Melanoma is an aggressive disease with few effective treatment options. Non-toxic, anti-tumor therapies and prophylactic approaches are currently being investigated to identify treatment options that will control and remove late-stage melanoma. The overall goal of this project was to establish an effective delivery method for a plasmid encoding human interleukin (phIL-15) into mouse melanoma cells (B16.F10) using the gene transfer technique electroporation (EP)1. The EP delivery phIL-15 was optimized using an in vitro 3D tumor model. The purpose was to translate these IL-15 delivery conditions into an in vivo mouse melanoma model to study IL-15 signal transduction and stimulate immune cells to destroy tumor antigens as well as promote an anti-tumor immune memory response. The in vitro 3D tumor model and the mouse model demonstrated similar expression patterns when delivering phIL-15 with different EP conditions. Intra-tumoral delivery using 500V/cm 20ms enhanced gene delivery and increased IL-15 protein expression compared to 1300V/cm 100μs. There was also a visible increase in transfection efficacy between tumor cells compared to skin cells when delivering pmIL-12 and phIL-15 plasmid constructs in vivo. The plasmid+EP groups 1300V/cm and 500V/cm stimulated increased expression of cytokines IL-1β, IL-6, INFγ, MIP-1β and TNFα. These EP groups also promoted tumor regression by up-regulating CD8+ T cells and CD4+ T cells which targeted melanoma. Regression and survival studies demonstrated that 73.3% of mice cleared B16.F10 cells when treated with phIL-xi15+1300V/cm and pVax+500V/cm. In addition, 53% of the mice responded to the phIL-15+500V/cm treatment group. Furthermore, 75% of the mice from group phIL-15+500V/cm survived secondary inoculation and tumor challenge. In conclusion, plasmid with encoding gene insert phIL-15 delivered by EP has the potential to act as an anti-tumor therapy because it promotes melanoma regression and enhances mouse survival through innate and adaptive cell-mediated immune responses.

>Magister Scientiae - MSc / Tyrosinase is an enzyme widely distributed in the biosphere and is found in many species of bacteria, fungi, animals, and plants; it is associated with melanin production. Even though it possesses many beneficial properties such as photoprotection, but overproduction causes undesirable effects such melasma, solar lentigines etc. Therefore, tyrosinase enzyme inhibitors are of far-ranging importance in cosmetics, medicinal products, and food industries. This study is aimed to test anti-tyrosinase activity in 37 plants from 20 families using mushroom tyrosinase inhibition method; each plant was extracted with methanol. The results showed that 17 plant extracts, exerted a considerable level of in vitro tyrosinase inhibition comparable to positive controls of kojic acid in the same solvent systems when evaluated spectrophotometrically. Among plant extracts, those that showed an inhibition rate >50 % at 50 μg/ml and ˃60 % at 200 μg/ml were A. karroo (Hayne.), A. afra Jacq. Ex Willd, C. geifolia (L.), E. racemosa (L.), H. petiolare Hilliard & B.L.Burt, M. quercifolia (L.), M. communis (L.), P. rigida (Wikstr.), P. ecklonii (Benth.), P. ericoides (L.), S. Africanacaerulea (L.), S. Africana-lutea (L.), S. antarcticus (Willd.), S. lucida (L.) F.A.Barkley, S. hamilifolius (L.), S. furcellata R.Br and T riparia which exhibited great anti-tyrosinase activity.

Le mélanome cutané est le cancer de la peau le plus agressif et malgré d’importants progrès dans sa prise en charge thérapeutique, la recherche de nouvelles stratégies se poursuit pour les patients non éligibles à la chimiothérapie ciblée ou non répondeurs aux thérapies actuelles. Le laboratoire UMR990 travaille sur une stratégie de radiothérapie interne vectorisée (RIV) utilisant des ligands ciblant spécifiquement et durablement la mélanine. Parmi ceux-ci la molécule ICF01012 couplée à l’iode 131 induit une inhibition significative de la croissance tumorale de tumeurs murines et de xénogreffes humaines pigmentées, associée à un allongement de la médiane de survie des animaux. Les objectifs de la thèse ont eu pour but i) de réaliser des études de dosimétrie dans un modèle de mélanome humain ii) d’approfondir les mécanismes de réponse à la RIV dans les modèles murins et humains iii) de potentialiser l’effet de cette stratégie par la co-injection de molécules radiosensibilisantes. L’étude dosimétrique réalisée sur le modèle de xénogreffe SK-Mel 3 montre ainsi que la croissance de ces tumeurs, qui possèdent environ 3 fois moins de mélanines que les tumeurs B16, est contrôlée de façon significative par 3 injections successives de 25 MBq. Ceci conforte l’idée d’approche théranostique avec la nécessité de caractériser les lésions d’un patient par imagerie en termes de cibles disponibles pour adapter le traitement. Nous montrons que dans les tumeurs SK-Mel 3, l’augmentation de la quantité de mélanines chez les animaux traités avec [131I]ICF01012 est similaire pour la phéo et l’eumélanine. De façon intéressante dans le modèle B16Bl6, le traitement avec [131I]ICF01012 réduit le nombre de métastases spontanées ou de colonies pulmonaires. Ce traitement est donc performant sur les petites tumeurs ou les cellules circulantes. Les mécanismes associés à la RIV sont superposables avec ceux induits par des irradiations externes à savoir cassures de l’ADN et arrêt de cycle. Les altérations moléculaires sont cependant différentes en fonction du statut du gène p53 des cellules tumorales. Le traitement avec [131I]ICF01012 induit un contrôle tumoral limité dans le temps, l’utilisation de radiosensibilisants est donc d’intérêt. Le coDbait est une petite molécule d’ADN qui mime une cassure double-brin d’ADN et sert ainsi de leurre pour les enzymes de réparation. Les modèles B16Bl6 et SK-Mel 3 ont été traités par [131I]ICF01012 associé à des injections intratumorales de coDbait. Les résultats obtenus sur la croissance tumorale et la survie des animaux montrent que l’association des deux molécules est synergique dans le modèle SK-Mel 3 à croissance lente et additive dans le modèle B16Bl6. Nous avons ainsi montré la possibilité d’augmenter les effets de la RIV dans les modèles précliniques par la déstabilisation des processus de réparation. Des travaux préliminaires montrent également un effet positif des nanoparticules de gadolinium sur l’efficacité d’[131I]ICF01012 dans le modèle B16Bl6. Les mécanismes mis en jeu restent à être définis. En conclusion, nos travaux montrent que la molécule [131I]ICF01012 constitue une thérapie alternative aux traitements conventionnels pour le mélanome métastatique pigmenté. [131I]ICF01012 est en cours de transfert clinique chez l’homme. / Melanoma is a highly aggressive skin cancer and despite the significant progress in its therapeutic management, the research keeps on developing new strategies for non-eligible patients for targeted chemotherapy or without successful therapy. The UMR990 is working on targeted radiotherapy (TRT) using ICF01012 vector labeled with iodine 131, with a high affinity for melanin and therefore for pigmented melanoma. [131I]ICF01012 induces a significant decrease of the tumoral growth in syngenic B16 models and human melanoma xenografts and a significant improvement of the survival median. The aims of the present work were i) to evaluate the dosimetry in human melanoma model, ii) to study mechanisms after TRT injection in xenografts and murin pigmented melanoma, iii) to potentiate the efficiency of this strategy using radiosensitizer. The dosimetric study showed that in SK-Mel 3 model, less pigmented than B16Bl6 tumors, the tumoral growth was significant controlled by 3 successive injections of 25 MBq. The theranostic approach is then necessary to characterize the available quantity of targets in the patient before treatment. The melanin content demonstrated that repeated injections of [131I]ICF01012 in xenografts induced an increase of melanin without any enrichment of the pheomelanin, an oxydant pigment. Interestingly, the treatment with [131I]ICF01012 reduced the number of spontaneous metastases or lung colonies in the B16Bl6 model. This treatment was then effective on small tumors or circulating cells. Consecutive mechanisms to the TRT were similar to those induced by external irradiation: DNA breaks and cell cycle arrest. However, they differed depending on p53 status of tumoral cells. The tumor regression was uncompleted after [131I]ICF01012 treatment and co-injection with radiosensitizers could improve its efficiency. We used small DNA molecules, called coDbait, mimicking the double-strand breaks to trap repairing enzymes. Syngenic model B16Bl6 and xenograft SK-Mel 3 were treated by [131I]ICF01012 and coDbait intratumoral injection. A synergistic effect of the two molecules was observed on SK-Mel 3 model, while the effect was additive in B16Bl6, a model with a fast doubling time. The coDbait destabilized the repairing process after treatment with [131I]ICF01012. Preliminary studies showed a positive effect of gadolinium nanoparticles on the efficiency of [131I] ICF01012 towards B16Bl6 tumors. The mechanisms involved remained to be defined. In conclusion, these data suggest that [131I]ICF01012 is a promising treatment for patients with pigmented metastatic melanoma.

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.

Proopiomelanocortin (POMC) is a 241 amino acids precursor protein, which encodes various neuropeptides including corticotropin (ACTH), a-melanocyte-stimulating hormone (a-MSH), and b-endorphin (b-EP). POMC plays an important role in stress response, metabolism, energy homeostasis and anti-inflammation. Recent studies demonstrated that systemic POMC gene delivery potently suppresses the tumor growth and metastasis of B16-F10 melanoma in vitro and in vivo via inhibition of NF-£eB/COX2 pathway. However, systemic POMC expression also led to elevated urine excretion and water intake in mice. This was attributed to enhanced steroidgenesis as evidence by elevated plasma corticosteroids levels in animals and increased cortisol production in adrenal H295R cells after POMC gene delivery. Since corticosteroids are also potent anti-inflammatory agents, it remains unclear whether the ACTH-mediated cortisol synthesis also contributed to the POMC-induced tumor suppression. To address this issue, we generated a series of adenovirus vectors encoding POMC genes with mutation or deletion in ACTH domain including ACTH (K15A/R17A). Unlike the wild type POMC, gene delivery of ACTH (K15A/R17A) resulted in significantly lower cortisol production, CYP11B1 mRNA level, and glucocorticoid responsive element (GRE)-driven luciferase activities in H295R cells. ACTH (K15A/R17A) gene delivery did not affect the urination and water intake in mice. Above all, ACTH (K15A/R17A) gene delivery remained capable of inhibiting the colonies formation and invasiveness of B16-F10 melanoma cells. In summary, steroidgenesis is not essential to POMC-mediated melanoma suppression. In addition, ACTH (K15A/R17A) gene delivery may provide a better alternative for melanoma control.

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No. of bitstreams: 2 Dissertação - Hellen Karine Paes Porto - 2012.pdf: 3126397 bytes, checksum: db30e536f55c8163ffbd3cd680c86ca9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / It is well known that metal complexes have been used as therapeutic agents since ancient times. However, with the success of cisplatin development as an antitumor agent in 1960inorganic drugs come to mainstream again. Despite the success of platinum compounds, serious problems are encountered when administering these drugs, such as nephrotoxicity, neurotoxicity and acquired resistance. In face of these problems other chemotherapeutic agents, less toxic to the organism and more efficient, become necessary. Several studies have been shown that ruthenium compounds present high selectivity for tumor cells and low systemic toxicity when compared to platinum (II) compounds. The present study evaluate antimor activity of two new ruthenium(II) compounds associated with amino acids, alanine and tryptophan. Ruthenium(II) compound were tested against B16-F10 and Ehrlich tumor cell lines and L-929 basal line using MTT assay, at different concentrations (0.2 - 200 mM) for 48 hours of treatment. Cell cycle analysis and apoptosis induction analyses by flow citometry and comet assay for DNA damage were also performed The IC50 values were estimated as 16.17 mM (RuAla) and 7.75 mM (RuTrp). The compound RuAla proved to be specific for the B16-F10 tumor cell line and showed a significant ability to change cell cycling profiles, arresting cells inG0/G1 phase, and also inducing cell death by apoptosis within 48 hours of treatment. The compound RuTrp showed high cytotoxic potential against Ehrlich tumor, interfering cell cycle kinetics,causing cell cycle arrest in G0/G1 phase and inducing cell death by apoptosis. Comet assay presented damage to genetic material only when cells were trated with high concentrations of RuTrp. , RuAla and RuTrp presented relevant cytotoxic activities towards tumor lineages tested in vitro. Thus, more specific tests are needed to elucidate the mechanism of action of these promising The ruthenium(II) compounds. / Sabe-se que complexos metálicos têm sido usados como agentes terapêuticos desde a antiguidade. No entanto, o reaparecimento de drogas inorgânicas iniciou-se em 1960 com o desenvolvimento e o sucesso da cisplatina como agente antitumoral. Apesar do sucesso dos compostos de platina, sérios problemas são enfrentados durante a administração dessas drogas, como nefro e neurotoxicidade e resistência. Em vista dos problemas relacionados com o tratamento a base de platina, outros quimioterápicos que sejam menos tóxicos ao organismo e mais eficientes tornam-se necessários. O estudo da atividade antitumoral se destaca com os complexos de rutênio, os quais têm demonstrado alta seletividade para células tumorais e baixa toxicidade sistêmica, quando comparados aos compostos de platina (II). O presente estudo teve como objetivo avaliar dois novos compostos de Rutênio (II), associados a aminoácidos, Alanina e Triptofano, com potencial antitumoral. No ensaio de citotoxicidade, os dois complexos de Rutênio foram avaliados frente a duas linhagens tumorais (B16-F10, Tumor de Ehrlich) e uma linhagem basal (L-929) através do teste de MTT, em diferentes concentrações dos compostos (0,2 – 200 μM) por 48 horas de tratamento. Foram realizados também análises de ciclo celular, ensaio cometa e teste Anexina V para avaliação do mecanismo de morte. Análise estatística para comparação entre os grupos tratados e controle foi utilizado Anova segundo um único critério e pós-teste Dunnet’s (software GraphPad Prism V4). Os valores de IC50 estimados foram 16,17μM (RuAla) e 7,75μM (RuTrp). O composto RuAla mostrou-se específico para a linhagem B16-F10 e apresentou capacidade de alterar o ciclo celular das células, parando a ciclagem em fase G0/G1, e também demonstrou induzir morte celular por apoptose em 48 horas de tratamento. O composto RuTrp apresentou alto potencial citotóxico frente ao Tumor de Ehrlich, interferiu na cinética do ciclo celular, parando o ciclo celular em fase G0/G1. O RuTrp também induziu morte celular por apoptose, entretanto somente apresentou dano ao material genético em altas concentrações. Todavia, teste mais específicos são necessários para a elucidação do mecanismo de ação desses dois novos composto a base de Rutênio (II) com promissores resultados anti-câncer.

La chimie médicinale est l’application des techniques de recherche en chimie pour synthétiser les molécules pharmaceutiques. Au cours des premiers stades de développements de chimie médicinale, les scientistes ont été principalement concernés par l’isolement des agents médicinaux présents dans les plantes, mais aujourd’hui, ils sont également impliqués dans la création et la synthèse de nouveaux composés pharmaceutiques, parfois basées sur des mécanismes récemment découverts. / Medicinal chemistry is the application of chemical research techniques to the synthesis of pharmaceuticals. During this PhD project, we tried to synthesize small molecules based on terminal alkyne and propargyl alcohols. These molecules were prepared by departing from a simple aldehyde and by doing reactions like Bestmann-Ohira in order to prepare terminal alkynes or reaction with ethynylmagnesium bromide to prepare propargyl alcoholsB. These prepared small molecules were then subjected into the 1,3-dipolar Huisgen cycloaddition reaction in the presence of organic azides in order to prepare small triazolic derivatives. Cytotoxicity property of these small triazoles was then tested against B16 melanoma cell lines. Primary results showed some activities for some of our synthesized molecules that need more study like adding functionalities on the structure to improve their activities.In the last part of this dissertation, a methodology study on the synthesis of propargyl ethers from their corresponding propargyl esters under microwave irradiations has been described. To the best of our knowledge preparation of propargyl ethers from propargyl esters or nucleophilic substitution of propargyl esters in order to find propargyl ethers has been reported in the literature only by use of catalysts such as Lewis acids or metal complexes. The advantage of our method was the absence of such catalysts and the time of the reaction which was less than 1h in comparison with the reported synthesis. Synthesis of such molecules can also be interesting in the synthesis of natural products.

Abstract : CD8 + T cells can be programmed in their naïve state with pro-inflammatory cytokines such as IL-15 and IL-21. IL-15 induces the proliferation of CD8 + T cells and allows the generation of memory cells. IL-21 programs CD8 + T cells to become more cytotoxic while retaining a memory type phenotype. In the laboratory, we studied the effect of these two cytokines in different contexts by using the mouse model MHC-I-restricted Pmel-1 transgenic TCR specific to the melanoma-derived gp10025-33 antigen, which is also expressed by normal melanocytes. First, we elucidated the effect of IL-15 on CD8 + T cells in the Pmel-1 transgenic model lacking the protein Suppressor of cytokine signaling 1 (SOCS1). SOCS1 is a critical regulator of T cell homeostasis. We have found that these mice have CD8 + T-cells expressing surface proteins characteristic of memory T cells (CD44, Ly6C, CD122 and CD62L). However, these cells decrease the expression of the TCR and increase that of CD5, indicative of TCR activation in vivo. When stimulated in vitro, these cells displayed a highly cytotoxic phenotype but very low proliferation. Adoptive cell transfer studies in Rag1 - / - mice showed that these cells can undergo homeostatic proliferation under lymphopenic conditions. This proliferation was characterized by severe inflammatory lesions in the skin, extremities and eyes. This study demonstrates the importance of IL-15 and SOCS1 in the regulation of self-reactive cells that can be activated under lymphopenic conditions and can cause autoimmune diseases. Second, we studied the synergistic effect of IL-15 and IL-21 in native CD8+ T cells for cancer immunotherapy. We used the mouse melanoma model B16-F10 (B16) which expresses very weakly MHC-I molecules. In parallel, we studied the effect of NOD-like receptor CARD domain containing 5 (NLRC5) overexpression, the trans-activator of MHC-I genes, in B16 cells in order to increase their immunogenicity and restore anti-tumor immunity. We generated stable lines of B16 cells expressing NLRC5 (B16-5); the co-stimulatory molecule of T cells, CD80 (B16-CD80), or both (B16-5 / 80). The over-expressing NLRC5 cells positively regulated the MHC-I and LMP2, LMP7 and TAP1 genes. B16-5 cells efficiently presented gp10025-33 to CD8+ Pmel-1 T cells and induced their proliferation. This proliferation was very robust when Pmel-1 naive cells were pre-stimulated with IL-15 and IL-21 prior to activation. In the presence of CD80, B16-5 cells stimulate Pmel-1 cells even without the addition of gp100, indicating that NLRC5 facilitates the treatment and presentation of endogenous tumor antigens. During subcutaneous implantation, B16-5 cells showed a significant reduction in tumor growth in C57BL/6 hosts but not in immunodeficient hosts, indicating that tumor cells expressing NLRC5 generated an anti-tumor immunity. This response is dependent on CD8 + T cells since in mice depleted of these cells, B16-5 cells formed large subcutaneous and pulmonary tumors. Finally, immunization with irradiated B16-5 cells allowed anti-tumor protection during challenge of parental B16 cells. Collectively, our results indicate that NLRC5 could be exploited to restore tumor immunogenicity and to stimulate protective antitumor immunity. / Les cellules T CD8+ peuvent être programmées à leur état naïf avec des cytokines pro-inflammatoires telles que l’IL-15 et l’IL-21. IL-15 induit la prolifération de cellules T CD8+ et permet la génération de cellules T CD8+ mémoire. IL-21 programme les cellules T CD8+ à devenir plus cytotoxiques tout en conservant un phénotype de type mémoire. Dans le laboratoire, nous avons étudié l’effet de ces deux cytokines dans différent contextes en utilisant le modèle de souris transgénique Pmel-1 qui possède des récepteurs de cellules T (TCR) spécifiques envers le peptide gp10025-33, exprimé par des cellules de mélanome et aussi par des mélanocytes. Premièrement, nous avons élucidé l’effet de l’IL-15 sur les cellules T CD8+ dans le modèle transgénique Pmel-1 déficient dans la protéine Suppressor of cytokine signaling 1 (SOCS1). SOCS1 est un régulateur critique de l’homéostasie de lymphocytes T. Nous avons trouvé que ces souris ont de cellules T CD8+ exprimant des protéines de surface caractéristique de cellules T mémoire (CD44, Ly6C, CD122 et CD62L). Cependant, ces cellules diminuent l’expression du TCR et augmentent celle de CD5, ce qui témoigne d’une activation du TCR in vivo. Lorsque stimulées in vitro, ces cellules montrent un phénotype hautement cytotoxique mais une prolifération très faible. Lorsque nous avons fait des études de transfert cellulaire adoptive dans de souris Rag1-/-, ces cellules ont proliféré de façon importante causant des lésions inflammatoires sévères dans la peau, les extrémités et les yeux. Cette étude démontre l’importance de l’IL-15 et de SOCS1 dans la régulation de cellules auto-réactives qui peuvent être activées sous des conditions lymphopéniques et qui peuvent causer de maladies auto-immunitaires. Deuxièmement, nous avons étudié l’effet synergique d’IL-15 et d’IL-21 dans les cellules T CD8+ naïves pour l’immunothérapie du cancer. Nous avons utilisé le modèle B16-F10 (B16) de mélanome de souris qui exprime très faiblement des molécules de CMH-I. En parallèle, nous avons étudié l’effet de la surexpression de NOD-like receptor CARD domain containing 5 (NLRC5), le trans-activateur de gènes de CMH-I dans les cellules B16, dans le but d’augmenter leur immunogénicité et de restaurer l’immunité anti-tumorale. Nous avons généré des lignées stables de cellules B16 exprimant NLRC5 (B16-5); la molécule co-stimulatrice de cellules T, CD80 (B16-CD80), ou les deux (B16-5 / 80). Les cellules sur-exprimant NLRC5 ont régulé positivement de manière constitutive les gènes MHC-I et LMP2, LMP7 et TAP1. Les cellules B16-5 ont efficacement présenté gp10025-33 aux cellules T CD8+ Pmel-1 et ont induit leur prolifération. Cette prolifération a été très robuste lorsque les cellules naïves Pmel-1 étaient pré-stimulées avec IL-15 et IL-21 avant leur activation. En présence de CD80, les cellules B16-5 stimulent les cellules Pmel-1 même sans l'addition de gp100, ce qui indique que NLRC5 facilite l’apprêtement et la présentation des antigènes tumoraux endogènes. Lors de l'implantation sous-cutanée, les cellules B16-5 ont montré une réduction significative de la croissance tumorale chez des hôtes C57BL/6, mais pas chez des hôtes immuno-déficients, ce qui indique que les cellules tumorales exprimant NLRC5 ont provoqué une immunité anti-tumorale. Cette réponse est dépendante de cellules T CD8+ puisque chez les souris déplétées de ces dernières, les cellules B16-5 ont formé de grandes tumeurs sous-cutanées et pulmonaires. Enfin, l'immunisation avec des cellules B16-5 irradiées a permis une protection anti-tumorale lors de la réimplantation des cellules B16 parentales. Collectivement, nos résultats indiquent que NLRC5 pourrait être exploité pour restaurer l'immunogénicité tumorale et pour stimuler l’immunité anti-tumorale protectrice.

No description available.

<p>Sea cucumbers have been found to contain bioactive compounds such as saponin, fucoidan, frondoside, and glycosides that have pharmacological properties like antitumor, antibacterial, anti-inflammation, and antihyperglycemic activity. Although several species of sea cucumbers have been studied and reared for the food and medicinal industries, not much research has been conducted on the species in the waters of the Eastern United States. In this research, physiological and immunological parameters of coelomic fluid from <em>Cucumaria</em> <em>frondosa</em>, <em>Isostychopus</em> <em>badionotus</em>, and <em>Pentacta</em> <em>pygmaea</em> were compared to find the most promising candidate with these properties and pharmacological benefits. We found that <em>C. frondosa</em> was the species with the best immunological and physiological parameters among the three studied. <em>C. frondosa</em> illustrated that its coelomic fluid contains the highest concentrations of cells and lysozymes that had the highest activity. Using <em>C. frondosa</em>’s tissue extracts and coelomic fluid, the ability of the extracts and coelomic fluid to inhibit murine melanoma cells (B16-F10) and modulate T-lymphocytes <em>in vitro</em> were investigated. Although no significant differences were seen statistically, the experiments illustrated that T-lymphocytes were highly activated at higher concentrations (0.001g/uL-0.0002g/uL) for tissue extracts and at lower concentrations (0.000008g/uL) for coelomic fluid. On the other hand, melanoma cells were inhibited highest at lower concentrations (0.000008g/uL-0.0000016/uL). In addition to these studies, the antibacterial activity of <em>C. frondosa</em> extract was tested on ten pathogenic bacterial species. Antibacterial activity of the <em>C. frondosa</em> extract was not seen in this experiment. However, hemolytic activity by compounds present in <em>C. frondosa</em> extracts was seen in blood agars culturing <em>Streptococcus pneumoniae</em> and <em>Enterococcus faecalis</em> in our experiment. Lastly, an <em>in vivo </em>study was conducted to see if <em>C. frondosa</em> extract can modulate stress in Nile tilapia. In our experiment, we observed that <em>C. frondosa</em> extract was able to enhance the activity of one of the parameters, phagocytic capacity significantly. However, we are not able to conclude that <em>C. frondosa</em> extract was able to mitigate chronic stress from the results obtained. Overall, observing the results from the projects, we cannot conclude that <em>C. frondosa</em> extracts illustrated pharmacological properties. Extensive studies are recommended and required to use <em>C. frondosa</em> extract for medicinal purposes. </p>