Tcm) alors que l’IL-15 semble plutôt supprimer celle des T effecteurs mémoires (Tem) pendant la phase chronique. Pour finir, nous avons identifié la voie JAK3/STAT5 comme étant la principale voie intracellulaire par laquelle l’IL-2 et l’IL-15 dirigent l’épuisement des LT CD8+. Au cours de cette thèse, nous avons donc mis en évidence un nouveau rôle de l’IL-2 et l’IL-15 dans l’épuisement des LT CD8+ au cours d’une infection virale chronique. Nos résultats apportent une meilleure compréhension du processus d’épuisement des LT CD8+ et démontrent pour la première fois une implication des cytokines. Nous espérons que ces travaux contribueront à améliorer les stratégies thérapeutiques actuelles contre le cancer et les infections virales chroniques. / CD8+ T cell exhaustion is a unique differentiation pathway which occurs during particular pathological contexts such as chronic viral infections (i.e. HIV, HCV and HBV) and cancers in which antigen (Ag) persists in the host. It appears clear now that this mechanism provokes the failure of adaptive responses against these pathologies and is particularly harmful to humans. The study of this process has led to the discovery of relevant molecules (“immune checkpoints”) that can be targeted to prevent and/or reverse exhaustion. Ensuing clinical trials have provided extremely promising results in the treatment of several cancers. However, although these targeted therapies allow a temporary regain of CD8+ T cell functions they still fail at reversing the exhaustion process. It is thus crucial to investigate the causative factors of such process that remain to be identified. The common gamma-chain (gamma c) family of cytokines which includes IL-2, -4, -7, -9, -15, and -21 are key soluble mediators involved in the development of adaptive immunity. These cytokines are intimately linked to T cell development, homeostasis, differentiation and maintenance. Among them, IL-2 and IL-15 display important functions on CD8+ T cell differentiation during an acute viral infection. However, impact of these cytokines on CD8+ T cell responses during a chronic viral infection remains to be investigated. Based on current knowledge of the functions of IL-2 and IL-15 on CD8+ T cell differentiation during an acute viral infection, we hypothesized that these cytokines promote CD8+ T cell exhaustion during a chronic viral infection. We first demonstrate in a mouse model of chronic viral infection (LCMV clone 13) and patients with chronic HCV that the IL-2-receptor beta chain (IL2R beta [CD122]) a cytokine receptor chain which binds to both IL-2 and IL-15 is selectively expressed on exhausted CD8+ T cells during a chronic viral infection. The intensity of CD122 expression positively correlates with severe exhaustion of CD8+ T cells in mice and humans. Using a mouse model in which CD8+ T cells lack the expression of the IL2R beta-chain, we demonstrate that IL-2 and IL-15 control several aspects of exhaustion. IL-2 and IL-15-dependent signals sustain the expression of several inhibitory receptors (characteristic of exhaustion) on CD8+ T cells and directly control the expression of some of them (e.g. 2B4 and TIM-3). IL-2 and IL-15 also direct the terminal exhaustion of CD8+ T cells and irreversibly abrogate their developmental plasticity toward memory T cell development. Together, we show for the first time key functions of IL-2 and IL-15 in directing CD8+ T cell exhaustion during a chronic viral infection. Next, we investigated the unique and redundant functions of IL-2 and IL-15 on CD8+ T cell exhaustion. We also determined individual time-frames of these cytokines and intracellular pathways by which they control CD8+ T cell exhaustion. IL-2 and IL-15 cooperate to promote 2B4 and TIM-3 expression on CD8+ T cells, and these cytokines likely collaborate to direct terminal exhaustion. In contrast, IL-2-dependent signals during priming preclude subsequent differentiation into central memory cells (Tcm) while prolonged exposure to IL-15 upon viral persistence likely suppresses effector memory cell (Tem) developmental potential. Finally, we demonstrate that the JAK3/STAT5 pathway is the dominant pathway by which IL-2 and IL-15 direct CD8+ T cell exhaustion. This thesis provides evidence of novel functions of IL-2 and IL-15 in directing CD8+ T cell exhaustion during a chronic viral infection. These results increase our understanding of the CD8+ T cell exhaustion process and demonstrate for the first time the involvement of cytokines. We hope that this work will contribute to the improvement of actual therapeutic strategies against chronic viral infections and cancers.

The fatty acid amides, a structurally diverse endogenous congener of molecules active in cell signaling, may prove to have diverse activity due to their interface with a number of receptor systems, including, but not limited to cannabinoid receptor 2 (CB2) and PPARγ. Select extracts of Echinacea spp. contain the fatty acid amides known as alkylamides. These extracts were a previously popular remedy relied on by U.S. physicians, one of the top sellers in the natural products industry and are currently a frequently physician prescribed remedy in Germany. In the series of experiments contained within, Galenic ethanolic extracts of Echinacea spp. root were used for the quantification, identification, degradation and bioactivity studies. On extraction, depending on the ratio of plant to solvent and fresh or dry, the data indicate that there is variability in the alkylamide classes extracted. For example the acetylene alkylamides appear to extract under different concentrations, as well as degrade faster than the olefinic alkylamides. In addition, the alkylamides are found to degrade significantly in both cut/sift and powdered forms of echinacea root. Human liver microsome oxidation of the major alkylamide dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide generate hydroxylated, caboxylated and epoxidized metabolites. The carboxylated metabolite has, thus far, shown different immune activity than the native tetraene isobutylamide. Bioactivity studies, based on cytokine modulation of the alkylamides have been assumed to be due to a classic CB2 response. However, the results of experiments contained herein suggest that IL-2 inhibition by the alkylamide undeca-2E-ene-8,10-diynoic acid isobutylamide, which does not bind to CB2, is due to PPARγ activation. These data, combined with data generated by other groups, suggest that the alkylamides of Echinacea spp. are polyvalent in effect, in that they modulate multiple biochemical pathways.

Successful treatment of cancer with adoptive immunotherapy (AIT) is dependent on the ability to produce large numbers of tumor-specific, functional T cells. The purpose of this thesis is to explore ways in which T cell expansion could be augmented. We have focused on exploring alternate gamma chain cytokines as stimulators of T cell proliferation and differentiation in addition to investigating the potential usefulness of gemcitabine (GEM) in abrogating the immunosuppressive effects of myeloid derived suppressor cells (MDSCs). B16 melanoma sensitized draining lymph node cells that have been activated in vitro with bryostatin-1 and ionomycin (B/I) were expanded in either IL-7/15 or in IL-2. We found that IL-7/15 was superior to IL-2 in expanding T cells for AIT of pulmonary metastases. Expansion of antitumor T cells was also improved by suppressing accumulation of MDSCs in mice bearing 4T1 mammary carcinoma using GEM. GEM directly inhibits both 4T1 mammary carcinoma cells and MDSCs. Its inhibition of MDSCs rescued tolerant T cells, augmenting both expansion and response to tumor antigen.

vi ABSTRACT IL-2 has been used in cancer therapy and also for other applications like treatment of chronic viral infections or as an adjuvant for vaccines. However, treatment with IL-2 is rather difficult due to its severe side effects. These toxicities, associated with high-dose treatment necessary for IL-2 to function, have been found the most limiting factor for IL- 2 applications. Further, particular anti-IL-2 monoclonal antibodies (mAb) can actually increase biological activity of IL-2 rather than block it. Binding of IL-2 to anti-IL-2 mAb creates a superagonistic immunocomplexes which have dramatically higher and selective biological activity in comparison to free IL-2 in vivo. Such approach may finally over- come the difficulties associated with administration of IL-2, thus opening brand new scopes for IL-2 and its application not only in the field of tumor therapy. We have shown that IL-2 immunocomplexes composed of IL-2 and anti-IL-2 mAb S4B6 (IL-2/S4B6) stimulate predominantly cells expressing CD122 and CD132 (dimeric IL-2 receptor), i.e. NK and MP CD8+ T cells, with Treg,  T and NKT cells being expanded as well. IL-2/S4B6 are able to drive the expansion of activated naive CD8+ T cells into functional memory-like CD8+ T cells. Moreover, these immunocomplexes exert therapeu- tical potential alone...

Vascular stiffening is defined as the reduced ability of the blood vessels to expand in response to an increase in blood pressure. Vascular stiffening is often not appreciated as a disease in and of itself but is important to recognize because it is considered a predictor of many cardiovascular disease states. Mechanisms of vascular stiffening remain largely unknown; however the immune system has been found to play major roles in cardiovascular disease and arterial remodeling. This dissertation therefore seeks to illustrate the role of the adaptive immune system in vascular stiffening. This has been done by modeling vascular stiffness in transgenic mice lacking an adaptive immune system as well as immunosuppression in normal mice using a novel method to stimulate regulatory T cells with a cytokine immune complex. We have found that inhibition of the immune system by the use of a genetic knockout (RAG 1 ⁻/⁻ mice) or suppression of an existing immune system with an IL-2/anti-IL-2 complex reduces the development of angiotensin II-induced vascular stiffening. This dissertation supports the role of the adaptive immune system, and particularly CD4⁺T cells, in the development of vascular stiffening as well as the protective roles of Tregs in the disease. It also highlights the use of the IL-2/anti-IL-2 complex as a new potential therapy for vascular stiffness. Therapeutics that suppress adaptive immune function may be beneficial in the treatment of vascular stiffening.

No / The interleukin-2-deficient (IL-2¿/¿) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2¿/¿ mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2¿/¿ mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-B (p65) activation with the inhibitory p50 form of NF-B predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.

5 Abstract Interleukin-2 possesses strong stimulatory activity for activated T and NK cells and thus it is an attractive molecule for immunotherapy. However, its unfavourable pharmacological properties, extremely short half-life and severe toxicities associated with high-dose IL-2 are the most serious and limiting drawbacks. Moreover, IL-2 has been also implicated in the homeostasis of T regulatory cells where it plays a decisive role as an essential growth factor of these cells. Several different approaches to improve the therapeutic potential of IL-2 have been studied. Recently described IL-2/anti-IL-2 mAb immunocomplexes which show much higher and selective biological activity in contrast with free IL-2 in vivo are probably the most promising of them. In this study, we compared the biological activity of free IL-2 with IL-2/anti-IL-2 mAb immunocomplexes in order to demonstrate their benefits over free IL-2. We also demonstrated that IL-2/anti-IL-2 mAb immunocomplexes possess noticeable antitumor activity in two syngeneic mouse tumor models, namely EL4 T lymphoma and B16F10 melanoma, if administered early in tumor progression. Therefore, we justified potential use of IL-2/anti-IL-2 mAb immunocomplexes in tumor immunotherapy. We covalently conjugated IL-2 to synthetic semitelechelic polymeric carrier based...

No description available.

It is now clear that human and animal exposure to estrogenic compound occurs through several sources. This include: i) naturally occurring endogenous estrogens, ii) exogenous or intentional estrogens for prophylactic (e.g. oral contraceptive) and therapeutic (e.g. as replacement therapy for ovulation in nulliparous women and in menopausal women, and in some men suffering from prostate cancer) purposes, iii) accidental via estrogenic chemical exposure (e.g. pesticides, industrial byproducts) and phytoestrogens (e.g. soybeans). It has long been recognized that estrogen, a female sex hormone, functions not only on the reproductive system, but also on various other systems including the immune system. Estrogens are thought to be of both physiologic and pathologic importance. Female in general, have better immune capabilities than males, a phenomenon attributed to the action of sex hormones on the immune system. There is also a female-gender bias in susceptibility to autoimmune diseases. Estrogens have been linked either directly or indirectly to the etiology and pathogenesis of various female-predominant autoimmune diseases. Estrogens have also been linked to the onset of cancer, and conditions where the immune system often malfunctions. Estrogen affects the functions of both B and T cells, possibly by regulating such factors as lymphokine gene expression and/or cellular death by apoptosis. However, the functioning of both B and T cells under the influence of long-term exposure to estrogen has not been fully understood. The primary aim of this thesis was to investigate the effect of long-term exposure to 17β-estradiol on lymphokine and bcl-2/fas (proto-oncogenes) mRNA expression. We evaluated the effects of estrogen on the expression of genes for lymphokines, which are essential for the immune response. It is hypothesized that estrogen may regulate the immune system by modifying the expression of lymphokine genes and/or genes that regulate apoptosis. The results demonstrated that long-term 17β-estradiol exposure reduced the viability of lymphocytes when compared to lymphocytes from placebo-treated mice. IL-2 and IFN-g mRNA was consistently higher in ConA-stimulated lymphocytes from estrogen-treated mice (P < 0.05). The mRNA for TGF-β₁ lymphokine was also increased but was not consistent at all time points of incubation. The expression of IL-4 mRNA was not noticeably affected by estrogen treatment of mice. Long-term exposure to 17β-estradiol appear to have some influence on the mRNA expression of proto-oncogenes fas and bcl-2 in splenic and thymic T lymphocytes. There was a trend of increased bcl-2 mRNA expression in estrogen-treated mice compared to placebo-treated mice, whereas the mRNA expression of fas gene appeared to be lower compared to controls. Overall, these findings suggest that 17β-estradiol may selectively influence lymphokine and proto-oncogene mRNA expression. These results suggest that the one mode of modulation of the immune response by 17β-estradiol may be through alterations in the lymphokine and proto-oncogene expression. Since estrogen-treatment markedly induces atrophy of the thymus and diminishes the cellularity of the lymphoid organs (e.g. Spleen), it became necessary to perform multiple assays on the same cells, particularly lymphokine and apoptosis gene expression. A secondary objective of this thesis was to investigate whether lymphocytes, which have undergone proliferation in Lympho-Pro™ assay (Alamar Blue assay), could be utilized for further analysis. In this regard, we found that a non-radioactive assay that utilizes Alamar Blue had significant advantages over the conventional ³H-thymidine incorporation assay. By using cells from estrogen and placebo-treated mice in the Alamar Blue assay, we found that this assay not only allowed determination of lymphocyte proliferation, but also the assessment of mRNA expression, cytogenetics, apoptosis and immunophenotyping of the same lymphocytes. / Master of Science