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CD45 isoforms have been identified in a variety of different species and mab against various isoforms have been instrumental to define cellular subsets. In the process of generating novel mab against chicken γδ T cells two mab with specificity for CD45 were identified and characterized. The analysis of the chicken CD45 genomic structure suggested three exons being involved in alternative splicing. We cloned and expressed the full length CD45 isoform and three shorter isoforms. While the 7D12 mab reacted with all of these isoforms, the 8B1 mab selectively reacted with two short isoforms lacking either exons 3 and 5 or exons 3, 5 and 6. As expected, the reactivity of 7D12 included all leukocyte subsets, also including thrombocytes. In contrast, the 8B1 mab only reacted with lymphocytes and monocytes. 8B1 expression was found on almost all blood αβ T cells, while a γδ T cell subset and virtually all B cells lacked 8B1 reactivity. The fraction of 8B1 - αβ and γδ cells was larger in splenocytes as compared to PBL and there was also a population of 8B1 + splenic B cells. CD3 stimulation of splenic T cells resulted in upregulation of the 8B1 antigen on all T cells. Three-color immunofluorescence revealed differences in CD28 expression between the 8B1⁺ and 8B1¯ γδ T cell subsets with a higher CD28 expression level on 8B1¯ cells. The CD28 antigen was upregulated upon stimulation of the cells with IL-2 and IL-12. This novel mab will be a useful tool to further analyze chicken γδ T cells in more detail. [ABSTRACT FROM AUTHOR]

Retinal Müller glial cells (RMG) are involved in virtually every retinal disease; however, the role of these glial cells in neuroinflammation is still poorly understood. Since cell surface proteins play a decisive role in immune system signaling pathways, this study aimed at characterizing the changes of the cell surface proteome of RMG after incubation with prototype immune system stimulant lipopolysaccharide (LPS). While mass spectrometric analysis of the human Müller glia cell line MIO-M1 revealed 507 cell surface proteins in total, with 18 proteins significantly more abundant after stimulation (ratio ≥ 2), the surfaceome of primary RMG comprised 1425 proteins, among them 79 proteins with significantly higher abundance in the stimulated state. Pathway analysis revealed notable association with immune system pathways such as "antigen presentation", "immunoregulatory interactions between a lymphoid and a non-lymphoid cell" and "cell migration". We could demonstrate a higher abundance of proteins that are usually ascribed to antigen-presenting cells (APCs) and function to interact with T-cells, suggesting that activated RMG might act as atypical APCs in the course of ocular neuroinflammation. Our data provide a detailed description of the unstimulated and stimulated RMG surfaceome and offer fundamental insights regarding the capacity of RMG to actively participate in neuroinflammation in the retina. [ABSTRACT FROM AUTHOR]

Desialylation of cell surface glycoproteins carried out by sialidases affects various immunological processes. However, the role of neuraminidase 1 (NEU1), one of the four mammalian sialidases, in inflammation and autoimmune disease is not completely unraveled to date. In this study, we analyzed the retinal expression of NEU1 in equine recurrent uveitis (ERU), a spontaneous animal model for autoimmune uveitis. Mass spectrometry revealed significantly higher abundance of NEU1 in retinal Müller glial cells (RMG) of ERU-diseased horses compared to healthy controls. Immunohistochemistry uncovered NEU1 expression along the whole Müller cell body in healthy and uveitic states and confirmed higher abundance in inflamed retina. Müller glial cells are the principal macroglial cells of the retina and play a crucial role in uveitis pathogenesis. To determine whether higher expression levels of NEU1 in uveitic RMG correlate with the desialylation of retinal cells, we performed lectin-binding assays with sialic acid-specific lectins. Through these experiments, we could demonstrate a profound loss of both α2-3- and α2-6-linked terminal sialic acids in uveitis. Hence, we hypothesize that the higher abundance of NEU1 in uveitic RMG plays an important role in the pathogenesis of uveitis by desialylation of retinal cells. As RMG become activated in the course of uveitis and actively promote inflammation, we propose that NEU1 might represent a novel activation marker for inflammatory RMG. Our data provide novel insights in the expression and implication of NEU1 in inflammation and autoimmune disease. [ABSTRACT FROM AUTHOR]

Aquaporins (AQPs) are small integral membrane proteins with 13 members in mammals and are essential for water transport across membranes. They are found in many different tissues and cells. Currently, there are conflicting results regarding retinal aquaporin expression and subcellular localization between genome and protein analyses and among various species. AQP4, 7, 9 and 11 were described in the retina of men; whereas AQP6, 8 and 10 were earlier identified in rat retinas and AQP4, 5 and 11 in horses. Since there is a lack of knowledge regarding AQP expression on protein level in retinas of different animal models, we decided to analyze retinal cellular expression of AQP4, 5 and 11 in situ with immunohistochemistry. AQP4 was detected in all 15 explored species, AQP5 and AQP11 in 14 out of 15. Interestingly, AQP4 was unambiguously expressed in Muller glial cells, whereas AQP5 was differentially allocated among the species analyzed. AQP11 expression was Muller glial cell-specific in 50% of the animals, whereas in the others, AQP11 was detected in ganglion cell layer and at photoreceptor outer segments. Our data indicate a disparity in aquaporin distribution in retinas of various animals, especially for AQP5 and 11. [ABSTRACT FROM AUTHOR]

• Plant lectins bind to MAP with high affinity. • Several of these lectins showed no interference with milk and non-target bacteria. • Concanavalin A, lentil and banana lectin are able to bind to intact MAP cells. [ABSTRACT FROM AUTHOR]

Background: Müller glial cells are important regulators of physiological function of retina. In a model disease of retinal inflammation and spontaneous recurrent uveitis in horses (ERU), we could show that retinal Müller glial cells significantly change potassium and water channel protein expression during autoimmune pathogenesis. The most significantly changed channel protein in neuroinflammatory ERU was aquaporin 11 (AQP11). Aquaporins (AQP, 13 members) are important regulators of water and small solute transport through membranes. AQP11 is an unorthodox member of this family and was assigned to a third group of AQPs because of its difference in amino acid sequence (conserved sequence is only 11 %) and especially its largely unknown function.Methods: In order to gain insight into the distribution, localization, and function of AQP11 in the retina, we first developed a novel monoclonal antibody for AQP11 enabling quantification, localization, and functional studies.Results: In the horse retina, AQP11 was exclusively expressed at Müller glial cell membranes. In uveitic condition, AQP11 disappeared from gliotic Müller cells concomitant with glutamine synthase. Since function of AQP11 is still under debate, we assessed the impact of AQP11 channel on cell volume regulation of primary Müller glial cells under different osmotic conditions. We conclude a concomitant role for AQP11 with AQP4 in water efflux from these glial cells, which is disturbed in ERU. This could probably contribute to swelling and subsequent severe complication of retinal edema through impaired intracellular fluid regulation.Conclusions: Therefore, AQP11 is important for physiological Müller glia function and the expression pattern and function of this water channel seems to have distinct functions in central nervous system. The significant reduction in neuroinflammation points to a crucial role in pathogenesis of autoimmune uveitis. [ABSTRACT FROM AUTHOR]

Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease. [ABSTRACT FROM AUTHOR]

Objective In equine recurrent uveitis (ERU), immune reactions are directed toward known antigens like S-antigen, interphotoreceptor retinoid-binding protein, and cellular retinalaldehyde-binding protein, and anti-retinal antibodies were detected in vitreous samples. The aim of this study was the investigation of intraocular immunoglobulin M (IgM) reactivities to retinal proteome. Procedures Retina was separated by one- and two-dimensional gel electrophoresis and blotted semidry on PVDF membranes. To identify intraocular IgM antibody responses to retinal tissue, blots were incubated with vitreous samples of ERU-diseased horses ( n = 50) and healthy controls ( n = 30), followed by an HRP-labeled secondary antibody specific for equine IgM. Noticeable 2D western blot signals were aligned on a 2D gel of retinal proteome, excised, and subsequently identified by tandem mass spectrometry. Results Interestingly, frequent and very miscellaneous IgM response patterns to the retinal proteome in 68% of ERU vitreous samples were detected. Binding of IgM antibodies was localized at 17 different molecular weights. The most frequently detected signal, in 21 of the 50 samples, was located at 49 kDa. Comparing the samples interindividually between one and up to nine different signals in one sample could be observed. All healthy vitreous samples were devoid of IgM antibodies. Analysis of targeted spots with mass spectrometry led to the clear identification of 11 different proteins (corresponding to 16 different spots). One candidate could not be discovered so far. Conclusion The considerable IgM response to retinal proteins demonstrates an ongoing immune response, which might contribute to the remitting relapsing character of ERU. Novel identified target proteins point to a diverse response pattern of individual ERU cases. [ABSTRACT FROM AUTHOR]

Objective--To characterize and compare the urine protein content in cats without urinary tract disease and cats with idiopathic cystitis (IdC), bacterial urinary tract infection (UTI), or urolithiasis. Animals--Control cats (n = 18) and cats with IdC (18), UTI (12), and urolithiasis (12) from which urine samples were obtained and 2 cats with obstructive IdC and 4 additional control cats from which postmortem urinary bladder biopsy specimens were obtained. Procedures--Protein contents in urine samples obtained via cystocentesis or catheterization were measured via the Bradford method. Urine proteins were separated by means of 1-dimensional gel electrophoresis. Evaluation of fibronectin content was performed via western blotting and immunohistochemical analysis. Urinary bladder biopsy specimens were examined histologically and analyzed immunohistochemically for fibronectin. Results--Urine fibronectin content was significantly greater in cats with IdC, compared with control cat findings. Urine fibronectin contents did not differ significantly among controls and cats with UTI or urolithiasis. Histologic examination of bladder biopsy specimens obtained from 2 cats with obstructive IdC revealed destruction of the urothelial lining of the urinary bladder and Severe fibrosis; immunohistochemical analysis revealed few fluorescence signals for fibronectin, unlike findings in control bladder biopsy specimens. Conclusions and Clinical Relevance--Results indicated that urine fibronectin content in cats with IdC was greater than that in controls, cats with UTI, or cats with urolithiasis. In cats with IdC, increased permeability of damaged urothelium may result in detachment and leakage of fibronectin into urine. Urine fibronectin might serve as a biomarker for diagnosis of IdC in cats. [ABSTRACT FROM AUTHOR]

OBJECTIVE To obtain new insights into aetiological backgrounds, and to search for diagnostic biomarkers by assessing the difference in urinary proteins between cats with spontaneous feline idiopathic cystitis (FIC) and healthy controls. MATERIALS AND METHODS Urine supernatants of 18 cats with FIC and 18 healthy control cats, and bladder biopsies of two FIC diseased cats and four healthy controls were included in the study. The Bradford method was used to determine protein quantity in urine supernatants. Urine was separated by two-dimensional (2-D) gel electrophoresis. Selected protein spots were excised from two-dimensional gels and analysed with tandem mass spectrometry. Validation of Trefoil factor 2 expression was realized with Western blot and immunohistochemistry. Western blot signal intensities were quantified with IMAGE QUANT software. RESULTS Eleven differentially expressed protein spots were identified between the 2-D gels of cats with FIC and control cats. Ten spots (only visible in the FIC gel) were identified as albumin and one spot (only visible in the control gel) was identified as Trefoil factor 2.Using quantification of Western blot signal intensities and immunohistochemistry a decrease in Trefoil factor 2 (TFF2) in cats with FIC could be revealed for the first time. CONCLUSION Deficiency in TFF2 possibly leads to impaired repairing abilities and immune response of the urothelium. The result could be a greater susceptibility to injury, inflammation and relapse. Therefore TFF2 deficiency might be an important event in FIC pathogenesis. Detection of a decrease in urinary TFF2 could serve as diagnostic biomarker, facilitating diagnosis. As FIC can serve as an animal model for human painful bladder syndrome/ interstitial cystitis, the findings of this study might also be valuable for interstitial cystitis research and should be further investigated. [ABSTRACT FROM AUTHOR]

In life sciences, antibodies are among the most commonly used tools for identifying, tracking, quantifying and isolating molecules, mainly proteins. However, it has recently become clear that antibodies often fall short with respect to specificity and selectivity and in many cases target proteins are not even known. When commercial availability of antibodies is scarce, e.g. for targeting proteins from farm animals, researchers face additional challenges: they often have to rely on cross-reactive antibodies, which are poorly characterized for their exact target, their actual cross-reactivity and the desired application. In this study, we aimed at identifying the true target of mouse monoclonal antibody 8F2, which was generated against chicken PBMC and used for decades in research, while it's actual target molecule remained unknown. We used 8F2 antibody for immunoprecipitation in chicken PBMC and subsequently identified its true target as CD11d, which was never described in chicken lymphocytes before, by quantitative LC-MSMS. The most abundant interactor of CD11d was identified as integrin beta 2. The existence of this alpha integrin was therefore clearly proven on protein level and provides a first basis to further assess the role of CD11d in chickens in future studies. Data are available via ProteomeXchange with identifier PXD017248. Our studies determined CD11d as the true target of a previously uncharacterized mouse monoclonal antibody 8F2, generated against chicken peripheral blood derived mononuclear cells (PBMC). This is therefore now first member of alpha integrins in chickens, that existence was now clearly identified on protein level. The additional identification of CD11d interactors provides information on integrin-dependent regulation of signaling networks, allowing further functional studies. Unlabelled Image [ABSTRACT FROM AUTHOR]