Human MIKO-1, a Hybrid Protein That Regulates Macrophage Function, Suppresses Lung Fibrosis in a Mouse Model of Bleomycin-Induced Interstitial Lung Disease.

Although interstitial lung disease (ILD) is a life-threatening pathological condition that causes respiratory failure, the efficiency of current therapies is limited. This study aimed to investigate the effects of human MIKO-1 (hMIKO-1), a hybrid protein that suppresses the abnormal activation of macrophages, on murine macrophage function and its therapeutic effect in a mouse model of bleomycin-induced ILD (BLM-ILD). To this end, the phenotype of thioglycolate-induced murine peritoneal macrophages co-cultured with hMIKO-1 was examined. The mice were assigned to normal, BLM-alone, or BLM + hMIKO-1 groups, and hMIKO-1 (0.1 mg/mouse) was administered intraperitoneally from day 0 to 14. The mice were sacrificed on day 28, and their lungs were evaluated by histological examination, collagen content, and gene expression levels. hMIKO-1 suppressed the polarization of murine macrophages to M2 predominance in vitro. The fibrosis score of lung pathology and lung collagen content of the BLM + hMIKO-1 group were significantly lower than those in the BLM-alone group. The expression levels of TNF-α, IL-6, IL-1ß, F4/80, and TIMP-1 in the lungs of the BLM + hMIKO-1 group were significantly lower than those in the BLM-alone group. These findings indicate that hMIKO-1 reduces lung fibrosis and may be a future therapeutic candidate for ILD treatment.

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.

Clinical usefulness of the PAXgene™ bone marrow RNA system for stabilizing total RNA.

The collection of clinical samples, such as bone marrow (BM) and peripheral blood, is an important procedure for the extraction of the cellular RNA. It is essential to preserve the extracted RNA during and after the collection of clinical samples to ensure the accurate analysis of gene expression. To date, the PAXgene™ Blood RNA System has been proven useful for stabilizing RNA extracted from peripheral blood; however, a problem concerning the stability of the total RNA stored using the system has been identified. The PAXgene™ Bone Marrow RNA System (BM system) is a newly developed system, and its clinical usefulness as a stabilizer for the cellular RNA in BM and peripheral blood was investigated with respect to the quality of RNA extracted using this system. A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was carried out using total RNA extracted with the BM system, which showed that total RNA was more stable in the BM system than in the conventional system, indicating that the BM system can be applied to RT-PCR. The BM system enabled us to detect Wilms' tumor suppressor gene (WT1) more effectively than the conventional system. In conclusion, the BM system is clinically valuable for extracting and stabilizing total RNA of high quality.

S100A8/A9 complex as a new biomarker in prediction of mortality in elderly patients with severe heart failure.

BACKGROUND: S100A8/A9 complex is a new inflammation-related protein and has a positive correlation with C-reaction protein level. However its role in chronic heart failure (CHF) remains unclear. METHODS AND RESULTS: Circulating levels of S100A8/A9 complex and other biomarkers (IL-6, IL-8, TNF-α, and BNP) were measured in CHF (n = 54) and hypertensive without CHF (n = 31) as well as healthy subjects (n = 23), with follow up to 1480 days. During follow-up, cumulative mortality rate for CHF patients was 63%. Plasma levels of S100A8/A9 complex, IL-6, IL-8 and TNF-α were significantly higher in CHF than the hypertensive patients and healthy subjects. A significant positive correlation was found between S100A8/A9 complex and IL-6 and IL-8 respectively. Cox regression analysis showed that IL-6 and IL-8 were predictors for mortality for 6 months, and S100A8/A9 complex, IL-6, IL-8 and age were predictors for mortality for one year whereas BNP, TNF-α, IL-6 and IL-8 remained predictors for mortality for two years. A combination of S100A8/A9 complex and IL-6 provided powerful predictive value in mortality for both 6 and 12 months. CONCLUSIONS: S100A8/A9 complex is a useful biomarker as a predictor for one year mortality and its combination with IL-6 is able to provide additive prognostic information in this vulnerable heart failure population in the elderly.

Dynamic mobility of immunological cells expressing S100A8 and S100A9 in vivo: a variety of functional roles of the two proteins as regulators in acute inflammatory reaction.

The immunological properties of rat S100A8 (r-S100A8) and S100A9 (r-S100A9) in immune cells are poorly understood. Enzyme-linked immunosorbent assay (ELISA) for r-S100A9 enabled us to discuss the differential functional roles of the two proteins, and their localization in the cells was observed microscopically. Recombinant human S100A8 (rh-S100A8) or S100A9 (rh-S100A9) were intravenously administrated into rats with LPS-induced liver damage. ELISA was used to measure the serum concentration of S100A9 in the rats. Western blotting and a preparative ELISA were used to prove specificity and avidity of monoclonal antibodies for r-S100A8 and r-S100A9. Immunohistochemical staining was carried out to visualize intracellular localization of the two proteins in the immune cells using the antibodies. When rh-S100A8 was intravenously injected in the rats (B group), the serum concentration of r-S100A9 apparently decreased as compared with that of the positive control rats (A group). The activities of AST, ALT, and LD in the rat sera (B group) also significantly went down in comparison with those of the rats (A group). Although both the S100A8 and S100A9 were abundantly expressed in activated immune cells, quite difference of not only their intracellular localization but also distribution of the cells expressing the two proteins was microscopically observed. In the rats (B group), less number of the immune cells or less amount of r-S100A8 and r-S100A9 in the cells than those of the rats (A group) was also seen. The r-S100A8 could serve as a regulator of acute inflammatory reaction in the rats with LPS-induced damage.

Identification of serum proteins that bind with S100A8, S100A9 and S100A8/A9: clinical significance of using proteins for monitoring the postoperative condition of liver recipients.

BACKGROUND: Serum proteins that non-specifically bind with human S100A8/A9 (h-S100A8/A9) have been proposed. Our aim was to isolate and identify these proteins, and verify their clinical significance for monitoring the postoperative condition of liver recipients, and further to discuss the transportation of human fibronectin (h-FN) with h-S100A8/A9 and its functional role in vivo. METHODS: To isolate the serum proteins, recombinant human S100A8, S100A9 and S100A8/A9 affinity columns were used. Proteins were identified by mass spectrometry. Two enzyme-linked immunosorbent assays (ELISA) were used to measure h-S100A8/A9 and h-FN in the sera of liver recipients. Flow cytometry was employed to detect h-S100A8/A9 and h-FN on immunological cells. Western blotting was used to confirm serum constituents using antibodies specific to each constituent. RESULTS: One of the proteins was identified with h-FN, and its fluctuation pattern in the serum of the recipient was in contrast to that of CRP. Flow cytometry showed a positive reaction for h-S100A8/A9 and h-FN on neutrophils and monocytes, indicating that both proteins exist on these cells. CONCLUSIONS: The h-FN could be transported with S100A8/A9 in blood and/or on immunological cells, and effectively prevent further attack by various internal oxidants or repair damaged liver tissue in vivo.

[Possible mechanism for regulation of inflammatory responses with the S100A8/A9 protein].

We have described a possible mechanism for the regulation of excessive inflammatory responses with S100A8/A9 protein in damaged rat livers. Recombinant human S100A8(r-S100A8) and S100A9 (r-S100A9) were expressed in E. coli cells, and their heterodimer (r-S100A8/A9) with 90% approximate purity was also prepared successfully. The effect of the r-S100A8/A9 on suppression of acute inflammatory changes in rat livers with LPS-induced damage was microscopically observed. Indeed, the liver damage diminished as the dose of the r-S100A8/A9 increased, and the minimum requirement of the protein was estimated to be 1,000 microg/rat in this study. Observation of superoxide anions was positively observed in control rats treated with LPS alone, but almost not in the livers of rats treated with the r-S 100A8/A9 1h after injection of LPS. This fact strongly suggests that the r-S100A8/A9 could indirectly suppress production of such internal oxidants according to unknown pathway (s) in acute inflammation. Expression of mRNAs of several kinds of inflammatory cytokines, such as TNF-alpha, IL-6 and IL-1beta, was also significantly suppressed, which was of much note. Therefore, the possibility that the r-S100A8/A9 partly inhibits the process of signal transduction of inflammatory responses in the immunological cells leading to down regulation of inflammatory changes in vivo was suggested in this study. Conclusively, S100A8/A9 is not necessarily an inflammatory-induced factor, and preferably effective on suppression of excessive inflammatory reaction in vivo dose-dependently, although the mechanism is still unclear.

Enhanced expression of the S100A8/A9 complex in acute myocardial infarction patients.

BACKGROUND: S100A8/A9 complex (S100A8/A9) is expressed in activated human neutrophils and macrophages. Enhanced expression of S100A8/A9 in atherosclerotic plaque of patients with unstable angina pectoris (UAP) has been demonstrated, but its profile in acute myocardial infarction (AMI) has not been clarified. METHODS AND RESULTS: Serum S100A8/A9 levels were serially measured in patients with AMI (n=55) and UAP (n=16) during the acute period. The expression of S100A8/A9 was examined immunohistochemically in the infarcted myocardium of 7 autopsied patients with AMI. Serum S100A8/A9 levels on the 1st day were 1,118+/-115 (SE) ng/ml in AMI patients as compared with 787+/-147 ng/ml in UAP patients. On days 3-5, serum S100A8/A9 levels in AMI patients reached a peak value and were significantly higher than the values in UAP patients (1,690+/-144 ng/ml vs 844+/-100 ng/ml; P<0.0001). In AMI patients, peak S100A8/A9 levels positively correlated with peak white blood cell and neutrophil counts, and peak creatine kinase-MB and peak C-reactive protein levels. Double immunostaining revealed that S100A8/A9 was specifically expressed in neutrophils and macrophages infiltrating the infarcted myocardium. CONCLUSIONS: S100A8/A9 is implicated in the pathophysiology of AMI and may be an additional biomarker of the local inflammatory response following AMI.

Serum ornithine carbamyltransferase reflects hepatic damage in diabetic obese mice.

BACKGROUND AND AIM: As ornithine carbamyltransferase (OCT) has proved to be a sensitive serum marker in the detection of hepatotoxicity in several models, it is important to confirm its application to the diagnosis of non-alcoholic fatty liver disease. METHODS: C57BL/6, KK-Ta and KK-Ay mice were fed a high-fat diet for 8 weeks and serum enzyme markers were examined. Serum OCT and alanine aminotransferase (ALT) were also measured in diabetic obese ob/ob and db/db mice fed a normal diet. Liver damage in these mice was evaluated by the hepatic content of tumor necrosis factor-alpha. RESULTS: Serum levels of OCT increased in KK-Ay fed a high-fat diet compared with the normal diet-fed group, whereas C57BL/6 and KK-Ta mice were not affected. In ob/ob mice, the relative increase was always greater in OCT than in ALT. In contrast, in db/db mice, the relative increase was always greater in ALT. Hepatic tumor necrosis factor-alpha was significantly elevated in ob/ob mice, but not in db/db mice. CONCLUSIONS: Serum OCT seemed to reflect tumor necrosis factor-alpha-mediated hepatic damage when compared with ALT in diabetic obese mice and could be useful in the application for non-alcoholic fatty liver disease with features of metabolic syndrome, such as obesity and diabetes.

[Possibility of formation of the S100A8/A9-proinflammatory cytokine complexes in vivo in acute inflammation and their functional roles].

We previously hypothesized that S100A8/A9 binds with several kinds of proinflammatory cytokines, such as TNF-alpha, IL-6 and IL-1beta, to form the S100A8/A9-proinflammatory cytokine complexes in vivo in acute inflammation, leading to subsidence of inflammatory responses. Our goal was to verify the presence of these complexes in liver tissues of rats with lipopolysaccharide (LPS) induced damage. We firstly prepared two kinds of the full-length cDNA encoding amino acid sequences of human S100A8 and S100A9 proteins, and constructed their pCold-I expression vectors. The recombinant S100A8 and S100A9 were successfully expressed in E. coli, and then purified by Ni-agarose columns, respectively. The S100A8/A9 was noncovalently synthesized in 2.0 mol/1 Tris-NaOH solution (pH 12) using the purified S100A8 and S100A9. After purification, this heterodimer (1 mg) was intraperitoneally injected into a rat 1h after injection of LPS. Two kinds of ELISA systems were used to detect the S100A8/A9-inflammatory cytokine complexes in the rat liver tissue. As determined by the ELISA-A and B, the reaction was apparently positive and quantitative. Immunohistochemistry provided such complexes-positive cells in the liver with damage. The S100A8/A9-positive cells almost corresponded to the cytokines-positive ones morphologically, strongly suggested the presence of the S100A8/A9-proinflammatory cytokine complexes. In conclusion, the possibility that these complexes were formed in vivo and accumulated to the immunological cells, such as macrophages and/or activated neutrophils, was indicated. Our effort is currently addressed to isolate the S100A8/A9-proinflammatory cytokine complexes using biochemical techniques, and to comprehensively resolute their clinical significance in the differential diagnosis of inflammatory diseases.

Suppression of inflammation in rat autoimmune myocarditis by S100A8/A9 through modulation of the proinflammatory cytokine network.

AIMS: S100A8/A9 is expressed in activated monocytes/macrophages and assumed to be heavily involved in the pathogenesis of acute inflammation. Although several studies have asserted that S100A8/A9 has a proinflammatory function, the exact biological function of S100A8/A9 is yet to be described. We examined the anti-inflammatory effects of S100A8/A9 on experimental autoimmune myocarditis (EAM) in rats. METHODS AND RESULTS: Experimental autoimmune myocarditis was induced in Lewis rats by immunization with porcine cardiac myosin. The recombinant (R-) S100A8/A9 was injected intraperitoneally into EAM rats. R-S100A8/A9 attenuated the severity of myocarditis, as evidenced by echocardiographic and histological findings. In addition, we found that not only the mRNA expression of proinflammatory cytokines [interleukin (IL)-1beta, IL-6, and tumour necrosis factor (TNF)-alpha] in the myocardium, but also their serum concentrations were suppressed in EAM rats treated with R-S100A8/A9. Nuclear factor-kappa B expression in inflammatory cells was also suppressed in the treated rats. To elucidate the mechanistic function of S100A8/A9 on proinflammatory cytokines in vivo, we used an ELISA on the supernatant of homogenized heart tissue treated with R-S100A8/A9. The findings revealed high-affinity binding of R-S100A8/A9 with IL-1beta, IL-6, and TNF-alpha in the myocardium, suggesting the trapping of proinflammatory cytokines by R-S100A8/A9. CONCLUSION: S100A8/A9 attenuates EAM through modulation of the proinflammatory cytokine network.

Ornithine carbamyltransferase is a sensitive marker for alcohol-induced liver injury.

BACKGROUND: Although mitochondrion-derived markers such as ornithine carbamyltransferase (OCT) and glutamate dehydrogenase (GLDH) have been reported to be good markers for alcohol-induced hepatic injury, their use has been limited due to the notion that mitochondrial markers are less sensitive than cytosol-derived markers. We determined the clinical importance of mitochondrion-derived markers in the evaluation of alcohol-induced hepatotoxicity. METHODS: Rats were administered alcohol chronically (5-30% ethanol in drinking water with or without high fat diet feeding for 15 weeks) and hepatic damages were evaluated by serum OCT and GLDH, together with other liver enzymes such as alanine aminotransferase and aspartate aminotransferase. Hepatic content of the enzymes was also evaluated in the chronic ethanol feeding model to confirm whether induction of the enzyme in the liver reflects the serum activity. RESULTS: The serum activities of OCT and GLDH increased significantly by chronic ethanol feeding while other markers did not. Although the hepatic content of OCT and GLDH also increased, the serum activities did not correlate with the hepatic activities and the extent of increase in the liver was much less than in serum. CONCLUSIONS: Mitochondrion-derived markers, especially OCT, appeared superior to cytosol-derived markers in the detection of alcohol-induced liver injury.