Transplantation of human mesenchymal stem cells has been shown to reduce infarct size and improve functional outcome in animal models of stroke. Here, we report a study designed to assess feasibility ...and safety of transplantation of autologous human mesenchymal stem cells expanded in autologous human serum in stroke patients. We report an unblinded study on 12 patients with ischaemic grey matter, white matter and mixed lesions, in contrast to a prior study on autologous mesenchymal stem cells expanded in foetal calf serum that focused on grey matter lesions. Cells cultured in human serum expanded more rapidly than in foetal calf serum, reducing cell preparation time and risk of transmissible disorders such as bovine spongiform encephalomyelitis. Autologous mesenchymal stem cells were delivered intravenously 36-133 days post-stroke. All patients had magnetic resonance angiography to identify vascular lesions, and magnetic resonance imaging prior to cell infusion and at intervals up to 1 year after. Magnetic resonance perfusion-imaging and 3D-tractography were carried out in some patients. Neurological status was scored using the National Institutes of Health Stroke Scale and modified Rankin scores. We did not observe any central nervous system tumours, abnormal cell growths or neurological deterioration, and there was no evidence for venous thromboembolism, systemic malignancy or systemic infection in any of the patients following stem cell infusion. The median daily rate of National Institutes of Health Stroke Scale change was 0.36 during the first week post-infusion, compared with a median daily rate of change of 0.04 from the first day of testing to immediately before infusion. Daily rates of change in National Institutes of Health Stroke Scale scores during longer post-infusion intervals that more closely matched the interval between initial scoring and cell infusion also showed an increase following cell infusion. Mean lesion volume as assessed by magnetic resonance imaging was reduced by >20% at 1 week post-cell infusion. While we would emphasize that the current study was unblinded, did not assess overall function or relative functional importance of different types of deficits, and does not exclude placebo effects or a contribution of recovery as a result of the natural history of stroke, our observations provide evidence supporting the feasibility and safety of delivery of a relatively large dose of autologous mesenchymal human stem cells, cultured in autologous human serum, into human subjects with stroke and support the need for additional blinded, placebo-controlled studies on autologous mesenchymal human stem cell infusion in stroke.
The prognosis of patients with malignant glioma is extremely poor, despite the extensive surgical treatment that they receive and recent improvements in adjuvant radio- and chemotherapy. In the ...present study, we propose the use of gene-modified mesenchymal stem cells (MSCs) as a new tool for gene therapy of malignant brain neoplasms. Primary MSCs isolated from Fischer 344 rats possessed excellent migratory ability and exerted inhibitory effects on the proliferation of 9L glioma cell in vitro. We also confirmed the migratory capacity of MSCs in vivo and showed that when they were inoculated into the contralateral hemisphere, they migrated towards 9L glioma cells through the corpus callosum. MSCs implanted directly into the tumor localized mainly at the border between the 9L tumor cells and normal brain parenchyma, and also infiltrated into the tumor bed. Intratumoral injection of MSCs caused significant inhibition of 9L tumor growth and increased the survival of 9L glioma-bearing rats. Gene-modification of MSCs by infection with an adenoviral vector encoding human interleukin-2 (IL-2) clearly augmented the antitumor effect and further prolonged the survival of tumor-bearing rats. Thus, gene therapy employing MSCs as a targeting vehicle would be promising as a new therapeutic approach for refractory brain tumor.
Prasugrel is a novel orally active thienopyridine prodrug with potent and long-lasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y(12) receptors by its active metabolite ...(AM). Previous studies have shown that the antiplatelet potency of prasugrel is at least 10 times higher than that of clopidogrel in rats and humans, but the mechanism of its higher potency has not yet been fully elucidated.
Oral administration of prasugrel to rats resulted in dose-related and time-related inhibition of ex vivo platelet aggregation, and its effect was about 10 times more potent than that of clopidogrel. The plasma concentration of prasugrel AM was higher than that of clopidogrel AM despite tenfold higher doses of clopidogrel, indicating more efficient in vivo production of prasugrel AM than of clopidogrel AM. In rat platelets, prasugrel AM inhibited in vitro platelet aggregation induced by adenosine 5'-diphosphate (ADP) (10 microm) with an IC(50) value of 1.8 microm. Clopidogrel AM similarly inhibited platelet aggregation with an IC(50) value of 2.4 microm. Similar results were also observed for ADP-induced (10 microm) decreases in prostaglandin E(1)-stimulated rat platelet cAMP levels. These results indicate that both AMs have similar in vitro antiplatelet activities.
The greater in vivo antiplatelet potency of prasugrel as compared to clopidogrel reflects more efficient in vivo generation of its AM, which demonstrates similar in vitro activity to clopidogrel AM.
We investigated whether FNIII14, a 22-mer peptide derived from fibronectin (FN) that potently impairs interaction of FN with beta1-integrin, could overcome cell adhesion-mediated drug resistance ...(CAM-DR) induced by very late antigen (VLA)-4-to-FN interaction in acute myelogenous leukemia (AML). Two AML cell lines, U937 cells and HL-60 cells, and fresh leukemic cells from six AML patients with high alpha4-integrin expression exhibited CAM-DR to cytosine arabinoside (Ara C) through VLA-4-to-FN interaction, while fresh leukemic cells from two AML patients with low alpha4-integrin expression did not display CAM-DR to Ara C. FNIII14 impaired VLA-4-to-FN interaction and restored sensitivity to Ara C in the CAM-DR leukemic cells. In these CAM-DR leukemic cells, upregulation of Bcl-2, which was induced through the focal adhesion kinase/Akt signal pathway upon VLA-4-to-FN interaction, was inhibited by FNIII14 treatment. In a mouse model of minimal residual disease (MRD) in bone marrow, 100% survival was achieved by combining FNIII14 with Ara C, whereas Ara C alone prolonged survival only slightly. The myelosuppression induced by Ara C was not augmented by the combination of FNIII14 in mouse experiments. Thus, the combination of anticancer drugs and FNIII14 holds promise to eradicate MRD in bone marrow after chemotherapy.
Background: Hepatitis C virus (HCV) infection is a major cause of human hepatocellular carcinoma (HCC). The precise mechanism of hepatocarcinogenesis in humans by HCV is currently unclear. It was ...recently shown, however, that transgenic mice with the HCV core gene often develop HCC, suggesting tumorigenic activity of the HCV core protein. Further, the HCV core protein expressed in HepG2 cells transfected with the core gene was shown to stimulate proliferation of transfectants through activation of nuclear factor-κB (NF-κB). The downstream target molecule(s) of NF-κB activated by the HCV core protein to evoke cell proliferation is not yet identified. Transforming growth factor (TGF) α, which is often overexpressed in various tumour tissues such as HCC, has been shown to stimulate hepatocyte proliferation through activation of the mitogen-activated protein kinase or extracellular signal-related protein kinase (MAPK/ERK) cascade. Aims: To explore the possibility that TGFα might be a target molecule for NF-κB activated by the HCV core, and that TGFα participates in the growth promotion of the core transfectants in an autocrine manner, activating the MAPK/ERK pathway. Methods: A HCV core expression vector was transfected into human hepatoma Huh-7, HepG2 and Hep3B cells. NF-κB activity was examined by an electrophoretic mobility shift assay. TGFα transcription was assessed by a luciferase reporter assay. TGFα protein was determined by immunoblot and ELISA. MAPK/ERK activity was examined by an in vitro kinase assay. Cell proliferation was assessed by a water-soluble tetrazolium salt-1 assay. Results: In the HCV core transfectants, NF-κB bound to the κB site in the TGFα proximal promoter region, resulting in an increase in TGFα transcription. Immunoblot as well as ELISA showed increased TGFα expression in the HCV core transfectants. SN50, a specific inhibitory peptide for NF-κB, cancelled HCV core-induced TGFα expression. HCV core protein increased cell proliferation as well as ERK activity of the HCV core transfectants as compared with the mock transfectants. The growth-promoting activity and activation of ERK by the HCV core protein were negated by treatment with anti-TGFα antibodies. Conclusions: These results suggest that the HCV core protein promotes proliferation of human hepatoma cells by activation of the MAPK/ERK pathway through up regulation of TGFα transcription via activation of NF-κB. Our finding provides a new insight into the mechanism of hepatocarcinogenesis by HCV infection.
The significance of serum concentrations of tumor necrosis factor-alpha (TNF-alpha) in the pathogenesis of inflammatory bowel disease (IBD) is uncertain. We measured TNF-alpha in serum from IBD ...patients by immuno-PCR to analyze the relationship between TNF-alpha and pathophysiologic state in IBD.
Serum samples were collected from 54 healthy blood donors, 29 patients with ulcerative colitis (UC; 46 samples), and 7 patients with Crohn disease (CD; 8 samples). DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to biotinylated third antibody. TNF-alpha sandwiched by antibodies was detected by PCR amplification of the DNA label.
TNF-alpha could be measured in all samples. The median serum concentration in IBD patients overall was approximately 390-fold higher than in healthy donors (median increase, 380-fold for UC, 640-fold for CD). The median serum TNF-alpha concentration was 1.7-fold higher in the active stage of UC than in the inactive stage (P <0.05), and this difference could be detected in individual patients.
Sensitive measurement of serum TNF-alpha could provide an important pathophysiologic marker for the presence and activity of IBD.
The efficacy of hypothermic intervention for permanent focal ischemia has yet to be clarified. This study investigated the effect of a prolonged moderate or mild hypothermia on permanent focal ...ischemia in rats.
Two permanent focal ischemia models in male Sprague-Dawley rats were used. Moderate (30 degrees C, in experiment 1) or mild (33 degrees C, in experiment 2) hypothermia was achieved at the time of the induction of focal ischemia and was maintained for 2 hours under general anesthesia. Thereafter, the hypothermic condition was maintained by means of a cold room for a total of 24 hours. The infarct volume and neurological function were analyzed for a maximum of 21 days and compared with that of the normothermia group. Regional cerebral blood flow was monitored for 6 hours in the ischemic core and penumbra region.
In experiment 1, the total infarct volume in the normothermic group was 368+/-59 mm(3); in contrast, it was significantly smaller in the hypothermia group: 169+/-33 mm(3) at 48 hours (mean+/-SEM, P:<0.05). In experiment 2, the infarct volume was 211+/-19 mm(3) in the normothermia group and 88+/-15 mm(3) in the hypothermia group at 21 days (P:<0.05). There were significant differences in neurological function from days 2 through 21 between the two groups. Mean regional cerebral blood flow in the penumbra region increased to a level >50% of baseline.
Prolonged mild hypothermia suppressed the development of cerebral infarct and neurological deficit chronically after the induction of permanent focal ischemia.
The aim of this dose escalation study was to determine the maximum-tolerated dose (MTD), dose-limiting toxicities (DLTs) and preliminary efficacy of docetaxel, S-1 and cisplatin combination ...chemotherapy in patients with unresectable metastatic gastric cancer. Seventeen patients received oral S-1 (40 mg m(-2) bid) on days 1-14, intravenous cisplatin (60 mg m(-2)) and docetaxel (60, 70 or 80 mg m(-2) depending on DLT) on day 8 every 3 weeks. The MTD of this combination was presumed to be docetaxel 70 mg m(-2). At this dose level, 40% of the patients (two of five) developed grade 4 neutropenia and 20% (one of five) exhibited grade 3 nausea during the first course. Therefore, the recommended dose of docetaxel was defined as 60 mg m(-2). The DLT was neutropenia. The response rate (RR) was 88.2% (15 of 17), consisting of one complete response and 14 partial responses. There were two stable diseases but no progressive disease. Of these 15 responders, four (23.5%) with high VEGF expression showed rapid tumour regression and achieved downstaging, leading to subsequent curative gastrectomy. Three of these have been disease free for about 3 years, suggesting a complete cure. In conclusion, this regimen was tolerable and showed a quite high RR, with an appreciable downstaging rate in metastatic gastric cancer.
Background: In recent years, there has been an increasing number of cases of early gastric cancer (T1, NX) with intramucosal invasion, which are untreatable by surgical or endoscopic mucosal ...resection (EMR) because of their high risk. Currently, no adequate treatment is available for such patients. Aim: The main objective of this study was to evaluate whether argon plasma coagulation (APC) is an effective and safe modality for treating early gastric cancer untreatable by surgical resection or EMR. Methods: The study group comprised 20 men and seven women diagnosed with gastric cancer with intramucosal invasion who were considered poor candidates for surgical resection or EMR due to risk factors such as severe cardiac failure or thrombocytopenia. Irradiation conditions for APC treatment were determined using swine gastric mucosa. We used an argon gas flow of 2 l/min at a power setting of 60 W and a maximum irradiation time of 15 s/cm2. The follow up period of the 27 patients ranged from 18 to 49 months (median 30 months). Results: All lesions were irradiated easily, including areas anatomically difficult for EMR such as the gastric cardia or the posterior wall of the upper gastric body. In 26 of 27 patients (96%) there was no evidence of recurrence during the follow up period (median 30 months). One patient showed recurrence six months after the treatment but was successfully retreated. No serious complications were found in any of the 27 patients but three patients (11%) experienced a feeling of abdominal fullness. Interpretation: APC is a safe and effective modality for treatment of early gastric cancer with intramucosal invasion untreatable by surgical resection or EMR. However, further observations are necessary to determine the long term prognosis of patients undergoing this treatment.
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