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Clinical examination, bronchoalveolar lavage fluid (BALF) cytology, acute- phase protein, and pulmonary hemostasis and fibrinolysis marker (fibrinogen, serum amyloid A [SAA], and D-dimer) results were compared between control and respiratory disease-affected horses. Using a clinical scoring system, horses (n = 58) were classified as respiratory disease-free (Controls, n = 15) or with recurrent airway obstruction (RAO; n = 18), inflammatory airway disease (n = 14) or chronic interstitial pneumopathy (n = 11). There were no significant differences in fibrinogen concentrations among groups, but there was a trend toward a lower value in controls (median 0.0024 g/L) than in horses with chronic pneumopathies (median 0.0052 g/L), in particular, those with RAO (median 0.0062 g/L). Fibrinogen concentration was positively correlated with percentage of neutrophils in BALF (rs = 0.377, p = 0.004). SAA concentrations were low; 65.5% of samples were below the detection limit. D-dimer concentrations were also low and quantifiable concentrations were only obtained after ultrafiltration and only in RAO (median 0.1 mg/L). In conclusion, there was limited evidence of increased coagulatory activity in chronic pneumopathies, apart from RAO. It is uncertain whether fibrinogen and D-dimer concentrations increased due to their role as acute-phase proteins or as a misbalance of coagulation and fibrinolysis.

This study was aimed to evaluate the concentrations of interleukin-1 receptor antagonist (IL-1ra) and interleukin-1 beta (IL-1β) in normal and osteoarthritic (OA) joints as well as the influence of joint location and arthrocentesis on these concentrations. Interleukin-1 receptor antagonist and IL-1β levels were determined in the synovial fluid (SF) of 18 normal and 18 OA joints. In all normal joints, arthrocentesis was repeated after 1 hour. No significant difference of SF IL-1ra and IL-1β levels between metacarpophalangeal/metatarsophalangeal, radiocarpal, and talocrural joints was observed. There was no significant change in SF IL-1ra and IL-1β levels between first and second arthrocentesis detectable. Synovial fluid IL-1ra and IL-1β levels were significantly increased in OA joints compared to normal joints. Synovial fluid WBC count and protein concentration were not significant different between normal and OA joints. Synovial fluid WBC count and protein concentration as well as IL-1ra and IL-1β concentration were positive correlated. The anatomic location of high motion joints seems to have no influence on SF IL-1ra and IL-1β levels. Arthrocentesis did not increase SF IL-1ra and IL-1β levels within 1 hour after joint puncture. Increased SF IL-1ra, IL-1β, and protein concentrations as well as WBC counts seem to be indicators of joint inflammation, but on their own are not allowing an exact differentiation between healthy and mild OA joints due to great value ranges and value overlap. Yet it has to be further investigated if in combination with other biomarkers, a clearer differentiation of pathologic processes in the joint can be made.

The objective of this study was to investigate the concentrations of interleukin-1 receptor antagonist (IL-1Ra) and interleukin-1s (IL-1s) in synovial fluid (SF) and serum (SE) of horses with different grades of osteoarthritis (OA) and septic arthritis. Based on SF analysis, radiographic, and arthroscopic scores, 40 horses were classified into three groups as follows: mild OA, advanced OA, and septic arthritis. Horses without orthopedic problems served as a control group. Equine-specific antibody enzyme-linked immunosorbent assays were used to determine the concentration of IL-1Ra and the catabolic cytokine IL-1s in SF and SE. Results were further compared with levels of the previously described biomarkers C-telopeptide fragments of type II collagen (CTX-II) and myeloperoxidase. In the present study, the SF of healthy joints, those with nonseptic OA, and those with septic arthritis contained significantly different levels of IL-1Ra. Serum concentrations of IL-1Ra were only significantly elevated in horses with septic arthritis when compared with the control group. Different levels of IL-1s were detected in SE of control horses compared with those with various degrees of joint disease. Synovial fluid concentrations of IL-1s were only moderately elevated in the groups of horses with joint disease. In addition to lameness examination and standard diagnostic procedures, the determination of IL-1Ra concentration in SF, in combination with further biomarkers, might be useful to assess the extent of intrasynovial inflammation.

The exocrine pancreas plays an important role in zinc homeostasis. Feeding very high (2000-3000mgzinc/kg diet) levels of zinc oxide to piglets for short periods is a common practice in the swine industry to improve performance and prevent diseases. The impact on pancreatic function and possible side effects during long-term feeding of high dietary zinc levels are still poorly understood. A total of 54 weaned piglets were either fed with low (57mg/kg, LZn), normal (164mg/kg, NZn) or high (2425mg/kg, HZn) zinc concentration in the diets. After 4 weeks of feeding, ten piglets per treatment were euthanized and pancreas samples were taken. Tissue zinc concentration and metallothionein abundance was greater with HZn compared with NZn and LZn (P0.05). Similarly, activity of α-amylase, lipase, trypsin and chymotrypsin was higher with HZn as compared with NZn and LZn diets (P0.05), whereas elastase activity was unchanged. Total trolox equivalent antioxidative capacity of pancreas tissue was higher with HZn diets compared with the other treatments (P0.05). Pancreatic protein profiles of NZn and HZn fed piglets were obtained by 2D-DIGE technique and revealed 15 differentially expressed proteins out of 2100 detected spots (P0.05). The differentially expressed proteins aldose reductase, eukaryotic elongation factor II and peroxiredoxin III were confirmed by immunoblotting. Identified proteins include zinc finger-containing transcription factors and proteins mainly associated with oxidative stress response and signal transduction in HZn compared with NZn pigs. Histologic examination however showed no morphologic changes. The results suggest that long-term supply of very high dietary zinc increases zinc and metallothionein concentration, and digestive enzyme activity, but also triggers oxidative stress reactions in the pancreas of young pigs. The data provide new insights into pancreatic function under outbalanced zinc homeostasis.

Background Overexpression of matrix-metalloproteinases (MMPs) has been shown to lead to tissue damage in equine recurrent airway obstruction (RAO), as a misbalance with their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), occurs. This favors irreversible pulmonary fibrosis formation. Increased levels of MMPs, TIMPs or altered ratios between them can be used as biomarkers of respiratory disease. We hypothesized that levels of MMPs, TIMPs and their ratios correlate with improvement in clinical findings and bronchoalveolar lavage fluid (BALF) cytology after 10 days of inhalative glucocorticoid therapy and environmental dust reduction (EDR) and may be used to monitor treatment success. Ten horses with a history of RAO participated in a prospective clinical study. Clinical and cytological scoring was performed before and after inhalative therapy using budesonide (1500 μg BID over 10 days) and EDR (bedding of wood shavings and wet hay as roughage). Gelatin zymography was performed for qualitative and semi-quantitative evaluation of MMP-2 and MMP-9 in BALF supernatant, while fluorimetry was used to evaluate MMP-8 activity. Additionally, specific equine ELISA assays were used for quantitative assessment of MMP-2, MMP-9, TIMP-1 and TIMP-2. Results A significant reduction in the total and several single parameters of the clinical score were found after 10 days of inhalative therapy and EDR. The concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 (ELISA) as well as their activities (MMP-2 and MMP-9 zymography and MMP-8 fluorimetry) were significantly decreased after therapy. Significant improvements in MMP-8/TIMP-1 and MMP-8/TIMP-2 ratios were also found, differences between other ratios before and after therapy were insignificant. Conclusions Metalloproteinases and their inhibitors, in particular MMP-9 and TIMP-2, are valuable markers for clinical improvement in RAO.

To compare hyaluronic acid (HA) concentrations measured in synovial fluid (SF) of joints with different stages of canine secondary osteoarthritis (OA), clinical-orthopedic, radiographic, macroscopic intra-operative and SF findings of 49 joints were assessed. The sum of single findings was correlated to HA concentrations measured by a commercially available ELISA. Joints were categorized into three OA-groups: non-osteoarthritic, mildly osteoarthritic, and severely osteoarthritic. A significant negative correlation was found between severity of OA and HA concentrations (r=-0.696; P0.001). Median values of HA concentrations decreased with increasing severity of the disease. Statistically significant differences in HA concentrations were observed between the OA-groups (P0.001). Due to overlapping values between groups, it was concluded that synovial HA concentrations may only indicate a trend of osteoarthritic disease activity, but is not suitable for staging the disease.

Cutaneous mast cell tumours (MCTs) are the most common skin tumours in dogs. However, the molecular differences between benign tumours with a good prognosis and highly malignant, invasive and metastatic tumours with short survival times are for the most part unclear. In the present study the proteome of low-grade MCTs with a good prognosis was compared with that of poor-prognosis high-grade tumours independent of their mutational status of exon 11 of the KIT gene. Using two-dimensional difference gel electrophoresis and mass spectrometry, 13 proteins with a significant differential expression between the two groups were identified. Four stress response proteins (HSPA9, PDIA3, TCP1A and TCP1E) were significantly up-regulated in high-grade tumours, while proteins mainly associated with cell motility and metastasis had either increased (WDR1, ACTR3, ANXA6) or decreased (ANXA2, ACTB) expression levels. High-grade tumours also had a paradox down-regulation of transferrin, a protein that is usually up-regulated in neoplastic cells. The histologically observable dedifferentiation of high-grade tumours was reflected by decreased tryptase protein expression levels. Results of quantitative real-time RT-PCR analysis indicated that the differences in protein expression levels of most proteins were regulated at the transcript level. Based on these findings, it is hypothesized that high-grade MCT cells have a higher resistance to cellular stress and thus are able to better cope with the adverse environment in highly proliferating tumours independent of increased KIT signalling. It is noteworthy that some of the proteins identified have been proposed as therapeutic targets for human oncology and it will be interesting to evaluate their therapeutic and diagnostic potential for canine MCTs.

Autoimmune lymphoproliferative syndrome (ALPS) is mainly caused by defects in the CD95 pathway. Raised CD3+TCRαβ+CD4-CD8- double negative T cells and impaired T cell apoptosis are hallmarks of the disease. In contrast, the B cell compartment has been less well studied. We found an altered distribution of B cell subsets with raised transitional B cells and reduced marginal zone B cells, switched memory B cells and plasma blasts in most of 22 analyzed ALPS patients. Moreover, 5 out of 66 ALPS patients presented with low IgG and susceptibility to infection revealing a significant overlap between ALPS and common variable immunodeficiency (CVID). In patients presenting with lymphoproliferation, cytopenia, hypogammaglobulinemia and impaired B cell differentiation, serum biomarkers were helpful in addition to apoptosis tests for the identification of ALPS patients. Our observations may indicate a role for apoptosis defects in some diseases currently classified as CVID.

We hypothesised that shorter treatment intervals of intraarticular autologous conditioned serum (ACS) injections would more beneficially affect the synovial fluid (SF) concentrations of IL-1ra, IL-1β and cartilage biomarkers, compared with the traditional weekly treatment intervals in joints suffering from natural OA. In a randomised comparative study, 12 horses with OA were allocated to two groups (n = 6). The horses in group 1 received three intraarticular ACS injections at weekly intervals, whereas the horses in group 2 received three intraarticular ACS injections at two-day intervals. The levels of IL-1ra, IL-1β, CPII, C12C and CS 846 were determined in SF before and after ACS treatment using commercially available ELISA kits. The SF IL-1ra concentration 1 hour and 4 hours after ACS injection was significantly increased compared to baseline levels and decreased back to it within 48 hours. Comparing the SF IL-1ra, IL-1β, C12C, CS 846 and CP II levels before and 42 days after ACS treatment, group 2 showed a significant decrease in all parameters and an approximation on the levels in normal joints. These results indicate that the long-time effect of an ACS treatment given at two-day intervals is characterized by decreased SF IL1ra, IL-1β, C12C, CP II and CS 846 concentrations, which might indicate an improvement in joint inflammation and cartilage degrading processes . Further investigations with greater sample sizes have to prove if the two-day treatment interval is preferable to the widely used treatment protocol of weekly intraarticular ACS injections.

The aim of this study was to develop a specific myeloperoxidase (MPO) activity assay in the synovial fluid of horses and investigate whether MPO activity is increased in different forms of joint diseases. Synovial fluid samples were taken from affected joints from horses with osteoarthritis, chronic non-septic arthritis and septic arthritis, and from healthy control horses. MPO activity was measured using a specific modified o-dianisidine-assay containing 4-aminobenzoic acid hydrazide as a potent and specific inhibitor of the MPO. This assay is characterized by high reproducibility. The results reveal only a slight elevation of MPO activity in the synovial fluid of horses with osteoarthritis and chronic non-septic arthritis. However, in the cases of septic arthritis a significant increase in MPO activity was found when compared to the controls. In conclusion the first field study suggests that synovial fluid MPO may be used as a marker for septic arthritis in horses.

The purpose of this study was to analyze the acute effect of whole body vibration exercise (WBVE) on clinical parameters and blood values in horses. Seven horses were exposed to a 10 min WBVE at a frequency of 15-21 Hz. Clinical parameters and venous blood samples were taken before and directly after WBVE. Acute short-term WBVE produced a decrease in serum cortisol (p=0.02) and creatine-kinase (p=0.02) values. Clinical parameters, hematology, fibrinogen, lactate, IGF-I, GGT, creatinine, myeloperoxidase activity and bone marker values were not significantly changed by WBVE. In adult sound horses WBVE was well tolerated and did not cause any sign of measured discomfort.

The aim of the present work was to evaluate whether concentrations of the carboxy-terminal cross-linked fragment of type II collagen (CTX-II), the activities of matrix metalloproteinase-2 and -9 (MMP-2/-9) and Myeloperoxidase (MPO) in canine synovial fluids (SF) can reflect structural alterations of articular cartilage in dogs with fragmented medial coronoid process (FMCP). Elbow joints with FMCP underwent radiographic and arthroscopic examination. Commercially available assays were used to analyze SF for CTX-II concentration and MMP-2/-9 activity. MPO activity was measured by o-dianisidine-assay. The MMPs were further evaluated by zymography. CTX-II concentration and MMP-2 activity showed age-dependent trends in controls. Increased enzyme activities of MPO and MMP-2/-9 were found in diseased dogs. MMP-9activity seems suitable to underline the subjective assessment of the degree of cartilage damage. These initial data of the study suggest that MPO and MMP-2/9 may be used as objective biomarkers in the diagnosis of canine osteoarthritis due to FMCP.

High dietary zinc concentrations are used to prevent or treat diarrhea in piglets and humans, but long-term adaptation to high zinc supply has yet not been assessed. Intestinal zinc uptake is facilitated through members of zinc transporter families SLC30 (ZnT) and SLC39 (ZIP). Whereas in rodents, regulation of zinc homeostasis at low or adequate zinc supply has been described, such mechanisms are unclear in piglets. A total of 54 piglets were fed diets containing 57 [low dietary zinc (LZn)], 164 [normal dietary zinc (NZn)], or 2425 [high dietary zinc (HZn)] mg/kg dry matter zinc. After 4 wk, 10 piglets/group were killed and jejunal tissues taken for analysis of zinc transporters SLC30A1 (ZnT1), SLC30A2 (ZnT2), SLC30A5 (ZnT5), SLC39A4 (ZIP4), divalent metal transporter 1 (DMT1), and metallothionein-1 (MT). Weight gain was higher (P < 0.05) in pigs fed HZn than in the LZn and NZn groups during the first 2 wk. Food intake did not differ between groups. The digesta and jejunal tissue zinc concentrations were higher (P < 0.05) in the HZn pigs than in NZn and LZn pigs. Expression of ZnT1 was higher (P < 0.05) and ZIP4 lower (P < 0.05) in HZn pigs than in the 2 other groups, whereas expression of ZnT5 and DMT1 did not differ between treatments. Expression of ZnT2 was lower (P < 0.05) in the LZn group than in the HZn and NZn groups. The mRNA expression and protein abundance of MT was higher (P < 0.05) in the HZn group than in the NZn and LZn groups. Studies with intestinal porcine cell line intestinal epithelial cell-J2 confirmed the dose-dependent downregulation of ZIP4 and upregulation of ZnT1 and MT (P < 0.05) with increasing zinc concentration within 24 h. In conclusion, high dietary zinc concentrations increase intracellular zinc, promote increased zinc export from intestinal tissues into extracellular compartments, and decrease zinc uptake from the gut lumen. The adaptive process appears to be established within 24 h; however, it does not prevent tissue zinc accumulation.

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P

Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER) and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ) proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS) or species-specific (porcine serum, PS) conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS), compared to conventional FBS culture (IPEC-J2/FBS), the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line's initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function.

Shortened estrous cycles, embryonic death, and abortion might be associated with insufficient secretion of progesterone by the canine corpora lutea. The aim of this study was to investigate the concentration of progesterone, prolactin, and relaxin in hypoluteoid and control bitches during pregnancy and the nonpregnant cycle. Moreover, canine antibodies against progesterone were analyzed because of a possible connection between embryonic loss associated with hormone changes related to a hormone antibody response. An enzyme-linked immunosorbent assay (ELISA) was developed and optimized for these purposes. Serum samples from 20 short-cycling and 18 control bitches were analyzed. Animals were assigned to pregnant and nonpregnant groups after ultrasound examination. The results show that the nonpregnant, short-cycling bitches had significantly lower progesterone concentrations than the animals of the control group. In German Shepherd dogs, prolactin concentrations were significantly higher in the hypoluteoid pregnant bitches compared with the control group. Relaxin concentrations did not differ significantly between the pregnant hypoluteoid and pregnant control group at any time of measurement. Moreover, increased levels of IgE antibodies against progesterone were found in the serum of six bitches (five short-cycling bitches, one control animal) out of a total of 38 animals. The results indicated a specific binding of the antibodies to progesterone, but a cross-reaction with estradiol and testosterone might occur. The presented data verify the existence of antibodies against progesterone and suggest that these antibodies might play a role in some cases of unexplained pregnancy failure and shortened cycle length.

Background Familial hemophagocytic lymphohistiocytosis is a genetic disorder of lymphocyte cytotoxicity that usually presents in the first two years of life and has a poor prognosis unless treated by hematopoietic stem cell transplantation. Atypical courses with later onset and prolonged survival have been described, but no detailed analysis of immunological parameters associated with typical versus atypical forms of familial hemophagocytic lymphohistiocytosis has been performed. Design and Methods We analyzed disease manifestations, NK-cell and T-cell cytotoxicity and degranulation, markers of T-cell activation and B-cell differentiation as well as Natural Killer T cells in 8 patients with atypical familial hemophagocytic lymphohistiocytosis due to mutations in UNC13D and STXBP2 . Results All but one patient with atypical familial hemophagocytic lymphohistiocytosis carried at least one splice-site mutation in UNC13D or STXBP2 . In most patients episodes of hemophagocytic lymphohistiocytosis were preceded or followed by clinical features typically associated with immunodeficiency, such as chronic active Epstein Barr virus infection, increased susceptibility to bacterial infections, granulomatous lung or liver disease, encephalitis or lymphoma. Five of 8 patients had hypogammaglobulinemia and reduced memory B cells. Most patients had a predominance of activated CD8+ T cells and low numbers of Natural Killer T cells. When compared to patients with typical familial hemophagocytic lymphohistiocytosis, NK-cell cytotoxicity and NK-cell and CTL degranulation were impaired to a similar extent. However, in patients with an atypical course NK-cell degranulation could be partially reconstituted by interleukin-2 and cytotoxic T-cell cytotoxicity in vitro was normal. Conclusions Clinical and immunological features of atypical familial hemophagocytic lymphohistiocytosis show an important overlap to primary immunodeficiency diseases (particularly common variable immunodeficiency and X-linked lymphoproliferative syndrome) and must, therefore, be considered in a variety of clinical presentations. We show that degranulation assays are helpful screening tests for the identification of such patients.

Alterations of the phospholipid (PL) compositions of body fluids are assumed to be indicative of inflammatory diseases, e.g. rheumatoid arthritis (RA). Recently, we have shown that particularly the phosphatidylcholine/lysophosphatidylcholine (PC/LPC) ratio determined in human synovial fluids (SF) and sera represents a reliable measure of the inflammatory state in RA patients. However, it is not yet clear to what extent the PC/LPC ratio is also affected by nutrition habits. In the present study, the PL and the corresponding acyl chain compositions of human body fluids (SF and serum of RA patients as well as serum from healthy volunteers) are compared with those of two other mammalian species (horses and dogs suffering from degenerative joint diseases as well as healthy controls) by high-resolution 31P-nuclear magnetic resonance (NMR) spectroscopy and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). The most important result of this study is that the PL compositions of SF and serum of horse and dog are comparable with those of human body fluids. Compared with humans, however, the horse body fluid contains less PCs with highly unsaturated arachidonoyl residues, while that of dogs possesses the highest content of arachidonoyl-containing PC. These species-related differences stem primarily from different nutrition habits (meat vs. plants).