Juo-Song Wang

Detection ofHelicobacter pylori in gastric biopsy tissue by polymerase chain reaction

To evaluate the sensitivity of a polymerase chain reaction (PCR) assay using nested primers in detectingHelicobacter pylori, gastric tissue biopsy specimens were collected on endoscopy from 17 patients with a duodenal ulcer. DNA was extracted by phenol/chloroform treatment or boiling in water, and then subjected to a nested PCR using two primer pairs from the urease gene ofHelicobacter pylori. Fourteen of the 17 patients were positive forHelicobacter pylori using DNA samples extracted by either method. The PCR results correlated well with the results of an enzyme immunoassay to detect IgG antibody. However, there were two culture negative patients. The three PCR negative patients were both culture negative and serologically negative. DNA from 9 of the 14 patients was randomly selected and subjected to semiquantification by serial dilutions, and then PCR. The results showed that phenol/chloroform extraction yielded 10–1000 times more DNA than the boiling method. It is concluded that the PCR assay is a rapid and sensitive method for detectingHelicobacter pylori, and that phenol/chloroform extraction is superior to simple boiling in obtaining DNA samples for PCR.

Chromosomal Abnormality in Hepatocellular Carcinoma by Comparative Genomic Hybridisation in Taiwan

The elucidation of the genetic changes of hepatocellular carcinoma (HCC) is very important for understanding the molecular mechanism of liver carcinogenesis. In order to identify the gains or losses in DNA sequence copy number in HCC, we used comparative genomic hybridisation to study 40 cases (44 tumours) of HCC. Tumour DNA and DNA from non-neoplastic liver tissue were labelled with different fluorochromes and then simultaneously hybridised to normal metaphase spread chromosomes. An image acquisition system was used to quantitate signal intensities contributed by tumour and reference DNA along the entire length of each chromosome. Regions of amplification and deletion were demonstrated as quantitative alterations. Losses were prevalent on chromosome regions 16q (43%), 17p (20%), 13q (20%), 4q (15%) and 8p (15%). Gains frequently occurred on 8q (30%), 1q (20%), 6p (20%) and 17q (18%). Hepatitis B virus carriers had a significantly higher frequency of losses on chromosome 16q. Furthermore, the minimal region of losses was narrowed down to 16q11-q22. This study confirms the presence of previously known chromosomal aberrations in HCC and highlights a new significant correlation between losses on chromosome 16q and hepatitis B virus carriers.

Microsatellite instability in gastric carcinoma with special references to histopathology and cancer stages

To study the molecular mechanism of gastric carcinogenesis, the frequencies of microsatellite instability were evaluated with seven dinucleotide repeat loci in 59 patients with gastric carcinoma. Microsatellite instability at two or more loci was found in 41.5% (17/41) of advanced gastric carcinoma, 21.4% (3/14) of early gastric carcinoma, but not in remnant gastric carcinoma (0/4), with an overall frequency of 33.9% (20/59). Diffuse gastric carcinoma had a similar prevalence (32.1%, 9/28) to intestinal gastric carcinoma (40.7%, 11/27). The frequency of microsatellite instability in gastric carcinoma was not significantly different with respect to age, sex and Helicobacter pylori infection. Microsatellite instability tended to occur more frequently in cancers of the cardia (62.5%, 5/8) compared with cancers of other stomach regions (31.9%, 15/47), but the difference was not statistically significant. These data suggest that microsatellite instability occurs in early gastric carcinoma and its occurrence increases during tumour progression. Furthermore, its frequency was independent of age, gender, histological types and Helicobacter pylori infection.

Differential Regulation of Interleukin-6 and Inducible Cyclooxygenase Gene Expression by Cytokines Through Prostaglandin-Dependent and -Independent Mechanisms in Human Dental Pulp Fibroblasts

Increased levels of interleukin-1 (IL)-1, tumor necrosis factor-alpha (TNF-alpha), IL-6, and prostaglandin E2 (PGE2) have been detected in inflamed pulp tissue. To gain further insight into the molecular pathogenesis of pulpitis, we investigated the effects of IL-1alpha or TNF-alpha and PGE2, either alone or in combination on IL-6 and cyclooxygenase (COX)-2 messenger RNA (mRNA) production in cultured human dental pulp (HDP) fibroblasts. Exposure of HDP fibroblasts to IL-1alpha or TNF-alpha resulted in elevated levels of IL-6 (approximately 3.4 to approximately 10.4-fold) and COX-2 (approximately 5 to approximately 6.2-fold) mRNA. Simultaneous addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 to the cultures significantly reduced the cytokine-induced IL-6 mRNA synthesis ranging from 45% to 65%. However, indomethacin enhanced the cytokine-stimulated IL-6 mRNA synthesis by approximately 1.7 to approximately 3.4-fold. This action could be reversed by exogenous PGE2. In contrast, PGE2 or indomethacin failed to modify the stimulatory effect of IL-1alpha or TNF-alpha on COX-2 gene expression. Because excessive levels of IL-6 and prostaglandins have been connected with the pathogenesis of several inflammatory diseases, our results suggest the involvement of HDP fibroblasts in the development of pulpitis via producing IL-6 and COX-2. Furthermore, expression of IL-6 and COX-2 genes in this cell seems to be differentially regulated by cytokines through prostaglandin-dependent and -independent pathways.

Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-1 Gene Expressions and Their Differential Regulation by Proinflammatory Cytokines and Prostaglandin in Nasal Polyp Fibroblasts

Chronic inflammation of the paranasal sinus leads to nasal polyp (NP) formation. In this study, we investigated the effect of stimulation of the proinflammatory cytokines interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α) and prostaglandin (PG) E2 on the production of messenger RNA (mRNA) of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in nasal polyp fibroblasts (NPFs) and nasal mucosa fibroblasts (NFs). The mRNAs of IL-1α, TNF-α, MMP-1, and TIMP-1 in 40 surgical specimens of NPs were studied by in situ hybridization to corroborate the in vitro findings. The results indicated a significant amount of constitutive MMP-1 mRNA in NPFs and cytokine-induced MMP-1 steady-state mRNAs in NFs. The effect of stimulation of cytokines on TIMP-1 mRNA synthesis was unremarkable in NPFs and NFs. Exogenous PGE2 enhanced cytokine-stimulated MMP-1 mRNA synthesis in NPFs. In situ hybridization revealed that cells expressing MMP-1 and TIMP-1 mRNAs (primarily plasma cells, fibroblasts, and endothelial cells) gathered around areas with loose stroma, suggestive of rapid extracellular matrix degradation. These data suggest that the pathogenesis of nasal polyposis could be related to production of MMP-1 and consequent promotion of matrix collagenolysis.

Simvastatin inhibits cytokine-stimulated Cyr61 expression in osteoblastic cells: A therapeutic benefit for arthritis

OBJECTIVE To examine the effects of proinflammatory cytokines on Cyr61 expression in osteoblastic cells and the modulatory action of simvastatin, to assess the role of CREB in Cyr61 induction, and to investigate the relationship of osteoblastic expression of Cyr61 to disease progression in experimental arthritis. METHODS Cyr61 expression and CREB phosphorylation at serine 133 were examined by Western blotting. Promoter activity of Cyr61 was assessed by luciferase assay with promoter deletion/mutagenesis and forced expression/gene silencing of CREB. Interaction between CREB and the Cyr61 promoter was evaluated by electrophoretic mobility shift assay and chromatin immunoprecipitation. CCL2 expression was examined by Northern blotting and enzyme-linked immunosorbent assay. In rats with collagen-induced arthritis (CIA), osteoblastic expression of Cyr61 was examined by immunohistochemistry, and disease progression was assessed by clinical, radiographic, and histologic examination. RESULTS In primary human osteoblasts and U2OS cells, Cyr61 expression stimulated by tumor necrosis factor α, interleukin-1β (IL-1β), oncostatin M (OSM), and other IL-6-family cytokines was suppressed by simvastatin. In U2OS cells, simvastatin inhibited OSM-induced CREB phosphorylation and CREB-DNA binding. Knockdown of CREB by short hairpin RNA reduced Cyr61 synthesis. OSM-induced Cyr61 promoter activation was dependent on CRE-CREB interaction and inhibited by simvastatin. Cyr61 enhanced CCL2 expression by U2OS cells. Intraarticular injection of simvastatin inhibited CIA progression and diminished the number of Cyr61+ osteoblasts and infiltrating macrophages. CONCLUSION Simvastatin inhibited cytokine-stimulated Cyr61 expression in osteoblastic cells and suppressed disease progression and osteoblastic expression of Cyr61 in inflammatory arthritis. This finding indicates that simvastatin may have potential as a therapeutic agent for inflammatory arthritis.

Cloning, expression and characterization of CCL21 and CCL25 chemokines in zebrafish.

Chemokines are a large group of proteins implicated in migration, activation, and differentiation of leukocytes. They are well-surveyed in mammals, but less is known in lower vertebrates about their spatiotemporal expressions and functions. From an evolutionary point of view, comparative analyses may provide some fundamental insights into these molecules. In mammals, CCL21 and CCL25 are crucial for thymocyte homing. Herein, we identified and cloned the zebrafish orthologues of CCL21 and CCL25, and analyzed their expression in embryos and adult fish by in situ hybridization. We found that CCL21 was expressed in the craniofacial region, pharynx, and blood vessels in embryos. In adult fish, CCL21 transcripts were located in the kidney, spinal cord, and blood cells. In contrast, expression of CCL25 was only detected in the thymus primordia in embryos. In adult fish, transcripts of CCL25 were maintained in the thymus, and they were also found in the brain and oocytes. Furthermore, we performed an antisense oligonucleotide experiment to evaluate the biological function of CCL25. Results showed that the recruitment of thymocytes was impeded by morpholino-mediated knockdown of CCL25, suggesting that CCL25 is essential for colonization of T-cells in the thymus in early development. Together, our results demonstrate the basic profiles of two CCL chemokines in zebrafish. The tissue-specific expression patterns may pave the way for further genetic dissection in this model organism.

The effect of implant design and bone quality on insertion torque, resonance frequency analysis, and insertion energy during implant placement in low or low- to medium-density bone.

PURPOSE This study investigated the effect of implant design and bone quality on insertion torque (IT), implant stability quotient (ISQ), and insertion energy (IE) by monitoring the continuous change in IT and ISQ while implants were inserted in artificial bone blocks that simulate bone of poor or poor-to-medium quality. MATERIALS AND METHODS Polyurethane foam blocks (Sawbones) of 0.16 g/cm³ and 0.32 g/cm³ were respectively used to simulate low density and low- to medium-density cancellous bone. In addition, some test blocks were laminated with a 1-mm 0.80 g/cm³ polyurethane layer to simulate cancellous bone with a thin cortical layer. Four different implants (Nobel Biocare Mk III-3.75, Mk III-4.0, Mk IV-4.0, and NobelActive-4.3) were placed into the different test blocks in accordance with the manufacturers instructions. The IT and ISQ were recorded at every 0.5-mm of inserted length during implant insertion, and IE was calculated from the torque curve. The peak IT (PIT), final IT (FIT), IE, and final ISQ values were statistically analyzed. RESULTS All implants showed increasing ISQ values when the implant was inserted more deeply. In contrast to the ISQ, implants with different designs showed dissimilar IT curve patterns during the insertion. All implants showed a significant increase in the PIT, FIT, IE, and ISQ when the test-block density increased or when the 1-mm laminated layer was present. Tapered implants showed FIT or PIT values of more than 40 Ncm for all of the laminated test blocks and for the nonlaminated test blocks of low to medium density. Parallel-wall implants did not exhibit PIT or FIT values of more than 40 Ncm for all of the test blocks. NobelActive-4.3 showed a significantly higher FIT, but a significantly lower IE, than Mk IV-4.0. CONCLUSIONS While the existence of cortical bone or implant designs significantly affects the dynamic IT profiles during implant insertion, it does not affect the ISQ to a similar extent. Certain implant designs are more suitable than others if high IT is required in bone of poor quality. The manner in which IT, IE, and ISQ represent the implant primary stability requires further study.

Simvastatin suppresses osteoblastic expression of Cyr61 and progression of apical periodontitis through enhancement of the transcription factor Forkhead/winged helix box protein O3a.

INTRODUCTION In this study, the role of transcription factor Forkhead/winged helix box protein O3a (FoxO3a) in Cyr61 expression and its modulation by simvastatin were investigated in cultured murine osteoblasts and a rat model of induced apical periodontitis. We also examined the effects of simvastatin on the synthesis of chemokine CCL2 and chemotaxis of macrophages in vitro. METHODS We assessed tumor necrosis factor (TNF)-α-stimulated expression of Cyr61 and phosphorylated inactive FoxO3a (p-FoxO3a) in MC3T3-E1 murine osteoblasts by Western analysis. Forced expression of FoxO3a by lentiviral-based gene transduction was performed, and its effect on Cyr61 expression was evaluated. The modulation of CCL2 secretion and macrophage chemotaxis by simvastatin were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced apical periodontitis, the relation between disease progression and osteoblastic expression of Cyr61, p-FoxO3a, and CCL2 and macrophage recruitment were studied by radiographic and immunohistochemistry analyses. RESULTS Western blot analysis showed enhanced expression of Cyr61 and p-FoxO3a after TNF-α treatment in a time-dependent manner. Simvastatin significantly counteracted the actions of TNF-α. Forced expression of FoxO3a reduced TNF-α-stimulated Cyr61 synthesis. Simvastatin and FoxO3a diminished TNF-α-induced CCL2 secretion and macrophage recruitment, whereas Cyr61 partially restored the stimulating action. In rat periapical lesions, simvastatin significantly attenuated bone resorption, reduced osteoblastic expressions of Cyr61, p-FoxO3a, and CCL2, and suppressed macrophage recruitment. CONCLUSIONS Simvastatin may alleviate periapical lesions by enhancing FoxO3a activity to suppress the synthesis of Cyr61 in osteoblasts. Moreover, the downstream effector mechanism of Cyr61 may involve CCL2 production and macrophage recruitment.

Epigallocatechin-3-gallate diminishes cytokine-stimulated Cyr61 expression in human osteoblastic cells: a therapeutic potential for arthritis

OBJECTIVE To assess the effects of epigallocatechin-3-gallate (EGCG) on cytokine-induced Cyr61 synthesis in human osteoblastic cells and the associated signalling pathways. The therapeutic effect of EGCG on CIA in rats was also studied. METHODS The expression of Cyr61 and NF-κB pathway molecules was examined by western blotting. CCL2 expression was assessed by northern blotting and ELISA. Interaction between NF-κB and Cyr61 promoter was evaluated by electrophoretic mobility shift assay. In rat CIA, osteoblastic expression of Cyr61 was examined by immunohistochemistry and disease progression was assessed by clinical, radiographic and histological examinations. RESULTS EGCG inhibited Cyr61 expression stimulated by cytokines in primary human osteoblasts and human osteoblastic cell line U2OS. In U2OS, oncostatin M (OSM) induced IκB-α degradation through the mTOR/rictor/Akt pathway, and EGCG attenuated the action. Electrophoretic mobility shift assay revealed that the OSM-enhanced NF-κB/DNA binding was reduced by EGCG, possibly through abrogating nucleus localization of p65 and p50. Cyr61 enhanced OSM-induced expression of CCL2. Moreover, EGCG diminished OSM-stimulated CCL2 expression at least partially via suppressing Cyr61 induction. Co-distribution of CD68(+) macrophages and Cyr61(+) osteoblasts in osteolytic areas was obvious in the CIA model. Clinical, radiographic and immunohistochemical analyses revealed that administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the number of Cyr61-synthesizing osteoblasts. CONCLUSION By modulating the mTOR/rictor/Akt/NF-κB pathway, EGCG attenuated Cyr61 production in osteoblastic cells and in turn diminished macrophage chemotaxis. Our data support the therapeutic potential of EGCG on arthritis.