Modulation of Human Immune Responses by Bovine Interleukin-10

Cytokines can be functionally active across species barriers. Bovine IL-10 has an amino acid sequence identity with human IL-10 of 76.8%. Therefore, the aim of this study was to evaluate whether bovine IL-10 has immunomodulatory activities on human monocytes and dendritic cells. Peripheral blood monocytes were isolated from healthy donors, and used directly or allowed to differentiate to dendritic cells under the influence of IL-4 and GM-CSF. Recombinant bovine IL-10 inhibited TLR induced activation of monocytes, and dose-dependently inhibited LPS-induced activation of monocyte-derived DCs comparable to human IL-10. By using blocking antibodies to either bovine IL-10 or the human IL-10 receptor it was demonstrated that inhibition of monocyte activation by bovine IL-10 was dependent on binding of bovine IL-10 to the human IL-10R. These data demonstrate that bovine IL-10 potently inhibits the activation of human myeloid cells in response to TLR activation. Bovine IL-10 present in dairy products may thus potentially contribute to the prevention of necrotizing enterocolitis and allergy, enhance mucosal tolerance induction and decrease intestinal inflammation and may therefore be applicable in infant foods and in immunomodulatory diets

震災豫防調査會報告第十八號正誤

<p>Insect bite hypersensitivity (IBH), which is a cutaneous allergic reaction to antigens from Culicoides spp., is the most prevalent skin disorder in horses. Misdiagnosis is possible, as IBH is usually diagnosed based on clinical signs. Our study is the first to employ IgE levels against several recombinant Culicoides spp. allergens as an objective, independent, and quantitative phenotype to improve the power to detect genetic variants that underlie IBH. Genotypes of 200 Shetland ponies, 127 Icelandic horses, and 223 Belgian Warmblood horses were analyzed while using a mixed model approach. No single-nucleotide polymorphism (SNP) passed the Bonferroni corrected significance threshold, but several regions were identified within and across breeds, which confirmed previously identified regions of interest and, in addition, identifying new regions of interest. Allergen-specific IgE levels are a continuous and objective phenotype that allow for more powerful analyses when compared to a case-control set-up, as more significant associations were obtained. However, the use of a higher density array seems necessary to fully employ the use of IgE levels as a phenotype. While these results still require validation in a large independent dataset, the use of allergen-specific IgE levels showed value as an objective and continuous phenotype that can deepen our understanding of the biology underlying IBH.</p

CXCL8 Chemokines in Teleost Fish: Two Lineages with Distinct Expression Profiles during Early Phases of Inflammation

Contains fulltext : 126584.pdf (publisher's version ) (Open Access

Cyprinid Herpesvirus 3 Il10 Inhibits Inflammatory Activities of Carp Macrophages and Promotes Proliferation of Igm+ B Cells and Memory T Cells in a Manner Similar to Carp Il10.

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease of carp and encodes for an Il10 homolog (ORF134). Our previous studies with a recombinant ORF134-deleted strain and the derived revertant strain suggested that cyprinid herpesvirus 3 Il10 (CyHV-3 Il10 [cyhv3Il10]) is not essential for viral replication in vitro, or virulence in vivo. In apparent contrast, cyhv3Il10 is one of the most abundant proteins of the CyHV-3 secretome and is structurally very similar to carp Il10 and also human IL10. To date, studies addressing the biological activity of cyhv3Il10 on cells of its natural host have not been performed. To address the apparent contradiction between the presence of a structurally conserved Il10 homolog in the genome of CyHV-3 and the lack of a clear phenotype in vivo using recombinant cyhv3Il10-deleted viruses, we used an in vitro approach to investigate in detail whether cyhv3Il10 exerts any biological activity on carp cells. In this study, we provide direct evidence that cyhv3Il10 is biologically active and, similarly to carp Il10, signals via a conserved Stat3 pathway modulating immune cells of its natural host, carp. In vitro, cyhv3Il10 deactivates phagocytes with a prominent effect on macrophages, while also promoting proliferation of Igm(+) B cells and memory T cells. Collectively, this study demonstrates a clear biological activity of cyhv3Il10 on cells of its natural host and indicates that cyhv3Il10 is a true viral ortholog of carp Il10. Furthermore, to our knowledge, this is the first report on biological activities of a nonmammalian viral Il10 homolog

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions

Differential immunomodulation of porcine bone marrow derived dendritic cells by E. coli Nissle 1917 and β-glucans.

In early life and around weaning, pigs are at risk of developing infectious diseases which compromise animal welfare and have major economic consequences for the pig industry. A promising strategy to enhance resistance against infectious diseases is immunomodulation by feed additives. To assess the immune stimulating potential of feed additives in vitro, bone marrow-derived dendritic cells were used. These cells play a central role in the innate and adaptive immune system and are the first cells encountered by antigens that pass the epithelial barrier. Two different feed additives were tested on dendritic cells cultured from fresh and cryopreserved bone marrow cells; a widely used commercial feed additive based on yeast-derived β-glucans and the gram-negative probiotic strain E. coli Nissle 1917. E. coli Nissle 1917, but not β-glucans, induced a dose-dependent upregulation of the cell maturation marker CD80/86, whereas both feed additives induced a dose-dependent production of pro- and anti-inflammatory cytokines, including TNFα, IL-1β, IL-6 and IL-10. Furthermore, E. coli Nissle 1917 consistently induced higher levels of cytokine production than β-glucans. These immunomodulatory responses could be assessed by fresh as well as cryopreserved in vitro cultured porcine bone marrow-derived dendritic cells. Taken together, these results demonstrate that both β-glucans and E. coli Nissle 1917 are able to enhance dendritic cell maturation, but in a differential manner. A more mature dendritic cell phenotype could contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved bone marrow-derived dendritic cells can be used as in vitro pre-screening tools which enable an evidence based prediction of the potential immune stimulating effects of different feed additives

Establishment and characterization of canine parvovirus-specific murine CD4+ T cell clones and their use for the delineation of T cell epitopes.

Canine parvovirus (CPV)-specific T cell clones were generated by culturing lymph node cells from CPV-immunized BALB/c mice at limiting dilutions in the presence of CPV antigen and interleukin-2 (IL-2). All isolated T cell clones exhibited the cell surface phenotype Thy1+, CD4+, CD8- and proliferated specifically in response to CPV antigen. After stimulation with CPV antigen in culture the T cell clones produced IL-2 and proliferated in the absence of exogenous IL-2. Naive mice to which CPV-specific T cell clones had been adoptively transferred developed a CPV-specific delayed type hypersensitivity reaction upon simultaneous intracutaneous injection of CPV in their ears. The ability of recombinant viral fusion proteins, representing the VP2 capsid protein of the antigenically closely related feline panleukopenia virus and of synthetic peptides derived from the amino acid sequence of the VP2 of CPV, to stimulate these T cell clones enabled the identification of T cell epitopes

Systematic Comparison of Zwitterionic and Non-Zwitterionic Antifouling Polymer Brushes on a Bead-Based Platform

Nonspecific adsorption of biomolecules to solid surfaces, a process called biofouling, is a major concern in many biomedical applications. Great effort has been made in the development of antifouling polymer coatings that are capable of repelling the nonspecific adsorption of proteins, cells, and micro-organisms. In this respect, we herein contribute to understanding the factors that determine which polymer brush results in the best antifouling coating. To this end, we compared five different monomers: two sulfobetaines, a carboxybetaine, a phosphocholine, and a hydroxyl acrylamide. The antifouling coatings were analyzed using our previously described bead-based method with flow cytometry as the read-out system. This method allows for the quick and automated analysis of thousands of beads per second, enabling fast analysis and good statistics. We report the first direct comparison made between a sulfobetaine with opposite charges separated by two and three methylene groups and a carboxybetaine bearing two separating methylene groups. It was concluded that both the distance between opposite charges and the nature of the anionic groups have a distinct effect on the antifouling performance. Phosphocholines and simple hydroxyl acrylamides are not often compared with the betaines. However, here we found that they perform equally well or even better, yielding the following overall antifouling ranking: HPMAA ≥ PCMA-2 ≈ CBMAA-2 > SBMAA-2 > SBMAA-3 ≫ nonmodified beads (HPMAA being the best).</p

Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi

Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi-infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity