Takuma Ishizaki

miR126 positively regulates mast cell proliferation and cytokine production through suppressing Spred1

The protein known as Spred1 (Sprouty‐related Ena/VASP homology‐1 domain‐containing protein) has been identified as a negative regulator of growth factor‐induced ERK/mitogen‐activated protein kinase activation. Spred1 has also been implicated as the target of microRNA‐126 (miR126), a miRNA located within the Egfl7 gene, and is involved in the regulation of vessel development through its role in regulating VEGF signaling. In this study, we examined the role of miR126 and Spred1 in the hematopoietic system, as miR126 has been shown to be overexpressed in leukemic cells. miR126 levels were down‐regulated during mast cell differentiation from bone marrow cells, whereas Spred1 expression was inversely up‐regulated. Overexpression of miR126 suppressed Spred1 expression and enhanced ERK activity in primary bone marrow cells and MC9 mast cells, which were associated with elevated FcεRI‐mediated cytokine production. To confirm the effect of Spred1 reduction in vivo, we generated hematopoietic cell‐specific Spred1‐conditional knockout mice. These mice showed increased numbers of mast cells, and Spred1‐deficient bone marrow‐derived mast cells were highly activated by cross‐linking of Fcε‐R stimulation as well as by IL‐3 and SCF stimulation. These results suggest that Spred1 negatively regulates mast cell activation, which is modulated by miR126.

SOCS3 in T and NKT Cells Negatively Regulates Cytokine Production and Ameliorates ConA-Induced Hepatitis

Suppressor of cytokine signaling 3 (SOCS3), a negative-feedback molecule for cytokine signaling, has been implicated in protection against liver injury. Previous studies have shown that overexpression of SOCS3 in the liver by adenovirus or membrane permeable recombinant protein protected the liver from various injuries. However it remained uncertain in which type of cells SOCS3 suppresses liver injury. In this study, we demonstrated that forced expression of SOCS3 in T and NKT cells suppressed ConA-induced hepatitis using T and NKT cell-specific SOCS3 transgenic (Lck-SOCS3 Tg) mice. IFN-γ and IL-4 production was reduced in Lck-SOCS3 Tg mice as well as splenocytes treated with ConA. IFN-γ and IL-4 levels were also reduced in Lck-SOCS3 Tg mice administrated with α-galactosylceramide, suggesting that SOCS3 in NKT cells has suppressive function. Sustained expression of SOCS3 in an NKT cell line also resulted in reduced expression of various cytokines and transcription factors. In contrast, T and NKT cell-specific SOCS3 conditional knockout (Lck-SOCS3 cKO) mice were hypersensitive to ConA-mediated hepatitis. Isolated SOCS3-deficient NKT cells produced higher levels of IFN-γ and IL-4. These data indicate that SOCS3 plays a negative regulatory role in NKT cell activation and that forced expression of SOCS3 in NKT cells is effective in preventing hepatitis.

Sprouty4 deficiency potentiates Ras-independent angiogenic signals and tumor growth

Sprouty proteins have been shown to negatively regulate a variety of receptor tyrosine kinase (RTK) signaling pathways and are considered to be tumor suppressor proteins. The pathophysiological functions of Sproutys in vivo remain to be investigated. In this study, we examined the physiological function of Sprouty4 as an angiogenic regulator, using Sprouty4 knockout (KO) mice and cells. We found that transplanted tumor cells grow much faster in Sprouty4 KO mice than in wild type (WT) mice, which we associate with enhanced neovascularization in the tumors transplanted into Sprouty4 KO mice. Moreover, vascular endothelial growth factor (VEGF)‐A‐induced angiogenesis and vascular permeability in vivo were enhanced in Sprouty4 KO mice compared with WT mice. Ex vivo angiogenesis, which we induced by VEGF‐A, basic fibroblast growth factor (bFGF), and sphingosine‐1‐phosphate (S1P), was also enhanced in the aortas of Sprouty4 KO mice. We demonstrated that Sprouty4 suppresses Ras‐independent VEGF‐A and S1P signaling, while it does not affect Ras‐dependent VEGF‐C signaling. These data indicate that Sprouty4 selectively suppresses Ras‐independent angiogenic factor signals and is an important negative regulator of pathophysiological angiogenesis. (Cancer Sci 2009; 100: 1648–1654)

Legius syndrome in fourteen families

Legius syndrome presents as an autosomal dominant condition characterized by café‐au‐lait macules with or without freckling and sometimes a Noonan‐like appearance and/or learning difficulties. It is caused by germline loss‐of‐function SPRED1 mutations and is a member of the RAS‐MAPK pathway syndromes. Most mutations result in a truncated protein and only a few inactivating missense mutations have been reported. Since only a limited number of patients has been reported up until now, the full clinical and mutational spectrum is still unknown. We report mutation data and clinical details in fourteen new families with Legius syndrome. Six novel germline mutations are described. The Trp31Cys mutation is a new pathogenic SPRED1 missense mutation. Clinical details in the 14 families confirmed the absence of neurofibromas, and Lisch nodules, and the absence of a high prevalence of central nervous system tumors. We report white matter T2 hyperintensities on brain MRI scans in 2 patients and a potential association between postaxial polydactyly and Legius syndrome.

Prostaglandin E2 is a major soluble factor produced by stromal cells for preventing inflammatory cytokine production from dendritic cells

Dendritic cells (DCs) are specialized antigen-presenting cells that play pivotal roles in initiating immune responses. However, DC maturation is usually strongly restricted by the stromal microenvironment, especially in non-lymphoid tissues, such as skin and mucosa. Although suppression of DC maturation by stromal cells has been well documented, the molecular basis of this suppression has not been established. In this study, we examined the role of fibroblasts for DC maturation in vitro. The mouse embryonic fibroblasts (MEFs) strongly suppressed LPS-induced DC maturation. Although suppression of class II MHC and CD40 required DC-MEF contact, soluble factors in the culture supernatant of MEFs were sufficient for the suppression of IL-12 and tumor necrosis factor-alpha production. Using molecular-size selection and HPLC, we determined that prostaglandin E2 (PGE2) is a major soluble inhibitory factor secreted by MEFs. This was confirmed by the fact that cyclooxygenase inhibitors inhibited the production of the suppressive factor by MEFs. These results suggest that PGE2 is a major soluble factor produced by MEFs for the suppression of inflammatory cytokine production from DCs, while a contact mechanism between MEFs and DCs is required for the suppression to induce T cell-stimulating molecules.

Suppression of Sproutys Has a Therapeutic Effect for a Mouse Model of Ischemia by Enhancing Angiogenesis

Sprouty proteins (Sproutys) inhibit receptor tyrosine kinase signaling and control various aspects of branching morphogenesis. In this study, we examined the physiological function of Sproutys in angiogenesis, using gene targeting and short-hairpin RNA (shRNA) knockdown strategies. Sprouty2 and Sprouty4 double knockout (KO) (DKO) mice were embryonic-lethal around E12.5 due to cardiovascular defects. The number of peripheral blood vessels, but not that of lymphatic vessels, was increased in Sprouty4 KO mice compared with wild-type (WT) mice. Sprouty4 KO mice were more resistant to hind limb ischemia and soft tissue ischemia than WT mice were, because Sprouty4 deficiency causes accelerated neovascularization. Moreover, suppression of Sprouty2 and Sprouty4 expression in vivo by shRNA targeting accelerated angiogenesis and has a therapeutic effect in a mouse model of hind limb ischemia. These data suggest that Sproutys are physiologically important negative regulators of angiogenesis in vivo and novel therapeutic targets for treating peripheral ischemic diseases.

Complete remission achieved by steroid pulse therapy following rituximab treatment in a case with autoimmune haemorrhaphilia due to anti-factor XIII antibodies

Complete remission achieved by steroid pulse therapy following rituximab treatment in a case with autoimmune haemorrhaphilia due to anti-factor XIII antibodies -

Successful Management of a Patient with Autoimmune Hemorrhaphilia due to Anti-Factor XIII/13 Antibodies Complicated by Pulmonary Thromboembolism

Autoimmune hemophilia-like disease (hemorrhaphilia) due to anti-factor XIII (FXIII) antibodies (AH13) is a very rare, life-threatening bleeding disorder. A 77-year-old woman developed macrohematuria and a right renal pelvic hematoma. The coagulation times were not prolonged, but FXIII activity and antigen levels were severely and moderately reduced to 9 and 29% of normal values, respectively. Accordingly, the FXIII-specific activity turned out to be low. FXIII inhibitor and anti-FXIII-A subunit autoantibodies were detected by a 1:1 crossmixing test and immunoblot and immunochromatographic assays. She was therefore diagnosed with “definite AH13” and treated with plasma-derived FXIII concentrates to arrest the hemorrhage. In addition to a highly compressed inferior vena cava by a huge renal pelvic hematoma, deep vein thrombosis (DVT) and pulmonary thromboembolism (PE) were identified by systemic computed tomography. The patient was immediately started on anticoagulation therapy with low-dose heparin. Emboli disappeared quickly, probably because under-crosslinked thrombi caused by severe FXIII deficiency are vulnerable to fibrinolysis. After about 1.5 years, anti-FXIII-A subunit autoantibodies still remained despite the use of rituximab, steroid pulse therapy, oral prednisolone, and oral cyclophosphamide treatments. In conclusion, an extremely rare AH13 case complicated by DVT and PE was successfully managed by balancing anticoagulation therapy with hemostatic therapy.

Successful Treatment of Epstein-Barr Virus-Associated Lymphoproliferative Disorder with Rituximab in a Patient Undergoing Immunosuppressive Therapy for Aplastic Anemia.

Epstein-Barr virus-associated lymphoproliferative disorder (EBV-LPD) is a currently emerging serious complication in immunosuppressed patients, especially in allogeneic transplant recipients. Several fatal cases of EBV-LPD have been reported in aplastic anemia (AA) patients receiving immunosuppressive therapy (IST) with antithymocyte globulin (ATG) plus cyclosporine A (CsA), but no appropriate prophylactic or therapeutic strategy has been established. Herein, we describe a 29-year-old man whose EBV-LPD was successfully treated with rituximab. He received IST with ATG plus CsA for hepatitis-associated AA. EBV-DNA in plasma, which was not detectable before IST, gradually increased after IST initiation. A high fever and systemic lymphadenopathy developed 31 days after IST initiation. An EBV-DNA titer of 5.7 × 105 copies/μl was detected, and a diagnosis of EBV-LPD was made. Although discontinuation of IST was not effective, a single dose of rituximab on day 33 resolved the clinical symptoms and completely eliminated EBV-DNA. Even after restarting CsA administration, no elevation of EBV-DNA was observed, and his complete blood cell count had fully recovered 1 year after IST. This case suggests that this treatment strategy for EBV-LPD with EBV-DNA monitoring and rituximab administration, which has been recommended in allogeneic transplant recipients, may also be useful in the context of AA patients receiving IST.