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Abstract Background In dogs with idiopathic epilepsy (IE), 33% develop resistance to conventional anti‐seizure medication (ASM) despite adequate treatment. In human medicine, an immune‐mediated etiology is suspected in a subset of ASM‐resistant patients with epilepsy and cerebrospinal fluid (CSF)‐specific immunoglobulin G (IgG)‐type oligoclonal bands (OCBs) have been detected. In dogs, cases of autoimmune encephalitis recently were reported. Neuroinflammation may provide an additional explanation for the lack of response of certain dogs with IE to ASM. Hypothesis Cerebrospinal fluid‐specific OCBs are found in a subgroup of dogs with ASM‐resistant IE. Animals Eighty‐four dogs with IE were recruited from 3 referral centers and classified based on their response to ASM treatment (responsive, n = 56; resistant, n = 28). Methods Detection of OCBs was performed using isoelectric focusing (IEF) followed by immunoblotting. Associations of CSF‐specific OCBs with seizure type, severity, and response to ASM were calculated using logistic regression models. Results The overall frequency of CSF‐specific OCBs in dogs with IE was 15.5% (95% confidence interval [CI], 8.5%‐25%). In dogs with ASM‐resistant IE, 21.4% (6/28) had CSF‐specific OCBs compared with only 12.5% (7/56) in those responsive to ASM, but no evidence of an association was detected (odds ratio, 1.9; 95% CI, 0.57‐6.35; P = .29). Conclusions and Clinical Importance Cerebrospinal fluid‐specific OCBs were detected in a subgroup of dogs with IE. This finding could indicate that intrathecal IgG synthesis as a sign of neuroinflammation may play a role in disease pathogenesis.

IntroductionInsect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis of horses caused by bites of Culicoides spp., sharing some common features with human atopic dermatitis. Allergen immunotherapy (AIT) using Culicoides whole-body extracts has limited efficacy. This study aimed to evaluate AIT with a pool of major Culicoides recombinant allergens in a prospective, double-blinded, placebo-controlled study.MethodsThe IBH lesion score was assessed during a pre-treatment year and first treatment year (May–October) in 17 horses and in May and July of a second treatment year. Nine horses were immunized subcutaneously 3× with a combination of nine r-allergens (20 μg each/injection) in alum and monophosphoryl lipid A (MPLA). Eight horses received a placebo. The immunization was repeated twice the following year. The specific antibody response to one of the AIT Culicoides r-allergens was assessed.ResultsIn the first treatment year, the decrease in average IBH lesion score was significantly larger in the AIT compared to the placebo group, with 67% of the AIT group and 25% of the placebo horses reaching >50% improvement of the average IBH lesion score. The response to the AIT was enhanced in the 2nd treatment year when 89% of the AIT vs. 14% of the placebo horses showed an improvement (p ≤ 0.01). IgG antibodies of all subclasses were induced, with IgG4/7 showing the most significant differences between groups. The post-AIT sera showed IgE blocking activity.DiscussionAIT using only a few injections of small amounts of r-allergens in alum and MPLA as immunomodulators seems a promising approach for the treatment of insect bite allergy.

Abstract Background Severe asthma (SA) in horses, resembling human asthma, is a prevalent, debilitating allergic respiratory condition marked by elevated allergen‐specific immunoglobulin E (IgE) against environmental proteins; however, research exploring the exposome's influence on IgE profiles is currently limited but holds paramount significance for diagnostic and therapeutic developments. Animals Thirty‐five sports horses were analyzed, consisting of environmentally matched samples from France (5 SA; 6 control), the United States (6 SA; 6 control), and Canada (6 SEA; 6 control). Methods This intentional cross‐sectional study investigated the sensitization profiles of SA‐affected and healthy horses via serological antigen microarray profiling. Partial least square‐discriminant analysis (PLS‐DA) was used to identify and rank the importance of allergens for class separation (ie, affected/non‐affected) as variable influence of projection (VIP), and allergen with commonality internationally established via frequency analysis. Results PLS‐DA models showed high discriminatory power in predicting SA in horses from Canada (area under the curve [AUC] 0.995) and France (AUC 0.867) but poor discriminatory power in horses from the United States (AUC 0.38). Hev b 5.0101, Cyn D, Der p 2, and Rum cr were the only shared allergens across all geographical groups. Conclusions and Clinical Importance Microarray profiling can identify specific allergenic components associated with SA in horses, while mathematical modeling of this data can be used for disease classification, highlighting the variability of sensitization profiles between geographical locations and emphasizing the importance of local exposure to the prevalence of different allergens. Frequency scoring analysis can identify important variables that contribute to the classification of SA across different geographical regions.

IntroductionSevere equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA.MethodsAiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses.Results and discussionFor 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.

Abstract Background The diagnostic value of allergen‐specific immunoglobulin E (IgE) in horses with asthma is uncertain. A recently developed protein microarray detected abnormally high latex‐specific IgE concentrations in the serum of horses with severe asthma. Objectives The main objective was to characterize the IgE profiles of asthmatic horses in Switzerland using a protein microarray platform in serum and bronchoalveolar lavage fluid (BALF). The secondary objective was to determine whether serological and BALF allergen‐specific IgE concentrations correlated. Animals Forty‐four asthmatic and 39 control horses ≥5 years of age. Methods This prospective cross‐sectional study investigated the sensitization profiles of horses with asthma compared with environmentally matched healthy controls. Both serum and BALF were analyzed using the protein microarray. Partial least square‐discriminant analysis (PLS‐DA) was used to identify and rank the importance of the allergens for class detection (ie, asthma vs control), with a variable influence on the projection (VIP) >1 considered significant. Results The allergens that best discriminated (VIP >1) asthmatic horses from controls were proteins derived from fungi (Aspergillus fumigatus), insects (Culicoides spp.), and latex (Hevea brasiliensis). The serological model predictive ability was markedly inferior (area under the curve [AUC] 0.585, 95% confidence interval [CI]: 0.454‐0.747) to that of the BALF (AUC 0.751, 95% CI: 0.582‐0.866). The two models shared nine allergens, of which eight showed significant weak to moderate correlations. Conclusion and Clinical Importance The concentrations of several allergen‐specific IgE were higher in asthmatic horses. The protein microarray performed better on BALF than serum for detection of asthma. Serological IgE concentrations do not closely correlate with BALF concentrations and should be interpreted with caution.

BackgroundGastric dilatation volvulus (GDV) can lead to organ failure including acute kidney injury (AKI). Due to its cytoprotective, antioxidant and anti-inflammatory effects, lidocaine has a potential to prevent AKI in dogs with GDV.Design and settingProspective, observational cohort study in client-owned dogs with GDV.ObjectiveTo determine concentrations of renal biomarkers for AKI in dogs with GDV with and without intravenous (IV) lidocaine therapy.MethodsThirty-two dogs were randomized to receive either IV lidocaine (2 mg/kg, followed by a lidocaine constant rate infusion at a dose of 50 μg/kg/min over 24 h; n = 17) or no lidocaine (n = 15). Blood and urine samples were taken at admission (T0) (only blood), during or immediately after surgery (T1), and 24 (T24) and 48 (T48) h after surgery. Plasma creatinine (pCr), plasma neutrophil gelatinase-associated lipocalin (pNGAL), urinary NGAL (uNGAL), uNGAL to creatinine ratio (UNCR), and urinary gamma-glutamyl transferase to creatinine ratio (uGGT/uCr) were evaluated. Biomarker concentrations were compared between dogs with and without IV lidocaine and the course of each marker was determined in comparison to its admission value.ResultsIn the entire population, a significantly higher pCr at T0 (median, 95 μmol/L, interquartile range, 82–105) compared with T1 (69 μmol/L, 60–78), T24 (63 μmol/L, 52–78), and T48 (78 μmol/L, 65–87) (P < 0.001) was found. Plasma NGAL increased significantly between T0 (5.66 ng/mL, 3.58–7.43) and T24 (7.50 ng/mL, 4.01–11.89) (P = 0.006) and T48 (9.86 ng/mL, 5.52–13.92) (P < 0.001), respectively. Urinary NGAL increased significantly between T1 (0.61 ng/mL, 0.30–2.59) and T24 (2.62 ng/mL, 1.86–10.92) (P = 0.001) and T48 (4.79 ng/mL, 1.96–34.97 (P < 0.001), respectively. UNCR increased significantly between T1 (0.15 μg/mmol, 0.09–0.54) and T24 (1.14 μg/mmol, 0.41–3.58) (P = 0.0015) and T48 (1.34 μg/mmol, 0.30–7.42) (P < 0.001), respectively. Concentrations of uGGT/uCr increased significantly from T0 highest at T24 (6.20 U/mmol, 3.90–9.90) and significantly decreased at T48 (3.76 U/mmol, 2.84–6.22) (P < 0.001). No significant differences in any renal biomarker concentration were found between dogs with and without IV lidocaine therapy.Conclusion and clinical relevancePlasma NGAL, uNGAL and UNCR remained increased up to 48 h post-surgery. No evidence of lidocaine-associated renoprotection was found.

BackgroundMeningoencephalitis of unknown origin (MUO) is an inflammatory disease of the canine central nervous system (CNS) that shares several features with multiple sclerosis (MS) in humans. In approximately 95% of MS patients, ≥ two immunoglobulin G (IgG) oligoclonal bands (OCBs) are detectable exclusively in the cerebrospinal fluid (CSF).Hypothesis/objectivesTo investigate OCBs in CSF and serum in dogs affected by MUO, intervertebral disc disease (IVDD), idiopathic epilepsy (IE), intracranial neoplasia (IN), steroid-responsive meningitis-arteritis (SRMA), and diseases outside the CNS. We hypothesize that the highest prevalence of CSF-specific OCBs (≥ two OCBs uniquely in the CSF) would be found in dogs affected by MUO.AnimalsClient-owned dogs (n = 121) presented to the neurology service due to neurological deficits.MethodsProspective study. Measurement of IgG concentration in CSF and serum via a canine IgG ELISA kit. OCB detection via isoelectric focusing (IEF) and immunoblot.ResultsPresence of CSF-specific OCBs was significantly higher in dogs with MUO (57%) compared to 22% in IN, 6% in IE, 15% in SRMA, 13% in IVDD, and 0% in the non-CNS group (p < .001). Dogs with MUO were 9.9 times more likely to show CSF-specific OCBs than all other diseases together (95% confidence interval, 3.7-26.4; p < .001).Conclusions and clinical importanceMUO showed the highest prevalence of CSF-specific OCBs, indicating an inflammatory B cell response. Future studies are needed to evaluate the prevalence in the specific MUO subtypes and a possible similarity with human MS.

Abstract Background Endothelial dysfunction might contribute to the development of leptospiral pulmonary hemorrhage syndrome (LPHS). Hypothesis Serum concentrations of markers of endothelial activation and dysfunction are higher in dogs with leptospirosis and correlate with the occurrence of LPHS and a higher case fatality rate. Animals Clinically healthy dogs (n = 31; 10/31 dogs confirmed healthy based on no detected abnormalities on blood work), dogs with leptospirosis with LPHS (n = 17) and without LPHS (n = 15), dogs with acute kidney injury not due to leptospirosis (AKI‐nL, n = 34). Methods Observational study. Serum concentrations of soluble intercellular adhesion molecule 1 (sICAM‐1), vascular endothelial growth factor (VEGF), and angiopoietin‐2 (Ang‐2) at admission were compared between groups. Correlations with outcome and the accuracy to predict LPHS were examined. Results Soluble intercellular adhesion molecule (sICAM‐1), VEGF, and Ang‐2 concentrations were higher in dogs with AKI‐nL (sICAM‐1 34.7 ng/mL, interquartile range [IQR] = 24.4‐75.5; VEGF 43.1 pg/mL, IQR = 12.3‐79.2; Ang‐2 8.5 ng/mL, IQR = 6.2‐12.3), leptospirosis without LPHS (sICAM‐1 45.1 ng/mL, IQR = 30.6‐59.0; VEGF 32.4 pg/mL, IQR = 12.5‐62.6; Ang‐2 9.6 ng/mL, IQR = 6.9‐19.3), and LPHS (sICAM‐1 69.7 ng/mL, IQR = 42.1‐89.1; VEGF 51.8 pg/mL, IQR = 26.3‐96.7; Ang‐2 8.0 ng/mL, IQR = 5.6‐12.2) compared to controls (P

Thirty controls (C) and 30 IBH-affected (T) Lusitano horses were evaluated. T horses were included based on anamnesis and physical examination, supported by questionnaires. All horses were submitted to skin tests, Intrademal (IDT) and Skin Prick Tests (SPT), on the neck with 14 specific allergens, 13 recombinant proteins (r-proteins) from Culicoides nubeculosus (Cul n) and Culicoides obsoletus (Cul o) salivary glands and Culicoides nubeculosus Whole Body Extract (Cul n WBE). Addicionally, a cluster of six T and six C horses were also tested with Cul n 3 and Cul n 4 produced in insect cells and barley, as well as E. coli produced Cul o 3 and Cul o WBE. Allergen concentrations were 10 µg/mL for IDT and 100 µg/mL for SPT, and wheal diameters assessed at 20 min, 6 and 48 h. IDTs were considered positive when wheal diameter was ≥50% of the histamine wheal and SPT’s ≥ 0.9 cm. In vitro tests, allergen-specific serum IgE and sulfidoleukotriene (sLT) release assay were also carried out. Results showed that Cul n WBE, Cul n 7, 8, 9, Cul o1P and Cul o 2P were the best performing allergens for SPTs (p ≤ 0.0001) for the 1st allergen panel and Cul o WBE, Cul n 3 Bar and Cul n 4 Bac (p ≤ 0.05) for the 2nd, presenting a higher discriminatory diagnostic potential than IDTs, at a concentration of 100 µg/mL, with readings assessed at 20 min. Regarding in vitro tests overall, the sLT release assay performed best.

Isoelectric focusing followed by immunoblotting is a method routinely used in human medicine to assess the presence of oligoclonal bands (OCBs) in cerebrospinal fluid (CSF) and serum. The detection of OCBs is a valuable diagnostic test, especially important in patients with the suspicion of multiple sclerosis (MS), in which at least two OCBs are found in the CSF not present in paired serum samples in up to 95% of patients. So far, presence of OCBs in CSF and serum of dogs has only been investigated in a small cohort of dogs diagnosed with degenerative myelopathy and healthy dogs. The main objective of the current study was to describe the method used for OCB detection and compare two different canine anti-IgG antibodies: a canine rabbit-anti-IgG antibody (Jackson ImmunoResearch) vs. a canine goat-anti-IgG antibody (Bio-Rad). The method was performed according to the instructions of the commercial kit used. The canine goat-anti-IgG antibody showed a better performance than the canine rabbit-anti-IgG antibody. The availability of the technique of OCB detection in the dog paves the way for further studies, especially in the field of inflammatory diseases of the canine central nervous system, and comparison between specific human and canine diseases.

Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horses by day 7 after vaccination. CD154 is an established indicator of antigen-reactive T helper cells in other species, but has not been characterized in horses. CD154 detection in equine PBMC by an anti-human CD154 antibody (clone 5C8) was confirmed by Western blots and then applied for flow cytometry. As a common indicator of equine T cell activation, cytokine induction was studied in parallel. T cells were analyzed by multicolor flow cytometry of PBMC after re-stimulation with TT in vitro. Reactive T helper (Th) cells were characterized by increased frequencies of CD4+CD154+ lymphocytes in in vitro TT-re-stimulated PBMC on day 14 after vaccination of the horses compared to pre-vaccination. The majority of all CD154+ cells after TT re-stimulation were CD4+ Th cells, but CD154 was also induced on CD4- cells albeit in lower frequencies. CD154+CD4+ Th cells were enriched in cytokine-expressing cells compared to CD154-CD4+ Th cells. Similar to the CD4+CD154+ frequencies, CD4+IL-4+, CD4+IFN-γ+ and CD4+TNF-α+ were increased after vaccination, but IL-4+ increased later than IFN-γ+ and CD4+TNF-α+, which already exceeded pre-vaccination frequencies on day 7. CD4+CD154+ frequencies correlated positively with those of CD4+IL-4+ (Th2) on day 14, and negatively with CD4+IFN-γ+ induction on day 7, but did not correlate with CD4+TNF-α+ frequencies or TT-specific antibody concentrations. CD154 appears to be a useful marker of antigen-reactive equine Th cells in combination with cytokine expression. The T cell analyses established here with TT can be applied to other antigens relevant for infections or allergies of horses and in horse models for translational research.

Equine insect bite hypersensitivity (IBH) is the most common skin disease affecting horses. It is described as an IgE-mediated, Type I hypersensitivity reaction to salivary gland proteins of Culicoides insects. Together with Th2 cells, epithelial barrier cells play an important role in development of Type I hypersensitivities. In order to elucidate the role of equine keratinocytes in development of IBH, we stimulated keratinocytes derived from IBH-affected (IBH-KER) (n = 9) and healthy horses (H-KER) (n = 9) with Culicoides recombinant allergens and extract, allergic cytokine milieu (ACM) and a Toll like receptor ligand 1/2 (TLR-1/2-L) and investigated their transcriptomes. Stimulation of keratinocytes with Culicoides allergens did not induce transcriptional changes. However, when stimulated with allergic cytokine milieu, their gene expression significantly changed. We found upregulation of genes encoding for CCL5, -11, -20, -27 and interleukins such as IL31. We also found a strong downregulation of genes such as SCEL and KRT16 involved in the formation of epithelial barrier. Following stimulation with TLR-1/2-L, keratinocytes significantly upregulated expression of genes affecting Toll like receptor and NOD-receptor signaling pathway as well as NF-kappa B signaling pathway, among others. The transcriptomes of IBH-KER and H-KER were very similar: without stimulations they only differed in one gene (CTSL); following stimulation with allergic cytokine milieu we found only 23 differentially expressed genes (e.g. CXCL10 and 11) and following stimulation with TLR-1/2-L they only differed by expression of seven genes. Our data suggests that keratinocytes contribute to the innate immune response and are able to elicit responses to different stimuli, possibly playing a role in the pathogenesis of IBH.

Abstract Background Severe asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance‐limiting disease associated with increased allergen‐specific immunoglobulin E (IgE) against a range of environmental aeroallergens. Objective To develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA‐affected horses. Animals Six SEA‐affected and 6 clinically healthy Warmblood performance horses. Methods Developed a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, bacteria, fungi, and arthropods associated with the horses, environment. Conditions were optimized and assessed for printing, incubation, immunolabeling, biological fluid source, concentration techniques, reproducibility, and specificity. Results This method identified a number of novel allergens, while also identifying an association between SEA and pollen sensitization. Immunolabeling methods confirmed the accuracy of a commercially available mouse anti‐horse IgE 3H10 source (R2 = 0.91). Biological fluid source evaluation indicated that sera and bronchoalveolar lavage fluid (BALF) yielded the same specific IgE profile (average R2 = 0.75). Amicon centrifugal filters were found to be the most efficient technique for concentrating BALF for IgE analysis at 40‐fold. Overnight incubation maintained the same sensitization profile while increasing sensitivity. Reproducibility was demonstrated (R2 = 0.97), as was specificity using protein inhibition assays. Arthropods, fungi, and pollens showed the greatest discrimination for SEA. Conclusions and Clinical Importance We have established that protein microarrays can be used for large‐scale IgE mapping of allergens associated with the environment of horses. This technology provides a sound platform for specific diagnosis, management, and treatment of SEA.

Abstract Background Hemorrhage in the spinal canal leads to further damage of the spinal cord influencing outcome in dogs with intervertebral disk (IVD) extrusion. The aim of the study was to evaluate blood degradation products and ferritin in the cerebrospinal fluid (CSF) of dogs with thoracolumbar IVD extrusion, and their association to clinical parameters and MRI findings. Results In the CSF of dogs with IVD extrusion, both net oxyhemoglobin absorption (NOA) and net bilirubin absorption (NBA) were significantly higher compared to the control groups of dogs with steroid responsive meningitis arteritis (SRMA) and idiopathic epilepsy (IE) (P

Background: The degree of systemic inflammation, reperfusion injury and endothelial activation are potentially important determinants of clinical outcomes in dogs with gastric dilatation volvulus (GDV).Objective: To evaluate plasma concentrations and kinetics of inflammatory markers in dogs with GDV over a time frame of 48 h, and to compare to healthy dogs.Design and Setting: Prospective, observational cohort study in client-owned dogs with GDV.Materials and Methods: Fifteen dogs with GDV and 9 healthy control dogs were enrolled. Plasma concentrations of interleukin (IL)-6, IL-7, IL-8, IL-10, IL-15, IL-18, interferon gamma (IFN-γ), keratinocyte chemotactic-like, monocyte chemotactic protein (MCP)-1, Angiopoietin (Ang)-2, and C-reactive protein (CRP) were measured at admission (prior any therapeutic intervention, (T0), immediately after surgery (T1), 24 ± 4 h (T24), and 48 ± 4 h (T48) post-surgery. Cytokines were measured using multiplex magnetic bead assay. Plasma Ang-2 was measured with a commercial human ELISA test kit validated for dogs.Results: Dogs with GDV had significantly higher plasma concentrations of IFN-γ and IL-10 compared to healthy control dogs at all time points. Concentrations of IL-6 were significantly higher at T1 and T24, concentrations of MCP-1 at T24, and concentrations of CRP at T24 and T48. A significant increase between T0 and T1 was found for IL-6, IL-10, and CRP, between T1 and T24 for IL-8, IFN-γ, MCP-1, and CRP, and between T24 and T48 for IL-15, Ang-2, and CRP. A significant decrease between T0 and T1 was found for IL-7, IL-8, IL-15, IL-18, and Ang-2; between T1 and T24 for IL-6 and KC-like; and between T24 and T48 for IL-6.Conclusion: In GDV dogs, a mild pro-inflammatory reaction was present at admission, which peaked immediately after and up to 24 h post-surgery, mainly represented by IL-6, IFN-γ, MCP-1, and CRP, and which decreased at T48. In addition, the anti-inflammatory IL-10 was increased in GDV dogs at all time points.

BackgroundInsect 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.AimsTo 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.MethodIgE 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 (7 years) on Culicoides-specific IgE was evaluated. Sero-positivity rates and IgE levels were compared.ResultsAt 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.ConclusionSignificant 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.

Background Genetic, epidemiologic, and clinical evidence suggests that, in horses, there are manifestations of hypersensitivity that can occur together. Objectives To investigate whether concurrent insect bite hypersensitivity (IBH) and severe equine asthma (EA) is associated with higher allergen‐specific and total serum immunoglobulin E (IgE) concentrations than only EA or IBH. Animals Healthy control horses (C, n = 40), horses with IBH (IBH, n = 24), severe EA (EA, n = 18), and both conditions (IBH/EA, n = 23) were included. Methods In our retrospective comparative study, sera from horses with signs of severe EA, IBH, and control animals were used. IgE specific for 15 recombinant (r) allergens as well as total serum IgE concentrations were measured by enzyme‐linked immunosorbent assay. Results Group IBH (median sum r‐Culicoides IgE: optical density at 405 nm [OD405] = 3.54 [0.48‐15.07]) and group IBH/EA (OD405 = 4.55 [0.46‐17.15]) had significantly (P .05) differences between group IBH and group IBH/EA. No significant differences among the groups were found for the other r‐allergens or total serum IgE concentration. Compared to controls, horses with severe IBH had significantly increased IgE concentration to 5 Culicoides r‐allergens (P

Abstract Background Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of Culicoides spp., which occurs frequently in horses imported from Iceland to continental Europe. IBH does not occur in Iceland because Culicoides species that bite horses are not present. However, Simulium vittatum (S. vittatum) are found in Iceland. In Europe, blood basophils from IBH-affected horses release significantly more sulfidoleukotrienes (sLT) than those from healthy controls after in vitro stimulation with Culicoides nubeculosus (C. nubeculosus) and S. vittatum. Aims of the study were: (I) using the sLT release assay, to test if horses living in Iceland were sensitized to S. vittatum and (II) to determine in a longitudinal study in horses imported from Iceland to Switzerland whether the sLT release assay would allow to predict which horses would develop IBH. Results Horses in Iceland, even when living in high S. vittatum areas, were usually not sensitized to S. vittatum or C. nubeculosus. Incidence of IBH in the 145 horses from the longitudinal study was 51% and mean time until IBH developed was 2.5 ± 1 year. Before import and after the first summer following import, there were no significant differences in sLT release between the endpoint healthy (H) and IBH groups. After the 2nd summer, when the number of clinically affected horses increased in the endpoint IBH group, a significantly higher sLT release after stimulation with C. nubeculosus but not with S. vittatum was observed. After the 3rd and 4th summer, the endpoint IBH group had a significantly higher sLT release with C. nubeculosus and S. vittatum than the endpoint H group. Some of the horses that remained healthy became transiently positive in the sLT release assay upon stimulation of their peripheral blood leucocytes with C. nubeculosus. Conclusions Horses in Iceland are not sensitized to S. vittatum. In horses that develop IBH, sensitization to S. vittatum is secondary to sensitization to C. nubeculosus and probably a result of an immunological cross-reactivity. A sLT release assay cannot be used to predict which horses will develop IBH. A transient positive reaction in the sLT release assay observed in horses that remained healthy suggests that immunoregulatory mechanisms may control an initial sensitization of the healthy horses.

Background: Insect bite hypersensitivity (IBH) is an eosinophilic allergic dermatitis of horses caused by type I/IVb reactions against mainly Culicoides bites. The vaccination of IBH-affected horses with equine IL-5 coupled to the Cucumber mosaic virus-like particle (eIL-5-CuMVTT) induces IL-5-specific auto-antibodies, resulting in a significant reduction in eosinophil levels in blood and clinical signs. Objective: the preclinical and clinical safety of the eIL-5-CuMVTT vaccine. Methods: The B cell responses were assessed by longitudinal measurement of IL-5- and CuMVTT-specific IgG in the serum and plasma of vaccinated and unvaccinated horses. Further, peripheral blood mononuclear cells (PBMCs) from the same horses were re-stimulated in vitro for the proliferation and IFN-γ production of specific T cells. In addition, we evaluated longitudinal kidney and liver parameters and the general blood status. An endogenous protein challenge was performed in murine IL-5-vaccinated mice. Results: The vaccine was well tolerated as assessed by serum and cellular biomarkers and also induced reversible and neutralizing antibody titers in horses and mice. Endogenous IL-5 stimulation was unable to re-induce anti-IL-5 production. The CD4+ T cells of vaccinated horses produced significantly more IFN-γ and showed a stronger proliferation following stimulation with CuMVTT as compared to the unvaccinated controls. Re-stimulation using E. coli-derived proteins induced low levels of IFNγ+CD4+ cells in vaccinated horses; however, no IFN-γ and proliferation were induced following the HEK-eIL-5 re-stimulation. Conclusions: Vaccination using eIL-5-CuMVTT induces a strong B-cell as well as CuMVTT-specific T cell response without the induction of IL-5-specific T cell responses. Hence, B-cell unresponsiveness against self-IL-5 can be bypassed by inducing CuMVTT carrier-specific T cells, making the vaccine a safe therapeutic option for IBH-affected horses.

Two types of gammaherpesviruses (γEHV) are known to infect horses, EHV-2 and EHV-5. Foals become infected early in life, probably via the upper respiratory tract, despite maternal antibodies. In this study, we analyzed samples from a herd of mares and their foals. The foals were followed from birth to 22 months of age and the dams during the first 6 months postpartum. Blood and nasal swab samples were taken regularly for evaluation of antibody responses, virus isolation and viral load by qPCR. EHV-2 was isolated on day 5, and EHV-5 on day 12, earlier than previously reported. γEHV specific antibodies were not detectable in serum of foals before colostrum intake but peaked a few days after colostrum. Overall, EHV-2 viral load peaked in nasal swab at three to four months of age, paralleled with decline in maternal antibodies, but EHV-5 viral load did not peak until month 12. Maternal antibodies had a notable effect on the viral load and induction of endogenous antibody production. Foals were grouped in two groups depending on the mare's γEHV specific total IgG levels in serum at birth, group-high and group-low. Group-high had higher levels of maternal γEHV specific total IgG and IgG4/7 for the first 3 months, but when the endogenous production had superseded maternal antibodies, group-low was higher. The maternal antibodies had an effect on the γEHV viral load. Group-low peaked in EHV-2 viral load one month earlier than group-high. These effects were more evident for EHV-5, as there were seven months between the viral load peaks for the groups. The study provides information on how maternal antibody transfer affects γEHV shedding and antibody production in offspring. It also extends our knowledge on the occurrence of EHV-2 and EHV-5 infection in foals during the first two years of life.

Immunoglobulin A deficiency (IgAD) is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS) to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei) identified 35 genomic loci suggestively associated (p

Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome.

The immune system of mammals is subject to continuous development during the postnatal phase of life. Studies following the longitudinal development of the immune system in healthy children are limited both by ethical considerations and sample volumes. Horses represent a particular valuable large animal model for T regulatory (Treg) cells and allergy research. We have recently characterised Treg cells from horses, demonstrated their regulatory capability and showed both their expansion and induction in vitro. Insect bite hypersensitivity (IBH) is a common allergy in horses resembling atopic dermatitis and studies have shown that first exposure to allergens in adult life results in an increased incidence of IBH. The aim of the present study was to characterize circulating CD4+CD25highFoxP3+cells in foals, evaluate their suppressive capability and their in vitro induction compared to adult horses. 19 foals (age range, 1-5 months), their adult mothers and six one-year-old horses (yearlings) were included in the study. The proportion of FoxP3+ cells within the circulating CD4+CD25high population was significantly higher in foals (47%) compared to their mothers (18%) and to yearlings (26%). Treg cells from foals also displayed a higher suppressive capability. Furthermore, CD4+CD25high cells in foals could be induced in vitro from CD4+CD25- cells in a significantly higher proportion compared to mares. These cells also displayed a significantly enhanced suppressive capability. In summary these findings support the notion that exposure of horses to allergens during maturation of the immune system assists the establishment of induced (i)Treg driven tolerance.

Recurrent airway obstruction (RAO) is a common and potentially debilitating lower airway disease in horses, which shares many similarities with human asthma. In susceptible horses RAO exacerbation is caused by environmental allergens and irritants present in hay dust. The objective of this study was the identification of genes and pathways involved in the pathology of RAO by global transcriptome analyses in stimulated peripheral blood mononuclear cells (PBMCs). We performed RNA-seq on PBMCs derived from 40 RAO affected and 45 control horses belonging to three cohorts of Warmblood horses: two half-sib families and one group of unrelated horses. PBMCs were stimulated with hay dust extract, lipopolysaccharides, a recombinant parasite antigen, or left unstimulated. The total dataset consisted of 561 individual samples. We detected significant differences in the expression profiles between RAO and control horses. Differential expression (DE) was most marked upon stimulation with hay dust extract. An important novel finding was a strong upregulation of CXCL13 together with many genes involved in cell cycle regulation in stimulated samples from RAO affected horses, in addition to changes in the expression of several HIF-1 transcription factor target genes. The RAO condition alters systemic changes observed as differential expression profiles of PBMCs. Those changes also depended on the cohort and stimulation of the samples and were dominated by genes involved in immune cell trafficking, development, and cell cycle regulation. Our findings indicate an important role of CXCL13, likely macrophage or Th17 derived, and the cell cycle regulator CDC20 in the immune response in RAO.

Crosslinking of immunoglobulin E antibodies (IgE) bound at the surface of mast cells and subsequent mediator release is considered the most important trigger for allergic reactions. Therefore, the genetic control of IgE levels is studied in the context of allergic diseases, such as asthma, atopic rhinitis, or atopic dermatitis (AD). We performed genome-wide association studies in 161 Labrador Retrievers with regard to total and allergen-specific immunoglobulin E (IgE) levels. We identified a genome-wide significant association on CFA 5 with the antigen-specific IgE responsiveness to Acarus siro. We detected a second genome-wide significant association with respect to the antigen-specific IgE responsiveness to Tyrophagus putrescentiae at a different locus on chromosome 5. A. siro and T. putrescentiae both belong to the family Acaridae and represent so-called storage or forage mites. These forage mites are discussed as major allergen sources in canine AD. No obvious candidate gene for the regulation of IgE levels is located under the two association signals. Therefore our studies offer a chance of identifying a novel mechanism controlling the host's IgE response.

En el artículo compartimos los primeros hallazgos de un proceso investigativo que pretende analizar las experiencias en el contexto de crisis sociosanitaria de un determinado grupo de jóvenes. Como parte del ejercicio interpretativo, consideramos que estos jóvenes han configurado sus trayectorias biográficas desde una posición conflictuada con determinadas disposiciones institucionales, donde enfrentan sus contextos marcados por la precarización e injusticia, pero a la vez, han construido acciones alternativas que fracturan determinados mandatos sociales. Nuestra apuesta investigativa propone relevar esta multiplicidad de posiciones de los mundos juveniles que se contrapone con parámetros normativos y homogeneizadores que predominan al momento de investigar juventudes.

Se presenta una revisión documental de la producción académica e institucional entre octubre 2019 y abril 2021 con relación a las juventudes chilenas durante la Revuelta y la Pandemia. Con ello, se busca generar conocimiento acerca de la construcción de las juventudes desde el saber académico e institucional con respecto a estos acontecimientos. El análisis se desarrolló a partir de tres categorías que se evidencian como centrales en esta producción: control social, acción política y afectividades. Finalizamos compartiendo algunas pistas analíticas y epistémicas que consideramos de relevancia para abordar las tensiones y reflexiones en torno a las juventudes en este momento de crisis.

O projeto profissional crítico do Serviço Social brasileiro tem na formação uma importante estratégia, que frente a conjuntura, representa um grande desafio. O presente artigo apresenta reflexões para o debate sobre formação e o ensino teórico-prático no Serviço Social. Para isso, retoma a análise da construção do projeto de formação crítica da profissão e os impactos do contexto neoliberal. Apresenta os dados de pesquisa com o estado da arte do debate sobre o ensino teórico-prático no Serviço Social com análise da relação de ensino e aprendizagem a partir da formação e suas expressões: na docência, ensino e método; instrumentalidade, instrumentos e a relação teoria e prática; as diretrizes curriculares e os fundamentos da profissão. Por fim, apresenta elementos do debate sobre as teorias pedagógicas contra hegemônicas e formação em saúde que trazem contribuições para o Serviço Social.

Abstract Background Studies have shown that radiation from radiotherapy increases the yeast colonization of patients. However it is not clear, if such radiation alters the yeast itself. The aim of the present study was therefore to report the direct impact of gamma radiation on Candida tropicalis. Methods C. tropicalis was obtained from a patient with a carcinoma, a suspension of this yeast containing 2.0 × 103 colony forming units per milliliter was prepared. It was submitted to gamma radiation dosage similar to that used in the treatment of head and neck cancer. After a cumulative dose of 7200 cGy some virulence attributes of C. tropicalis, including macro and micromorphological characteristics, adhesion and biofilm abilities, murine experimental infection and phagocytosis resistance were evaluated on irradiated and non-irradiated yeasts. Results After irradiation the colony morphology of the yeast was altered from a ring format to a smooth appearance in most colonies. Scanning electron microscopy revealed notable differences in the structures of both these colonies and the yeast cells, with the loss of pseudohyphae following irradiation and an increase in extracellular matrix production. The adherence and biofilm production of the yeast was greater following irradiation, both in terms of the number of yeasts and total biomass production on several abiotic surfaces and TR146 cells. The phagocytic index of the irradiated yeasts was not statistically different; however, the presence of cellular debris was detected in the kidneys of infected animals. Mice infected with irradiated yeasts developed an infection at the site of the yeast inoculation, although systemic infection was unchanged. Conclusions Our findings show for the first time that C. tropicalis, one of the most important yeasts from colonization, which cause fatal candidemia in cancer patients, is affected by gamma irradiation, with changes to its virulence profile.

Resumo: O presente artigo apresenta o debate sobre as expressões conservadoras no trabalho em saúde a partir da análise da abordagem familiar e comunitária na Saúde da Família. Toma como referência de análise o movimento de disputa hegemônica existente na política de saúde e a direção social que trabalho em saúde assume nesse contexto. Situa os impactos da contrarreforma na política de saúde no processo de trabalho, na análise e intervenção das relações familiares e comunitárias e na educação em saúde.

Este estudo é parte de uma pesquisa em andamento (2017-2019), cujo objetivo é elaborar uma cartografia literária de contos latino-americanos tradicionais para uma viagem imaginária a ser realizada com uma turma do 5º ano do ensino fundamental de uma escola pública na região do Triângulo Mineiro. Destina-se a professores e alunos do ensino fundamental para o ensino da leitura literária e de suas práticas. Tem como metodologia a abordagem cartográfica cuja problemática se insere no campo multidisciplinar para abarcar a diversidade cultural dos povos latino-americanos historicamente construída nas relações interétnicas, e situada nas relações entre dominantes colonizadores e povos dominados. A escolha dos contos latino-americanos para desenvolver à intervenção pedagógica focou diferentes identidades culturais do continente latino-americano por meio do texto literário, situando-se na cultura literária. Os contos fazem parte de uma coleção bilíngue (português e espanhol), publicada por editoras latino-americanas nos anos de 1980 e 1990, apoiadas pelo Centro Regional para o Fomento do Livro na América Latina e Caribe -CERLALC e da UNESCO. Quanto ao gênero literário, a opção pelos contos tradicionais atendeu aos objetivos estabelecidos: trazer a diversidade cultural e a pluralidade de identidades coletivamente construídas pelos povos latino-americanos. A elaboração da cartografia literária, tem como eixo a problemática social presente nos contos que se desenrola no cotidiano realidade/magia. O resultado é a elaboração de uma cartografia literária multidisciplinar com os contos latino-americanos que trazem semelhanças e diferenças, aproximações e distanciamentos da cultura latino-americana.

O presente texto visa compreender como os espaços de instrução e leitura, existentes na antiga capital goiana (Goiás), na segunda metade do século XIX e início do século XX, foram (re)apropriados por algumas mulheres de forma a lhes garantir a expressão de suas experiências. Para tanto, partindo da documentação do Gabinete Litterario Goyano, criado em 1864, buscamos, por meio do protagonismo de Silvina Ermelinda Xavier de Britto e Jacintha Luiza do Couto Brandão Peixoto, compreender questões em torno da instrução das mulheres, de seu exercício da leitura, de um ensaio de emancipação, representado pelos pedidos de voto das goianas em 1889 e das táticas manejadas por estas mulheres na expectativa de exercitar sua intelectualidade, ainda que lhes fosse apresentado como um caminho cheio de obstruções.

Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria (e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

No presente texto, procuramos compreender as representações de feminilidade presentes no discurso da Justiça nos casos em que esta instância foi chamada a intervir em processos envolvendo homens como agressores e mulheres como vítimas sejam nos casos de crimes contra a pessoa (homicídios, tentativas de homicídios e lesões corporais) seja nos crimes contra a honra, ou chamados “crimes sexuais” (estupros e defloramentos). O texto está organizado em três partes que focalizam: o formato que a prática dos crimes adquiriu na Comarca de Catalão no período de 1890-1941; um estudo de caso envolvendo um crime contra a honra (defloramento), na tentativa de compreender o modelo normativo de família e principalmente de mulher; e, por fim, outro estudo de caso de um crime contra a pessoa (tentativa de homicídio), caracterizado com “crime passional”, com o intuito de problematizar os limites da Justiça em compreender a violência física contra a mulher como parte de um processo mais amplo de opressão.