Comparison of TGF-beta 1 concentrations in bronchoalveolar fluid of horses affected with heaves and of normal controls

Airway remodeling may play an important role in heaves pathophysiology. Transforming growth factor-beta 1 (TGF-beta1) is a potent profibrotic cytokine, which might contribute to airway wall thickening and fibrosis of bronchiolar and alveolar submucosa. An ELISA designed for the measurement of human TGF-beta1 was used to measured total TGF-beta1 released in bronchoalveolar lavage fluid (BALF) of normal horses and of those affected with heaves in remission. The specificity of the assay for TGF-beta1 of the horse was confirmed using recombinant equine TGF-beta1. The influence of hay exposure on TGF-beta1 release in the airways was also examined by stabling horses in a dusty environment. TGF-beta1 was found in the BALF of all horses. However, no significant difference between basal concentration of TGF-beta1 in BALF of control horses versus that of horses affected with heaves was found. Furthermore, no differences were identified in these populations 1 and 9 days after allergen challenge. In conclusion, these data indicate that TGF-beta1 is released in BALF fluid of horses in biologically active concentrations. Other studies are necessary for a better definition of the role of this cytokine within the lung, as our study does not establish a causal relationship between TGF-beta1 and the pathophysiology of heaves in the horse.     

Monoclonal antibodies to equine CD23 identify the low-affinity receptor for IgE on subpopulations of IgM+ and IgG1+ B-cells in horses

CD23, also called FcεRII, is the low-affinity receptor for IgE and has first been described as a major receptor regulating IgE responses. In addition, CD23 also binds to CD21, integrins and MHC class II molecules and thus has a much wider functional role in immune regulation ranging from involvement in antigen-presentation to multiple cytokine-like functions of soluble CD23. The role of CD23 during immune responses of the horse is less well understood. Here, we expressed equine CD23 in mammalian cells using a novel IL-4 expression system. Expression resulted in high yield of recombinant IL-4/CD23 fusion protein which was purified and used for the generation of monoclonal antibodies (mAbs) to equine CD23. Seven anti-CD23 mAbs were further characterized. The expression of the low-affinity IgE receptor on equine peripheral blood mononuclear cells was analyzed by flow cytometric analysis. Cell surface staining showed that CD23 is mainly expressed by a subpopulation of equine B-cells. Only a very few equine T-cells or monocytes expressed CD23. CD23(+) B-cells were either IgM(+) or IgG1(+) cells. All CD23(+) cells were also positive for cell surface IgE staining suggesting in vivo IgE binding by the receptor. Two of the CD23 mAbs detected either the complete extracellular region of CD23 or a 22kDa cleavage product of CD23 by Western blotting. The new anti-CD23 mAbs provide valuable reagents to further analyze the roles of CD23 during immune responses of the horse in health and disease.     

Generation and characterization of monoclonal antibodies to equine CD16

The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the closely related Fc receptor CD32 expressed in the same system. In flow cytometric analysis with equine peripheral leukocytes, the mAbs labeled cells in the granulocyte, monocyte, and lymphocyte populations in a pattern consistent with other species. Monocytes that were strongly labeled with CD16 mAb 9G5 were also positive for the LPS receptor CD14. Cytospins made with peripheral leukocytes were immunohistochemically labeled and showed mAb recognition of primarily mononuclear cells. ELISA revealed that the nine mAbs can be grouped into three patterns of epitope recognition. These new antibodies will serve as useful tools in the investigation of equine immune responses and inflammatory processes.     

Development and characterization of mouse monoclonal antibodies reactive with chicken CD83

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein containing the extracellular region of chCD83 was expressed in Chinese Hamster Ovary (CHO) cells and isolated from the spent cell culture medium by protein G affinity chromatography. The extracellular region of the chCD83 protein was purified and used to immunize mice. A cell fusion was performed, from which 342 hybridomas were screened for mAbs to chCD83. Two mAbs, chCD83-159 and chCD83-227, stained the greatest percentage of chCD83-transfected CHO cells and were selected for further characterization. By flow cytometry, both mAbs reacted with a chicken macrophage cell line, HD11. Both mAbs also recognized a single 53 kDa protein on Western blots of lysates from lipopolysaccharide-stimulated spleen mononuclear cells or unstimulated HD11 cells. Immunostaining of chicken secondary lymphoid organs identified chCD83(+) cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen mononuclear cells with concanavalin A (Con A) decreased the percentage of chCD83(+) cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII(+) cells compared with cells treated with an isotype-matched negative control mAb. These chCD83 mAbs may be useful for future investigations of chicken immune cell maturation and mechanisms of action.     

Sward patterns created by patch grazing are stable over more than a decade

Under extensive grazing, a mosaic pattern of frequently defoliated short patches and rarely defoliated tall patches is often formed. The agronomic and ecological consequences of this patch‐grazing pattern strongly depend on its stability between successive years. We assessed patch structure and temporal stability under three intensities of cattle stocking (moderate, lenient and very lenient) in a cattle grazing experiment established in 2002. Aerial images of the whole area taken in 2005, 2010, 2013 and 2015 were classified into short and tall patches using random forest classification. These were complemented by annual sward height measurements (2007‐2017) at 10 permanent plots per paddock, which were classified into sward height classes. The mean proportion (0.72, 0.32, 0.19) and size of short patches decreased with stocking intensity, while size of tall patches increased. Inter‐annual stability depended on patch type and stocking intensity and was particularly high for the respective dominant patch type. Of the short patch area in 2015, 0.62, 0.29 and 0.30 were classified as short in all four aerial images under moderate, lenient and very lenient grazing, respectively; the corresponding proportions for tall patches were 0.10, 0.53 and 0.65. Our results imply that short and tall patches experience persistent differences in local grazing intensity over extended periods. The long‐term effects of this heterogeneity on soil properties and vegetation composition need to be monitored to assess agronomic sustainability and ecological potential of patch‐grazed pastures.     

Cover Image,Volume 16, Issue 1

The cover image, by Alexandra Keller et al., is based on the Original Article The JAK2/STAT5 signaling pathway as a potential therapeutic target in canine mastocytoma, DOI: 10.1111/vco.12311

     

Improved cadmium sorption isotherms by the determination of initial contents using the radioisotope 109Cd

Cadmium (Cd) sorption isotherms were estimated by two different analytical approaches to assess the influence of initial Cd concentrations of soil matrix on the sorption of added Cd. For the laboratory experiments a heterogeneous set of samples was collected to include a wide range of different initial Cd concentrations. Comparison of both analytical methods (conventional analysis, radioanalysis) resulted in a strong conformity of Cd contents in solution at equilibrium. The calculated Cd concentrations in the soil solid phase differ according to the analytical approach for considering the initial contents. The determination of the initial contents by the proposed radioanalytical method with ¹⁰⁹Cd resulted in long linear Freundlich‐isotherms, even in the low concentration range. Thus, radioanalysis seems to be the most suitable method to recognise the initial contents of Cd in soil. EDTA extractable Cd represent the initial concentrations, which are averaged over solid and liquid phase. However, depending on the sorption characteristics of the soil these rates vary. In the investigated set of soil samples 52.3 to 99.3% of Cd must be added to the solid phase.