An NMR and molecular modeling study of the site-specific binding of histamine by heparin, chemically modified heparin, and heparin-derived oligosaccharides

The diprotonated form of histamine binds site-specifically to heparin, a highly sulfated 1-->4 linked repeating copolymer comprised predominantly of 2-O-sulfo-alpha-L-iduronic acid (the I ring) and 2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl (the A ring). The binding is mediated by electrostatic interactions. The structural features of histamine and heparin, which are required for the site-specific binding, have been identified from the results of (1)H NMR studies of the binding of histamine by six heparin-derived oligosaccharides and four chemically modified heparins and molecular modeling studies. The results indicate that the imidazolium ring of diprotonated histamine is critical for directing site-specific binding, while the ammonium group increases the binding affinity. The imidazolium ring binds within a cleft, with the A ring of an IAI triad at the top of the cleft, and the I rings forming the two sides. The H3 proton of the A ring is in the shielding cone of the imidazolium ring. The carboxylate group of the I-ring at the reducing end of the IAI triad and possibly the sulfamido group of the A-ring are essential for site-specific binding, whereas the 2-O-sulfate group of the I ring and the 6-O-sulfate group of the A ring are not. The results indicate that histamine binds to the IAI triad with the I rings in the (1)C(4) conformation. Also, the configuration of the carboxylate group is critical, as indicated by the absence of site-specific binding of histamine by the related IAG sequence, where G is alpha-D-glucuronic acid. The molecular modeling results indicate that the N1H and N3H protons of the imidazolium ring of site-specifically bound histamine are hydrogen bonded to the carboxylates of the I rings at the nonreducing and reducing ends of the IAI trisaccharide sequence.     

Determinants for Achieving the LDL-C Target of Lipid Control for Secondary Prevention of Cardiovascular Events in Taiwan

Background

Epidemiological and clinical studies have clearly established the link between low-density lipoprotein cholesterol (LDL-C) and atherosclerosis-related cardiovascular consequences. Although it has been a common practice for physicians to prescribe lipid-lowering therapy for patients with dyslipidemia, the achievement rate is still not satisfied in Taiwan. Therefore, the determinants for achieving the LDL-C target needed to be clarified for better healthcare of the patients with dyslipidemia.

Method

This registry-type prospective observational study enrolled the patients with cardiovascular diseases (coronary artery disease (CAD) and cerebrovascular disease (CVD)) from 18 medical centers across Taiwan, and clinically followed them for five years. At every clinical visit, vital signs, clinical endpoints, adverse events, concurrent medications and laboratory specimens were obtained as thoroughly as possible. The lipid profile (total cholesterol, high-density lipoprotein cholesterol, LDL-C, triglyceride), liver enzymes, and creatinine phosphokinase were evaluated at baseline, and every year thereafter. The cross sectional observational data was analyzed for this report.

Result

Among the 3,486 registered patients, 54% had their LDL-C < 100 mg/dL. By univariate analysis, the patients achieving the LDL-C target were associated with older age, more male sex, taller height, lower blood pressure, more under lipid-lowering therapy, more smoking cessation, more history of CAD, DM, physical activity, but less history of CVD. The multivariate analysis showed statin therapy was the most significant independent determinant for achieving the treatment target, followed by age, history of CAD, diabetes, blood pressure, and sex. However, most patients were on regimens of very-low to low equipotent doses of statins.

Conclusion

Although the lipid treatment guideline adherence is improving in recent years, only 54% of the patients with cardiovascular diseases have achieved their LDL-C target in Taiwan, and the most significant determinant for this was statin therapy.     

Clinical and Prognostic Implications of Roundabout 4 (Robo4) in Adult Patients with Acute Myeloid Leukemia

Background

Robo4 is involved in hematopoietic stem/progenitor cell homeostasis and essential for tumor angiogenesis. Expression of Robo4 was recently found in solid tumors and leukemia stem cells. However, the clinical implications of Robo4 expression in patients with acute myeloid leukemia (AML) remain unclear.

Methods

We investigated the clinical and prognostic relevance of mRNA expression of Robo4 in bone marrow (BM) mononuclear cells from 218 adult patients with de novo AML. We also performed immunohistochemical staining to assess the Robo4 protein expression in the BM biopsy specimens from 30 selected AML patients in the cohort.

Results

Higher Robo4 expression was closely associated with lower white blood cell counts, expression of HLA-DR, CD13, CD34 and CD56 on leukemia cells, t(8;21) and ASXL1 mutation, but negatively correlated with t(15;17) and CEBPA mutation. Compared to patients with lower Robo4 expression, those with higher expression had significantly shorter disease-free survival (DFS) and overall survival (OS). This result was confirmed in an independent validation cohort. Furthermore, multivariate analyses showed that higher Robo4 expression was an independent poor prognostic factor for DFS and OS in total cohort and patients with intermediate-risk cytogenetics, irrespective of age, WBC count, karyotype, and mutation status of NPM1/FLT3-ITD, and CEBPA.

Conclusions

BM Robo4 expression can serve as a new biomarker to predict clinical outcomes in AML patients and Robo4 may serve as a potential therapeutic target in patients with higher Robo4 expression.     

Caveolin-1 siRNA对与乳腺癌细胞共培养的成纤维细胞自噬体表达影响的研究

为了探索乳腺癌相关的人成纤维细胞中窖蛋白-1(Caveolin-1,Cav-1)与自噬体的相关性及其对乳腺癌细胞的作用,该研究采用si RNA技术干扰成纤维细胞株ESF表达Cav-1,q RTPCR和Western blot确定si RNA干扰Cav-1表达的效果;Transwell insert方法共培养乳腺癌细胞株BT474和ESF细胞,单丹磺酰戊二胺(monodansylcadaverin,MDC)染色、激光共聚焦显微镜观察Cav-1 si RNA对自噬体表达的影响;q RT-PCR和Western blot检测Cav-1 si RNA对微管相关蛋白1轻链3II(microtubule-associated protein 1 light chain 3 II,LC3II)表达的影响;CCK-8方法检测BT474细胞的增殖和活力。结果显示,靶向Cav-1的si RNA下调了ESF细胞中Cav-1的表达;Cav-1 si RNA促进ESF细胞自噬体和LC3II的表达,转染了Cav-1 si RNA的ESF细胞与BT474细胞共培养对自噬体和LC3II的作用更为显著;BT474细胞在ESFsi Cav-1(ESF cells transfected with Cav-1 si RNA)细胞共培养条件下增殖显著加快。研究表明,Cav-1 si RNA促进了与乳腺癌细胞共培养的成纤维细胞自噬体和LC3II的表达,同时加快了与成纤维细胞共培养的乳腺癌细胞的增殖。     

Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.     

The Beneficial Effects of P2X7 Antagonism in Rats with Bile Duct Ligation-induced Cirrhosis

Splanchnic angiogenesis in liver cirrhosis often leads to complications as gastroesophageal variceal hemorrhage and the treatment efficacy is adversely affected by poor portal-systemic collateral vasoresponsiveness related to nitric oxide (NO). Purinergic receptor subtype P2X₇ participates in the modulation of inflammation, angiogenesis, fibrogenesis and vasoresponsiveness, but the relevant influence in cirrhosis is unknown. Common bile duct-ligated (CBDL) or sham-operated Spraque-Dawley rats received brilliant blue G (BBG, a P2X₇ antagonist and food additive) or vehicle from the 15^th to 28^th day after operations, then hemodynamics, mesenteric angiogenesis, portal-systemic shunting, liver fibrosis, and protein expressions of angiogenic and fibrogenic factors were evaluated. The influence of oxidized ATP (oATP, another P2X₇ receptor antagonist) on the collateral vasoresponsiveness to arginine vasopressin (AVP) was also surveyed. BBG decreased superior mesenteric artery (SMA) flow, portal-systemic shunting, mesenteric vascular density, and mesenteric protein expressions of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), phospho (p)-VEGFR2, platelet-derived growth factor (PDGF), PDGF receptor beta (PDGFRβ), cyclooxygenase (COX)-1, COX-2, and endothelial NO synthase (eNOS) in CBDL rats. BBG also ameliorated liver fibrosis and down-regulated hepatic interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), PDGF, IL-1β, transforming growth factor-beta (TGF-β), p-extracellular-signal-regulated kinases (ERK), and alpha-smooth muscle actin (α-SMA) expressions in CBDL rats. The collateral vasocontractility to AVP was enhanced by oATP. oATP down-regulated eNOS, inducible NOS (iNOS), VEGF, Akt, p-Akt, and nuclear factor-kappa B (NF-κB) expressions in splenorenal shunt, the most prominent intra-abdominal collateral vessel in rodents. P2X₇ antagonism alleviates splanchnic hyperemia, severity of portal-systemic shunting, mesenteric angiogenesis, liver fibrosis, and enhances portal-systemic collateral vasoresponsiveness in cirrhotic rats. P2X₇ blockade may be a feasible strategy to control cirrhosis and complications.     

Innate Immunity Drives the Initiation of a Murine Model of Primary Biliary Cirrhosis

Invariant natural killer T (iNKT) cells play complex roles in bridging innate and adaptive immunity by engaging with glycolipid antigens presented by CD1d. Our earlier work suggested that iNKT cells were involved in the initiation of the original loss of tolerance in primary biliary cirrhosis (PBC). To address this issue in more detail and, in particular, to focus on whether iNKT cells activated by a Th2-biasing agonist (2s,3s,4r)-1-O-(α-_D-galactopyranosyl)-N-tetracosanoyl-2-amino-1,3,4-nonanetriol (OCH), can influence the development of PBC in a xenobiotic-induced PBC murine model. Groups of mice were treated with either OCH or, as a control, α-galactosylceramide (α-GalCer) and thence serially followed for cytokine production, markers of T cell activation, liver histopathology and anti-mitochondrial antibody responses. Further, additional groups of CD1d deleted mice were similarly studied. Our data indicate that administration of OCH has a dramatic influence with exacerbation of portal inflammation and hepatic fibrosis similar to mice treated with α-GalCer. Further, iNKT cell deficient CD1d knockout mice have decreased inflammatory portal cell infiltrates and reduced anti-mitochondrial antibody responses. We submit that activation of iNKT cells can occur via overlapping and/or promiscuous pathways and highlight the critical role of innate immunity in the natural history of autoimmune cholangitis. These data have implications for humans with PBC and emphasize that therapeutic strategies must focus not only on suppressing adaptive responses, but also innate immunity.     

Antimicrobial Non-Susceptibility of Escherichia coli from Outpatients and Patients Visiting Emergency Rooms in Taiwan

Longitudinal nationwide surveillance data on antimicrobial non-susceptibility and prevalence of extended-spectrum β-lactamases (ESBLs) as well as AmpC β-lactamases producers among Escherichia coli from different sources in the community settings are limited. Such data may impact treatment practice. The present study investigated E. coli from outpatients and patients visiting emergency rooms collected by the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program. A total of 3481 E. coli isolates were studied, including 2153 (61.9%) from urine and 1125 (32.3%) from blood samples. These isolates were collected biennially between 2002 and 2012 from a total of 28 hospitals located in different geographic regions of Taiwan. Minimum inhibitory concentrations (MIC) were determined using methods recommended by the Clinical Laboratory Standards Institute (CLSI). The prevalence and factors associated with the presence of ESBL and AmpC β-lactamase-producers were determined. Significant increases in non-susceptibility to most β-lactams and ciprofloxacin occurred during the study period. By 2012, non-susceptibility to cefotaxime and ciprofloxacin reached 21.1% and 26.9%, respectively. The prevalence of ESBL- and AmpC- producers also increased from 4.0% and 5.3%, respectively, in 2002–2004, to 10.7% for both in 2010–2012 (P < 0.001). The predominant ESBL and AmpC β-lactamase genes were CTX-M and CMY-types, respectively. Non-susceptibility of urine isolates to nitrofurantoin remained at around 8% and to fosfomycin was low (0.7%) but to cefazolin (based on the 2014 CLSI urine criteria) increased from 11.5% in 2002–2004 to 23.9% in 2010–2012 (P <0.001). Non-susceptibility of isolates from different specimen types was generally similar, but isolates from elderly patients were significantly more resistant to most antimicrobial agents and associated with the presence of ESBL- and AmpC- β-lactamases. An additional concern is that decreased ciprofloxacin susceptibility (MIC 0.12–1 mg/L) was as high as 25% in isolates from all age groups, including those from pediatric patients. Our data indicated that there is a need to re-evaluate appropriate treatment selection for community-acquired infections in Taiwan. Identification of community reservoirs of multidrug-resistant E. coli is also warranted.     

Emergence of OXA-48-Producing Klebsiella pneumoniae in Taiwan

The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four bla _OXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured bla _CTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the bla _OXA-48 gene in all four isolates was identical to pKP_OXA-48N1, a bla _OXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify bla _OXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of bla _OXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern.