The purpose of this study was to determine and analyse student achievement as a result of the use of an experimental edition of a programmed business English textbook in selected classes. The participants in the study were 202 post high school and junior college students distributed in seven classes in four states. The textbook used was the experimental edition of Programmed Business English, developed by Harry Huffman of Virginia Polytechnic Institute, Blacksburg, Virginia, and Syrell Rogovin, a linguist at the Thomas J. Watson Research Center. Yorktown Heights, New York. The students took a pretest before initiating their study of the textbook. Four interim tests were taken during the study, and a posttest was taken at the conclusion of the study. A comparison of posttest over pretest achievement represented students’ business English achievement as a result of their study of the textbook. Specific problems and findings in the study were: 1: Problem: Reliability of the pretest and the posttest used in the study. Findings: By using Tests A and B (pretests and posttests) the investigator found a .66 positive correlation between Tests A and B. By using the split-half method on Tests A and B the investigator found a .82 positive correlation on Test A and a .87 positive correlation on Test B. 2: Problem: Business English achievement of students before they used the program business English textbook. Findings: The achievement of individual students before they used the programmed business English textbook ranged from a low of 23 percent for a student in Class 1 to a high of 82 percent for students in Classes 3 and 7. 3. Problem: Business English achievement of selected classes of students after they used the programmed business English textbook. Findings: The achievement of individual students after they used the programmed business English Textbook ranged from a low of 31 percent for a student in Class 2 to a high of 99 percent for a student in Class 3. 4. Problem: Analysis and interpretation of the business English achievement of each class after the programmed business English textbook had been used. Findings: The mean gains in points were 20.41, 24.39, 25.00, 22.31, 19.09, 36.18, and 19.14 for the seven classes who used the textbook. 5. Problem: Comparison of the business English achievement among the classes that used the programmed business English textbook. Findings: Class 6 had the highest mean achievement (88.41) after completing the textbook used in the study, and Class 1 had the lowest mean achievement (64.59). The mean posttest achievement for all the classes was 74.11 percent. 6. Problem: Participating instructors’ opinions as to the strengths and weaknesses of the programmed business English textbook. Findings: Five of the six instructors who returned questionnaires to the investigator had favorable opinions of the textbook. They stated that it was beneficial for more detailed work and left the teacher free to work with slower students. All the replies stated that the textbook provides for more hours or periods of instruction than were used. The major objection to the textbook was the testing program. The tests, according to the instructors, were too infrequent and covered too much material. The advantages the instructors cited were: the provision for individual differences, the immediate verification of response, and the opportunity provided the teacher to work with individual students. / Master of Science

The principal purpose of the study was to determine if the programmed unit on the mathematics of interest, developed by Dr. Harry Huffman, Virginia Polytechnic Institute, could be adapted to the use of high school students at the junior and senior level. To accomplish this objective, extensive revision was necessary. / Master of Science

The interactions of tumours with normal host tissue are key determinants of cancer growth and progression. The ability or inability of the patient’s immune system to mount a response against the tumour is tightly correlated with prognosis. One of the ways tumours avoid detection and elimination by the immune system is by expressing programmed death ligand 1 (PD-L1). PD-L1 binds to its receptor programmed death 1 (PD1) on T cells, inhibiting T cell responsiveness to antigenic stimuli. Blockade of the PD1/PD-L1 pathway removes this negative signal and restores anti-tumour immunity. While this blockade of PD1/PD-L1 is well established through the use of antibodies, small molecule inhibitors of PD1/PD-L1 are relatively unknown. We employed in silico docking in order to find small molecules capable of binding to either PD1 or PD-L1, and the highest-ranked compounds were tested in biophysical assays for their ability to inhibit PD1/PD-L1 binding. A thermal shift assay identified a pyrazole compound as a possible binding partner for PD-L1, but follow-up assays showed that it had no effect on the PD1/PD-L1 interaction and that its apparent binding was probably due to aggregation. An ELISA assay identified a tryptophan diamine compound as an apparent stabilizer of the PD1/PD-L1 interaction. However this compound, too, was later identified to be inactive in orthogonal assays. We identified a family of salicylic acid derivatives that interfered with TR-FRET measurements – an unusual observation, given that TR-FRET is touted as being insensitive to most mechanisms of compound interference. This discovery should help other fragment- screening groups identify false positives more easily. We also probed the mechanism of inhibition of a recently disclosed family of small molecule PD1/PD-L1 inhibitors from Bristol-Myers Squibb. Concurrently with other groups, we used protein NMR, size exclusion chromatography, and SPR to determine that the compounds were inducing homodimerization through the PD1-binding face of PD-L1. Furthermore, using cellular crosslinking and live cell imaging, we showed that these first generation inhibitors are fairly ineffective at inhibiting this interaction on the cell surface. More potent compounds will be needed to see any cellular effect from this mechanism of action. / Graduate / 2019-02-15

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21303号 / 医博第4392号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 生田 宏一, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13557号 / 論医博第2286号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 伊藤 能永, 教授 森信 暁雄 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les cellules Natural Killer (NK) sont des effecteurs de l’immunité innée et constituent de véritables sentinelles dans l’immuno-surveillance contre les virus et les processus tumoraux. Pour échapper à la reconnaissance NK, les virus ainsi que les cellules tumorales utilisent de nombreux subterfuges. Au cours de ce travail, nous nous sommes intéressés à deux facteurs régulateurs de la fonction des cellules NK, la molécule PD-1 et la prostaglandine E2 (PGE2). Programmed death 1 (PD-1) est une molécule régulatrice exprimée sur les lymphocytes T et B activés. L’engagement de PD-1 par ses ligands inhibe leurs fonctions effectrices et prolifératives. Au cours de certaines infections virales chroniques ou de tumeurs, l’expression de PD-1 est associée à l'épuisement fonctionnel des lymphocytes T effecteurs. Le blocage de l’axe PD-1/PD-1 ligands restaure les fonctions effectrices des lymphocytes et représente ainsi une approche thérapeutique prometteuse. Nous avons mis en évidence de manière fortuite une expression inhabituelle du récepteur PD-1 sur les cellules NK au cours de certaines infections virales chroniques (HHV8, VIH ou VHC). L’expression de PD-1 sur les cellules NK caractérise une population récemment activée (CD69++, CD25 + et Nkp44 +), susceptible à l’apoptose (Annexin V+), exprimant une moindre quantité du récepteur de cytotoxicité naturelle NKp46. L’analyse fonctionnelle montre que les cellules NK PD-1+ ont des capacités de cytotoxicité (dégranulation CD107a) et de production de cytokines (IFNγ) réduites en comparaison avec leurs homologues PD-1-. De façon intéressante, l’IL-2 et l’IL-15 peuvent restaurer les fonctions effectrices des cellules NK PD-1+. Contrairement aux cellules T dont l’expression de PD-1 est induite par divers stimuli, seule la stimulation des récepteurs NKp46 et NKp30, en synergie avec l’action de l’IL-15 ou l’IL-2, induit in vitro de façon reproductible l’expression de PD-1 sur les cellules NK de témoins sains. Pour pouvoir disposer d’un modèle in vitro de cellules NK PD-1+, nous avons généré des cellules exprimant PD-1 de manière stable par transduction lentivirale de la lignée NKL. En comparaison avec les cellules transduites par le vecteur vide, les cellules NKL PD-1+ ont des capacités cytotoxiques réduites, confirmant nos résultats chez les patients. Nous avons aussi étudié les mécanismes par lesquels la PGE2, une autre molécule immuno-modularice, régule les fonctions des cellules NK. Nos résultats suggèrent que la PGE2 agit à travers ses récepteurs EP2 et EP4 pour inhiber l’expression de NKG2D et de l’IL-15Rγ induite par l’IL-15 sur les cellules NK. Ce travail doit nous permettre de mieux comprendre comment la PGE2 s’oppose aux effets activateurs de l’IL-15 sur les cellules NK, et représente un mécanisme de rétrocontrôle de l’inflammation. En conclusion, nos résultats montrent que l’expression de PD-1 sur les cellules NK représente un mécanisme supplémentaire d’échappement viral à la réponse immune. La suite de l’étude sur la PGE2 devrait nous permettre d’évaluer l’intérêt de l’utilisation d’antagonistes spécifiques dans l’immunothérapie anti-tumorale par l’IL-15. Le lien entre la PGE2 et l’expression de PD-1 sur les cellules NK est en cours d’investigation. / Natural Killer (NK) cells are effectors of the innate immune system, and play a crucial role in virus and cancer immunesurveillance. To escape NK-cell mediated elimination of infected or transformed cells, viruses and tumors have developed multiple strategies to interfere with NK-cell functions. In the present study, we investigated the role of two regulatory molecules, Programmed Death-1 (PD-1) and Prostaglandine E2 (PGE2), in controlling NK cell activation and effector functions. PD-1 is a key immune checkpoint receptor expressed by activated T and B lymphocytes. Upon interaction with its cognate ligands, PD-1 inhibits lymphocyte proliferation and functions. During cancer or chronic viral infections, PD-1 expression is associated with functional exhaustion of effector T cells. Blockade of PD-1 signaling restores T-cell functions, and represents a promising therapeutic tool. We fortuitously observed unusual expression of PD-1 on a subset of CD56dim NK cells in some patients with persistent viral infection (HHV8, HIV or HCV). We show that PD-1 expression on NK cells characterizes a subpopulation of recently activated cells (CD69+, CD25+, Nkp44+) that are sensitive to apoptosis (Annexin V+) and not senescent (CD57-). NKp46 expression was also markedly decreased on PD-1+ NK cells. In vitro functional experiments showed that PD-1+ NK cells had impaired cytotoxic capacity (CD107a degranulation) and reduced IFN-γ production compared to their PD-1- counterpart, suggesting that they might represent functionally exhausted NK cells. Interestingly, exogenous IL-2 and IL-15 could restore PD-1+ NK cell effector functions. While strong non-specific stimulation by PMA/ionomycin transiently induced PD-1 on control NK cells, only activation through NKp46 or NKp30 receptors in the presence of IL-15 could reproducibly induce stable PD-1 expression. To investigate the effect of PD-1 expression on NK cells in the absence of any confounding factor related to the underlying disease, we generated NK cells stably expressing PD-1 after lentiviral transduction. Compared to NK cells transduced with the control vector, PD-1+ cells showed a constitutively decreased CD107a degranulation, thus confirming our findings in NK cells from infected patients. In the second part of this study, we investigated the immunosuppressive role of PGE2 in the control of IL-15-mediated NK cell activation. Our results suggest that PGE2 acts through EP2 and EP4 receptors to inhibit IL-15 induced NKG2D and IL-15Rγ expression on NK cells. These findings allow a better comprehension of PGE2/IL-15 antagonism in the regulation of NK cell responses. In conclusion, our results indicate that PD-1 expression on NK cells could represent a supplementary mechanism of immune evasion strategy, and allow introducing the concept of exhausted NK cells, similar to exhausted PD-1+ T cells. Our results also demonstrate that PGE2 exerts a negative feedback on IL-15-mediated effects on NK cells. Blocking PGE2 or its receptors could be of interest in IL-15 tumor immunotherapy to potentiate IL-15-induced cytotoxic functions of NK cells.

Les pathogènes respiratoires sont parmi les causes majeures de décès dans le monde entier. Déchiffrer les mécanismes d'évasion immune employés par les pathogènes est essentiel pour le développement de stratégies thérapeutiques contre les pathogènes respiratoires. Dans ce contexte, la vole de signalisation PDL-1 (programmed death ligand 1)-PD-1 (programmed death 1) a été impliquée dans l'évasion immune par les cellules tumorales et des virus. Par conséquent, j'ai voulu étudier le rôle de la voie PD-L1 dans la modulation de la réponse immunitaire contre le Mycobacterium tuberculosis et l'Aspergillus fumigatus. J'ai trouvé que l'α-(1,3)-glucan dérivé de l'A. fumigatus activait les cellules dendritiques (CDs) ; la maturation des CDs était partiellement dépendante du Toll like receptor (TLR)-2. L'analyse de la polarisation des cellules T CD4+ a révélé que les CDs éduquées par l'α-(1,3)-glucan induisent la génération de cellules T régulatrices (Treg) CD4+ CD25+FoxP3+, ceci étant en partie lié à l'expression de PD-L1 sur les CDs. De façon importante, le blocage de PD-L1 sur les CDs augmente la sécrétion d'IFN-γ sans moduler la réponse Th17. De manière similaire, PD-L1 induit par M. tuberculosis freine la réponse Th1 sans moduler la réponse Th17. L'analyse des voies de signalisation en aval a indiqué que la voie sonic hedgehog (SHH) en réponse au mycobacterium médiait l'induction de PD-L1 en inhibant des microARNs spécifiques, miR-324-5p et miR-338-5p qui ciblent PD-L1. De plus, SHH induit la cyclooxygénase (COX)-2 qui catalyse la synthèse de la prostaglandine E2 (PGE2) qui agit en synergie avec PD-L1 pour coordonner l'expansion des Treg. / SummaryPulmonary infections caused by respiratory pathogens are among the major causes of death worldwide. The outcome of infection depends on the ability of the host to respond to the challenge posed by the pathogens. Of note, the host needs to sense the pathogen, mount an efficient immune response and finally clear the ensuing inflammatory response to avoid tissue damage. In this context pathogens have adapted numerous strategies that hijack the host mechanisms to dampen the immune response and as a consequence causing infection. The programmed death-ligand 1 (PD-L1) – programmed death 1 (PD-1) pathway is a key pathway involved in mediating self-tolerance thereby maintaining homeostasis. Elegant reports have demonstrated that the PD-L1 – PD-1 pathway is exploited by cancer cells and viruses as an immune evasion mechanism to suppress effector T cell responses. Thus, I aimed at investigating the role of PD-L1 pathway in modulating immune response to Mycobacterium tuberculosis a bacterial pathogen and Aspergillus fumigatus an opportunistic fungal pathogen. I found that A. fumigatus-derived α-(1,3)-glucan induces maturation of DCs and secretion of various immunoregulatory cytokines that was partially dependant on Toll like receptor (TLR)-2. Analysis of CD4+ T cell polarization revealed that α-(1,3)-glucan-educated DCs induced CD4+ CD25+FoxP3+ regulatory T cell (Treg) generation that was in part dependent on the PD-L1 expression on DCs. Importantly, blocking PD-L1 on DCs enhanced IFN-γ secretion without modulating Th17 response. Similarly, M. tuberculosis induced PD-L1 dampened Th1 response without modulating Th17 response. Analysis of downstream signalling pathways indicated that, mycobacterium-responsive sonic hedgehog (SHH) mediated PD-L1 induction by inhibiting specific microRNAs, miR-324-5p and miR-338-5p that target PD-L1. Additionally, SHH induced cyclooxygenase (COX)-2 catalysed the synthesis of prostaglandin E2 (PGE2) that synergize with PD-L1 to coordinate the expansion of Tregs. My results thus demonstrate that respiratory pathogens either directly or by harbouring imuunoregulatory antigens highjack the PD-L1 pathway to suppress the protective Th1 response and orchestrate Treg generation without modulating Th17 response. Importantly, my results provide a rational for exploiting immunotherapeutic approaches that target PD-1 – PD-L1 co-stimulatory axis to restore effector T cell response to respiratory pathogens.

The administration of antibodies blocking the immune checkpoint molecules programmed cell death protein 1 (PD-1) or programmed cell death 1 ligand 1 (PD-L1) has evolved as a very promising treatment option for cancer patients. PD-1/PD-L1 inhibition has significantly enhanced expansion, cytokine secretion, and cytotoxic activity of CD4+ and CD8+ T lymphocytes, resulting in enhanced antitumor responses. Anti-PD-1 or anti-PD-L1 therapy has induced tumor regression and improved clinical outcome in patients with different tumor entities, including melanoma, non-small-cell lung cancer, and renal cell carcinoma. These findings led to the approval of various anti-PD-1 or anti-PD-L1 antibodies for the treatment of tumor patients. However, the majority of patients have failed to respond to this treatment modality. Comprehensive immune monitoring of clinical trials led to the identification of potential biomarkers distinguishing between responders and non-responders, the discovery of modes of treatment resistance, and the design of improved immunotherapeutic strategies. In this review article, we summarize the evolving landscape of biomarkers for anti-PD-1 or anti-PD-L1 therapy.

published_or_final_version / Education / Master / Master of Education

The present investigation concerned itself with the efficiency of the audio-tutorial method of teaching biology as compared with the more conventional method usually seen in college biology, which involves the use of lectures and laboratory periods.