Hepatitis B virus X stimulates redox signaling through activation of ataxia telangiectasia mutated kinase

Hepatitis B virus X (HBX) protein plays a crucial role in carcinogenesis, but its mechanism is unclear. The involvement of ataxia telangiectasia mutated (ATM) kinase in the enhanced redox system was investigated by examining the phosphorylation level of ATM in HBX gene-transfected cells and in transgenic mice following redox system manipulation by treatment with hydrogen peroxide (H₂O₂) or antioxidant. Western blotting and immunostaining showed that phospho-ATM was significantly increased by HBX both in vitro (3.2-fold; p<0.05) and in vivo (4-fold; p<0.05), and this effect was abrogated by antioxidant treatment. The level of PKC-δ in HBX-expressing cells was increased 3.5-fold compared to controls. Nuclear localized NF-E2-related factor 2 (Nrf2) was increased in HBX-expressing cells exposed to H₂O₂, but remained at lower levels after the treatment with rottlerin, KU55933, or caffeine. The levels of anti-oxidant molecules were increased in HBX expressing cells and in transgenic mice, indicating that HBX stimulates the Nrf2-mediated redox system. The levels of intracellular reactive oxygen species (ROS) were significantly increased in HBX-expressing cells treated with hydrogen peroxide in the presence of ATM inhibitor KU55933 or caffeine. Treatment of HBX-expressing cells with KU55933 or caffeine before the exposure to H₂O₂ increased the ratio of cell apoptosis to 33 ± 4% (p<0.05) and 22 ± 4% (p<0.05), respectively. Collectively, HBX stimulates the ATM-mediated PKC-δ/Nrf2 pathway, and maintains the enhanced activity of the redox system. Therefore, manipulating ATM kinase activity might be a useful strategy for treating HBX-induced carcinogenesis.     

Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14

Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS-TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, but is not coprecipitated with LPS-TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS-TLR4-MD-2 complexes was approximately 3 nM, which is approximately 10-20 times lower than the reported Kd for LPS-MD-2 or LPS-CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.     

Small hypervascular hepatocellular carcinoma revealed by double arterial phase CT performed with single breath-hold scanning and automatic bolus tracking

OBJECTIVE: The purpose of this study was to evaluate the usefulness of double arterial phase CT for the detection of small hypervascular hepatocellular carcinomas, using an automated bolus-tracking technique to initiate the hepatic arterial phase CT. MATERIALS AND METHODS: Double arterial and late phase contrast-enhanced helical CT scans were obtained on 287 consecutive patients suspected of having hepatocellular carcinoma. These included 56 patients with 90 small (< or 3 cm) hepatocellular carcinomas and 50 patients with no hepatocellular carcinomas. CT scans of these patients were interpreted by three reviewers. The first arterial phase scan was initiated automatically 10 sec after the bolus-tracking program detected the threshold enhancement of 50 H in the abdominal aorta. Three reviewers interpreted the late phase CT scans in combination with the first, second, or both hepatic arterial phases. Measures of the reviewers' detection of hepatocellular carcinoma included analysis of interobserver variation, sensitivity, specificity, and area under receiver operating characteristic curve (A(z)). RESULTS: The time elapsed from bolus initiation to threshold aortic enhancement ranged from 10 to 24 sec (mean, 13 sec), resulting in initiation of the first arterial phase CT scan from 20 to 34 sec (mean, 23 sec). The combination of late phase CT and both first and second arterial phase images showed significantly better performance than the combination of the late phase and either the first or second arterial phases, although the difference was most evident in comparison with the combination of second arterial and late phases. CONCLUSION: An automated bolus-tracking program can be used to optimize the timing of hepatic arterial phase CT. Multiphasic CT performed using this technique is useful in detection of small hepatocellular carcinoma.     

Attenuation of Postmenopausal Bone Loss in Patients with Transient Hypoparathyroidism After Total Thyroidectomy

Background

Increased bone mineral density (BMD) has been reported in patients with postsurgical permanent hypoparathyroidism. Hypoparathyroidism may attenuate the high-turnover bone loss in postmenopausal women. We reported previously that patients who had transient hypoparathyroidism postoperatively were at subclinical hypoparathyroid (hP) status even 5 years after surgery. We hypothesized that patients with transient hypoparathyroidism (ThP) may have altered BMD.

Methods

A total of 140 women who underwent total thyroidectomy had BMD measurements of the lumbar spine, femoral neck, and radius 3 years after surgery. At surgery, 99 patients were ≥50 years and 41 were <50 years. They were divided into three groups according to their postoperative parathyroid function: There were 80 patients in the no hP (NhP) group, 54 in the ThP group, and 6 in the permanent hP (PhP) group.

Results

Among the 99 patients aged ≥50 years, 36 ThP patients had median Z scores of the BMD in all three areas (lumbar spine, femoral neck, radius) that were significantly higher (by 1.083, 0.533, and 1.047, respectively) than those in the 60 NhP patients aged ≥50 years. The BMDs in the three PhP patients ≥50 years were higher than those in the NhP and ThP patients, but the difference did not reach significance except for in the femoral neck. Multivariate logistic regression analyses showed that Z scores > 0 were significantly associated only with the presence of ThP postoperatively. In the patients <50 years, the BMD values were not significantly different among the three groups except at the radius in PhP patients, which was significantly lower than those of the other patients.

Conclusions

We found that ThP was associated with increased BMD in postmenopausal women. This may be due to attenuation of the high-turnover bone loss in postmenopausal women.     

Prognosis of Patients with Benign Thyroid Diseases Accompanied by Incidental Papillary Carcinoma Undetectable on Preoperative Imaging Tests

Background Despite the recent wide availability of ultrasonography and fine-needle aspiration biopsy, endocrine surgeons often encounter incidental papillary carcinoma (IPC), that is a papillary carcinoma that had gone undetected by preoperative imaging studies but was identified by pathological examination of surgical specimens resected for benign thyroid diseases. Methods The present study was developed to investigate the prognoses of 317 patients who underwent surgery for benign diseases involving IPC in comparison with the prognoses of 1,674 patients with clinically apparent papillary carcinoma detected preoperatively and diagnosed. Results None of the patients underwent further surgery such as completion total thyroidectomy and node dissection immediately after the diagnosis of IPC. To date, 7 patients (2.2%) have had recurrences; 6 of those were locoregional recurrences and one was a bone metastasis. None of the patients have died of thyroid carcinoma. The disease-free survival of IPC patients was significantly better than that of clinically apparent papillary carcinoma patients and was similar to that of the subset of patients with papillary microcarcinoma without clinically apparent metastasis. Conclusions Because IPC is associated with good prognosis, further surgery, such as completion total thyroidectomy or lymph node dissection immediately after the diagnosis of IPC is not necessary.     

A highly potent 26,27-Hexafluoro-1a,25-dihydroxyvitamin D3 on calcification in SV40-transformed human fetal osteoblastic cells

26,27-hexafluoro-1a,25-dihydroxyvitamin D3 (F6-D3) has been reported to be 5-10 times more potent than 1a,25-dihydroxyvitamin D3[1,25(OH)2D3] in biological systems in vivo and in vitro. However, the effect of F6-D3 on bone formation has yet to be clarified. In the present study, we investigated the effect of F6-D3 on SV40-transfected human fetal osteoblastic cells (SV-HFO) and found it to be about 100 times greater than that of 1,25(OH)2D3 in stimulating calcification. F6-D3 was also about 100 times more effective than 1,25(OH)2D3 in enhancing the expression of mRNA for alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In the presence of 10?8 M F6-D3 and 10?6 M 1,25(OH)2D3, the calcification began on day 9 and increased up to day 19. Expression of mRNA for ALP and OCN reached a maximum on day 4 and thereafter declined. On the other hand, when osteoblastic cells were incubated with a low level of [1b-3H]-F6-D3- or [1b-3H]-1,25(OH)2D3, each radioactive peak could not be detected. However, on the incubation of osteoblastic cells and radioactive substrate in the presence of ketoconazole, a selective inhibitor of CYP24, a clear peak for each substrate was detected. This suggested that F6-D3 as well as 1,25(OH)2D3 is metabolized by CYP24. Osteoblastic cells were incubated with 10?8 M[1b-3H]-F6-D3 or 10?8 M[1b-3H]-1,25(OH)2D3 for 4, 9, and 14 days. A small peak of 1,25(OH)2D3 was observed and thereafter its level decreased. In addition, two unknown peaks increased when the culture period was extended. In the case of F6-D3, peaks of F6-D3 and 26,27-hexafluoro-23-oxo-1a,25(OH)2D3(23-oxo-F6) were clearly detected, the latter being about 4 times higher than the former. Both peaks was retained up to day 14. The amount of unlabeled F6-D3 and 23-oxo-F6 calculated from the specific radioactivity in the cells may be similar to the amount of 1,25(OH)2D3 and its metabolites. The strong activity of F6-D3 in stimulating calcification may be due to the fact that F6-D3 is much more potent than 1,25(OH)2D3 in enhancing the expression of mRNA for ALP, OCN, and OPN and that the amount of F6-D3 and 23-oxo-F6 accumulated in the cells is much greater than that of 1,25(OH)2D3 and its metabolite.