Natural Killer T Cells Infiltrate Neuroblastomas Expressing the Chemokine CCL2

CD1d-restricted Vα24-Jα18–invariant natural killer T cells (iNKTs) are potentially important in tumor immunity. However, little is known about their localization to tumors. We analyzed 98 untreated primary neuroblastomas from patients with metastatic disease (stage 4) for tumor-infiltrating iNKTs using TaqMan® reverse transcription polymerase chain reaction and immunofluorescent microscopy. 52 tumors (53%) contained iNKTs, and oligonucleotide microarray analysis of the iNKT+ and iNKT− tumors revealed that the former expressed higher levels of CCL2/MCP-1, CXCL12/SDF-1, CCL5/RANTES, and CCL21/SLC. Eight tested neuroblastoma cell lines secreted a range of CCL2 (0–21.6 ng/ml), little CXCL12 (≤0.1 ng/ml), and no detectable CCL5 or CCL21. CCR2, the receptor for CCL2, was more frequently expressed by iNKT compared with natural killer and T cells from blood (P < 0.001). Supernatants of neuroblastoma cell lines that produced CCL2 induced in vitro migration of iNKTs from blood of patients and normal adults; this was abrogated by an anti-CCL2 monoclonal antibody. CCL2 expression by tumors was found to inversely correlate with MYCN proto-oncogene amplification and expression (r = 0.5, P < 0.001), and MYCN-high/CCL2-low expression accurately predicted the absence of iNKTs (P < 0.001). In summary, iNKTs migrate toward neuroblastoma cells in a CCL2-dependent manner, preferentially infiltrating MYCN nonamplified tumors that express CCL2

Echocardiographic evaluation of right ventricular diastolic function in pulmonary hypertension

Background: Right ventricular (RV) diastolic dysfunction may be prognostic in pulmonary hypertension (PH). However, its assessment is complex and relies on conductance catheterisation. We aimed to evaluate echocardiography-based parameters as surrogates of RV diastolic function, provide validation against the gold standard, end-diastolic elastance (Eed), and define the prognostic impact of echocardiography-derived RV diastolic dysfunction. Methods: Patients with suspected PH who underwent right heart catheterisation including conductance catheterisation were prospectively recruited. In this study population, an echocardiography-based RV diastolic function surrogate was derived. Survival analyses were performed in patients with precapillary PH in the Giessen PH Registry, with external validation in patients with pulmonary arterial hypertension at Sapienza University (Rome). Results: In the derivation cohort (n=61), the early/late diastolic tricuspid inflow velocity ratio (E/A) and early tricuspid inflow velocity/early diastolic tricuspid annular velocity ratio (E/e') did not correlate with Eed (p&gt;0.05). Receiver operating characteristic analysis revealed a large area under the curve (AUC) for the peak lateral tricuspid annulus systolic velocity/right atrial area index ratio (S'/RAAi) to detect elevated Eed (AUC 0.913, 95% confidence interval (CI) 0.839-0.986) and elevated end-diastolic pressure (AUC 0.848, 95% CI 0.699-0.998) with an optimal threshold of 0.81 m2·s-1·cm-1. Subgroup analyses demonstrated a large AUC in patients with preserved RV systolic function (AUC 0.963, 95% CI 0.882-1.000). Survival analyses confirmed the prognostic relevance of S'/RAAi in the Giessen PH Registry (n=225) and the external validation cohort (n=106). Conclusions: Our study demonstrates the usefulness of echocardiography-derived S'/RAAi for noninvasive assessment of RV diastolic function and prognosis in PH

The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia reveals an organism heavily shielded by drug resistance determinants

The complete Stenotrophomonas maltophilia genome sequence suggests that it can act as a reservoir of antibiotic resistance determinants

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli

Gram-negative bacteria, such as Escherichia coli, expel toxic chemicals via tripartite efflux pumps spanning both the inner and outer membranes. The three parts are: 1) a membrane fusion protein connecting 2) a substrate-binding inner membrane transporter to 3) an outer membrane-anchored channel in the periplasmic space. A crystallographic model of this tripartite efflux complex has been unavailable simply because co-crystallization of different components of the system has proven to be extremely difficult. We previously described the crystal structures of both the inner membrane transporter CusA1 and membrane fusion protein CusB2 of the CusCBA efflux system3,4 from E. coli. We here report the co-crystal structure of the CusBA efflux complex, revealing the trimeric CusA efflux pump interacts with six CusB protomers at the upper half of the periplasmic domain. These six CusB molecules form a channel extending contiguously from the top of the pump. The affinity of the CusA and CusB interaction was found to be in the micromolar range. Finally, we predicted a three-dimensional structure of the trimeric CusC outer membrane channel, and develop a model of the tripartite efflux assemblage. This CusC3-CusB6-CusA3 model presents a 750 kDa efflux complex spanning the entire bacterial cell envelope to export Cu(I)/Ag(I) ions

A controlled trial of rivaroxaban after transcatheter aortic-valve replacement

Background: whether the direct factor Xa inhibitor rivaroxaban can prevent thromboembolic events after transcatheter aortic-valve replacement (TAVR) is unclear. Methods: we randomly assigned 1644 patients without an established indication for oral anticoagulation after successful TAVR to receive rivaroxaban at a dose of 10 mg daily (with aspirin at a dose of 75 to 100 mg daily for the first 3 months) (rivaroxaban group) or aspirin at a dose of 75 to 100 mg daily (with clopidogrel at a dose of 75 mg daily for the first 3 months) (antiplatelet group). The primary efficacy outcome was the composite of death or thromboembolic events. The primary safety outcome was major, disabling, or life-threatening bleeding. The trial was terminated prematurely by the data and safety monitoring board because of safety concerns. Results: after a median of 17 months, death or a first thromboembolic event (intention-to-treat analysis) had occurred in 105 patients in the rivaroxaban group and in 78 patients in the antiplatelet group (incidence rates, 9.8 and 7.2 per 100 person-years, respectively; hazard ratio with rivaroxaban, 1.35; 95% confidence interval [CI], 1.01 to 1.81; P = 0.04). Major, disabling, or life-threatening bleeding (intention-to-treat analysis) had occurred in 46 and 31 patients, respectively (4.3 and 2.8 per 100 person-years; hazard ratio, 1.50; 95% CI, 0.95 to 2.37; P = 0.08). A total of 64 deaths occurred in the rivaroxaban group and 38 in the antiplatelet group (5.8 and 3.4 per 100 person-years, respectively; hazard ratio, 1.69; 95% CI, 1.13 to 2.53). Conclusions: in patients without an established indication for oral anticoagulation after successful TAVR, a treatment strategy including rivaroxaban at a dose of 10 mg daily was associated with a higher risk of death or thromboembolic complications and a higher risk of bleeding than an antiplatelet-based strategy. (Funded by Bayer and Janssen Pharmaceuticals; GALILEO ClinicalTrials.gov number, NCT02556203.)

Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell.1,2 The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions.3,4 No prior structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide important new structural information about the HME sub-family of RND efflux pumps. The structures suggest that the metal binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. Intriguingly, this cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal binding site, four methionine pairs - three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, utilizing these methionine pairs/clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites