Flagellar filament structural protein induces Sjögren's syndrome-like sialadenitis in mice.

OBJECTIVE: Sjögren's syndrome (SS) is a systemic autoimmune disease that primarily affects lacrimal and salivary glands. We previously reported that FliC derived from Escherichia coli could induce autoimmune pancreatitis-like lesions. From these results, we speculated that FliC could also induce SS-like exocrinopathy. In this study, we investigated the effects of chronic exposure to FliC on lacrimal and salivary glands and the possibility that it might lead to an autoimmune response. METHODS: C57BL/6 mice were repeatedly injected with FliC and histological changes, serum levels of cytokine/chemokines and autoantibodies were evaluated at different time points after the final injection. The presence of sialadenitis was diagnosed by histological methods. RESULTS: In FliC-treated groups, 57% of subjects developed inflammatory cell infiltrates around ducts in mandibular salivary glands, but not lacrimal glands. In addition, serum levels of total IgG, IgG1, and IgG2a were significantly higher in FliC-treated groups. Intriguingly, serum anti-SSA/Ro levels were also significantly higher in FliC-treated groups. Cytokine analysis revealed that serum levels of IL-1ß, IL-12p70, IL-13, IFN-γ, IL-15, and IL-23 seemed to be higher in FliC-treated mice. CONCLUSIONS: Our data suggest that FliC-treated mice develop an SS-like phenotype. Our model may elucidate the relationship between commensal bacteria and SS.

Effect of Lipopolysaccharide on the Progression of Non-Alcoholic Fatty Liver Disease in High Caloric Diet-Fed Mice.

The incidence of non-alcoholic steatohepatitis (NASH) is increasing. Because gut microbiota have been highlighted as one of the key factors in the pathogenesis of metabolic syndrome, we investigated the involvement of the bacterial component in the progression of non-alcoholic fatty liver (NAFL) to NASH. C57BL/6 mice were fed with maintenance food (MF, groups A and B) or a high caloric diet (HCD, groups C and D) for 1 month. Mice were then divided into four groups: Groups A and C were inoculated with PBS, while groups B and D were inoculated with lipopolysaccharide (LPS) plus complete Freund's adjuvant (CFA). The inoculations were performed a total of 3 times over 3 months. At 6 months, while hepatic steatosis was observed in groups C and D, cellular infiltration and fibrosis were less evident in group C than in group D. Inflammatory cytokines were upregulated in groups B and D. 16S rRNA pyrosequencing of whole colon homogenates containing faeces showed that certain bacterial groups, such as Bacteroidaceae, Peptostreptococcaceae and Erysipelotrichaceae, were increased in groups C and D. Although loading of bacterial components (LPS) resulted in hepatic inflammation in both MF- and HCD-fed mice, HCD feeding was more crucial in the progression of NAFL during the triggering phase.

The A-domain of beta 2 integrin CR3 (CD11b/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF.

The A-domain is a approximately 200-amino acid peptide present within structurally diverse proadhesive proteins including seven integrins. A recombinant form of the A-domain of beta 2 integrins CR3 and LFA-1 has been recently shown to bind divalent cations and to contain binding sites for protein ligands that play essential roles in leukocyte trafficking to inflammatory sites, phagocytosis and target cell killing. In this report we demonstrate that the neutrophil adhesion inhibitor, NIF produced by the hookworm Ancyclostoma caninium is a selective CD11b A-domain binding protein. NIF bound directly, specifically and with high affinity (Kd of approximately 1 nM) to recombinant CD11b A-domain (r11bA). The binding reaction was characterized by rapid association and very slow dissociation, and was blocked by an anti-r11bA monoclonal antibody. No binding was observed to rCD11aA. The NIF-r11bA interaction required divalent cations, and was absent when the mutant r11bA D140GS/AGA (that lacks divalent cation binding capacity) was used. The NIF binding site in r11bA was mapped to four short peptides, one of which being an iC3b binding site. The interaction of NIF with CR3 in intact cells followed similar binding kinetics to those with r11bA, and occurred with similar affinity in resting and activated human neutrophils, suggesting that the NIF epitope is activation independent. Binding of NIF to CR3 blocked its ability to bind to its ligands iC3b, fibrinogen, and CD54, and inhibited the ability of human neutrophils to ingest serum opsonized particles. NIF thus represents the first example of a disintegrin that targets the integrin A-domain, and is likely to be used by the hookworm to evade the host's inflammatory response. The unique structure of NIF, which lacks a disintegrin motif, emphasizes basic structural differences in antagonists targeting A+ and A- integrins, that should be valuable in drug design efforts aimed at generating novel therapeutics. Identification of the region in NIF mediating A-domain binding should also be useful in this regard, and may, as in the case of disintegrins, unravel a new structural motif with cellular counterparts mediating important physiologic functions.

Clinical implication of vascular cell adhesion molecule-1 and very late activation antigen-4 interaction, and matrix metalloproteinase-2 production in patients with liver disease.

OBJECTIVES: To clarify the role of adhesion molecule in liver cell injury. PATIENTS AND METHODS: The serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), and the expression of VCAM-1 and its ligand, very late activation antigen-4 (VLA-4), were examined in patients with various liver diseases. In addition, the presence of matrix metalloproteinase-2 (MMP-2) was investigated because the release of MMP-2 is thought to be mediated by VLA-4-positive cells. sVCAM-1 and MMP-2 were measured by ELISA assay, and VCAM-1 and VLA-4 were studied by immuno-histological methods. RESULTS: In acute hepatitis (AH) patients, the serum level of sVCAM-1 was significantly elevated compared with that in other cohorts. VCAM-1 was expressed on sinusoidal lining cells but not on hepatocytes. In patients with chronic liver disease, sVCAM-1 levels rose in concert with the progression of chronic hepatitis (CH), and VCAM-1 was also expressed. VLA-4 was detected in both mononuclear cells and Kupffer cells in AH livers, but mainly in Kupffer cells in patients with CH. In AH patients, MMP-2 levels were similar to those in control subjects, but in CH and liver cirrhosis patients, MMP-2 level was elevated in association with CH progression. CONCLUSIONS: The immune response through the VCAM-1 and VLA-4 pathways is important in hepatocyte injury, especially in AH patients, to attach VLA-4-positive mononuclear cells to VCAM-1-positive sinusoidal lining cells. The distribution of VLA-4-positive cells differs between AH and CH patients. VLA-4-positive Kupffer cells in chronic liver diseases might be involved in the progression of CH, perhaps through the mechanism of upregulation of MMP-2 production.

[The analysis of peripheral blood lymphocyte subsets in patients with Epstein Barr virus induced hepatitis].

Both humoral and cell-mediated immunity to Epstein Barr virus related antigen have recently been reported and are thought to be important for the generation of Epstein Barr virus induced human diseases. For this reason, to study immunological features in Epstein Barr virus induced hepatitis (EBH), we studied the ratio of various immunocompetent cells in peripheral blood and compared them to that of healthy control and acute hepatitis (disease control). We found that a significant elevation of CD8+ CD11- cells existed in EBH patients (48.9 +/- 18.3% in EBH, 17.0 +/- 6.9% in AH and 13.6 +/- 7.0% in healthy control) and atypical lymphocytes which are frequently observed in EBH belong to CD8+ CD11- cells. Regarding the study of surface expression of activated T cell markers, we found that these CD8+ cells also expressed HLA-DR antigen on their cell surface. Furthermore, the percentage of CD8+ CD11- cells returned to normal level with the recovery of their liver function. From these results, we can assume that activated CD8+ CD11- cells may have an important roles in generation of EBH.

Effect of alpha-interferon on hepatitis B virus-specific cytotoxic T cells.

To study the mechanism of the effects of alpha-interferon (alpha-IFN) on chronic hepatitis B, we examined its effect on hepatitis B virus (HBV)-specific cytotoxic T cells (CTL). Using two different HBV-DNA transfected human myeloma cell lines, one expressing hepatitis B core antigen (HBcAg; C4) and the other expressing hepatitis B surface antigen (HBsAg; S6) as targets in cytotoxic tests in vitro, peripheral blood mononuclear cells obtained from chronic hepatitis B patients who were treated with alpha-IFN were examined for their cytotoxic activity against these transfectants. During the treatment with alpha-IFN, in association with a decline of serum alanine amino transferase levels, CTL activities were significantly reduced. An inhibition study in vitro revealed that alpha-IFN did not directly inhibit these CTL activities, indicating that alpha-IFN may inhibit the induction of CTL, and thereby may be related to the reduction of hepatocyte injury.

Antibody dependent cell-mediated cytotoxicity using hepatocellular carcinoma reactive monoclonal antibody.

We have established monoclonal antibodies from mice immunized with the human hepatocellular carcinoma cell line, hu-H2. One of the antibodies, designated 523(KY-3), was reactive with this hepatocellular carcinoma cell line as well as with the human pancreatic cancer cell line, MIA. Another monoclonal antibody, 512(KY-2), only reacted with the hepato-cellular carcinoma cell lines. Neither antibody reacted with the colon cancer cell line CW3. Pretreatment of peripheral blood mononuclear cells with 523 resulted in enhancement of their natural cytotoxicity to hu-H2 (9.0 vs 18.4% in subject 1, 3.5 vs 14.7% in subject 2, and 14.2 vs 31.0% in subject 3). In contrast, such antibody mediated enhanced natural cytotoxicity was not found by pretreatment of the same peripheral blood mononuclear cell with 512. With the similarity in reactivity of 523, this antibody dependent enhancement was found in natural cytotoxicity to hu-H2 and MIA but not to CW3. Based on the facts that 523 did not have a direct cytopathic effect on these tumours and that this 523-mediated enhanced natural cytotoxicity was inhibited by anti-FcgammaRIII antibody, we concluded that the 523-mediated enhanced cytotoxicity reflects its activity to induce antibody dependent cytotoxic cells. Thus, these results demonstrate that several distinct tumour-specific antigens exist in hepatocellular carcinoma (HCC) and that one of them represents a potentially useful target for immunotherapy of human hepatocellular carcinoma.

Analytical study of the clinical response to two distinct adoptive immunotherapies for advanced hepatocellular carcinoma: comparison between LAK cell and CTL therapy.

To evaluate the effect of two distinct adoptive immunotherapies, tumor-specific cytotoxic T-cell (CTL) therapy and lymphokine-activated killer (LAK) cell therapy, the clinical responses of patients with stage IV primary hepatocellular carcinoma (HCC) treated with these therapies were studied. Of 18 patients treated with CTL, 3 had complete regression (CR), 2 had partial regression, and 3 had minor regression (MR). Their median survival was 21 months after the end of therapy, and 1 CR patient survived for > 6 years. On the other hand, in the LAK-cell-treated group of eight patients, four had MR and their median survival was only 2 months. No survival was observed 27 months after the end of LAK cell therapy. These results indicate that tumor-specific CTL therapy is more effective than LAK cell therapy and that it might be a promising therapeutic tool for advanced HCC patients.

Analysis of adhesion molecules in patients with idiopathic portal hypertension.

BACKGROUND: The aetiology of idiopathic portal hypertension (IPH) is unknown. However, some evidence of immunological abnormalities in IPH patients has been reported. METHODS: As adhesion molecules are important in the interaction between lymphocytes and accessory and target cells, the expression and release of the soluble form of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule (ICAM-1) were examined in this study. RESULTS: In IPH patients, the serum level of soluble VCAM-1 was found to be increased, compared with that of healthy subjects, fatty liver patients and chronic hepatitis patients. The level of soluble ICAM-1 of IPH patients was found to be slightly increased, compared with that of healthy subjects; however, it was not different from the level in patients with other diseases. The expression of VCAM-1 was observed in the sinusoidal lining cells and endothelial cells around the liver vessels of several IPH patients. In contrast, ICAM-1 was weakly expressed in sinusoidal lining cells and hepatocytes in the liver tissue of only one of four IPH patients. CONCLUSIONS: This differential pattern of VCAM-1 and ICAM-1 was found in IPH patients and it was suggested that VCAM-1 might be an important molecule in the occurrence of IPH.

Balloon-occluded retrograde transvenous obliteration (BRTO), a promising nonsurgical therapy for ectopic varices: a case report of successful treatment of duodenal varices by BRTO.

We present a patient with massive tarry stool, diagnosed as duodenal varices bleeding. Endoscopic ligation (EL) therapy was carried out for the varices, but because the possibility of re-bleeding remained, we treated the varices with balloon-occluded retrograde transvenous obliteration (BRTO), and duodenal varices were successfully decompressed without any complications. BRTO might be a novel therapeutic approach for ectopic varices, including duodenal varices.

A novel therapeutic approach for rectal varices: a case report of rectal varices treated with double balloon-occluded embolotherapy.

We present a patient with continuous melena, diagnosed as rectal varices bleeding. She had a history of esophageal varices, which was treated by endoscopic ligation therapy. Eight years after the treatment of esophageal varices, the continuous melena began. Colonoscopic examination showed that the melena was caused by rectal varices, which were so severe that they could not be treated by either endoscopic sclerotherapy or surgical devascularization. Taking into considering the overall risk of treating rectal varices, we chose the approach of double balloon-occluded embolotherapy (DBOE) with 5% ethanolamine oleate with iopamodol as a liquid embolic material. DBOE is one of the interventional radiology techniques (Morita et al., Acta Hepatol Jpn 1994;35:109-120), but in this case was a completely new and novel clinical procedure for rectal varices. After the DBOE therapy, the condition of rectal varices was markedly improved. Thus, DBOE might be a new tool for treating inoperable rectal varices.

Association of AIM, a novel apoptosis inhibitory factor, with hepatitis via supporting macrophage survival and enhancing phagocytotic function of macrophages.

A hallmark of many inflammatory diseases is the destruction of tissue cells by infiltrating hematopoietic cells including lymphocytes, neutrophils, and macrophages. The regulation of apoptosis of both target tissue cells and the infiltrating cells is one of the key events that defines the initiation and the progression of inflammation. However, the precise picture of the apoptosis regulation of the cells at the inflammatory sites is still unclear. We recently isolated a novel apoptosis inhibitory factor, termed AIM, which is secreted exclusively by tissue macrophages. In this report, we present unique characteristics of AIM associated with liver inflammation (hepatitis), identified by introducing an experimental hepatitis in both AIM-transgenic mice, which overexpress AIM in the body, and normal mice. First, endogenous AIM expression in macrophages is rapidly increased in response to inflammatory stimuli. Second, AIM appears to inhibit the death of macrophages in the inflammatory regions, judging by the remarkably increased number of macrophages observed in the liver from transgenic mice. In addition, we show that AIM also enhances the phagocytosis by macrophages, which emphasizes the multifunctional character of AIM. All these findings strongly provoke an idea that AIM may play an important role in hepatitis pathogenesis in a sequential manner; first AIM expression is up-regulated by inflammatory stimuli, and then in an autocrine fashion, AIM supports the survival of infiltrating macrophages as well as enhances phagocytosis by macrophages, which may result in an efficient clearance of dead cells and infectious or toxic reagents.

Fatal propionic acidemia in mice lacking propionyl-CoA carboxylase and its rescue by postnatal, liver-specific supplementation via a transgene.

Propionic acidemia (PA) is an inborn error of metabolism caused by the genetic deficiency of propionyl-CoA carboxylase (PCC). By disrupting the alpha-subunit gene of PCC, we created a mouse model of PA (PCCA(-/-)), which died in 24-36 h after birth due to accelerated ketoacidosis. A postnatal, liver-specific PCC expression via a transgene in a far lower level than that in wild-type liver, allowed PCCA(-/-) mice to survive the newborn and early infant periods, preventing a lethal fit of ketoacidosis (SAP(+)PCCA(-/-) mice). Interestingly, SAP(+)PCCA(-/-) mice, in which the transgene expression increased after the late infant period, continued to grow normally while mice harboring a persistent low level of PCC died in the late infant period due to severe ketoacidosis, clearly suggesting the requirement of increased PCC supplementation in proportion to the animal growth. Based on these results, we propose a two-step strategy to achieve an efficient PA prevention in human patients: a partial PCC supplementation in the liver during the newborn and early infant periods, followed by a larger amount of supplementation in the late infant period.