Paraoxonase-1 suppresses experimental colitis via the inhibition of IFN-γ production from CD4 T cells.

Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract, where excessive Th1 cell responses are observed. We performed experiments to identify immunologically bioactive proteins in human plasma and found that paraoxonase (PON)-1, which has esterase activity and is associated with high-density lipoproteins, inhibited the IFN-γ production by both murine and human differentiating Th1 cells. Trinitrobenzene sulfonic acid-induced colitis was attenuated by the administration of PON-1. The beneficial effects of PON-1 were associated with a reduced ratio of IFN-γ-producing CD4 T cells in the mesenteric lymph nodes and decreased production of T cell-related cytokines in the colon. PON-1 inhibited the TCR-induced activation of ERK-MAPK signaling and the nuclear translocation of NF-κB in CD4 T cells. Interestingly, an excessive CD4 T cell response was observed in PON-1-deficient mice under physiological and pathological conditions. Additionally, the efficacy of PON-1 or G3C9-C284A (G3C9), which shows a higher esterase activity than PON-1, on colitis was similar to that of an anti-TNF-α mAb, which is a clinically used CD treatment. Moreover, G3C9 more effectively suppressed CD4(+)CD45RB(high) cell transfer-induced chronic colitis in mice than did PON-1, and the efficacy of G3C9 against the colitis was similar to that of the anti-TNF-α mAb. Therefore, PON-1 (or G3C9) administration may be clinically beneficial for CD patients.

The proportion of lymphocytic inflammation with CD123-positive cells in lupus erythematous profundus predict a clinical response to treatment.

Lupus erythematosus profundus is a rare inflammatory disorder of subcutaneous fat in patients with lupus ery-thematosus. Previous reports suggested that plasmacytoid dendritic cells, which expressed CD123 and CD303 antigens, play a central proinflammatory role in the patho-genesis of lupus erythematosus. To find the factors that determine the response to treatment, we analysed 23 skin specimens from the patients with lupus erythematosus profundus. The patients with considerable lymphocytic inflammation with high percentages of CD123+ cells in dermis and subcutaneous fat significantly responded to the systemic corticosteroid therapies. On the other hand, the patients with minor lymphocytic inflammation with low percentages of CD123+ cells showed poor response to treatments. The mean percentage of CD123+ cells in patients who showed good response to therapy was significantly higher than those that showed poor response (p = 0.027). These results suggest that the clinical response to treatment of lupus erythematosus profundus could be predicted from the histological features.

[Acetylcholine Receptor Antibody Neutralization and Selective B-Cell Suppression Using Fc Fusion Protein].

Myasthenia gravis (MG) is an autoimmune disease characterized by formation of autoantibodies against the nicotinic acetylcholine receptor (AChR). Some patients do not show sufficient improvement and develop adverse effects following administration of conventional immune therapy; therefore, the development of new treatments is important. Based on the concept of "selective removal of pathogenic antibodies and cells without suppression of normal immunity," we are developing a fusion protein referred to as AChR-Fc (composed of the AChR alpha subunit and Fc region of human immunoglobulin G1), which shows the following mechanisms of action: selective neutralization of AChR antibodies and cytotoxic activity against AChR antibody-producing pathogenic B cells. Treatment with AChR-Fc is a novel therapeutic approach that may be useful in the management of MG.

Serum levels of leptin receptor in patients with malignant melanoma as a new tumor marker.

Leptin is known to be abnormally expressed in a variety of cancers, and leptin receptors have been reported to be expressed on human melanoma cells. In this study, we evaluated the possibility that the serum levels of leptin receptor could be a tumor marker of malignant melanoma (MM). Serum samples were obtained from 71 patients with MM, and the serum levels of leptin receptor were measured by double-determinant ELISA. Interestingly, serum levels of leptin receptor decreased gradually with the stages of MM, being highest at in situ and lowest at stage IV. There was also a trend of reverse correlation between tumor thickness and serum levels of leptin receptor. To our knowledge, this is the first report investigating the serum levels of leptin receptor in MM, and serum leptin receptor levels may be used as a useful tumor marker of MM.

Apolipoprotein A-II suppressed concanavalin A-induced hepatitis via the inhibition of CD4 T cell function.

Con A-induced hepatitis has been used as a model of human autoimmune or viral hepatitis. During the process of identifying immunologically bioactive proteins in human plasma, we found that apolipoprotein A-II (ApoA-II), the second major apolipoprotein of high-density lipoprotein, inhibited the production of IFN-γ by Con A-stimulated mouse and human CD4 T cells. Con A-induced hepatitis was attenuated by the administration of ApoA-II. The beneficial effect of ApoA-II was associated with reduced leukocyte infiltration and decreased production of T cell-related cytokines and chemokines in the liver. ApoA-II inhibited the Con A-induced activation of ERK-MAPK and nuclear translocation of NFAT in CD4 T cells. Interestingly, exacerbated hepatitis was observed in ApoA-II-deficient mice, indicating that ApoA-II plays a suppressive role in Con A-induced hepatitis under physiological conditions. Moreover, the administration of ApoA-II after the onset of Con A-induced hepatitis was sufficient to suppress disease. Thus, the therapeutic effect of ApoA-II could be useful for patients with CD4 T cell-related autoimmune and viral hepatitis.

Limited influence of aspirin intake on mast cell activation in patients with food-dependent exercise-induced anaphylaxis: comparison using skin prick and histamine release tests.

Food-dependent exercise-induced anaphylaxis (FDEIA) is a severe systemic syndrome induced by physical exercise after ingesting causative food. Aspirin is a well-known trigger for anaphylaxis in patients with FDEIA. Possible mechanisms by which symptoms are aggravated by aspirin include enhanced antigen absorption and mast cell activation. The aim of this study was to determine whether aspirin intake has an influence on mast cell/basophil activation in patients with FDEIA. Provocation tests revealed that adding aspirin to the causative food challenge in 7 of 9 (77.8%) patients with FDEIA provoked symptoms. In most cases, pretreatment with aspirin did not enhance skin tests (71.4%) or histamine release tests (88.9%) with food allergen challenges. The study confirms that histamine release and skin prick tests can be adjunctive tools for diagnosing FDEIA. In addition, our results suggest that exacerbation of FDEIA symptoms by aspirin is not mediated by direct effects of aspirin on mast cell/basophil activation.

Kinesin family member 20A is a novel melanoma-associated antigen.

It has been shown recently that immunotherapy for advanced melanoma is effective. However, in order to improve the efficacy of immunotherapy, the identification of more specific melanoma-associated antigens is urgently needed. Kinesin family member 20A (KIF20A) has been reported to be a promising immunotherapeutic target for pancreatic cancer. To investigate the expression of KIF20A in melanoma, we performed quantitative reverse transcript (RT)-PCR and western blotting analyses of melanoma cell lines. We also investigated primary melanomas and naevus tissues with immunohistochemistry and real-time RT-PCR. KIF20A expression was detected in 59% of melanomas and 12% of naevi by immunohisto-chemistry, and 64% of melanomas and 60% of naevi by real-time RT-PCR. The primary melanomas that were positive for KIF20A showed a significantly greater thickness than those that were negative, and patients with KIF20A+ melanoma tended to develop recurrence earlier. These results suggest that immunotherapy with KIF20A may be a novel treatment option for advanced melanoma.

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells.

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.

[Fc Fusion Protein as a Novel Treatment for Myasthenia Gravis].

Myasthenia gravis (MG) is an inflammatory disorder caused by autoantibodies against the nicotinic acetylcholine receptor (AChR). New therapeutic options for MG are required because the conventional treatments cannot always achieve long-term remission in MG patients. We developed a fusion protein, AChR-Fc, as a novel therapeutic agent for patients with MG. It is composed of the extracellular domain of human AChR-α1 subunit and human IgG1. AChR-Fc exhibited dual activities: it neutralized anti-AChR antibodies and demonstrated an enhanced cytotoxicity against autoantibody-producing B cells. Therefore, AChR-Fc is a novel biomolecule that can be used in the treatment of patients with MG, and potentially overcomes the disorder.

Mesenchymal stem cells cultured under hypoxic conditions had a greater therapeutic effect on mice with liver cirrhosis compared to those cultured under normal oxygen conditions.

BACKGROUND: Mesenchymal stem cells (MSCs) can be easily expanded. They can be acquired from medical waste such as adipose and umbilical cord tissues, are influenced by culturing conditions, and exert anti-inflammatory, antioxidant, anti-fibrotic, and angiogenic effects. We analyzed the multi-directional effects of MSCs cultured under hypoxic conditions and their underlying mechanisms in the treatment of liver cirrhosis in a mouse model. METHODS: Human bone marrow-derived MSCs cultured under hypoxic (5% O2; hypoMSCs) and normoxic (21% O2; norMSCs) conditions were compared by cap analysis of gene expression (CAGE) with or without serum from liver cirrhosis patients. The therapeutic effects of MSCs, including serum liver enzyme induction, fibrosis regression, and hepatic oxidative stress, were evaluated by injecting 1 × 106, 2 × 105, or 4 × 104 MSCs/mouse into the tail veins of mice with carbon tetrachloride (CCl4)-induced liver cirrhosis. Intravital imaging was performed with a two-photon excitation microscope to confirm the various MSC migration paths to the liver. RESULTS: CAGE analysis revealed that the RNA expression levels of prostaglandin E synthase (Ptges) and miR210 were significantly higher in hypoMSCs than in norMSCs. In vivo analysis revealed that both hypoMSCs and norMSCs reduced serum alanine aminotransferase, oxidative stress, and fibrosis compared to that in control mice in a dose-dependent manner. However, hypoMSCs had stronger therapeutic effects than norMSCs. We confirmed this observation by an in vitro study in which hypoMSCs changed macrophage polarity to an anti-inflammatory phenotype via prostaglandin E2 (PGE2) stimulation. In addition, miR210 reduced the rate of hepatocyte apoptosis. Intravital imaging after MSC administration showed that both cell types were primarily trapped in the lungs. Relatively a few hypoMSCs and norMSCs migrated to the liver. There were no significant differences in their distributions. CONCLUSION: The therapeutic effect of hypoMSCs was mediated by PGE2 and miR210 production and was greater than that of norMSCs. Therefore, MSCs can be manipulated to improve their therapeutic efficacy in the treatment of liver cirrhosis and could potentially serve in effective cell therapy. MSCs produce several factors with multidirectional effects and function as "conducting cells" in liver cirrhosis.

Protective effects of antithrombin on puromycin aminonucleoside nephrosis in rats.

We investigated the effects of antithrombin, a plasma inhibitor of coagulation factors, in rats with puromycin aminonucleoside-induced nephrosis, which is an experimental model of human nephrotic syndrome. Antithrombin (50 or 500 IU/kg/i.v.) was administered to rats once a day for 10 days immediately after the injection of puromycin aminonucleoside (50 mg/kg/i.v.). Treatment with antithrombin attenuated the puromycin aminonucleoside-induced hematological abnormalities. Puromycin aminonucleoside-induced renal dysfunction and hyperlipidemia were also suppressed. Histopathological examination revealed severe renal damage such as proteinaceous casts in tubuli and tubular expansion in the kidney of control rats, while an improvement of the damage was seen in antithrombin-treated rats. In addition, antithrombin treatment markedly suppressed puromycin aminonucleoside-induced apoptosis of renal tubular epithelial cells. Furthermore, puromycin aminonucleoside-induced increases in renal cytokine content were also decreased. These findings suggest that thrombin plays an important role in the pathogenesis of puromycin aminonucleoside-induced nephrotic syndrome. Treatment with antithrombin may be clinically effective in patients with nephrotic syndrome.

Severe bacterial sepsis results in delayed diagnosis of tuberculous lymphadenitis in a rheumatoid arthritis patient treated with adalimumab.

Although tumor necrosis factor (TNF)-α inhibitors are effective in patients with rheumatoid arthritis (RA), an increased risk of infections often becomes a serious problem. It is well known that TNF-α inhibitors increase the risk of tuberculosis, but extrapulmonary tuberculosis often induced by them is difficult to diagnose using routine imaging examinations. We described a case of delayed diagnosis of a tuberculous lymphadenitis in a patient with RA treated with TNF-α inhibitor because of the complications of severe bacterial sepsis. In this case, rescreening with the interferon-γ release assay and excisional biopsy were useful in confirming the diagnosis of extrapulmonary tuberculosis. In the case we presented, she had other risk factors, that is, advanced age at the start of anti- TNF-α treatment or concomitant use of corticosteroid, might contribute to the development of complex infections. We should keep in mind that careful follow-up and appropriate examinations are necessary in caring for patients administering immunosuppressive treatments including anti- TNF-α drugs.

Cell division cycle-associated protein 1 as a new melanoma-associated antigen.

Immune checkpoint inhibitors have increased the median survival of melanoma patients. To improve their effects, antigen-specific therapies utilizing melanoma-associated antigens should be developed. Cell division cycle-associated protein 1 (CDCA1), which has a specific function at the kinetochores for stabilizing microtubule attachment, is overexpressed in various cancers. CDCA1, which is a member of cancer-testis antigens, does not show detectable expression levels in normal tissues. Quantitative reverse transcription polymerase chain reaction and immunoblotting analyses revealed that CDCA1 was expressed in all of the tested melanoma cell lines, 74% of primary melanomas, 64% of metastatic melanomas and 25% of nevi. An immunohistochemical analysis and a Cox proportional hazards model showed that CDCA1 could be a prognostic marker in malignant melanoma (MM) patients. CDCA1-specific siRNA inhibited the cell proliferation of SKMEL2 and WM115 cells, but did not reduce the migration or invasion activity. These results suggest that CDCA1 may be a new therapeutic target of melanoma.

Immunotherapy against Metastatic Melanoma with Human iPS Cell-Derived Myeloid Cell Lines Producing Type I Interferons.

In recent years, immunotherapy for advanced melanoma has been gaining increased attention. The efficacy of anti-cytotoxic T-lymphocyte antigen 4 antibodies, anti-programmed cell death 1 antibodies, and the BRAF(V600E) kinase inhibitor has been proven in metastatic melanoma. At the same time, adoptive cell transfer has significant effects against metastatic melanoma; however, it is difficult to apply on a broad scale because of the problems related to cell preparation. To overcome these problems, we developed immune cell therapy using induced pluripotent stem (iPS) cells. The benefit of our method is that a large number of cells can be readily obtained. We focused on macrophages for immune cell therapy because macrophage infiltration is frequently observed in solid cancers. In this study, the efficacy of human iPS cell-derived myeloid cell lines (iPS-ML) genetically modified to express type I IFNs against human melanoma cells was examined. The morphology, phagocytic ability, and surface markers of iPS-ML were similar to those of macrophages. The iPS-ML that express type I IFNs (iPS-ML-IFN) showed significant effects in inhibiting the growth of disseminated human melanoma cells in SCID mice. The infiltration of iPS-ML into the tumor nests was confirmed immunohistologically. The iPS-ML-IFNs increased the expression of CD169, a marker of M1 macrophages that can activate antitumor immunity. The iPS-ML-IFNs could infiltrate into tumor tissue and exert anticancer effects in the local tumor tissue. In conclusion, this method will provide a new therapeutic modality for metastatic melanoma.

Hair shaft miRNA-221 levels as a new tumor marker of malignant melanoma.

miRNA-221 (miR-221) is known to be abnormally expressed in many human cancers. The serum levels of miR-221 have been reported as a tumor marker for malignant melanoma (MM). We hypothesized that the hair shaft miR-221 levels may be increased in patients with MM. We therefore assessed the possibility that hair shaft miR-221 levels could be a marker for MM. The hair shaft miR-221 levels were significantly higher in patients with MM than controls. The rates of increased hair shaft miR-221 levels above the cut-off value were comparable to those of serum 5-S-CD, which is a tumor marker commonly used for MM. Measurements of the hair shaft miR-221 levels could have potential clinical value in the detection of MM. This is the first report investigating the hair shaft levels of an miRNA in patients with MM. Our investigations offer new insight into the relationship between miR-221 and MM, and may provide a new, non-invasive way to screen for melanoma.

Investigation of FOXM1 as a Potential New Target for Melanoma.

Recent studies have shown that immunotherapies and molecular targeted therapies are effective for advanced melanoma. Non-antigen-specific immunotherapies such as immunocheckpoint blockades have been shown to be effective in the treatment of advanced melanoma. However, the response rates remain low. To improve their efficacy, they should be combined with antigen-specific immunotherapy. Elevated expression of the transcription factor, Forkhead box M1 (FOXM1), has been reported in various human cancers, and it has been shown to have potential as a target for immunotherapy. The purpose of this study was to investigate the FOXM1 expression in human melanoma samples and cell lines, to evaluate the relationship between the FOXM1 expression and the clinical features of melanoma patients and to investigate the association between the FOXM1 and MAPK and PI3K/AKT pathways in melanoma cell lines. We conducted the quantitative reverse transcription PCR (qRT-PCR) and Western blotting analyses of melanoma cell lines, and investigated melanoma and nevus tissue samples by qRT-PCR and immunohistochemistry. We performed MEK siRNA and PI3K/AKT inhibitor studies and FOXM1 siRNA studies in melanoma cell lines. We found that FOXM1 was expressed in all of the melanoma cell lines, and was expressed in 49% of primary melanomas, 67% of metastatic melanomas and 10% of nevi by performing immunohistochemical staining. Metastatic melanoma samples exhibited significantly higher mRNA levels of FOXM1 (p = 0.004). Primary melanomas thicker than 2 mm were also more likely to express FOXM1. Patients whose primary melanoma expressed FOXM1 had a significantly poorer overall survival compared to patients without FOXM1 expression (p = 0.024). Downregulation of FOXM1 by siRNA significantly inhibited the proliferation of melanoma cells, and blockade of the MAPK and PI3K/AKT pathways decreased the FOXM1 expression in melanoma cell lines. In conclusion, FOXM1 is considered to be a new therapeutic target for melanoma.

Effects of efonidipine hydrochloride on renal arteriolar diameters in spontaneously hypertensive rats.

Efonidipine, a calcium antagonist, has been reported to dilate not only afferent but also efferent arterioles, thereby reducing glomerular hydrostatic pressure. We investigated the effect of chronic treatment with efonidipine or lisinopril on the afferent and efferent arteriolar diameters by the vascular cast technique. Four-week-old spontaneously hypertensive rats (SHR) were divided into three groups: untreated, efonidipine (25 mg/kg/day)-treated, and lisinopril (3 mg/kg/day)-treated. At 22 weeks of age, the renal vasculatures were fixed at the maximally dilated condition. The morphometrical measurements showed that the treatments with efonidipine and lisinopril caused structural alteration of the vasculature, resulting in significantly greater efferent arteriolar diameters than in untreated SHR. In addition, lisinopril-treated rats had wider afferent lumina. The renoprotective effect of efonidipine and lisinopril might be partly due to the structurally larger efferent arteriolar lumen.

A novel and selective Na+/Ca2+ exchange inhibitor, SEA0400, improves ischemia/reperfusion-induced renal injury.

We evaluated the effects of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline), a novel and selective Na+/Ca2+ exchange inhibitor, on ischemic acute renal failure. Ischemic acute renal failure in rats was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after the contralateral nephrectomy. SEA0400 administration (0.3, 1 and 3 mg/kg, i.v.) before ischemia dose-dependently attenuated the ischemia/reperfusion-induced renal dysfunction and histological damage such as tubular necrosis. SEA0400 pretreatment at the higher dose suppressed the increment of renal endothelin-1 content after reperfusion. The ischemia/reperfusion-induced renal dysfunction was also overcome by post-ischemia treatment with SEA0400 at 3 mg/kg, i.v. In in vitro study, SEA0400 (0.2 and 1 microM) protected cultured porcine tubular cells (LLC-PK1) from hypoxia/reoxygenation-induced cell injury. These findings support the view that Ca2+ overload via the reverse mode of Na+/Ca2+ exchange, followed by endothelin-1 overproduction, plays an important role in the pathogenesis of ischemia/reperfusion-induced renal injury. The possibility exists that a selective Na+/Ca2+ exchange inhibitor such as SEA0400 is useful as effective therapeutic agent against ischemic acute renal failure in humans.