8,503 research outputs found

    Gluten Immunogenic Peptides as Standard for the Evaluation of Potential Harmful Prolamin Content in Food and Human Specimen

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    Gluten is a complex mixture of storage proteins in cereals like wheat, barley, and rye. Prolamins are the main components of gluten. Their high content in proline and glutamine makes them water-insoluble and difficult to digest in the gastrointestinal tract. Partial digestion generates peptide sequences which trigger immune responses in celiac and gluten-sensitive patients. Gluten detection in food is challenging because of the diversity, in various food matrices, of protein proportions or modifications and the huge number of immunogenic sequences with differential potential immunoactivity. Attempts to develop standard reference materials have been unsuccessful. Recent studies have reported the detection of a limited number of dominant Gluten Immunogenic Peptides (GIP) that share similarities to epitopes presented in the őĪ-gliadin 33-mer, which showed to be highly proteolytic resistant and is considered to be the most immunodominant peptide within gluten in celiac disease (CD). GIP were detectable and quantifiable in very different kind of difficult to analyze food, revealing the potential immunogenicity by detecting T-cell activity of celiac patients. But GIP were also found in stool and urine of celiac patients on a supposedly gluten-free diet (GFD), showing the capacity to resist and be absorbed and excreted from the body, providing the first simple and objective means to assess adherence to the GFD. Methods to specifically and sensitively detect the most active GIP in food and biological fluids are rational candidates may use similar analytical standard references for determination of the immunopathological risk of gluten exposure in gluten-related diseases.Espa√Īa, MINECO AGL2013-48946-CEspa√Īa, Ministerio de Ciencia, Innovaci√≥n y Universidades y Corporaci√≥n Tecnol√≥gica de Andaluc√≠a (CTA

    Delivery of mixed-lineage kinase domain-like protein by vapor nanobubble photoporation induces necroptotic-like cell death in tumor cells

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    Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis

    Patent Landscape of Influenza A Virus Prophylactic Vaccines and Related Technologies

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    Executive Summary: This report focuses on patent landscape analysis of technologies related to prophylactic vaccines targeting pandemic strains of influenza. These technologies include methods of formulating vaccine, methods of producing of viruses or viral subunits, the composition of complete vaccines, and other technologies that have the potential to aid in a global response to this pathogen. The purpose of this patent landscape study was to search, identify, and categorize patent documents that are relevant to the development of vaccines that can efficiently promote the development of protective immunity against pandemic influenza virus strains. The search strategy used keywords which the team felt would be general enough to capture (or ‚Äúrecall‚ÄĚ) the majority of patent documents which were directed toward vaccines against influenza A virus. After extensive searching of patent literature databases, approximately 33,500 publications were identified and collapsed to about 3,800 INPADOC families. Relevant documents, almost half of the total, were then identified and sorted into the major categories of vaccine compositions (about 570 families), technologies which support the development of vaccines (about 750 families), and general platform technologies that could be useful but are not specific to the problems presented by pandemic influenza strains (about 560 families). The first two categories, vaccines and supporting technologies, were further divided into particular subcategories to allow an interested reader to rapidly select documents relevant to the particular technology in which he or she is focused. This sorting process increased the precision of the result set. The two major categories (vaccines and supporting technologies) were subjected to a range of analytics in order to extract as much information as possible from the dataset. First, patent landscape maps were generated to assess the accuracy of the sorting procedure and to reveal the relationships between the various technologies that are involved in creating an effective vaccine. Then, filings trends are analyzed for the datasets. The country of origin for the technologies was determined, and the range of distribution to other jurisdictions was assessed. Filings were also analyzed by year, by assignee, and by inventor. Finally, the various patent classification systems were mapped to find which particular classes tend to hold influenza vaccine-related technologies. Besides the keywords developed during the searches and the landscape map generation, the classifications represent an alternate way for further researchers to identify emerging influenza technologies. The analysis included creation of a map of keywords, as shown above, describing the relationship of the various technologies involved in the development of prophylactic influenza A vaccines. The map has regions corresponding to live attenuated virus vaccines, subunit vaccines composed of split viruses or isolated viral polypeptides, and plasmids used in DNA vaccines. Important technologies listed on the map include the use of reverse genetics to create reassortant viruses, the growth of viruses in modified cell lines as opposed to the traditional methods using eggs, the production of recombinant viral antigens in various host cells, and the use of genetically-modified plants to produce virus-like particles. Another major finding was that the number of patent documents related to influenza being published has been steadily increasing in the last decade, as shown in the figure below. Until the mid-1990s, there were only a few influenza patent documents being published each year. The number of publications increased noticeably when TRIPS took effect, resulting in publication of patent applications. However, since 2006 the number of vaccine publications has exploded. In each of 2011 and 2012, about 100 references disclosing influenza vaccine technologies were published. Thus, interest in developing new and more efficacious influenza vaccines has been growing in recent years. This interest is probably being driven by recent influenza outbreaks, such as the H5N1 (bird flu) epidemic that began in the late 1990s and the 2009 H1N1 (swine flu) pandemic. The origins of the vaccine-related inventions were also analyzed. The team determined the country in which the priority application was filed, which was taken as an indication of the country where the invention was made or where the inventors intended to practice the invention. By far, most of the relevant families originated with patent applications filed in the United States. Other prominent priority countries were the China and United Kingdom, followed by Japan, Russia, and South Korea. France was a significant priority country only for supporting technologies, not for vaccines. Top assignees for these families were mostly large pharmaceutical companies, with the majority of patent families coming from Novartis, followed by GlaxoSmithKline, Pfizer, U.S. Merck (Merck, Sharpe, & Dohme), Sanofi, and AstraZeneca. Governmental and nonprofit institutes in China, Japan, Russia, South Korea and the United States also are contributing heavily to influenza vaccine research. Lastly, the jurisdictions were inventors have sought protection for their vaccine technologies were determined, and the number of patent families filing in a given country is plotted on the world map shown on page seven. The United States, Canada, Australia, Japan, South Korea and China have the highest level of filings, followed by Germany, Brazil, India, Mexico and New Zealand. However, although there are a significant number of filings in Brazil, the remainder of Central and South America has only sparse filings. Of concern, with the exception of South Africa, few other African nations have a significant number of filings. In summary, the goal of this report is to provide a knowledge resource for making informed policy decisions and for creating strategic plans concerning the assembly of efficacious vaccines against a rapidly-spreading, highly virulent influenza strain. The team has defined the current state of the art of technologies involved in the manufacture of influenza vaccines, and the important assignees, inventors, and countries have been identified. This document should reveal both the strengths and weaknesses of the current level of preparedness for responding to an emerging pandemic influenza strain. The effects of H5N1 and H1N1 epidemics have been felt across the globe in the last decade, and future epidemics are very probable in the near future, so preparations are necessary to meet this global health threat

    HLA Class-II Associated HIV Polymorphisms Predict Escape from CD4+ T Cell Responses.

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    Antiretroviral therapy, antibody and CD8+ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4+ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-ő≥ ELISpot assay, we determined that the magnitude of CD4+ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p<0.0001). However, regardless of the group, the magnitude of responses to AE was lower as compared to NAE (p<0.0001). CD4+ T cell responses in patients with acute HIV infection (AHI) demonstrated poor immunogenicity towards AE as compared to NAE encoded by their transmitted founder virus. Longitudinal data in AHI off antiretroviral therapy demonstrated sequence changes that were biologically confirmed to represent CD4+ escape mutations. These data demonstrate an innovative application of HLA-associated polymorphisms to identify biologically relevant CD4+ epitopes and suggests CD4+ T cells are active participants in driving HIV evolution

    Docosahexaenoic acid (DHA) promotes immunogenic apoptosis in human multiple myeloma cells, induces autophagy and inhibits STAT3 in both tumor and dendritic cells

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    Docosahexaenoic acid (DHA), a ŌČ-3 polyunsaturated fatty acid found in fish oil, is a multi-target agent and exerts anti-inflammatory and anticancer activities alone or in combination with chemotherapies. Combinatorial anticancer therapies, which induce immunogenic apoptosis, autophagy and STAT3 inhibition have been proposed for long-term therapeutic success. Here, we found that DHA promoted immunogenic apoptosis in multiple myeloma (MM) cells, with no toxicity on PBMCs and DCs. Immunogenic apoptosis was shown by the emission of specific DAMPs (CRT, HSP90, HMGB1) by apoptotic MM cells and the activation of their pro-apoptotic autophagy. Moreover, immunogenic apoptosis was directly shown by the activation of DCs by DHA-induced apoptotic MM cells. Furthermore, we provided the first evidence that DHA activated autophagy in PBMCs and DCs, thus potentially acting as immune stimulator and enhancing processing and presentation of tumor antigens by DCs. Finally, we found that DHA inhibited STAT3 in MM cells. STAT3 pathway, essential for MM survival, contributed to cancer cell apoptosis by DHA. We also found that DHA inhibited STAT3 in blood immune cells and counteracted STAT3 activation by tumor cell-released factors in PBMCs and DCs, suggesting the potential enhancement of the anti-tumor function of multiple immune cells and, in particular, that of DCs

    Targeted-pig trial on safety and immunogenicity of serum-derived extracellular vesicles enriched fractions obtained from Porcine Respiratory and Reproductive virus infections

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    The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the etiological agent of one of the most important swine diseases with a significant economic burden worldwide. Unfortunately, available vaccines are partially effective highlighting the need of novel approaches. Previously, antigenic viral proteins were described in serum-derived extracellular vesicles (EVs) from pigs previously infected with PRRSV. Here, a targeted-pig trial was designed to determine the safety and immunogenicity of such extracellular vesicles enriched fractions. Our results showed that immunizations with EV-enriched fractions from convalescence animals in combination with montanide is safe and free of virus as immunizations with up-to two milligrams of EV-enriched fractions did not induce clinical symptoms, adverse effects and detectable viral replication. In addition, this vaccine formulation was able to elicit specific humoral IgG immune response in vaccinated animals, albeit variably. Noticeably, sera from vaccinated animals was diagnosed negative when tested for PRRSV using a commercial ELISA test; thus, indicating that this new approach differentiates vaccinated from infected animals. Lastly, after priming animals with EV-enriched fractions from sera of convalescence animals and boosting them with synthetic viral peptides identified by mass spectrometry, a distinctive high and specific IFN-ő≥ response was elicited. Altogether, our data strongly suggest the use of serum EV-enriched fractions as a novel vaccine strategy against PRRSV.Anti-CD9, Anti-CD63 and anti-CD81 antibodies were kindly donated by Francisco S√°nchez-Madrid and Maria Ya√Īez-Mo, Hospital de la Princesa, Madrid, Spain. The authors wish to particularly thank Gl√≤ria Abella for her collaboration in conducting the field study and to Marta Alcob√©, Miriam Moron Font and Paula Crego Mendez for technical assistance. This study received support from Innovex Therapeutics S.L., Pinsos del Segre SA, Granja Casany√©, Grup de Sanejament Porci (GSP, Lleida, Spain) and the FEDER project (COMRDI16-1-0035-03). Sergio Montanter-Tarbes is an industrial doctorate awarded by the Government of Catalonia, Spain (No. 2014 DI 044). ISGlobal and IGTP are members of the CERCA Programme, Generalitat de Catalunya

    Tumor-reactive immune cells protect against metastatic tumor and induce immunoediting of indolent but not quiescent tumor cells

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    Two major barriers to cancer immunotherapy include tumor-induced immune suppression mediated by myeloid-derived suppressor cells and poor immunogenicity of the tumor-expressing self-antigens. To overcome these barriers, we reprogrammed tumor-immune cell cross-talk by combined use of decitabine and adoptive immunotherapy, containing tumor-sensitized T cells and CD25+ NKT cells. Decitabine functioned to induce the expression of highly immunogenic cancer testis antigens in the tumor, while also reducing the frequency of myeloid-derived suppressor cells and the presence of CD25+ NKT cells rendered T cells, resistant to remaining myeloid-derived suppressor cells. This combinatorial therapy significantly prolonged survival of animals bearing metastatic tumor cells. Adoptive immunotherapy also induced tumor immunoediting, resulting in tumor escape and associated disease-related mortality. To identify a tumor target that is incapable of escape from the immune response, we used dormant tumor cells. We used Adriamycin chemotherapy or radiation therapy, which simultaneously induce tumor cell death and tumor dormancy. Resultant dormant cells became refractory to additional doses of Adriamycin or radiation therapy, but they remained sensitive to tumor-reactive immune cells. Importantly, we discovered that dormant tumor cells contained indolent cells that expressed low levels of Ki67 and quiescent cells that were Ki67 negative. Whereas the former were prone to tumor immunoediting and escape, the latter did not demonstrate immunoediting. Our results suggest that immunotherapy could be highly effective against quiescent dormant tumor cells. The challenge is to develop combinatorial therapies that could establish a quiescent type of tumor dormancy, which would be the best target for immunotherapy

    Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella

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    Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS‚ÄČ+‚ÄČFliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responsesFil: Hiriart, Yanina. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Centro Cient√≠fico Tecnol√≥gico Conicet - La Plata. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos; ArgentinaFil: Rossi, Andr√©s Hugo. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Oficina de Coordinaci√≥n Administrativa Parque Centenario. Instituto de Investigaciones Bioqu√≠micas de Buenos Aires. Fundaci√≥n Instituto Leloir. Instituto de Investigaciones Bioqu√≠micas de Buenos Aires; ArgentinaFil: Biedma, Marina Elizabeth. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Centro Cient√≠fico Tecnol√≥gico Conicet - La Plata. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos; ArgentinaFil: Errea, Agustina Juliana. Universidad Nacional de La Plata; Argentina. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Centro Cient√≠fico Tecnol√≥gico Conicet - La Plata. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos; ArgentinaFil: Moreno, Griselda Noem√≠. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Centro Cient√≠fico Tecnol√≥gico Conicet - La Plata. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos; ArgentinaFil: Cayet, D.. Universidad Nacional de La Plata; ArgentinaFil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Oficina de Coordinaci√≥n Administrativa Parque Centenario. Instituto de Investigaciones Bioqu√≠micas de Buenos Aires. Fundaci√≥n Instituto Leloir. Instituto de Investigaciones Bioqu√≠micas de Buenos Aires; ArgentinaFil: Blanc√°, Bruno Martin. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Centro Cient√≠fico Tecnol√≥gico Conicet - La Plata. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos; ArgentinaFil: Sirard, J.C.. Centre National de la Recherche Scientifique; FranciaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Oficina de Coordinaci√≥n Administrativa Parque Centenario. Instituto de Investigaciones Bioqu√≠micas de Buenos Aires. Fundaci√≥n Instituto Leloir. Instituto de Investigaciones Bioqu√≠micas de Buenos Aires; ArgentinaFil: Berguer, Paula Mercedes. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Oficina de Coordinaci√≥n Administrativa Parque Centenario. Instituto de Investigaciones Bioqu√≠micas de Buenos Aires. Fundaci√≥n Instituto Leloir. Instituto de Investigaciones Bioqu√≠micas de Buenos Aires; ArgentinaFil: Rumbo, Mart√≠n. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Centro Cient√≠fico Tecnol√≥gico Conicet - La Plata. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunol√≥gicos y Fisiopatol√≥gicos; Argentin
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