Recombinant Human Soluble Thrombomodulin Suppresses Arteritis in a Mouse Model of Kawasaki Disease.

INTRODUCTION AND OBJECTIVE: Kawasaki disease (KD) is associated with diffuse and systemic vasculitis of unknown aetiology and primarily affects infants and children. Intravenous immunoglobulin (IVIG) treatment reduces the risk of developing coronary aneurysms, but some children have IVIG-resistant KD, which increases their risk of developing coronary artery injury. Here, we investigated the effect of recombinant human soluble thrombomodulin (rTM), which has anticoagulant, anti-inflammatory, and cytoprotective properties on the development of coronary arteritis in a mouse model of vasculitis. METHODS: An animal model of KD-like vasculitis was created by injecting mice with Candida albicans water-soluble fraction (CAWS). This model was used to investigate the mRNA expression of interleukin (IL)-10, tumour necrosis factor alpha (TNF-α), and tissue factor (TF), in addition to histopathology of heart tissues. RESULTS: rTM treatment significantly reduces cardiac vascular endothelium hypertrophy by 34 days after CAWS treatment. In addition, mRNA expression analysis revealed that rTM administration increased cardiac IL-10 expression until day 27, whereas expression of TNF-α was unaffected. Moreover, in the spleen, rTM treatment restores IL-10 and TF expression to normal levels. CONCLUSION: These findings suggest that rTM suppresses CAWS-induced vasculitis by upregulating IL-10. Therefore, rTM may be an effective treatment for KD.

SOCS1 Antagonist-Expressing Recombinant Bacillus Calmette-Guérin Enhances Antituberculosis Protection in a Mouse Model.

Suppressor of cytokine signaling 1 (SOCS1) plays a key role in the negative regulation of JAK/STAT signaling, which is involved in innate immunity and subsequent adaptive immunity. Bacillus Calmette-Guérin (BCG) induces upregulation of SOCS1 expression in host cells, which may lead to the suppression of immune responses by BCG via inhibition of the JAK/STAT signaling pathway. This might cause A reduction in the protective effect of a BCG vaccine. In the current study, we assessed the immune responses to and the protective efficacy of a recombinant BCG secreting a dominant negative mutant of the SOCS1 molecule (rBCG-SOCS1DN). C57BL/6 mice were immunized with rBCG-SOCS1DN or parental BCG Tokyo vaccine strain harboring an empty plasmid vector (rBCG-pSO). rBCG-SOCS1DN enhanced the activation of bone marrow-derived dendritic cells and the activation of T cells compared with those with rBCG-pSO. The amounts of IFN-γ, TNF-α, and IL-6 produced by splenocytes of rBCG-SOCS1DN-immunized mice were larger than those produced by splenocytes of rBCG-pSO-immunized mice. Moreover, the rBCG-SOCS1DN-immunized mice showed a substantial reduction in the number of CFU of Mycobacterium tuberculosis in the lungs and spleens compared with that in control BCG-immunized mice when the immunized mice were infected with a highly pathogenic M. tuberculosis strain by inhalation. These findings provide evidence for the possibility of rBCG-SOCS1DN being an effective M. tuberculosis vaccine with a novel concept of rBCG as a tool for immunomodulation in host cells.

Primary Role of Suppressor of Cytokine Signaling 1 in Mycobacterium bovis BCG Infection.

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of JAK/STAT signaling and is induced by mycobacterial infection. To understand the major function of SOCS1 during infection, we established a novel system in which recombinant Mycobacterium bovis bacillus Calmette-Guérin expressed dominant-negative SOCS1 (rBCG-SOCS1DN) because it would not affect the function of SOCS1 in uninfected cells. When C57BL/6 mice and RAG1-/- mice were intratracheally inoculated with rBCG-SOCS1DN, the amount of rBCG-SOCS1DN in the lungs was significantly reduced compared to that in the lungs of mice inoculated with a vector control counterpart and wild-type BCG. However, these significant differences were not observed in NOS2-/- mice and RAG1-/- NOS2-/- double-knockout mice. These findings demonstrated that SOCS1 inhibits nitric oxide (NO) production to establish mycobacterial infection and that rBCG-SOCS1DN has the potential to be a powerful tool for studying the primary function of SOCS1 in mycobacterial infection.

Cynomolgus monkey induced pluripotent stem cells established by using exogenous genes derived from the same monkey species.

Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.

Propionibacterium acnes vaccination induces regulatory T cells and Th1 immune responses and improves mouse atopic dermatitis.

Atopic dermatitis (AD) is a chronic disease characterized by a polarized Th2 immune response. Propionibacterium acnes (P. acnes) has been shown to elicit strong Th1 immune responses. We hypothesized that the host immune response to P. acnes will prevent the development of AD. To demonstrate this hypothesis, we investigated the effect of P. acnes vaccination on AD that occurs in keratin 14/driven caspase-1 transgenic mouse. Vaccination with low dose of P. acnes successfully prevented clinical manifestations in the skin of AD mice associated with systemic and cutaneous increased expression of Th1-type cytokines but without suppression of Th2 cytokines. Interestingly, the numbers of IFN-γ(+) T cells, FoxP3(+) CD4(+) CD25(+) T cells (nTreg) and IL-10(+) T cells (Tr1) were significantly increased in the spleen. P. acnes vaccination has effects to alter the cytokine milieu and may be useful for the improvement of atopic symptom.

Effects of mycobacteria major secretion protein, Ag85B, on allergic inflammation in the lung.

Many epidemiological studies have suggested that the recent increase in prevalence and severity of allergic diseases such as asthma is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination. However, the underlying mechanisms by which mycobacterial components suppress allergic diseases are not yet fully understood. Here we showed the inhibitory mechanisms for development of allergic airway inflammation by using highly purified recombinant Ag85B (rAg85B), which is one of the major protein antigens secreted from M. tuberculosis. Ag85B is thought to be a single immunogenic protein that can elicit a strong Th1-type immune response in hosts infected with mycobacteria, including individuals vaccinated with BCG. Administration of rAg85B showed a strong inhibitory effect on the development of allergic airway inflammation with induction of Th1-response and IL-17and IL-22 production. Both cytokines induced by rAg85B were involved in the induction of Th17-related cytokine-production innate immune cells in the lung. Administration of neutralizing antibodies to IL-17 or IL-22 in rAg85B-treated mice revealed that IL-17 induced the infiltration of neutrophils in BAL fluid and that allergen-induced bronchial eosinophilia was inhibited by IL-22. Furthermore, enhancement of the expression of genes associated with tissue homeostasis and wound healing was observed in bronchial tissues after rAg85B administration in a Th17-related cytokine dependent manner. The results of this study provide evidence for the potential usefulness of rAg85B as a novel approach for anti-allergic effect and tissue repair other than the role as a conventional TB vaccine.

Recombinant Ag85B vaccine by taking advantage of characteristics of human parainfluenza type 2 virus vector showed Mycobacteria-specific immune responses by intranasal immunization.

Viral vectors are promising vaccine candidates for eliciting suitable Ag-specific immune response. Since Mycobacterium tuberculosis (Mtb) normally enters hosts via the mucosal surface of the lung, the best defense against Mtb is mucosal vaccines that are capable of inducing both systemic and mucosal immunity. Although Mycobacterium bovis bacille Calmette-Guérin is the only licensed tuberculosis (TB) vaccine, its efficacy against adult pulmonary forms of TB is variable. In this study, we assessed the effectiveness of a novel mucosal TB vaccine using recombinant human parainfluenza type 2 virus (rhPIV2) as a vaccine vector in BALB/c mice. Replication-incompetent rhPIV2 (M gene-eliminated) expressing Ag85B (rhPIV2-Ag85B) was constructed by reverse genetics technology. Intranasal administration of rhPIV2-Ag85B induced Mtb-specific immune responses, and the vaccinated mice showed a substantial reduction in the number of CFU of Mtb in lungs and spleens. Unlike other viral vaccine vectors, the immune responses against Ag85B induced by rhPIV2-Ag85B immunization had an advantage over that against the viral vector. In addition, it was revealed that rhPIV2-Ag85B in itself has an adjuvant activity through the retinoic acid-inducible gene I receptor. These findings provide further evidence for the possibility of rhPIV2-Ag85B as a novel TB vaccine.

Intranasally administered antigen 85B gene vaccine in non-replicating human Parainfluenza type 2 virus vector ameliorates mouse atopic dermatitis.

Atopic dermatitis (AD) is a refractory and recurrent inflammatory skin disease. Various factors including heredity, environmental agent, innate and acquired immunity, and skin barrier function participate in the pathogenesis of AD. T -helper (Th) 2-dominant immunological milieu has been suggested in the acute phase of AD. Antigen 85B (Ag85B) is a 30-kDa secretory protein well conserved in Mycobacterium species. Ag85B has strong Th1-type cytokine inducing activity, and is expected to ameliorate Th2 condition in allergic disease. To perform Ag85B function in vivo, effective and less invasive vaccination method is required. Recently, we have established a novel functional virus vector; recombinant human parainfluenza type 2 virus vector (rhPIV2): highly expressive, replication-deficient, and very low-pathogenic vector. In this study, we investigated the efficacy of rhPIV2 engineered to express Ag85B (rhPIV2/Ag85B) in a mouse AD model induced by repeated oxazolone (OX) challenge. Ear swelling, dermal cell infiltrations and serum IgE level were significantly suppressed in the rhPIV2/Ag85B treated mouse group accompanied with elevated IFN-γ and IL-10 mRNA expressions, and suppressed IL-4, TNF-α and MIP-2 mRNA expressions. The treated mice showed no clinical symptom of croup or systemic adverse reactions. The respiratory tract epithelium captured rhPIV2 effectively without remarkable cytotoxic effects. These results suggested that rhPIV2/Ag85B might be a potent therapeutic tool to control allergic disorders.

Single systemic administration of Ag85B of mycobacteria DNA inhibits allergic airway inflammation in a mouse model of asthma.

The immune responses of T-helper (Th) and T-regulatory cells are thought to play a crucial role in the pathogenesis of allergic airway inflammation observed in asthma. The correction of immune response by these cells should be considered in the prevention and treatment of asthma. Native antigen 85B (Ag85B) of mycobacteria, which cross-reacts among mycobacteria species, may play an important biological role in host-pathogen interaction since it elicits various immune responses by activation of Th cells. The current study investigated the antiallergic inflammatory effects of DNA administration of Ag85B from Mycobacterium kansasii in a mouse model of asthma. Immunization of BALB/c mice with alum-adsorbed ovalbumin followed by aspiration with aerosolized ovalbumin resulted in the development of allergic airway inflammation. Administration of Ag85B DNA before the aerosolized ovalbumin challenge protected the mice from subsequent induction of allergic airway inflammation. Serum and bronchoalveolar lavage immunoglobulin E levels, extent of eosinophil infiltration, and levels of Th2-type cytokines in Ag85B DNA-administered mice were significantly lower than those in control plasmid-immunized mice, and levels of Th1-and T-regulatory-type cytokines were enhanced by Ag85B administration. The results of this study provide evidence for the potential utility of Ag85B DNA inoculation as a novel approach for the treatment of asthma.

Eplerenone attenuates myocardial fibrosis in the angiotensin II-induced hypertensive mouse: involvement of tenascin-C induced by aldosterone-mediated inflammation.

Tenascin-C is an extracellular matrix glycoprotein that is supposed to be a profibrotic molecule in various fibrogenic processes. To elucidate its significance for myocardial fibrosis in the hypertensive heart, we used a mouse model with infusion of angiotensin II and examined results by histology, immunohistochemistry, in situ hybridization, and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Angiotensin II treatment elevated blood pressure and expression of tenascin-C by interstitial fibroblasts in perivascular fibrotic lesions, and angiotensin II infusion caused accumulation of macrophages. It also upregulated expression of collagen Ialpha2; IIIalpha1; and proinflammatory/profibrotic mediators including transforming growth factor beta (TGFbeta), platelet-derived growth factor alpha (PDGF-A), PDGF-B, and PDGF-receptor alpha, but not IL-1beta and PDGF-receptor beta, in the myocardium. Treatment with an aldosterone receptor antagonist, eplerenone, significantly attenuated angiotensin II-induced fibrosis, expression of tenascin-C, and inflammatory changes without affecting the blood pressure level. In vitro, neither eplerenone nor aldosterone exerted any influence on tenascin-C expression of cardiac fibroblasts, whereas angiotensin II, TGF-beta1, and PDGF significantly upregulated expression of tenascin-C. These results suggest that, in the angiotensin II-induced hypertensive mouse heart: (1) tenascin-C may be involved in the progression of cardiac fibrosis and (2) aldosterone may elicit inflammatory reactions in myocardium, which might, in turn, induce tenascin-C synthesis of fibroblasts through at least 2 pathways mediated by TGF-beta and PDGF-A-B/PDGF-receptor alpha.

Expression of large tenascin-C splice variants by hepatic stellate cells/myofibroblasts in chronic hepatitis C.

BACKGROUND/AIMS: Earlier studies have suggested involvement of tenascin-C (TN-C) in liver fibrosis. Here, we examined expression of TN-C variants and types of alternatively spliced fibronectin-type III (FNIII) repeats in chronic hepatitis. METHODS: Using three monoclonal antibodies against TN-C variants, immunohistochemical staining was performed and the correlation with histological parameters of chronic hepatitis C was examined. The cellular source was also determined and variant expression and their types were tested using isolated rat hepatic stellate cells (HSCs), liver myofibroblasts, and/or LI90 cells. RESULTS: Large variants were not expressed in normal liver, but were up-regulated in chronic hepatitis, especially at sites of interface hepatitis and confluent necrosis, showing stronger correlations between staining intensity and these than with other parameters or fibrosis. TN-C deposition was closely correlated with increase in the number of alpha-smooth muscle actin-positive cells, i.e. activated HSCs/myofibroblasts, and in situ hybridization showed TN-C mRNA signals in the cells. Activated HSCs and myofibroblasts in culture highly expressed large variants of TN-C. In LI90 cells, sequencing of large variants revealed that the FNIII repeats D and A1/A4, followed by B, were preferentially included. CONCLUSIONS: TN-C and its variants are produced by HSCs/myofibroblasts, suggesting important roles in liver fibrogenesis.

Tenascin-C regulates recruitment of myofibroblasts during tissue repair after myocardial injury.

Tenascin-C (TN-C) is an extracellular matrix molecule that is expressed during wound healing in various tissues. Although not detectable in the normal adult heart, it is expressed under pathological conditions. Previously, using a rat model, we found that TN-C was expressed during the acute stage after myocardial infarction and that alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts appeared in TN-C-positive areas. In the present study, we examined whether TN-C controls the dynamics of myofibroblast recruitment and wound healing after electrical injury to the myocardium of TN-C knockout (TNKO) mice compared with wild-type (WT) mice. In TNKO mice, myocardial repair seemed to proceed normally, but the appearance of myofibroblasts was delayed. With cultured cardiac fibroblasts, TN-C significantly accelerated cell migration, alpha-SMA expression, and collagen gel contraction but did not affect proliferation. Using recombinant fragments of murine TN-C, the functional domain responsible for promoting migration of cardiac fibroblasts was mapped to the conserved fibronectin type III (FNIII)-like repeats and the fibrinogen (Fbg)-like domain. Furthermore, alternatively spliced FNIII and Fbg-like domains proved responsible for the up-regulation of alpha-SMA expression. These results indicate that TN-C promotes recruitment of myofibroblasts in the early stages of myocardial repair by stimulating cell migration and differentiation.

Tenascin-C upregulates matrix metalloproteinase-9 in breast cancer cells: direct and synergistic effects with transforming growth factor beta1.

Tenascin-C (TN-C) and matrix metalloproteinases (MMPs) are highly expressed in cancer tissues and probably promote cell migration during cancer progression. TN-C and MMPs are often co-localized in areas of active tissue remodeling in pathologic conditions, suggesting reciprocal regulation. To investigate whether TN-C regulates MMPs expression in cancer cells, we first exposed mammary cancer cells derived from TN-C-deficient mice to TN-C and examined MMPs expression. TN-C was then compared with fibronectin (FN), laminin (LN), basic fibroblast growth factor (b-FGF) and transforming growth factor-beta1 (TGF-beta1). Results of endpoint RT-PCR, quantitative real-time RT-PCR and gelatin zymography demonstrated that TN-C, strongly and dose dependently, upregulates MMP-9 expression in murine mammary cancer cells. TN-C weakly induced MMP-2, MMP-3 and MMP-13. FN and LN induced MMP-9 to lesser extents compared with TN-C. b-FGF had no effect on MMP-9 expression. TGF-beta1 induced MMP-9 expression in a dose-dependent manner, and this induction was significantly enhanced by addition of TN-C. TN-C and TGF-beta1 also upregulated MMP-9 expression in the human breast cancer cell line MDA-MB-231. Neutralization with specific anti-TGF-beta1 antibody showed decreased expression of MMP-9, indicating that TGF-beta controls the baseline MMP-9 expression by a direct autocrine mechanism. Under neutralization of TGF-beta, addition of TN-C still upregulated MMP-9. Conversely, neutralization of endogenous TN-C (in a TN-C-positive mammary cancer cell line) downregulated TGF-beta-induced MMP-9 expression. Thus, TN-C induces MMP-9 expression directly and by collaboration with TGF-beta. These findings reveal a novel role of TN-C in breast cancer progression.

Co-stimulation of human breast cancer cells with transforming growth factor-beta and tenascin-C enhances matrix metalloproteinase-9 expression and cancer cell invasion.

Transforming growth factor-beta (TGF-beta), tenascin-C (TN-C) and matrix metalloproteinases (MMPs) have been demonstrated independently to be associated with disease progression and poor prognosis in patients with breast cancer. The present study explored effects of TGF-beta and TN-C on MMP-9 expression and cancer invasion. An experimental study was designed to analyse MDA-MB-231 breast cancer cells, known for their high invasiveness, after stimulation with TGF-beta1 and/or TN-C. TGF-beta1 stimulated TN-C expression in the cells. Co-stimulation of MDA-MB-231 cells with TN-C and TGF-beta increased MMP-9 expression at both the gene (28-fold) and the protein levels. The in vitro invasion also increased (4-fold). GM6001 inhibited the invasion induced by the co-stimulation. The combined effect of TN-C and TGF-beta resulted in enhanced MMP-9 expression and cancer invasion in vitro.

Densin-180, a synaptic protein, links to PSD-95 through its direct interaction with MAGUIN-1.

BACKGROUND: Densin-180, a brain-specific protein highly concentrated at the postsynaptic density (PSD), belongs to the LAP [leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains] family of proteins, some of which play fundamental roles in the establishment of cell polarity. RESULTS: To identify new Densin-180-interacting proteins, we screened a yeast two-hybrid library using the COOH-terminal fragment of Densin-180 containing the PDZ domain as bait, and we isolated MAGUIN-1 as a Densin-180-binding protein. MAGUIN-1, a mammalian homologue of Drosophila connector enhancer of KSR (CNK), is known to interact with PSD-95 and has a short isoform, MAGUIN-2. The Densin-180 PDZ domain bound to the COOH-terminal PDZ domain-binding motif of MAGUIN-1. Densin-180 co-immunoprecipitated with MAGUIN-1 as well as with PSD-95 from the rat brain. In dissociated hippocampal neurones Densin-180 co-localized with MAGUINs and PSD-95, mainly at neuritic spines. In transfected cells, Densin-180 formed a ternary complex with MAGUIN-1 and PSD-95, whereas no association was detected between Densin-180 and PSD-95 in the absence of MAGUIN-1. MAGUIN-1 formed a dimer or multimer via the COOH-terminal leucine-rich region which is present in MAGUIN-1 but not in -2. Among the PDZ domains of PSD-95, the first was sufficient for interaction with MAGUIN-1. CONCLUSION: These results suggest that the potential to dimerize or multimerize allows MAGUIN-1 to bind simultaneously to both Densin-180 and PSD-95, leading to the ternary complex assembly of these proteins at the postsynaptic membrane.

Involvement of large tenascin-C splice variants in breast cancer progression.

Alternative splicing of fibronectin-like type III (FNIII) repeats of tenascin-C (Tn-C) generates a number of splice variants. The distribution of large variants, typical components of provisional extracellular matrices that are up-regulated during tumor stroma remodeling, was here studied by immunoblotting and immunohistochemistry using a monoclonal antibody against the FNIII B domain (named 4C8MS) in a series of human breast cancers. Large Tn-C variants were found at only low levels in normal breast tissues, but were highly expressed at invading sites of intraductal cancers and in the stroma of invasive ductal cancers, especially at invasion fronts. There was a positive correlation between the expression of large Tn-C variants and the cell proliferation rate determined by immunolabeling of the Ki-67 antigen. Of the Tn-C recombinant fragments (all FNIII repeats or mFNIII FL, the conserved FNIII domain only, the epidermal growth factor-like domain, and the fibrinogen-like domain) which were expressed by CHO-K1 cells transfected with mouse Tn-C cDNAs, only the mFNIII FL enhanced in vitro migration and mitotic activity of mammary cancer cells derived from a Tn-C-null mouse. Addition of 4C8MS blocked the function of mFNIII FL. These findings provide strong evidence that the FNIII alternatively spliced region has important roles in tumor progression of breast cancer.