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S100A12 and S100A8/A9 (calprotectin) are released from activated mononuclear cells and belong to the group of damage associated molecular patterns. Fecal S100A12 and S100A8/A9 concentrations have been suggested as biomarkers of intestinal inflammation in dogs with chronic inflammatory enteropathies (CIE). However, the mucosal cellular infiltrate in dogs with CIE is primarily lymphocytic-plasmacytic. Whether fecal S100A12 and S100A8/A9 levels reflect the number and/or activity of intestinal mucosal mononuclear cells, or whether these proteins are also produced by other cells has not been investigated. Thus, the aim of this study was to evaluate intestinal mucosal S100A12 and S100A8/A9 positivity and a potential relationship with the respective protein concentrations in serum and fecal samples in dogs with CIE. Serum (single sample), fecal samples (from 3 consecutive days), and gastrointestinal tissue biopsies (i.e., stomach, duodenum, ileum, and colon) were evaluated from 21 dogs with CIE. Serum and fecal S100A12 and S100A8/A9 concentrations were measured by analytically validated in-house ELISAs. Tissue biopsies underwent routine histopathology and immunohistochemical evaluation for S100A12 and S100A8/A9 positivity (S100A12 + and S100A8/A9 + , each recorded as positive cells/mm 2 ). S100A12 + and S100A8/A9 + cells were identified in all segments of the gastrointestinal tract, but were predominantly localized in the lamina propria (LP). Duodenal LP S100A12 positivity correlated statistically significantly with that in the stomach and ileum (ρ = 0.66 and 0.69, both p < 0.01), but was inversely correlated with the severity of macrophage infiltration in the duodenum (ρ=-0.47, p = 0.042). Ileal LP S100A8/A9 positivity correlated positively with the extent of ileal neutrophil and macrophage infiltration (ρ=0.61, p = 0.047). Fecal S100A12 concentrations strongly correlated with the number of S100A12 + cells along the entire gastrointestinal tract (ρ = 0.76, p = 0.028), whereas serum S100A12 concentrations were inversely correlated to colonic S100A12 + cell counts (ρ=-0.50, p = 0.043). Mucosal S100A8/A9 + cell counts were not associated with the corresponding fecal or serum S100A8/A9 concentrations. These results suggest that the intestinal mucosa in dogs with CIE contains an increased number of activated (pro-inflammatory) phagocytes expressing and secreting the S100A12 protein, but the macrophage population seen on routine histopathology is predominantly mature (anti-inflammatory) with a reduced or absent expression of S100A12 and a normal or increased expression of S100A8/A9. However, the distribution of intestinal S100A8/A9 expression requires further study., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Inflammatory bowel disease (IBD) is a common condition in dogs, and a dysregulated innate immunity is believed to play a major role in its pathogenesis. S100A12 is an endogenous damage-associated molecular pattern molecule, which is involved in phagocyte activation and is increased in serum/fecal samples from dogs with IBD. S100A12 binds to the receptor of advanced glycation end products (RAGE), a pattern-recognition receptor, and results of studies in human patients with IBD and other conditions suggest a role of RAGE in chronic inflammation. Soluble RAGE (sRAGE), a decoy receptor for inflammatory proteins (e.g., S100A12) that appears to function as an anti-inflammatory molecule, was shown to be decreased in human IBD patients. This study aimed to evaluate serum sRAGE and serum/fecal S100A12 concentrations in dogs with IBD. Serum and fecal samples were collected from 20 dogs with IBD before and after initiation of medical treatment and from 15 healthy control dogs. Serum sRAGE and serum and fecal S100A12 concentrations were measured by ELISA, and were compared between dogs with IBD and healthy controls, and between dogs with a positive outcome (i.e., clinical remission, n=13) and those that were euthanized (n=6). The relationship of serum sRAGE concentrations with clinical disease activity (using the CIBDAI scoring system), serum and fecal S100A12 concentrations, and histologic disease severity (using a 4-point semi-quantitative grading system) was tested. Serum sRAGE concentrations were significantly lower in dogs with IBD than in healthy controls (p=0.0003), but were not correlated with the severity of histologic lesions (p=0.4241), the CIBDAI score before (p=0.0967) or after treatment (p=0.1067), the serum S100A12 concentration before (p=0.9214) and after treatment (p=0.4411), or with the individual outcome (p=0.4066). Clinical remission and the change in serum sRAGE concentration after treatment were not significantly associated (p=0.5727); however, serum sRAGE concentrations increased only in IBD dogs with complete clinical remission. Also, dogs that were euthanized had significantly higher fecal S100A12 concentrations than dogs that were alive at the end of the study (p=0.0124). This study showed that serum sRAGE concentrations are decreased in dogs diagnosed with IBD compared to healthy dogs, suggesting that sRAGE/RAGE may be involved in the pathogenesis of canine IBD. Lack of correlation between sRAGE and S100A12 concentrations is consistent with sRAGE functioning as a non-specific decoy receptor. Further studies need to evaluate the gastrointestinal mucosal expression of RAGE in healthy and diseased dogs, and also the formation of S100A12-RAGE complexes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Idiopathic inflammatory bowel disease (IBD) in dogs can be challenging to diagnose and fecal markers of disease that correlate with its severity could potentially be clinically useful. Surrogate inflammatory markers, such as the concentration of fecal S100A12, are used to detect active IBD in humans. The aim of this study was to determine the relationship between fecal canine S100A12 concentrations and clinical, endoscopic, and histologic disease severity. Twenty-six dogs with IBD and 90 healthy control dogs were enrolled. Spot fecal samples were collected and fecal canine S100A12 concentrations measured by an in-house ELISA. The correlation of fecal canine S100A12 concentrations with clinical disease activity (using the canine chronic enteropathy clinical activity index scoring system) and with endoscopic and histologic disease severity (using semi-quantitative grading systems) was assessed in dogs with IBD. Concentrations of fecal canine S100A12 were significantly higher in dogs with IBD (median [interquartile range]: 223 [21-3477]ng/g) than in healthy controls (median [interquartile range]: 9 [5-31]ng/g; P<0.0001). Fecal canine S100A12 concentrations correlated with the CCECAI score (ρ=0.4778; P=0.0408) and the severity of endoscopic lesions in the duodenum (ρ=0.4703; P=0.0354) and colon (ρ=0.9747; P=0.0144), but not with the severity of histopathologic changes except for inflammatory lesions in the colon (ρ=0.8669; P=0.0230). A concentration of 273ng fecal canine S100A12/g feces or greater distinguished (a) dogs with moderate to severe endoscopic disease in any GI section from dogs with at most mild endoscopic disease, and (b) dogs with very severe clinical disease (i.e., a CCECAI score of ≥12) from dogs with a CCECAI score of <12, with a sensitivity of 71% and 90%, respectively, and a specificity of 89% and 75%, respectively. This study showed that fecal canine S100A12 concentrations are increased in dogs with IBD. Further, this study showed that fecal canine S100A12 is associated with the clinical disease activity, the severity of endoscopic lesions, and the severity of colonic inflammation in dogs with IBD. Fecal S100A12 concentrations are potentially useful as a biomarker of inflammation in dogs with IBD., (Copyright © 2014 Elsevier B.V. All rights reserved.)

S100A12 (calgranulin C) is a Ca(2+)-binding protein that has been proposed to play a central role in both innate and acquired immune responses. In humans, S100A12 has been reported to be increased in serum and/or plasma in patients with various inflammatory disorders, and this protein has been suggested to be a sensitive and specific marker for inflammatory bowel disease (IBD). An immunoassay for S100A12 is currently available for use in humans, but antibodies against the human protein do not cross-react with canine S100A12 (cS100A12). Both sensitive and specific markers for canine patients with systemic or localized inflammatory diseases are currently lacking, thus the aim of this study was to develop and analytically validate a radioimmunoassay (RIA) for the quantification of cS100A12 in serum and fecal specimens and to determine the biological variation of cS100A12 in serum from healthy dogs. A competitive liquid-phase RIA was developed and analytically validated by determining assay working range, dilutional parallelism, spiking recovery, and intra- and inter-assay variability. Reference intervals for serum and fecal concentrations of cS100A12 were established from 124 and 65 healthy dogs, respectively, and components of variation for serum cS100A12 were determined from 11 dogs over 2.6 months. The working range of the assay was 0.6-432.7 μg/L. No cross-reactivity was observed with the cS100A8/A9 protein complex, the closest structural analogues available. Observed-to-expected ratios (O/E) for the serial dilution of serum and fecal extracts ranged from 97.2 to 146.8% and from 75.3 to 129.8%, respectively. O/E for spiking recovery for serum and fecal extracts ranged from 87.8 to 130.4% and from 84.8 to 143.8%, respectively. Coefficients of variation (CV) for intra- and inter-assay variability for sera were ≤ 8.1% and ≤ 7.8%, respectively, and were ≤ 7.8% and ≤ 8.7%, respectively, for fecal extracts. Reference intervals for serum and fecal cS100A12 were 33.2-225.1 μg/L and <24-745 ng/g, respectively. For biological variability testing, analytical, intra-individual, inter-individual, and total CV were 5.7, 29.2, 31.2, and 66.0%, respectively, yielding an index of individuality of 0.95 and a minimum critical difference (p<0.05) for sequential values of 84.9%. The RIA for cS100A12 measurement described here is analytically sensitive and specific, linear, accurate, precise, and reproducible, and will facilitate further research into the clinical utility of quantifying serum and fecal cS100A12 in canine patients with inflammatory diseases. Moderate changes in serum cS100A12 concentrations may be clinically relevant; however, the use of a population-based reference interval may require caution., (Copyright © 2011 Elsevier B.V. All rights reserved.)