Novel Immunogens Of Cellular Immunity Revealed Using In Vitro Human Cell-based Approach

In the last 150 years, tremendous headway has been made in our understanding of the human immune system. Pioneers in the field such as Paul Ehrlich, Elie Metchnikoff, Louis Pasteur, Robert Koch and Walter Reed carried out seminal studies that established the groundwork for our understanding of humoral and cellular immunity in humans. However, this direct line of evidence into human immunology was diverted in the mid-20th century with the adoption of a model which allowed for investigators to use a reductionist-approach with the promise to resolve immunity at a molecular level. This revolutionary model was the scientific commercialization of various inbred strains of mice. It seems inconceivable how a four-legged nocturnal rodent managed to become the focus of billions of dollars of research to improve our understanding of human immunity. Nevertheless, this strange surrogate for human immunity did provide us with major conceptual advances in areas, such as identification of dendritic cell population heterogeneity, T cell help for B cell antibody production, MHC-restricted recognition of virusinfected cells, and even the discovery of cell types like NKT cells. However, these prior advances have now been prefaced with decades-worth of disappointing, non-translational findings. The best examples of such disappointments are in murine models of autoimmunity, cancer immunotherapy, and vaccinology where numerous studies have revealed promising outcomes in mice but were met with failure or limited success upon translation into humans. We do not look at this as a failure of the murine model; rather we consider it a call to arms to innovate in vitro surrogates to examine human immunity when otherwise bound by ethical limitation from working directly in humans. To overcome these challenges, we developed a system to interrogate novel immunogens that begins by generating human dendritic cells (DCs), a cell type necessary to mounting a protective immune response. DCs for research and clinical applications are typically derived from purified blood monocytes that are cultured in a cocktail of cytokines for a week or more. Because it has been iv suggested that these cytokine-derived DCs may be deficient in some important immunological functions and might not accurately represent antigen presenting cell (APC) populations found under normal conditions in vivo, there is an interest in developing methods that permit the derivation of DCs in a more physiologically relevant manner in vitro. Here, we describe a simple and reliable technique for generating large numbers of highly purified DCs that is based on a one-way migration of blood monocytes through a layer of human umbilical vein endothelial cells (HUVECs) that are cultured to confluency in the upper chamber of a Transwell device. The resultant APCs, harvested from the lower Transwell chamber, resemble other cultured DC populations in their expression of major histocompatibility (MHC) and costimulatory molecules, ability to phagocytose protein antigens and capacity to trigger primary antigen-specific T cell responses. This technique offers several advantages over the standard method of in vitro cytokine-driven DC development, including: (1) the rapidity of this approach, as DC differentiation occurs in only 2 days, (2) the differentiation process itself, which is more akin to the development of DCs under physiologic conditions and (3) the cost effectiveness of the system, since no monocyte pre-selection is required and DC development occurs in the absence of expensive recombinant cytokines. Taken together, this approach allows for the exploration of novel immunogens utilizing a physiologically representative population of APCs enriched from circulating blood. The outbreak of the swine-origin H1N1 influenza in the spring of 2009 took epidemiologists, immunologists, and vaccinologists by surprise and galvanized a massive worldwide effort to produce millions of vaccine doses to protect against this single virus strain. Of particular concern was the apparent lack of pre-existing antibody capable of eliciting cross-protective immunity against this novel virus, which fueled fears this strain would trigger a particularly far-reaching and lethal pandemic. Given that disease caused by the swine-origin virus was far less severe than expected, we hypothesized v cellular immunity to cross-conserved T cell epitopes might have played a significant role in protecting against the pandemic H1N1 in the absence of cross-reactive humoral immunity. We collaborated with bioinformaticians to develop an immunoinformatics approach to predict CD4+ T cell epitopes conserved between the 2008-2009 seasonal H1N1 vaccine strain and pandemic H1N1 (A/California/04/2009) hemagglutinin proteins that could act as novel immunogens and function as potential vaccine candidates or compliments to current vaccine formulations. We examined these peptides using T cells from human donors not exposed to the pandemic virus demonstrating that pre-existing CD4+ T cells can elicit cross-reactive effector responses against the pandemic H1N1 virus. As well, we showed the computational tools created by our collaborators were 80-90% accurate in predicting CD4+ T cell epitopes and their HLA-DRB1-dependent response profiles in donors that were chosen at random for HLA haplotype. Combined, these results confirm the power of coupling immunoinformatics to define broadly reactive CD4+ T cell epitopes with a highly sensitive in vitro model to verify these in silico predictions as a means to understand human cellular immunity, including cross-protective responses, and to define CD4+ T cell epitopes for potential vaccination efforts against future influenza viruses and other pathogens. It is thought that utilizing highly conserved peptides as novel immunogens of cellular immunity for future vaccination strategies may require an adjuvant for efficacy. However, the FDA has approved the use of only two adjuvant compounds (Alum or MPL®) which may not be compatible or offer effective immune enhancement in novel vaccine preparations, thereby soliciting the need for novel adjuvants. Nanoparticles have since been a topic of adjuvant potential. Nanoparticles harbor great potential because they possess unique physicochemical properties compared to their larger counter parts as a result of quantum-size effects and their inherent large surface area to volume ratio. These physicochemical properties govern how a nanoparticle will behave in its environment. However, vi researchers have only just begun to catalogue the biological effect these properties illicit. Moreover, little is known about the interaction between the immune system and NPs. However, in light of the recent development in new adjuvants that involves composites and coatings of polymers, lipids, ligands, TLR agonist, the ability of a simple metal oxide nanopowder to effectively induce or couple immunomodulation would provide researchers a basic alternative to costly and complex adjuvant development. Considering the evidence suggesting NPs can act as immunopotentiators, we questioned whether these materials can act not only as innate adjuvants, but as novel immunogens to cellular immunity. To accomplish this, we under took a set of studies to investigate any nanoparticle size-induced effects using TiO2, one of the most widely manufactured nanoparticles, as a model. We explored titanium dioxide synthesized into its three most commonly nanoarchitectures: anatase (7-10 nm), rutile (15-20 nm), and nanotube (10-15 nm diameters, 70-150 nm length) in comparison to a micron-sized formulation. We used the fully human autologous MIMIC® immunological construct has been utilized as a predictive, nonanimal alternative to diagnose nanoparticle immunogenicity. Cumulatively, treatment with titanium dioxide nanoparticles in the MIMIC® system led to elevated levels of proinflammatory cytokines and increased maturation and expression of costimulatory molecules on dendritic cells. Additionally, these treatments effectively primed activation and proliferation of naïve CD4+ T cells in comparison to dendritic cells treated with titanium dioxide microspheres, characteristic of an in vivo inflammatory response, providing evidence of a size induced difference between the nano-sized and micron-sized material, revealing novel immune cell recognition and activation by a crystalline nanomaterial in a size-dependent manner. Having identified nanomaterial size as a contributing feature of nanoparticle induced immunopotentiation, we became interested if additional physicochemical properties such as surface vii reactivity or catalytic behavior could also be immunostimulatory. Moreover, because we witnessed a stimulatory effect to dendritic cells following nanoparticle treatment, we were curious how these nanoparticle-touched dendritic cells would impact adaptive immunity. Since TiO2 acts as an oxidant we chose an antioxidant nanoparticle, CeO2, as a counterpart to explore how divergent nanoparticle surface reactivity impacts innate and adaptive immunity. We focused on the effect these nanoparticles had on human dendritic cells and TH cells as a strategy towards defining their impact to cellular immunity. Combined, we report that TiO2 nanoparticles potentiate DC maturation inducing the secretion of IL- 12p70 and IL-1B, while treatment with CeO2 nanoparticles induced IL-10, a hallmark of suppression. When delivered to T cells alone TiO2 nanoparticles induced stronger proliferation in comparison to CeO2 which also stimulated TReg differentiation. When co-cultured in allogeneic T cell assays, the materials directed alternate TH polarization whereby TiO2 drives largely a TH1 dominate response, whereas CeO2 drove a TH2 bias. Combined, we report a novel immunomodulatory capacity of nanomaterials with catalytic activity. While unintentional exposure to these nanomaterials could pose a serious health risk, development and targeted use of such immunomodulatory nanoparticles could provide researchers with new tools for novel adjuvant strategies or therapeutics

Strategies to overcome the age-old problem of immunosenescence

Immunosenescence, which is the deterioration of the immune system that comes with age, hampers the development of protective immunity to vaccine antigens. The molecular underpinnings of this process remain poorly understood. Given that systems immunology approaches have been successfully applied towards identifying important molecular features driving productive innate and adaptive immune responses to different vaccines, our goal was to use these approaches to understand, predict, and eventually overcome immunosenescence. To this end, we coupled these systems immunology approaches with highly sensitive in vitro human immune assays to gain insights on specific gene targets that could be modulated to restore the age-dependent decline of immune function

Androgen regulates Cdc6 transcription through interactions between androgen receptor and E2F transcription factor in prostate cancer cells

Androgen receptor plays a critical role in the development and maintenance of cancers in the prostate. Earlier, we have shown that Cdc6, a regulatory protein for initiation of DNA replication, is down regulated in androgen-insensitive prostate cancer cells. In this report, we studied the involvement of androgen, mediated through androgen receptor (AR) in regulation of Cdc6 expression. Our results demonstrated that androgen treatment stimulated Cdc6 expression in xenograft tumors and androgen-sensitive prostate cancer cells. We also showed that androgen treatment stimulated Cdc6 transcription through possible interaction of AR with the ARE sequence in the Cdc6 promoter and that the stimulatory effect of androgen required intact E2F binding sites in the promoter. Androgen treatment differentially altered nuclear availability of E2F1 and E2F3, and increased the amount of hypophosphorylated retinoblastoma protein (pRb) in the nucleus in a time dependent fashion. We further showed that AR interacted with E2F transcription factors in a ligand-independent manner and that ligand-bound AR was less efficient in interacting with E2F proteins. DNA-protein interaction assays indicated that androgen treatment altered binding of E2F1 to the Cdc6 promoter in prostate cancer cells. We conclude that AR regulates Cdc6 transcription through interaction with the Cdc6 promoter, and complex formation with E2F1 and E2F3 in a differential manner. (c) 2008 Elsevier B.V. All rights reserved

Immunomodulation and T Helper TH1/TH2 Response Polarization by CeO2 and TiO2 Nanoparticles

Immunomodulation by nanoparticles, especially as related to the biochemical properties of these unique materials, has scarcely been explored. In an in vitro model of human immunity, we demonstrate two catalytic nanoparticles, TiO2 (oxidant) and CeO2 (antioxidant), have nearly opposite effects on human dendritic cells and T helper (T-H) cells. For example, whereas TiO2 nanoparticles potentiated DC maturation that led towards T(H)1-biased responses, treatment with antioxidant CeO2 nanoparticles induced APCs to secrete the anti-inflammatory cytokine, IL-10, and induce a T(H)2-dominated T cell profile. In subsequent studies, we demonstrate these results are likely explained by the disparate capacities of the nanoparticles to modulate ROS, since TiO2, but not CeO2 NPs, induced inflammatory responses through an ROS/inflammasome/IL-1 beta pathway. This novel capacity of metallic NPs to regulate innate and adaptive immunity in profoundly different directions via their ability to modulate dendritic cell function has strong implications for human health since unintentional exposure to these materials is common in modern societies

Impact of Trivalent Arsenicals on Selenoprotein Synthesis

BACKGROUND: Exposure to arsenic has been associated with development of skin, lung, bladder, liver, and kidney cancer. Recent evidence suggests that an increase in oxidative stress in cells treated with arsenicals represents the molecular mechanism behind arsenic-induced carcinogenesis. Selenium, in the form of selenocysteine, is necessary for the activity of several enzymes with a role in defense against reactive oxygen species. A mutual sparing effect between arsenic and selenium has been shown in animal studies when both metalloids are present in high concentrations. OBJECTIVES: To determine whether changes in selenoprotein synthesis may be an underlying mechanism behind arsenic-induced carcinogenesis, we analyzed the new synthesis of selenoproteins within cells after exposure to inorganic or methylated arsenicals using a human keratinocyte cell model. RESULTS: Addition of arsenite to culture medium blocked new synthesis of selenoproteins when selenium was present in the form of selenite, and appeared to stimulate the use of serum-derived selenium. Monomethylarsonous acid (MMA(III)) treatment of cells, in contrast, did not block all new synthesis of selenoproteins but did result in an increase in cytosolic thioredoxin reductase (TrxR1) at both the mRNA and protein levels. MMA(III) also reduced the new synthesis of cellular glutatione peroxidase (cGpx) and other smaller selenoproteins. Dimethylarsinous acid (DMA(III)) stimulated selenoprotein synthesis by an as yet unknown mechanism. CONCLUSIONS: These results suggest that arsenite and MMA(III) are key metabolites that trigger higher levels of TrxR1, and both lead to a reduction in the expression of cGpx. Together these effects certainly could lead to carcinogenesis given the knowledge that many cancers have higher levels of TrxR, and reduced Gpx levels will reduce the cell’s ability to defend against reactive oxygen species. Based on these results, the impact of the trivalent arsenicals arsenite and MMA(III) on selenoprotein synthesis may indeed represent a potential molecular mechanism for the higher rates of cancer observed in populations exposed to high levels of arsenic

Immunomodulation and T Helper TH1/TH2 Response Polarization by CeO2 and TiO2 Nanoparticles

Immunomodulation by nanoparticles, especially as related to the biochemical properties of these unique materials, has scarcely been explored. In an in vitro model of human immunity, we demonstrate two catalytic nanoparticles, TiO2 (oxidant) and CeO2 (antioxidant), have nearly opposite effects on human dendritic cells and T helper (T-H) cells. For example, whereas TiO2 nanoparticles potentiated DC maturation that led towards T(H)1-biased responses, treatment with antioxidant CeO2 nanoparticles induced APCs to secrete the anti-inflammatory cytokine, IL-10, and induce a T(H)2-dominated T cell profile. In subsequent studies, we demonstrate these results are likely explained by the disparate capacities of the nanoparticles to modulate ROS, since TiO2, but not CeO2 NPs, induced inflammatory responses through an ROS/inflammasome/IL-1 beta pathway. This novel capacity of metallic NPs to regulate innate and adaptive immunity in profoundly different directions via their ability to modulate dendritic cell function has strong implications for human health since unintentional exposure to these materials is common in modern societies

Coupling sensitive \u3cem\u3ein vitro\u3c/em\u3e and in silico techniques to assess cross-reactive CD4\u3csup\u3e+\u3c/sup\u3e T cells against the swine-origin H1N1 influenza virus

The outbreak of the novel swine-origin H1N1 influenza in the spring of 2009 took epidemiologists, immunologists, and vaccinologists by surprise and galvanized a massive worldwide effort to produce millions of vaccine doses to protect against this single virus strain. Of particular concern was the apparent lack of pre-existing antibody capable of eliciting cross-protective immunity against this novel virus, which fueled fears this strain would trigger a particularly far-reaching and lethal pandemic. Given that disease caused by the swine-origin virus was far less severe than expected, we hypothesized cellular immunity to cross-conserved T cell epitopes might have played a significant role in protecting against the pandemic H1N1 in the absence of cross-reactive humoral immunity. In a published study, we used an immunoinformatics approach to predict a number of CD4+ T cell epitopes are conserved between the 2008–2009 seasonal H1N1 vaccine strain and pandemic H1N1 (A/California/04/2009) hemagglutinin proteins. Here, we provide results from biological studies using PBMCs from human donors not exposed to the pandemic virus to demonstrate that pre-existing CD4+ T cells can elicit cross-reactive effector responses against the pandemic H1N1 virus. As well, we show our computational tools were 80–90% accurate in predicting CD4+ T cell epitopes and their HLA-DRB1-dependent response profiles in donors that were chosen at random for HLA haplotype. Combined, these results confirm the power of coupling immunoinformatics to define broadly reactive CD4+ T cell epitopes with highly sensitive in vitro biological assays to verify these in silico predictions as a means to understand human cellular immunity, including cross-protective responses, and to define CD4+ T cell epitopes for potential vaccination efforts against future influenza viruses and other pathogens

A bird can not set my agenda : the role of source types on credibility in agenda setting on Twitter

This file was last viewed in Microsoft Edge.The literature on McCombs and Shaw's 1972 theory of agenda setting has a robust history with over 425 empirical studies as of McCombs and Reynolds 2009 review. The rise of social media has allowed scholarship on agenda setting to expand. Research, however, has not examined whether agenda setting and credibility vary based on the Twitter source. An 18-day experiment was conducted at a mid-sized Midwestern university to examine the agenda setting effects of three different Twitter sources based on Manheim and Albritton's 1984 three agenda conceptualization: media, government and peer. Results demonstrated no agenda setting effects; however, a mock government organization was found as more credible than a mock news organization or peer. This indicates a shift in credibility paradigms of the early 21st century, as political figures and media outlets combat each other's credibility. These findings warrant further examination of the credibility of different sources on Twitter, and how they fit into the broader media landscape

Transcriptional Regulation Of Mammalian Mirna Genes

MicroRNAs (miRNAs) are members of a growing family of non-coding transcripts, 21-23 nucleotides long, which regulate a diverse collection of biological processes and various diseases by RNA-mediated gene-silencing mechanisms. While currently many studies focus on defining the regulatory functions of miRNAs, few are directed towards how miRNA genes are themselves transcriptionally regulated. Recent studies of miRNA transcription have elucidated RNA polymerase II as the major polymerase of miRNAs, however, little is known of the structural features of miRNA promoters, especially those of mammalian miRNAs. Here, we review the current literature regarding features conserved among miRNA promoters useful for their detection and the current novel methodologies available to enable researchers to advance our understanding of the transcriptional regulation of miRNA genes. © 2010 Elsevier Inc

Functional Characterisation Of The Regulatory Subunit Of Cyclic Amp-Dependent Protein Kinase A Homologue Of Giardia Lamblia: Differential Expression Of The Regulatory And Catalytic Subunits During Encystation

To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3′5′-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation. © 2006 Australian Society for Parasitology Inc