COX-2-mediated stimulation of the lymphangiogenic factor VEGF-C in human breast cancer

Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (EP) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. EP1 as well as EP4 receptor antagonists inhibited VEGF-C production indicating the roles of EP1 and EP4 in VEGF-C upregulation by endogenous PGE2. Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by EP1/EP4 receptors

Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection

BACKGROUND Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients. Recurrences are common after antibiotic therapy. Actoxumab and bezlotoxumab are human monoclonal antibodies against C. difficile toxins A and B, respectively. METHODS We conducted two double-blind, randomized, placebo-controlled, phase 3 trials, MODIFY I and MODIFY II, involving 2655 adults receiving oral standard-of-care antibiotics for primary or recurrent C. difficile infection. Participants received an infusion of bezlotoxumab (10 mg per kilogram of body weight), actoxumab plus bezlotoxumab (10 mg per kilogram each), or placebo; actoxumab alone (10 mg per kilogram) was given in MODIFY I but discontinued after a planned interim analysis. The primary end point was recurrent infection (new episode after initial clinical cure) within 12 weeks after infusion in the modified intention-to-treat population. RESULTS In both trials, the rate of recurrent C. difficile infection was significantly lower with bezlotoxumab alone than with placebo (MODIFY I: 17% [67 of 386] vs. 28% [109 of 395]; adjusted difference, −10.1 percentage points; 95% confidence interval [CI], −15.9 to −4.3; P<0.001; MODIFY II: 16% [62 of 395] vs. 26% [97 of 378]; adjusted difference, −9.9 percentage points; 95% CI, −15.5 to −4.3; P<0.001) and was significantly lower with actoxumab plus bezlotoxumab than with placebo (MODIFY I: 16% [61 of 383] vs. 28% [109 of 395]; adjusted difference, −11.6 percentage points; 95% CI, −17.4 to −5.9; P<0.001; MODIFY II: 15% [58 of 390] vs. 26% [97 of 378]; adjusted difference, −10.7 percentage points; 95% CI, −16.4 to −5.1; P<0.001). In prespecified subgroup analyses (combined data set), rates of recurrent infection were lower in both groups that received bezlotoxumab than in the placebo group in subpopulations at high risk for recurrent infection or for an adverse outcome. The rates of initial clinical cure were 80% with bezlotoxumab alone, 73% with actoxumab plus bezlotoxumab, and 80% with placebo; the rates of sustained cure (initial clinical cure without recurrent infection in 12 weeks) were 64%, 58%, and 54%, respectively. The rates of adverse events were similar among these groups; the most common events were diarrhea and nausea. CONCLUSIONS Among participants receiving antibiotic treatment for primary or recurrent C. difficile infection, bezlotoxumab was associated with a substantially lower rate of recurrent infection than placebo and had a safety profile similar to that of placebo. The addition of actoxumab did not improve efficacy. (Funded by Merck; MODIFY I and MODIFY II ClinicalTrials.gov numbers, NCT01241552 and NCT01513239.

Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A

Background: Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme's active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016). Results: We performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1. Conclusions: The amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA

Low-temperature Near-field Scanning Optical Microscope for UV-visible Spectroscopy of Nanostructures

A low-temperature near-field scanning optical microscope (LT-NSOM) is designed and realized in order to study the optical properties of nanostructures at cryogenic temperatures in the UV-visible wavelength range. Due to the simple and extensive optical design, any type of NSOM operation mode is accessible. In particular, the localized photoluminescence spectra of nanostructures can be measured at temperatures from 13 K up to 300 K. We successfully demonstrate the performance of our LT-NSOM by investigating InGaN/GaN multiple quantum wells grown on a sapphire substrate at 15 K