Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma.

Introduction: Equine asthma (EA) is a common lower airway disease in horses, but whether its pathogenesis is allergic is ambiguous. Extrinsic stimuli like hay dust induce acute exacerbation of clinical signs and sustained local neutrophilic inflammation in susceptible horses. Aspergillus fumigatus is an EA stimulus, but it is unclear if it merely acts as an IgE-provoking allergen. We aimed to comprehensively analyze immunoglobulin (Ig) isotypes in EA, elucidating their binding to different A. fumigatus antigens, and their quantities systemically in serum and locally in bronchoalveolar lavage fluid (BALF). Methods: Serum and BALF from healthy horses (HE, n = 18) and horses with mild-moderate asthma (MEA, n = 20) or severe asthma (SEA, n = 24) were compared. Ig isotype (IgG1, IgG3/5, IgG4/7, IgG6, IgA, and IgE) binding to nine antigens (A. fumigatus lysate, and recombinant Asp f 1, Asp f 7, Asp f 8, dipeptidyl-peptidase 5, class II aldolase/adducin domain protein, glucoamylase, beta-hexosaminidase, and peptide hydrolase) was compared by enzyme-linked immunosorbent assays. Total Ig isotype contents were determined by bead-based assays. Results: MEA and SEA differed from HE but hardly from each other. Compared to HE, asthmatic horses showed increased anti-A. fumigatus binding of IgG (BALF and serum) and IgA (BALF). Serum and BALF IgE binding and total IgE contents were similar between HE and EA. Single antigens, as well as A. fumigatus lysate, yielded similar Ig binding patterns. Serum and BALF IgG1 binding to all antigens was increased in SEA and to several antigens in MEA. Serum IgG4/7 binding to two antigens was increased in SEA. BALF IgA binding to all antigens was increased in SEA and MEA. Total BALF IgG1 and IgG4/7 contents were increased in SEA, and serum IgG4/7 content was increased in MEA compared to HE. Yet, total isotype contents differentiated EA and HE less clearly than antigen-binding Ig. Discussion: A. fumigatus immunogenicity was confirmed without identification of single dominant antigens here. A. fumigatus provoked elevated BALF IgG1 and IgA binding, and these isotypes appear relevant for neutrophilic EA, which does not support allergy. BALF Ig isotype differentiation beyond IgE is crucial for a comprehensive analysis of immune responses to fungi in EA pathogenesis.

Monoclonal antibodies for equine CD25 improve detection of regulatory T cells in horses.

CD25, the interleukin-2 receptor α-chain, is expressed on cell surfaces of different immune cells and is commonly used for phenotyping of regulatory T cells (Tregs). CD25 has essential roles in the maintenance of hemostasis and immune tolerance and Treg cell involvement has been shown in human diseases and murine models for allergy, autoimmunity, cancer, chronic inflammation, and many others. In horses, a cross-reactive anti-human CD25 antibody has previously been used for characterizing Tregs. Here, we developed monoclonal antibodies (mAbs) to equine CD25 and compared their staining pattern with the anti-human CD25 antibody by flow cytometry. The comparison of the two reagents was performed by two separate analyses in independent laboratories. Overall, similar staining patterns for equine peripheral blood lymphocytes were obtained with the anti-human CD25 antibody and equine CD25 mAb 15-1 in both laboratories. Both reagents identified comparable CD4+CD25+ and CD4+CD25+FOXP3+ percentages after stimulation of peripheral blood mononuclear cells (PBMC) with pokeweed mitogen. However, when compared to the anti-human CD25 antibody, the equine CD25 mAb 15-1 resulted in a better staining intensity of the equine CD25+ cells and increased the percentages of Tregs and other CD25+ cells ex vivo and after culturing of PBMC without stimulation. In summary, the equine CD25 mAbs provide new, improved reagents for Tregs and CD25+ cell phenotyping in horses.

Monoclonal antibody development advances immunological research in horses.

Host immune analyses require specific reagents to identify cellular and soluble components of the immune system. These immune reagents are often species-specific. For horses, various immunological tools have been developed and tested by different initiatives during the past decades. This article summarizes the development of well characterized monoclonal antibodies (mAbs) for equine immune cells, immunoglobulin isotypes, cytokines, and chemokines.

Neonatal and maternal upregulation of antileukoproteinase in horses.

Introduction: The end of gestation, ensuing parturition, and the neonatal period represent highly dynamic phases for immunological changes in both mother and offspring. The regulation of innate immune cells at the maternal-fetal interface during late term pregnancy, after birth, and during microbial colonization of the neonatal gut and other mucosal surfaces, is crucial for controlling inflammation and maintaining homeostasis. Innate immune cells and mucosal epithelial cells express antileukoproteinase (SLPI), which has anti-inflammatory and anti-protease activity that can regulate cellular activation. Methods: Here, we developed and validated new monoclonal antibodies (mAbs) to characterize SLPI for the first time in horses. Peripheral blood and mucosal samples were collected from healthy adults horses and a cohort of mares and their foals directly following parturition to assess this crucial stage. Results: First, we defined the cell types producing SLPI in peripheral blood by flow cytometry, highlighting the neutrophils and a subset of the CD14+ monocytes as SLPI secreting immune cells. A fluorescent bead-based assay was developed with the new SLPI mAbs and used to establish baseline concentrations for secreted SLPI in serum and secretion samples from mucosal surfaces, including saliva, nasal secretion, colostrum, and milk. This demonstrated constitutive secretion of SLPI in a variety of equine tissues, including high colostrum concentrations. Using immunofluorescence, we identified production of SLPI in mucosal tissue. Finally, longitudinal sampling of clinically healthy mares and foals allowed monitoring of serum SLPI concentrations. In neonates and postpartum mares, SLPI peaked on the day of parturition, with mares returning to the adult normal within a week and foals maintaining significantly higher SLPI secretion until three months of age. Conclusion: This demonstrated a physiological systemic change in SLPI in both mares and their foals, particularly at the time around birth, likely contributing to the regulation of innate immune responses during this critical period.

Equine herpesvirus type 1 (EHV-1) replication at the upper respiratory entry site is inhibited by neutralizing EHV-1-specific IgG1 and IgG4/7 mucosal antibodies.

Equine herpesvirus type 1 (EHV-1) is a contagious respiratory pathogen that infects the mucosa of the upper respiratory tract (URT). Mucosal immune responses at the URT provide the first line of defense against EHV-1 and are crucial for orchestrating immunity. To define host-pathogen interactions, we characterized B-cell responses, antibody isotype functions, and EHV-1 replication of susceptible (non-immune) and clinically protected (immune) horses after experimental EHV-1 infection. Nasal secretion and nasal wash samples were collected and used for the isolation of DNA, RNA, and mucosal antibodies. Shedding of infectious virus, EHV-1 copy numbers, viral RNA expression, and host B-cell activation in the URT were compared based on host immune status. Mucosal EHV-1-specific antibody responses were associated with EHV-1 shedding and viral RNA transcription. Finally, mucosal immunoglobulin G (IgG) and IgA isotypes were purified and tested for neutralizing capabilities. IgG1 and IgG4/7 neutralized EHV-1, while IgG3/5, IgG6, and IgA did not. Immune horses secreted high amounts of mucosal EHV-1-specific IgG4/7 antibodies and quickly upregulated B-cell pathway genes, while EHV-1 was undetected by virus isolation and PCR. RNA transcription analysis reinforced incomplete viral replication in immune horses. In contrast, complete viral replication with high viral copy numbers and shedding of infectious viruses was characteristic for non-immune horses, together with low or absent EHV-1-specific neutralizing antibodies during viral replication. These data confirm that pre-existing mucosal IgG1 and IgG4/7 and rapid B-cell activation upon EHV-1 infection are essential for virus neutralization, regulation of viral replication, and mucosal immunity against EHV-1.IMPORTANCEEquine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion storms, and neurologic outbreaks known as equine herpes myeloencephalopathy (EHM). EHV-1 is transmitted with respiratory secretions by nose-to-nose contact or via fomites. The virus initially infects the epithelium of the upper respiratory tract (URT). Host-pathogen interactions and mucosal immunity at the viral entry site provide the first line of defense against the EHV-1. Robust mucosal immunity can be essential in protecting against EHV-1 and to reduce EHM outbreaks. It has previously been shown that immune horses do not establish cell-associated viremia, the prerequisite for EHM. Here, we demonstrate how mucosal antibodies can prevent the replication of EHV-1 at the epithelium of the URT and, thereby, the progression of the virus to the peripheral blood. The findings improve the mechanistic understanding of mucosal immunity against EHV-1 and can support the development of enhanced diagnostic tools, vaccines against EHM, and the management of EHV-1 outbreaks.

Optimization of a bovine cytokine multiplex assay using a new bovine and cross-reactive equine monoclonal antibodies.

Cytokines are important markers for immune activation, regulation, and homeostasis. The lack of monoclonal antibodies (mAbs) and sensitive assays to evaluate cytokine secretion has hindered research of bovine inflammation and immune regulation. We recently developed a fluorescent bead-based multiplex assay (multiplex assay) for bovine IL-10, TNF-α, and IFN-γ. Although the original assay covers a broad concentration range for the 3 targets, analytical sensitivity for IL-10 and IFN-γ could be improved to facilitate detection of these cytokines in their physiological low pg/mL range. To optimize the multiplex assay, we generated a new bovine IL-10 mAb and explored its use for the detection of intracellular and secreted bovine IL-10. The new bovine IL-10 mAb 130 recognized recombinant bovine IL-10 fusion protein and did not react with the fusion protein tag, or the TNF-α and IFN-γ standards in the multiplex assay. For improving IFN-γ detection, we explored cross-reactivity of anti-equine IFN-γ mAbs by intracellular staining of bovine stimulated peripheral blood mononuclear cells (PBMC). Equine IFN-γ mAb 3 showed excellent cross-reactivity with bovine IFN-γ by intracellular detection. Adding IL-10 mAb 130 and IFN-γ mAb 3 to the bovine multiplex assay substantially improved the analytical sensitivity with lower limits of detection in the low pg/mL range for all analytes. The detection ranges for the optimized multiplex assay were determined as 2 - 134,000 pg/mL for IL-10, 8 - 127,000 pg/mL for IFN-γ, and 12 - 193,000 pg/mL for TNF-α. The assay was next used to measure cytokine concentrations in cell culture supernatants from PBMC stimulated in plasma from whole blood stimulation to confirm native IL-10, TNF-α, and IFN-γ recognition and to explore the upper detection limits of the assay. In PBMC stimulation with a mix of phorbol myristate acetate (PMA) and ionomycin resulted in highest cytokine concentrations, while in plasma from whole blood stimulation, highest concentrations were observed in samples stimulated with a mix of lipopolysaccharide (LPS), phytohemagglutinin (PHA), and the TLR-2/6 agonist Pam2Csk4. PBMC and whole blood stimulation protocols showed that the optimized multiplex assay covers a wide linear detection range for measuring cytokine concentrations in bovine samples. For whole blood stimulation, a cocktail of pathogen associated molecular patterns elicited a stronger cytokine response than a mix of PMA and ionomycin, but response varied considerably between individual cattle. In conclusion, optimizing the bovine cytokine assay with new reagents improved the lower detection limits and widened the linear detection ranges while lowering the background of the multiplex assay.

Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.

Introduction: Severe 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. Methods: Aiming 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 discussion: For 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.

SARS-CoV-2 serosurvey of healthy, privately owned cats presenting to a New York City animal hospital in the early phase of the COVID-19 pandemic (2020-2021).

Both domestic and non-domestic cats are now established to be susceptible to infection by SARS-CoV-2, the cause of the ongoing COVID-19 pandemic. While serious disease in cats may occur in some instances, the majority of infections appear to be subclinical. Differing prevalence data for SARS-CoV-2 infection of cats have been reported, and are highly context-dependent. Here, we report a retrospective serological survey of cats presented to an animal practice in New York City, located in close proximity to a large medical center that treated the first wave of COVID-19 patients in the US in the Spring of 2020. We sampled 79, mostly indoor, cats between June 2020 to May 2021, the early part of which time the community was under a strict public health "lock-down". Using a highly sensitive and specific fluorescent bead-based multiplex assay, we found an overall prevalence of 13/79 (16%) serologically-positive animals for the study period; however, cats sampled in the Fall of 2020 had a confirmed positive prevalence of 44%. For SARS-CoV-2 seropositive cats, we performed viral neutralization test with live SARS-CoV-2 to additionally confirm presence of SARS-CoV-2 specific antibodies. Of the thirteen seropositive cats, 7/13 (54%) were also positive by virus neutralization, and 2 of seropositive cats had previously documented respiratory signs, with high neutralization titers of 1:1024 and 1:4096; overall however, there was no statistically significant association of SARS-CoV-2 seropositivity with respiratory signs, or with breed, sex or age of the animals. Follow up sampling of cats, while limited in scope, showed that positive serological titers were maintained over time. In comparison, we found an overall confirmed positive prevalence of 51% for feline coronavirus (FCoV), an endemic virus of cats, with 30% confirmed negative for FCoV. We demonstrate the impact of SARS-CoV in a defined feline population during the first wave of SARS-CoV-2 infection of humans, and suggest that human-cat transmission was substantial in our study group. Our data provide a new context for SARS-CoV-2 transmission events across species.

Representative Public Health Surveys Pose Several Challenges: Lessons Learned Across 9 Communities During the COVID-19 Pandemic.

Community surveillance surveys offer an opportunity to obtain important and timely public health information that may help local municipalities guide their response to public health threats. The objective of this paper is to present approaches, challenges, and solutions from SARS-CoV-2 surveillance surveys conducted in different settings by 2 research teams. For rapid assessment of a representative sample, a 2-stage cluster sampling design was developed by an interdisciplinary team of researchers at Oregon State University between April 2020 and June 2021 across 6 Oregon communities. In 2022, these methods were adapted for New York communities by a team of veterinary, medical, and public health practitioners. Partnerships were established with local medical facilities, health departments, COVID-19 testing sites, and health and public safety staff. Field staff were trained using online modules, field manuals describing survey methods and safety protocols, and in-person meetings with hands-on practice. Private and secure data integration systems and public awareness campaigns were implemented. Pilot surveys and field previews revealed challenges in survey processes that could be addressed before surveys proceeded. Strong leadership, robust trainings, and university-community partnerships proved critical to successful outcomes. Cultivating mutual trust and cooperation among stakeholders is essential to prepare for the next pandemic.

Inflammatory markers for differentiation of endometritis in the mare.

BACKGROUND: Endometrial biopsy is required to diagnose mares with chronic endometritis and endometrial degenerative fibrosis. An increase in understanding of equine reproductive immunology could be utilised to create less-invasive, time-efficient diagnostic tools especially when evaluating mares for chronic endometritis. OBJECTIVES: To evaluate inflammatory cytokine and chemokine concentrations in uterine fluid samples collected by low-volume lavage (LVL) as a potential screening diagnostic biomarker for endometritis. STUDY DESIGN: Prospective cross-sectional clinical study. METHODS: Forty-six mares underwent a LVL and subsequently endometrial biopsy. Mares were split in three groups: healthy, acute endometritis, and chronic endometrial fibrosis (CEF) based on cytological and histological evaluation. A fluorescent bead-based multiplex assay for IFN-γ, IFN-α, IL-1ß, IL-4, IL-10, IL-17, sCD14, TNF-α, CCL2, CCL3, CCL5 and CCL11 were carried out on the LVL fluid. The endometrial biopsy was utilised for histology and qPCR of IFN-γ, IL-1ß, IL-6, IL-8, IL-17, TNF-α, CCL2 and CCL3 genes. Statistical analyses examined differences in inflammatory markers and predictive modelling for diseased endometrium. RESULTS: Secreted concentrations of IFN-γ were lower in LVL fluid from reproductively healthy mares compared with acute endometritis (p = 0.04) and CEF (p = 0.006). Additionally, IL-17, IL-10, IL-1ß, TNF-α, CCL2, CCL3, CCL5 and CCL11 were significantly increased (p ≤ 0.04) in LVL from CEF mares compared with healthy mares. Mares with CCL2 concentrations ≥550 pg/mL (14/14) had 100% probability of having CEF and/or acute endometritis. Healthy mares had lower relative abundance of IL-17 mRNA compared with mares in CEF group [median (interquartile rage) = 14.76 (13.3, 15.3) and 12.4 (10.54, 13.81)], respectively (p = 0.02). MAIN LIMITATIONS: Limited sample size: larger numbers of mares with and without endometritis are required and reference intervals in LVL samples have to be established. CONCLUSIONS: Inflammatory chemokines and cytokines concentrations differed between healthy mares and mares with acute endometritis or CEF in LVL.

Comparison of antibody and antigen response to intranasal and intramuscular EHV-1 modified-live vaccination in healthy adult horses.

During neurological EHV-1 outbreaks, modified-live vaccines (MLV) are often administrated intranasally in an off-label fashion to healthy cohort horses in order to achieve rapid mucosal immunity. Thus, the goal of the present study was to determine if a commercially available EHV-1 MLV given intranasally to healthy horses would trigger a measurable systemic and/or mucosal antibody response. Eight healthy adult horses were given the EHV-1 MLV vaccine intranasally, while 8 healthy adult horses received the vaccine intramuscularly. An additional 8 healthy horses served as unvaccinated controls. EHV-1 specific antibodies (total IgG, IgG4/7, IgG1 and IgA) were measured in blood and nasal secretions prior to vaccine administration and 14- and 30-days post-vaccine administration. Further, nasal secretions and whole blood were tested for the presence of EHV-1 DNA by qPCR prior to and 5 days after vaccine administration. EHV-1 was detected by qPCR for the first 48 hours post-intranasal vaccine administration in nasal secretions in a total of three horses. Total EHV-1 IgG and IgG4/7 antibody values in serum increased only in horses receiving the intramuscular MLV. Antibody values at 14- and 30-days post vaccine administration were not different from values prior to vaccine administration in horses receiving the intranasal vaccine. The results support the intramuscular use of the EHV-1 MLV as recommended by the manufacturer. Intranasal vaccination with the study-specific EHV-1 MLV did not induce an increase in systemic or nasal antibodies, therefore, this vaccine route seems suboptimal and should not be used to vaccinate adult horses that have received multiple EHV-1 vaccinations and have pre-existing antibodies against EHV-1.

IgE-binding monocytes upregulate the coagulation cascade in allergic horses.

IgE-binding monocytes are a rare peripheral immune cell type involved in the allergic response through binding of IgE on their surface. IgE-binding monocytes are present in both healthy and allergic individuals. We performed RNA sequencing to ask how the function of IgE-binding monocytes differs in the context of allergy. Using a large animal model of allergy, equine Culicoides hypersensitivity, we compared the transcriptome of IgE-binding monocytes in allergic and non-allergic horses at two seasonal timepoints: (i) when allergic animals were clinical healthy, in the winter "Remission Phase", and (ii) during chronic disease, in the summer "Clinical Phase". Most transcriptional differences between allergic and non-allergic horses occurred only during the "Remission Phase", suggesting principal differences in monocyte function even in the absence of allergen exposure. F13A1, a subunit of fibrinoligase, was significantly upregulated at both timepoints in allergic horses. This suggested a role for increased fibrin deposition in the coagulation cascade to promote allergic inflammation. IgE-binding monocytes also downregulated CCR10 expression in allergic horses during the "Clinical Phase", suggesting a defect in maintenance of skin homeostasis, which further promotes allergic inflammation. Together, this transcriptional analysis provides valuable clues into the mechanisms used by IgE-binding monocytes in allergic individuals.

A linear SARS-CoV-2 DNA vaccine candidate reduces virus shedding in ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 760 million cases and over 6.8 million deaths as of March 2023. Vaccination has been the main strategy used to contain the spread of the virus and to prevent hospitalizations and deaths. Currently, two mRNA-based vaccines and one adenovirus-vectored vaccine have been approved and are available for use in the U.S. population. The versatility, low cost, and rapid production of DNA vaccines provide important advantages over other platforms. Additionally, DNA vaccines efficiently induce both B- and T-cell responses by expressing the antigen within transfected host cells, and the antigen, after being processed into peptides, can associate with MHC class I or II of antigen-presenting cells (APCs) to stimulate different T cell responses. However, the efficiency of DNA vaccination needs to be improved for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been used successfully in the field of veterinary oncology, resulting in high rates of response after electrochemotherapy. Here, we evaluate the safety, immunogenicity, and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate delivered by intramuscular injection followed by electroporation (Vet-ePorator™) in ferrets. The linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects. Additionally, the vaccine elicited neutralizing antibodies and T cell responses on day 42 post-immunization using a low dose of the linear DNA construct in a prime-boost regimen. Most importantly, vaccination significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model.

IgE+ plasmablasts predict the onset of clinical allergy.

Introduction: IgE+ plasmablasts develop following allergen exposure and B cell activation. They secrete IgE and therefore are directly linked to maintain the mechanisms of IgE-mediated allergies. Here, we show that the presence of IgE+ plasmablasts in peripheral blood not only coincides with clinical allergy, but also predicts the upcoming development of clinical disease. Methods: Using an equine model of naturally occurring allergy, we compared the timing of allergen exposure, arrival of IgE+ plasmablasts in peripheral blood, and onset of clinical disease. Results: We found that IgE+ plasmablasts predict the development of clinical allergy by at least 3 weeks and can be measured directly by flow cytometry or by IgE secretion following in vitro culture. We also compared the IgE secretion by IgE+ plasmablasts with total plasma IgE concentrations and found that while IgE secretion consistently correlates with clinical allergy, total plasma IgE does not. Discussion: Together, we describe IgE+ plasmablasts as a reliable and sensitive predictive biomarker of allergic disease development.

Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study.

Introduction: Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses. Methods: Proteins from Dermatophagoides pteronyssinus (Der p) were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses' sera (n=5 per group). Results: Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses' sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics' sera on four but higher binding with healthy horses' sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses' serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA. Discussion: Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.

Populations of NK Cells and Regulatory T Cells in the Endometrium of Cycling Mares-A Preliminary Study.

Endometrial immune cells are essential to support uterine functions across the estrous cycle and in preparation for pregnancy. It has been acknowledged that changes in phenotype and/or numbers of lymphocytes, such as regulatory T cells (Tregs) and NK cells, might result in lower fertility in women and mice. Little is known about equine endometrial immune cells across the estrous cycle. Here, we compared the populations of endometrial Tregs and NK cells in estrus and diestrus in mares. Endometrial biopsy and blood samples were taken in estrus and diestrus from 11 mares ages 4-12 years. Flow cytometry with anti-CD4, -CD25 and -FOXP3 and anti-NKp46 and -CD3 antibodies was used to determine the populations of Tregs and NK cells, respectively. The concentration of progesterone was measured with chemiluminescence immunoassay. The results were analyzed with paired Student t tests. The mean percentage of endometrial CD4+FOXP3+ Tregs was 13.7 ± 6.2% in diestrus and 14.5 ± 5.9% in estrus, while the mean percentage of endometrial CD4+FOXP3+CD25+ Tregs changed from 3.6 ± 2.1% in diestrus to 2 ± 2% in estrus (p = 0.0947). The mean proportion of CD3-NKp46+ lymphocytes in the endometrium was not significantly different, with 6 ± 1% in estrus and 6.5 ± 1.4% in diestrus. There was a large variation in the percentage of NK cells between mares of 2.1-12.7%. This study showed, for the first time, the presence of CD4+FOXP3+CD25+ Tregs and CD3-NKp46+ NK cells in the endometrium of non-pregnant cycling mares. The percentage of Tregs, and to a greater extent NK cells, showed large fluctuations between mares. Both Tregs and NK cells might be important for the preparation of the endometrium for semen deposition and pregnancy; however, further research is required.

Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant.

The quantification of cytokines can improve our understanding of immune response and inflammation dynamics in dairy cows. Bead-based assays provide a sensitive, high-throughput platform, allowing for simultaneous quantification of multiple cytokines within a wide linear detection range. Our objective was to develop a multiplex bead-based assay using monoclonal antibodies for simultaneous quantification of bovine tumor necrosis factor (TNF)-α, IL-10, and IFN-γ in plasma and peripheral blood mononuclear cell (PBMC) culture supernatants. Recombinant cytokine standards produced in mammalian cells were used to determine the lower limit of detection and the linear detection range for each cytokine. The lower limit of detection was 110 pg/mL for IL-10, 95 pg/mL for TNF-α, and 20 pg/mL for IFN-γ. The linear quantification range was 110 to 241,000 pg/mL for IL-10, 95 to 620,000 pg/mL for TNF-α, and 20 to 130,000 pg/mL for IFN-γ. All 3 monoclonal capture and detection antibodies were specific for their respective cytokine analyte when using the recombinant IL-10, TNF-α, and IFN-γ standards. Intraassay and interassay coefficients of variation (CV) were <10% and <12%, respectively, for all analytes and samples matrices. Next, concentrations of native cytokines were determined in PBMC culture supernatants (n = 4) and in plasma from whole-blood samples (n = 6) with or without stimulation with Escherichia coli lipopolysaccharide or a mix of phorbol myristate acetate (PMA) and ionomycin. Peak concentrations of all 3 cytokines were secreted from PBMC after PMA/ionomycin stimulation (TNF-α, 8 h, range: 39,266-506,422 pg/mL; IL-10, 18 h, range: 15,770-63,415 pg/mL; IFN-γ 18 h, range: 189,977-492,659 pg/mL). In contrast, the highest concentrations in plasma from whole-blood stimulation were observed for IL-10 and TNF-α after LPS stimulation (TNF-α, 4 h, range: 1,764-13,460 pg/mL; IL-10, 24 h, range: 2,401-6,371 pg/mL), whereas PMA and ionomycin induced the highest secretion of IFN-γ (18 h, range: 53-20,215 pg/mL). In conclusion, the multiplex assay can quantify native IL-10, TNF-α, and IFN-γ across a broad concentration range in bovine plasma and cell culture supernatant, thereby providing a novel tool to evaluate inflammatory profiles in cattle and especially in dairy cows with inflammatory conditions. The existing multiplex assay can be expanded in the future by adding bead assays for additional bovine cytokines.

Influence of sperm cryopreservation on sperm motility and proAKAP4 concentration in mice.

Background: The protein proAKAP4 is crucial for sperm motility and has been suggested as an indicator of male fertility. We determined the relationship between proAKAP4 concentration and sperm motility parameters in mice, and investigated the effects of cryopreservation on these variables. Methods: Computer-assisted sperm analysis and ELISA were applied to determine sperm motility and proAKAP4 concentration in fresh and frozen-thawed epididymal sperm of SWISS, B6D2F1, C57BL/6N, and BALB/c mice. Results: ProAKAP4 levels ranged between 12 and 89 ng/ml and did not differ between fresh and frozen-thawed samples, or between strains. We found a negative relationship between proAKAP4 levels and some sperm motility parameters. Sperm traits differed between strains, and cryopreservation negatively affected sperm velocity but not sperm direction parameters. Conclusion: ProAKAP4 levels in epididymal mouse spermatozoa were unaffected by cryopreservation, highlighting the robustness of this parameter as a potentially time-independent marker for sperm motility and fertility. The high individual variation in proAKAP4 levels supports the potential role of proAKAP4 as a marker for sperm quality, though we found no positive, and even negative relationships between proAKAP4 levels and some sperm motility parameters. Future studies have to investigate the significance of proAKAP4 as an indicator for fertility in mice.

Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences.

Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were analyzed. Using FC, BAL cells and PBMC were comprehensively characterized by cell surface markers ex vivo. An increased percentage of DH24A+ polymorphonuclear cells, and decreased percentages of CD14+ macrophages were detected in BAL from horses with sEA compared to healthy horses or horses with mEA, while lymphocyte proportions were similar between all groups. Independently of EA, macrophages in BAL were CD14+CD16+, which contrasts the majority of CD14+CD16- classical monocytes in PBMC. Percentages of CD16-expressing BAL macrophages were reduced in BAL from horses with sEA compared to healthy horses. While PBMC lymphocytes predominantly contain CD4+ T cells, B cells and few CD8+ T cells, BAL lymphocytes comprised mainly CD8+ T cells, fewer CD4+ T cells and hardly any B cells. These lymphocyte subsets' distributions were similar between all groups. After PMA/ionomycin stimulation in vitro, lymphocyte activation (CD154 and T helper cell cytokine expression) was analyzed in BAL cells of 26 of the horses and group differences were observed (p=0.01-0.11). Compared to healthy horses' BAL, CD154+ lymphocytes from horses with mEA, and CD4+IL-17A+ lymphocytes from horses with sEA were increased in frequency. Activated CD4+ T helper cells were more frequent in asthmatics' (mEA, sEA) compared to healthy horses' PBMC lymphocytes. In summary, FC analysis of BAL cells identified increased polymorphonuclear cells frequencies in sEA as established, while macrophage percentages were mildly reduced, and lymphocyte populations remained unaffected by EA. Cytokine production differences of BAL lymphocytes from horses with sEA compared to healthy horses' cells point towards a functional difference, namely increased local type 3 responses in sEA.

Peripheral CD23hi/IgE+ Plasmablasts Secrete IgE and Correlate with Allergic Disease Severity.

Production and secretion of IgE by B cells, plasmablasts, and plasma cells is a central step in the development and maintenance of allergic diseases. IgE can bind to one of its receptors, the low-affinity IgE receptor CD23, which is expressed on activated B cells. As a result, most B cells bind IgE through CD23 on their surface. This makes the identification of IgE producing cells challenging. In this study, we report an approach to clearly identify live IgE+ plasmablasts in peripheral blood for application by both flow cytometry analysis and in vitro assay. These IgE+ plasmablasts readily secrete IgE, upregulate specific mRNA transcripts (BLIMP-1 IRF4, XBP1, CD138, and TACI), and exhibit highly differentiated morphology all consistent with plasmablast differentiation. Most notably, we compared the presence of IgE+ plasmablasts in peripheral blood of allergic and healthy individuals using a horse model of naturally occurring seasonal allergy, Culicoides hypersensitivity. The model allows the comparison of immune cells both during periods of clinical allergy and when in remission and clinically healthy. Allergic horses had significantly higher percentages of IgE+ plasmablasts and IgE secretion while experiencing clinical allergy compared with healthy horses. Allergy severity and IgE secretion were both positively correlated to the frequency of IgE+ plasmablasts in peripheral blood. These results provide strong evidence for the identification and quantification of peripheral IgE-secreting plasmablasts and provide a missing cellular link in the mechanism of IgE secretion and upregulation during allergy.