An integrated analysis of fecal microbiome and metabolomic features distinguish non-cirrhotic NASH from healthy control populations.

BACKGROUND AND AIMS: There is great interest in identifying microbiome features as reliable noninvasive diagnostic and/or prognostic biomarkers for non-cirrhotic NASH fibrosis. Several cross-sectional studies have reported gut microbiome features associated with advanced NASH fibrosis and cirrhosis, where the most prominent features are associated with cirrhosis. However, no large, prospectively collected data exist establishing microbiome features that discern non-cirrhotic NASH fibrosis, integrate the fecal metabolome as disease biomarkers, and are unconfounded by BMI and age. APPROACH AND RESULTS: Results from shotgun metagenomic sequencing performed on fecal samples prospectively collected from 279 US patients with biopsy-proven NASH (F1-F3 fibrosis) enrolled in the REGENERATE I303 study were compared to those from 3 healthy control cohorts and integrated with the absolute quantification of fecal bile acids. Microbiota beta-diversity was different, and BMI- and age-adjusted logistic regression identified 12 NASH-associated species. Random forest prediction models resulted in an AUC of 0.75-0.81 in a receiver operator characteristic analysis. In addition, specific fecal bile acids were significantly lower in NASH and correlated with plasma C4 levels. Microbial gene abundance analysis revealed 127 genes increased in controls, many involving protein synthesis, whereas 362 genes were increased in NASH many involving bacterial environmental responses (false discovery rate < 0.01). Finally, we provide evidence that fecal bile acid levels may be a better discriminator of non-cirrhotic NASH versus health than either plasma bile acids or gut microbiome features. CONCLUSIONS: These results may have value as a set of baseline characteristics of non-cirrhotic NASH against which therapeutic interventions to prevent cirrhosis can be compared and microbiome-based diagnostic biomarkers identified.

Distinct community structures of the fungal microbiome and respiratory health in adults with cystic fibrosis.

BACKGROUND: The respiratory tract fungal microbiome in cystic fibrosis (CF) has been understudied despite increasing recognition of fungal pathogens in CF lung disease. We sought to better understand the fungal communities in adults with CF, and to define relationships between fungal profiles and clinical characteristics. METHODS: We enrolled 66 adults with CF and collected expectorated sputum, spirometry, Cystic Fibrosis Questionnaire-revised, and clinical data. Fungi were molecularly profiled by sequencing of the internal transcribed spacer (ITS) region. Total fungal abundance was measured by quantitative PCR. Relative abundance and qPCR-corrected abundances were determined. Selective fungus culture identified cultivable fungi. Alpha diversity and beta diversity were measured and relationships with clinical parameters were interrogated. RESULTS: Median age was 29 years and median FEV1 percent predicted 58%. Members of the Candida genus were the most frequent dominant taxa in CF sputum. Apiotrichum, Trichosporon, Saccharomyces cerevisiae, and Scedosporium were present in high relative abundance in few samples; whereas, Aspergillus species were detected at low levels. Higher FEV1% predicted and CFTR modulator use were associated with greater alpha-diversity. Chronic azithromycin use was associated with lower alpha-diversity. Patients with acute pulmonary had distinct fungal community composition compared to clinically stable subjects. Differing yeast species were mainly responsible for the community differences. CONCLUSION: The respiratory tract fungal microbiome in adults with CF is associated with lung function, pulmonary exacerbation status, macrolide use, and CFTR modulator use. Future work to better understand fungal diversity in the CF airway and its impact on lung health is necessary.

Effects of conventional and heated tobacco product smoking on discoloration of artificial denture teeth.

STATEMENT OF PROBLEM: Cigarette smoke can cause discoloration of artificial denture teeth. However, studies on the effects of heated tobacco product smoke on artificial denture teeth are lacking. PURPOSE: The purpose of this in vitro study was to evaluate the effects of conventional cigarette and heated tobacco product smoke on the color stability of artificial denture teeth. MATERIAL AND METHODS: Ninety maxillary central incisor denture teeth (Endura Anterior HC5 A3; Shofu) were randomly divided into 3 groups (n=30). Teeth in the control group were exposed to air; those in group CC were exposed to conventional cigarette (Marlboro Medium; Philip Morris) smoke, and those in group HT were exposed to heated tobacco product (IQOS 2.4 plus holder, Marlboro Heets Silver; Philip Morris) smoke. Before the experiment, the shade of the artificial denture teeth was evaluated in accordance with the Commission International de I'Eclairage (CIELab) color system by using a spectrophotometer (Shadepilot; DeguDent GmbH). The average CIELab value was estimated by scanning the entire labial surface of each specimen. To simulate smoking, standard conditions described by the Coresta Recommended Method N°22 were used-the puff duration was 2 seconds, with a 60-second interval between puffs. For each cigarette, 6 puffs and 6 intervals were simulated across 372 seconds. A total of 105 cigarettes were used based on a smoking simulation of 15 cigarettes each day for 7 days. The teeth in the control group were stored in fresh air in the smoke chamber for the same period as those in the experimental groups. After the experiment, L∗, a∗, and b∗ values were measured, and ΔE was calculated to evaluate the color change. All statistical analyses were performed with a statistical software program using a paired t test to determine discoloration after exposure to cigarette smoke. One-way ANOVA and the Tukey test were used to evaluate the significant differences between groups (α=.05). RESULTS: Lightness was significantly lower in the CC and HT groups (P<.001). All CIELab values showed statistically significant differences in the CC group. The greatest color change was observed in the CC group (ΔE=6.93 ±0.59), whereas the HT group showed a clinically imperceptible color change (ΔE=0.79 ±0.21). Discoloration was minimal in the CC group (ΔE=0.34 ±0.13). CONCLUSIONS: Conventional cigarette and heated tobacco product smoke can change the color of denture teeth. Heated tobacco product smoke causes less discoloration of denture teeth.

Early stool microbiome and metabolome signatures in pediatric patients undergoing allogeneic hematopoietic cell transplantation.

BACKGROUND: The contribution of the gastrointestinal tract microbiome to outcomes after allogeneic hematopoietic cell transplantation (HCT) is increasingly recognized. Investigations of larger pediatric cohorts aimed at defining the microbiome state and associated metabolic patterns pretransplant are needed. METHODS: We sought to describe the pretransplant stool microbiome in pediatric allogenic HCT patients at four centers. We performed shotgun metagenomic sequencing and untargeted metabolic profiling on pretransplant stool samples. Samples were compared with normal age-matched controls and by clinical characteristics. We then explored associations between stool microbiome measurements and metabolite concentrations. RESULTS: We profiled stool samples from 88 pediatric allogeneic HCT patients, a median of 4 days before transplant. Pretransplant stool samples differed from healthy controls based on indices of alpha diversity and in the proportional abundance of specific taxa and bacterial genes. Relative to stool from healthy patients, samples from HCT patients had decreased proportion of Bacteroides, Ruminococcaeae, and genes involved in butyrate production, but were enriched for gammaproteobacterial species. No systematic differences in stool microbiome or metabolomic profiles by age, transplant indication, or hospital were noted. Stool metabolites demonstrated strong correlations with microbiome composition. DISCUSSION: Stool samples from pediatric allogeneic HCT patients demonstrate substantial dysbiosis early in the transplant course. As microbiome disruptions associate with adverse transplant outcomes, pediatric-specific analyses examining longitudinal microbiome and metabolome changes are imperative to identify causal associations and to inform rational design of interventions.

Decreased Intestinal Microbiome Diversity in Pediatric Sepsis: A Conceptual Framework for Intestinal Dysbiosis to Influence Immunometabolic Function.

OBJECTIVES: The intestinal microbiome can modulate immune function through production of microbial-derived short-chain fatty acids. We explored whether intestinal dysbiosis in children with sepsis leads to changes in microbial-derived short-chain fatty acids in plasma and stool that are associated with immunometabolic dysfunction in peripheral blood mononuclear cells. DESIGN: Prospective observational pilot study. SETTING: Single academic PICU. PATIENTS: Forty-three children with sepsis/septic shock and 44 healthy controls. MEASUREMENTS AND MAIN RESULTS: Stool and plasma samples were serially collected for sepsis patients; stool was collected once for controls. The intestinal microbiome was assessed using 16S ribosomal RNA sequencing and alpha- and beta-diversity were determined. We measured short-chain fatty acids using liquid chromatography, peripheral blood mononuclear cell mitochondrial respiration using high-resolution respirometry, and immune function using ex vivo lipopolysaccharide-stimulated whole blood tumor necrosis factor-α. Sepsis patients exhibited reduced microbial diversity compared with healthy controls, with lower alpha- and beta-diversity. Reduced microbial diversity among sepsis patients (mainly from lower abundance of commensal obligate anaerobes) was associated with increased acetic and propionic acid and decreased butyric, isobutyric, and caproic acid. Decreased levels of plasma butyric acid were further associated with lower peripheral blood mononuclear cell mitochondrial respiration, which in turn, was associated with lower lipopolysaccharide-stimulated tumor necrosis factor-α. However, neither intestinal dysbiosis nor specific patterns of short-chain fatty acids were associated with lipopolysaccharide-stimulated tumor necrosis factor-α. CONCLUSIONS: Intestinal dysbiosis was associated with altered short-chain fatty acid metabolites in children with sepsis, but these findings were not linked directly to mitochondrial or immunologic changes. More detailed mechanistic studies are needed to test the role of microbial-derived short-chain fatty acids in the progression of sepsis.

Dynamic Changes in the Nasal Microbiome Associated With Disease Activity in Patients With Granulomatosis With Polyangiitis.

OBJECTIVE: Little is known about temporal changes in nasal bacteria in granulomatosis with polyangiitis (GPA). This study was undertaken to examine longitudinal changes in the nasal microbiome in association with relapse in GPA patients. METHODS: Bacterial 16S ribosomal RNA gene sequencing was performed on nasal swabs from 19 patients with GPA who were followed up longitudinally for a total of 78 visits, including 9 patients who experienced a relapse and 10 patients who remained in remission. Relative abundance of bacteria and ratios between bacteria were examined. Generalized estimating equation models were used to evaluate the association between bacterial composition and 1) disease activity and 2) levels of antineutrophil cytoplasmic antibody (ANCA) with specificity for proteinase 3 (PR3), adjusted for medication. RESULTS: Corynebacterium and Staphylococcus were the most abundant bacterial genera across all nasal samples. Patients with quiescent disease maintained a stable ratio of Corynebacterium to Staphylococcus across visits. In contrast, in patients who experienced a relapse, a significantly lower ratio was observed at the visit prior to relapse, followed by a higher ratio at the time of relapse (adjusted P < 0.01). Species-level analysis identified an association between a higher abundance of nasal Corynebacterium tuberculostearicum and 1) relapse (adjusted P = 0.04) and 2) higher PR3-ANCA levels (adjusted P = 0.02). CONCLUSION: In GPA, significant changes occur in the nasal microbiome over time and are associated with disease activity. The occurrence of these changes months prior to the onset of relapse supports a pathogenic role of nasal bacteria in GPA. Our results uphold existing hypotheses implicating Staphylococcus as an instigator of disease and have generated a novel finding involving Corynebacterium as a potential mediator of disease in GPA.

Gut Microbiome Profile After Pancreatectomy in Infants With Congenital Hyperinsulinism.

OBJECTIVES: The objective of this study was to characterize gut microbiome profiles of infants with congenital hyperinsulinism (HI) who underwent near-total or partial pancreatectomy for hypoglycemia management, as compared with healthy controls. METHODS: A prospective observational cohort study was performed. Subjects were infants (0-6 months) with HI who underwent removal of pancreatic tissue for management of intractable hypoglycemia from February 2017 to February 2018 at the Children's Hospital of Philadelphia. Fecal samples were collected postoperatively, on full enteral nutrition. The gut microbiome of HI subjects was analyzed and compared with age-matched samples from healthy infants. RESULTS: Seven subjects with ≥50% pancreatectomy and 6 with <50% pancreatectomy were included. α (within-sample) diversity was lowest among infants with ≥50% pancreatectomy (richness: false discovery rate, 0.003; Shannon index: false discovery rate, 0.01). ß (between-sample) diversity (Bray-Curtis dissimilarity, P = 0.02; Jaccard distance, P = 0.001) differed across groups (≥ or <50% pancreatectomy, controls). Bifidobacteria and Klebsiella species were least abundant among infants with ≥50% pancreatectomy but did not differ between infants with <50% pancreatectomy and historical controls. CONCLUSIONS: Infants with HI who underwent ≥50% pancreatectomy differed from age-matched infants in gut microbiome profile, whereas those with <50% pancreatectomy more closely resembled control profiles. The durability of this difference should be investigated.

Highly brain-permeable apoferritin nanocage with high dysprosium loading capacity as a new T2 contrast agent for ultra-high field magnetic resonance imaging.

High sensitivity at ultra-high field (UHF) and sufficient potential to penetrate the brain are the most desirable characteristics in the development of contrast agents (CAs) for magnetic resonance imaging (MRI). However, incorporating such qualities into a single nanocarrier is challenging. Herein, we report a new strategy for a highly brain-permeable MR CA with high sensitivity at UHF by loading dysprosium chelates (DyL) in apoferritin cavities (Apo-DyL). We also design the chelate ligand structure to increase DyL loading capacity within the apoferritin cavity. Using the intracerebroventricular (ICV) injection approach as a new delivery route for Apo-DyL, we demonstrate that apoferritin loaded with DyL can penetrate the brain-ventricular barrier and diffuse into the brain. This brain-permeable capability is unique to Apo-DyL, compared with other types of nanoparticles used in MRI. Apo-DyL also shows significant increase in MR sensitivity of DyL at UHF. Furthermore, based on brain tumor imaging at UHF, Apo-DyL can significantly enhance the tumor for a lower dose of the CA than the previously reported Gd- or Mn-loaded apoferritin nanoplatform. Therefore, Apo-DyL can be a novel nanoplatform that is a highly sensitive and versatile MR CA for UHF brain imaging.

Antiplatelet Activity of Acylphloroglucinol Derivatives Isolated from Dryopteris crassirhizoma.

Platelets are an important component of the initial response to vascular endothelial injury; however, platelet dysfunction induces the acute clinical symptoms of thrombotic disorders, which trigger severe cardiovascular diseases such as myocardial infarction, ischemia, and stroke. In this study, we investigated the Dryopteris crassirhizoma's antiplatelet activity. A water extract of D. crassirhizoma (WDC) was partitioned into dichloromethane (DCM), ethyl acetate, n-butyl alcohol, and water. Among these four fractions, the DCM fraction potently inhibited the collagen-stimulated platelet aggregation in a concentration-dependent manner. From this fraction, five different acylphloroglucinol compounds and one flavonoid were isolated by activity-guided column chromatography. They were identified by comparing their mass, 1H-, and 13C-NMR spectral data with those reported in the literature. Quantifying the six compounds in WDC and its DCM fraction by high-performance liquid chromatography (HPLC) revealed that butyryl-3-methylphloroglucinol (compound 4) was the most abundant in these samples. Additionally, butyryl-3-methylphloroglucinol showed the strongest inhibitory activity in the collagen- and arachidonic acid (AA)-induced platelet aggregation, with inhibition ratios of 92.36% and 89.51% in the collagen and AA-induced platelet aggregation, respectively, without cytotoxicity. On the active concentrations, butyryl-3-methylphloroglucinol significantly suppressed the convulxin-induced platelet activation. Regarding the structure-activity relationships for the five acylphloroglucinol compounds, our results demonstrated that the functional butanonyl, methoxy, and hydroxy groups in butyryl-3-methylphloroglucinol play important roles in antiplatelet activity. The findings indicate that acylphloroglucinols, including butyryl-3-methylphloroglucinol from D. crassirhizom, possess an antiplatelet activity, supporting the use of this species for antiplatelet remedies.

Improved detectability of sex steroids from frozen sections of breast cancer tissue using GC-triple quadrupole-MS.

Sex steroids in clinical endocrinology have been mainly investigated with peripheral blood and urine samples, while there is limited information regarding the local levels within tissues. To improve analytical properties of sex steroids from trace amounts of tissue samples, two-phase extractive ethoxycarbonlyation and subsequent pentafluoropropionyl derivatization coupled to gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed. The optimized analytical conditions led to excellent chromatographic separation of 15 estrogens, 6 androgens, and 2 progestins. The quantitative results were calculated based on in-house control samples as the steroid-free tissues, and the precision and accuracy were 4.2%-26.8% and 90.8%-116.4%, respectively. The on-column limit of quantification was from 180 fg to 0.5 pg for androgens and estrogens, and 1.25 pg for progestins, which were found to be linear (r2 > 0.990). The validated method was then applied to quantify 7 sex steroids from three 100-µm-thick frozen breast tissue slices from postmenopausal patients with breast cancer. This is the first report on the improved GC-MS/MS method for the detection of androgens and pregnenolone from breast cancer tissues, and it can be a useful technique to measure the local levels of sex steroids, thus, enhancing our understanding of the pathophysiological significances of steroidogenesis.

Protective Effects of Spatholobi Caulis Extract on Neuronal Damage and Focal Ischemic Stroke/Reperfusion Injury.

Neuronal apoptotic cell death plays an important role in many neurological disorders, including Alzheimer's disease, Parkinson's disease, and ischemic stroke. Spatholobi Caulis (SC) has been widely used in traditional herbal medicine for the treatment of cancer, inflammation, viral infection, and anemia. However, the protective effects of SC extract (SCE) against apoptotic cell death in the brain have not been reported. We investigated the protective effects of SCE against neuronal injury etoposide-induced neurotoxicity and in rats subjected to focal transient ischemic stroke middle cerebral artery occlusion (MCAO) for 45 min, followed by 7 days of reperfusion. The in vitro study demonstrated that SCE protected cells against etoposide-induced cell viability loss in SH-SY5Y cells. Apoptotic phenotypes, such as cleaved PARP and caspase-3, and oxidative stress in etoposide-treated cells were ameliorated by SCE treatment. In MCAO-reperfusion injury, SCE promoted neuronal survival and level of brain-derived neurotrophic factor (BDNF) by reducing glial activation, oxidative stress, and apoptosis in the ipsilateral cortex. These results indicated that SCE exerted protective effects under etoposide treatment and in a MCAO-reperfusion model by reducing JNK and p38 MAPK activation. This study presents the first evidence that SCE has therapeutic potential for the treatment of ischemic stroke or neurological disorder-related cell death.

KBH-1, an herbal composition, improves hepatic steatosis and leptin resistance in high-fat diet-induced obese rats.

BACKGROUND: KBH-1 is an herbal mixture of Saururus chinensis, Curcuma longa and Polygala tenuifolia. Each herb has been reported to have various pharmaceutical activities; however, the synergistic effect of this herbal composition on obesity has not yet been determined. We investigated the alleviation effect of KBH-1 and its possible molecular mechanism in obesity-induced hepatic steatosis and leptin resistance in the hypothalamus. METHODS: We used HepG2 cells, primary neuronal cells and a high-fat diet (HFD)-induced obesity rat model to determine the effect of KBH-1 in vitro and in vivo on hepatic steatosis and leptin resistance accompanied by obesity. To identify the alleviation effect on lipid accumulation, HepG2 cells stimulated by FFA were stained with Oil Red O; in addition, immunoblotting and qPCR were performed to determine the effect of KBH-1 on the activation of proteins and nuclear enzymes in HepG2 cells and the steatotic liver of HFD-induced obesity rats. To examine the effect of KBH-1 on the leptin resistance of the hypothalamus and its possible molecular mechanism, we examined the effect of KBH-1 on the activation of the leptin resistance-related protein in primary cultured cortical neuron cells and the hypothalamus of an HFD-induced obesity rat model. In addition, we used HPLC analysis to identify the standard compound of KBH-1. RESULTS: KBH-1 not only suppressed the lipid deposition in HepG2 cells exposed to free fatty acids (FFA) but also significantly down-regulated major factors in lipogenesis and up-regulated major factors in lipolysis. Similarly, in a HFD-induced obesity model, KBH-1 improved hepatic steatosis by alleviating the effects on lipogenic genes and kinases. In addition, KBH-1 significantly improved the leptin-mediated signals impaired by obesity or FFA in the obesity model and primary cultured cortical neuron cells. In addition, KBH-1 was analyzed to include six standard compounds using HPLC analysis, among these compounds, onji-saponin B and curcumin were potently suppressed the level of triglycerides. CONCLUSIONS: KBH-1 exhibits alleviating effects by improving hepatic steatosis and leptin resistance by up-regulating the activation of AMPK and suppressing the expression of PPARγ. These findings show the potential of KBH-1 as a functional food supplement or preventive agent in the treatment of obesity.

The effects of adiponectin and inflammatory cytokines on diabetic vascular complications in obese and non-obese patients with type 2 diabetes mellitus.

AIMS: To evaluate the associations between inflammatory cytokines and adiponectin and various vascular complications in type 2 diabetes mellitus (T2DM). METHODS: A total of 761 patients with T2DM were divided into a non-obese group and an obese group to enable the effects of obesity and T2DM on vascular complications to be differentiated. The serum levels of circulating inflammatory cytokines, that is, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, total adiponectin, and high molecular weight (HMW) adiponectin were measured, and carotid intima media thickness (IMT), the presence of carotid plaque, and the severities of retinopathy and nephropathy, were assessed. RESULTS: The obese group had significantly lower serum total and HMW adiponectin levels than the non-obese group. In the obese group, serum levels of total and HMW adiponectin, and TNF-α were significantly higher in patients with proliferative retinopathy than in those without retinopathy after adjusting for covariates. In the non-obese group, only IL-6 levels were significantly higher in patients with proliferative retinopathy than in those without. Serum levels of total and HMW adiponectin were significantly higher in patients with macroalbuminuria than in those with normoalbuminuria. No significant difference of three cytokines levels were observed depending on the carotid IMT or the presence of plaque. Logistic regression analysis revealed that serum total adiponectin (OR=1.209, P=0.038), diabetes duration (OR=1.230, P=0.014), and HbA1c (OR=2.359, P=0.006) were significantly associated with proliferative retinopathy in the obese group. CONCLUSION: The study shows total adiponectin may influence proliferative retinopathy in obese patient with T2DM.

The Herbal Medicine KBH-1 Inhibits Fat Accumulation in 3T3-L1 Adipocytes and Reduces High Fat Diet-Induced Obesity through Regulation of the AMPK Pathway.

The aim of this study was to investigate whether a novel formulation of an herbal extract, KBH-1, has an inhibitory effect on obesity. To determine its anti-obesity effects and its underlying mechanism, we performed anti-obesity-related experiments in vitro and in vivo. 3T3-L1 preadipocytes were analyzed for lipid accumulation as well as the protein and gene expression of molecular targets involved in fatty acid synthesis. To determine whether KBH-1 oral administration results in a reduction in high-fat diet (HFD)-induced obesity, we examined five groups (n = 9) of C57BL/6 mice as follows: 10% kcal fat diet-fed mice (ND), 60% kcal fat diet-fed mice (HFD), HFD-fed mice treated with orlistat (tetrahydrolipstatin, marketed under the trade name Xenical), HFD-fed mice treated with 150 mg/kg KBH-1 (KBH-1 150) and HFD-fed mice treated with 300 mg/kg KBH-1 (KBH-1 300). During adipogenesis of 3T3-L1 cells in vitro, KBH-1 significantly reduced lipid accumulation and down-regulated the expression of master adipogenic transcription factors, including CCAAT/enhancer binding protein (C/EBP) ß, C/EBP α and peroxisome proliferation-activity receptor (PPAR) γ, which led to the suppression of the expression of several adipocyte-specific genes and proteins. KBH-1 also markedly phosphorylated the adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). In addition, KBH-1-induced the inhibition effect on lipid accumulation and AMPK-mediated signal activation were decreased by blocking AMPK phosphorylation using AMPK siRNA. Furthermore, daily oral administration of KBH-1 resulted in dose-dependent decreases in body weight, fat pad mass and fat tissue size without systemic toxicity. These results suggest that KBH-1 inhibits lipid accumulation by down-regulating the major transcription factors of the adipogenesis pathway by regulating the AMPK pathway in 3T3-L1 adipocytes and in mice with HFD-induced obesity. These results implicate KBH-1, a safe herbal extract, as a potential anti-obesity therapeutic agent.

Murrayafoline A Induces a G0/G1-Phase Arrest in Platelet-Derived Growth Factor-Stimulated Vascular Smooth Muscle Cells.

The increased potential for vascular smooth muscle cell (VSMC) growth is a key abnormality in the development of atherosclerosis and post-angioplasty restenosis. Abnormally high activity of platelet-derived growth factor (PDGF) is believed to play a central role in the etiology of these pathophysiological situations. Here, we investigated the anti-proliferative effects and possible mechanism(s) of murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa Guillamin (Rutaceae), on PDGF-BB-stimulated VSMCs. Murrayafoline A inhibited the PDGF-BB-stimulated proliferation of VSMCs in a concentration-dependent manner, as measured using a non-radioactive colorimetric WST-1 assay and direct cell counting. Furthermore, murrayafoline A suppressed the PDGF-BB-stimulated progression through G0/G1 to S phase of the cell cycle, as measured by [(3)H]-thymidine incorporation assay and cell cycle progression analysis. This anti-proliferative action of murrayafoline A, arresting cell cycle progression at G0/G1 phase in PDGF-BB-stimulated VSMCs, was mediated via down-regulation of the expression of cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, and proliferating cell nuclear antigen (PCNA), and the phosphorylation of retinoblastoma protein (pRb). These results indicate that murrayafoline A may be useful in preventing the progression of vascular complications such as restenosis after percutaneous transluminal coronary angioplasty and atherosclerosis.

Change of Bone Mineral Density and Biochemical Markers of Bone Turnover in Patients on Suppressive Levothyroxine Therapy for Differentiated Thyroid Carcinoma.

Untreated hyperthyroidism and high-dose thyroid hormone are associated with osteoporosis, and increased bone mineral density (BMD) has been demonstrated in postmenopausal females with hypoparathyroidism. Studies on the effect of suppressive levothyroxine (LT4) therapy on BMD and bone metabolism after total thyroidectomy in patients with differentiated thyroid carcinoma have presented conflicting results, and few studies in relation to the status of hypoparathyroidism have been studied. One hundred postmenopausal women and 24 premenopausal women on LT4 suppression therapy were included in this study. BMD of lumbar spine and femur and bone turnover markers were measured at the baseline and during the follow-up period up to 18 months using dual energy X-ray absorptiometry. Biochemical marker of bone resorption was measured by urine deoxypyridinoline and bone formation by serum osteocalcin. The age ranged from 36 to 64 years old. Thyroid stimulating hormone (TSH) was suppressed during the study. The results showed that BMD of femur and lumbar spine were not significantly changed in both pre- and postmenopausal women except femur neck in postmenopausal women without hypoparathyroidism. Patients with hypoparathyroidism had higher BMD gain than those without hypoparathyroidism in total hip (1.25 vs. -1.18%, P=0.015). Biochemical markers of bone turnover, serum osteocalcin, and urine deoxypyridinoline did not show significant change. In conclusion, patients with well differentiated thyroid carcinoma are not at a great risk of bone loss after LT4 suppressive therapy. The state of hypoparathyroidism is associated with increased BMD, particularly in postmenopausal women.

Evaluation of effect of galvanic corrosion between nickel-chromium metal and titanium on ion release and cell toxicity.

PURPOSE: The purpose of this study was to evaluate cell toxicity due to ion release caused by galvanic corrosion as a result of contact between base metal and titanium. MATERIALS AND METHODS: It was hypothesized that Nickel (Ni)-Chromium (Cr) alloys with different compositions possess different corrosion resistances when contacted with titanium abutment, and therefore in this study, specimens (10×10×1.5 mm) were fabricated using commercial pure titanium and 3 different types of Ni-Cr alloys (T3, Tilite, Bella bond plus) commonly used for metal ceramic restorations. The specimens were divided into 6 groups according to the composition of Ni-Cr alloy and contact with titanium. The experimental groups were in direct contact with titanium and the control groups were not. After the samples were immersed in the culture medium - Dulbecco's modified Eagle's medium[DMEM] for 48 hours, the released metal ions were detected using inductively coupled plasma mass spectrometer (ICP-MS) and analyzed by the Kruskal-Wallis and Mann-Whitney test (P<.05). Mouse L-929 fibroblast cells were used for cell toxicity evaluation. The cell toxicity of specimens was measured by the 3-{4,5-dimethylthiazol-2yl}-2,5-diphenyltetrazolium bromide (MTT) test. Results of MTT assay were statistically analyzed by the two-way ANOVA test (P<.05). Post-hoc multiple comparisons were conducted using Tukey's tests. RESULTS: The amount of metal ions released by galvanic corrosion due to contact between the base metal alloy and titanium was increased in all of the specimens. In the cytotoxicity test, the two-way ANOVA showed a significant effect of the alloy type and galvanic corrosion for cytotoxicity (P<.001). The relative cell growth rate (RGR) was decreased further on the groups in contact with titanium (P<.05). CONCLUSION: The release of metal ions was increased by galvanic corrosion due to contact between base metal and titanium, and it can cause adverse effects on the tissue around the implant by inducing cytotoxicity.

Antiplatelet action of indirubin-3'-monoxime through suppression of glycoprotein VI-mediated signal transduction: a possible role for ERK signaling in platelets.

We investigated the antiplatelet activity of indirubin-3'-monoxime (I3O) and the underlying mechanisms. In a rat carotid artery injury model, oral administration (20 mg/kg/day) of I3O for 3 days significantly prolonged occlusion time, and ADP- and collagen-induced platelet aggregation. In washed platelets in vitro, I3O potently inhibited collagen-induced platelet aggregation by suppressing phospholipase Cγ2 (PLCγ2) phosphorylation, subsequently blocking diacylglycerol and arachidonic acid (AA) formation, P-selectin secretion and the production of thromboxane B2. Platelet aggregation induced by phorbol-12-myristate 13-acetate, a protein kinase C (PKC) activator, was inhibited by I3O. Both I3O and U0126, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, markedly reduced collagen-induced phosphorylation of ERK1/2 and p47, resulting in the blockade of cyclooxygenase (COX)-mediated AA metabolite production in AA-treated platelets. I3O suppressed phosphorylation of JNK, p38, GSK-3ß, and AKT. I3O inhibited glycoprotein VI (GPVI), as a collagen receptor, by suppressing the phosphorylation of tyrosine kinase Syk of GPVI and the phosphorylation of PLCγ2 and ERK1/2 stimulated by convulxin, as a specific stimulator. Our results indicate that an antiplatelet effect of I3O is due to the suppression of GPVI-mediated signaling pathways. In collagen-stimulated platelets, ERK1/2 phosphorylation is adenylyl cyclase-dependent and leads to the modulation of PKC-p47 signaling and COX-1-mediated AA-metabolic pathways.

Extract of Ulmus macrocarpa Hance prevents thrombus formation through antiplatelet activity.

Ulmus macrocarpa Hance (Ulmaceae) has been used as a traditional oriental medicine for the treatment of edema, mastitis, gastric cancer and inflammation. The aim of this study was to investigate the effects of Ulmus macrocarpa extract (UME) on thrombus formation in vivo, platelet activation ex vivo and fibrinolytic activity in vitro. To identify the antithrombotic activity of UME in vivo, we used an arterial thrombosis model. UME delayed the occlusion time by 13.4 and 13.9 min at doses of 300 and 600 mg/kg, respectively. UME significantly inhibited ex vivo platelet aggregation induced by collagen and adenosine 5'-diphosphate (ADP), respectively, but did not affect the coagulation times following activated partial thromboplastin and prothrombin activation. Therefore, to investigate the antiplatelet effect of UME, the effect of UME on collagen and ADP-induced platelet aggregation in vitro was examined. UME exhibited antiplatelet aggregation activity, induced by ADP and collagen. Furthermore, the fibrinolytic activity of UME was investigated. The results showed that UME significantly increased fibrinolysis at 1,000 mg/ml. In conclusion, the results suggested that UME may significantly inhibit artery thrombus formation in vivo, potentially due to antiplatelet activity, and also exhibits potential as a clot­dissolving agent for thrombolytic therapy.

5,8-Dimethoxy-2-Nonylamino-Naphthalene-1,4-Dione Inhibits Vascular Smooth Muscle Cell Proliferation by Blocking Autophosphorylation of PDGF-Receptor ß.

As the abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis and vascular restenosis, a candidate drug with antiproliferative properties is needed. We investigated the antiproliferative action and underlying mechanism of a newly synthesized naphthoquinone derivative, 5,8-dimethoxy-2-nonylamino-naphthalene-1,4-dione (2-nonylamino-DMNQ), using VSMCs treated with platelet-derived growth factor (PDGF). 2-Nonylamino-DMNQ inhibited proliferation and cell number of VSMCs induced by PDGF, but not epidermal growth factor (EGF), in a concentration-dependent manner without any cytotoxicity. This derivative suppressed PDGF-induced [(3)H]-thymidine incorporation, cell cycle progression from G0/G1 to S phase, and the phosphorylation of phosphor-retinoblastoma protein (pRb) as well as the expression of cyclin E/D, cyclin-dependent kinase (CDK) 2/4, and proliferating cell nuclear antigen (PCNA). Importantly, 2-nonylamino-DMNQ inhibited the phosphorylation of PDGF receptorß(PDGF-Rß) enhanced by PDGF at Tyr(579), Tyr(716), Tyr(751), and Tyr(1021) residues. Subsequently, 2-nonylamino-DMNQ inhibited PDGF-induced phosphorylation of STAT3, ERK1/2, Akt, and PLCγ1. Therefore, our results indicate that 2-nonylamino-DMNQ inhibits PDGF-induced VSMC proliferation by blocking PDGF-Rß autophosphorylation, and subsequently PDGF-Rß-mediated downstream signaling pathways.