Longitudinal analysis of allergen-specific IgE and IgG subclasses as potential predictors of insect bite hypersensitivity following first exposure to Culicoides in Icelandic horses.

BACKGROUND: Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland because of the absence of Culicoides, but the prevalence is high in horses imported from Iceland to environments where Culicoides are present. HYPOTHESIS/OBJECTIVE: Test, in a longitudinal study before and after Culicoides exposure, whether a primary sensitizing Culicoides allergen can be identified and if an increase of allergen-specific immunoglobulin (Ig)E or IgG subclasses precedes clinical signs of IBH. ANIMALS: Thirty two horses imported from Iceland to Europe; 16 developed IBH and 16 remained healthy. METHODS: Determination of IgE and IgG subclasses against recombinant (r)-Culicoides allergens and Culicoides extract in sera taken before first exposure to Culicoides and yearly over a period of 3-4 years. RESULTS: Before Culicoides exposure, there were no significant differences in Culicoides-specific serum IgE levels between horse that developed IBH or remained healthy. Culicoides exposure induced an individual IgE response pattern (to a median of 4.5 r-allergens) in the IBH but not in the healthy end-point group. The increase in serum IgE levels to Culicoides r-allergens was concurrent with the initial onset of clinical signs of IBH. IBH-affected horses displayed significantly higher allergen-specific IgG1 and IgG5 levels than healthy controls. Recombinant Culicoides obsoletus 1 (Cul o1) and Cul o3-specific IgG5 was significantly higher in the IBH compared to the healthy end-point group, before clinical signs of IBH. CONCLUSION/CLINICAL RELEVANCE: Allergen-specific serum IgE cannot be used as predictor for IBH, whereas allergen-specific IgG5 levels may have a predictive value.

Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum.

BACKGROUND: Dendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including horses. Currently, the gold standard protocol for generating dendritic cells from monocytes across various species relies upon a combination of GM-CSF and IL-4 added to cell culture medium which is supplemented with FBS. The aim of this study was to substitute FBS with heterologous horse serum. For this purpose, equine monocyte-derived dendritic cells (eqMoDC) were generated in the presence of horse serum or FBS and analysed for the effect on morphology, phenotype and immunological properties. Changes in the expression of phenotypic markers (CD14, CD86, CD206) were assessed during dendritic cell maturation by flow cytometry. To obtain a more complete picture of the eqMoDC differentiation and assess possible differences between FBS- and horse serum-driven cultures, a transcriptomic microarray analysis was performed. Lastly, immature eqMoDC were primed with a primary antigen (ovalbumin) or a recall antigen (tetanus toxoid) and, after maturation, were co-cultured with freshly isolated autologous CD5+ T lymphocytes to assess their T cell stimulatory capacity. RESULTS: The microarray analysis demonstrated that eqMoDC generated with horse serum were indistinguishable from those generated with FBS. However, eqMoDC incubated with horse serum-supplemented medium exhibited a more characteristic dendritic cell morphology during differentiation from monocytes. A significant increase in cell viability was also observed in eqMoDC cultured with horse serum. Furthermore, eqMoDC generated in the presence of horse serum were found to be superior in their functional T lymphocyte priming capacity and to elicit significantly less non-specific proliferation. CONCLUSIONS: EqMoDC generated with horse serum-supplemented medium showed improved morphological characteristics, higher cell viability and exhibited a more robust performance in the functional T cell assays. Therefore, horse serum was found to be superior to FBS for generating equine monocyte-derived dendritic cells.

An allergen-fused dendritic cell-binding peptide enhances in vitro proliferation of equine T-cells and cytokine production.

Allergen-specific immunotherapy (AIT) constitutes the only curative approach for allergy treatment. There is need for improvement of AIT in veterinary medicine, such as in horses suffering from insect bite hypersensitivity, an IgE-mediated dermatitis to Culicoides. Dendritic cell (DC)-targeting represents an efficient method to increase antigen immunogenicity. It is studied primarily for its use in improvement of cancer therapy and vaccines, but may also be useful for improving AIT efficacy. Immunomodulators, like the Toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid-A (MPLA) has been shown to enhance the IL-10 response in horses, while CpG-rich oligonucleotides (CpG-ODN), acting as TLR-9 agonists, have been shown to induce Th1 or regulatory responses in horses with equine asthma. Our aim was to evaluate in vitro effects of antigen-targeting to equine DC with an antigen-fused peptide known to target human and mouse DC and investigate whether addition of MPLA or CpG-ODN would further improve the induced immune response with regard to finding optimal conditions for equine AIT. For this purpose, DC-binding peptides were fused to the model antigen ovalbumin (OVA) and to the recombinant Culicoides allergen Cul o3. Effects of DC-binding peptides on cellular antigen uptake and induction of T cell proliferation were assessed. Polarity of the immune response was analysed by quantifying IFN-γ, IL-4, IL-10, IL-17 and IFN-α in supernatants of antigen-stimulated peripheral blood mononuclear cells (PBMC) in presence or absence of adjuvants. Fusion of DC-binding peptides to OVA significantly enhanced antigen-uptake by equine DC. DC primed with DC-binding peptides coupled to OVA or Cul o3 induced a significantly higher T-cell proliferation compared to the corresponding control antigens. PBMC stimulation with DC-binding peptides coupled to Cul o3 elicited a significant increase in the pro-inflammatory cytokines IFN-γ, IL-4, IL-17, as well as the anti-inflammatory IL-10, but not of IFN-α. Adjuvant addition further enhanced the effect of the DC-binding peptides by significantly increasing the production of IFN-γ, IL-4, IL-10 and IFN-α (CpG-ODN) and IL-10 (MPLA), while simultaneously suppressing IFN-γ, IL-4 and IL-17 production (MPLA). Targeting equine DC with allergens fused to DC-binding peptides enhances antigen-uptake and T-cell activation and may be useful in increasing the equine immune response against recombinant antigens. Combination of DC-binding peptide protein fusions with adjuvants is necessary to appropriately skew the resulting immune response, depending on intended use. Combination with MPLA is a promising option for improvement of AIT efficacy in horses, while combination with CpG-ODN increases the effector immune response to recombinant antigens.

First clinical expression of equine insect bite hypersensitivity is associated with co-sensitization to multiple Culicoides allergens.

BACKGROUND: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis in horses incited by salivary allergens from Culicoides spp. IBH does not occur in Iceland, as the causative agents are absent, however a high prevalence is seen in horses exported to Culicoides-rich environments. AIMS: To study the natural course of sensitization to Culicoides allergens and identify the primary sensitizing allergen(s) in horses exported from Iceland utilizing a comprehensive panel of Culicoides recombinant (r-) allergens. METHOD: IgE microarray profiling to 27 Culicoides r-allergens was conducted on 110 serological samples from horses imported to Switzerland from Iceland that subsequently developed IBH or remained healthy. Furthermore, a longitudinal study of 31 IBH horses determined IgE profiles the summer preceding first clinical signs of IBH (TIBH-1), the summer of first clinical signs (TIBH) and the following summer (TIBH+1). In a group of Icelandic horses residing in Sweden, effects of origin (born in Iceland or Sweden) and duration of IBH (<4 years, 4-7 years, >7 years) on Culicoides-specific IgE was evaluated. Sero-positivity rates and IgE levels were compared. RESULTS: At TIBH, horses were sensitized to a median of 11 r-allergens (range = 0-21), of which nine were major allergens. This was significantly higher than TIBH-1 (3, 0-16), as well as the healthy (1, 0-14) group. There was no significant increase between TIBH and TIBH+1(12, 0-23). IBH-affected horses exported from Iceland had a significantly higher degree of sensitization than those born in Europe, while duration of IBH did not significantly affect degree of sensitization. CONCLUSION: Significant sensitization is only detected in serum the year of first clinical signs of IBH. Horses become sensitized simultaneously to multiple Culicoides r-allergens, indicating that IgE-reactivity is due to co-sensitization rather than cross-reactivity between Culicoides allergens. Nine major first sensitizing r-allergens have been identified, which could be used for preventive allergen immunotherapy.

In vivo phosphoenolpyruvate carboxykinase promoter mapping identifies disrupted hormonal synergism as a target of inflammation during sepsis in mice.

UNLABELLED: In mammals, proper maintenance of blood glucose levels within narrow limits is one of the most critical prerequisites for healthy energy homeostasis and body function. Consequently, hyper- and hypoglycemia represent hallmarks of severe metabolic pathologies, including type II diabetes and acute sepsis, respectively. Although the liver plays a crucial role in the control of systemic glucose homeostasis, the molecular mechanisms of aberrant hepatic glucose regulation under metabolic stress conditions remain largely unknown. Here we report the development of a liver-specific adenoviral in vivo system for monitoring promoter activity of the key gluconeogenic enzyme gene phosphoenolpyruvate carboxykinase (PEPCK) in mice. By employing in vivo promoter deletion technology, the glucocorticoid response unit (GRU) and the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) were identified as critical cis-regulatory targets of proinflammatory signaling under septic conditions. In particular, both elements were found to be required for inhibition of PEPCK transcription during sepsis, thereby mediating endotoxic hypoglycemia. Indeed, expression of nuclear receptor cofactor peroxisome proliferator-activator receptor coactivator 1alpha (PGC-1alpha), the molecular mediator of GRU/CRE synergism on the PEPCK promoter, was found to be specifically repressed in septic liver, and restoration of PGC-1alpha in cytokine-exposed hepatocytes blunted the inhibitory effect of proinflammatory signaling on PEPCK gene expression. CONCLUSION: The dysregulation of hormonal synergism through the repression of PGC-1alpha as identified by in vivo promoter monitoring may provide a molecular rationale for hypoglycemia during sepsis, thereby highlighting the importance of hepatic glucose homeostasis for metabolic dysfunction in these patients.

Candidatus Amarolinea and Candidatus Microthrix Are Mainly Responsible for Filamentous Bulking in Danish Municipal Wastewater Treatment Plants.

Filamentous bulking is a common serious operational problem leading to deteriorated sludge settling that has long been observed in activated sludge biological wastewater treatment systems. A number of bacterial genera found therein possess filamentous morphology, where some have been shown to be implicated in bulking episodes (e.g., Ca. Microthrix), the impact of many others is still not clear. In this study we performed a survey of 17 Danish municipal wastewater treatment plants (WWTPs) with nutrient removal using 16S rRNA amplicon sequencing over a period of 13 years, where all known filamentous bacteria from 30 genera were analyzed. The filamentous community constituted on average 13 ± 6%, and up to 43% of total read abundance with the same genera common to all plants. Ca. Microthrix and several genera belonging to phylum Chloroflexi were among the most abundant filamentous bacteria. The effect of filamentous bacteria on sludge settling properties was analyzed using measurements of the diluted sludge volume index (DSVI). Strong positive correlations with DSVI were observed only for Ca. Microthrix and Ca. Amarolinea, the latter being a novel, recently characterized genus belonging to the phylum Chloroflexi. The bulking potential of other filamentous bacteria was not significant despite their presence in many plants. Low phylogenetic diversity was observed for both Ca. Microthrix and Ca. Amarolinea, making physiological characterization of individual species and potential development of control strategies more feasible. In this study we show that, despite the high diversity of filamentous phylotypes in Danish WWTPs, only few of them were responsible for severe bulking episodes.

Nuclear receptor cofactor receptor interacting protein 140 controls hepatic triglyceride metabolism during wasting in mice.

UNLABELLED: In mammals, triglycerides (TG) represent the most concentrated form of energy. Aberrant TG storage and availability are intimately linked to the negative energy balance under severe clinical conditions, such as starvation, sepsis, or cancer cachexia. Despite its crucial role for energy homeostasis, molecular key determinants of TG metabolism remain enigmatic. Here we show that the expression of nuclear receptor cofactor receptor interacting protein (RIP) 140 was induced in livers of starved, septic, and tumor-bearing mice. Liver-specific knockdown of RIP140 led to increased hepatic TG release and alleviated hepatic steatosis in tumor-bearing, cachectic animals. Indeed, hepatic RIP140 was found to control the expression of lipid-metabolizing genes in liver. CONCLUSION: By preventing the mobilization of hepatic TG stores, the induction of RIP140 in liver provides a molecular rationale for hepatic steatosis in starvation, sepsis, or cancer cachexia. Inhibition of hepatic RIP140 transcriptional activity might, thereby, provide an attractive adjunct scheme in the treatment of these conditions.

Identification and characterization of equine blood plasmacytoid dendritic cells.

Dendritic cells (DC) are antigen-presenting cells that can be classified into three major cell subsets: conventional DC1 (cDC1), cDC2 and plasmacytoid DCs (pDC), none of which have been identified in horses. Therefore, the objective of this study was to identify and characterize DC subsets in equine peripheral blood, emphasizing on pDC. Surface marker analysis allowed distinction of putative DC subsets, according to their differential expression of CADM-1 and MHC class II. Equine pDC were found to be Flt3(+) CD4(low) CD13(-) CD14(-) CD172a(-) CADM-1(-) MHCII(low). The weak expression of CD4 on equine pDC contrasts with findings in several other mammals. Furthermore, pDC purified by fluorescence-activated cell sorting were found to be the only cell subset able to produce large amounts of IFN-α upon TLR9-agonist stimulation. The pDC identity was confirmed by demonstrating high-levels of PLAC8, RUNX2 and TCF4 expression, showing pDC-restricted expression in other mammals.

Dynamics of the Fouling Layer Microbial Community in a Membrane Bioreactor.

Membrane fouling presents the greatest challenge to the application of membrane bioreactor (MBR) technology. Formation of biofilms on the membrane surface is the suggested cause, yet little is known of the composition or dynamics of the microbial community responsible. To gain an insight into this important question, we applied 16S rRNA gene amplicon sequencing with a curated taxonomy and fluorescent in situ hybridization to monitor the community of a pilot-scale MBR carrying out enhanced biological nitrogen and phosphorus removal with municipal wastewater. In order to track the dynamics of the fouling process, we concurrently investigated the communities of the biofilm, MBR bulk sludge, and the conventional activated sludge system used to seed the MBR system over several weeks from start-up. As the biofilm matured the initially abundant betaproteobacterial genera Limnohabitans, Hydrogenophaga and Malikia were succeeded by filamentous Chloroflexi and Gordonia as the abundant species. This study indicates that, although putative pioneer species appear, the biofilm became increasingly similar to the bulk community with time. This suggests that the microbial population in bulk water will largely determine the community structure of the mature biofilm.

Back to Basics--The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities.

DNA extraction and primer choice have a large effect on the observed community structure in all microbial amplicon sequencing analyses. Although the biases are well known, no comprehensive analysis has been conducted in activated sludge communities. In this study we systematically explored the impact of a number of parameters on the observed microbial community: bead beating intensity, primer choice, extracellular DNA removal, and various PCR settings. In total, 176 samples were subjected to 16S rRNA amplicon sequencing, and selected samples were investigated through metagenomics and metatranscriptomics. Quantitative fluorescence in situ hybridization was used as a DNA extraction-independent method for qualitative comparison. In general, an effect on the observed community was found on all parameters tested, although bead beating and primer choice had the largest effect. The effect of bead beating intensity correlated with cell-wall strength as seen by a large increase in DNA from Gram-positive bacteria (up to 400%). However, significant differences were present at lower phylogenetic levels within the same phylum, suggesting that additional factors are at play. The best primer set based on in silico analysis was found to underestimate a number of important bacterial groups. For 16S rRNA gene analysis in activated sludge we recommend using the FastDNA SPIN Kit for Soil with four times the normal bead beating and V1-3 primers.

The glucocorticoid receptor controls hepatic dyslipidemia through Hes1.

Aberrant accumulation of lipids in the liver ("fatty liver" or hepatic steatosis) represents a hallmark of the metabolic syndrome and is tightly associated with obesity, type II diabetes, starvation, or glucocorticoid (GC) therapy. While fatty liver has been connected with numerous abnormalities of liver function, the molecular mechanisms of fatty liver development remain largely enigmatic. Here we show that liver-specific disruption of glucocorticoid receptor (GR) action improves the steatotic phenotype in fatty liver mouse models and leads to the induction of transcriptional repressor hairy enhancer of split 1 (Hes1) gene expression. The GR directly interferes with Hes1 promoter activity, triggering the recruitment of histone deacetylase (HDAC) activities to the Hes1 gene. Genetic restoration of hepatic Hes1 levels in steatotic animals normalizes hepatic triglyceride (TG) levels. As glucocorticoid action is increased during starvation, myotonic dystrophy, and Cushing's syndrome, the inhibition of Hes1 through the GR might explain the fatty liver phenotype in these subjects.

Signal-dependent control of gluconeogenic key enzyme genes through coactivator-associated arginine methyltransferase 1.

Together with impaired glucose uptake in skeletal muscle, elevated hepatic gluconeogenesis is largely responsible for the hyperglycemic phenotype in type II diabetic patients. Intracellular glucocorticoid and cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent signaling pathways contribute to aberrant hepatic glucose production through the induction of gluconeogenic enzyme gene expression. Here we show that the coactivator-associated arginine methyltransferase 1 (CARM1) is required for cAMP-mediated activation of rate-limiting gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and glucose-6-phosphatase genes. Mutational analysis showed that CARM1 mediates its effect via the cAMP-responsive element within the PEPCK promoter, which is identified here as a CARM1 target in vivo. In hepatocytes, endogenous CARM1 physically interacts with cAMP-responsive element binding factor CREB and is recruited to the PEPCK and glucose-6-phosphatase promoters in a cAMP-dependent manner associated with increased promoter methylation. CARM1 might, therefore, represent a critical component of cAMP-dependent glucose metabolism in the liver.