Дисертації з теми "Tcell"

Cancer immunotherapy using chimeric antigen receptor (CAR) T-cells has shown exceptional promise in the treatment of patients with refractory B-cell malignancy. In this approach, patient-derived peripheral blood T-cells are engineered to express a cell surface receptor, which confers specificity for a tumour-associated (TA) antigen. Mucin-1 (MUC1) is a large transmembrane glycoprotein that is overexpressed in 90% of breast cancers. A further important characteristic of this mucin is the fact that it is under-glycosylated in cancer cells. This holds the potential for CAR T-cell mediated targeting of MUC1 epitopes in tumour-cells which are not exposed in normal cells. Antibodies such as TAB004 and HMFG2 are considered to bind preferentially to TA-MUC1. The aim of this PhD project was the development of a CAR Tcell approach for MUC1-positive breast cancer. Herein, the anti-tumour potential of a novel 2nd generation MUC1-specific CAR, named TAB28z, has been investigated. The binding domain of this CAR is derived from the TAB004 anti-MUC1 antibody. TAB28z is being compared with two other previously developed MUC1-specific CARs, H28z and HDF28z, both of which are derived from the HMFG2 antibody. TAB28z CAR T-cells demonstrated significant anti-tumour activity against MUC1-positive breast cancer cell lines in the in vitro setting. Nevertheless, it became apparent throughout this project that MUC1 expressed on activated T-cells is detected by both HMFG2-based and TAB004-based CAR T-cells during the T-cell expansion period. This background recognition resulted in tonic signalling by CARs, which was accompanied by constitutive production of IFN-γ, CAR T-cell enrichment, reduced T-cell expansion and a trend towards upregulation of T-cell activation and exhaustion markers. Despite these observations, the activity of the three MUC1-specific signalling CARs was investigated in two different breast cancer xenograft models. No significant anti-tumour responses were observed in either of the two models, which could possibly be attributed to the effects of tonic signalling.

Complement is a key part of innate immunity and vital in the first line of defence against invading pathogens. The understanding of the role of complement in the host’s immune defence has undergone its first major change when it became clear that complement is also required for the optimal induction and function of adaptive immunity, particularly B cell activation and T cell effector responses. It is now also becoming increasingly clear that complement plays a key role in the contraction of T cell responses and thus, by definition, in immune homeostasis. The first part of this thesis focuses on such least understood role of complement. Firstly, evidence is reported for a novel mechanism by which the complement regulatory protein CD46 regulates the key T helper type 1 cytokine IFN-y and the canonical immunosuppressive cytokine IL-10. The production of both IFN-y and IL-10 is shown to be temporally regulated by TCR and CD46 signals in CD4+ T cells in an IL-2-dependent fashion and is shown to be defective in rheumatoid arthritis patients. The molecular pathways linked to the CD46-induced switch of TR! cells are largely undefined and were investigated. Gene array studies highlighted the asparagine endopeptidase (AEP) as a candidate molecule involved in the regulation of the switch of TH1 cells towards IL-10 production. The CD46-driven IFN-y secretion, but not the intracellular expression, was found to be substantially reduced by AEP inhibition; IL-10 production was consequently affected confirming the importance of IFN-y signalling in the CD46-induced "switch" mechanism. The cytokines produced by T cells activated via CD46 may also be important in the crosstalk with surrounding tissues. Here the functional consequences of CD46 signals were further investigated in the context of intestinal epithelial cells to allow the discrimination of the effects of CD46 signals in the two different cells types. A novel role for CD46 in the promotion of cellular proliferation and wound healing was reported.

Malignant pleural mesothelioma (MPM) is an incurable cancer that commonly presents at an advanced stage. Although surgery, chemotherapy and radiotherapy treatment may be used, median survival from diagnosis is less than 12 months. Consequently, new therapeutic approaches are essential. Chimeric antigen receptors (CARs) are fusion molecules that couple the HLA-independent binding of a cell surface target to the delivery of a tailored T-cell activating signal. The receptor tyrosine kinase c-Met is overexpressed in >80% of MPM making it an attractive candidate for CAR T-cell immunotherapy. To target c-Met, three candidate CARs were developed named N28z, M28z and cM28z. All contained a CD28/CD3ζ endodomain fused to one of three stabilised peptides based on the N and K1 domains of hepatocyte growth factor, which is the only natural ligand for c-Met. Specificity and functionality of c-Met re-targeted CAR+ T-cells was confirmed by co-cultivation with c-Met-expressing NIH 3T3 and MPM cell lines. This was indicated by target-dependent cytotoxicity and enhanced cytokine release (IL-2 and IFN-γ), when compared to appropriate controls. Anti-tumour activity of all three candidate CARs could be further enhanced by pre-treatment of tumour cells with poorly cytotoxic doses of chemotherapy (cisplatin and pemetrexed) or by co-incubation with the PD-1 blocker, pembrolizumab. No differences between function of the three candidate CARs were evident in these studies. To evaluate in vivo anti-tumour activity, an intraperitoneal MPM xenograft model was established that was amenable to monitoring using bioluminescence imaging. Candidate c-Met re-targeted CARs were coexpressed with the chimeric cytokine receptor, 4αβ, enabling IL-4 mediated, selective enrichment of CAR+ T-cells. In mice with an established tumour burden, I found that cM28z/4αβ+ T-cells were superior to other c-Met re-targeted or control T-cells in eliciting sustained disease control. Together, these findings demonstrate proof of concept for the utility of c-Met re-targeted CAR+ T-cells to recognise and destroy mesothelioma tumour cells.

The aim of this thesis was to characterise the T-cell component of the lymph node (LN) microenvironment in chronic lymphocytic leukaemia (CLL). It is well established that the proliferation of the neoplastic clone in CLL occurs primarily within the secondary lymphoid tissues and that within LNs these cells are in close contact with T-cells and other elements of the microenvironment. First I present a phenotypic study of the T-cells of the CLL LN obtained by fine needle aspiration. I found that, compared to CLL peripheral blood (PB), CLL LNs contain an excess of effector memory CD4+ T-cells and that the LN T-cells express more PD-1 than those from the PB; suggesting ongoing activation. Multiparameter immunofluorescence microscopy on LN biopsy sections showed that proliferating Ki67+ CLL cells are in contact with CD4+ PD-1+ T-cells. Next I sought to characterise the T-cell receptor (TCR) repertoire of flow sorted LN and PB CD4+ T-cells by spectratyping. This revealed oligoclonality in both compartments, with a significant reduction in TCR diversity in the PD-1hi subset. All subsets were skewed compared to healthy age matched PB. The spectratype profiles were distinct between the LN and PB and therefore likely a result of interaction with different antigens. This data was complemented by high throughput sequencing of the TCR, which showed more sequence overlap between 2 LNs taken from the same CLL patient, than between the LN and PB. Finally I present a preliminary in-vitro study aimed at recapitulating the events in CLL LNs. This study showed that PD-L1 can be upregulated on PB CLL cells by CD3/CD28 stimulation of accompanying T-cells, and that blocking PD-1 can lead to increased secretion of interferon gamma. Taken together these findings suggest that CLL CD4+ T-cells are subject to chronic activation and have undergone antigen driven oligoclonal expansion within the LN.

Uno de los mecanismos genéticos que facilitan la innovación evolutiva es la formación de nuevos genes. De entre los procesos que permiten generar nuevos genes, la domesticación molecular de genes transposónicos es uno de los más desconocidos. Sin embargo, los transposones domesticados (TDs) han sido cruciales para el desarrollo de innovaciones evolutivas tan relevantes como el sistema inmune adaptativo de los gnatóstomos o la placenta de los mamíferos terios. Sólo recientemente se ha intentado una identificación sistemática de TDs. El primer punto de esta tesis consistió en la búsqueda de TDs en el genoma humano, mediante un sencillo programa informático que clasificaba como candidatos a los genes cuya región codificante solapara >50% con un transposón. Uno de los candidatos identificados fue Tceal7, un miembro de la familia génica específica de mamíferos euterios Bex/Tceal. Así, pudimos trazar el origen de esta familia a la domesticación de una serie de fragmentos de retrotransposones L1 en el ancestro de los placentarios. Este es el segundo caso de domesticación molecular de un L1 en metazoos, y el primero que origina una familia multigénica. El siguiente paso fue caracterizar el proceso de exaptación de las regiones reguladoras que permitieron el nacimiento de la familia Bex/Tceal. Así, se puso de manifiesto la relación existente entre el origen de esta familia y otros genes específicos de euterios situados en el mismo locus del cromosoma X: la familia ArmcX y el gen Hnrnph2. La región que da sentido a esta relación es el motivo BGW. Los motivos BGW de diversos genes fueron testados mediante un ensayo de transgen reportero. De este modo pudimos determinar que el motivo BGW no es la encargada de conferir especificidad de tejido a la expresión de estos genes, sino que, probablemente, es una secuencia con una elevada capacidad de sensar señales regulatorias del ecosistema genómico circundante. En estudios recientes, las proteínas BEX han sido catalogadas como proteínas intrínsecamente desordenadas que forman hubs de interacciones proteína-proteína. El ORF de los genes Bex/Tceal deriva del extremo amino-terminal del ORF1 de un retrotransposón L1 llamado HAL1b. Esta región de la proteína transposónica se corresponde con el coiled-coil, una región típicamente desordenada. Encontramos que todas las proteínas BEX/TCEAL tienen una estructura desordenada y algunas de ellas un coiled-coil en su extremo carboxi-terminal, con lo que es altamente probable que esta característica, conservada del transposón ancestral, haya determinado la promiscuidad de estas proteínas en cuanto a sus interacciones. Existe poca o ninguna información sobre la función de la mayoría de los genes Bex/Tceal. Por lo tanto, se decidió generar y caracterizar una línea de ratones mutantes para Bex3, uno de los miembros de esta familia. Para ello, se utilizó el sistema CRISPR/Cas9. La caracterización de una de las dos líneas de ratones mutantes arrojo una serie de alteraciones comportamentales asociadas a un comportamiento autístico, así como malformaciones esqueléticas. De forma relevante, se detectó una hiperactivación de lá vía mTOR en el encéfalo de los ratones mutantes, lo que brinda una primera pista sobre las bases moleculares del fenotipo comportamental. Como conclusión, se ha identificado e investigado un nuevo caso de domesticación molecular de un transposón en el origen de los euterios, que dio lugar a la familia multigénica Bex/Tceal. Además, se ha generado y empezado a caracterizar el fenotipo de una línea de ratones mutante para Bex3, uno de los genes de esta familia. Queda para futuros estudios profundizar en las implicaciones evolutivas de la aparición de esta familia.
One of the genetic mechanisms that facilitate evolutionary innovation is the originaton of new genes. The molecular domestication of transposable elements is among the least known sources of new genes. This study began with the design of a simple bioinformatic pipeline to identify new events of molecular domestication in the human genome. Genes with more than 50% of their coding sequence overlapping with an annotated transposon were considered as candidates. With this method we identified the Tceal7 gene, which belongs to an eutherian-specific family called Bex/Tceal. Thus, we could trace the origin of this family to the molecular domestication of L1 retrotransposon fragments in the ancestor of placental mammals. This is the second case of this kind in metazoans, and the first to give rise to a multigenic family. The next step was to characterize the exaptation process of regulatory regions that allowed the birth of this family. In this way, we could find that the origin of the Bex/Tceal family is tightly related to other eutherian-specific genes that lay in the same locus. To investigate the function of this family, we aimed to generate and characterize Bex3-mutant transgenic mice using CRISPR/Cas9 technology. We found that these mice had autistic-like behaviors and several skeletal malformations. Furthermore, the mTOR pathway was hyperactivated in the brain of adult Bex3-mutant mice. This is a first step towards the understanding of the molecular basis of the observed phenotype. In conclusion, we have identified and investigated a new molecular domestication in the origin of eutherian mammals that gave rise to the multigene family Bex/Tceal. Moreover, we have started to analyze the function of this family and the evolutionary implications of its birth.

PURPOSE: Identification of novel Common Lymphoid Precursors (CLP) cells in peripheral blood and tissue derived from cancer patients characterized by the expression of Lin-CD34+DNAM-1bright CXCR4+ markers and Lin-CD34-CD16+CD7- markers, previously identified in peripheral blood of patients with chronic inflammation and not found in healthy donors. In vitro culture of CLPs cells derived from tumor samples. METHODS: Phenotypic and functional analyses on novel CLPs were performed by multiparametric cytofluorimetric assays. RESULTS: Analysis of peripheral blood and tissue samples of cancer patients revealed the presence of Lin-CD34+DNAM-1bright CXCR4+ and Lin-CD34-CD16+CD7- inflammatory CLPs. In addition, an increase of Lin-CD34DNAM-1brightCXCR4+ inflammatory lymphoid precursors is shown in peripheral blood of cancer patients following NSCLC therapeutic treatment. Characterization of in vitro derived progenies from CLP cells derived from cancer patients highlighted the presence of mature and functional T progenies. DISCUSSION: The identification of novel CLPs not only in peripheral blood of patients with chronic inflammation but also in peripheral blood and tissue of cancer patients (Lymphoma, Kaposi’s Sarcoma, NSCLC) suggests that these lymphoid precursors are released in peripheral blood during inflammatory condition and could be involved in tumor progression control. CONCLUSION: The presence of precursor cells in tumor samples is a starting point for discovering their role in tumor pathogenesis.

Objetivo: Analisar a influência da espessura corneana central na acuidade visual (AV) corrigida após transplante de córnea endotelial lamelar profundo (TCELP). Métodos: Foram estudados de forma prospectiva 155 olhos de 127 pacientes portadores de ceratopatia bolhosa ou distrofia endotelial de Fuchs no sexto mês de pós-operatório do TCELP, entre março de 2000 e março de 2005. Foram excluídos pacientes com outras alterações oculares que justificassem baixa AV. Todos os pacientes foram submetidos à avaliação oftálmica, quando foram determinadas AV corrigida, por meio de exame refratométrico, e espessura corneana central, através da paquimetria ultra-sônica. As técnicas usada foram previamente descritas. Os olhos foram agrupados de acordo com as medidas de AV: grupo I (20/20 - 20/30), grupo II (20/40 - 20/50), grupo III (20/60 - 20/80), grupo IV (20/100 - 20/400). Para correlação com paquimetria e análise estatística, as medidas de AV foram convertidas da tabela de Snellen para a tabela logarítmica (logMAR). Foram criadas variáveis categóricas para expressar status de faixa de normalidade de espessura corneana (entre 495 e 651 ?m), usando como pontos de corte valores encontrados na literatura. Resultados: A média, o desvio padrão e a variação da paquimetria foi: grupo I (n=38) 571 ±80 um, 408 a 784 um; grupo II (n=79) 598 ±80 um, 437 a 816 um; grupo III (n=30) 605 ±99 um, 454 a 945 um e grupo IV (n=8) 607 ±120 ?m, 410 a 781 ?m. Analisando o resultado da AV e a porcentagem de casos com espessura corneana acima de 651 um, foi observada associação linear significativa (P=0,037; ?2 de tendência linear) entre o aumento da paquimetria e a piora da AV. Analisando a associação entre os grupos de AV e a porcentagem de casos com espessura corneana abaixo da faixa de normalidade (<495 um), não foi encontrada significância estatística (P=0,92; x2 de Pearson). Quando analisado o resultado visual do grupo I em relação ao resultado dos grupos II+III+IV em conjunto, observou-se que somente 13% dos casos do grupo I e 30% dos casos dos demais grupos apresentaram espessura corneana maior do que 651 ?m. Essa correlação demonstrou significância estatística limítrofe (P=0,066; x2 de Pearson com correção de Yates). Conclusão: Observou-se associação linear significativa entre piora da AV corrigida e aumento da espessura corneana central. Quando analisados somente casos com paquimetria abaixo da faixa de normalidade, não foi observada associação significativa entre piora da AV corrigida e espessura corneana central
Purpose: To analyze the influence of central corneal thickness in the corrected visual acuity (VA) after deep lamellar endothelial corneal keratoplasty (DLEK). Methods: Retrospective study of 155 eyes of 127 patients 6 months post-op DLEK between March 2000 and March 2005. These patients had been previously diagnosed with either bullous keratopathy or Fuch\'s endothelial dystrophy. Patients with other ophthalmic conditions that could cause loss of vision were excluded. All patients underwent ophthalmic evaluation to determine corrected VA by means of refraction and central corneal thickness by means of ultrasonic pachymetry. Eyes were grouped according to visual acuity into 4 groups: I (20/20 - 20/30), II (20/40 - 20/50), III (20/60 - 20/80), IV (20/100 - 20/400). For statistical analysis and corelation with pachymetry, VA measurements were converted to logMAR. Categorical variables were created to express normal range corneal thickness status (from 495 to 651 um) using values published on the literature. Results: Mean and standart deviation pachymetry values were: group I (n=38) 571 ±80 ?m, ranging from 408 to 784 um; group II (n=79) 598 ±80 um, ranging from 437 to 816 ?m; group III (n=30) 605 ±99 um, ranging from 454 to 945 um and group IV (n=8) 607 ±120 um, ranging from 410 to 781 ?m. Analyzing the VA results and the percentage of cases with corneal thickness above 651 um, a significant linear correlation between higher pachymetry and worse VA was observed (P=0.037; linear trend). Analyzing the association between the different groups and the percentage of cases with corneal thickness bellow 495 um, there was no statistical significance (P=0.92; Pearson\'s x2). When analyzing the visual results of group I compared to groups II+III+IV together, it was observed that only 13% of group I cases and 30% of cases from the other groups presented corneal thickness greater then 651 um. This correlation showed borderline statistical significance (P=0.066; Pearson\'s x2 with Yates\' correction). Conclusions: A significant linear correlation between increased corneal thickness and worse VA was observed. When analyzing only cases bellow normal pachymetry, there was no correlation between corneal thickness and worse VA

Primary cutaneous T-cell lymphoma (CTCL) is a clinically heterogeneous malignancy of mature skin-homing T-cells. Mycosis fungoides (MF) is an indo¬lent subtype of CTCL whilst Sezary syndrome (SS) is an aggressive leukaemic variant characterised by the presence of malignant Sezary cells in the periph¬eral blood. A role for epigenetic mechanisms in the pathogenesis of CTCL has been proposed but not extensively investigated. In this thesis, the contribution of DNA methylation to the regulation of SHP-1, Fas and PLS3, which have been implicated in the pathogenesis of CTCL, was examined. Gene expression was assessed on the mRNA and protein levels using qPCR and flow cytometry re¬spectively whilst DNA methylation was evaluated using bisulphite conversion and Pyrosequencing. Contrary to results in CTCL cell lines, primary malignant cells from CTCL patients did not display aberrant DNA methylation or dysregu-lated expression of SHP-1, a negative regulator of STATS signalling. Dysregula-tion of Fas, a key regulator of apoptosis, was observed in 34/47 SS patients with 13/47 showing over-expression compared to healthy controls and 21/47 showing under-expression, attributed to the positional hypermethylation of five CpG din-ucleotides in the Fas CpG island. PLS3, an actin-binding protein not normally expressed in any haematopoietic cell, was aberrantly expressed in malignant cells from 21/35 SS patients and 3/8 MF patients with demonstrated clonal blood in¬volvement. CpG dinucleotides 95-99 in the PLS3 CpG island were differentially methylated between healthy lymphocytes and keratinocytes, and hypomethyla-tion of these CpG dinucleotides was observed in SS patients expressing PLS3. To investigate expression of PLS3 on the protein level a novel anti-PLS3 anti¬body was raised and optimised for use in Western blotting, flow cytometry and immunofluorescence. In conclusion, these data demonstrate that SHP-1 is not regulated by methylation in primary CTCL cells whilst Fas is dysregulated by hypermethylation of five key CpG dinucleotides and PLS3 is dysregulated by hy-pomethylation of CpG dinucleotides 95-99.

The adoptive transfer of human regulatory T cells (Tregs) in transplantation offers an attractive therapeutic alternative in the current struggle to improve long-term outcomes. CD4+CD25+FOXP3+ (Tregs) play an important role in immunoregulation and have been shown in animal models to promote transplantation tolerance. Phase I trials in bone marrow transplantation and type I diabetes have already shown that ex vivo expanded Tregs have an excellent safety profile, which is encouraging for the broader application of these cells. The clinical trials initiated at King’s College London, ThRIL and the ONE study, are the leading trials of autologous Treg immunotherapy worldwide in the setting of liver and kidney transplantation, respectively. The success of these trials is reliant on the implementation of protocols that comply with Good Manufacturing Practice (GMP) guidelines centered on the successful isolation and expansion of a functional and stable human Treg population from prospective transplant recipients. The main focus of this thesis has been the adoptive cell therapy of Tregs in the context of liver transplantation. In this regard, it was first pertinent to study the biology of Tregs from patients with alcohol related cirrhosis (ARC), representing the majority of patients on the liver transplant waiting list. As such, an in-depth phenotypic and functional characterisation of the isolated Tregs from these patients was carried out. The results shown herein demonstrate that Tregs from ARC patients display impaired suppressive function. Based on this finding, a series of experiments were conducted in order to delineate the mechanism behind the apparent defect in Treg suppressive function. This led to a novel discovery of a defect in the expression of the cytoprotective enzyme, heme oxgenase-1 (HO-1), by patient Tregs, in correlation with the apparent Treg dysfunction. Subsequently, adherence to a GMP compatible 36-day expansion protocol resulted in the enrichment of a pure population of Tregs (91.3% CD4+CD25+ and 0.153% CD8+ cells), reaching numbers needed for their clinical translation. In addition, the protocol ensured the maintenance of FOXP3 expression (94.6% of the CD4+CD25+ cells expressed FOXP3 at the end of expansion) with an increase in the frequency of FOXP3Hi cells throughout expansion. Culture in the presence of rapamycin also confirmed the stability of the expanded Tregs, whereby the cells did not convert to Th17 cells when cultured in the presence of pro-inflammatory stimuli.

碩士
國立中正大學
通訊工程研究所
95
Intrusion detection system (IDS) plays a vital part in network security. There are many proposals for IDS in MANET at present; however, there remains some incompleteness to be fulfilled—IDS response, for instance. In this paper we propose an IDS response protocol inspired from the concept of immune system of the human body. Based on SecAODV IDS, the protocol can enhance its functions. We expect to accelerate the spreading of IDS information of SecAODV IDS and to control as many fault messages as possible by isolating malicious nodes as soon as they are detected. That way, we can prevent network malicious nodes from spreading unreal IDS messages which lead to network crash. In this paper we will illustrate how the protocol functions and describe the procedures with several examples. We will also analyze why the protocol can accelerate the spreading of IDS information and avoid fault messages. Finally, the protocol is put to the proof with network simulator and a conclusion is drawn.

碩士
國立清華大學
分子醫學研究所
92
One new strain of Lactobacillus, identified as Lactobacillus rhamnosus TCELL-1, was isolated from the healthy adult rectum biopsies in our laboratory. A new food-grade shuttle cloning vector for Lb. rhamnosus TCELL-1 and E. coli was constructed using the nisin immunity gene nisI as a selection marker. The food-grade shuttle cloning vector, pNI, was constructed using the pAMβ1 replicon, the pUC origin, the nisI gene, the promoter Lac for nisI expression, and the nucT reporter gene. Electroporation into Lb. rhamnosus TCELL-1 was selected with the nucT reporter gene. Plasmid pNI was confirmed in Lb. rhamnosus TCELL-1 with southern hybridization. Lb. rhamnosus TCELL-1 carrying pNI was shown to be able to grow in medium containing a maximum of 60 IU nisin/ml. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in Lb. rhamnosus TCELL-1 in order to construct improved starter bacteria for food applications.

碩士
國立清華大學
生物科技研究所
93
Abstract Lactobacillus is a kind of probiotic bacteria , which is often applied to milk products。In this study, we tried to investigate the production of sorbitol how to produce it through metabolic engineering。Sorbitol, that possess the physiological function , has been proved to effectively reducing dental caries。If we timely add sorbitol to our daily milk-food，we could go a step further to raise its additional value。 According to Hugenholtz et. al.(2002)，within the sorbitol pathway sorbitol production is catalized by the Stl-6P-DHase gene (sorbitol-6 phosphate dehydrogenase ) and we’ll isolate the enzymatic gene from a new strain of Lactobacillus rhamnosus TCELL-1 in our laboratory , ligated with a shuttle vector pHY300PLK via electroporation into Lb. rhamnosus TCELL-1。 The result shows that the encoding Stl-6P-DHase gene is overexpressive and has been observed higher production about four fold of sorbitol than without the enzymatic gene does in Lb. rhamnosus TCELL-1。

碩士
國立清華大學
分子醫學研究所
94
In recent year food industry trend to reduce the using of preservative, and change to use lactic acid bacteria or enzyme to preserved food, so we could go a step further to raise its additional value. Diacetyl is one kind of aromatic product in the metabolic process of lactic acid bacteria, which has the fragrance and also has inhibition effect to some bacteria. According to the past research, overexpression of ilvBN gene in lactic acid bacteria may effectively increase diacetyl of lactic acid bacteria. Therefore, this research we will isolate the enzymatic gene from a new strain Lactobacillus rhamnosus TCELL-1 by overlapping PCR method, ligated with a shuttle vector pHY300PLK via electroporation into Lb. rhamnosus TCELL-1. The results of GC/MS show that the encoding ilvBN gene is overexpressive and has been observed higher production about four fold of diacetyl than without the enzymatic gene does in Lb. rhamnosus TCELL-1. In deferred agar spot assay, we observed that overexpression ilvBN gene in TCELL-1 can inhibit food pathogens effectively.

碩士
國立清華大學
生命科學系
91
One strain of Lactobacillus, identified as Lactobacillus rhamnosus TCELL-1, was isolated from the human rectum biopsies in our laboratory. In this study, we tried to investigate the production of bacteriocin or bacteriocin-like inhibitory substance by TCELL-1. By using of deferred agar spot assay, we observed that TCELL-1 can inhibit food pathogens effectively and inhibit some lactobacilli test strains to a certain extent. But there is no obvious inhibition effect with regard to TCELL-1 neutralized supernatant when tested by agar well diffusion assay. We turned to use Bioscreen assay to evaluate inhibition effect of TCELL-1 supernatant. Compared with control, Enterobacter aerogenes showed obvious growth difference in TCELL-1 supernatant. According to the growth phenomenon of E. aerogenes, it was suitable to call “growth retardation” effect rather than “growth inhibition”. Addition of catalase demonstrated that hydrogen peroxide was not the cause of growth retardation. Serial dilution of TCELL-1 supernatant assay also proved that the growth retardation does not cause by phage and nutrient depletion. The growth-retardation factor was heat-stable, not sensitive to proteolytic enzyme and could be precipitated by ammonium sulfate, suggested that it may be a protein complex.

Dissertação de mest., Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011
T-cell acute lymphoblastic leukemia (T-ALL) is a thymocyte malignancy. Although the identification of several genetic alterations in this leukemia has allowed a better understanding of how it develops, more research is required to develop more efficient therapies. The pre-T cell receptor (pre-TCR) complex has been characterized structurally and functionally and is an essential player in T-cell development. However, its contribution to T-cell leukemogenesis remains controversial. Indeed, reports have noticed the importance of the pre-TCR in some mouse models of T-cell leukemia. T-ALL mouse models are useful to study the cellular and molecular mechanisms underlying this disease. Regarding the TEL-JAK2 transgenic mouse model, it was found that the absence of the pre-TCR complex from T lymphocytes led to a significant delay in T-ALL onset. A genomic DNA analysis by array comparative genomic hybridization (aCGH) revealed that late-onset pre-TCR-negative leukemias presented more genomic alterations (gains and losses of DNA) than early-onset pre-TCR-positive leukemias, indicating that these alterations can occur and be selected to compensate for the absence of pre-TCR. The goal of this project was to identify genes aberrantly expressed in pre-TCR-negative leukemia. To this end, the copy number of candidate genes were compared between leukemic cells from regular TEL-JAK2 mice and TEL-JAK2 mice without the Rag2 gene, which is essential for the pre-TCR complex formation. The Cdkn2a, Cdkn2b, Myc, Ikaros, Pten, Bcl11b, and Fbxw7 genes, which are localized in genomic regions previously found to be altered in pre-TCR-deficient leukemic cells and/or are involved in cancer, have been analyzed. By performing quantitative PCR (qPCR) we found DNA copy number alterations in TEL-JAK2;Rag2-/- leukemia DNA as compared to regular TEL-JAK2 leukemia DNA in the same genomic regions that showed chromosomal abnormality in aCGH analysis. Further studies need to be performed to better understand the precise role of pre-TCR in T-ALL and the role of compensatory mechanisms occurring in the absence of pre-TCR.

La pantetina, un tiolo a basso peso molecolare, rappresenta la forma stabile disulfidica della panteteina, il substrato metabolico che costituisce la parte attiva del coenzima A. Grazie alla capacità di abbassare l’indice lipidico senza effetti collaterali documentati, per decenni la pantetina è stata somministrata a pazienti con disfunzioni metaboliche. Studi recenti hanno dimostrato che la somministrazione di pantetina inibisce l’insorgenza della sindrome infiammatoria cerebrale in un modello animale di malaria, determinando la down-regolazione delle principali risposte cellulari pro-infiammatorie. Inoltre, il trattamento con pantetina è in grado di migliorare il decorso clinico in altri modelli animali di malattie neurologiche, tra le quali la malattia di Parkinson. Considerato che l’immunometabolismo è una disciplina che rappresenta una nuova frontiera nell’immunologia, lo scopo di questo studio è quello di studiare l’effetto metabolico associato alla somministrazione di pantetina sulla patogenesi dell’encefalomielite sperimentale autoimmune (EAE), il modello animale della sclerosi multipla (MS) umana. I nostri esperimenti in vivo hanno dimostrato che il trattamento con pantetina è in grado di interferire con l’evoluzione della forma recidivante - remittente dell’EAE (RR-EAE), ritardando l’esordio della malattia e riducendo la severità del quadro clinico. Inoltre, la somministrazione del farmaco a seguito del manifestarsi dei primi sintomi di malattia, ha determinato un indicativo miglioramento del decorso e della severità della RR-EAE. Come dimostrato da analisi neuropatologiche, questi rilevanti dati clinici sono associati ad una diminuzione degli infiltrati infiammatori e del grado di demielinizzazione a livello del midollo spinale di topi trattati con pantetina. Utilizzando un nuovo sistema di imaging in vivo (IVIS 200; Caliper Life Science), è stato inoltre dimostrato che gli animali trattati con pantetina presentano un ridotto leakage della barriera emato-encefalica (BEE) durante la fase pre-clinica della RR-EAE. Un ulteriore dato molto importante è la significativa riduzione della capacità proliferativa antigene-specifica delle cellule T isolate dai linfonodi drenanti di animali trattati con pantetina, se paragonato alle cellule isolate da animali non-trattati. I linfociti ottenuti da topi trattati hanno presentato anche una diminuzione nella produzione di citochine pro-infiammatorie durante il picco iniziale di malattia. Inoltre, i studi condotti in vitro suggeriscono che il pre-trattamento con pantetina ha un effetto bloccante sull’adesione integrino-dependente delle cellule T encefalitogeniche. La pantetina ha inoltre un effetto inibitorio anche sull’adesione in vivo delle cellule T attivate, effetto dimostrato tramite l’utilizzo di microscopia intravitale in un modello di infiammazione dei vasi cerebrali. Analisi eseguite tramite ImageStream System hanno evidenziato come l’inibizione sulla capacità adesiva delle cellule T PLP-specifiche non possa essere spiegata tramite cambiamenti di espressione integrinica o della capacità delle integrine di formare cluster indotti tramite attivazione chemochinica. Dato che la funzionalità delle integrine non dipende solo dalla loro avidità per i ligandi, ma anche dalla loro localizzazione all’interno di rafts lipidici ricchi di colesterolo presenti sulla superficie cellulare, si è analizzata l’influenza del trattamento con pantetina sulla conformazione dei rafts lipidici sulle cellule T attivate. Sorprendentemente, gli esperimenti condotti hanno evidenziato che il pre-trattamento con pantetina è in grado di ridurre notevolmente la formazione di rafts lipidici e la loro clusterizzazione sulle cellule T, nonostante le integrine fossero ancora organizzate in grandi caps polarizzati come sulle cellule T non trattate. Per analizzare più a fondo l’effetto globale della pantetina sulle cellule T attivate, si sono effettuati studi di metabolomica, i quali hanno evidenziato un effetto metabolico molto significativo della pantetina sulle cellule T trattate. In particolare, il trattamento con pantetina ha un effetto molto marcato su cellule T memory, se paragonate a cellule T naïve, modulando almeno 45 diversi pathways metabolici e causando un forte aumento della quantità di coenzima A disponibile, il quale va principalmente ad aumentare l’efficienza del ciclo di Krebs. Questi dati sembrerebbero indicare una capacità da parte della pantetina di regolare molto finemente il metabolismo delle cellule T patogeniche responsabili del danno al sistema nervoso centrale osservato nella MS. In conclusione i nostri risultati dimostrano che la pantetina ha un effetto immuno-modulatorio sull’EAE attraverso la riduzione dell’attivazione e dell’adesività delle cellule T patogeniche ed il mantenimento dell’integrità’ della BEE. Considerando il basso costo del farmaco, la sua sicura somministrazione in assenza di effetti collaterali e i nostri nuovi risultati, questo tiol a basso peso molecolare potrebbe rappresentare un approccio terapeutico efficace nel trattamento della MS.
Pantethine is a low molecular weight thiol and represents the stable disulfate form of pantetheine, the metabolic substrate constituting the active part of coenzyme A (CoA). For decades, pantethine has been administrated to patients with metabolic disorders for its lipid lowering activity, without any side effect reported. Recent data showed that pantethine prevents the occurrence of cerebral malaria, with the down-regulation of key cellular responses to the inflammatory cerebral syndrome. Immunometabolism represents a new frontier in immunology and the aim of this study was to investigate the effect of metabolic intervention with pantethine on experimental autoimmune encephalomyelitis (EAE), the animal model of human multiple sclerosis (MS). Our in vivo experiments demonstrated that pantethine treatment prevents the development of chronic and relapsing-remitting EAE by delaying the disease onset and reducing the clinical score. Furthermore, pantethine treatment started after disease onset significantly ameliorated disease course and severity. The surprising clinical results were accompanied by a decrease in inflammatory infiltrates and in demyelination in pantethine treated-mice, proved by our neuropathological studies. Moreover, using IVIS 200 system we found that pantethine treated-mice had a reduced BBB leakage during the pre-clinical phase of relapsing-remitting EAE. Importantly, T-cells isolated from draining lymph nodes of mice treated with pantethine showed a significant reduction in antigen-specific proliferation and pro-inflammatory cytokines production at disease peak when compared with untreated-mice. Furthermore, our data from in vitro experiments demonstrated that pantethine pre-treatment of encephalitogenic T-cells blocked T-cell adhesion on purified integrin ligands. In addition, pantethine inhibited activated T-cell adhesion in vivo using an intravital microscopy model performed in inflamed brain venules. Importantly, pantethine had no effect on chemokine-induced naïve T-cell adhesion, proving that pantethine treatment has a selective effect only on activated T-cells. On the other hand, ImageStream analysis show that the important inhibition of proteolipid-specific T-cells adhesion could not be explain by changes on integrin expression or on their ability to form clusters upon chemokine-induce activation. Integrin functionality does not depend only on their avidity for their ligands but also on its localization into cholesterol-rich membrane rafts on cell surface. Thus, we next investigated the role of pantethine treatment on lipid rafts conformation on activated T-cells. Surprisingly, our in vitro experiments demonstrated that pantethine pre-treatment strongly reduced lipid rafts formation and raft clusters on encephalitogenic T-cells, although integrins were still present in big polarized caps. These results suggest that pantethine dissolve lipid rafts on activated T-cell, possibly interfering with the signal transduction necessary to support integrin-dependent firm adhesion. To further understand pantethine effect on in T-cell functions, we performed metabolomics analysis on pantethine-treated activated T-cells. Our data suggested a global metabolic effect of pantethine on T-cells. Overall, pantethine treatment significantly and differently affected the metabolism of memory T lymphocytes with respect to naïve T lymphocytes, leading to the modulation of at least 45 metabolic pathways with a huge availability of CoA that greatly enhances the Krebs cycle efficiency. These final consideration made us believe that pantethine could be able to perform a fine metabolic tuning of the pathogenic T-cells involved in demyelinating diseases, as MS. In conclusion, our results demonstrate that pantethine has an immuno-modulatory effect on EAE by reducing T-cell activation and adhesiveness, and maintaining BBB integrity. As pantethine has a low-cost and has successfully administered in different context to man in the absence of side effects, our results suggest that this low molecular weight thiol may represent a valuable new therapeutic approach in MS.

碩士
國立清華大學
分子醫學研究所
94
Lactic acid bacteria, groups of Gram positive bacteria, have thick peptidoglycan layers in their cell wall, which make the process of cell wall lysis in genomic DNA isolation difficult. A novel method, XS method to isolate genomic DNA from lactobacillus is introduced, which is different from conventional isolation protocol. This method is rapid, requires no enzymatic or mechanical cell disruption, nor multiple organic solvent extractions. Isolated DNA are proven can be used in various molecular biology analysis, such as PCR, restriction enzyme digestion, and cloning. Isolation of genomic DNA from Lactobacillus rhamnosus TCELL-1 using XS method faces the low yield problem, and hence, lysozyme treatment prior to XS treatment (XSL method) is introduced. DNA isolated from Lb. rhamnosus TCELL-1 via XSL method are used in cloning of alr gene. The partial length of this gene is 843 bp, which can be translated into ALR protein with 281 residues. Comparison of deduced amino acid sequence reveals 85.8 % identity with that of Lb. casei ATCC334. A highly conserved region in N-terminal, AVVKANGYGH, is found.

碩士
國立臺灣大學
免疫學研究所
105
Naïve B cells could act as antigen-presenting cells to induce a subpopulation of Foxp3- regulatory T cells, called Treg-of-B cells. Naïve B-cell-primed T cells is through cell-cell contact and independent of IL-10. The suppressive function of Treg-of-B cells partly requires close cell-cell proximity. Therefore, Treg-of-B cells is a population different from natural Treg cells (nTreg) and Type 1 regulatory T cells (Tr1). Recent studies had demonstrated that Treg-of-B cells could be developed as a therapeutic approach against transplant rejection, allergy and rheumatological diseases. However, our studies showed that the differentiation of Treg-of-B cells involved STAT6 phosphorylation and IL-4 secretion. Hence, IL-4 might play a role in induction and/or function of Treg-of-B cells. This study was performed to examine the role of IL-4 signaling pathway in the development and function of Treg-of-B cells. We blocked IL-4 by anti-IL4 antibodies to study the suppressive function and cytokines profile of Treg-of-B cells. However, the result showed no significant difference between experimental group and control group in the suppressive ability, but the levels of IL-4 and IL-10 were lower in neutralized group. We next used Il4-/- mice for further investigation by suppressive function analysis, cytokines profile comparison. the result showed nosignificant difference between WT, Il4-/- Treg-of-B cells in suppressive ability but the level of IL-10 was lower in Il4-/- Treg-of-B(WT) cells. These results might indicate that IL-4 did not affect the induction and function of Treg-of-B cells. Previous study had demonstrated that IL-4 control several immune cell growth and survival. Therefore, we also examined whether IL-4 might maintain the survival of Treg-of-B cells. After induced cell death, we compared the cell death rate experimental group and control group by Annexin V/ PI staining or analyzed apoptosis-related gene expression. The results showed that more cell death induced in IL-4 blocking Treg-of-B cells and Il4-/- Treg-of-B cells. Although IL-2 and IL-4 could rescue the cell death in control group, IL- 2 also partially rescued the cell death of experimental group, but the combination of these two cytokines might enlarged the gap of cell death rate between IL-4 neutralization or not. Furthermore, we analyzed bcl-2 gene family, including anti- apoptotic gene bcl-x, bcl-w and pro-apoptotic gene bax. The results showed that IL-4 blocking Treg-of-B cells expressed slightly lower level of bcl-x. Il4-/- Treg-of-B cells showed more significant difference with lower levels of bcl-x and bcl-w gene expression. The results hinted that IL-4 might involve in maintaining Treg-of-B cells survival. In the deficient of IL-4, more apoptosis might be induced on Treg-of-B cells. In summary, our studies suggested that IL-4 might not affect the induction and function of Treg-of-B cells. In the deficient of IL-4, Treg-of-B cells might tend to go on apoptosis. Therefore, IL-4 might maintain the survival of Treg-of-B cells. In the future, the regulatory mechanism of cytokines production and apoptosis by IL-4-STAT6 signaling pathway might require further researches.

Enquanto a grande maioria das células T expressa um recetor de células T (TCR) composto de heterodímeros αβ, uma população menor expressa um TCR γδ. Estas células diferem das células T αβ, pois o seu TCR pode reconhecer antigénios mesmo na ausência de apresentação pelas moléculas de MHC (complexo principal de histocompatibilidade). As células T γδ, estão particularmente presentes nas mucosas e superfícies epiteliais e respondem a moléculas associadas a infeção, danificação tecidular e stress celular. Além disso, vários subconjuntos de células T γδ mostraram atividades anti tumorais e imunorreguladoras. Possuem um papel ativo nas infeções pulmonares bacterianas, víricas e fúngicas, assim como alergias e fibrose. Recentemente foi estudado o seu potencial na reparação tecidular em doenças inflamatórias crónicas, associado à produção da citoquina IL-17, como a Diabetes Mellitus tipo 2, Obesidade, Psoríase e Asma. Alguns subconjuntos desta população celular revelaram também um papel determinante no desenvolvimento tumoral direcionando estudos no sentido de desenvolver imunoterapias baseadas nas mesmas.
While the vast majority of T cells express a T cell receptor (TCR) composed of αβ heterodimers, a smaller population expresses a γδ TCR. These cells differ from αβ T cells, as their TCR can recognize antigens even in the absence of presentation by the MHC molecules. T γδ T cells are particularly present in the mucosal and epithelial surfaces and respond to molecules associated with infection, tissue damage and cell stress. In addition, several subsets of γδ T cells showed antitumor and immunoregulatory activities. They play an active role in bacterial, viral and fungal lung infections, as well as allergies and fibrosis. Recently it was studied its potential in tissue repair in chronic inflammatory diseases, associated with the production of IL-17 cytokine, such as Type 2 Diabetes Mellitus, Obesity, Psoriasis and Asthma. Some subsets of this cell population also revealed a determining role in tumor development directing studies to develop specific immunotherapies.