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.

all I Know Is What I Read In The Papers - Will Rogers -- essays resear

"All I Know Is What I Read In The Papers" - Will Rogers There have been many criteria over the past few centuries that measured one's political clout and influence: divine right, property, money, and acquaintances. In the twentieth century, particularly the past two decades, the political power to influence others resides in information: the more information you have and the more you know how to use it, the more potential influence you have. People rely on the media for their information, as it is the most easily accessible, efficient, and passive way of acquiring knowledge. Unfortunately, the media is not completely reliable as it can and has been manipulated by politicians, their parties, and their governments. This makes the media a powerful weapon as politicians use it to effect voters political choices through advertising, change popular opinion on issues of state, and debasing political campaigns through smear tactics. "You can make a candidate someone they aren't. You can protect them from someone they are, or make them more of what they are".-Senator Norm Atkins(1) "An election is like a one day sale†¦the product (candidate) in a sale (campaign) is only available a few hours on one day".(2) The main goal one hopes to achieve by advertising something is to make it marketable so people will purchase it. Since what a politician hopes to ultimately do is persuade people to vote for, or buy, their political platform, they would be foolish to not take advantage of the captive and passive audience of the advertising mass media. Unfortunately politicians and their management take advantage of this medium to manipulate voters' choices. Two cases of advertising manipulation on voters was during the Canadian National Referendum of 1992 and the Quebec Referendum of 1995. During the National Referendum of 1992 over the Charlottetown Accord "three hours of free broadcast time was made available during prime time on every radio and television network that met the statutory criteria"(3) according to the Referendum Act. The act also states that "half (of the time) is allocated to the ‘Yes' and half to the ‘No' side"(4). This allotment of advertising time did not take into account the print advertisement that was plastered al... ... in that matter"(22). Truedeau floored Bouchard by saying that "the federalists would have done better in the recent Quebec referendum "(23) if the Yes side didn't "make Quebeckers, especially former premier Rene"Levesque, look like victims"(24), Politics is a very dirty game, and if you don't develop a thick skin to deal with the rhetoric then you will not survive the smear campaigns. "I fear three newspapers more than a hundred thousand bayonets"(25) The mass media in all its manifestations has a mandate to be a forum for views both directly and indirectly through advertising and journalist reporting, This massive forum has been the place, for many years, that politicians have had their voice. Like many other institutions, the mass media has been utilized as a tool of the political world with which politicians, their parties, and their governments capture the fixated and passive audience, thus making the media a powerful device to affect voters political choices through advertising, change popular opinion on issues of state, and debasing political campaigns with smear tactics.

How Does Infection Enter the Body? Essay

How does infection enter the body? Infection control is about controlling the spread of communicable diseases between people. Some of the individuals we support will be particularly vulnerable to infection and this means you need to be extra vigilant! In order to spread infection, there needs to be a source, a host, a means of transmission and a point of entry. The source could be a person or the surroundings; the host is the person at risk. Transmission could be through contact with another person, equipment, food and drink or pests. The point of entry could be through breathing, eating, drinking, cuts, wounds or medical procedures like catheters or injections. The five main routes are: * Contact – * Direct transmission involves direct body surface-to-body surface contact and physical transfer of microorganisms from an infected or colonised person to a susceptible host. This may occur between patient and carer during patient care that involves direct personal contact, or between any two persons (patients, carers, others) in the health care setting. * Indirect transmission involves the contamination of an inanimate object (such as instruments or dressings) by an infected or colonised person. The contaminated item or environment may then transmit the infection to a susceptible host via contact. * Droplet – Coughing, sneezing and talking can generate droplets. Procedures such as suctioning and bronchoscopy are also a source of droplets. Transmission occurs when an infected or colonised person generates droplets containing microorganisms which are propelled a short distance through the air and deposited on the conjunctivae, nasal mucosa or mouth of a host. Droplets do not remain suspended in the air; so special air handling and ventilation are not required to prevent droplet transmission. * Airborne – transmission occurs when either airborne droplet nuclei or dust particles disseminate infectious agents. * Common vehicle – Common vehicle transmission applies to micro-organisms transmitted by contaminated items such as food, water, medications, devices and equipment. * Vector borne transmission – Vector-borne transmission occurs when vectors such as insects (mosquitoes, flies) or vermin (rats, mice) transmit microorganisms; Sometimes a microorganism can be transmitted by more than one route. With our clients there are additional points of  entry – the stoma sites and it is your responsibility as a HCSW to prevent cross contamination and promote good infection control procedures. The following diagram outlines how following the practices and protocols put in place by the company can help to ensure infection is not introduced by breaking the chain of infection by being conscious of your actions – safeguarding the health of yourself, your clients and others.

Who or That †That Is The Question

Who or That – That Is The Question Who or That – That Is The Question Who or That – That Is The Question By Guest Author This is a guest post by Charles Ray. If you want to write for Daily Writing Tips check the guidelines here. Some might think me an old fashioned, stuffy person when it comes to grammar. I realize that language, whether written or spoken, is a living thing, and that it changes with time; but, there are some modern conventions that I have problems with. Some things that young people say and write these days grate on my ear, and I resist them with all my might. One of the conventions that really gets my dander up is the use of ‘that’ in sentences when logic, and my ear, tells me that ‘who’ would be more appropriate. Here, for instance, is a sentence I encountered recently in a paper written by a college graduate: â€Å"The judge that decided the case came from the lower court.† Now, I assume the judge in this sentence is human, and when I struggled with English grammar many decades ago, this sentence would have earned red marks all over the page – and quite likely a failing grade. I would have been told in no uncertain terms that the correct formulation is, â€Å"The judge who. . .† I have been chided by many of my colleagues for my fussiness over this particular issue; and it is just one of many modern grammatical conventions that send me into orbit when I encounter them. They’ve pointed out that this is not ‘incorrect,’ and besides, it has become accepted usage among a large number of writers. Well, not this writer. Correct, it might be, and I’m not entirely convinced of that, but it just doesn’t sound right. It grates on my ear when I hear or read it. Just because a lot of people do something doesn’t make it the right and proper thing to do. In my dictionary, one of the definitions of ‘that’ is, â€Å"Adj. Being the one singled out or understood.† ‘Who,’ on the other hand, is defined as â€Å"What or which persons used as a relative pronoun to introduce a clause when the antecedent is a human, or is understood to be a human.† From these definitions, I will grant there is some merit to the argument that using ‘that’ instead of ‘who’ is not incorrect, per se. But, when we write, the purpose is to communicate, and when a ‘correct’ convention gets in the way of communication, it is ‘wrong.’ The language and its grammar are constantly changing, but this is no reason for us to blithely accept each change. We should in fact firmly resist any change until it is proven to be not merely correct, but right. I have not been convinced that substituting ‘that’ for ‘who’ is right, and until I am, I will continue to be the grammar cop who stands in the middle of the street with hands out saying, â€Å"Stop!† And, that is all I have to say about ‘that.’ Want to improve your English in five minutes a day? Get a subscription and start receiving our writing tips and exercises daily! Keep learning! Browse the Grammar category, check our popular posts, or choose a related post below:7 Classes and Types of PhrasesBest Websites to Learn English30 Nautical Expressions

Writer#8217;s Manual for Research Paper on Child Development

Writer#8217;s Manual for Research Paper on Child Development Writing a research paper is not as daunting as some people like to believe. If you have the required information, you can create a compelling paper in no time. And to be able to do so, you need to learn the methods and techniques that outline an exemplary research paper. However, knowing how a research paper is written alone is not really that beneficial. To compose a perfect research paper on child development, you have to read our previous two guides i.e. 10 facts for a research paper on child development and 20 child development research paper topics. These two guidelines will play a vital role in allowing you to write quickly and effectively – leaving you worry free at the time of need. Composing an Attractive Title A title is what really attracts your audience to read your research paper. It is the first thing they will notice about it. You should compose a persuasive title by providing insights to what the reader is going to find inside the paper. You should also try to indicate the variables, both independent and dependent ones. Writing the Abstract The abstract, in general terms, is a brief summary of your content in your research paper. It contains your research question, hypothesis (if there is any) and the discoveries along with the studies you have done on that particular topic. You can also discuss the resources and samples that you’ll use in your paper. Composing the Introduction of Your Paper Introduction is one of the most important paragraphs of your research paper. If your title is attractive, but your introduction isn’t convincing, the reader would not want to read further. To compel the reader to read further, you have to speak clearly. Tell your readers about the problem you’ll be discussing. Describe the research that you did on the problem or topic and sprinkle some information on sources and evidence that support your research and point of view. Concluding everything very briefly would allow the reader to know what’s coming in the next paragraphs and he’ll be interested to read it further. To refine the introduction even further, you have to define issues and problems that would later lead to you research. Explain the variables (independent and dependent) of your research/experiment. Define why you were eager to study this phenomenon in the first place and add some little personal touch to it while describing it. You can also include the hypothesis right after that. Plus, you can define some significant concepts that will be mentioned in your paper but it’s considered optional. Finally, don’t forget to conclude your introduction with a clear and concise delimitation focus. Writing the Body of Your Research Paper Once you have gone through the beginning, it’s time that you start working on the main body of your paper. These paragraphs should be properly organized and must be full of information. Each paragraph should only talk about a particular research that you have done with proper source. Concluding the Paper with Documentation Documenting the sources in your research paper is necessary because it clarifies that your research is original and authentic and it is not a copied version of the sources you used. It also helps the reader know that you have a good understanding of the topic that you have chosen to research on. And of course, if you forget to include the documentation then your research may be suspected as plagiarized content. Don’t forget to include the bibliography of the sources that you have use. It’s a must to let readers know that your research is authentic and has been borrowed from some credible sources. In other words, it helps define your research as an unbiased version of your opinion, based mostly on facts and nothing else. That’s about it! Once you have concluded the paper. It’s time to take a break. Once reenergized, go through your paper and correct mistakes to have a perfect output. Remove parts that might seem a bit excessive, add information that would make your paper better. Don’t forget to look for outline mistakes and grammatical errors and consider revising again. The more you revise, the better your research paper would become. Now you’re reading to write an exemplary research paper on child development. Be sure to read our first and second guide as well, if you haven’t already. Happy writing!

History Assignment Example | Topics and Well Written Essays - 250 words - 32

History - Assignment Example For instance, the American mixed martial arts (MMA) is one such sport whereby two competitors fight it out to see who has the better skill, determination and athleticism to beat down their opponent, just the way the gladiators used to do in Rome (DeCosemo 1). As events of modern day sport, it’s not all about the game, there are other things included such as involvement of characters, determination, technical skill and the personal drama (DeCosemo 1). A few good examples of this would be present day football or soccer, American football, Basketball, and baseball. So during the Roman Empire it was gladiator fights that brought spectators or people together to sit and watch gladiators fight and kill each other. While everything else remains the same from the Roman times to present day sports such as the intensity, cheering, and shouts of spectators or fans, the only difference in modern sports is that they is no killings in the name of being victorious as was the case in gladiator fights (DeCosemo 1). It can safely be said that sports have changed over the centuries since the era of the Roman Empire. Not any more do we find men sword fighting or even fighting wild animals. Yet, there are still several similarities between the Roman Empire sports and the modern day sports, especially in terms of spectators and fans. In both the ancient and modern day sports, there are people who adore the game and

Juvenile Justice Project (outline) How Teenagers Should be Punished Assignment

Juvenile Justice Project (outline) How Teenagers Should be Punished - Assignment Example These minor violations result in harsh and brutal punishments from them. This is the simplest parenting trap many parents may fall into as at this point parents are trying to win their battle of ego and being superior rather than giving some learning to their child about how to choose good and bad. Parents must remember that this is not the wrestling match or a tug of war but only the time to demonstrate their role in the best possible way. Parents do have some authority over their child but that does not mean to change this into a power struggle scenario. (ii). Negative Impact Of Physical Aggression: Child who suffers a constant hitting by their parents may end up having violent behavior issues later with their friends, siblings or even with their better halves. Conclusion: Thus physical assaults practice on teenagers must be condemned as this may lead to loss of self confidence of child and respect of parents in the eye of child. Certain alternatives for combating the discipline issues with child must be kept in mind for the proper upbringing of the