Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo.

Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.

A role for FcγRIIB in the development of murine bleomycin-induced fibrosis.

BACKGROUND: Systemic sclerosis (SSc) is a systemic autoimmune disease characterized by excessive fibrosis. FcγRIIB is a low-affinity receptor for the Fc fragment of IgG. FcγRIIB is expressed on the surface of various leukocyte subsets and signals negative feedback pathways to down-regulate B-cell antigen receptor signaling. OBJECTIVE: The aim of the present study was to investigate the role of FcγRIIB in the development of a murine bleomycin-induced scleroderma model. METHODS: The experimental fibrosis model was generated by the intradermal injection of bleomycin into wild-type (WT) and FcγRIIB-deficient (FcγRIIB-/-) mice. We histologically assessed skin and lung fibrosis as well as inflammatory cell infiltration. Cytokine and chemokine expression levels were measured with RT-PCR. RESULTS: The severity of fibrosis in the skin and lung was significantly worse in FcγRIIB-/- mice than in WT mice. In the skin of bleomycin-treated mice, the numbers of CD8+ T cells, F4/80+ macrophages, MPO+ neutrophils, NK1.1+NK cells, and B220+ B cells were significantly higher in FcγRIIB-/- mice than in WT mice. The expression of TNF-α and IL-1ß was significantly higher in FcγRIIB-/- mice than in WT mice as was the expression of ICAM-1, CXCL2, and CCL3 in the affected skin. An adoptive transfer of splenic leukocytes from FcγRIIB-/- mice into WT mice showed exacerbated skin and lung fibrosis compared to WT mice without an adoptive transfer. CONCLUSION: These results indicate that FcγRIIB plays an inhibitory role in skin and lung fibrosis. Moreover, modulating FcγRIIB signaling has potential as a therapeutic approach for SSc.

Classification of Japanese patients with mild/early systemic sclerosis (SSc) by the 2013 ACR/EULAR classification criteria for SSc.

OBJECTIVE: To classify Japanese patients with mild/early systemic sclerosis (SSc) by the 2013 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) Classification Criteria (new 2013 criteria). METHODS: We assessed 120 patients who visited Kanazawa University Hospital suspected of SSc and who did not meet the 1980 ACR preliminary classification criteria for SSc. We clinically diagnosed 16 patients with primary Raynaud's disease and 104 with mild/early SSc prior to being assessed by the new 2013 criteria. RESULTS: None of the 16 patients with primary Raynaud's disease met the new 2013 criteria. On the other hand, 94 out of the 104 patients (90.3%) with mild/early SSc by our clinical diagnosis met the new 2013 criteria. Among the 94 SSc patients, sclerodactyly was detected in 58 (62%), puffy fingers in 62 (66%), abnormal nailfold capillaries in 89 (95%), Raynaud's phenomenon in 93 (99%), and SSc-related autoantibodies (Abs) in 85 (90%). The median (range) score of these 94 patients was 12 (9-14). Ten mild/early SSc patients who did not meet the new 2013 criteria had the following clinical features: puffy fingers in 1 (10%), abnormal nailfold capillaries in 8 (80%), Raynaud's phenomenon in 9 (90%), and SSc-related autoAbs in 8 (80%). The median (range) score of these 10 patients was 7 (5-8). CONCLUSION: The new 2013 criteria can classify most mild/early Japanese SSc patients, which may contribute to early treatment interventions.

Chemokine receptors CCR2 and CX3CR1 regulate skin fibrosis in the mouse model of cytokine-induced systemic sclerosis.

BACKGROUND: Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM), and develop under the influence of certain cytokines. We previously established a mouse model of skin fibrosis induced by exogenous application of cytokines. We have revealed that both the number of macrophages and the levels of macrophage chemoattractant protein-1 (MCP-1) mRNA positively correlate with the extent of skin fibrosis. Macrophages can be divided into two subsets, the first expressing CCR2, and the second expressing CX3CR1. OBJECTIVE: To elucidate the role of skin infiltrating macrophages based on CCR2 and CX3CR1 in this cytokine-induced murine fibrosis model. METHODS: We examined the amounts of collagen deposited in granulation tissues, the numbers of macrophages and the levels of several mRNA in wild type (WT) mice, CCR2(-/-) mice, and CX3CR1(-/-) mice during injections of transforming growth factor-ß (TGF-ß) followed by injections of connective tissue growth factor (CTGF). RESULTS: TGF-ß injection increased the expressions of MCP-1, fractalkine, CCR2 and CX3CR1 mRNA in WT mice. The overproduction of collagen induced by TGF-ß was significantly reduced by CCR2 deficiency, while collagen contents induced by CTGF were restored to wild-type levels. In contrast, overproduction of collagen in CX3CR1-deficient mice decreased nearly 50% by both TGF-ß and CTGF stimulations. CONCLUSION: The involvement of CCR2/MCP-1 interaction (CCR2-dependent loop) was during the TGF-ß phase. In contrast, the fractalkine/CX3CR1 interaction contributes to the initiation of fibrosis by TGF-ß and its maintenance by CTGF. Collectively, two subsets of macrophages both cooperatively and independently play important roles in the development of fibrosis.

Role of connective tissue growth factor and its interaction with basic fibroblast growth factor and macrophage chemoattractant protein-1 in skin fibrosis.

Activation of the immune system and abnormal growth of skin fibroblasts cause systemic sclerosis. Growth factors have various biological activities, including mediation of immune reactions. The growth factor family includes basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and connective tissue growth factor (CTGF). CTGF, an important downstream mediator of TGF-beta in fibrosis, has been suggested to play a specific role in fibrotic disorders. We have directed our attention to the role of CTGF in sustaining skin fibrosis. To better understand its effects in vivo, we established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, bFGF transiently induced subcutaneous fibrosis. Simultaneous injection of bFGF and CTGF increased skin fibrosis compared with a single injection of bFGF. Serial injections of bFGF for 3 days followed by CTGF for 4 days, or of CTGF followed by bFGF, did not cause skin fibrosis but simultaneous injections increased macrophage chemoattractant protein-1 (MCP-1) mRNA expression levels. To further define the mechanisms of skin fibrosis in vivo, bFGF and CTGF were injected simultaneously into MCP-1 knockout mice, resulting in decreased collagen levels in granulation tissues on day 8. The number of inflammatory cells, such as mast cells, macrophages and lymphocytes, was significantly decreased in MCP-1 knockout mice compared with wild-type mice. These results suggest that bFGF induces collagen production by stimulating skin fibroblasts and CTGF cooperates with bFGF. Our results indicate that the induction of MCP-1 is necessary for infiltration of inflammatory cells.

Neutralizing monoclonal antibody to human connective tissue growth factor ameliorates transforming growth factor-beta-induced mouse fibrosis.

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM) and are understood to develop under the influence of fibrogenic growth factors. To better understand the detailed mechanisms of persistent fibrosis in SSc, we have previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, transforming growth factor-beta (TGF-beta) transiently induced subcutaneous fibrosis and serial injections of connective tissue growth factor (CTGF) after TGF-beta caused persistent fibrosis. These results suggest that CTGF plays an important role in the development of persistent skin fibrosis and that CTGF may be a potential and specific therapeutic target in skin fibrosis. Therefore, the aim of the current study is to develop a neutralizing monoclonal antibody against human CTGF. We also investigated the neutralizing effect of the antibodies in our animal model. Firstly, by using the DNA immunization method, we developed a panel of anti-CTGF antibodies recognizing the native conformation of human CTGF. Next, to examine the anti-fibrosing effects of these antibodies, newborn B6 mice received subcutaneous injections of TGF-beta for 3 days with either anti-CTGF neutralizing antibodies or control purified immunoglobulin. Anti-CTGF antibodies significantly reduced skin fibrosis and collagen contents compared with the control group. These results suggest that our anti-CTGF antibodies are capable of blocking the development of skin fibrosis at least partially and these anti-CTGF neutralizing antibodies may be useful as the feasible strategy to treat skin fibrotic diseases as SSc.