Transcriptional activities of two newly identified Haemaphysalis longicornis tick-derived promoter regions in the Ixodes scapularis tick cell line (ISE6).

Ticks are obligate haematophagous ectoparasites considered to be second to mosquitoes as vectors of human diseases and the most important vector for animals. Despite efforts to control tick infestations, they remain a serious health problem. Gene manipulation has been established in mosquitoes and led to the control of mosquito populations and of mosquito-borne pathogens. Therefore, gene manipulation could be useful for controlling ticks and tick-borne pathogens. To investigate effective gene expression vectors for ticks, the promoter activities of commercial plasmids were evaluated in a tick cell line (ISE6). Dual luciferase assays revealed that pmirGLO, the human phosphoglycerate kinase promoter contained plasmid vector, showed the highest activity in ISE6 cells amongst the tested plasmids. Moreover, we identified the promoter regions of the Haemaphysalis longicornis actin (HlAct) and the intracellular ferritin (HlFer1) genes. To construct a more effective expression vector for ticks, these promoter regions were inserted into pmirGLO (pmirGLO-HlAct pro and pmirGLO-HlFer1 pro). The pmirGLO-HlAct pro vector showed significantly higher promoter activity than pmirGLO, whereas the pmirGLO-HlFer1 pro vector demonstrated significantly lower promoter activity than pmirGLO in ISE6 cells. The HlAct promoter region may have high promoter activity in ISE6 cells. The results of the present study provide useful information for the development of a genetic modification system in ticks.

Meta-Analysis of the Risk of Immune-Related Adverse Events With Anticytotoxic T-Lymphocyte-Associated Antigen 4 and Antiprogrammed Death 1 Therapies.

We assessed the risks of immune-related adverse events with anticytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and antiprogrammed death 1 (PD1) therapies by meta-analysis. Twenty-one studies including 11,144 patients were found. Anti-CTLA4 therapy was associated with a significantly higher risk of overall immune-related adverse events: diarrhea, immune-related colitis, pruritus, and rash compared to control therapies (relative risk (RR) = 2.43, 2.10, 11.39, 3.88, 3.87, 95% confidence interval (CI) = 1.77-3.34, 1.52-2.45, 6.30-20.59, 2.37-6.37, 2.39-6.27, P < 0.001 for all outcomes). Anti-PD1 therapy was associated with a significantly higher risk of pruritus (RR = 4.01, 95% CI = 1.97 to 8.17, P < 0.001); however, it did not increase the risks of other adverse events. Anti-CTLA4 and anti-PD1 therapies have distinct features of immune-related adverse events. The results of our study would aid the surveillance and management of immune-related adverse events in patients receiving these therapies.

Systematic review with meta-analysis: the efficacy and safety of CT-P13, a biosimilar of anti-tumour necrosis factor-α agent (infliximab), in inflammatory bowel diseases.

BACKGROUND: Biosimilars of anti-tumour necrosis factor (TNF)-α agents have now become clinically available for the treatment of inflammatory bowel diseases (IBD). AIM: To perform a systematic review and meta-analysis to evaluate the efficacy and safety of biosimilars of anti-TNF-α agents in patients with IBD. METHODS: Electronic databases were searched. The outcomes were the pooled rates of clinical response or remission, sustained clinical response or remission, and adverse events in patients with IBD induced with or switched to biosimilars of anti-TNF-α agents. RESULTS: Eleven observational studies reporting outcomes in 829 patients treated with biosimilar of infliximab (CT-P13) were identified. The pooled rates of clinical response among Crohn's disease (CD) and ulcerative colitis (UC) at 8-14 weeks were 0.79 (95% confidence interval (CI) = 0.65-0.88) and 0.74 (95% CI = 0.65-0.82), respectively, and at 24-30 weeks were 0.77 (95% CI = 0.63-0.86) and 0.77 (95% CI = 0.67-0.85) respectively. Adverse events were rare (CD, 0.08 (95% CI = 0.02-0.26); UC, 0.08 (95% CI = 0.03-0.17)). The pooled rates of sustained clinical response among CD and UC after switching from infliximab to CT-P13 at 30-32 weeks were 0.85 (95% CI = 0.71-0.93) and 0.96 (95% CI = 0.58-1.00), respectively, and at 48-63 weeks were 0.75 (95% CI = 0.44-0.92) and 0.83 (95% CI = 0.19-0.99) respectively. Adverse events were rare (CD, 0.10, 95% CI = 0.02-0.31; UC, 0.22, 95% CI = 0.04-0.63). CONCLUSIONS: CT-P13 was associated with excellent clinical efficacy and safety profile, supporting its use in the treatment of IBD.

Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human alpha-fetoprotein gene promoter.

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but is reactivated in hepatocellular carcinoma. It has been shown that the positive and negative transcriptionally regulatory elements of the human AFP gene, which play an important role in its developmental regulation, exist over the quite extended region (4 kb). We constructed a hybrid gene consisting of herpes simplex virus thymidine kinase (HSV-tk) gene under the control of the 0.3-kb human AFP gene promoter and inserted it into a retroviral vector. When AFP-producing hepatoma cells were infected with this recombinant retrovirus (LNAF0.3TK virus), the cells expressed HSV-tk gene and exhibited increased sensitivity to ganciclovir parallel with the ability of AFP production. On the other hand, the retroviral infection had little effect on ganciclovir-mediated cytotoxicity in AFP-nonproducing hepatoma or non-hepatoma cells. Moreover, the addition of dexamethasone increased the cytotoxicity of aciclovir to the virus-infected, AFP-producing cells through a glucocorticoid-responsive element in the AFP promoter, although aciclovir, by itself, had little cytotoxicity. These results demonstrate that the AFP promoter sequence alone can provide enough tumor-specific activity for therapeutic gene expression and induce selective growth inhibition by ganciclovir in the virus-infected, AFP-producing human hepatoma cells. In addition, it is possible that expression of the therapeutic gene is modulated by administration of dexamethasone or other agents that alter AFP promoter activity after gene transduction.

Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter.

We previously reported that the retroviral vector expressing the herpes simplex virus-thymidine kinase gene under the control of 0.3-kb human alpha-fetoprotein (AFP) gene promoter (AF0.3) provided the cytotoxicity to ganciclovir (GCV) in high-AFP-producing human hepatoma cells but not in low-AFP-producing cells. Therefore, specific enhancement of AFP promoter activity is likely to be required to induce enough cytotoxicity in low-AFP-producing hepatoma cells. In this study, we constructed a hybrid promoter, [HRE]AF, in which a 0.4-kb fragment of human vascular endothelial growth factor 5'-flanking sequences containing hypoxia-responsive element (HRE) was fused to AF0.3 promoter. By means of the reporter gene transfection assay, hypoxia-inducible transcriptions that were mediated by [HRE]AF promoter were detected in low- and non-AFP-producing human hepatoma cells, but not in nonhepatoma cells. When the herpes simplex virus-thymidine kinase gene controlled by [HRE]AF promoter was transduced into hepatoma and nonhepatoma cells by a retroviral vector, the exposure to 1% O2 induced GCV cytotoxicity specifically in the hepatoma cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors consisting of the virus-infected hepatoma cells gradually disappeared by GCV administration. These results indicate that the hypoxia-inducible enhancer of the human vascular endothelial growth factor gene, which is directly linked to human AFP promoter, involves selective and enhanced tumoricidal activity in gene therapy for hepatocellular carcinoma.

Transduction of antisense cyclin D1 using two-step gene transfer inhibits the growth of rat hepatoma cells.

Cyclin D1, one of the G(1) cyclins, is frequently overexpressed in several types of carcinomas and is thought to play an important role in tumorigenesis and tumor progression including hepatocellular carcinoma. We constructed a retrovirus vector-carrying rat cyclin D1 cDNA in the reverse orientation, resulting in expression of antisense (AS) cyclin D1 mRNA. For efficient transduction of this recombinant retrovirus, two-step gene transfer was performed. The rat hepatoma cell line (dRLh84) was infected with this recombinant retrovirus after preinfection with adenovirus expressing the retrovirus receptor. In the rat hepatoma cells, AS cyclin D1 mRNA was expressed, inducing a decrease in the expression of endogenous cyclin D1 mRNA and an inhibition of cell growth. Moreover, two-step gene transfer of AS cyclin D1 into s.c. hepatoma xenografts resulted in inhibition of tumor growth and prolonged animal survival. In the virus-infected tumor xenografts, expression of cyclin D1 was immunohistochemically inhibited, and apoptosis of hepatoma cells was detected. These findings suggest that transduction of AS cyclin D1 is useful as an adjunct to standard treatments for hepatocellular carcinoma.

Evaluation of a Proton Pump Inhibitor for Sleep Bruxism: A Randomized Clinical Trial.

Bruxism is a repetitive jaw-muscle activity characterized by clenching or grinding of the teeth and/or bracing or thrusting of the mandible. Recent advances have clarified the relationship between gastroesophageal reflux and sleep bruxism (SB). However, the influence of pharmacological elimination of gastric acid secretion on SB has not been confirmed. The authors aimed to assess the efficacy of a proton pump inhibitor (PPI) on SB and to examine the gastrointestinal (GI) symptoms and endoscopic findings of the upper GI tract in SB patients. The authors performed a randomized double-blind placebo-controlled crossover study at Kagoshima University Hospital. Twelve patients with polysomnography (PSG)-diagnosed SB underwent an assessment of GI symptoms using the frequency scale for the symptoms of gastroesophageal reflux disease (FSSG) and esophagogastroduodenoscopy. At baseline (i.e., before interventions), the mean frequencies of electromyography (EMG) bursts and rhythmic masticatory muscle activity (RMMA) episodes were 65.4 ± 49.0 bursts/h and 7.0 ± 4.8 episodes/h, respectively, and at least 1 RMMA episode with grinding noise was confirmed in all participants. The mean FSSG score was 8.4 ± 5.6, and 41.7% of patients were diagnosed with gastroesophageal reflux disease. Mild reflux esophagitis was confirmed in 6 patients. PSG, including EMG of the left masseter muscle and audio-video recording, was performed on days 4 and 5 of administration of 10 mg of the PPI (rabeprazole) or placebo. PPI administration yielded a significant reduction in the frequency of EMG bursts, RMMA episodes, and grinding noise. No significant differences were observed regarding the swallowing events and sleep variables. Since the clinical application of PPI for SB treatment should remain on hold at present, the results of this trial highlight the potential application of pharmacological gastroesophageal reflux disease treatment for SB patients. Larger scale studies are warranted to corroborate these findings. (UMIN Clinical Trials Registry: UMIN000004577).

DNA/RNA-dependent ATPase activity is associated with ATBF1, a multiple homeodomain-zinc finger protein.

The AT motif-binding factor 1 (ATBF1)-A is a large transcription factor containing four homeodomains and 23 zinc finger motifs. It has a number of motifs involved in transcriptional regulation, and in addition, several motifs found in enzymes, such as ATPases and helicases. In this study, we examined whether ATPase activity is associated with the ATBF1-A molecule. A 263-amino acid segment of the ATBF1-A molecule, termed AHZ, which contains the ATPase A-motif, homeodomain IV and zinc finger 21, was expressed in Escherichia coli in the form of glutathione S-transferase fusion protein and analyzed for ATPase activity. We found that AHZ was able to hydrolyze ATP with K(m) 10.6 microM and K(cat) 0.055 min(-1) at 5 mM Mg(2+) and pH 7.75. AHZ retained bacterial DNA and removal of the DNA resulted in 70% decrease in ATPase activity. The addition of double- or single-stranded DNAs restored 70-75% ATPase activity and that of RNA restored 50-55% activity. Site-directed mutagenesis of the A-motif resulted in 34% reduction of ATPase activity with no significant loss of bound DNA. In contrast, mutation of homeodomain IV and zinc finger 21 resulted in 90 and 80% reduction of ATPase, respectively, with the loss of the ability to bind to DNA and RNA. These results show that ATBF1 has at least one enzyme activity in addition to regulation of DNA transcription. The ATPase activity associated with ATBF1-A is DNA/RNA-dependent and unique in that it requires both homeodomain and zinc finger motifs.

Cloning of the cDNA encoding the mouse ATBF1 transcription factor.

We have isolated a mouse ATBF1 cDNA which is 12-kb long and capable of encoding a 406-kDa protein containing four homeodomains and 23 zinc-finger motifs. Mouse ATBF1 is 94% homologous to the human ATBF1-A transcription factor. Northern blot and RNase protection analysis showed that levels of ATBF1 transcripts were low in adult mouse tissues, but high in developing brain, consistent with a role for ATBF1 in neuronal differentiation.

Retrovirus-mediated gene therapy for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by human alpha-fetoprotein enhancer directly linked to its promoter.

We have previously reported that a retrovirus vector (LNAF0.3TK) carrying a herpes simplex virus thymidine kinase gene regulated only by the 0.3-kb human alpha-fetoprotein (AFP) promoter provides ganciclovir (GCV)-mediated cytotoxicity in high AFP-producing human hepatoma cells but not in low AFP-producing cells. In the present study, a retrovirus vector (LNAF0.3(E+)TK), in which herpes simplex virus thymidine kinase gene expression is under the control of a human AFP enhancer directly linked to its promoter, was constructed and compared with LNAF0.3(E+)TK. In the intermediate and low AFP-producing human hepatoma cells PLC/PRF/5 and huH1/cl.2, respectively, as well as in the high AFP-producing human hepatoma cells (HepG2), LNAF0.3(E+)TK sensitized these cells to GCV in vitro but did not affect cell growth in nonhepatoma cells (HeLa). In an animal model using athymic mice harboring PLC/PRF/5 cells, GCV treatment resulted in more pronounced growth inhibition in the LNAF0.3(E+)TK virus-infected cells than in the LNAF0.3(E+)TK virus-infected cells. These results indicate that the human AFP enhancer that is directly linked to its promoter involves selective and enhanced tumoricidal activity in gene therapy for hepatocellular carcinoma.

Enhanced expression of hepatitis B surface antigen by sodium butyrate in PLC/PRF/5 human hepatoma cells.

The effect of butyric acid, a natural fermentation product of colonic bacterial flora, on hepatitis B surface antigen (HBsAg) expression was investigated in HBsAg-positive PLC/PRF/5 human hepatoma cells. By Northern blot analysis, the levels of HBsAg mRNA increased dose-dependently using sodium butyrate (0-2 mmol/l). In transient chloramphenicol acetyltransferase plasmid transfection experiments, the HBsAg-preS2 promoter activity as well as the HBV enhancer 1 activity was stimulated by sodium butyrate, whereas the HBsAg-preS1 promoter activity was not. These results indicate that butyric acid functions as a physiological regulator of HBsAg expression through the portal blood flow and possibly contributes to increased expression ratio of preS2/S to preS1 polypeptides recognized in persistant HBV infection.

Differential regulation of glucose transporter-1 and -2 gene expression by hepatocyte growth factor in human hepatoblastoma cells.

Glucose transporters, GLUT-1 and GLUT-2, are key factors involved in facilitative glucose transport to hepatocytes. The aim of the present study was to clarify how hepatocyte growth factor (HGF) regulates expression of both genes in HepG2 human hepatoblastoma cells. HGF dose-dependently suppressed cell growth, but enhanced cellular glucose uptake together with increased expression of GLUT-1 protein. This increase resulted from elevation of its transcript. In contrast, no changes were found in expression of GLUT-2 protein or its transcript by HGF treatment. These results indicate that HGF stimulates glucose incorporation through the selective up-regulation of GLUT-1 gene expression in HepG2 cells.

Developmental changes in expression of the ATBF1 transcription factor gene.

The expression of the ATBF1 gene in developing brain was analyzed in mice from 13 days of gestation to 28 days after birth using RNase protection and in situ hybridization methods. The level of ATBF1 transcripts was the highest on embryonic day 13-15 and then progressively decreased to a hardly detectable level on postnatal day 28. Throughout the period examined, ATBF1 mRNA was expressed consistently in the basal telencephalon, diencephalon, and mesencephalon, with the highest levels in the inferior colliculus and thalamus. On the other hand, no significant expression was observed in cerebellum, neocortex, hippocampus, and olfactory bulb. These results illustrate significant regional differences in the ATBF1 expression and suggest a role of the ATBF1 gene in the formation of some specific cell populations in developing central nervous system.

A case of chronic hepatitis C developing insulin-dependent diabetes mellitus associated with various autoantibodies during interferon therapy.

We report a case of chronic hepatitis C presenting insulin-dependent diabetes mellitus (IDDM) associated with various autoantibodies including possible anti-insulin receptor antibody (AIRA) during interferon (IFN) therapy. A 57-year-old man having chronic hepatitis C virus (HCV) infection with chronic thyroiditis received IFN therapy. The thyroid function was well-controlled by administration of thyroid hormone, although thyroid autoantibodies were positive. At 15 weeks after starting IFN (reaching 530 million units of total dose), marked thirst happened, with increased fasting plasma glucose level (488 mg/dl) and decreased daily urinary C peptide immunoreactivity level (less than 4.2 microg/day). IDDM occurred with anti-nuclear antibody (ANA), anti-DNA antibody and possible AIRA, and thyroid autoantibodies titers increased, but without pancreatic islet cell antibody and anti-glutamic acid decarboxylase antibody. Administration of IFN was stopped and insulin treatment was started, but plasma glucose level was not controlled well. AIRA became negative 2 months later, however, insulin antibody (IA) was positive when tested after 18 months. Serum HCV RNA has been negative, and a normal level of serum transaminase has been observed since IFN therapy. It is likely that IFN therapy induced the immunological disturbance and resulted in occurrence of various autoantibodies and IDDM in the patient.

Retrovirus-mediated gene therapy for human hepatocellular carcinoma transplanted in athymic mice.

Gene therapy using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of the 0.3-kb human alpha-fetoprotein (AFP) gene promoter (LNAF0.3TK virus) in combination with ganciclovir (GCV) treatment was performed in athymic mice harboring AFP-producing HuH-7 human hepatoma cells. GCV treatment resulted in pronounced growth inhibition of the virus-infected HuH-7 xenograft in mice, but did not affect growth of the parental xenograft. These results indicate that the AFP gene promoter sequence allows enough therapeutic gene expression to induce the GCV-mediated cytotoxicity in vivo in AFP-producing human hepatoma cells.

Gene Targeting to Hepatomas (AFP).

Hepatocellular carcinoma (HCC) is one of the most common malignancies with poor prognosis worldwide, especially in eastern Asia and Africa (1). Recent advances in delivering genes to mammalian cells stimulate the possibility of gene therapy for human diseases, including cancer gene therapy (2). One approach of gene therapy for cancers is the transduction of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells, because the killing effect of the HSV-tk product on the virus-infected cells is seen in only proliferating cells (3). HSV-tk can efficiently phosphorylate nucleoside analogs, and the phosphorylated products act as a chain terminator of DNA synthesis, leading to cell death (4). In addition, successful application of suicide gene therapy for cancer, in part, relies on the bystander effect, where the active chemotherapeutic agent produced in target cells diffuses from cells to neighboring malignant cells in sufficient concentrations to induce growth inhibition (5).

[Systemic and regional myocardial distribution of 123I-BMIPP in normal subjects].

We estimated the systemic and left ventricular (LV) regional myocardial distribution of 123I-BMIPP (beta-methyl-p-iodophenyl-pentadecanoic-acid) in normal subjects (n = 13, mean age 43.9). In planar studies, the count ratios of heart, lung and liver to mediastinum were 2.63, 1.28 and 3.80 in the early images, and 2.23, 1.20 and 2.26 in the delayed images, respectively. The uptake in liver was almost identical with that in heart in the delayed images. In SPECT studies, the regional relative counts in anterior, septal, posterior and lateral LV walls were 100, 98, 96 and 108 (%) in the initial images, and 100, 98, 99 and 107 (%) in the delayed images, respectively. The regional relative uptake was significantly higher in the lateral wall than those in the other parts of LV walls in both images. The relative counts in the basal, mid- and apical portions were 100, 111 and 87 (%) in the initial images, and 100, 113, 92 (%) in the delayed images, respectively. These results suggest that the myocardial regional distribution of BMIPP is not always uniform even in normal subjects. Thus, it is necessary to interpret with caution in the light of these findings, especially for detect in a myocardial lesion in an early phase of cardiomyopathy or a mild myocardial ischemia.

[Clinical significance of 123I-MIBG myocardial scintigraphy in patients with hypertrophic cardiomyopathy].

We studied the abnormality of myocardial sympathetic nervous system in patients with hypertrophic cardiomyopathy using 123I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy in comparison with the parameters of other clinical examinations. In 50 patients with HCM, the heart to mediastinum 123I-MIBG uptake ratio (H/M) was significantly low and washout rate (WR) of 123I-MIBG was significantly high respectively compared with normal subjects (n = 8). H/M was negatively correlated with serum norepinephrine level, wall thickness of left ventricle, left ventricular mass index, left ventricular end diastolic pressure respectively, and WR was positively correlated with those parameters respectively. On the other hand, LF/HF calculated by spectral analysis in holter electrocardiogram was positively correlated with H/M, and negatively correlated with WR. In HCM, H/M in patients with subjective symptoms was significantly lower than that without subjective symptoms, and WR in patients with paroxysmal atrial fibrillation was significantly higher than that without paroxysmal atrial fibrillation. This study revealed that H/M and WR reflected the severity and the difference of disease type in HCM. In conclusion, 123I-MIBG contributes to evaluating more details in diagnosis and pathophysiology of HCM.

[Effect of bisoprolol, a beta 1-selective beta-blocker, on lipid and glucose metabolism and quality of life in elderly patients with essential hypertension].

The present study investigated the effect of bisoprolol, a beta 1-selective beta-blocker without intrinsic sympathomimetic activity (ISA), on lipid and glucose metabolism and quality of life (QOL) in elderly patients with essential hypertention. Bisoprolol at doses of 5-10 mg was administered once daily for 12 weeks to 60 non-elderly and 21 elderly outpatients with mild to moderate essential hypertension. In both groups bisoprolol significantly decreased both systolic and diastolic blood pressures and significantly reduced pulse rates to the same extent. The levels of serum cholesterol, HDL-cholesterol and triglyceride, and the response of plasma glucose and insulin to 75 g oral glucose load, were not changed in either group by the bisoprolol treatment. Bisoprolol significantly improved QOL in both groups. Bradycardia, a side effect attributable to bisoprolol, was noted in only one patient in the elderly group. These results suggest that bisoprolol is a safe and useful antihypertensive drug in elderly and non-elderly patients with essential hypertension.

[Left ventricular aneurysm with normal coronary arteriogram after myocardial infarction].

Left ventricular aneurysms after myocardial infarction are generally considered one of the complications of severe coronary artery disease. Postinfarction ventricular aneurysms with normal coronary arteriogram are rare. Only a few cases have been reported previously in Japan. We examined the incidence and the clinical characteristics of postinfarction ventricular aneurysms without coronary obstruction. Among the consecutive 1800 patients studied in our laboratory with selective coronary cineangiography and left ventriography, we found 5 (4 male, 1 female) patients with left ventricular aneurysms with no or minimal coronary arterial obstruction. The patient's ages ranged from 34 to 83 with a mean of 59. Interestingly, no patient had prior anginal history, and every case occurred with a first sudden attack of chest pain. The likely mechanisms causing the development of myocardial infarction were coronary spasm and/or thromboembolic accident. One patient, in whom a coronary induction test was performed, showed positive findings. It is possible that poor collateral circulation and well preserved contraction of viable myocardium in these patients bring about the formation of left ventricular aneurysm after myocardial infarction with normal coronary arteriogram.