Development of New Antibodies and an ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine.

Food poisoning can be caused by the potato alkaloids α-solanine (SO) and α-chaconine (CHA). Therefore, this study aimed to establish new enzyme-linked immunosorbent assays (ELISAs) for detecting these two toxins in biological samples and potato extracts. Two antibodies that bind to solanidine, a chemical compound found in both SO and CHA, were newly developed, and two types of ELISAs (Sold1 ELISA and Sold2 ELISA) were constructed. We measured SO and CHA diluted in phosphate-buffered saline (PBS), serum, and urine. The detection performance of the two ELISAs for SO and CHA in PBS was higher than in serum and urine, and the sensitivity of Sold2 ELISA was lower than that of Sold1 ELISA. Thus, we used these ELISAs to measure SO and CHA in potato part extracts and found that potato sprouts contained approximately 80-fold more SO and CHA than tubers and 8-fold more SO and CHA than peels. Although the detection sensitivity of SO and CHA depends on the sample types, these ELISAs may be effective as future clinical and food testing methods after further improvements.

Nicotine Exerts a Stronger Immunosuppressive Effect Than Its Structural Analogs and Regulates Experimental Colitis in Rats.

Ulcerative colitis (UC) is an intractable disease that causes persistent colonic inflammation. Numerous studies have reported that smoking can afford clinical benefits in UC. This study aimed to elucidate whether nicotine, the main component in cigarettes, can exert pharmacological effects against experimental UC. To achieve this objective, we compared the effects of nicotine with those of structural nicotine analogs in a UC rodent model (Slc: Wistar rats, male, 9-week-old, and 220-250 g/rat). Nicotine, or a respective structural analog (nornicotine, cotinine, anabasine, myosmine, and anatabine), was administered intraperitoneally daily to rats (n = 6/group) exhibiting dextran sulfate sodium-induced experimental colitis. Examining the colon tissues of model rats, we compared disease severity, cytokine secretion, and α7 nicotine acetylcholine receptor (nAChR7) expression. We observed that nicotine administration induced weight loss at 2.35% in 10 days. Notably, the reduction in histological severity (score) of UC was more pronounced in rats treated with nicotine (score = 4.83, p = 0.042) than in untreated rats (score = 8.17). Nicotine administration increased nAChR7 expression 6.88-fold (p = 0.022) in inflammatory sites of the colon, mainly by suppressing the production of interleukin (IL)-1ß and IL-6. Moreover, the secretion of these cytokines was suppressed in lipopolysaccharide-stimulated rat macrophages (MΦ) treated with nicotine. In conclusion, nicotine better alleviates experimental UC than the examined structural analogs by activating nAChR7 expression and suppressing proinflammatory cytokines in MΦ.

Carbonic Anhydrase III Has Potential as a Biomarker for Experimental Colitis and Functions as an Immune Regulator by Inhibiting Inflammatory Cytokine Secretion.

Ulcerative colitis (UC) is characterized by chronic inflammation of the large intestine, repeated remissions, and symptom relapses. Although unknown components in colonic regions are deeply involved in the pathogenesis of UC, the causes of UC development and aggravation have not yet been elucidated in detail. To identify key factors, we investigated the changes in protein components in the large intestine of rats with dextran sulfate sodium-induced experimental colitis (UCR). The components that differed in their concentration between normal rats (WT) and UCR were carefully investigated by electrophoretic separation and mass spectrometry. Based on these results, seven proteins with different expression levels between the WT and UCR were observed. Among them, we focused on carbonic anhydrase III (CA-III) in the pathogenesis of UC. CA-III concentrations in the colon tissue and serum were quantitatively measured using an enzyme-linked immunosorbent assay (ELISA) and real-time PCR, and the levels significantly decreased in both the colon tissue and serum of UCR with the aggravation of experimental UC. In an in vitro assay, CA-III function in peritoneal macrophages (MΦ) from rats was investigated. Upon stimulation of MΦ with lipopolysaccharide (LPS), the CA-III concentration significantly decreased in the cytoplasm of these cells. MΦ treated with an anti-CAIII antibody followed by stimulation with LPS actively secreted inflammatory cytokines, particularly interleukin-6 and tumor necrosis factor-α. Therefore, CA-III in MΦ appears to be an immune regulator that suppresses the secretion of inflammatory cytokines.

A New Hybrid Protein Is a Novel Regulator for Experimental Colitis in Rats.

We newly developed a hybrid protein, tentatively named rMIKO-1, using gene technology. We herein investigated the effects of rMIKO-1 on activated macrophages and discussed its potential as a suppressor of experimental colitis. Fluorescent microscopy was used to observe the dynamic mobility of rMIKO-1 in macrophages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, fluorescent immunochemical staining, flow cytometry, enzyme-linked immunosorbent assays, a polymerase chain reaction/quantitative polymerase chain reaction, and hematoxylin and eosin staining were conducted to assess the potential activity of rMIKO-1. A large amount of bleeding was observed in rats treated with 5% dextran sulfate sodium (DSS) alone on day 8 after treatment initiation, but not in those treated with 5% DSS plus rMIKO-1. In the in vitro assay, rMIKO-1 rapidly bound to macrophages, immediately entered cells by an unknown mechanism, and then migrated inside the nucleus. This result suggests that rMIKO-1 plays important immunological roles in the nucleus. Despite the activation of macrophages by lipopolysaccharide, the mRNA expression of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß, was significantly suppressed in macrophages preliminarily treated with rMIKO-1 for 1 h. Complexes of rMIKO-1 with nuclear factor-kappa B (NF-κB)/p65 and ß-actin formed in activated macrophages, which attenuated experimental colitis in rats. These results strongly suggest that rMIKO-1 negatively regulates excessively activated macrophages through the NF-κB/p65 signaling pathway. Therefore, rMIKO-1 is a novel suppressor of experimental colitis in rats through the negative regulation of activated macrophages.

Serum complement C3 and α2-macroglobulin are potentially useful biomarkers for inflammatory bowel disease patients.

AIMS: Ulcerative colitis (UC), characterized by chronic inflammation and its recurrence in the large intestine, is well known as inflammatory bowel disease (IBD). Suitable biomarkers specific for UC are poorly understood till date. We aimed to discover novel serum biomarkers for UC and identify good indicators that reflected the severity of UC. MAIN METHODS: Serum samples were obtained from out-patients with IBD (n = 101) and healthy volunteers (HVs, n = 101). Serum proteins were subjected to high performance liquid chromatography (HPLC) and sodium dodecyl sulfate-electrophoresis (SDS-PAGE) analysis. After electrophoresis, proteins in the gel were identified by mass spectrometry. Further, the protein concentration was measured by enzyme-linked immunosorbent assays (ELISAs). Based on the results, correlations between the serum levels of these proteins and the disease activity index scores for UC were statistically evaluated. PRINCIPAL FINDINGS: HPLC showed that chromatograms of serum proteins from HVs apparently differed from those of patients with IBD. Eleven protein bands, which were different in their protein concentrations from those in HVs, were separated by SDS-PAGE accordingly. Among them, complement C3 (c-C3) and α2-macroglobulin (α2-MG), with high protein scores, were identified by mass spectrometry. The serum concentration of c-C3 in patients with IBD was higher than that in HVs. However, the level of α2-MG in patients with IBD was significantly lower than that in HVs. Hence, the serum levels of c-C3 and α2-MG could be good indicators of the severity of UC. CONCLUSION: Serum c-C3 and α2-MG are suitable biomarkers for monitoring the condition of patients with UC.

Circulating S100A8/A9 is potentially a biomarker that could reflect the severity of experimental colitis in rats.

AIMS: The clinical significance of circulating S100A8/A9 (calprotectin) in patients with ulcerative colitis (UC) is poorly understood. We examined whether serum S100A8/A9 is a good biomarker for UC, and whether the serum level is a useful index for the severity of the disease. MAIN METHODS: Experimental animal (rats) were used to verify clinical significance of serum S100A8/A9 as a biomarker. Rats treated with 5% dextran sulfate sodium (DSS) alone (UCR) or with 5%DSS plus tacrolimus (TMR) were subjected to the experiment. The serum concentrations of rat S100A8/A9 (r-S100A8/A9) and other inflammatory biomarkers, such as C-reactive protein (CRP) and inflammatory cytokines, in the both groups were measured using enzyme-linked immunosorbent assays (ELISAs). The tissue damage in the large intestinal tract was visualized by hematoxylin-eosin staining. The relationship between the serum concetrations of these inflammatory biomarkers and the histological scores of the rectal tissue was statistically analyzed. PRINCIPLE FINDINGS: As determined by the ELISAs, the serum concentration of r-S100A8/A9 in the UCR hardly correlated with those of not only CRP but also some inflammatory cytokines. The deterioration of the rectal tissue, mainly epithelium structure of a large intestine, in the UCR was clearly observed, but was not so severe as that in the TMR. The histological scores of the rectal tissue in the UCR significantly correlated with the serum level of r-S100A8/A9, but not with other inflammatory biomarkers. Furthermore, macrophages actively produced r-S100A8/A9 in response to stimulation with lipopolysaccharide and quickly secreted it in circulation. Therefore, the serum level of r-S100A8/A9 suggestively changes in accordance with the severity of experimental UC. CONCLUSION: Circulating r-S100A8/A9 is a useful biomarker for experimental UC, and its serum level correlates with the disease severity as judged by histological score.

Serum S100A8/A9 as a Potentially Sensitive Biomarker for Inflammatory Bowel Disease.

BACKGROUND: The clinical significance of human S100A8/A9 (h-S100A8/A9) in patients with inflammatory bowel disease (IBD) is poorly understood. OBJECTIVE: To clarify whether serum S100A8/A9 is a sensitive biomarker for IBD. METHODS: Serum specimens from outpatients with IBD (n = 101) and healthy volunteers (HVs) (n = 101) were used in this study. Enzyme-linked immunosorbent assays for h-S100A8/A9 and inflammatory cytokines were performed using these specimens. Further, correlation analysis was performed to investigate the significance of h-S100A8/A9 fluctuation in patients with IBD. RESULTS: The average of serum h-S100A8/A9 concentration in outpatients with IBD was significantly higher than that in HVs. The concentration of h-S100A8/A9 in patients with IBD was barely correlated with that of CRP and inflammatory cytokines. Despite that finding, the serum level of h-S100A8/A9 in patients with ulcerative colitis (UC) was correlated with the severity of IBD, compared with other inflammatory proteins. CONCLUSION: Serum h-S100A8/A9 is superior to CRP as a sensitive biomarker for IBD.

Establishment of S100A8 Transgenic Rats to Understand Innate Property of S100A8 and Its Immunological Role.

The innate properties of S100A8 as a regulator in acute inflammation have not yet been elucidated in detail. Our aims are to newly establish S100A8 transgenic rats (Tg-S100A8) and to elucidate the immunological functions of S100A8. Following the treatment with 5% dextran sulfate sodium for 1 week, the body weight in Tg-S100A8 weakly decreased after the start; however, that in Japanese Wistar rats (WT) significantly decreased in the end. The serum level of CRP in Tg-S100A8 was significantly lower than that in WT, although the concentration of CRP apparently increased in both Tg-S100A8 and WT. The dynamic mobility of S100A8 and S100A9 in macrophages was microscopically observed using fluorescent immunological staining, in which the S100A9 was dominantly expressed in many macrophages in the rectal tissue of WT. As determined by PCR and real-time PCR, the levels of S100A8 messenger RNA (mRNA) in several organ tissues of the Tg-S100A8, such as heart and small intestine, were apparently higher than those of WT, respectively. The expression of IL-6 and TNF-α mRNAs was negatively regulated in main organ tissues of the large colon of Tg-S100A8 followed by down-regulation of IL-6 protein. An important result was that the expression of S100A8 mRNA was strongly induced in many macrophages of Tg-S100A8, whereas that of some inflammatory cytokine mRNAs described above were significantly reduced. Tg-S100A8 has potential as a useful experimental model rat not only for investigating the innate properties of S100A8 as a regulator, but also for clarifying its functional role in immune cells from a myeloid origin, particularly macrophages.

Enzyme-linked immunosorbent assay for S100A9 in the stool of rats with dextran sulfate sodium-induced colitis.

Calprotectin, a heterodimer of S100A8 and S100A9, has been reported to be a useful biomarker in inflammatory bowel disease (IBD); however, the relationship between the fecal level of S100A9 and the extent of inflammation in IBD remains unclear. Our aim was to develop a new enzyme-linked immunosorbent assay (ELISA) for rat S100A9, and to investigate whether changes in fecal S100A9 levels reflect the inflammatory conditions in the intestinal tracts of rats with dextran sulfate sodium (DSS)-induced colitis. Anti-rat S100A9 monoclonal antibodies were raised in mice and used for the development of a novel ELISA for rat S100A9. The performance of our ELISA was assessed by dilution and recovery tests, and the detection range was 3.75-240ng/mL. The dilution test showed good linearity. The recovery of fecal S100A9 was 95.1% (mean), with a range of 86.1%-108.8%. Colitis was induced in rats by oral administration of 3% DSS/drinking water (DW) for 11days (D group), while DW alone was provided to rats of the control group (C group) during the same period. The extent of inflammation was evaluated with the disease activity index (DAI), and the concentration of fecal S100A9 was determined by ELISA. Both the DAI scores and the fecal S100A9 levels were significantly higher in the D group than in the C group. Microscopic observation revealed that S100A9 was dominantly produced in many immune cells of myeloid origin in rat rectal tissues. These results indicate that the novel ELISA may be applied to clinically evaluate IBD in rats with high sensitivity. In conclusion, our ELISA is useful in toxicological and pharmacological evaluations.

CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells' immune functions.

S100A8 and S100A9 (S100 proteins) are regulators of immune cells of myeloid origin. Whereas S100 proteins are found at high concentrations in such cells, their immunologic roles remain unclear. We focused on cluster of differentiation 68 (CD68). The aim of this study is to investigate whether CD68 binds to extracellular S100A8 and/or S100A9 and subsequently participates in the regulation of the cells' immune functions. ELISA and affinity chromatography showed that both recombinant rat S100A8 (r-S100A8) and r-S100A9 bound to r-CD68, but not to r-CD14. Flow cytometry clearly showed evidences supporting above the 2 results. As analyzed by flow cytometry, a less amount of r-S100A8 or r-S100A9 bound to the macrophages treated with some deglycosylation enzymes. In an in vitro assay, the expression levels of S100A8 and S100A9 were significantly suppressed after the macrophages had been treated with an anti-CD68 antibody (ED1). As stimulated macrophages with r-S100A9, the expression of IL-1ß mRNA in macrophages, which were treated with anti-TLR4 or -RAGE antibodies, was significantly suppressed. r-S100A8 up-regulated IL-6 and IL-10 mRNAs, while r-S100A9 did TNF-α and IL-6 mRNAs, although these regulations were not statistically significant. Small interfering CD68 also significantly suppressed activation of macrophages through an autocrine pathway by r-S100A8 or r-S100A9. In macrophages stimulated with LPS, fluorescent immunologic staining showed that most CD68 colocalized with S100A8 or S100A9 and that the levels of all 3 molecules were markedly increased. In conclusion, CD68 on macrophages binds to S100A8 and S100A9 and thereby, plays a role in the regulation of the cells' immune functions.

Autocrine pathways involving S100A8 and/or S100A9 that are postulated to regulate the immunological functions of macrophages in rats.

The development of ulcerative colitis (UC) is closely associated with abnormally functioning macrophages. Rat S100A8 (r-S100A8) and r-S100A9 (S100 proteins) is abundantly expressed in immune cells of myeloid origin, macrophages; however, it remains unclear why r-S100A9 is dominantly expressed in the macrophages of UC rats (UCR). The purpose of this study was to verify the immunological roles of S100 proteins in UCR. We observed the distribution of S100 protein-positive macrophages in the large colons of UCR using a fluorescent immunological staining method, so that S100 protein-positive macrophages were restricted to the rectal tissues of the UCR, and that the mRNA levels of r-S100A8 and r-S100A9 were up-regulated by stimulation with recombinant rat S100A8 (rr-S100A8) alone and rr-S100A9 alone, respectively. When the changes in the mRNA levels of r-S100A8 and r-S100A9 in macrophages were examined in in vitro study by PCR and real-time PCR, the mRNA levels of anti-inflammatory and inflammatory cytokines increased selectively after stimulation with rr-S100A8 alone and rr-S100A9 alone, respectively. These results suggest that autocrine signal transduction pathways involving S100 proteins regulate the immunological functions of macrophages to maintain homeostasis in the gastrointestinal tract. This may be depended on expression balance of S100 proteins in macrophages. It is strongly suggested that in UCR the immune functions of macrophages are regulated in a complex manner by r-S100A8 and/or r-S100A9 through undefined autocrine pathways on the cells.