Yohta Shimada

We generated iPS cells from human dermal fibroblasts (HDFs) of Fabry disease using a Sendai virus (SeVdp) vector; this method has been established by Nakanishi et al. for pathogenic evaluation. We received SeVdp vector from Nakanishi and loaded it simultaneously with four reprogramming factors (Klf4, Oct4, Sox2, and c-Myc) to HDFs of Fabry disease; subsequently, we observed the presence of human iPS-like cells. The Sendai virus nucleocapsid protein was not detected in the fibroblasts by RT-PCR analysis. Additionally, we confirmed an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. Moreover, ultrastructural features of these iPS cells included massive membranous cytoplasmic bodies typical of HDFs of Fabry disease. Thus, we successfully generated human iPS cells from HDFs of Fabry disease that retained the genetic conditions of Fabry disease; also, these abnormal iPS cells could not be easily differentiated into mature cell types such as neuronal cells, cardiomyocytes, etc. because of a massive accumulation of membranous cytoplasmic bodies in lysosomes, possibly the persistent damages of intracellular architecture.

Mucopolysaccharidosis type II (MPS II) is a neuropathic lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), which leads to the accumulation of glycosaminoglycans (GAGs). We demonstrated that biochemical alterations in the brains of MPS II mice are not corrected by bone marrow transplantation (BMT) or enzyme replacement therapy, although BMT has been shown to be effective for other neurodegenerative MPSs, such as Hurler syndrome. In this study, we demonstrated that lentiviral isogeneic hematopoietic stem cell (HSC) gene therapy corrected neuronal manifestations by ameliorating lysosomal storage and autophagic dysfunction in the brains of MPS II mice. IDS-transduced HSCs increased enzyme activity both in various visceral organs and the CNS. Decreased levels of GAGs were observed in many organs, including cerebra, after transplantation of IDS-transduced HSCs. In addition, lentiviral HSC gene therapy normalized the secondary accumulation of autophagic substrates, such as p62 and ubiquitin-protein conjugates, in cerebra. Furthermore, in contrast to naive MPS II mice, there was no deterioration of neuronal function observed in transplant recipients. These results indicated that lentiviral HSC gene therapy is a promising approach for the treatment of CNS lesions in MPS II.

We report 4 cases of late onset glycogen storage disease type II (GSD II) or Pompe disease (OMIM #232300), under enzyme replacement therapy (ERT) with recombinant human acid alpha glucosidase (rh-GAA, OMIM *606800). In these 4 cases, we focused on the case of a 28-years-old man, whose condition at the ERT starting was the worst and resulted in poor prognosis. The autopsy was done under his family's permission, and revealed severe accumulation of glycogen in his muscle, especially diaphragm or iliopsoas, and pulmonary veno-occlusive disease (PVOD) which resulted in severe pulmonary hypertension (PH). This is the first report of PVOD as the cause of PH in Pompe disease. We studied this case comparing to another 3 cases of late onset Pompe disease under the same course of ERT in our hospital, and the average data of the group of late onset Pompe disease with severe pulmonary insufficiency receiving ERT, supposed that low score of the body mass index (BMI) on the baseline, the presence of specific genotype (p.R600C), and signs of pulmonary dysfunction suggesting PH (tachypnea, ultrasound cardiography data) were factors that influenced the prognosis. For a better prognosis in the late onset Pompe disease, an early diagnosis for the early start of ERT before the onset of respiratory failure should be important, and the deliberate management and care should be needed even after the ERT start, especially for severe cases including pulmonary dysfunction.

Pompe disease (glycogen storage disease type II) is an autosomal recessive myopathic disorder arising from the deficiency of lysosomal acid α-glucosidase (GAA). Activation of autophagy is a key pathophysiological feature in skeletal muscle fibers and fibroblasts from patients with Pompe disease. The accumulation of autophagic vacuoles has been shown to interfere with the efficacy of enzyme replacement therapy with recombinant human GAA. However, the induction mechanism of autophagy in Pompe disease is still unclear. In this study, we show that misfolded GAA-induced endoplasmic reticulum (ER) stress triggers autophagy in a manner regulated by p38 MAPK signaling pathways in fibroblasts from late-onset patients with Pompe disease. By studying normal fibroblasts and patient fibroblasts carrying a c.546G>T mutation, we uncovered that mutant GAA was rapidly degraded by proteasome. In addition, we found both activation of ER stress response and autophagy in these patient fibroblasts. Treatment with N-butyl-deoxynojirimycin (NB-DNJ), which acts as a pharmacological chaperone for certain mutant forms of GAA, led to attenuation of not only ER stress, but also autophagy in patient fibroblasts. Levels of phosphorylated p38 MAPK observed in patient fibroblasts were decreased after treatment with NB-DNJ. The autophagic response in patient fibroblasts was also negatively regulated by treatment with the p38 MAPK inhibitor SB203580. These findings define a critical role for ER stress in the activation of autophagy due to GAA mutation, and provide evidence that chaperone therapy may be a useful treatment for alleviation of autophagy in Pompe disease patients carrying a chaperon-responsive mutation.

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and is characterized by the accumulation of glycosaminoglycans (GAGs). MPS II has been treated by hematopoietic stem cell therapy (HSCT)/enzyme replacement therapy (ERT), but its effectiveness in the central nervous system (CNS) is limited because of poor enzyme uptake across the blood–brain barrier (BBB). To increase the efficacy of ERT in the brain, we tested an intraventricular ERT procedure consisting of repeated administrations of IDS (20 μg/mouse/3 weeks) in IDS-knockout, MPS II model mice. The IDS enzyme activity and the accumulation of total GAGs were measured in mouse brains. The IDS activity was significantly increased, and the accumulation of total GAGs was decreased in the MPS II mouse brains treated with multiple administrations of IDS via intraventricular ERT. Additionally, a high level of IDS enzyme activity was appreciated in other MPS II mouse tissues, such as the liver, spleen, testis and others. A Y-maze was used to test learning and memory after repeated intraventricular ERT with IDS. The IDS-treated mouse groups recovered the capacity for short-term memory and activity. Although large and small vacuoles were found at the margin of the cerebellar Purkinje cells in the disease-control mice, these vacuoles disappeared upon treated with IDS. Loss of vacuoles was also observed in other tissues (liver, kidney and testis). These results demonstrate the possible efficacy of an ERT procedure with intraventricular administration of IDS for the treatment of MPS II.

Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid α-glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart, and liver. Hypertrophic cardiomyopathy is frequently seen in the infantile onset Pompe disease. On the other hand, cardiovascular complication of the late-onset Pompe disease is considered as less frequent and severe than that of infantile onset. There are few investigations which show cardiovascular complication of late onset Pompe disease due to the shortage of appropriate disease model. We have generated late-onset Pompe disease-specific induced pluripotent stem cell (iPSC) and differentiated them into cardiomyocytes. Differentiated cardiomyocyte shows glycogen accumulation and lysosomal enlargement. Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC. The efficacy of gene therapy is maintained following the cardiomyocyte differentiation. Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote. It is suggested that Pompe disease iPSC-derived cardiomyocyte is replicating disease-specific changes in the context of disease modeling, drug screening, and cell therapy.

We previously demonstrated that mesenchymal stem cells (MSCs) differentiate into functional kidney cells capable of urine and erythropoietin production, indicating that they may be used for kidney regeneration. However, the viability of MSCs from dialysis patients may be affected under uremic conditions. In this study, we isolated MSCs from the adipose tissues of end-stage kidney disease (ESKD) patients undergoing long-term dialysis (KD-MSCs; mean: 72.3 months) and from healthy controls (HC-MSCs) to compare their viability. KD-MSCs and HC-MSCs were assessed for their proliferation potential, senescence, and differentiation capacities into adipocytes, osteoblasts, and chondrocytes. Gene expression of stem cell-specific transcription factors was analyzed by PCR array and confirmed by western blot analysis at the protein level. No significant differences of proliferation potential, senescence, or differentiation capacity were observed between KD-MSCs and HC-MSCs. However, gene and protein expression of p300/CBP-associated factor (PCAF) was significantly suppressed in KD-MSCs. Because PCAF is a histone acetyltransferase that mediates regulation of hypoxia-inducible factor-1α (HIF-1α), we examined the hypoxic response in MSCs. HC-MSCs but not KD-MSCs showed upregulation of PCAF protein expression under hypoxia. Similarly, HIF-1α and vascular endothelial growth factor (VEGF) expression did not increase under hypoxia in KD-MSCs but did so in HC-MSCs. Additionally, a directed in vivo angiogenesis assay revealed a decrease in angiogenesis activation of KD-MSCs. In conclusion, long-term uremia leads to persistent and systematic downregulation of PCAF gene and protein expression and poor angiogenesis activation of MSCs from patients with ESKD. Furthermore, PCAF, HIF-1α, and VEGF expression were not upregulated by hypoxic stimulation of KD-MSCs. These results suggest that the hypoxic response may be blunted in MSCs from ESKD patients.

Pompe disease (PD) is a lysosomal storage disease that is caused by a deficiency of the acid α-glucosidase, which results in glycogen accumulation in the lysosome. The major clinical symptoms of PD include skeletal muscle weakness, respiratory failure, and cardiac hypertrophy. Based on its severity and symptom onset, PD is classified into infantile and late-onset forms. Lysosomal accumulation of glycogen can promote many types of cellular dysfunction, such as autophagic dysfunction, endoplasmic reticulum stress, and abnormal calcium signaling within skeletal muscle. However, the disease mechanism underlying PD cardiomyopathy is not fully understood. Several researchers have shown that PD induced pluripotent stem cell (iPSC)-derived cardiomyocytes successfully replicate the disease phenotype and are useful disease models. We have analyzed the metabolomic profile of late-onset PD iPSC-derived cardiomyocytes and found that oxidative stress and mitochondrial dysfunction are likely associated with cardiac complications. Furthermore, we have validated that these disease-specific changes were also observed in the cardiomyocytes and skeletal muscle of a genetically engineered murine PD model. Oxidative stress may contribute to skeletal muscle and cardiomyocyte dysfunction in PD mice; however, NF-E2-related factor 2 was downregulated in cardiomyocytes and skeletal muscle, despite evidence of oxidative stress. We hypothesized that oxidative stress and an impaired antioxidative stress response mechanism may underlie the molecular pathology of late-onset PD. Stem Cells Translational Medicine 2017;6:31-39.

Pompe disease (PD) is a lysosomal disorder caused by acid α-glucosidase (GAA) deficiency. Progressive muscular weakness is the major symptom of PD, and enzyme replacement therapy can improve the clinical outcome. However, to achieve a better clinical outcome, alternative therapeutic strategies are being investigated, including gene therapy and pharmacological chaperones. We previously used lentiviral vector-mediated GAA gene transfer in PD patient-specific induced pluripotent stem cells. Some therapeutic efficacy was observed, although glycogen accumulation was not normalized. Transcription factor EB is a master regulator of lysosomal biogenesis and autophagy that has recently been associated with muscular pathology, and is now a potential therapeutic target in PD model mice. Here, we differentiated skeletal muscle from PD patient-specific induced pluripotent stem cells by forced MyoD expression. Lentiviral vector-mediated GAA and transcription factor EB gene transfer independently improved GAA enzyme activity and reduced glycogen content in skeletal muscle derived from PD-induced pluripotent stem cells. Interestingly, GAA and transcription factor EB cooperatively improved skeletal muscle pathology, both biochemically and morphologically. Thus, our findings show that abnormal lysosomal biogenesis is associated with the muscular pathology of PD, and transcription factor EB gene transfer is effective as an add-on strategy to GAA gene transfer.

Constitutive activation of nuclear factor kappa-B (NF-κB) contributes to the aggressive behavior of pancreatic cancer. Over-expression of downstream target genes of NF-κB such as intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) leads to the promotion of cell adhesion, angiogenesis, invasion and metastasis. We previously reported that nafamostat mesilate, a synthetic serine protease inhibitor, blocks NF-κB activation in pancreatic cancer. We hypothesized that nafamostat mesilate may inhibit cell adhesion, angiogenesis, invasion and metastases in peritoneal dissemination of pancreatic cancer.In vitro, we assessed inhibition of NF-κB, phosphorylated IκBα, ICAM-1, VEGF and MMP-9 activity by nafamostat mesilate using human pancreatic cancer cell lines (AsPC-1, BxPC-3 and PANC-1). Changes in adhesion and invasion abilities of cancer cells were then evaluated by nafamostat mesilate treatment. In vivo, the efficacy of nafamostat mesilate treatment was assessed using peritoneal dissemination of pancreatic cancer in mice.In vitro, nafamostat mesilate inhibited activities of NF-κB, phosphorylated IκBα, ICAM-1, VEGF and MMP-9. Moreover, nafamostat mesilate not only inhibited cell adhesion and invasion but also increased the sensitivity of anoikis. In vivo, tumor growth using AsPC-1 cells of the treatment group was significantly slower, and survival rate was significantly better, than those in control group (p < 0.05).Nafamostat mesilate reduced peritoneal metastasis and prolonged survival of pancreatic cancer-bearing mice.

Our study is the first to demonstrate the ability to generate iPS cells from a mouse model of Pompe disease. Initially, mouse tail tip fibroblasts were harvested from male, 8-week-old (GAA) knockout mice, and three reprogramming factors (Oct3/4, Sox2 and Klf4) were transfected into the isolated donor cells using a retroviral vector. These iPS cells also showed decreased levels of GAA enzymatic activity and strong positive staining with periodic acid-Schiff (indicating the accumulation of glycogen) and acid phosphatase (lysosomal activation marker). Pompe-iPS cells were differentiated into skeletal muscle cells in Matrigel®-coated plates. Spindle-shaped skeletal muscle cells were successfully generated from Pompe-iPS cells and showed spontaneous contraction and positive staining with the myosin heavy chain antibody. Electron microscopic analysis of the skeletal muscle cells showed typical morphological features, including Z-bands, I-bands, A-bands and H-bands, which were visible in wild-type and Pompe cells. Furthermore, Pompe skeletal muscle cells accumulated massive glycogen in lysosomes. This study indicates that the iPS and skeletal muscle cells generated in this study could also be a useful disease model for studies investigating the pathogenesis and treatment of skeletal muscle in Pompe disease.

Pompe disease (PD), which is also called glycogen storage disease type II (GSDII), is one of the lysosomal storage diseases (LSDs) caused by a deficiency in acid-α-glucosidase (GAA) in the lysosome and is characterized by the accumulation of glycogen in various cells. PD has been treated by enzyme replacement therapy (ERT). We generated induced pluripotent stem cells (iPSCs) from the cells of patients with infantile-type and late-onset-type PD using a retrovirus vector to deliver transgenes encoding four reprogramming factors, namely, OCT4, SOX2, c-MYC, and KLF4. We confirmed that the two types of PD-iPSCs exhibited an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. The PD-iPSCs exhibited strong positive staining with Periodic acid-Schiff (PAS). Moreover, ultrastructural features of these iPSCs exhibited massive glycogen granules in the cytoplasm, particularly in the infantile-type but to a lesser degree in the late-onset type. Glycogen granules of the infantile-type iPSCs treated with rhGAA were markedly decreased in a dose-dependent manner. Human induced pluripotent stem cell provides an opportunity to build up glycogen storage of Pompe disease in vitro. It represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies for Pompe disease.

Pompe disease is an autosomal recessive myopathic disorder caused by the deficiency of lysosomal acid α-glucosidase (GAA). Recently, we showed that function of mutant GAA in fibroblasts derived from Pompe disease patient carrying c.546G>T mutation is improved by treatment with proteasome inhibitor bortezomib as well as pharmacological chaperone (PC). However, bortezomib-responsive GAA mutations are not fully characterized. In this study, we showed the effect of bortezomib on different mutants of GAA in patient fibroblasts and transiently expressed HEK293T cells. Bortezomib increased the maturation and residual activity of GAA in patient fibroblasts carrying PC-responsive M519V and PC-unresponsive C647W mutations. Enhanced colocalization of GAA with lysosomal marker LAMP2 was also observed in patient fibroblasts after treatment with bortezomib. When four distinct mutant GAAs, which show different response to PC, were overexpressed in HEK293T cells, bortezomib improved the activity of M519V, S529V, and C647W in them (1.3-5.9-fold). These results indicate that bortezomib enhances the activity of some PC-unresponsive GAA mutants as well as PC-responsive mutants.

Small molecules called pharmacological chaperones have been shown to improve the stability, intracellular localization, and function of mutated enzymes in several lysosomal storage diseases, and proposed as promising therapeutic agents for them. However, a chaperone compound for mucopolysaccharidosis type II (MPS II), which is an X-linked lysosomal storage disorder characterized by a deficiency of iduronate-2-sulfatase (IDS) and the accumulation of glycosaminoglycans (GAGs), has still not been developed. Here we focused on the Δ-unsaturated 2-sulfouronic acid-N-sulfoglucosamine (D2S0), which is a sulfated disaccharide derived from heparin, as a candidate compound for a pharmacological chaperone for MPS II, and analyzed the chaperone effect of the saccharide on IDS by using recombinant protein and cells expressing mutated enzyme. When D2S0 was incubated with recombinant human IDS (rhIDS) in vitro, the disaccharide attenuated the thermal degeneration of the enzyme. This effect of D2S0 on the thermal degeneration of rhIDS was enhanced in a dose-dependent manner. D2S0 also increased the residual activity of mutant IDS in patient fibroblasts. Furthermore, D2S0 improved the enzyme activity of IDS mutants derived from six out of seven different mutations in HEK293T cells transiently expressing them. These results indicate that D2S0 is a potential pharmacological chaperone for MPS II.

Background Thrombomodulin, an anticoagulant that inhibits thrombin-induced growth factor promotion, also has an anti-inflammatory effect. Furthermore, thrombomodulin inhibits nuclear factor-kappa B activation, which plays a crucial role in cancer progression. We assessed the antitumor activity of recombinant thrombomodulin for pancreatic cancer. Methods A xenograft orthotopic model was established in mice by implantation of human pancreatic cancer cells. The animals were treated with intraperitoneal injection of recombinant thrombomodulin 5 times a week for 4 weeks. Nuclear factor-kappa B activation was evaluated by measuring nuclear localization of the p65. Efficacy of recombinant thrombomodulin on the signal transduction of nuclear factor-kappa B was measured in vitro under preconditioning with thrombin or epidermal growth factor. Results Tumor growth was suppressed by recombinant thrombomodulin (P < .05). Recombinant thrombomodulin inhibited the expression of IκB kinase β (P < .05) and pIκBα (P < .01), as well as the activation of nuclear factor-kappa B NF-κB (P < .001). Furthermore, recombinant thrombomodulin inhibited thrombin-induced protease activate receptor 1 and nuclear factor-kappa B activation in vitro (P < .05). The number of Ki67-positive cells was decreased by recombinant thrombomodulin (P < .01). Recombinant thrombomodulin also suppressed body weight loss associated with pancreatic cancer (P < .05). No obvious adverse effects were observed. Conclusion Recombinant thrombomodulin significantly suppressed tumor growth against human pancreatic cancer by blocking thrombin-induced nuclear factor-kappa B activation without adverse effects. Thrombomodulin, an anticoagulant that inhibits thrombin-induced growth factor promotion, also has an anti-inflammatory effect. Furthermore, thrombomodulin inhibits nuclear factor-kappa B activation, which plays a crucial role in cancer progression. We assessed the antitumor activity of recombinant thrombomodulin for pancreatic cancer. A xenograft orthotopic model was established in mice by implantation of human pancreatic cancer cells. The animals were treated with intraperitoneal injection of recombinant thrombomodulin 5 times a week for 4 weeks. Nuclear factor-kappa B activation was evaluated by measuring nuclear localization of the p65. Efficacy of recombinant thrombomodulin on the signal transduction of nuclear factor-kappa B was measured in vitro under preconditioning with thrombin or epidermal growth factor. Tumor growth was suppressed by recombinant thrombomodulin (P < .05). Recombinant thrombomodulin inhibited the expression of IκB kinase β (P < .05) and pIκBα (P < .01), as well as the activation of nuclear factor-kappa B NF-κB (P < .001). Furthermore, recombinant thrombomodulin inhibited thrombin-induced protease activate receptor 1 and nuclear factor-kappa B activation in vitro (P < .05). The number of Ki67-positive cells was decreased by recombinant thrombomodulin (P < .01). Recombinant thrombomodulin also suppressed body weight loss associated with pancreatic cancer (P < .05). No obvious adverse effects were observed. Recombinant thrombomodulin significantly suppressed tumor growth against human pancreatic cancer by blocking thrombin-induced nuclear factor-kappa B activation without adverse effects.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease (LSD) caused by a deficiency of the iduronate-2-sulfatase (IDS) that catabolizes glycosaminoglycans (GAGs). Abnormal accumulations of GAGs in somatic cells lead to various manifestations including central nervous system (CNS) disease. Enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are the currently available therapy for MPS II, but both therapies fail to improve CNS manifestations. We previously showed that hematopoietic stem cell targeted gene therapy (HSC-GT) with lethal irradiation improved CNS involvement in a murine model of MPS II which lacks the gene coding for IDS. However, the strong preconditioning, with lethal irradiation, would cause a high rate of morbidity and mortality. Therefore, we tested milder preconditioning procedures with either low dose irradiation or low dose irradiation plus an anti c-kit monoclonal antibody (ACK2) to assess CNS effects in mice with MPS II after HSC-GT. Mice from all the HSC-GT groups displayed super-physiological levels of IDS enzyme activity and robust reduction of abnormally accumulated GAGs to the wild type mice levels in peripheral organs. However, only the mice treated with lethal irradiation showed significant cognitive function improvement as well as IDS elevation and GAG reduction in the brain. These results suggest that an efficient engraftment of genetically modified cells for HSC-GT requires strong preconditioning to ameliorate CNS involvement in cases with MPS II.

Mucopolysaccharidosis type II is a disease caused by organ accumulation of glycosaminoglycans due to iduronate 2-sulfatase deficiency. This study investigated the pathophysiology of the bone complications associated with mucopolysaccharidosis II and the effect of lentivirus-mediated gene therapy of hematopoietic stem cells on bone lesions of mucopolysaccharidosis type II mouse models in comparison with enzyme replacement therapy. Bone volume, density, strength, and trabecular number were significantly higher in the untreated mucopolysaccharidosis type II mice than in wild-type mice. Accumulation of glycosaminoglycans caused reduced bone metabolism. Specifically, persistent high serum iduronate 2-sulfatase levels and release of glycosaminoglycans from osteoblasts and osteoclasts in mucopolysaccharidosis type II mice that had undergone gene therapy reactivated bone lineage remodeling, subsequently reducing bone mineral density, strength, and trabecular number to a similar degree as that observed in wild-type mice. Bone formation, resorption parameters, and mineral density in the diaphysis edge did not appear to have been affected by the irradiation administered as a pre-treatment for gene therapy. Hence, the therapeutic effect of gene therapy on the bone complications of mucopolysaccharidosis type II mice possibly outweighed that of enzyme replacement therapy in many aspects.

Abstract Lysosomal degradation plays a crucial role in the metabolism of biological macromolecules supplied by autophagy. The regulation of the autophagy‐lysosome system, which contributes to intracellular homeostasis, chemoresistance, and tumor progression, has recently been revealed as a promising therapeutic approach for pancreatic cancer (PC). However, the details of lysosomal catabolic function in PC cells have not been fully elucidated. In this study, we show evidence that suppression of acid alpha‐glucosidase (GAA), one of the lysosomal enzymes, improves chemosensitivity and exerts apoptotic effects on PC cells through the disturbance of expression of the transcription factor EB. The levels of lysosomal enzyme were elevated by gemcitabine in PC cells. In particular, the levels of GAA were responsive to gemcitabine in a dose–dependent and time–dependent manner. Small interfering RNA against the GAA gene (si GAA ) suppressed cell proliferation and promoted apoptosis in gemcitabine‐treated PC cells. In untreated PC cells, we observed accumulation of depolarized mitochondria. Gene therapy using adenoviral vectors carrying shRNA against the GAA gene increased the number of apoptotic cells and decreased the tumor growth in xenograft model mice. These results indicate that GAA is one of the key targets to improve the efficacy of gemcitabine and develop novel therapies for PC.

Vascular endothelial growth factor (VEGF) and its receptors VEGF-R1, -R2 and -R3 play important roles in tumor angiogenesis and are associated with poor prognosis in several solid tumors. However, their functional significance remains unclarified. Here, we investigated the associations between the expression of these receptors and the clinical outcomes of colorectal cancer (CRC) patients.An immunohistochemical approach was used to detect VEGF-R1, -R2 and -R3 expression in 91 CRC patients who underwent surgery and received chemotherapy at the National Cancer Center Hospital. Statistical analysis was performed to determine the prognostic significance of these biomarkers.Immunoreactivity for VEGF-R2 and -R3 was localized in microvessels and that for VEGF-R1 in cancer cells and stromal microvessels. VEGF-R1 staining in cancer cells (>10% staining) was found in 84 patients (92%) and in stromal vessels in 75 patients (82%). VEGF-R2 staining in tumor vessels (>10% staining) was found in 84 patients (92%), whereas VEGF-R3 staining was found in 85 patients (93%). Strong positive staining (>60% staining) of VEGF-R1 in tumor cells, and VEGF-R1, -R2 and -R3 in vessels was identified in 58 (64%), 33 (36%), 52 (57%) and 60 (66%) patients, respectively. Univariate analysis revealed that VEGF-R1 strong positive staining correlated with shorter post-operative survival in patients with Stage II/III disease (P = 0.01), but neither VEGF-R2 nor R3 expression correlated with survival.VEGF-R1, -R2 and -R3 were highly expressed in CRC cells and stromal vessels. VEGF-R1 strong positive staining correlated with shorter survival after CRC surgery.

Mucolipidosis type III (MLIII) is a mild form of Mucolipidosis type II (MLII, I-cell disease) of late onset, of which almost no pathological study has been reported, as it is a very rare disease. We encountered the case of a 23-year-old man of Japanese and Caucasian mixed parentage diagnosed with MLIII by enzyme assay and genotyping. He died suddenly due to severe dilated cardiomyopathy. On the day after his death, autopsy was performed, and accumulation of Luxol Fast Blue (LFB) positive material was found to be most severe in the neuronal cells of dorsal root ganglions (DRG). Electromicroscopic DRG revealed the neuronal cytoplasm was filled with a zebra-body-like membranous matrix. We tried immunohistochemistry to investigate the mechanism of such accumulation in the DRG that resulted in double positive anti-ubiquitin antibody (FK-2) and anti-LC3 antibody (as specific marker for autophagy) staining, and speculated activating of autophagosome pathway, and ‘zebra-body’ should be suspected as dysfunctional autophagosome. We also detected foamy cell proliferation in the dura mater, Auerbach's plexus (peripheral nervous system), podocytes of almost all glomeruli, cartilage tissue in lumbar discs, and in cardiac muscle. We tried FK-2 and anti-LC3 antibody staining also for the podocytes, the area with the most marked proliferation of foamy cells, but the result was negative. This led us to speculate that these pathological findings, namely, accumulation of LFB-positive material and foamy fibroblast proliferation, might be the forms of dysfunctional autophagosome at various stages of development. This pathological study of MLIII supports the theory that MLIII is a mild type of MLII because of the close similarity of their pathological findings.

Pompe disease (glycogen storage disease type II) is an autosomal recessive myopathic disorder arising from the deficiency of lysosomal acid α-glucosidase (GAA). Recently, we found that mutant GAA in patient fibroblasts carrying c.546G>T mutation is stabilized by treatment with proteasome inhibitor as well as pharmacological chaperon N-butyl-deoxynojirimycin. In this study, we characterized the effect of two proteasome inhibitors, bortezomib and MG132, on maturation, subcellular localization and residual activity of mutant GAA in the patient fibroblasts carrying c.546G>T mutation. Each proteasome inhibitor promoted the stabilization of patient GAA and processing of them to mature forms without cytotoxic effect. Immunocytochemical analysis showed increased colocalization of GAA with the lysosomal marker LAMP2 in patient fibroblasts treated with proteasome inhibitors. Furthermore, bortezomib and MG132 also increased enzyme activity in the patient fibroblasts (about 4-fold and 2-fold, respectively). These findings indicate that proteasome inhibitor may be a novel drug as potential pharmacological chaperone therapy for Pompe disease patient carrying chaperon-responsive mutation.

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Pompe disease (glycogen storage disease type II) is an autosomal recessive neuromuscular disorder arising from a deficiency of lysosomal acid α-glucosidase (GAA). Accumulation of autophagosomes is a key pathological change in skeletal muscle fibers and fibroblasts from patients with Pompe disease and is implicated in the poor response to enzyme replacement therapy (ERT). We previously found that mutant GAA-induced endoplasmic reticulum (ER) stress initiated autophagy in patient fibroblasts. However, the mechanism of induction of autophagy in fibroblasts from Pompe disease patients lacking ER stress remains unclear. In this study, we show that inactivated Akt induces ER stress-independent autophagy via mTOR suppression in patient fibroblasts. Activated autophagy as evidenced by increased levels of LC3-II and autophagic vesicles was observed in patient fibroblasts, whereas PERK phosphorylation reflecting the presence of ER stress was not observed in them. These patient fibroblasts showed decreased levels of not only phosphorylated Akt, but also phosphorylated p70 S6 kinase. Treatment with insulin, which acts as an activator of the Akt signaling pathway, resulted in increased phosphorylation of both Akt and p70 S6 kinase and suppression of autophagy in patient fibroblasts. In addition, following combination treatment with recombinant human GAA plus insulin, enhanced localization of the enzymes with lysosomes was observed in patient fibroblasts. These findings define a critical role of Akt suppression in the induction of autophagy in fibroblasts from patients with Pompe disease carrying an ER stress non-inducible mutation, and they provide evidence that insulin may potentiate the effect of ERT.

Animal and human studies of enzyme replacement therapy (ERT) for Pompe disease (PD) have indicated that antibodies (Abs) generated against infused recombinant human α-glucosidase (rhGAA) can have a negative impact on the therapeutic outcome and cause hypersensitivity reactions. We showed that parenteral administration of anti-CD3 Abs into mice can reduce the titer of anti-human GAA Abs in wild-type mice administered the enzyme. Mice that had been treated with anti-CD3 Abs and then subjected to a secondary challenge with rhGAA showed a lower increase in Ab titers than control mice. Moreover, the administration of anti-CD3 Abs also reduced the levels of pre-existing Abs. Treatment with anti-CD3 Abs also prevented a lethal hypersensitivity reaction and reduced the Ab titers in a mouse model of PD. Mice treated with anti-CD3 Abs showed reduced numbers of CD4(+) and CD8(+) cells, and an increased ratio of CD4(+)CD25(+)/CD4(+) and CD4(+)CD25(+)FoxP3(+)/CD4(+) cells. When the CD4(+)CD25(+) cells were depleted using anti-CD25 Abs, the observed reduction in Abs against the enzyme by anti-CD3 Abs was abrogated. This suggests that CD4(+)CD25(+) cells are important for the immune suppressive activity of anti-CD3 Abs. In summary, anti- CD3 Abs are useful for inducing immune tolerance to ERT for PD.

Before the availability of an enzyme replacement therapy (ERT) for mucopolysaccharidosis type II (MPS II), patients were treated by bone marrow transplantation (BMT). However, the effectiveness of BMT for MPS II was equivocal, particularly at addressing the CNS manifestations. To study this further, we subjected a murine model of MPS II to BMT and evaluated the effect at correcting the biochemical and pathological aberrations in the viscera and CNS. Our results indicated that BMT reduced the accumulation of glycosaminoglycans (GAGs) in a variety of visceral organs, but not in the CNS. With the availability of an approved ERT for MPS II, we investigated and compared the relative merits of the two strategies either as a mono or combination therapy. We showed that the combination of BMT and ERT was additive at reducing tissue levels of GAGs in the heart, kidney and lung. Moreover, ERT conferred greater efficacy if the immunological response against the infused recombinant enzyme was low. Finally, we showed that pathologic GAGs might potentially represent a sensitive biomarker to monitor the therapeutic efficacy of therapies for MPS II.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by the deficient activity of iduronate 2-sulfatase (IDS), which is involved in the lysosomal catabolism of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. Such a deficiency leads to the accumulation of undegraded GAGs in some organs. Although enzyme replacement therapy is available as a treatment of MPS II, there are some limitations, such as the requirement of weekly administration for whole life. To avoid such limitations, hematopoietic cell transplantation (HSCT) is a possible alternative. In fact, some report suggested positive effects of HSCT for MPS II. However, HSCT has also some limitations. Strong conditioning regimens can cause severe side effects. For overcome this obstacle, we studied the efficacy of ACK2, an antibody that blocks KIT, followed by low-dose irradiation as a preconditioning regimen for HSCT using a murine model of MPS II. This protocol achieves 58.7 ± 4.92% donor chimerism at 16 weeks after transplantation in the peripheral blood of recipient mice. GAG levels were significantly reduced in liver, spleen, heart and intestine. These results indicated that ACK2-based preconditioning might be one of the choices for MPS II patients who receive HSCT.

Abstract Although the inhibition of acid ceramidase (AC) is known to induce antitumor effects in various cancers, there are few reports in pancreatic cancer, and the underlying mechanisms remain unclear. Moreover, there is currently no safe administration method of AC inhibitor. Here the effects of gene therapy using siRNA and shRNA for AC inhibition with its mechanisms for pancreatic cancer were investigated. The inhibition of AC by siRNA and shRNA using an adeno‐associated virus 8 (AAV8) vector had antiproliferative effects by inducing apoptosis in pancreatic cancer cells and xenograft mouse model. Acid ceramidase inhibition elicits mitochondrial dysfunction, reactive oxygen species accumulation, and manganese superoxide dismutase suppression, resulting in apoptosis of pancreatic cancer cells accompanied by ceramide accumulation. These results elucidated the mechanisms underlying the antitumor effect of AC inhibition in pancreatic cancer cells and suggest the potential of the AAV8 vector to inhibit AC as a therapeutic strategy.

Certain proteins insoluble in aqueous salt solutions are difficult to separate from impurities by immunoaffinity techniques, even when the proteins are solubilized with denaturants due to interference of the antigen-antibody reaction. Representative examples of such proteins are the ubiquitin-protein conjugates that accumulate in neuronal tissues of neurodegenerative diseases, the hallmark of such disorders. In this study, we developed a novel sample preparation method comprising two successive steps: Sodium dodecyl sulfate (SDS) removal from the SDS-containing extracts and renaturation of the denatured proteins. The application of this method was tested on ubiquitin-protein conjugates in the brains of Niemann-Pick type C disease mouse and in heat-shocked K562 erythroleukemia cells. The ubiquitin-protein conjugates in both cases are insoluble in Tris-buffered saline but soluble in 2% SDS. The SDS-solubilized fractions prepared from each of the samples were further pretreated by the method mentioned above, and the ubiquitin-protein conjugates were efficiently immunoprecipitated with the anti-ubiquitin antibody from them. This method was also applied successfully to the immunoprecipitation of flotillin-1, a lipid raft protein, from mouse brain extract prepared with 2% SDS. These results indicate that this simple protocol has potential applications for excellent immunoaffinity separation of the less-soluble proteins in diverse cells and tissues.

Animal and human studies of enzyme replacement therapy for Pompe disease have indicated that antibodies generated against the infused recombinant human acid α-glucosidase (rhGAA) can negatively impact therapeutic outcome. In this study, we show that oral administration of rhGAA into mice can reduce the titer of anti-rhGAA antibody following immunization with rhGAA. Oral administration of rhGAA is safe and antigen specific, it offers advantages over other immunosuppressive drugs.

Degradation in the lysosome/vacuole is not the final step of autophagy. In particular, for starvation-induced autophagy it is necessary to release the breakdown products back into the cytosol. However, some researchers ignore this last step and simply refer to the endpoint of autophagy as degradation, or perhaps even cargo delivery. In many cases this is not a serious issue; however, the analysis of autophagy's role in certain diseases makes clear that this can be a significant error.

Neoadjuvant chemoradiotherapy followed by radical surgery is the standard treatment for patients with locally advanced low rectal cancer. However, several studies have reported that ionizing radiation (IR) activates nuclear factor kappa B (NF-κB) that causes radioresistance and induces matrix metalloproteinase (MMP)-2/-9, which promote tumor migration and invasion. Nafamostat mesilate (FUT175), a synthetic serine protease inhibitor, enhances the chemosensitivity to cytotoxic agents in digestive system cancer cells by inhibiting NF-κB activation. Therefore, we evaluated the combined effect of IR and FUT175 on cell proliferation, migration and invasion of colorectal cancer (CRC) cells. IR-induced upregulation of intranuclear NF-κB, FUT175 counteracted this effect. Moreover, the combination treatment suppressed cell viability and induced apoptosis. Similar effects were also observed in xenograft tumors. In addition, FUT175 prevented the migration and invasion of cancer cells caused by IR by downregulating the enzymatic activity of MMP-2/-9. In conclusion, FUT175 enhances the anti-tumor effect of radiotherapy through downregulation of NF-κB and reduces IR-induced tumor invasiveness by directly inhibiting MMP-2/-9 in CRC cells. Therefore, the use of FUT175 during radiotherapy might improve the efficacy of radiotherapy in patients with CRC.

Objectives: Paclitaxel (PTX) is a useful treatment for peritoneal dissemination of malignant tumors. However, chemoresistance due to PTX-induced nuclear factor κB (NF-κB) activation is an important cause of suboptimal therapeutic effect. We previously reported nafamostat mesilate (FUT175) inhibits NF-κB activation and promotes apoptosis in pancreatic cancer. We hypothesized that addition of FUT175 to PTX may enhance the antitumor effect in peritoneal dissemination of pancreatic cancer. Methods: In vitro, we assessed NF-κB activity and apoptosis by the combination of FUT175 and PTX using human pancreatic cancer cell line (AsPc-1). In vivo, we established peritoneal dissemination in nude mice by intraperitoneal injection of AsPc-1 cells. The animals were treated with intraperitoneal injection thrice a week of FUT175, once a week of PTX, or a combination of thrice a week of FUT175 and once a week of PTX (combination group). Results: In the combination groups, PTX-induced NF-κB activation was inhibited, and apoptosis was enhanced in comparison with other groups both in vitro and in vivo. In the combination group, tumor growth, serum tumor marker, and survival rate were significantly better than those in other groups (P < 0.05). Conclusions: Combination chemotherapy using PTX with FUT175 exerts an antitumor effect for peritoneal dissemination of pancreatic cancer.

Abstract Background Fabry disease (FD) is a lysosomal storage disorders characterized by a deficiency of the lysosomal enzyme, α‐galactosidase A. This results in the accumulation of glycolipids, mainly globotriaosylceramide (GL‐3), in the lysosomes of various organs. Although bone marrow transplantation and hematopoietic stem cell‐based gene therapy can offer the potential of a curative therapeutic outcome for FD, the minimum requirement of donor cells or gene‐corrected cells to reduce GL‐3 levels is not known. Methods Lethally‐irradiated FD mice were transplanted intravenously with normal bone marrow cells (Ly5.1 positive) mixed with those of FD mice (Ly5.2 positive) at various ratios to investigate the level of engraftment and enzyme activity necessary to effect a reduction in GL‐3 storage. Results Chimerism of whole white blood cells of recipients' peripheral blood remained stable at 8 weeks after transplantation, and chimerism of granulocytes, monocytes, B cells and T cells was equal to that of white blood cells. GL‐3 levels were significantly reduced in the lung and heart of animals with a 30% and 50% chimera, respectively. The extent of reduction in these mice was almost identical to that with 100% chimera. Conclusions In FD mice, reconstitution with 100% donor cells is not required to obtain a therapeutic effect following bone marrow transplantation. These results suggest that a 30% gene correction might be sufficient to reverse disease manifestations in FD. Copyright © 2011 John Wiley &amp; Sons, Ltd.

GM1-gangliosidosis is a progressive neurodegenerative glycosphingolipidosis resulting from a GLB1 gene mutation causing a deficiency of the lysosomal enzyme β-galactosidase, which leads to the abnormal accumulation of GM1 ganglioside in the central nervous system. In the most severe early infantile phenotype, excessive ganglioside accumulation results in a rapid decline in neurological and psychomotor functions, and death occurs within 2 years of age. Currently, there is no effective therapy for GM1-gangliosidosis. In this study, we evaluated the therapeutic efficacy of ex vivo gene therapy targeting hematopoietic stem cells using a lentiviral vector to increase enzyme activity, reduce substrate accumulation, and improve astrocytosis and motor function. Transplanting GLB1-transduced hematopoietic stem cells in mice increased β-galactosidase enzyme activity in the central nervous system and visceral organs. Specifically, this gene therapy significantly decreased GM1 ganglioside levels in the brain, especially in the cerebrum. More important, this gene therapy rectified astrocytosis in the cerebrum and improved motor function deficits. Furthermore, the elevation of serum β-galactosidase activity in secondary-transplanted mice suggested the ability of transduced hematopoietic stem cells to repopulate long term. These data indicate that ex vivo gene therapy with lentiviral vectors is a promising approach for the treatment of brain deficits in GM1 gangliosidosis.

Abstract Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficient activity of the iduronate‐2‐sulfatase. This leads to accumulation of glycosaminoglycans (GAGs) in the lysosomes of various cells. Although it has been proposed that bone marrow transplantation (BMT) may have a beneficial effect for patients with MPS II, the requirement for donor‐cell chimerism to reduce GAG levels is unknown. To address this issue, we transplanted various ratios of normal and MPS II bone marrow cells in a mouse model of MPS II and analyzed GAG accumulation in various tissues. Chimerism of whole leukocytes and each lineage of BMT recipients’ peripheral blood was similar to infusion ratios. GAGs were significantly reduced in the liver, spleen, and heart of recipients. The level of GAG reduction in these tissues depends on the percentage of normal‐cell chimerism. In contrast to these tissues, a reduction in GAGs was not observed in the kidney and brain, even if 100 % donor chimerism was achieved. These observations suggest that a high degree of chimerism is necessary to achieve the maximum effect of BMT, and donor lymphocyte infusion or enzyme replacement therapy might be considered options in cases of low‐level chimerism in MPS II patients.

Anderson-Fabry (FD) disease is an inborn error of metabolism caused by a deficiency of α-galactosidase A (GLA), a lysosomal enzyme. Many male FD patients display a classic FD phenotype; however, some female patients have neither reduced leukocyte GLA enzyme activity level nor FD symptoms. Thus, GLA gene analysis is especially important for diagnosing suspected FD in female subjects. In this study, we revealed 4 novel GLA gene mutations in 5 independent families using GLA cDNA analysis and multiplex ligation-dependent probe amplification (MLPA) analysis. These distinct mutations included a large deletion mutation from intron 1 to exon 5 (c.195-471_c.691del5.5k, corresponding to g.8508_g.14069del5.5k), an insertion mutation of splicing enhancer sequence in intron 4 (c.639+329_c.639+330ins113, corresponding to g.12627_g.12628ins113), an insertion mutation of retrotransposon L1 in exon 4 (c.634_c.635, corresponding to g.12293_g.12294), and a non-SNP deep intronic point mutation in intron 3 (c.547+395G>C, corresponding to g.11727G>C). It is difficult to detect these mutations with direct sequencing of only the exonic element. When exonic mutations are not found in the GLA gene from suspected FD patients, GLA cDNA and MLPA analyses should be performed to detect large deletion/insertion and intronic mutations including transcription abnormalities.

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder arising from deficiency of iduronate-2-sulfatase (IDS), which results in progressive accumulation of glycosaminoglycans (GAGs) in multiple tissues. Accumulated GAGs are generally measured as the amount of total GAGs. However, we recently demonstrated that GAG accumulation in the brain of MPS II model mice cannot be reliably detected by conventional dye-binding assay measuring total GAGs. Here we developed a novel quantitative method for measurement of disease-specific GAGs based on the analysis of 2-sulfoiduronic acid levels derived from the non-reducing terminal end of the polysaccharides by using recombinant human IDS (rhIDS) and recombinant human iduronidase (rhIDUA). This method was evaluated on GAGs obtained from the liver and brain of MPS II mice. The GAGs were purified from tissue homogenates and then digested with rhIDS and rhIDUA to generate a desulfated iduronic acid from their non-reducing terminal end. HPLC analysis revealed that the generated iduronic acid levels were markedly increased in the liver and cerebrum of the MPS II mice, whereas the uronic acid was not detected in wild-type mice. These results indicate that this assay clearly detects the disease-specific GAGs in tissues from MPS II mice.

Histones are key components of chromatin. We investigated histone H2A-immunoreactive proteins in acute monocytic leukemia THP-1 cells using three polyclonal antibodies raised against peptides corresponding to distinct regions of H2A. Two unknown immunoreactive proteins (9- and 12-kDa proteins), H2A (14kDa) and ubiquitinated H2A (23kDa) were found in the cell lysates prepared by immediate direct addition of SDS-PAGE sample buffer to the cells as well as in the nuclear and chromatin fractions. However, they were not found in the cytoplasmic fraction. The unknown proteins were successfully purified by immunoaffinity chromatography from the cell nucleus extract and identified as 9-kDa H2A(1-87) and 12-kDa H2A(1-114), suggesting that both were produced by limited proteolysis of intact H2A(1-129). The truncated forms of H2A probably persisted as chromatin constituents, since the stability of H2A(1-87) in the chromatin fraction was sensitive to treatment with micrococcal nuclease, and H2A(1-114) was solubilized with lower ionic strength from the chromatin fraction obtained by micrococcal nuclease treatment. Truncated H2A proteins in THP-1 cells were transiently increased in amount by short-term treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce macrophage-like differentiation. Furthermore, these increases were suppressed by preceding treatment with carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132) but not with carbobenzoxy-l-isoleucyl-gamma-t-butyl-l-glutamyl-l-alanyl-l-leucinal (PSI), both of which are generally known as proteasome inhibitors. Our results suggest that histone H2A is cleaved at least at two sites by protease(s) that remain obscure, and might affect chromatins in the early stage of THP-1 cell differentiation.

Abstract We report an autopsy case of advanced late‐onset Pompe disease (juvenile type). At 15 years‐of‐age, the patient experienced proximal weakness and subsequently required non‐invasive positive pressure ventilation. Enzyme replacement therapy was initiated at 37 years‐of‐age, which improved pinch power and alleviated respiratory distress. At age 41 years, repetitive pneumothorax led to respiratory failure and death. Autopsy excluding the brain showed few vacuoles and periodic acid–Schiff‐positive fibers in the left rectus femoris, compared with the contralateral muscle biopsy carried out at 21 years‐of‐age. Vacuolar changes were also less evident than previous autopsy cases without enzyme replacement therapy. The findings suggest that enzyme replacement therapy might have suppressed myovacuolopathy in our patient with advanced late‐onset Pompe disease. Apart from vacuolopathy, the autopsied muscle showed marked myofiber atrophy and fibrosis, suggesting that disuse atrophy could have contributed to the muscle weakness.

Key Points Type I IFN preconditioning enhances HSC engraftment efficiency. IFN-based pre-transplant conditioning is applicable to the treatment of Sly syndrome.

Abstract Pancreatic cancer is often resistant to chemotherapy. We previously showed the efficacy of combination treatment using gemcitabine and nafamostat mesilate ( FUT ‐175) for patients with unresectable pancreatic cancer. However, the mechanisms that affect the sensitivity of FUT ‐175 are not fully understood. The purpose of the present study was to clarify the mechanism of the sensitivity to FUT ‐175, with a focus on the activity of glycogen synthase kinase‐3β ( GSK ‐3β). In vitro, we assessed sensitivity to FUT ‐175 in human pancreatic cancer cell lines ( PANC ‐1 and MIAP aCa‐2) and difference of signaling in these cells by cell proliferation assay, Western blot analysis and microarray. Next, we assessed cell viability, apoptotic signal and nuclear factor‐kappa B ( NF ‐κB) activity in response to treatment with FUT ‐175 alone and in combination with GSK ‐3 inhibitor or protein phosphatase 2A ( PP 2A) by cell proliferation assay, Western blot analysis and enzyme‐linked immunosorbent assay. Phosphorylated GSK ‐3β level was significantly higher in MIAP aCa‐2 (high sensitivity cell) than in PANC ‐1 (low sensitivity cell). Cell viability and NF ‐κB activity were significantly decreased by addition of GSK ‐3 inhibitor to FUT ‐175, and levels of cleaved caspase‐8 were increased by inhibition of GSK ‐3. PP 2A inhibitor increased the levels of phosphorylated GSK ‐3β and sensitized both cell lines to FUT ‐175 as measured by cell viability and apoptotic signal. The results indicate that GSK ‐3β activity plays a key role in the antitumor effect of FUT ‐175 in pancreatic cancer cells, and regulation of GSK ‐3β by PP 2A inhibition could be a novel therapeutic approach for pancreatic cancer.

Abstract Background Pompe disease is caused by a deficiency of acid alpha‐glucosidase. Its prevalence varies depending on ethnicity, and is less prevalent in Japan as compared with other countries. Because of the wide spectrum of clinical features in late‐onset cases, some patients might be misdiagnosed with another myopathy, thus the actual prevalence in Japan might not be accurately reported. Aim To clarify the actual prevalence of late‐onset Pompe disease, we investigated acid alpha‐glucosidase activity in patients with undiagnosed myopathies. Methods Of 42 patients with undiagnosed myopathies, 41 underwent assessment of acid alpha‐glucosidase enzyme activity using dried blood spot analysis. As a second step, reassessment of acid alpha‐glucosidase activity was carried out using cultured skin fibroblasts. We also determined acid alpha‐glucosidase activity in biopsied muscle tissue obtained from the one other patient, who had previously undergone a muscle biopsy. Finally, gene analysis was carried out to confirm diagnosis. Results Four patients showed reduced acid alpha‐glucosidase activity in dried blood spot findings, of whom one possessed a pseudo‐deficiency allele. Furthermore, one patient who showed reduced acid alpha‐glucosidase activity in a biopsied muscle specimen was diagnosed with late‐onset Pompe disease based on gene analysis. Conclusions Of the present 42 patients, only one patient was diagnosed with late‐onset Pompe disease. The prevalence of Pompe disease in Japan does not seem to be as high as in other countries, though a certain number of patients might exist among those with undiagnosed myopathies. A larger and more systematic survey is necessary to elucidate the actual prevalence of late‐onset Pompe disease in Japan.

[This corrects the article DOI: 10.1371/journal.pone.0102311.].

Mucolipidosis type II is an autosomal recessive lysosomal storage disease caused by N-acetylglucosamine-1-phosphotransferese deficiency. We report here pathological findings of an autopsy case of mucolipidosis type II. The patient was an 8-year-old boy with mucolipidosis type II and was complicated with hypertrophic cardiomyopathy. He suddenly developed progressive respiratory failure and finally died. At autopsy, systemic accumulation of undigested lysosomal metabolites was prominent, particularly in the heart, lungs, and dorsal root ganglion. In cardiomyocyte, LC3, an autophagy marker, was positive in the cytoplasm. Ubiquitin, p62, K48 polyubiquitin, and K63 polyubiquitin were also positive in the cytoplasm. Our findings suggest that autophagic dysfunction might be associated with the cardiomyopahty of mucolipidosis type II.

Shirai, Yoshihiro MD, PhD; Shiba, Hiroaki; Uwagawa, Tadashi MD, PhD; Shimada, Yohta; Horiuchi, Takashi MD; Saito, Nobuhiro MD; Sugano, Hiroshi; Furukawa, Kenei MD, PhD; Ohashi, Toya MD; Yanaga, Katsuhiko MD, PhD, FACS Author Information

3209 Background: Cetuximab is a monoclonal antibody that selectively binds to the Epidermal Growth Factor Receptor (EGFR). Cetuximab as a single agent is effective in patients with colorectal cancer refractory to irinotecan and oxaliplatin (Lenz ASCO 2004). This phase I study assessed the safety and PK profile of cetuximab as a single agent in Japanese patients with solid tumors. Methods: Monotherapy with cetuximab was examined across a 100–500mg/m2 dose range. Thirty patients with chemotherapy refractory, EGFR -expressing solid tumors were enrolled in cohorts of five to receive weekly IV infusions of 100 mg/m2 cetuximab following an initial dose of 100mg/m2 (cohort 1), or weekly IV infusions of 250mg/m2 following initial doses of 250 (cohort 2), 400 (cohort 3) or 500 mg/m2 (cohort 4). To investigate the PK profile, cetuximab infusion was skipped on day 8 in the 1st to 4th cohort. Following an initial dose of 400mg/m2, cetuximab was administered weekly IV infusion of 250mg/m2 in the last cohort (cohort 5). Blood samples for PK analysis were collected for up to eight weeks after the initial dose. Results: Thirty pts were enrolled in total (CRC 29 pts, NSCLC 1 pt), median age 54 (range: 37-73) with a median of 3 prior chemotherapy regimens (range: 1–5). Twenty-four pts (up to cohort 4) were evaluable, and no DLT has been observed to date. The most frequent adverse events were rash (71%), acne (58%), anorexia (50%), diarrhea (46%), fatigue (46%) and fever (38%), which were of Grade 1–2 in severity. The preliminary PK results in the first cohort were virtually identical to those observed in Caucasians. In preliminary efficacy results of 18 evaluable pts, 2 CRC pts had PR, 10 pts SD. Conclusions: Preliminary results suggest that Cetuximab is well tolerated as a single agent in Japanese patients with solid tumors at the cohort 1–4. An updated analysis will be provided. No significant financial relationships to disclose.

Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid alpha glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart and liver. Autophagic buildup is associated to skeletal muscle pathology and known to inhibit the efficacy of enzyme replacement therapy. Myogenic conversion by MyoD forced expression is one of the feasible approaches towards disease modeling of neuromascular disorders. We have generated skeletal myocyte from Pompe disease fibroblast by direct reprogramming by lentiviral MyoD transfer. We have cloned human MYOD1 by RT-PCR from normal control fibroblast (AG08498). EcoR1 site was added and then cloned into 3rd generation lentiviral vector (CSII-EF1α-MCS). High titer lentiviral vector was produced by ultracentrifuge. Next we have infected CS-EF1α-MYOD1 at MOI50 to healthy control fibroblast. 48 hours after transfection, we have confirmed MYOD1 expression in cytoplasm and nucleus of control fibroblast by immunofluorescence. Other than MyoD, other myogenic factors are expressed and confirmed by immunofluorescence (MyoD, MyoG, Myf5, Pax7 and MHC). We have also infected CS-EF1α-MYOD1 to patient derived fibroblast (GM20124) and confirmed the expressions of myogenic factors, MyoD, MyoG, Myf5, Pax7 and MHC by immunofluorescence. Moreover, we have checked gene expression by RT-PCR and confirmed myogenic markers (Pax7, Pax3, Myf5, MyoD, AchR and MHC) are positive both in healthy control and patient derived fibroblasts. To visualize autophagic accumulation, we have infected RFP-GFP-LC3B vector (Promo™ Autophagy Sensor Kit) to induced myoblasts. It allows us to detect LC-3 accumulation which is suggesting autophagic buildup in pompe disease patient compared to healthy control. We have investigated autophagic buildup seen in skeletal myoblasts of Pompe disease at the live-cell imaging. Moreover, we have cloned lentiviral vector which expresses TFEB, a master gene for lysosomal biogenesis, to treat autophagic buildup and infected to MyoD induced myoblast. Massive accumulation of LC-3 is decreased after CSII-EF1α-TFEB transfection. High titer lentiviral MyoD transfer is useful in terms of direct reprogramming of fibroblast into skeletal myoblast. In addition, disease modeling of Pompe disease allows live-cell imaging of autophagic buildup of skeletal myocyote.

458 Background: The KRAS mutation has been associated with resistance to cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in metastatic colorectal cancer (mCRC). However, the predictive biomarkers of cetuximab resistance in KRAS wild-type mCRC remain unknown. We explored the possible roles of PTEN methylation and mutation of the EGFR L2 domain, which is the site of binding to cetuximab, in cetuximab resistance in KRAS wild-type mCRC. Methods: The subjects were 247 mCRC patients screened for KRAS status at the National Cancer Center Hospital between September 2008 and April 2010. Genomic DNA was extracted from formalin- fixed paraffin-embedded colorectal cancer tissue samples. Mutation analysis of KRAS and the EGFR L2 domain was performed by direct sequencing. Methylation analysis of PTEN was performed by quantitative real-time methylation-specific PCR with a set of primers specific to the methylated and unmethylated sequences, using sodium bisulfate-modified DNA. Results: Of the 247 mCRC patients, 136 patients had wild-type KRAS (55%). In 9 of these patients, the quality of the DNA was sufficient for analysis of PTEN methylation levels. Eight of the 9 patients received a cetuximab-based regimen (with irinotecan: 4, monotherapy: 4). The best response was PR in 4 patients (25%), SD in 2 (12.5%), and PD in 2 (12.5%). The best response of the one patient with methylated PTEN treated with cetuximab and irinotecan was SD. Mutation analysis of the EGFR L2 domain was performed in 65 of the 136 patients with wild-type KRAS. All of them received a cetuximab-based regimen (with irinotecan: 50, monotherapy: 15). The best response was PR in 13 patients (20%), SD in 26 (40%), PD in 20 (31%). The one patient who had a mutation at exon 9 showed a partial response to cetuximab and irinotecan. Conclusions: Methylation of PTEN and mutation of the EGFR L2 domain were analyzed in Japanese mCRC patients. Our findings do not provide sufficient evidence that EGFR L2 domain mutation and methylation of PTEN are correlated with resistance to cetuximab. No significant financial relationships to disclose.

ABSTRACT Background Previous studies showed that gene mutations (NRAS, BRAF, PIK3CA) are associated with a poor prognosis or resistance of anti-EGFR antibody in metastatic colorectal cancer (mCRC) patients with wild-type (WT) of KRAS codon 12/13 (KRAS-WT). However, the significance of these biomarkers has not been clarified. In addition, EGFR immunohistochemistry (IHC) and EGFR gene amplification to evaluate the efficacy of anti-EGFR antibody treatment have not been reported for mCRC. Method We evaluated tumor response and survival in patients who received anti-EGFR antibody by mutation analysis of KRAS, NRAS, BRAF, and PIK3CA in KRAS-WT patients with mCRC. Tumor DNA samples are obtained from patients treated in our hospital with anti-EGFR antibody between August 2008 and August 2011. Results A total of 117 patients were enrolled in this analysis, including 100 KRAS-WT patients. Seventy-one patients (60.7%) were all WT for KRAS, NRAS, BRAF, and PIK3CA, and 46 patients (39.3%) had at least one mutation or had insufficient DNA samples to analyze. Mutations of KRAS codon 61 (2 patients), KRAS codon 146 (5), BRAF V600E (2), PIK3CA exon9 (8), NRAS codon 12/13 (2), and NRAS codon 61 (5) were detected. No patients had a mutation of PIK3CA exon 20. Patients with at least one mutation had no response. Mutations of KRAS codon 146, NRAS codon 61, and BRAF V600E were associated with a shorter progression-free survival (PFS) compared with all WT patients (P = 0.049, P = 0.004, P = 0.036, respectively). Twelve patients (12% of KRAS-WT patients) with a mutation of KRAS codon 146, BRAF V600E, NRAS codon 61 had poor prognosis compared with the other KRAS-WT patients (PFS, 6.4 versus 2.0 months, P Conclusion Mutations of KRAS codon 146, NRAS codon 61, and BRAF V600E could be a strong prognostic factor of anti-EGFR antibody in patients with mCRC. Combination of IHC and DISH of EGFR could identify patients with a tumor response to anti-EGFR antibody in patients that are all wild type for KRAS, NRAS, BRAF, and PIK3CA.

Abstract Sphingolipid metabolism plays an important role in the formation of cellular membranes and is associated with malignant potential and chemosensitivity of cancer cells. Sphingolipid degradation depends on multiple lysosomal glucosidases. We focused on acid β-glucosidase (GBA), a lysosomal enzyme the deficiency of which is related to mitochondrial dysfunction. We analyzed the function of GBA in pancreatic ductal adenocarcinoma (PDAC). Human PDAC cell lines (PANC-1, BxPC-3 and AsPC-1) were examined under conditions of GBA knockdown via the short interfering RNA (siRNA) method. We assessed the morphological changes, GBA enzyme activity, GBA protein expression, cell viability, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) and mitophagy flux of PDAC cells. The GBA protein level and enzyme activity differed among cell lines. GBA knockdown suppressed cell proliferation and induced apoptosis, especially in PANC-1 and BxPC-3 cells, with low GBA enzyme activity. GBA knockdown also decreased the MMP and impaired mitochondrial clearance. This impaired mitochondrial clearance further induced dysfunctional mitochondria accumulation and ROS generation in PDAC cells, inducing apoptosis. The antiproliferative effects of the combination of GBA suppression and gemcitabine were higher than those of gemcitabine alone. These results showed that GBA suppression exerts a significant antitumor effect and may have therapeutic potential in the clinical treatment of PDAC.

(Molecular Therapy: Methods & Clinical Development 25, 448–460; June 2022) In the originally published version of this article, the following was included as part of the procedure for GFAP analysis in this study: "Cerebral tissues were homogenized in nine volumes of 50 mM Tris-HCl, pH 7.5 (Tris buffer), and protease inhibitor cocktail (PIC; Roche Diagnostics, Indianapolis, IN), followed by sonication and centrifugation at 18,000 × g for 1 h at 4 °C. Residual pellets were lysed with seven volumes of Tris buffer containing 2% sodium dodecyl sulfate (SDS) and PIC, followed by sonication and centrifugation.” This is partly incorrect, and the proper procedure is as follows: “Cerebral tissues were homogenized in nine volumes of 50 mM Tris-HCl, pH 7.5 (Tris buffer), and protease inhibitor cocktail (PIC; Roche Diagnostics, Indianapolis, IN), followed by sonication. Sonicated sample is diluted with 10% sodium dodecyl sulfate (SDS) to make final solution of 2% SDS concentration, followed by sonication and centrifugation.” This change has been corrected online, and the authors apologize for this error. Hematopoietic stem cell gene therapy ameliorates CNS involvement in murine model of GM1-gangliosidosisTsunogai et al.Molecular Therapy - Methods & Clinical DevelopmentApril 27, 2022In BriefEx vivo gene therapy using lentiviral vector for GM1 gangliosidosis model mice increased β-galactosidase enzyme activity in the central nervous system, reduced substrate accumulation, and improved astrocytosis and motor function. This gene therapy is a promising approach for the treatment of brain deficits in GM1 gangliosidosis. Full-Text PDF Open Access

Autophagy refers to bulk degradation processes responsible for the turnover of long-lived proteins, disposal of damaged organelles, and clearance of aggregate prone proteins. Aberrant autophagy causes the formation of cytoplasmic inclusion bodies, leading to liver injury and neurodegeneration. However, details of abnormalities related to impaired autophagy are largely unknown. The efficiency of the autophagy pathway relies on cargo receptors to identify the ubiquitinated targets destined for the degradation pathway. For instance, p62 promotes the formation of protein aggregates and their association with the autophagosome. Recent studies showed that murine p62 contains a highly conserved LC3 recognition sequence (LRS). Structural analysis of the LC3–LRS complex revealed an interaction between Trp340 and Leu343 of p62 and two hydrophobic pockets (hp1 and hp2) on the ubiquitin fold of LC3. The LRS motif of NBR1, autophagy receptor, presents differences to this classical LRS motif with a tyrosine residue and an isoleucine residue substituting Trp and Leu, respectively. NMR studies of NBR1–LRS complexed with GABARAPL, another Atg8 homologue, indicated that the presence of tryptophan residue in the LRS motif increases the binding affinity, but other substitutions have little effect on the binding affinity due to enthalpy–entropy compensation. The aforementioned results indicate that each autophagic receptor has a unique interaction form. Most recently, it has been demonstrated that the selectivity of the autophagy receptor NDP52 for LC3C is crucial for innate immunity. Other than those listed above, many autophagy receptors and Atg8 homologue binding proteins are reported.In vivo experiments showed that cells expressing p62 mutants lacking LC3 binding ability accumulate ubiquitin-positive inclusion bodies, instead of autophagosomes, as in hepatitis and neurodegenerative diseases. These data demonstrate that cellular levels of p62 are tightly regulated by autophagy through direct interaction with LC3, and that selective turnover of p62 via autophagy prevents inclusion body formation.

The authors would like to make a correction to their published paper [...].

Taniai, Tomohiko MD; Shirai, Yoshihiro MD, PhD; Shimada, Yohta PhD; Hamura, Ryoga MD; Yanagaki, Mitsuru MD; Takada, Naoki MD; Tsuboi, Kazuhito PhD; Okamoto, Yasuo MD, PhD; Ohashi, Toya MD, PhD; Yanaga, Katsuhiko MD, PhD, FACS Author Information

Yanagaki, Mitsuru MD; Shirai, Yoshihiro MD, PhD; Hamura, Ryoga MD; Taniai, Tomohiko MD; Shimada, Yohta PhD; Horiuchi, Takashi MD, PhD; Takada, Naoki MD; Saito, Nobuhiro MD, PhD; Ohashi, Toya MD, PhD; Yanaga, Katsuhiko MD, PhD, FACS Author Information

We report a 42-year-old woman with acute lymphoblastic leukemia who received allogeneic bone marrow transplantation (BMT) in the first remission from her 75-year-old, HLA-identical, MLC-non-reactive mother. Considering the difficulty to obtain a sufficient number of bone marrow cells from such an old donor, we harvested the cells (2.31 x 10(8)/kg) on day -37 and cryopreserved them until use. BMT was performed on June 3rd, 1993 after conditioning regimen with total body irradiation, high-dose AraC and cyclophosphamide. Graft-versus-host disease (GVHD) prophylaxisis was attempted by cyclosporin A and short-term methtrexate. Her hematopoietic recovery was favorable with no signs and symptoms of GVHD as far as day 218.