Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer’s gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.

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The production of hydrogen peroxide (H2O2) by neutrophilic polymorphonuclear leukocytes (PMN) after stimulation and the infiltration of PMN in glomeruli were determined in 20 patients with primary IgA nephropathy. The H2O2 production of PMN after the stimulation was measured with a spectrophotometer using horseradish peroxidase as substrate. The results were as follows: 1) when PMN were pretreated with cytochalasin B, H2O2 production after stimulation with heat-aggregated IgG (IgG) or serum-treated zymosan (STZ) was significantly higher in patients with IgA nephropathy than in controls, and 2) there was an increased amount of PMN localized in glomeruli in patients with IgA nephropathy using immunofluorescence of monoclonal anti-PMN antibody. It appeared that the increased renal infiltration of PMN which have a high potential for production of reactive oxygen species might induce the glomerular injuries in patients with IgA nephropathy.

Although it is difficult in Western medicine to eliminate edema occurring in the lower extremities after intrapelvic lymph node dissection for malignant gynecologic tumors, we successfully treated or prevented this postoperative complication with moxibustion and acupuncture, initiated after the occurrence of lymphedema in 12 patients and as soon as possible after surgery in 12 others. An increase in deep body temperature with acupuncture or moxibustion was found to be essential for successful treatment.

ObjectivesTo investigate the effects of irbesartan on inflammation and apoptosis in atherosclerotic plaques by histochemical examination and molecular imaging using 14C-FDG and 99mTc-annexin A5.BackgroundIrbesartan has a peroxisome proliferator-activated receptor gamma (PPARγ) activation property in addition to its ability to block the AT1 receptor. Accordingly, irbesartan may exert further anti-inflammatory and anti-apoptotic effects in atherosclerotic plaques. However, such effects of irbesartan have not been fully investigated. Molecular imaging using 18F-FDG and 99mTc-annexin A5 is useful for evaluating inflammation and apoptosis in atherosclerotic plaques.MethodsFemale apoE−/− mice were treated with irbesartan-mixed (50 mg/kg/day) or irbesartan-free (control) diet for 12 weeks (n = 11/group). One week after the treatment, the mice were co-injected with 14C-FDG and 99mTc-annexin A5, and cryostat sections of the aortic root were prepared. Histochemical examination with Movat's pentachrome (plaque size), Oil Red O (lipid deposition), Mac-2 (macrophage infiltration), and TUNEL (apoptosis) stainings were performed. Dual-tracer autoradiography was carried out to evaluate the levels of 14C-FDG and 99mTc-annexin A5 in plaques (%ID×kg). In vitro experiments were performed to investigate the mechanism underlying the effects.ResultsHistological examination indicated that irbesartan treatment significantly reduced plaque size (to 56.4%±11.1% of control), intra-plaque lipid deposition (53.6%±20.2%) and macrophage infiltration (61.9%±20.8%) levels, and the number of apoptotic cells (14.5%±16.6%). 14C-FDG (43.0%±18.6%) and 99mTc-annexin A5 levels (45.9%±16.8%) were also significantly reduced by irbesartan treatment. Irbesartan significantly suppressed MCP-1 mRNA expression in TNF-α stimulated THP-1 monocytes (64.8%±8.4% of un-treated cells). PPARγ activation was observed in cells treated with irbesartan (134%±36% at 3 µM to 3329%±218% at 81 µM) by a PPARγ reporter assay system.ConclusionsRemissions of inflammation and apoptosis as potential therapeutic effects of irbesartan on atherosclerosis were observed. The usefulness of molecular imaging using 18F-FDG and 99mTc-annexin A5 for evaluating the therapeutic effects of irbesartan on atherosclerosis was also suggested.