Angiographic Safety and Efficacy of the ReSolv Flow-Diverting Stent in a Rabbit Model.

BACKGROUND: Bioresorbable polymer-based flow-diverting stents have potential benefits over existing metal devices. This study aimed to evaluate the safety and efficacy of the novel ReSolv device, which is a primarily polymer-based flow-diverting stent, using the in vivo rabbit sidewall saccular aneurysm model. METHODS: ReSolv stents were deployed in 14 New Zealand White rabbits that had undergone aneurysm creation procedures. Animals were allocated to follow-up time points of 1, 3, 6, 9, 12, 16, or 18 months. Angiographic images were evaluated by an independent neurointerventionalist blinded to follow-up time points for (1) in-stent stenosis, (2) parent vessel and jailed side branch patency, (3) wall apposition, and (4) aneurysm occlusion using the Raymond-Roy Occlusion Classification (RROC), O'Kelly Marotta grading scale, and the 4F flow diversion predictive score. Primary efficacy outcome was defined as RROC Class I or II. RESULTS: At a median follow-up time of 7.5 months, parent vessel (14/14) and jailed side (33/33) branches were patent in all cases. There was no development of thrombus on the stent or cases of significant in-stent stenosis, and all stents had good wall apposition. Adequate occlusion was found in 85.7% (n = 12) of animals, including an RROC Class I in 64.3% (n = 9) and RROC Class II in 21.4% (n = 3). CONCLUSIONS: The ReSolv stent shows encouraging angiographic safety and efficacy outcomes after placement in a rabbit sidewall saccular aneurysm model. Longer term studies are ongoing to determine eventual fate of the aneurysm, parent vessel, and jailed side branches after absorption of the polymer component of the stent.

Dynamic reprogramming of signaling upon met inhibition reveals a mechanism of drug resistance in gastric cancer.

The Met receptor tyrosine kinase is activated or genetically amplified in some gastric cancers, but resistance to small-molecule inhibitors of Met often emerges in patients. We found that Met abundance correlated with a proliferation marker in patient gastric tumor sections, and gastric cancer cell lines that have MET amplifications depended on Met for proliferation and anchorage-independent growth in culture. Inhibition of Met induced temporal changes in gene expression in the cell lines, initiated by a rapid decrease in the expression of genes encoding transcription factors, followed by those encoding proteins involved in epithelial-mesenchymal transition, and finally those encoding cell cycle-related proteins. In the gastric cancer cell lines, microarray and chromatin immunoprecipitation analysis revealed considerable overlap between genes regulated in response to Met stimulation and those regulated by signal transducer and activator of transcription 3 (STAT3). The activity of STAT3, extracellular signal-regulated kinase (ERK), and the kinase Akt was decreased by Met inhibition, but only inhibitors of STAT3 were as effective as the Met inhibitor in decreasing tumor cell proliferation in culture and in xenografts, suggesting that STAT3 mediates the pro-proliferative program induced by Met. However, the phosphorylation of ERK increased after prolonged Met inhibition in culture, correlating with decreased abundance of the phosphatases DUSP4 and DUSP6, which inhibit ERK. Combined inhibition of Met and the mitogen-activated protein kinase kinase (MEK)-ERK pathway induced greater cell death in cultured gastric cancer cells than did either inhibitor alone. These findings indicate combination therapies that may counteract resistance to Met inhibitors.

Decreased expression of BRCA1 in SK-BR-3 cells is the result of aberrant activation of the GABP Beta promoter by an NRF-1-containing complex.

BACKGROUND: BRCA1 has recently been identified as a potential regulator of mammary stem/progenitor cell differentiation, and this function may explain the high prevalence of breast cancer in BRCA1 mutation carriers, as well as the downregulation of BRCA1 in a large proportion of sporadic breast cancers. That is, loss of BRCA1 function results in blocked differentiation with expansion of the mammary stem/progenitor cells. Because BRCA1 also maintains genomic integrity, its loss could produce a pool of genetically unstable stem/progenitor cells that are prime targets for further transforming events. Thus, elucidating the regulatory mechanisms of BRCA1 expression is important to our understanding of normal and malignant breast differentiation. RESULTS: Loss of BRCA1 expression in the ErbB2-amplified SK-BR-3 cell line was found to be the result of loss of activity of the ets transcription factor GABP, a previously characterized regulator of BRCA1 transcription. The expression of the non-DNA binding GABPß subunit was shown to be deficient, while the DNA binding subunit, GABPα was rendered unstable by the absence of GABPß. Deletion analysis of the GABPß proximal promoter identified a potential NRF-1 binding site as being critical for expression. Supershift analysis, the binding of recombinant protein and chromatin immunoprecipitation confirmed the role of NRF-1 in regulating the expression of GABPß. The siRNA knockdown of NRF-1 resulted in decreased GABPß and BRCA1 expression in MCF-7 cells indicating that they form a transcriptional network. NRF-1 levels and activity did not differ between SK-BR-3 and MCF-7 cells, however the NRF-1 containing complex on the GABPß promoter differed between the two lines and appears to be the result of altered coactivator binding. CONCLUSIONS: Both NRF-1 and GABP have been linked to the regulation of nuclear-encoded mitochondrial proteins, and the results of this study suggest their expression is coordinated by NRF-1's activation of the GABPß promoter. Their linkage to BRCA1, a potential breast stem cell regulator, implies a connection between the induction of mitochondrial metabolism and breast differentiation.

Modified bacterial mutation test procedures for evaluation of peptides and amino acid-containing material.

Biological materials can release amino acids during the course of bacterial mutation testing. Low levels of released amino acids from soluble materials can cause moderate increases in the number of revertant colonies on the plate, whereas higher levels lead to overgrowth of the background lawn, making counting of revertant colonies impossible. For poorly soluble material, the released amino acids can be present at high levels in localized spots on the plate, leading to the growth of 'pseudorevertant' colonies. The 'treat and wash' modified preincubation method employed here is an adaptation of the treat and plate method (used for evaluation of antibiotics) and involves washing the bacteria free of test compound after a 90 min exposure prior to plating out on minimal plates. The MC overlay method is a modified version of the standard plate incorporation assay, in which a top overlay containing 4% high viscosity methylcellulose is used in place of agar to stabilize the test compound in solution, preventing precipitation and subsequent localized amino acid release. Both modified methods produce the expected results for negative and positive controls. Peptides [synthetic curtailed analogs of human parathyroid hormone, PTH(1-34) and Ostabolin-C] that produced false positive results or could not be evaluated owing to overgrowth of the background lawn using standard methods, showed no artifacts and no evidence of genotoxicity using the modified methods. It is concluded that the treat and wash and MC overlay methods are valid versions of the bacterial mutation test for avoiding complications associated with released amino acids.

Examination of the potential genotoxicity of pure capsaicin in bacterial mutation, chromosome aberration, and rodent micronucleus tests.

There is widespread dietary exposure to capsaicin in the form of chili peppers, while capsaicin's analgesic qualities have led to increased use of a topical herbal remedy in various impure forms. Most recently, injection of pure capsaicin has been proposed as a means of relieving a variety of debilitating diseases, in which case tissues would receive relatively high and direct exposure. The purpose of the present study, where a series of standard assays were performed in accordance with the Organisation for Economic Cooperation and Development guidance, was to clarify earlier conflicting reports concerning potential genotoxicity of capsaicin prior to administering it to patients in an injectable form. The results confirm the absence of genotoxic activity of high-purity capsaicin in the bacterial mutation and chromosome aberration tests. In addition, no evidence of cytotoxicity or genotoxicity was seen in the rat bone marrow micronucleus test, where systemic exposure to pure capsaicin was achieved using the subcutaneous route and a rising dose toleration protocol. It is concluded that pure capsaicin is not active in the standard battery of genotoxicity assays recommended by the International Conference on Harmonisation for evaluation of new medicines; earlier reported in vitro genotoxic activity is probably associated with mutagenic impurities in commercial grades of the material.

Identification of peptide inhibitors of Pseudomonas aeruginosa exotoxin A function using a yeast two-hybrid approach.

The yeast two-hybrid system was used to identify peptide inhibitors of exotoxin A of Pseudomonas aeruginosa with the goal of using these to design peptide-based drugs against the toxin. A random peptide library consisting of 10(7) peptides ranging in length from 16 to 63 residues was screened for peptides that interact with the C-domain of exotoxin A. From the 10(7) transformants screened, three unique peptides of 63, 61 and 25 amino acids in length were found to specifically interact with the enzyme domain. The genes encoding these peptides were cloned and expressed as fusion proteins with the maltose-binding protein. In vitro inhibition measurements indicated that two of the peptides were modest inhibitors of toxin enzyme activity. These peptides now provide the basis for the development of more potent inhibitors, which will serve as lead inhibitors for evolution of potent peptide-based therapeutics.