Anti-tumour Immunity through GP-100-TLR Agonist Conjugation

Anti-tumour Immunity through GP-100-TLR Agonist Conjugation Enhancing anti-tumour immunity through gp-100-TLR agonist conjugation. Introduction Soluble cancer vaccines remain an area of high interest to researchers with the ability to enhance immune responses against present cancers and induce protective immunity against future cases. In developing new vaccines finding ways to increase the immunogenicity of cancer antigens is a major challenge(1-3). The addition of Toll-like receptor (TLR) agonists is one strategy which can successfully boost immune cell activation and response to cancer antigens. By stimulating TLRs, these agonists increase expression of several co-stimulatory molecules on antigen presenting cells (APCs) such as CD80/86 and CD40(4-6).They also increase tumour peptide loading onto type 1 2 Major histocompatibility complex (MHC) proteins. Together this leads to greater activation of tumour-specific effector immune cells such as CD4+ and CD8+ T-cells resulting in increased tumour clearance via their cytotoxic activity. Vaccine formulations which have included antigen and TLR agonists as a mixture have had pro mising results with many in clinical trials(4, 7, 8). Despite this, few have assessed the effect of chemically conjugating these constituents, a strategy which could increase efficiency of both TLR activation and peptide loading onto MHC(9-12). Many conjugation strategies that do exist today capitalise on the use of pH and redox sensitive linkers. Differences in pH and redox environments intracellularly enable triggerable release of these vaccines whilst protecting antigen and agonist from degradation extracellularly where they are administered. Research into the use of Glutathione-sensitive disulphide linkers has demonstrated that the immune response to model antigen Ovalbumin'(OVA) could be increased through linkage to the TLR agonist, CpG oligodeoxynucleotide (ODN)(10, 11). Our research aims to repeat this using both stable and reversible linkers as well as a more clinically relevant, tumour associated antigen (TAA) called gp-100 expressed on melanomas. In addition, we aim to ass ess the effectivity of different TLR agonists within conjugates including Polyinosinic polycytidylic acid (Poly I:C) and two different classes of CpG ODNs, B and C respectively. Each of these agonists activate different signalling pathways within antigen presenting cells leading to unique cytokine profiles and T-cell responses. Poly I:C for example, is a potent activator of TLR3 which activates the TRIF pathway inducing release of type 1 interferons such as IFN Beta(6, 13, 14). This increases MHC-I expression and stimulates a Th1 type immune response which favours cell-mediated immunity including CD8+ T-cell activation. In comparison, CpG class B and C stimulate TLR 9 activating the MYD88 pathway and release of proinflammatory cytokines IL-6 and IL-17. This results in enhanced CD4+ and CD8+ T-cell responses, B cell activation and antibody production(10, 11, 15). Both types of response have potential to give clinical benefit in different ways highlighting the potential of these conju gates in tumour treatment. Finally, we will also assess how the composition of the TAA effects its presentation on MHC. To assess this, a smaller Gp-100 peptide which does not require intracellular processing will be compared to a longer peptide requiring processing. This project will assess which conjugates enhance anti-tumour responses in mice and how they achieve this looking specifically at Dendritic cell activation and CD8+ T-cell proliferation and cytokine production. Hypotheses In terms of CD8+ responses, T cell proliferation and cytokine release, I hypothesize that Poly I:C reversibly linked to processed gp-100 will be the most effective inducing a strong Th1 response and IFN-B cytokine release. This is because Poly I:C stimulates several intracellular signals in addition to TLR3 including RIG-1 and MDA-5. This would lead to increased release of proinflammatory cytokines including IL-6, IL-12, IL-1B and IFN-B. Specifically the release of IFN-B would induce a strong anti-viral-like Th1 T cell response through increased expression of MHC-1 molecules on APCs and inducing release of IFN-Y, TNF-A and IL-2 from CD4+ T cells. As well as this factor, TLR3 is exclusively expressed on myeloid dendritic cells, the most effective dendritic cell subset in presenting antigen, and not expressed on plasmacytoid dendritic cells. Secondly, I Hypothesize that CpG class C will induce the most cytokine production in dendritic cells including the cytokines IL-6, IL-12, IFN-A and IL-1B. CpG molecules stimulate TLR9 which is expressed in the endolysosomal compartment of plasmacytoid dendritic cells exclusively. As a dendritic cell subset, plasmacytoid DCs are well known for their proinflammatory cytokine production at levels much higher than other DC subsets. CpG class C in particular stimulates the release of IFN-a in addition to IL-6, IL-12 etc. stimulating both a Th1 response and a B cell response. Aims and objectives Aims Produce gp-100-CpG ODN and gp-100-Poly I:C reversible and stable conjugates with either processed or non-processed Gp-100 peptides Measure dendritic cell subset activation through expression of MHC-II, CD40 and CD86 molecules and cytokine release (IL-12, IL-6, IL-1B, IFN-A and IFN-B ) Measure Tumour specific T cell activation (CD4+ and CD8+), proliferation using carboxyfluorecein succinimidyl ester (CSFE) and cytokine release (IFN-Y, IL-2,TNF-A) Methods The proposed project for the year will focus on three main objectives 1) Produce gp-100-CpG ODN and gp-100-Poly I:C reversible and stable conjugates with either processed or non-processed Gp-100 peptides. First, we will modify free amino groups on the lysine residues of each gp-100 peptide (processed amino acid sequence: KVPRNQDWL vs unprocessed: CAVGALKVPRNQDWLGVPRQL) and TLR agonists (suspended in a modification buffer ph. 8). Then we will link these together with either the stable linker (HYN) or the reversible linker (HYN-SS) in a ph. 6 conjugation buffer separately. Product concentration after each individual modification step will be measured using Nanodrop1000 at 280 m after desalting excess product using vivspin 500 filter. Final product conjugation will be confirmed using the reversed phase liquid chromatograph at the School of Pharmacy which will allow us to visualise each individual product according to their differing polarities, and quantify their ratio. Our second objective is to Measure dendritic cell subset activation through expression of MHC-II, CD40 and CD86 molecules and cytokine release (IL-12, IL-6, IL-1B, IFN-B, IFN-A). To achieve this, we will isolate bone marrow cells from C57BL/6 mice and treat with GM-CSF to produce CD11c+ dendritic cells. These will then be treated with either individual TLR agonists, TLR agonist-gp100 mixtures or TLR agonist-gp100 conjugates (reversible or non-reversible). After 24hrs of treatment these cells with be stained with fluorescent antibodies for CD80, CD40, CD11C, and MHC-II, viewed on the Gallios flow cytometer in Pathology and analysed using Kaluza software. This experiment will be repeated at least three times to enable statistical analysis, which will be performed using Graph Pad prism software. Cytokine release from these cells will be measured using an enzyme linked immunosorbent assay (ELISA) for IL-6, IL-12, IFN-B and IFN-a. Our third objective is to Measure Tumour specific CD3+ T cell: activation (CD8+), proliferation (CSFE) and cytokine release (IFN-Y, IL-2). This will be achieved through isolation of splenocytes from Pmel (T-cells specific to gp-100) transgenic mice and sorting of CD8+ cells using the Automacs machine at Pathology. These cells will then be stained using CSFE and co-cultured separately with C57BL/6 BMDCs treated according to objective 2. After 72hrs cells will be analysed using the Gallios flow cytometer to measure T-cell activation (CD3+) and proliferation (CSFE). To measure cytokine release, cell cultures will undergo an ELISA for IFN-Y and IL-2. Proposed Budget Mice C57BL/6 x 10 @ $50 each$500 PMEL x 10 @ $50 each$500 Antibodies CD86-PE$300 CD11c-APC$300 CD40-PECy7$300 CD8a-APC$300 CD3-PE$300 MHC-I$300 MHC-II FITC$300 Cell culture reagents IMDM Media$400 Foetal calf serum$500 Cytokine detection Cytokine detection kit$2000 Conjugation reagents S4FB Linker$450 S-SS-4FB Linker$350 S-HYNIC cross linker$850 2-Hydrazinopyradine.dihydrochloride$450 2-Sulphobenzaldehyde$450 CpG class B$500 CpG class C$500 Poly I:C$500 Vivspin filters$200 Total$9250 References 1.Obeid JM, Hu Y, Slingluff CL. Vaccines, adjuvants and dendritic cell activators Current Status and Future Challenges. Seminars in oncology. 2015;42(4):549-61. 2.Guo C, Manjili MH, Subjeck JR, Sarkar D, Fisher PB, Wang X-Y. Therapeutic Cancer Vaccines: Past, Present and Future. Advances in cancer research. 2013;119:421-75. 3.Schlom J. Therapeutic Cancer Vaccines: Current Status and Moving Forward. JNCI Journal of the National Cancer Institute. 2012;104(8):599-613. 4.Kaczanowska S, Joseph AM, Davila E. TLR agonists: our best frenemy in cancer immunotherapy. Journal of leukocyte biology. 2013;93(6):847-63. 5.Pradere J-P, Dapito DH, Schwabe RF. The Yin and Yang of Toll-like Receptors in Cancer. Oncogene. 2014;33(27):3485-95. 6.Maruyama K, Selmani Z, Ishii H, Yamaguchi K. Innate immunity and cancer therapy. International immunopharmacology. 2011;11(3):350-7. 7.Iribarren K, Bloy N, Buque A, Cremer I, Eggermont A, Fridman WH, et al. Trial Watch: Immunostimulation with Toll-like receptor agonists in cancer therapy. Oncoimmunology. 2016;5(3):e1088631. 8.Dowling JK, Mansell A. Toll-like receptors: the swiss army knife of immunity and vaccine development. Clinical Translational Immunology. 2016;5(5):e85. 9.Flanary S, Hoffman AS, Stayton PS. Antigen delivery with poly(propylacrylic acid) conjugation enhances MHC-1 presentation and T-cell activation. Bioconjugate chemistry. 2009;20(2):241-8. 10.Herbath M, Szekeres Z, Kovesdi D, Papp K, Erdei A, Prechl J. Coadministration of antigen-conjugated and free CpG: effects of in vitro and in vivo interactions in a murine model. Immunology letters. 2014;160(2):178-85. 11.Kramer K, Shields NJ, Poppe V, Young SL, Walker GF. Intracellular Cleavable CpG Oligodeoxynucleotide-Antigen Conjugate Enhances Anti-tumor Immunity. Molecular Therapy. 2017;25(1):62-70. 12.Slutter B, Soema PC, Ding Z, Verheul R, Hennink W, Jiskoot W. Conjugation of ovalbumin to trimethyl chitosan improves immunogenicity of the antigen. Journal of controlled release : official journal of the Controlled Release Society. 2010;143(2):207-14. 13.Ammi R, De Waele J, Willemen Y, Van Brussel I, Schrijvers DM, Lion E, et al. Poly(I:C) as cancer vaccine adjuvant: knocking on the door of medical breakthroughs. Pharmacology therapeutics. 2015;146:120-31. 14.Cho HI, Barrios K, Lee YR, Linowski AK, Celis E. BiVax: a peptide/poly-IC subunit vaccine that mimics an acute infection elicits vast and effective anti-tumor CD8 T-cell responses. Cancer immunology, immunotherapy : CII. 2013;62(4):787-99. 15.Scheiermann J, Klinman DM. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer. Vaccine. 2014;32(48):6377-89.

Coconut: the most economically important member of the great palm family Essay

CHAPTER 1 (BACKGROUND OF THE STUDY) In this modern world considered as the era of comforts, we also face poverty and scarcity of resources because of over population. So, people today are searching for some easier and better ways to save money through substituting commercialized products with improvised and homemade products, economizing, etc. In short people today are just being practical on what they will buy or what they will do to meet their needs. The coconut is the fruit of the most economically important member of the great palm family, Palmae. The genus Cocos are Southeast Asians and contain only one species, Cocos Nucifera.  Cultivated in tropical lowlands, almost always near the sea, the coconut has long been distributed throughout Southeast Asia and along the Tropical African and American coasts. The coconut is known for its great versatility as seen in the many uses of its different parts. For centuries, the coconut pal has supplied the people of the Pacific Islands with food, drink, shelter, and most of their needs. The durian is the fruit of several tree species belonging to the genus Durio. There are 30 recognised Durio species, at least nine of which produce edible fruit. Durio zibethinus is the only species available in the international market: other species are sold in their local regions. Regarded by many people in Southeast Asia as the â€Å"king of fruits†, the  durian is distinctive for its large size, strong odor, and formidable thorn-covered husk. The fruit can grow as large as 30 centimeters (12 in) long and 15 centimeters (6 in) in diameter, and it typically weighs one to three kilograms (2 to 7 lb.). Its shape ranges from oblong to round, the color of its husk green to brown, and its flesh pale yellow to red, depending on the species. Corn (Zea mays) has been grown in the northeast for generations, and is a demanding crop but one that is highly-valued for its use. Corn, Zea mays, is an annual grass in the family Poaceae and is a staple food crop grown all over the world. Corn is the second most important crop in the Philippines. About 14 million Filipinos prefer white corn as their main staple and yellow corn accounts for about 50% of livestock mixed feeds. Some 600,000 farm households depend on corn as a major source of livelihood, in addition to transport services, traders, processors and agricultural input suppliers who directly benefit from corn production, processing, marketing and distribution. Shoe shining is the process of applying an external substance to the surface of a shoe to improve the materials and make it shinier. Shoe shining has been a part of shoe care for hundreds of years. Adding a shine to a shoe brings polish to an outfit. Shoe polish products are low-value items are frequently purchased as a single but might last for several days. The researchers wanted to produce shoe polish out of the coconut husks, durian husks and corn cobs because we found another use for them. This leads to conduct an experiment using the ashes of coconut husk, durian husks and corn cobs for shoe polish. If these products would be successful, it can help in recycling coconut husks, durian husks and corn cobs and can lessen them to avoid them scattering all over our community. STATEMENT OF THE PROBLEM This study aims to make an effective shoe polish out of ashes from burned coconut husks, durian skins, and corn cobs. Specifically, the study would like to answer the following: * What are the components that are present in the ashes? * How effective is the shoe polish in terms of: – Shine that it could give – Life Span – Color of Polish compared to other brands * What is the difference between the commercial shoe polish from the shoe polish out of ashes from the coconut husks, durian skins, and corn cobs? ASSUMPTION The researchers believed that Coconut husks, Corn Cobs and Durian Skins are effective alternative shoe polish and can make it a source of income. HYPOTHESIS There was no significant difference between the efficiency of our product to the commercial shoe polish that was sold on the market. SIGNIFICANCE OF THE STUDY One of the main benefits of a shoe shine is that it helps preserve the material that shoes are made out of. Polishing products also provides the coating of wax on the leather that helps in keeping it waterproof and reduces the dirt accumulated on the leather. Shoe polish also gives a moisturizing effect to the leather and proper care may help in lasting the shoes for several years. Since coconut, durian, and corn is abundant in our country, people can make it a source of livelihood. SCOPE AND LIMITATION The study is focused on the effectiveness of the alternative shoe polish and aims to shine shoes at a long period of time. Furthermore, this study is only limited to the effectiveness, color, shine, duration, and the odor it can give. METHODOLOGY The purpose of this chapter is to present the experimental assumptions underpinning this research, as well as to introduce the research strategy and empirical techniques applied then the materials used in conducting this  study. The chapter defines the scope and limitations of the research design. Materials The researchers utilized the following materials in accomplishing the project: Coconut husks, Corn Cobs, Durian Skins, Coals, Matchsticks, Ashes, Tongs, Strainer, 3 Basins, Pitcher, Water, Measuring Cups, Detergent Bar Soap, Knife, Frying Pan, Spoon, Citric Acid, Glycerin, Dye, and Kerosene. The sun was used as the source of heat to dry the Coconut husks, Corn cobs and Durian skins. The coals and matchsticks are used to burn the three different fruit shells and tongs was used to protect our hands from getting burned. The strainer was used to remove big particles and for us to gather fine ashes. The 3 basins were used as containers of the ashes. The pitcher was used as a container for the water. The measuring cups were used to measure the quantity of the water, citric acid, glycerin, dye and kerosene needed. The knife was used to cut the bar soap. The frying pan and spoon were used in heating up the whole mixture. Procedure The researchers gathered (1) one sack coconut husk, (1) one sack corn cobs and (1) one sack durian skin. After collecting the three different fruit shells the researchers let them stay under the sun for three days to get totally dry. Then after three days the researchers prepared the materials for the burning process. The researchers burned the three different dried fruit shells separately and collected the ashes. Afterwards, they inspect the ashes and they remove the big particles using a strainer. 1.Burn the dried coconut husk, corn cob, and durian skin separately and collect the ashes. 2.Inspect the ashes and remove the big particles. 3.Measure a certain amount of water. 4.Cut the bar soap into small pieces and dissolve it on water. 5.Add the ashes. 6.Heat the mixture until it boils, then stir evenly. 7.After few minutes add the citric acid and the glycerin. 8.Pour into a container. The mixture needs to settle for a number of hours before used.

Crucible essay Essay

The Crucible takes place in Salem Massachusetts during 1692. It takes place during the tragic time of the so called Salem witch hunts when many innocent people were accused of being a witch or dealing with the devil. As a result of these convictions many people confessed to save their lives, others who would not confess to a lie were hung or executed by other methods such as being pressed. The play the Crucible was wrote by Arthur Miller during the â€Å"Red Scare†, which was almost parallel with the Salem witch hunts in that many innocent people were accused of actions of communism and espionage that they did not take part in. The Crucible is not so much about a witch hunt as it is an illumination of human weakness, hypocrisy, and vindictiveness. In the Crucible there is quite a bit of human weakness. This can easily be seen through all of the people in the story who admitted to being a witch or dealing with the devil instead of being strong and denying the fact that were not a witch and be put to death. When this all begins, Abigail, the reverends own niece, blames Tituba, the reverends slave from Barbados of being a witch. When she is accused on page 847 she first denies it, â€Å"I don’t compact with no devil,† later on after Mr. Putnam says â€Å"this woman must be hanged†, Tituba gives into her natural human weakness and cries out â€Å"†¦ I tell him I don’t desire to work for him(the devil) sir.† That is just one of the many cases where human weakness is illuminated in the play, it is also the most common, many people gave in to the pressure so they wouldn’t be hung. Another way human weakness was illuminated in the Crucible is that John Proctor will not admit his affair with Abigail because he is afraid he will lose his farm and ruin his name. Mary Warren also gives into human weakness when John Proctor asks her to go to the court and tell that the girls are all faking there so called sickness caused by the accused witches. When she tries to tell Judge Danforth Abigail interferes and Mary pretends to go crazy again. Amongst all the human weakness there was a lot of hypocrisy mostly amongst the people who seemed strong and pure. The Crucible may take place during the Salem witch trials of 1692, however the illumination of weakness, hypocrisy, and vindictiveness are what the  play is actually about. Just like what was going on during the Red Scare during Arthur Miller’s time.

Workers of the Progressive Era - 1047 Words

Progressive Era: Working Class Workers during the gilded age were marginalized by their working conditions, low income, and limited working hours. To overcome the marginalization for the working class, they created labor movements and went on strikes. Although the workers had created many strikes and labor unions, they were at the least successful. Workers were marginalized by the poor working conditions they had. A lot of the time the workers feared going to their workshops because they knew what they were getting themselves into. In 1906, Upton Sinclair, a writer during the gilded age, wrote a novel, The Jungle, in which took place inside work factories. He expressed the fact that the work was†¦show more content†¦They would have no nails,-they had worn them off pulling hides; their knuckles were swollen so that their fingers spread out like a fan.†(Sinclair, 1906) He stated this to point out that the workers had horrible conditions in the workshops and they needed to be justified in that state. Similarly, a recent article ,Labor in Progressive Era Politics, expressed an event of deaths in a workshop located in New York in 1911; this event is well known by the name The Triangle Fire. In the article it states that the â€Å"Triangle Shirtwaist Company in New York killed 146 garment workers in 1911, public outrage prompted the creation of a state commission to study the origins of the fire and the conditions of industrial workplace.†(Unknown) This event was not only tragic, but also a huge spark of the idea that the marginalization shall be no more. They were going to do what they needed to overcome the working conditions. To overcome the harsh and terrible working conditions, workers decided to go on strikes to catch the attention of the â€Å"big guys† so they can make improvements in the workshops. Many of the strikes were unsuccessful, stopped, or ignored. The strikes went on for about twenty years, a few of them were: The Great Strike,1877; Haymarket Riot,1886; Homestead Strike, 1892; Pullman Strike, 1894. In a Speech given by Eugene Debs he had said â€Å"To realize this great social ideal is a work of education, and organization. The working classesShow MoreRelatedThe Progressive Era And The New Deal1103 Words   |  5 Pagestraditions of the Progressive Era. When examining the New Deal, Progressive influence is evident based first off of the social and political issues addressed by reforms. Second, the reforms from the two times themselves are uncannily similar, again due to the focus on the same problems existing in the United States. 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