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Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.

For the treatment of cancer, immunotherapy has some inherent advantages over other treatment modalities: disseminated disease can be eradicated due to the systemic nature of immunity, the immune system is effective against a wide range of targets, long-term memory can offer added protection against disease relapse, immunotherapy should be relatively non-toxic, and it can be synergistically combined with other treatment platforms such as radiation and chemotherapy. Type 1 immune responses are thought to be superior for the treatment of cancer and, as the quintessential Th1 polarizing cytokine, interleukin-12 (IL-12) holds much promise; however, optimal therapeutic protocols have yet to be developed and clinical results have fallen short of this promise. The in vivo IL-12 experiments described here highlight a characteristic of cellular therapy that has not previously been appreciated. That is, the effect of cell-mediated cytokine delivery on the immediate microenvironment and how that affects the immune response initiated. This observation has implications for the clinical application of IL-12 therapy but may also prove to be an important consideration when studying other immunostimulants. I have herein developed a novel in vitro assay system that I have used to dissect the cellular responses to IL-12 and to identify the signals that are required for activation of a cluster of differentiation 4 (CD4)+ effector population that affects leukaemia cell clearance both in vitro and in vivo. This work, and the future studies proposed, will expand our understanding of the potential of IL-12 immunotherapy and enhance our ability to manipulate therapeutic conditions to favour the desired response. Moreover, the in vitro assay system offers a method for further characterization of CD4+ effector cells and the development of protocols to initiate their potent anticancer activity.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Resumo: A terapia pró-droga/enzima direcionada por anticorpo (ADEPT) consiste em uma primeira etapa , no direcionamento de uma enzima veiculada por anticorpo à uma célula tumoral. Numa segunda etapa uma pró-droga inócua é administrada, e, na presença da enzima, produz compostos citotóxicos restritos à localização do tumor. O par enzima/pró-droga horseradish peroxidase (HRP)/ ácido 3- indol acético (IAA) tem sido aplicada nas estratégias ADEPT. Nesta combinação, o hormônio de planta não tóxico IAA é ativado para espécies citotóxicas pela ação catalítica da HRP. A elucidação das etapas e produtos da reação IAA/HRP levou uma série de moléculas produto a serem apontadas como responsáveis pelos efeitos citotóxicos sem que, até o presente momento, o mecanismo de citotoxicidade tenha sido elucidado. Nesse trabalho, utilizando-se células McCoy como alvo, foi constatado um efeito citotóxico dose dependente do sistema IAA/HRP, por necrose. Esse efeito é quase completamente abolido com a utilização de substâncias antioxidantes ou em anaerobiose. Também foi estudado o uso de um Ester derivado do IAA, o Etil Ester do IAA, como uma nova combinação citotóxica pró-droga/ enzima. Foi constatado que a HRP isolada não consegue catalizar a oxidação do Etil Ester do IAA na ausência de uma enzima adicional (esterase). Dessa forma, pode-se controlar a citotoxicidade do IAA pelo uso de duas enzimas, HRP e esterase. Finalmente, foram apresentadas evidências da aplicação potencial da tríade: Etil Ester IAA/ esterase/ HRP como uma estratégia potencial para a metodologia ADEPT e correlata. / Abstract: The antibody-directed enzyme pro-drug therapy (ADEPT) in a first stage, it's directed to an enzyme carried to an antibody to a tumor cell. In a second stage a pro-drug harmless is administered, and in the presence of the enzyme, produces cytotoxic compounds restricted the location of the tumor. The pair enzyme / pro-drug horseradish peroxidase (HRP)/ 3 - indole acetic acid (IAA) has been applied in ADEPT strategies. In this combination, the nontoxic plant hormone nontoxic IAA is activated for cytotoxic species by the action of catalytic HRP. The elucidation of the steps and products of the reaction IAA/ HRP led to a series of product molecules identified as being responsible for cytotoxic effects, without, so far, the mechanism of cytotoxicity has been elucidated. In this work, using cells McCoy as a target, we have seen a cytotoxic effect dosedependent system IAA/ HRP, for necrosis. This effect is almost completely abolished with the use of antioxidant substances or oxygen depletion. We also studied the use of an Ester derived from the IAA, the Ethyl Ester of the IAA, as a new combination cytotoxic pro-drug/ enzyme. We have seen that the HRP alone can not catalyze the oxidation of Ethyl Ester of the IAA in the absence of an additional enzyme (esterase). Thus, we can control the cytotoxicity of the IAA for the use of two enzymes, HRP and esterase. Finally, we showed evidence of the potential application of the triad: Ethyl Ester IAA/esterase/ HRP as a potential strategy for the methodology ADEPT and correlates. / Orientador: Luiz Marcos da Fonseca / Coorientador: Valdecir Farias Ximenes / Banca: Maria das Graças Carvalho / Banca: Eliana Aparecida Varanda / Mestre

The importance of Cytotoxic T-Cell (CTL) reponses during the course of intracellular infections has received a lot of attention during the past few decades. CTLs respond to epitopes presented by the Major Histocompatibility Complex (MHC) originating from intracellular proteins for which they have an appropriate T-Cell Receptor (TCR) for. This response is crucial for the control of pathogens such as Influenza, Hepatitis, HIV and others by destroying the cell in which the pathogen replicates. Due to the extreme polymorphism of MHC molecules, Computational Immunology techniques have been developed to detect potential MHC ligands and as a consequence, potential CTL epitopes. The polymorphism factor needs to be taken into account especially when concerning the design of vaccines with a CTL response component to maximize population coverage. Tools have been constructed that combine the predictions tools concerning major steps in this pathway, that is, proteasomal cleavage, Transporter associated with Antigen Presentation (TAP) affinity, Major Histocompatibility Complex (MHC) affinity and Immunogenicity. In this study, a novel method is developed to combine the different steps in the pathway, which includes the development of a novel TAP predictor. Furthermore, by using a BLOSUM-based score in conjunction with the epitope prediction results, a novel CTL epitopebased clustering method was developed. Two pathogens with major CTL epitope components, but vastly different mutation rates were chosen to infer whether the aforementioned methods can be used to detect potential CTL epitopes and group sequences together based on shared immunogenicity. / Dissertation (MSc)--University of Pretoria, 2011. / Bioinformatics and Computational Biology Unit / unrestricted

Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.

No / The plant derived indoloquinoline alkaloid cryptolepine was investigated for its cytotoxic properties in 12 human tumour cell lines and in primary cultures of tumour cells from patients. The fluorometric microculture cytotoxicity assay was used to assess cytotoxicity and DNA mocro-array analysis to evaluate gene expression. Cryptolepine mean IC50 in the cell line panel was 0.9 microM compared with 1.0 and 2.8 microM in haemaotological and solid tumour malignancies respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as sensitive as haematological malignancies, respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as senstive as haematological malignancies. Cryptolepine activity showed highest correlations to topoisomerase II and microtubule targetting drugs. In the cell lines cryptolepine activity was essentially unaffected by established mechanisms of drug resistance. A number of genes were identified as associated with cryptolepine activity. In conclusion, cryptolepine shows interesting in vitro cytotoxic properties and its further evaluation as an anticancer drug seems warranted.

Compounds from the marine environment exhibit a wide variety of biological activities, and thus hold much promise as potential drugs. The halichondrins, isolated from the Kaikoura sponge Lissodendoryx sp. are no exception to this, demonstrating potent anticancer activity. Novel cytotoxic compounds have also been isolated from the Chatham Rise sponge Lamellomorpha strongylata. Knowledge of the cellular origins of such compounds is desirable, in order to establish if the sponge or associated micro-organisms are producing the compounds of interest. Siderophores are also important molecules, which are produced on demand by bacteria in order to obtain sufficient iron necessary for their growth. Knowledge of the biosynthesis of these compounds has potential for the control of undesirable bacteria, such as the anthrax-causing pathogen Bacillus anthracis. Cell separation studies have been carried out on Lamellomorpha strongylata, locating a swinholide in sponge-associated filamentous bacteria and theonellapeptolides in sponge-associated unicellular bacteria. A microscopic analysis of dissociated cells from Lissodendoryx sp. was also undertaken. The structures of four new halichondrins (3.13 - 3.16), isolated from Lissodendoryx sp., have been determined from spectral data. All of these compounds are very similar to known B series halichondrins, with differences occurring only beyond carbon 44. As biological activity has been shown to be derived from the portion of the molecule between carbons 1 and 35, they all retain good activity in the P388 assay as expected. A new siderophore, petrobactin sulfonate (4.2), was characterised, along with three cyclic imide siderophore derivatives (4.3 - 4.5). Petrobactin sulfonate is the first marine siderophore containing a sulfonated 3,4-dihydroxy aromatic ring. The structures were elucidated from spectral data, resulting in a revision of the NMR assignments of petrobactin.

Previous studies have shown that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite T. parva. The present study set out to determine the role of granule enzymes in mediating killing of parasitized cells, first by characterising the granzymes expressed by bovine lymphocytes and, second, by investigating their involvement in killing of target cells. Experiments using the perforin inhibitor concanamycin A confirmed that CD8+ T cell killing of T. parva-infected cells is dependent on granule exocytosis, a process that involves release of granzymes into the target cell, resulting in activation of apoptotic pathways. Analysis of the bovine genome sequence identified orthologues of granzymes A, B, H, K and M, as well as another gene O, most closely related to granzyme A. The genes were found within 3 loci in the genome. Using specific PCR assays, all of these granzymes were shown to be expressed in Theileria-specific CD8+ T cells. Further studies were undertaken to study the role of granzyme B in killing. DNA constructs encoding functional and non-functional forms of bovine granzyme B were produced and the proteins expressed in COS cells were used to establish an enzymatic assay to detect and quantify expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parvaspecific CD8+ T cell clones were found to be significantly correlating with levels of granzyme B protein expression. Moreover, the granzyme B inhibitor III, Z-IETDFMK was shown to inhibit killing by CD8+ T cell clones.