Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

Prediction of clinically significant prostate cancer through urine metabolomic signatures: A large-scale validated study.

PURPOSE: Currently, there are no accurate markers for predicting potentially lethal prostate cancer (PC) before biopsy. This study aimed to develop urine tests to predict clinically significant PC (sPC) in men at risk. METHODS: Urine samples from 928 men, namely, 660 PC patients and 268 benign subjects, were analyzed by gas chromatography/quadrupole time-of-flight mass spectrophotometry (GC/Q-TOF MS) metabolomic profiling to construct four predictive models. Model I discriminated between PC and benign cases. Models II, III, and GS, respectively, predicted sPC in those classified as having favorable intermediate risk or higher, unfavorable intermediate risk or higher (according to the National Comprehensive Cancer Network risk groupings), and a Gleason sum (GS) of ≥ 7. Multivariable logistic regression was used to evaluate the area under the receiver operating characteristic curves (AUC). RESULTS: In Models I, II, III, and GS, the best AUCs (0.94, 0.85, 0.82, and 0.80, respectively; training cohort, N = 603) involved 26, 24, 26, and 22 metabolites, respectively. The addition of five clinical risk factors (serum prostate-specific antigen, patient age, previous negative biopsy, digital rectal examination, and family history) significantly improved the AUCs of the models (0.95, 0.92, 0.92, and 0.87, respectively). At 90% sensitivity, 48%, 47%, 50%, and 36% of unnecessary biopsies could be avoided. These models were successfully validated against an independent validation cohort (N = 325). Decision curve analysis showed a significant clinical net benefit with each combined model at low threshold probabilities. Models II and III were more robust and clinically relevant than Model GS. CONCLUSION: This urine test, which combines urine metabolic markers and clinical factors, may be used to predict sPC and thereby inform the necessity of biopsy in men with an elevated PC risk.

Second-line levofloxacin-based quadruple therapy versus bismuth-based quadruple therapy for Helicobacter pylori eradication and long-term changes to the gut microbiota and antibiotic resistome: a multicentre, open-label, randomised controlled trial.

BACKGROUND: Levofloxacin-based therapy or bismuth-based quadruple therapy are the recommended second-line regimens for Helicobacter pylori eradication after failure of clarithromycin-based therapy. However, resistance to levofloxacin has increased in the past decade. Furthermore, little is known about the long-term effects of H pylori eradication on the antibiotic resistome. In this study, we compared these second-line eradication therapies for efficacy, tolerability, and short-term and long-term effects on the gut microbiota, antibiotic resistome, and metabolic parameters. METHODS: We did a multicentre, open-label, parallel group, randomised controlled trial at eight hospitals in Taiwan. Adult patients (age ≥20 years) with persistent H pylori infection after first-line clarithromycin-based therapy were randomly assigned (1:1, permuted block sizes of four) to receive levofloxacin-based sequential quadruple therapy for 14 days (EAML14; esomeprazole 40 mg and amoxicillin 1 g for 7 days, followed by esomeprazole 40 mg, metronidazole 500 mg, and levofloxacin 250 mg for 7 days, all twice-daily) or bismuth-based quadruple therapy for 10 days (BQ10; esomeprazole 40 mg twice daily, bismuth tripotassium dicitrate 300 mg four times a day, tetracycline 500 mg four times a day, and metronidazole 500 mg three times a day). All investigators were masked to the randomisation sequence. The primary endpoint was H pylori eradication rate measured by 13C urea breath test 6 weeks after second-line treatment according to both intention-to-treat (ITT) and per-protocol analysis. The microbiota composition and antibiotic resistome of faecal samples collected at baseline (before treatment) and at 2 weeks, 8 weeks, and 1 year after eradication therapy was profiled by shotgun metagenomic sequencing and 16S rRNA gene sequencing. The frequency of adverse effects and changes in the gut microbiota and antibiotic resistome were assessed in all participants with available data. The trial is complete and registered with ClinicalTrails.gov, NCT03148366. FINDINGS: Between Feb 25, 2015, and Dec 11, 2020, 560 patients were randomly assigned to receive EAML14 or BQ10 (n=280 per group; 261 [47%] men and 299 [53%] women). Mean age was 55·9 years (SD 12·7) in the EAML14 group and 54·9 years (12·3) in the BQ10 group. Eradication of H pylori was achieved in 246 (88%) of 280 participants in the EAML14 group and 245 (88%) of 280 in the BQ10 group according to ITT analysis (risk difference -0·4%, 95% CI -5·8 to 5·1; p=0·90). In the per-protocol analysis, 246 (90%) of 273 participants in the EAML14 group and 245 (93%) of 264 participants in the BQ10 group achieved H pylori eradication (risk difference 2·7%, 95% CI -0·2 to 7·4; p=0·27). Transient perturbation of faecal microbiota diversity at week 2 was largely restored to basal state 1 year after EAML14 or BQ10. Diversity recovery was slower with BQ10, and recovery in species abundance was partial after both therapies. On shotgun sequencing, we observed significant increases in total resistome after EAML14 (p=0·0002) and BQ10 (p=4·3 × 10-10) at week 2, which were restored to pretreatment level by week 8. The resistance rates of Escherichia coli and Klebsiella pneumonia to levofloxacin, ciprofloxacin, ampicillin (ampicillin-sulbactam for K pneumonia), and various cephalosporins were significantly increased in the EAML14 group compared with in the BQ10 group at week 2, which were restored to pretreatment levels and showed no significant differences at week 8 and 1 year. The frequency of any adverse effects was significantly higher after BQ10 therapy (211 [77%] of 273 participants) than after EAML14 therapy (134 [48%] of 277; p<0·0001). INTERPRETATION: We found no evidence of superiority between levofloxacin-based quadruple therapy and bismuth-based quadruple therapy in the second-line treatment of H pylori infection. The transient increase in the antibiotic resistome and perturbation of faecal microbiota diversity were largely restored to pretreatment state from 2 months to 1 year after eradication therapy. FUNDING: The Ministry of Science and Technology of Taiwan, the Ministry of Health and Welfare of Taiwan, National Taiwan University Hospital, Taipei Veteran General Hospital, and the Australian Federal Government through the St George and Sutherland Medical Research Foundation. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Bismuth salts with versus without acid suppression for Helicobacter pylori infection: A transmission electron microscope study.

BACKGROUND: Bismuth oxychloride produced by interaction of bismuth compounds with gastric acid is believed to damage Helicobacter pylori. The effect of bismuth salts on H. pylori in the presence of strong acid suppression is unknown. This randomized trial aimed to determine effects of bismuth subcitrate on H. pylori with and without acid suppression. METHODS: H. pylori -positive participants were allocated (1:1:1) to receive (a) no treatment (control), (b) colloidal bismuth subcitrate (CBS, 125 mg/tab), or (c) CBS plus high-dose proton-pump inhibitor (PPI), esomeprazole 40 mg q.i.d. for 3 days. In the treatment groups, CBS was given: 1 dose, 1 hour before endoscopy, 1 dose, 4 hours before endoscopy, or q.i.d. 24 hours before endoscopy. The study end-points were evaluated using transmission electron microscopy to observe the morphological changes of H. pylori in antral and corpus biopsies. RESULTS: Twenty-seven H. pylori carriers were enrolled in this trial with qualitative end-points. In the no treatment group, active budding and replication of H. pylori were observed. In the CBS group, cellular swelling, vacuolization, structural degradation, and cell wall eruption of H. pylori were observed, with no apparent association with when the CBS was given. Among those receiving high-dose PPI-plus CBS or CBS only, there were no differences in number of H. pylori present or severity of bacterial damage whether CBS was given 1, 4, or 24 hours before endoscopy. CONCLUSIONS: Based on direct morphological evaluation, the toxic effect of CBS treatment on H. pylori was demonstrated independent of acid suppression with PPI.

14 day sequential therapy versus 10 day bismuth quadruple therapy containing high-dose esomeprazole in the first-line and second-line treatment of Helicobacter pylori: a multicentre, non-inferiority, randomized trial.

Background: Whether extending the treatment length and the use of high-dose esomeprazole may optimize the efficacy of Helicobacter pylori eradication remains unknown. Objectives: To compare the efficacy and tolerability of optimized 14 day sequential therapy and 10 day bismuth quadruple therapy containing high-dose esomeprazole in first-line therapy. Methods: We recruited 620 adult patients (≥20 years of age) with H. pylori infection naive to treatment in this multicentre, open-label, randomized trial. Patients were randomly assigned to receive 14 day sequential therapy or 10 day bismuth quadruple therapy, both containing esomeprazole 40 mg twice daily. Those who failed after 14 day sequential therapy received rescue therapy with 10 day bismuth quadruple therapy and vice versa. Our primary outcome was the eradication rate in the first-line therapy. Antibiotic susceptibility was determined. ClinicalTrials.gov: NCT03156855. Results: The eradication rates of 14 day sequential therapy and 10 day bismuth quadruple therapy were 91.3% (283 of 310, 95% CI 87.4%-94.1%) and 91.6% (284 of 310, 95% CI 87.8%-94.3%) in the ITT analysis, respectively (difference -0.3%, 95% CI -4.7% to 4.4%, P = 0.886). However, the frequencies of adverse effects were significantly higher in patients treated with 10 day bismuth quadruple therapy than those treated with 14 day sequential therapy (74.4% versus 36.7% P < 0.0001). The eradication rate of 14 day sequential therapy in strains with and without 23S ribosomal RNA mutation was 80% (24 of 30) and 99% (193 of 195), respectively (P < 0.0001). Conclusions: Optimized 14 day sequential therapy was non-inferior to, but better tolerated than 10 day bismuth quadruple therapy and both may be used in first-line treatment in populations with low to intermediate clarithromycin resistance.

AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis.

Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the ΔatlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans, which contributes to biofilm formation in infective endocarditis.

Dual Targeting of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase and Histone Deacetylase as a Therapy for Colorectal Cancer.

Statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) inhibitors decreasing serum cholesterol and have shown promise in cancer prevention. In this study, we demonstrated the oncogenic role of HMGR in colorectal cancer (CRC) by disclosing increased HMGR activity in CRC patients and its enhancement of anti-apoptosis and stemness. Our previous studies showed that statins containing carboxylic acid chains possessed activity against histone deacetylases (HDACs), and strengthened their anti-HDAC activity through designing HMGR-HDAC dual inhibitors, JMF compounds. These compounds exerted anti-cancer effect in CRC cells as well as in AOM-DSS and Apc(Min/+) CRC mouse models. JMF mostly regulated the genes related to apoptosis and inflammation through genome-wide ChIP-on-chip analysis, and Ingenuity Pathways Analysis (IPA) predicted their respective regulation by NR3C1 and NF-κB. Furthermore, JMF inhibited metastasis, angiogenesis and cancer stemness, and potentiated the effect of oxaliplatin in CRC mouse models. Dual HMGR-HDAC inhibitor could be a potential treatment for CRC.

Catechins and Sialic Acid Attenuate Helicobacter pylori-Triggered Epithelial Caspase-1 Activity and Eradicate Helicobacter pylori Infection.

The inflammasome/caspase-1 signaling pathway in immune cells plays a critical role in bacterial pathogenesis; however, the regulation of this pathway in the gastric epithelium during Helicobacter pylori infection is yet to be elucidated. Here, we investigated the effect of catechins (CAs), sialic acid (SA), or combination of CA and SA (CASA) on H. pylori-induced caspase-1-mediated epithelial damage, as well as H. pylori colonization in vitro (AGS cells) and in vivo (BALB/c mice). Our results indicate that the activity of caspase-1 and the expression of its downstream substrate IL-1 ß were upregulated in H. pylori-infected AGS cells. In addition, we observed increased oxidative stress, NADPH oxidase gp91phox, CD68, caspase-1/IL-1 ß , and apoptosis, but decreased autophagy, in the gastric mucosa of H. pylori-infected mice. We have further demonstrated that treatment with CASA led to synergistic anti-H. pylori activity and was more effective than treatment with CA or SA alone. In particular, treatment with CASA for 10 days eradicated H. pylori infection in up to 95% of H. pylori-infected mice. Taken together, we suggest that the pathogenesis of H. pylori involves a gastric epithelial inflammasome/caspase-1 signaling pathway, and our results show that CASA was able to attenuate this pathway and effectively eradicate H. pylori infection.

Aristolochic acid-induced upper tract urothelial carcinoma in Taiwan: clinical characteristics and outcomes.

Aristolochic acid (AA), a component of all Aristolochia-based herbal medicines, is a potent nephrotoxin and human carcinogen associated with upper urinary tract urothelial carcinoma (UUC). To investigate the clinical and pathological characteristics of AA-induced UUC, this study included 152 UUC patients, 93 of whom had been exposed to AA based on the presence of aristolactam-DNA adducts in the renal cortex. Gene sequencing was used to identify tumors with A:T-to-T:A transversions in TP53, a mutational signature associated with AA. Cases with both aristolactam-DNA adducts and A:T-to-T:A transversions in TP53 were defined as AA-UUC, whereas patients lacking both of these biomarkers were classified as non-AA-UUC. Cases with either biomarker were classified as possible-AA-UUC. Forty (26%), 60 (40%), and 52 (34%) patients were classified as AA-UUC, possible-AA-UUC and non-AA-UUC, respectively. AA-UUC patients were younger (median ages: 64, 68, 68 years, respectively; p=0.189), predominately female (65%, 42%, 35%, respectively; p=0.011), had more end-stage renal disease (28%, 10%, 12%, respectively; p=0.055), and were infrequent smokers (5%, 22%, 33%, respectively; p=0.07) compared to possible-AA-UUC and non-AA-UUC patients. All 14 patients who developed contralateral UUC had aristolactam-DNA adducts; ten of these also had signature mutations. The contralateral UUC-free survival period was shorter in AA-UUC compared to possible- or non-AA-UUC (p=0.019 and 0.002, respectively), whereas no differences among groups were observed for bladder cancer recurrence. In conclusion, AA-UUC patients tend to be younger and female, and have more advanced renal disease. Notably, AA exposure was associated with an increased risk for developing synchronous bilateral and metachronous contralateral UUC.

Efficacy of genotypic resistance-guided sequential therapy in the third-line treatment of refractory Helicobacter pylori infection: a multicentre clinical trial.

OBJECTIVES: The efficacy of sequential therapy and the applicability of genotypic resistance to guide the selection of antibiotics in the third-line treatment of Helicobacter pylori have not been reported. We aimed to assess the efficacy of genotypic resistance-guided sequential therapy in third-line treatment. METHODS: Genotypic and phenotypic resistances were determined in patients who failed at least two eradication therapies by PCR with direct sequencing and agar dilution test, respectively. The patients were retreated with sequential therapy containing esomeprazole and amoxicillin for the first 7 days, followed by esomeprazole and metronidazole plus clarithromycin, levofloxacin or tetracycline for another 7 days (all twice daily), according to genotypic resistance determined using gastric biopsy specimens. Eradication status was determined by the (13)C-urea breath test. Trial registered at clinicaltrials.gov (identifier: NCT01032655). RESULTS: The overall eradication rate was 80.7% (109/135, 95% CI 73.3%-86.5%) in the intention-to-treat analysis. The presence of amoxicillin resistance (OR 6.83, 95% CI 1.62-28.86, P = 0.009) and prior sequential therapy (OR 4.77, 95% CI 1.315-17.3, P = 0.017), but not tetracycline resistance (tetracycline group), were associated with treatment failure. The eradication rates in patients who received clarithromycin-, levofloxacin- and tetracycline-based sequential therapies were 78.9% (15/19), 92.2% (47/51) and 71.4% (25/35) in strains susceptible to clarithromycin, levofloxacin and tetracycline, respectively. CONCLUSIONS: A simple molecular method guiding sequential therapy can achieve a high eradication rate in the third-line treatment of refractory H. pylori infection.

Hypocalcemia and tetany caused by vitamin D deficiency in a child with intestinal lymphangiectasia.

Primary intestinal lymphangiectasia is a rare disease of children, which is characterized by chronic diarrhea and complicated with malnutrition, including fat-soluble vitamin deficiency. We report a girl aged 4 years and 8 months who was diagnosed with the disease by endoscopic duodenal biopsy at 8 months of age. She presented initially with chronic diarrhea at 4 months of age. Generalized edema with hypoalbuminemia frequently occurred despite regular albumin supplements. Multiple vitamins initially were not supplied regularly. Episodes of tetany caused by hypocalcemia developed 4 years after the diagnosis of intestinal lymphangiectasia. Imaging study (long-bone X-ray and dual-energy X-ray absorptiometry) revealed low bone density. Complicated vitamin D deficiency [low serum 25-hydroxy vitamin D concentration (< 12.48 mmol/L, the detection limit)] and secondary hyperparathyroidism were confirmed via blood testing. Vitamin D supplementation for 3 months improved her bone density, secondary hyperparathyroidism and frequent tetany. Vitamin D status should be monitored in patients with intestinal lymphangiectasia.

Hypoxia-stimulated vascular endothelial growth factor production in human nasal polyp fibroblasts: effect of epigallocatechin-3-gallate on hypoxia-inducible factor-1 alpha synthesis.

OBJECTIVE: To verify the inhibitory effects of epigallocatechin-3-gallate (EGCG) on the synthesis of hypoxia-induced vascular endothelial growth factor (VEGF) in nasal polyp fibroblasts (NPFs). DESIGN: Eight primary cultures of NPFs were established from nasal polyps. Effects of EGCG on the production of hypoxia-inducible factor (HIF)-1 alpha (the most potent VEGF stimulant) and VEGF by NPFs under hypoxic conditions were measured by Western blot analysis. Immunohistochemical staining was used to examine the in vivo expressions of HIF-1 alpha and VEGF in 20 sections of nasal polyps. RESULTS: Western blot analysis showed that cobalt chloride induced HIF-1 alpha and VEGF synthesis in NPFs in a time-dependent manner, reaching a plateau at 4 and 8 hours, respectively, following treatment. Epigallocatechin-3-gallate attenuated the level of HIF-1 alpha induced by cobalt chloride and also reduced cobalt chloride-stimulated VEGF production by suppressing HIF-1 alpha synthesis. Furthermore, oligomycin (a specific HIF-1 alpha inhibitor) combined with EGCG resulted in a more profound inhibition of VEGF synthesis compared with oligomycin or EGCG treatment alone. Nevertheless, the synergistic effect seemed smaller than the sum of their individual actions. Immunohistochemical analysis revealed the presence of HIF-1 alpha and VEGF in NPFs and mononuclear round cells. Intimate alignment of VEGF-positive fibroblasts and proliferating small capillaries was frequently found. CONCLUSIONS: Nasal polyp fibroblasts contribute to the pathogenesis of nasal polyps by producing VEGF to promote angiogenesis under hypoxic conditions. Epigallocatechin-3-gallate substantially diminishes HIF-1 alpha and VEGF synthesis in NPFs.

Modification effects of GSTM1, GSTT1 and CYP2E1 polymorphisms on associations between raw salted food and incomplete intestinal metaplasia in a high-risk area of stomach cancer.

Incomplete intestinal metaplasia (IM) is a precursor of stomach cancer. To identify risk factors of incomplete IM, a 2-stage survey was carried out in 1995 among 1,485 residents in Matzu, an area with highest mortality from stomach cancer in Taiwan. There were 312 study subjects including 174 men and 138 women sampled for the gastroendoscopic examination of IM. Information on personal and familial history of stomach cancer, cigarette smoking, alcohol consumption and intake frequency of various salted food items were obtained by personal interview based on a structured questionnaire. Blood samples were collected from each participant. Four biopsies per subject were taken from all subjects at gastroendoscopic examination to diagnose the status of IM pathologically. The Helicobacter pylori in biopsies was detected by the histomorphological or immunochemistry method, and antibodies against H. pylori in serum by the enzyme-linked immunosorbent assay. Plasma level of selenium was determined by atomic absorption spectrometry, plasma level of retinol, alpha-tocopherol, alpha-carotene, and beta-carotene by high performance liquid chromatography, genotypes of glutathione S-transferase (GST) M1 and T1 and cytochrome P450 (CYP) 2E1 by polymerase chain reaction. The significant association between history of stomach cancer among first-degree relatives and incomplete IM was found (odds ratio [OR] = 2.50; 95% confidence interval [CI] = 1.15-5.43). There was no association between H. pylori infection and incomplete IM. Alcohol drinkers for >20 years had an elevated risk compared to non-drinkers (OR = 3.34; 95% CI = 1.19-9.39). No associations between incomplete IM and plasma levels of selenium, retinol, alpha-tocopherol, alpha-carotene and beta-carotene were found. Salted food including salted meat, dehydrated salted vegetables and raw salted seafood consumed at ages of