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Special attention is given to cow’s milk and its variants, with ongoing discussions about health-related impacts primarily focusing on the A1 variant in contrast to the A2 variant. The difference between these variants lies in a single amino acid alteration at position 67 of β-casein. This alteration is presumed to make the A1 variant more susceptible to enzymatic breakdown during milk digestion, leading to an increased release of the peptide β-casomorphin-7 (BCM-7). BCM-7 is hypothesized to interact with µ-opioid receptors on immune cells in humans. Although BCM-7 has demonstrated both immunosuppressive and inflammatory effects, its direct impact on the immune system remains unclear. Thus, we examined the influence of A1 and A2 milk on Concanavalin A (ConA)-stimulated human peripheral blood mononuclear cells (PBMCs), as well as the effect of experimentally digested A1 and A2 milk, containing different amounts of free BCM-7 from β-casein cleavage. Additionally, we evaluated the effects of pure BCM-7 on the proliferation of ConA-stimulated PBMCs and purified CD4+ T cells. Milk fundamentally inhibited PBMC proliferation, independent of the β-casein variant. In contrast, experimentally digested milk of both variants and pure BCM-7 showed no influence on the proliferation of PBMCs or isolated CD4+ T cells. Our results indicate that milk exerts an anti-inflammatory effect on PBMCs, regardless of the A1 or A2 β-casein variant, which is nullified after in vitro digestion. Consequently, we deem BCM-7 unsuitable as a biomarker for food-induced inflammation.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of bovine paratuberculosis, a chronic granulomatous enteritis leading to economic losses and posing a risk to human health due to its zoonotic potential. The pathogen cannot reliably be detected by standard methods, and immunological procedures during the infection are not well understood. Therefore, the aim of our study was to explore host–pathogen interactions in MAP-infected dairy cows and to improve diagnostic tests. Serum proteomics analysis using quantitative label-free LC-MS/MS revealed 60 differentially abundant proteins in MAP-infected dairy cows compared to healthy controls from the same infected herd and 90 differentially abundant proteins in comparison to another control group from an uninfected herd. Pathway enrichment analysis provided new insights into the immune response to MAP and susceptibility to the infection. Furthermore, we found a higher abundance of Cathepsin S (CTSS) in the serum of MAP-infected dairy cows, which is involved in multiple enriched pathways associated with the immune system. Confirmed with Western blotting, we identified CTSS as a potential biomarker for bovine paratuberculosis. This study enabled a better understanding of procedures in the host–pathogen response to MAP and improved detection of paratuberculosis-diseased cattle.

The perception of circulating granulocytes as cells with a predetermined immune response mainly triggered by pathogens is evolving, recognizing their functional heterogeneity and adaptability, particularly within the neutrophil subset. The involvement of these cells in the pathophysiology of autoimmune uveitis has become increasingly clear, yet their exact role remains elusive. We used an equine model for autoimmune-mediated recurrent pan-uveitis to investigate early responses of granulocytes in different inflammatory environments. For this purpose, we performed differential proteomics on granulocytes from healthy and diseased horses stimulated with IL8, LPS, or PMA. Compared to healthy horses, granulocytes from the recurrent uveitis model significantly changed the cellular abundance of 384 proteins, with a considerable number of specific changes for each stimulant. To gain more insight into the functional impact of these stimulant-specific proteome changes in ERU pathogenesis, we used Ingenuity Pathway Analysis for pathway enrichment. This resulted in specific reaction patterns for each stimulant, with IL8 predominantly promoting Class I MHC-mediated antigen processing and presentation, LPS enhancing processes in phospholipid biosynthesis, and PMA, clearly inducing neutrophil degranulation. These findings shed light on the remarkably differentiated responses of neutrophils, offering valuable insights into their functional heterogeneity in a T-cell-driven disease. Raw data are available via ProteomeXchange with identifier PXD013648.

The majority of peptides presented by MHC class I result from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides to MHC class I loading non-canonically. We used an immunopeptidomics workflow combined with prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect the proteasomal processing of immune epitopes presented by A549 cells. The treatment of A549 cells with IFNγ enhanced the proteasomal cleavage probability of MHC class I ligands for both the constitutive proteasome and the immunoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated the HLA allotype-specific proteasomal cleavage probabilities for MHC class I ligands, the peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely, HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. The peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue, suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases.

The aim of the study was to gain deeper insights in the potential of polyclonal stimulation of PBMC with banana lectin (BanLec) from Musa paradisiaca. BanLec induced a marked proliferative response in cow and pig PBMC, but was strongest in pigs, where it induced an even higher proliferation rate than Concanavalin A. Molecular processes associated with respective responses in porcine PBMC were examined with differential proteome analyses. Discovery proteomic experiments was applied to BanLec stimulated PBMC and cellular and secretome responses were analyzed with label free LC-MS/MS. In PBMC, 3955 proteins were identified. After polyclonal stimulation with BanLec, 459 proteins showed significantly changed abundance in PBMC. In respective PBMC secretomes, 2867 proteins were identified with 231 differentially expressed candidates as reaction to BanLec stimulation. The transcription factor “E74 like ETS transcription factor 1 (ELF1)” was solely enriched in BanLec stimulated PBMC. BanLec induced secretion of several immune regulators, amongst them positive regulators of activated T cell proliferation and Jak-STAT signaling pathway. Top changed immune proteins were CD226, CD27, IFNG, IL18, IL2, CXCL10, LAT, ICOS, IL2RA, LAG3, and CD300C. BanLec stimulates PBMC of cows and pigs polyclonally and induces IL2 pathway and further proinflammatory cytokines. Proteomics data are available via ProteomeXchange with identifier PXD027505.

Laron syndrome (LS) is a rare genetic disorder characterized by low levels of insulin-like growth factor 1 (IGF1) and high levels of growth hormone (GH) due to mutations in the growth hormone receptor gene (GHR). A GHR-knockout (GHR-KO) pig was developed as a model for LS, which displays many of the same features as humans with LS-like transient juvenile hypoglycemia. This study aimed to investigate the effects of impaired GHR signaling on immune functions and immunometabolism in GHR-KO pigs. GHR are located on various cell types of the immune system. Therefore, we investigated lymphocyte subsets, proliferative and respiratory capacity of peripheral blood mononuclear cells (PBMCs), proteome profiles of CD4− and CD4+ lymphocytes and IFN-α serum levels between wild-type (WT) controls and GHR-KO pigs, which revealed significant differences in the relative proportion of the CD4+CD8α− subpopulation and in IFN-α levels. We detected no significant difference in the respiratory capacity and the capacity for polyclonal stimulation in PBMCs between the two groups. But proteome analysis of CD4+ and CD4− lymphocyte populations revealed multiple significant protein abundance differences between GHR-KO and WT pigs, involving pathways related to amino acid metabolism, beta-oxidation of fatty acids, insulin secretion signaling, and oxidative phosphorylation. This study highlights the potential use of GHR-KO pigs as a model for studying the effects of impaired GHR signaling on immune functions.

Müller cells are the main macroglial cells of the retina exerting a wealth of functions to maintain retinal homoeostasis. Upon pathological changes in the retina, they become gliotic with both protective and detrimental consequences. Accumulating data also provide evidence for a pivotal role of Müller cells in the pathogenesis of diabetic retinopathy (DR). While microglial cells, the resident immune cells of the retina are considered as main players in inflammatory processes associated with DR, the implication of activated Müller cells in chronic retinal inflammation remains to be elucidated. In order to assess the signaling capacity of Müller cells and their role in retinal inflammation, we performed in-depth proteomic analysis of Müller cell proteomes and secretomes after stimulation with INFγ, TNFα, IL-4, IL-6, IL-10, VEGF, TGFβ1, TGFβ2 and TGFβ3. We used both, primary porcine Müller cells and the human Müller cell line MIO-M1 for our hypothesis generating approach. Our results point towards an intense signaling capacity of Müller cells, which reacted in a highly discriminating manner upon treatment with different cytokines. Stimulation of Müller cells resulted in a primarily pro-inflammatory phenotype with secretion of cytokines and components of the complement system. Furthermore, we observed evidence for mitochondrial dysfunction, implying oxidative stress after treatment with the various cytokines. Finally, both MIO-M1 cells and primary porcine Müller cells showed several characteristics of atypical antigen-presenting cells, as they are capable of inducing MHC class I and MHC class II with co-stimulatory molecules. In line with this, they express proteins associated with formation and maturation of phagosomes. Thus, our findings underline the importance of Müller cell signaling in the inflamed retina, indicating an active role in chronic retinal inflammation.

Bovine paratuberculosis is a serious chronic disease of the gastrointestinal tract that causes economic losses and dramatically affects animal health in livestock. The underlying infectious agent, Mycobacterium avium subspecies paratuberculosis (MAP), cannot reliably be detected by standard diagnostic tests due to the long asymptomatic disease stage. The aim of this study was to detect proteomic changes in peripheral blood mononuclear cells (PBMC) from cows of the same herd with different MAP infection status after co-incubation with viable MAP in vitro using label-free LC-MS/MS. In our proteomic discovery experiment, we detected 2631 differentially regulated proteins between cows with negative MAP infection status (so-called MAP-resistant cows) and cows with positive MAP infection status (so-called persistently MAP-infected cows). In MAP-resistant cows, we detected enriched immune-related signaling pathways for TLR2 and MHC class II component proteins, among others, indicating a successful defensive immune response of the cows to MAP. In contrast, persistently MAP-infected cows were not directly enriched in immune-related signaling pathways associated with ITGA2B and KCNMA1, among others. The introduction of these distinct immune responses contributes to a better understanding of the bovine immune response and mechanisms of susceptibility to MAP.

In the pathophysiology of autoimmune-mediated uveitis, granulocytes have emerged as possible disease mediators and were shown to be pre-activated in equine recurrent uveitis (ERU), a spontaneous disease model. We therefore used granulocytes from ERU horses to identify early molecular mechanisms involved in this dysregulated innate immune response. Primary granulocytes from healthy and ERU horses were stimulated with IL8, and cellular response was analyzed with differential proteomics, which revealed significant differences in protein abundance of 170 proteins in ERU. Subsequent ingenuity pathway analysis identified three activated canonical pathways “PKA signaling”, “PTEN signaling” and “leukocyte extravasation”. Clustered to the leukocyte extravasation pathway, we found the membrane-type GPI-anchored protease MMP25, which was increased in IL8 stimulated ERU granulocytes. These findings point to MMP25 as a possible regulator of granulocyte extravasation in uveitis and a role of this molecule in the impaired integrity of the blood-retina-barrier. In conclusion, our analyses show a clearly divergent reaction profile of pre-activated granulocytes upon IL8 stimulation and provide basic information for further in-depth studies on early granulocyte activation in non-infectious ocular diseases. This may be of interest for the development of new approaches in uveitis diagnostics and therapy. Raw data are available via ProteomeXchange with identifier PXD013648.

As one of the leading causes of blindness worldwide, uveitis is an important disease. The exact pathogenesis of autoimmune uveitis is not entirely elucidated to date. Equine recurrent uveitis (ERU) represents the only spontaneous animal model for autoimmune uveitis in humans. As the metabolism of immune cells is an emerging field in research and gains more and more significance to take part in the pathogenesis of various diseases, we conducted experiments to investigate the metabolism of immune cells of ERU cases and healthy controls. To our knowledge, the link between a deviant immunometabolism and the pathogenesis of autoimmune uveitis was not investigated so far. We showed that PBMC of ERU cases had a more active metabolic phenotype in basal state by upregulating both the oxidative phosphorylation and the glycolytic pathway. We further revealed an increased compensatory glycolytic rate of PBMC and CD4+ T cells of ERU cases under mitochondrial stress conditions. These findings are in line with metabolic alterations of immune cells in other autoimmune diseases and basic research, where it was shown that activated immune cells have an increased need of energy and molecule demand for their effector function. We demonstrated a clear difference in the metabolic phenotypes of PBMC and, more specifically, CD4+ T cells of ERU cases and controls. These findings are another important step in understanding the pathogenesis of ERU and figuratively, human autoimmune uveitis.

Background The underlying pathomechanisms in diabetic retinopathy (DR) remain incompletely understood. The aim of this study was to add to the current knowledge about the particular role of retinal Müller glial cells (RMG) in the initial processes of DR. Methods Applying a quantitative proteomic workflow, we investigated changes of primary porcine RMG under short term high glucose treatment as well as glycolysis inhibition treatment. Results We revealed significant changes in RMG proteome primarily in proteins building the extracellular matrix (ECM) indicating fundamental remodeling processes of ECM as novel rapid response to high glucose treatment. Among others, Osteopontin (SPP1) as well as its interacting integrins were significantly downregulated and organotypic retinal explant culture confirmed the selective loss of SPP1 in RMG upon treatment. Since SPP1 in the retina has been described neuroprotective for photoreceptors and functions against experimentally induced cell swelling, it’s rapid loss under diabetic conditions may point to a direct involvement of RMG to the early neurodegenerative processes driving DR. Data are available via ProteomeXchange with identifier PXD015879.

Horses worldwide suffer from equine recurrent uveitis (ERU), an organ-specific, immune-mediated disease with painful, remitting-relapsing inflammatory attacks alternating with periods of quiescence, which ultimately leads to blindness. In course of disease, both eyes can eventually be affected and since blind horses pose a threat to themselves and their surroundings, these animals have to be killed. Therefore, this disease is highly relevant for veterinary medicine. Additionally, ERU shows strong clinical and pathological resemblance to autoimmune uveitis in man. The exact cause for the onset of ERU is unclear to date. T cells are believed to be the main effector cells in this disease, as they overcome the blood retinal barrier to invade the eye, an organ physiologically devoid of peripheral immune cells. These cells cause severe intraocular inflammation, especially in their primary target, the retina. With every inflammatory episode, retinal degeneration increases until eyesight is completely lost. In ERU, T cells show an activated phenotype, with enhanced deformability and migration ability, which is reflected in the composition of their proteome and downstream interaction pathways even in quiescent stage of disease. Besides the dysregulation of adaptive immune cells, emerging evidence suggests that cells of the innate immune system may also directly contribute to ERU pathogenesis. As investigations in both the target organ and the periphery have rapidly evolved in recent years, giving new insights on pathogenesis-associated processes on cellular and molecular level, this review summarizes latest developments in ERU research.

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap in precise knowledge about altered immune mechanisms in this disease. Since diabetic INSC94Y transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they provide a favorable model to explore functional alterations of immune cells in an early stage of diabetes mellitus in vivo. Hence, we investigated peripheral blood mononuclear cells (PBMC) of these diabetic pigs compared to non-diabetic wild-type littermates. We found a 5-fold decreased proliferative response of T cells in INSC94Y tg pigs to polyclonal T cell mitogen phytohemagglutinin (PHA). Using label-free LC-MS/MS, a total of 3,487 proteins were quantified, and distinct changes in protein abundances in CD4+ T cells of early-stage diabetic pigs were detectable. Additionally, we found significant increases in mitochondrial oxygen consumption rate (OCR) and higher basal glycolytic activity in PBMC of diabetic INSC94Y tg pigs, indicating an altered metabolic immune cell phenotype. Thus, our study provides new insights into molecular mechanisms of dysregulated immune cells triggered by permanent hyperglycemia.

In recent years, a lack of stability of dairy products with extended shelf life (e.g., yoghurt products, UHT desserts) has occurred, with the corresponding products liquefying significantly after days or weeks. This project aimed to identify the enzymes responsible for the liquefaction of the affected products based on differential proteomic analyses. No evidence was found for the presence of starch-degrading bacteria in the affected products. With zymography and proteome analysis, we detected the cause of liquefaction in a pudding by contamination of its aroma component with an engineered amylolytic enzyme, cyclomaltodextrin glucanotransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes. In addition, we detected contamination with Pseudomonas-derived proteolytic ATP-dependent Clp protease in one pudding batch and proteases in technically used amylases, which degraded β-caseins in another batch. Identification of these agents with liquefying properties in dairy products are useful for adjustment of production protocols and/or composition of additives, and thus shelf life extension.

We recently identified a deviant bovine immune phenotype characterized by hyperproliferation of lymphocytes after polyclonal stimulation. This phenotype was first discovered in dams that responded to PregSure BVD vaccination by producing pathological antibodies, triggering the fatal disease “bovine neonatal pancytopenia” in calves. The aim of the study was to gain deeper insights into molecular processes occurring in lymphocytes of immune phenotypes and the effect on their secretome after immune stimulation. Two discovery proteomic experiments were performed with unstimulated and Pokeweed Mitogen (PWM) stimulated lymphocytes, using label-free LC-MS/MS. In lymphocytes, 2447 proteins were quantified, and 1204 proteins were quantified in the secretome. Quantitative proteome analysis of immune deviant and control samples after PWM stimulation revealed clear differences. The increase in abundance of IL17A, IL17F, IL8, CCL5, LRRC59, and CLIC4 was higher in controls through mitogenic stimulation. In contrast, the abundance of IFNγ, IL2, IL2RA, CD83, and CD200 increased significantly more in immune deviant lymphocytes. Additional pathway enrichment analysis of differentially secreted proteins also yielded fundamental differences between the immune phenotypes. Our study provides a comprehensive dataset, which gives novel insights into proteome changes of lymphocytes from different bovine immune phenotypes. These differences point to the development of diverse immune responses of bovine immune phenotypes after immune stimulation.

The participating signals and structures that enable primary immune cells migrating within dense tissues are not completely revealed until now. Especially in autoimmune diseases, mostly unknown mechanisms facilitate autoreactive immune cells to migrate to endogenous tissues, infiltrating and harming organ-specific structures. In order to gain deeper insights into the migratory behavior of primary autoreactive immune cells, we examined peripheral blood-derived lymphocytes (PBLs) of horses with equine recurrent uveitis (ERU), a spontaneous animal model for autoimmune uveitis in humans. In this study, we used a three-dimensional collagen I hydrogel matrix and monitored live-cell migration of primary lymphocytes as a reaction to different chemoattractants such as fetal calf serum (FCS), cytokines interleukin-4 (IL-4), and interferon-γ (IFN-γ), and a specific uveitis autoantigen, cellular retinaldehyde binding protein (CRALBP). Through these experiments, we uncovered distinct differences between PBLs from ERU cases and PBLs from healthy animals, with significantly higher cell motility, cell speed, and straightness during migration of PBLs from ERU horses. Furthermore, we emphasized the significance of expression levels and cellular localization of septin 7, a membrane-interacting protein with decreased abundance in PBLs of autoimmune cases. To underline the importance of septin 7 expression changes and the possible contribution to migratory behavior in autoreactive immune cells, we used forchlorfenuron (FCF) as a reversible inhibitor of septin structures. FCF-treated cells showed more directed migration through dense tissue and revealed aberrant septin 7 and F-actin structures along with different protein distribution and translocalization of the latter, uncovered by immunochemistry. Hence, we propose that septin 7 and interacting molecules play a pivotal role in the organization and regulation of cell shaping and migration. With our findings, we contribute to gaining deeper insights into the migratory behavior and septin 7-dependent cytoskeletal reorganization of immune cells in organ-specific autoimmune diseases.

The molecular mechanisms driving specific regulation of neutrophils are not completely understood to date. In order to characterize fundamental granulocyte features on protein level, we analyzed changes in proteome composition as reaction to stress from cell activation processes. For this purpose, we isolated primary granulocytes from equine whole blood through density gradient centrifugation followed by sodium chloride lysis and stimulated cells for 30 min with interleukin-8 (IL8) due to its role as a chemotactic factor for neutrophils. We additionally used phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), which are primarily associated to neutrophil extracellular trap formation and release of reactive oxygen species. From mass spectrometry analysis, we identified a total of 2,032 proteins describing the whole granulocyte proteome, including 245 proteins (12% of identified proteome) newly associated to in vivo expression in primary equine granulocytes (hypothetical proteins). We also found distinct and different changes in protein abundance (ratio ≥ 2) after short stimulation of cells with various stimuli, pointing to rapid and differentiated reaction pattern. IL8 stimulation resulted in increased protein abundance of 58 proteins (3% of proteome), whereas PMA induced changed protein abundance of 207 (10 % of proteome) and LPS of 46 proteins (2% of proteome). Enrichment analyses clearly showed fundamental differences between stimuli, with primary association of IL8 stimulation to processes in immune response, receptor signaling and signal transduction. Top enrichment for PMA on the other hand pointed to vesicle mediated transport and exocytosis. Stimulation with LPS did not result in any significant enrichment. Although we detected 43% overlap of enrichment categories for IL8 and PMA stimulation, indicating that activation of neutrophils with different stimuli partly induces some similar biological processes and pathways, hierarchical clustering showed clear differences in distribution and biological relevance of clusters between the chosen stimuli. Our studies provide novel information on the granulocyte proteome and offer insights into early, differentiated granulocyte reaction to stimuli, which contribute to a better understanding of molecular mechanisms involved in activation and recruitment of neutrophils, through inflammatory stimuli.

Information about epileptogenesis-associated changes in protein expression patterns is of particular interest for future selection of target and biomarker candidates.Bioinformatic analysis of proteomic data sets can increase our knowledge about molecular alterations characterizing the different phases of epilepsy development following an initial epileptogenic insult.Here, we report findings from a focused analysis of proteomic data obtained for the hippocampus and parahippocampal cortex samples collected during the early post-insult phase, latency phase, and chronic phase of a rat model of epileptogenesis.The study focused on proteins functionally associated with cell stress, cell death, extracellular matrix (ECM) remodeling, cell-ECM interaction, cell-cell interaction, angiogenesis, and blood-brain barrier function. The analysis revealed prominent pathway enrichment providing information about the complex expression alterations of the respective protein groups.In the hippocampus, the number of differentially expressed proteins declined over time during the course of epileptogenesis. In contrast, a peak in the regulation of proteins linked with cell stress and death as well as ECM and cell-cell interaction became evident at later phases during epileptogenesis in the parahippocampal cortex. The data sets provide valuable information about the time course of protein expression patterns during epileptogenesis for a series of proteins. Moreover, the findings provide comprehensive novel information about expression alterations of proteins that have not been discussed yet in the context of epileptogenesis. These for instance include different members of the lamin protein family as well as the fermitin family member 2 (FERMT2). Induction of FERMT2 and other selected proteins, CD18 (ITGB2), CD44 and Nucleolin were confirmed by immunohistochemistry.Taken together, focused bioinformatic analysis of the proteomic data sets completes our knowledge about molecular alterations linked with cell death and cellular plasticity during epileptogenesis. The analysis provided can guide future selection of target and biomarker candidates.

Mycobacterium avium subspecies paratuberculosis (MAP) are detectable viable in milk and other dairy products. The molecular mechanisms allowing the adaptation of MAP in these products are still poorly understood. To obtain information about respective adaptation of MAP in milk, we differentially analyzed the proteomes of MAP cultivated for 48 h in either milk at 37 °C or 4 °C or Middlebrook 7H9 broth as a control. From a total of 2197 MAP proteins identified, 242 proteins were at least fivefold higher in abundance in milk. MAP responded to the nutritional shortage in milk with upregulation of 32% of proteins with function in metabolism and 17% in fatty acid metabolism/synthesis. Additionally, MAP upregulated clusters of 19% proteins with roles in stress responses and immune evasion, 19% in transcription/translation, and 13% in bacterial cell wall synthesis. Dut, MmpL4_1, and RecA were only detected in MAP incubated in milk, pointing to very important roles of these proteins for MAP coping with a stressful environment. Dut is essential and plays an exclusive role for growth, MmpL4_1 for virulence through secretion of specific lipids, and RecA for SOS response of mycobacteria. Further, 35 candidates with stable expression in all conditions were detected, which could serve as targets for detection. Data are available via ProteomeXchange with identifier PXD027444.

Despite intense research efforts, the knowledge about the mechanisms of epileptogenesis and epilepsy is still considered incomplete and limited. However, an in-depth understanding of molecular pathophysiological processes is crucial for the rational selection of innovative biomarkers and target candidates.Here, we subjected proteomic data from different phases of a chronic rat epileptogenesis model to a comprehensive systems level analysis. Weighted Gene Co-expression Network analysis identified several modules of interconnected protein groups reflecting distinct molecular aspects of epileptogenesis in the hippocampus and the parahippocampal cortex. Characterization of these modules did not only further validate the data but also revealed regulation of molecular processes not described previously in the context of epilepsy development. The data sets also provide valuable information about temporal patterns, which should be taken into account for development of preventive strategies in particular when it comes to multi-targeting network pharmacology approaches.In addition, principal component analysis suggests candidate biomarkers, which might inform the design of novel molecular imaging approaches aiming to predict epileptogenesis during different phases or confirm epilepsy manifestation. Further studies are necessary to distinguish between molecular alterations, which correlate with epileptogenesis versus those reflecting a mere consequence of the status epilepticus.

Objective: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. Methods: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. Results: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. Conclusions: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.

The purpose of the current study was to analyze the binding patterns of serum autoantibodies from juvenile idiopathic arthritis (JIA) and JIA-associated uveitis (JIAU) patients to proteomes from different ocular tissues and to identify potential ocular autoantigens in JIAU. Proteomes from porcine iris, ciliary body, or retina tissue were isolated, separated using 2D-gel electrophoresis, and transferred to a blotting membrane. The binding pattern of serum antibodies from JIA or JIAU patients or healthy controls to ocular proteins was visualized by using anti-human IgG secondary antibodies and chemiluminescence reaction. Selected protein spots were excised from silver-stained 2D gels and subjected to mass spectrometry. Serum antibodies binding to ocular proteins were detected in all patient groups and healthy controls. Irrespective of the patient groups, serum antibodies bound to 49 different protein spots of the retina proteome, to 53 of the ciliary body proteome, and to 44 of the iris proteome. The relative binding frequency of sera to these iris protein spots was significantly higher in JIAU than in JIA patients or healthy controls. Particularly in JIAU patients, cluster analyses indicated a broad range of serum antibodies directed against ocular antigens, mostly in the iris proteome. Iris proteins frequently bound by serum antibodies in all groups were identified as tubulin beta chain, vimentin, ATP synthase subunit beta, actin, and L-lactate dehydrogenase B chain. Iris proteins exclusively bound by JIAU serum antibodies were heat shock cognate 71 kDa protein and keratin. Although serum autoantibody binding to ocular antigens was not disease-specific, a significant diversity of autoantibodies against a broad range of antigens, particularly from the iris tissue, was detected in JIAU patients. As the iris is a major site of inflammation in JIAU, the present data give further evidence that autoantibodies may be involved in JIAU immunopathology.

A novel vaccine against bovine viral diarrhea (BVD) induced pathogenic antibody production in 5–10% of BVD-vaccinated cows. Transfer of these antibodies via colostrum caused Bovine neonatal pancytopenia (BNP) in calves, with a lethality rate of 90%. The exact immunological mechanisms behind the onset of BNP are not fully understood to date. To gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (BNP cows) and non-responders. After in vitro stimulation of peripheral blood derived lymphocytes (PBL), we detected distinctly deviant expression levels of several master regulators of immune responses in BNP cells, pointing to a changed immune phenotype with severe dysregulation of immune response in BNP cows. Interestingly, we also found this response pattern in 22% of non-BVD-vaccinated cows, indicating a genetic predisposition of this immune deviant (ID) phenotype in cattle. We additionally analyzed the functional correlation of the ID phenotype with 10 health parameters and 6 diseases in a retrospective study over 38 months. The significantly increased prevalence of mastitis among ID cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections.

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.

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye. Since one typical feature of the autoimmune disease, equine recurrent uveitis (ERU), is the breakdown of this barrier, we recently performed comparative analysis of healthy and uveitic RPE. We identified for the first time peripherin 2, which is responsible for visual perception and retina development, to be localized in RPE. The purpose of this study was therefore to validate our findings by characterizing the expression patterns of peripherin 2 in RPE and retina. We also investigated whether peripherin 2 expression changes in ERU and if it is expressed by the RPE itself. Via immunohistochemistry, significant downregulation of peripherin 2 in uveitic RPE compared to the control was detectable, but there was no difference in healthy and uveitic retina. A further interesting finding was the clear distinction between peripherin 2 and the phagocytosis marker, rhodopsin, in healthy RPE. In conclusion, changes in the expression pattern of peripherin 2 selectively affect RPE, but not retina, in ERU. Moreover, peripherin 2 is clearly detectable in healthy RPE due to both phagocytosis and the expression by the RPE cells themselves. Our novel findings are very promising for better understanding the molecular mechanisms taking place on RPE in uveitis.

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.

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.

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.

Feline idiopathic cystitis (FIC) is the only spontaneous animal model for human interstitial cystitis (IC), as both possess a distinctive chronical and relapsing character. Underlying pathomechanisms of both diseases are not clearly established yet. We recently detected increased urine fibronectin levels in FIC cases. The purpose of this study was to gain further insight into the pathogenesis by assessing interacting partners of fibronectin in urine of FIC affected cats. Several candidate proteins were identified via immunoprecipitation and mass spectrometry. Considerable changes in FIC conditions compared to physiological expression of co-purified proteins were detected by Western blot and immunohistochemistry. Compared to controls, complement C4a and thioredoxin were present in higher levels in urine of FIC patients whereas loss of signal intensity was detected in FIC affected tissue. Galectin-7 was exclusively detected in urine of FIC cats, pointing to an important role of this molecule in FIC pathogenesis. Moderate physiological signal intensity of galectin-7 in transitional epithelium shifted to distinct expression in transitional epithelium under pathophysiological conditions. I-FABP expression was reduced in urine and urinary bladder tissue of FIC cats. Additionally, transduction molecules of thioredoxin, NF-κB p65 and p38 MAPK, were examined. In FIC affected tissue, colocalization of thioredoxin and NF-κB p65 could be demonstrated compared to absent coexpression of thioredoxin and p38 MAPK. These considerable changes in expression level and pattern point to an important role for co-purified proteins of fibronectin and thioredoxin-regulated signal transduction pathways in FIC pathogenesis. These results could provide a promising starting point for novel therapeutic approaches in the future.

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology.

Autoimmune uveitis is an intraocular inflammation that arises through autoreactive T-cells attacking the inner eye, eventually leading to blindness. However, the contributing molecular pathomechanisms within the affected tissues remain as yet elusive. The extracellular matrix (ECM) is a highly dynamic structure that varies tremendously and influences the encompassing tissue. In order to assess ECM re-modeling in autoimmune uveitis, we investigated the expression of ECM molecules fibronectin and osteopontin in vitreous and retina samples. This was carried out in the only spontaneous animal model for human autoimmue uveitis, namely equine recurrent uveitis (ERU) that resembles the human disease in clinical as well as in immunopathological aspects. ERU is a naturally occurring autoimmune disease in horses that develops frequently and has already proved its value to study disease-related pathomechanisms. Western blot analysis of fibronectin and osteopontin in healthy and uveitic vitreous revealed significant reduction of both proteins in uveitis. Immunohistochemical expression of fibronectin in healthy retinas was restricted to the inner limiting membrane abutting vimentin positive Müller cell endfeet, while in uveitic sections, a disintegration of the ILM was observed changing the fibronectin expression to a dispersed pattern extending toward the vitreous. Retinal expression of osteopontin in control tissue was found in a characteristic Müller cell pattern illustrated by co-localization with vimentin. In uveitic retinas, the immunoreactivity of osteopontin in gliotic Müller cells was almost absent. The ability of Müller cells to express fibronectin and osteopontin was additionally shown by immunocytochemistry of primary cultured equine Müller cells and the equine Müller cell line eqMC-7. In conclusion, severe ECM re-modeling in autoimmune uveitis reported here, might affect the adhesive function of fibronectin and thus the anchoring of Müller cell endfeet to the ILM. Furthermore, the absence of osteopontin in gliotic Müller cells might represent reduced neuroprotection, an osteopontin attribute that is intensively discussed.

Cellular retinaldehyde binding protein (CRALBP) is an autoantigen in spontaneous equine recurrent uveitis. In order to test whether CRALBP contributes to human autoimmune uveitis, the specificity of antibodies from human uveitis patient's sera was first evaluated in two-dimensional (2D) Western blot analysis. Subsequent identification of the immunoreactive proteins by mass spectrometry resulted in the identification of CRALBP as a putative autoantigen. Additionally, sera from human uveitis and control patients were by Western blot using purified human recombinant CRALBP. Anti-CRALBP autoantibodies occur more frequently (P