Characterization of a novel wood mouse virus related to murid herpesvirus 4.

Two novel gammaherpesviruses were isolated, one from a field vole (Microtus agrestis) and the other from wood mice (Apodemus sylvaticus). The genome of the latter, designated wood mouse herpesvirus (WMHV), was completely sequenced. WMHV had the same genome structure and predicted gene content as murid herpesvirus 4 (MuHV4; murine gammaherpesvirus 68). Overall nucleotide sequence identity between WMHV and MuHV4 was 85 % and most of the 10 kb region at the left end of the unique region was particularly highly conserved, especially the viral tRNA-like sequences and the coding regions of genes M1 and M4. The partial sequence (71 913 bp) of another gammaherpesvirus, Brest herpesvirus (BRHV), which was isolated ostensibly from a white-toothed shrew (Crocidura russula), was also determined. The BRHV sequence was 99.2 % identical to the corresponding portion of the WMHV genome. Thus, WMHV and BRHV appeared to be strains of a new virus species. Biological characterization of WMHV indicated that it grew with similar kinetics to MuHV4 in cell culture. The pathogenesis of WMHV in wood mice was also extremely similar to that of MuHV4, except for the absence of inducible bronchus-associated lymphoid tissue at day 14 post-infection and a higher load of latently infected cells at 21 days post-infection.

Cellular uptake and transport of gold nanoparticles incorporated in a liposomal carrier.

Recent interest in using gold nanoparticles (Au NPs) for therapy in radiation medicine has motivated development of a liposome-based system to enhance their delivery to cells. In this study, liposomes were demonstrated to perform like a "Trojan Horse" to deliver small (1.4 nm) Au NPs into tumor cells by overcoming the energetically unfavorable endocytosis process for small NPs. The results reveal that the liposomal approach provides a thousand-fold enhancement in the cellular uptake of the small Au NPs. Real-time intracellular tracking of the Au NP-liposomes revealed an average speed of 12.48 +/- 3.12 microm/hr for their intracellular transport. Analysis of the time-dependent intracellular spatial distribution of the Au NP-liposomes demonstrated that they reside in lysosomes (final degrading organelles) within 40 minutes of incubation. Knowledge gained in these studies opens the door to pursuing liposomes as a viable strategy for delivery of Au NPs in radiation therapy applications. FROM THE CLINICAL EDITOR: Gold nanoparticles (Au NPs) as part of an optimized liposome-based delivery system have been proposed for therapy in radiation medicine. The approach resulted in a thousand-fold enhancement in the cellular uptake of Au NPs compared to conventional delivery methods, with the nanoparticles residing in lysosomes within 40 minutes of incubation.

Coronary artery perforation successfully treated with tris-acryl gelatin microsphere embolisation.

We describe a case of coronary artery perforation in a 76-year-old man, successfully treated by tris-acryl gelatin microsphere embolisation. This novel interventional embolic material is used in interventional radiology for arterial embolisation. We believe that this is the first report of its use for a coronary artery perforation.

Spatial and temporal mapping of heterogeneity in liposome uptake and microvascular distribution in an orthotopic tumor xenograft model.

Existing paradigms in nano-based drug delivery are currently being challenged. Assessment of bulk tumor accumulation has been routinely considered an indicative measure of nanomedicine potency. However, it is now recognized that the intratumoral distribution of nanomedicines also impacts their therapeutic effect. At this time, our understanding of the relationship between the bulk (i.e., macro-) tumor accumulation of nanocarriers and their intratumoral (i.e., micro-) distribution remains limited. Liposome-based drug formulations, in particular, suffer from diminished efficacy in vivo as a result of transport-limiting properties, combined with the heterogeneous nature of the tumor microenvironment. In this report, we perform a quantitative image-based assessment of macro- and microdistribution of liposomes. Multi-scalar assessment of liposome distribution was enabled by a stable formulation which co-encapsulates an iodinated contrast agent and a near-infrared fluorescence probe, for computed tomography (CT) and optical microscopy, respectively. Spatio-temporal quantification of tumor uptake in orthotopic xenografts was performed using CT at the bulk tissue level, and within defined sub-volumes of the tumor (i.e., rim, periphery and core). Tumor penetration and relative distribution of liposomes were assessed by fluorescence microscopy of whole tumor sections. Microdistribution analysis of whole tumor images exposed a heterogeneous distribution of both liposomes and tumor vasculature. Highest levels of liposome uptake were achieved and maintained in the well-vascularized tumor rim over the study period, corresponding to a positive correlation between liposome and microvascular density. Tumor penetration of liposomes was found to be time-dependent in all regions of the tumor however independent of location in the tumor. Importantly, a multi-scalar comparison of liposome distribution reveals that macro-accumulation in tissues (e.g., blood, whole tumor) may not reflect micro-accumulation levels present within specific regions of the tumor as a function of time.

Coronary intervention with a heparin-coated stent and aspirin only.

OBJECTIVES: To determine the safety and efficacy of a post-stenting anti-platelet regimen of aspirin without additional ticlopidine or clopidogrel after successful heparin-coated stent implantation. METHODS: A prospective, non-randomized, multi-center pilot study of patients undergoing percutaneous coronary intervention, including those with acute coronary syndromes and small vessels with one-month clinical follow-up, was undertaken. Patients received a heparin-coated stent and were treated with aspirin only. RESULTS: Over a period of 6 months, a total of 122 patients were recruited in 6 centers. Their mean age was 57.2 10.0 years, 79% were male and 31% had unstable angina. Most (75%) had single-vessel disease, predominantly of the left anterior descending artery (51%), with a mean reference diameter of 2.44 mm 0.44 mm at baseline and 2.48 0.41 mm post stenting. At a 1-month clinical follow-up, no major adverse cardiovascular events (including subacute stent thrombosis) had occurred. Five patients were readmitted to hospital for symptoms unrelated to the interventional procedure. CONCLUSIONS: Heparin-coated stent implantation using an antiplatelet regimen of aspirin only, appears to be safe and feasible. A randomized trial of a larger number of patients appears warranted.

Streptococcus mutans levels and caries prevalence in low-income schoolchildren.

Previous studies have shown that persons having high Streptococcus mutans levels in the saliva are "at risk" for dental caries. Most investigators agree, that if high levels of S. mutans were identified early in the life of at-risk children, dental decay could be reduced or eliminated through intervention. The purpose of this study is to show an association between S. mutans levels and caries prevalence in a sample of elementary school children. The study group consisted of 242 school children, ages 5-13 years. The subjects were divided into two age groups, 5-8 years and 9-13 years. Approximately 59 percent were African Americans. The sample of 242 children were equally females and males, 50 percent in each group. The Dentocult SM Test was used to make S. mutans determinations. The df-t index was used to determine the number of decayed and filled teeth of children ages 5-8 years; the DMF-T Index estimated the number of decayed, missing or filled teeth of children ages 9-13 years. Dental caries were found in 58 percent of the children (mean = 2.67, and range of 1-11). Approximately 47 percent of the children with caries had high S. mutans levels (100K-1M). Females had higher S. mutans levels than males in the 9-13 age group, p < .05. Analysis of Variance Test indicated that S. mutans levels for older females (ages 9-13) were significantly higher than those observed in males the same age (p < .01). This trend was not observed in younger children, ages 5-8 years. In addition, no significant difference or interaction was noted by sex for S. mutans levels and decayed or filled teeth (df-t) for younger children. We conclude that high levels of Streptococcus mutans are related to increased number of decayed teeth and conversely, low Streptococcus mutans levels are related to fewer dental caries. This study was supported in part by Colgate-Palmolive Company and the National Dental Association Foundation, Inc.

Assessing dental caries prevalence in African-American youth and adults.

It has been well documented that dental caries affect millions of children in the USA with the majority experiencing decay by the late teens. This is especially true for low-income minorities. The objective of this descriptive study was to determine dental caries prevalence in a sample of low-income African-American youth and adults. A total of 1034 individuals were examined. They were divided into two age groups: youth, 9-19 years and adults, 20-39 years. Females comprised approximately 65 percent (64.5) of the study group. The DMFT Index was used to determine caries prevalence in this study population. The DMFT findings showed that approximately 73 percent (72.9 percent) of the youth had either decayed, missing or filled teeth. Male youth had slightly higher DMFT mean scores than female youth: male mean = 7.93, standard error = 0.77, female mean = 7.52, standard error = 0.36; however, as females reached adulthood their DMFT scores increased substantially, mean = 15.18, standard error = 0.36. Caries prevalence was much lower in male adults, DMFT, mean = 7.22, standard error of 0.33. The decayed component for female adults mean score was 6.81, a slight increase over adult males, mean = 6.58. Although there were few filled teeth in both age groups, female adults had slightly more filled teeth than male adults, females mean = 2.91 vs. males; however, adult males experienced slightly more missing teeth, mean = 5.62 as compared to adult females, mean = 5.46. n = 2.20. Both female and male adults had an increase in missing teeth. As age increased there was a significant correlation among decayed, missing and filled teeth as tested by Analysis of Variance (ANOVA), p < 0.01. A significant correlation was found between filled teeth by sex, p < .005. We conclude that caries prevalence was higher in female and male youth, but dental caries increased more rapidly in females as they reached adulthood.

Image-based analysis of the size- and time-dependent penetration of polymeric micelles in multicellular tumor spheroids and tumor xenografts.

While the heightened tumor accumulation of systemically administered nanomedicines relative to conventional chemotherapeutic agents has been well established, corresponding improvements in therapeutic efficacy have often been incommensurate. This observation may be attributed to the limited exposure of cancer cells to therapy due to the heterogeneous intratumoral distribution and poor interstitial penetration of nanoparticle-based drug delivery systems. In the present work, the spatio-temporal distribution of block copolymer micelles (BCMs) of different sizes was evaluated in multicellular tumor spheroids (MCTS) and tumor xenografts originating from human cervical (HeLa) and colon (HT29) cancer cells using image-based, computational techniques. Micelle penetration was found to depend on nanoparticle size, time as well as tumor and spheroid cell line. Moreover, spheroids demonstrated the capacity to predict relative trends in nanoparticle interstitial transport in tumor xenografts. Overall, techniques are presented for the assessment of nanoparticle distribution in spheroids and xenografts and used to evaluate the influence of micelle size and cell-line specific tissue properties on micelle interstitial penetration.

A next-generation bioresorbable coronary scaffold system: from bench to first clinical evaluation: 6- and 12-month clinical and multimodality imaging results.

OBJECTIVES: This study sought to perform clinical and imaging assessments of the DESolve Bioresorbable Coronary Scaffold (BCS). BACKGROUND: BCS, which is drug eluting, may have potential advantages compared with conventional metallic drug-eluting stents. The DESolve system, designed to provide vessel support and neointimal suppression, combines a poly-l-lactic acid-based scaffold with the antiproliferative myolimus. METHODS: The DESolve First-in-Man (a non-randomized, consecutive enrollment evaluation of the DESolve myolimus eluting bioresorbable coronary stent in the treatment of patients with de novo native coronary artery lesions) trial was a prospective multicenter study enrolling 16 patients eligible for treatment. The principal safety endpoint was a composite of cardiac death, myocardial infarction, and clinically indicated target lesion revascularization. The principal imaging endpoint was in-scaffold late lumen loss (LLL) assessed by quantitative coronary angiography (QCA) at 6 months. Intravascular ultrasound (IVUS) and optical coherence tomography (OCT) imaging was performed at baseline and 6 months; multislice computed tomography (MSCT) was performed at 12 months. RESULTS: Acute procedural success was achieved in 15 of 15 patients receiving a study scaffold. At 12 months, there was no scaffold thrombosis and no major adverse cardiac events directly attributable to the scaffold. At 6 months, in-scaffold LLL (by QCA) was 0.19 ± 0.19 mm; neointimal volume (by IVUS) was 7.19 ± 3.56%, with no evidence of scaffold recoil or late malapposition. Findings were confirmed with OCT and showed uniform, thin neointimal coverage (0.12 ± 0.04 mm). At 12 months, MSCT demonstrated excellent vessel patency. CONCLUSIONS: This study demonstrated the feasibility and efficacy of the DESolve BCS. Results showing low in-scaffold LLL, low % neointimal volume at 6 months, no chronic recoil, and maintenance of lumen patency at 12 months prompt further study. (DESolve First-in-Man; EudraCT number 2011-000027-32).

First-in-human evaluation of a bioabsorbable polymer-coated sirolimus-eluting stent: imaging and clinical results of the DESSOLVE I Trial (DES with sirolimus and a bioabsorbable polymer for the treatment of patients with de novo lesion in the native coronary arteries).

OBJECTIVES: This first-in-human multicenter study sought to examine prospectively the safety and efficacy of a new, cobalt chromium thin-strut, coronary absorbable polymer-coated, sirolimus-eluting stent. BACKGROUND: Bioabsorbable polymers on drug-eluting stents may lower the long-term risks of inflammation, delayed healing, and adverse events. METHODS: We enrolled patients with symptomatic coronary artery disease with stable or unstable angina pectoris and >50% diameter stenosis, amenable to coverage with a ≤23-mm long stent in a vessel 2.5 to 3.5 mm in diameter. All patients received dual antiplatelet therapy after implantation. Patients, in groups of 10, underwent repeat angiography, intravascular ultrasound, and optical coherence tomography at 4, 6, or 8 months, and all patients were seen or contacted at 18 months of follow-up. RESULTS: The median (range) in-stent late lumen loss (LLL) was 0.03 mm (-0.22 to 0.21 mm), 0.10 mm (-0.03 to 1.2 mm), and 0.08 mm (-0.01 to 0.28 mm), at 4, 6, and 8 months, respectively. At 18 months, the median in-stent LLL was 0.08 mm (-0.30 to 0.46 mm). On optical coherence tomography, the proportion of uncovered stent struts decreased from a median of 7.3% (range 0.4% to 46.3%) at 4 months to 0% (range: 0% to 3.4%) at 18 months. The percentage of neointimal volume obstruction by intravascular ultrasound increased from a median of 5.3% to 9.1% between 4 and 6 months and remained nearly unchanged thereafter through 18 months of follow-up. The only recorded major adverse cardiac event was a myocardial infarction. CONCLUSIONS: At 18 months of follow-up, this absorbable polymer-coated, cobalt chromium sirolimus-eluting stent was associated with a low and stable in-stent LLL, complete strut coverage, and no stent thrombosis. (First-In-Human Trial of the MiStent Drug-Eluting Stent [DES] in Coronary Artery Disease [DESSOLVE-I]; NCT01247428).

Feasibility, safety, and efficacy of a novel polymeric pimecrolimus-eluting stent: traditional pre-clinical safety end points failed to predict 6-month clinical angiographic results.

OBJECTIVES: The aim of this study was to determine the safety and efficacy of a novel pimecrolimus-eluting stent in a porcine coronary model and in a phase I clinical trial. BACKGROUND: Rapamycin- and paclitaxel-eluting stents reduce the need for repeat intervention by limiting neointimal hyperplasia but might cause delayed healing, pre-disposing patients to late stent thrombosis. Because inflammation plays a key role in restenosis, pimecrolimus, an anti-inflammatory drug, might reduce restenosis without adversely affecting re-endothelialization. METHODS: We evaluated a novel polymeric pimecrolimus-eluting stent covered with a thin parylene C diffusion barrier in a porcine coronary model and in a phase I human clinical trial. The clinical study was a prospective, nonrandomized, first-in-human hypothesis-generating study that enrolled 15 patients who had a single de novo native coronary stenosis. RESULTS: At 28 days and 3 months in the porcine model, histopathologic indicators predicted safety and biocompatibility when stents coated with polymer only, drug only, and 2 drug-polymer formulations were compared with bare-metal stents (BMS). In the phase I clinical trial, 15 patients had successful implantation of pimecrolimus-eluting stents. By 6 months, no patient suffered death, myocardial infarction, or stent thrombosis. However, the angiographic restenosis (61%), mean late loss (1.44 mm), and repeat target lesion revascularization (53%) were significantly higher than historical BMS controls. Whereas the primary end point was percent volume obstruction, restenosis was so severe that operators performed intravascular ultrasound examination in only 6 patients. CONCLUSIONS: Pimecrolimus-eluting stents induced an exaggerated neointimal hyperplasia at 6 months in comparison with historical controls.

The "crush" technique for coronary artery bifurcation stenting: insights from micro-computed tomographic imaging of bench deployments.

OBJECTIVES: This study provides insights into "crush" coronary bifurcation stenting through imaging of bench deployments. BACKGROUND: Although the strategy of provisional side-branch stenting is widely accepted for suitable bifurcation lesions, there is no consensus on the best option for elective stenting with 2 stents. The crush technique has the potential to scaffold and apply the drug to the side-branch ostium where restenosis is most common. METHODS: Sequential steps of crush stent deployment and post-dilation were undertaken in silicone phantoms and recorded on cine angiography and microcomputed tomography. We assessed the effect of deployment strategies, post-dilation strategies, and cell size on side-branch ostial area. RESULTS: Side-branch ostial coverage by metal struts was 53% (95% confidence interval [CI]: 46 to 59) after 1-step kissing post-dilation and was reduced by 2-step kissing post-dilation to 33% (95% CI: 28 to 37; p < 0.0001). Although the residual stenosis after the classical crush strategy was 47% (95% CI: 39 to 53), it was 36% (95% CI: 31 to 40; p = 0.002) after mini-crush deployment. Stents with larger cell size (>3.5 mm diameter) had a residual stenosis of 37% (95% CI: 32 to 42) after crush deployment that was less than the residual stenosis for stents with smaller cell size (52%; 95% CI: 44 to 60; p < 0.0001). CONCLUSIONS: Side-branch ostial stenosis after crush stenting was minimized by mini-crush deployment, 2-step kissing post-dilation, and the use of stents with larger cell size. It is unknown if optimizing stent deployment at bifurcation lesions will reduce clinical stent thrombosis and restenosis.

Enzymatic detergent treatment protocol that reduces protease-resistant prion protein load and infectivity from surgical-steel monofilaments contaminated with a human-derived prion strain.

The unconventional nature of the infectious agent of prion diseases poses a challenge to conventional infection control methodologies. The extraneural tissue distribution of variant and sporadic Creutzfeldt-Jakob disease has increased concern regarding the risk of prion disease transmission via general surgical procedures and highlighted the need for decontamination procedures that can be incorporated into routine processing. In this study, the ability of preparations of enzymatic medical instrument cleaners to reduce the infectivity associated with a rodent-adapted strain of human prion disease, previously reported to be resistant to decontamination, was tested. Efficient degradation of the disease-associated prion protein by enzymatic cleaning preparations required high treatment temperatures (50-60 degrees C). Standard decontamination methods (1 M NaOH for 1 h or autoclaving at 134 degrees C for 18 min) reduced infectivity associated with the human-derived prion strain by less than 3 log10 LD50. In contrast, a 30 min treatment with the optimized enzymatic cleaning preparation protocols reduced infectivity by more than 3 log10 LD50 and when used in conjunction with autoclave cycles eliminated detectable levels of infectivity. The development of prion decontamination procedures that are compatible with routine cleaning and sterilization of medical and surgical instruments may reduce the risk of the transmission of prion disease in general surgery.

Periodontal disease and diabetes mellitus.

Infections of the tissue surrounding the teeth (periodontitis) are usually caused by anaerobic gram-negative microorganisms. This infection causes destruction of the supporting alveolar bone and can lead to tooth loss. Removal of these microorganisms can slow or arrest the progression of periodontitis. Diabetes patients are at greater risk of developing periodontitis, may not respond as well to periodontal therapy as nondiabetic patients, and may require more aggressive treatment to manage periodontitis. Microorganisms that cause periodontitis and the host response to these may increase insulin resistance in diabetic patients. Treatment of periodontitis could improve glycemic control. A model is presented in which periodontal pathogens may cause increases in proinflammatory cytokines that mediate increases in insulin resistance, resulting in an increase in blood glucose. Following periodontal therapy, this process may be reversed.

Properties of GDP-mannose pyrophosphorylase, a critical enzyme and drug target in Leishmania mexicana.

Leishmania parasites synthesize a range of mannose-containing glycoconjugates thought to be essential for virulence in the mammalian host and sandfly vector. A prerequisite for the synthesis of these molecules is the availability of the activated mannose donor, GDP-Man, the product of the catalysis of mannose-1-phosphate and GTP by GDP-mannose pyrophosphorylase (GDP-MP). In contrast to the lethal phenotype in fungi, the deletion of the gene in Leishmania mexicana did not affect parasite viability but led to a total loss of virulence, making GDP-MP an ideal target for anti-Leishmania drug development. We show by immunofluorescence and subcellular fractionation that GDP-MP is a cytoplasmic protein, and we describe a colorimetric activity assay suitable for the high throughput screening of small molecule inhibitors. We expressed recombinant GDP-MP as a fusion with maltose-binding protein and separated the enzyme from maltose-binding protein by thrombin cleavage, ion-exchange, and size exclusion chromatography. Size exclusion chromatography and analytical ultracentrifugation studies demonstrate that GDP-MP self-associates to form an enzymatically active and stable hexamer. However, sedimentation studies show that the GDP-MP hexamer dissociates to trimers and monomers in a time-dependent manner, at low protein concentrations, at low ionic strength, and at alkaline pH. Circular dichroism spectroscopy reveals that GDP-MP is comprised of mixed alpha/beta structure, similar to its closest related homologue, N-acetyl-glucoseamine-1-phosphate uridyltransferase (Glmu) from Streptococcus pneumoniae. Our studies provide insight into the structure of a novel target for the development of anti-Leishmania drugs.