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1.  Therapeutic effect of intratumoral injections of dendritic cells for locally recurrent gastric cancer: a case report 
An 80-year-old man with a history of gastric cancer and pulmonary emphysema underwent a distal gastrectomy for gastric cancer in 1997. In 2010, an endoscopic examination revealed a depressed-type lesion at the oral side of the anastomosis, which was diagnosed as signet-ring adenocarcinoma. Surgical management was considered, but was rejected because of obstructive and restrictive respiratory events. Chemotherapy was terminated because of adverse events. Endoscopy was used to administer intratumoral injections of dendritic cells (DCs) targeting synthesized peptides of Wilms tumor 1 (WT1) and mucin 1, cell-surface associated (MUC1). An immunohistochemical analysis of the tumor samples indicated positivity for WT1 and MUC1. One month after seven cycles of DC had been administered (between November 2010 and April 2011), no suspicious lesions were evident, and his biopsy results were normal. The patient has been in remission for 30 months. Intratumoral injections of DCs showed therapeutic effects in this patient, who could not undergo endoscopic submucosal dissection or surgery.
PMCID: PMC4320508  PMID: 25526950
Dendritic cell; WT1; MUC1; Immunotherapy and recurrent gastric cancer
2.  Persistent Release of IL-1s from Skin Is Associated with Systemic Cardio-Vascular Disease, Emaciation and Systemic Amyloidosis: The Potential of Anti-IL-1 Therapy for Systemic Inflammatory Diseases 
PLoS ONE  2014;9(8):e104479.
The skin is an immune organ that contains innate and acquired immune systems and thus is able to respond to exogenous stimuli producing large amount of proinflammatory cytokines including IL-1 and IL-1 family members. The role of the epidermal IL-1 is not limited to initiation of local inflammatory responses, but also to induction of systemic inflammation. However, association of persistent release of IL-1 family members from severe skin inflammatory diseases such as psoriasis, epidermolysis bullosa, atopic dermatitis, blistering diseases and desmoglein-1 deficiency syndrome with diseases in systemic organs have not been so far assessed. Here, we showed the occurrence of severe systemic cardiovascular diseases and metabolic abnormalities including aberrant vascular wall remodeling with aortic stenosis, cardiomegaly, impaired limb and tail circulation, fatty tissue loss and systemic amyloid deposition in multiple organs with liver and kidney dysfunction in mouse models with severe dermatitis caused by persistent release of IL-1s from the skin. These morbid conditions were ameliorated by simultaneous administration of anti-IL-1α and IL-1β antibodies. These findings may explain the morbid association of arteriosclerosis, heart involvement, amyloidosis and cachexia in severe systemic skin diseases and systemic autoinflammatory diseases, and support the value of anti-IL-1 therapy for systemic inflammatory diseases.
PMCID: PMC4131904  PMID: 25119884
3.  The feasibility and clinical effects of dendritic cell-based immunotherapy targeting synthesized peptides for recurrent ovarian cancer 
Despite the increased rate of complete response to initial chemotherapy, most patients with advanced ovarian cancer relapse and succumb to progressive disease. Dendritic cell (DC)-based immunotherapy has been developed as a novel strategy for generating antitumor immunity as part of cancer treatments. The present study aimed to assess the feasibility and clinical effects of DC therapy for recurrent ovarian cancer (ROC).
This retrospective study included 56 ROC patients who initially received standard chemotherapy followed by DC-based immunotherapy targeting synthesized peptides at 2 institutions between March 2007 and August 2013. The adverse events (AEs) and clinical responses were examined.
No serious treatment-related AEs were observed. Seventy one percent of the enrolled patients developed an immunologic response. The median survival time (MST) from ROC diagnosis was 30.4 months, and that from the first vaccination was 14.5 months. Albumin levels of ≥4.0 g/dL and lactate dehydrogenase levels of <200 IU/L before vaccination were identified as significant independent factors by multivariate Cox proportional hazard analysis. The MST from the first vaccination in patients with albumin levels of ≥4.0 and <4.0 g/dL were 19.9 and 11.6 months, respectively. The corresponding disease control rates were 36% and 15%, respectively.
Our results demonstrated the feasibility and potential clinical effectiveness of DC-based immunotherapy for ROC patients. Additionally, a good nutritional status might be an important factor for further clinical effects.
PMCID: PMC4108140  PMID: 25298213
Dendritic cell; WT1; MUC1; CA125; Immunotherapy and recurrent ovarian cancer
4.  Alterations in the Corneal Nerve and Stem/Progenitor Cells in Diabetes: Preventive Effects of Insulin-Like Growth Factor-1 Treatment 
This study aimed to investigate whether corneal nerve and corneal stem/progenitor cells are altered in insulin-like growth factor-I (IGF-I-) treated individuals with diabetes. A group consisting of db/db mice with type 2 diabetes mellitus (DM) and a wild-type group were assessed by neural and corneal stem/progenitor cell markers immunostaining and real-time PCR. Moreover, the expression of corneal nerve and stem/progenitor cell markers was examined in IGF-1-treated diabetic mice. Compared with a normal cornea, swelling and stratification of the corneal epithelium were noted in db/db mice. Beta-III tubulin immunostaining revealed that the corneal subbasal plexuses in diabetic mice were thinner with fewer branches. mRNA expression levels of Hes1, Keratin15, and p75 (corneal stem/progenitor cell markers) and the intensity and number of positive cells of Hes1 and Keratin19 immunostaining diminished in the diabetic corneas. Compared with the subbasal nerve density in the normal group, a decrease in the diabetic group was observed, whereas the corneal subbasal nerve density increased in IGF-1-treated diabetic group. The decreased expression of Hes1 and Keratin19 was prevented in IGF-1-treated diabetic group. Our data suggest that corneal nerve and stem/progenitor cells are altered in type 2 DM, and IGF-I treatment is capable of protecting against corneal damage in diabetes.
PMCID: PMC3948593  PMID: 24696681
5.  Skewed Helper T-Cell Responses to IL-12 Family Cytokines Produced by Antigen-Presenting Cells and the Genetic Background in Behcet's Disease 
Behcet's disease (BD) is a multisystemic inflammatory disease and is characterized by recurrent attacks on eyes, brain, skin, and gut. There is evidence that skewed T-cell responses contributed to its pathophysiology in patients with BD. Recently, we found that Th17 cells, a new helper T (Th) cell subset, were increased in patients with BD, and both Th type 1 (Th1) and Th17 cell differentiation signaling pathways were overactivated. Several researches revealed that genetic polymorphisms in Th1/Th17 cell differentiation signaling pathways were associated with the onset of BD. Here, we summarize current findings on the Th cell subsets, their contribution to the pathogenesis of BD and the genetic backgrounds, especially in view of IL-12 family cytokine production and pattern recognition receptors of macrophages/monocytes.
PMCID: PMC3892938  PMID: 24490076
6.  Ablation of the mTORC2 component rictor in brain or Purkinje cells affects size and neuron morphology 
The Journal of Cell Biology  2013;201(2):293-308.
The mTOR complex 2 (mTORC2) is essential in the central nervous system for normal neuronal structure and function, potentially through effects on PKC signaling and independent of the related mTOR complex 1 (mTORC1).
The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. Whereas mTORC1 is known to regulate cell and organismal growth, the role of mTORC2 is less understood. We describe two mouse lines that are devoid of the mTORC2 component rictor in the entire central nervous system or in Purkinje cells. In both lines neurons were smaller and their morphology and function were strongly affected. The phenotypes were accompanied by loss of activation of Akt, PKC, and SGK1 without effects on mTORC1 activity. The striking decrease in the activation and expression of several PKC isoforms, the subsequent loss of activation of GAP-43 and MARCKS, and the established role of PKCs in spinocerebellar ataxia and in shaping the actin cytoskeleton strongly suggest that the morphological deficits observed in rictor-deficient neurons are mediated by PKCs. Together our experiments show that mTORC2 has a particularly important role in the brain and that it affects size, morphology, and function of neurons.
PMCID: PMC3628512  PMID: 23569215
7.  Motor dysfunction in cerebellar Purkinje cell-specific vesicular GABA transporter knockout mice 
γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the adult mammalian central nervous system and plays modulatory roles in neural development. The vesicular GABA transporter (VGAT) is an essential molecule for GABAergic neurotransmission due to its role in vesicular GABA release. Cerebellar Purkinje cells (PCs) are GABAergic projection neurons that are indispensable for cerebellar function. To elucidate the significance of VGAT in cerebellar PCs, we generated and characterized PC-specific VGAT knockout (L7-VGAT) mice. VGAT mRNAs and proteins were specifically absent in the 40-week-old L7-VGAT PCs. The morphological characteristics, such as lamination and foliation of the cerebellar cortex, of the L7-VGAT mice were similar to those of the control littermate mice. Moreover, the protein expression levels and patterns of pre- (calbindin and parvalbumin) and postsynaptic (GABA-A receptor α1 subunit and gephyrin) molecules between the L7-VGAT and control mice were similar in the deep cerebellar nuclei that receive PC projections. However, the L7-VGAT mice performed poorly in the accelerating rotarod test and displayed ataxic gait in the footprint test. The L7-VGAT mice also exhibited severer ataxia as VGAT deficits progressed. These results suggest that VGAT in cerebellar PCs is not essential for the rough maintenance of cerebellar structure, but does play an important role in motor coordination. The L7-VGAT mice are a novel model of ataxia without PC degeneration, and would also be useful for studying the role of PCs in cognition and emotion.
PMCID: PMC3893617  PMID: 24474904
cerebellum; Purkinje cells; VGAT; knockout mice; ataxia; mouse model
8.  Reduced bone morphogenetic protein receptor type 1A signaling in neural-crest-derived cells causes facial dysmorphism 
Disease Models & Mechanisms  2012;5(6):948-955.
Bone morphogenetic protein (BMP) receptor type 1A (BMPR1A) mutations are associated with facial dysmorphism, which is one of the main clinical signs in both juvenile polyposis and chromosome 10q23 deletion syndromes. Craniofacial development requires reciprocal epithelial/neural crest (NC)-derived mesenchymal interactions mediated by signaling factors, such as BMP, in both cell populations. To address the role of mesenchymal BMP signaling in craniofacial development, we generated a conditional knockdown mouse by expressing the dominant-negative Bmpr1a in NC-derived cells expressing the myelin protein zero(Mpz)-Cre transgene. At birth, 100% of the conditional mutant mice had wide-open anterior fontanelles, and 80% of them died because of cleft face and cleft palate soon after birth. The other 20% survived and developed short faces, hypertelorism and calvarial foramina. Analysis of the NC-derived craniofacial mesenchyme of mutant embryos revealed an activation of the P53 apoptosis pathway, downregulation of both c-Myc and Bcl-XL, a normal growth rate but an incomplete expansion of mesenchymal cells. These findings provide genetic evidence indicating that optimal Bmpr1a-mediated signaling is essential for NC-derived mesenchymal cell survival in both normal nasal and frontal bone development and suggest that our model is useful for studying some aspects of the molecular etiology of human craniofacial dysmorphism.
PMCID: PMC3484876  PMID: 22773757
9.  Aberrant Activation of Heat Shock Protein 60/65 Reactive T Cells in Patients with Behcet's Disease 
Autoimmune Diseases  2012;2012:105205.
Behcet's disease (BD) is a multisystemic inflammatory disease and is characterized by recurrent attacks on eyes, brain, skin, and gut. There is evidence that skewed T-cell responses contributed to its pathophysiology in patients with BD. We found that heat shock proteins (HSPs) reactive T cells were prevalent in patients with BD. Here, we summarize current findings on HSP reactive T cells and their contribution to the pathogenesis in patients with BD.
PMCID: PMC3467778  PMID: 23082245
11.  Role of SDF1/CXCR4 Interaction in Experimental Hemiplegic Models with Neural Cell Transplantation 
Much attention has been focused on neural cell transplantation because of its promising clinical applications. We have reported that embryonic stem (ES) cell derived neural stem/progenitor cell transplantation significantly improved motor functions in a hemiplegic mouse model. It is important to understand the molecular mechanisms governing neural regeneration of the damaged motor cortex after the transplantation. Recent investigations disclosed that chemokines participated in the regulation of migration and maturation of neural cell grafts. In this review, we summarize the involvement of inflammatory chemokines including stromal cell derived factor 1 (SDF1) in neural regeneration after ES cell derived neural stem/progenitor cell transplantation in mouse stroke models.
PMCID: PMC3317678  PMID: 22489115
neural stem/progenitor cells; chemokines; cell migration; chemokine receptor
14.  Development of Axon-Target Specificity of Ponto-Cerebellar Afferents 
PLoS Biology  2011;9(2):e1001013.
The function of neuronal networks relies on selective assembly of synaptic connections during development. We examined how synaptic specificity emerges in the pontocerebellar projection. Analysis of axon-target interactions with correlated light-electron microscopy revealed that developing pontine mossy fibers elaborate extensive cell-cell contacts and synaptic connections with Purkinje cells, an inappropriate target. Subsequently, mossy fiber–Purkinje cell connections are eliminated resulting in granule cell-specific mossy fiber connectivity as observed in mature cerebellar circuits. Formation of mossy fiber-Purkinje cell contacts is negatively regulated by Purkinje cell-derived BMP4. BMP4 limits mossy fiber growth in vitro and Purkinje cell-specific ablation of BMP4 in mice results in exuberant mossy fiber–Purkinje cell interactions. These findings demonstrate that synaptic specificity in the pontocerebellar projection is achieved through a stepwise mechanism that entails transient innervation of Purkinje cells, followed by synapse elimination. Moreover, this work establishes BMP4 as a retrograde signal that regulates the axon-target interactions during development.
Author Summary
Brain functions rely on highly selective neuronal networks which are assembled during development. Network assembly involves targeted neuronal growth followed by recognition of the appropriate target cells and selective synapse formation. How neuronal processes select their appropriate target cells from an array of interaction partners is poorly understood. In this study, we have addressed this question for the axons emerging from the pontine gray nucleus, a major brainstem nucleus that relays information between the cortex and the cerebellum, a brain area responsible for the control of skilled movements but also emotional processing. Using advanced microscopy techniques, we find that developing mossy fibers establish synaptic contacts rather promiscuously, and elaborate extensive synapses with Purkinje cells, an inappropriate target. These contacts are subsequently eliminated, and proper synaptic connectivity is then restricted to granule and Golgi neurons. We identify bone morphogenetic protein 4 (BMP4) as a regulator of these inappropriate mossy fiber-Purkinje cell contacts. BMP growth factors are best known for their functions in cell specification during embryonic development, and our results support an additional retrograde signaling function between axons and their target cells in early postnatal stages. In summary, we show that the specificity of the synaptic connections in the ponto-cerebellar circuit emerges through extensive elimination of transient synapses.
PMCID: PMC3035609  PMID: 21346800
15.  E3 Ubiquitin Ligase Synoviolin Is Involved in Liver Fibrogenesis 
PLoS ONE  2010;5(10):e13590.
Background and Aim
Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.
The expression and localization of synoviolin in the liver were analyzed in CCl4-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno−/− MEF cells.
In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.
Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.
PMCID: PMC2963597  PMID: 21049091
16.  Functional Roles of Otx2 Transcription Factor in Postnatal Mouse Retinal Development▿ † 
Molecular and Cellular Biology  2007;27(23):8318-8329.
We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2+/−; Crx−/− retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx−/− retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2+/−; Crx−/− retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cell-specific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.
PMCID: PMC2169187  PMID: 17908793
17.  Congenital Semilunar Valvulogenesis Defect in Mice Deficient in Phospholipase Cɛ†  
Molecular and Cellular Biology  2005;25(6):2191-2199.
Phospholipase Cɛ is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cɛ is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cɛ. These results suggest a crucial role of phospholipase Cɛ downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.
PMCID: PMC1061609  PMID: 15743817
18.  Txk, a Nonreceptor Tyrosine Kinase of the Tec Family, Is Expressed in T Helper Type 1 Cells and Regulates Interferon γ Production in Human T Lymphocytes 
The Journal of Experimental Medicine  1999;190(8):1147-1154.
Differentiation of human T cells into T helper (Th)1 and Th2 cells is vital for the development of cell-mediated and humoral immunity, respectively. However, the precise mechanism responsible for the Th1 cell differentiation is not fully clarified. We have studied the expression and function of Txk, a member of the Tec family of nonreceptor tyrosine kinases. We found that Txk expression is restricted to Th1/Th0 cells with IFN-γ producing potential. Txk transfection of Jurkat T cells resulted in a several-fold increase of IFN-γ mRNA expression and protein production; interleukin (IL)-2 and IL-4 production were unaffected. Antisense oligodeoxynucleotide of Txk specifically inhibited IFN-γ production of normal peripheral blood lymphocytes, antigen-specific Th1 clones, and Th0 clones; IL-2 and IL-4 production by the T cells was unaffected. Txk cotransfection led to the enhanced luciferase activity of plasmid (p)IFN-γ promoter/enhancer (pIFN-γ[-538])-luciferase–transfected Jurkat cells upon mitogen activation. Txk transfection did not affect IL-2 and IL-4 promoter activities. Thus, Txk specifically upregulates IFN-γ gene transcription. In fact, Txk translocated from cytoplasm into nuclei upon activation and transfection with a mutant Txk expression plasmid that lacked a nuclear localization signal sequence did not enhance IFN-γ production by the cells, indicating that nuclear localization of Txk is obligatory for the enhanced IFN-γ production. In addition, IL-12 treatment of peripheral blood CD4+ T cells enhanced the Txk expression, whereas IL-4 treatment completely inhibited it. These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-γ production by the cells through Th1 cell–specific positive transcriptional regulation of the IFN-γ gene.
PMCID: PMC2195662  PMID: 10523612
Txk; gene transcription; T helper 1/T helper 2 cells; human; interferon γ
19.  Recent advances in corneal regeneration and possible application of embryonic stem cell-derived corneal epithelial cells 
The depletion of limbal stem cells due to various diseases leads to corneal opacification and visual loss. The unequivocal identification and isolation of limbal stem cells may be a considerable advantage because long-term, functional recovery of corneal epithelium is linked to graft constructs that retain viable stem cell populations. As specific markers of limbal stem cells, the ATP-binding cassette, sub-family G, member2 (ABCG2), a member of the multiple drug-resistance (MDR) family of membrane transporters which leads to a side population phenotype, and transcription factor p63 were proposed recently. Conventional corneal transplantation is not applicable for patients with limbal stem cells deficiency, because the conventional allograft lacks limbal stem cells. The introduction of limbal epithelial cell transplantation was a major advance in the therapeutic techniques for reconstruction of the corneal surface. Limbal epithelial cell transplantation is clinically conducted when cultured allografts as well as autografts are available; however, allografts have a risk of immunologic rejection and autografts are hardly available for patients with bilateral ocular surface disorders. Embryonic stem (ES) cells are characterized by their capacity to proliferate indefinitely and to differentiate into any cell type. We induced corneal epithelial cells from ES cells by culturing them on type IV collagen or alternatively, by introduction of the pax6 gene into ES cells. Recent advances in our study supports the possibility of their clinical use as a cell source for reconstruction of the damaged corneal surface. This review summarizes the recent advances in corneal regeneration therapies and the possible application of ES cell-derived corneal epithelial cells.
PMCID: PMC2704521  PMID: 19668514
corneal epithelial cell; limbal stem cell; transplantation; embryonic stem cell

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