ECHOGENIC LIPOSOMES FOR NITRIC OXIDE DELIVERY AND BREAST CANCER TREATMENT

Liposomes, also known as nontoxic, biodegradable, and non-immunogenic therapeutic delivery vehicles, have been proposed as a carrier for drugs and antitumor agents in cancer chemotherapy. Echogenic liposomes (ELIP) have the potential to entrap air or bioactive gas to enhance acoustic reflectivity in ultrasound and are used as a contrast agent. The innovative part of this study is based on a novel concept to encapsulate nitric oxide (NO) gas into ELIP, deliver it to breast cancer cells, and control its release via direct ultrasound exposure. Studies on the effect of NO in tumor biology have shown that a high levels of NO (> 300 nM) leads to cytostasis or apoptosis by decreasing the translation of several cell cycle proteins and stimulating cancer cell death by activating the p53 pathway. The central hypothesis is that NO gas can be packaged and delivered through a delivery methodology to breast cancer cells to facilitate tumor regression with minimal systemic toxicity. The primary goal of this thesis is to develop an echogenic liposomal solution that has the ability to encapsulate NO, to release NO locally upon ultrasound exposure, and to induce breast cancer cell death. NO-containing echogenic liposomes (NO-ELIP) were prepared by the freezing-under-pressure method previously developed in our laboratory. It was necessary to evaluate stability of NO-ELIP and release of NO from NO-ELIP by measuring echogenicity using intravascular ultrasound images. Breast cancer cell lines, MDA-MB-231 and MDA-MB-468, were selected to investigate the cytotoxic effects of NO liberated from NO-ELIP and their response to NO concentration. Ultrasound-triggered NO release from NO-ELIP using ultrasound activation was studied. It was demonstrated that NO-ELIP remained stable for 5 hours in bovine serum albumin. Delivery of NO using NO-ELIP induced cytotoxicity and programmed cell death of MDA-MB-231 and MDA-MB-468 after 5 hours of incubation. Enhancement of the NO-ELIP effect for therapeutic application was observed with ultrasound activation. This work demonstrates that NO-ELIP can incorporate and deliver NO to breast cancer cells providing increased NO stability and ultrasound-controlled NO release. Improved therapeutic effect with the use of NO-ELIP is expected to be found for breast cancer treatment.

Mechanism of surgical stress impairment of murine natural killer cell cytotoxicity

Natural killer cells may provide an important first line of defense against metastatic implantation of solid tumors. This antitumor function occurs during the intravascular and visceral lodgment phase of cancer dissemination, as demonstrated in small animal metastasis models. The role of the NK cell in controlling human tumor dissemination is more difficult to confirm, at least partially because of ethical restraints on experimental design. Nonetheless, a large number of solid tumor patient studies have demonstrated NK cell cytolysis of both autologous and allogeneic tumors.^ Of the major cancer therapeutic modalities, successful surgery in conjunction with other treatments offers the best possibility of cure. However, small animal experiments have demonstrated that surgical stress can lead to increased rates of primary tumor take, and increased incidence, size, and rapidity of metastasis development. Because the physiologic impact of surgical stress can also markedly impair perioperative antitumor immune function in humans, we examined the effect of surgical stress on perioperative NK cell cytolytic function in a murine preclinical model. Our studies demonstrated that hindlimb amputation led to a marked impairment of postoperative NK cell cytotoxicity. The mechanism underlying this process is complex and involves the postsurgical generation of splenic erythroblasts that successfully compete with NK cells for tumor target binding sites; NK cell-directed suppressor cell populations; and a direct impairment of NK cell recycling capacity. The observed postoperative NK cell suppression could be prevented by in vivo administration of pyrimidinone biologic response modifiers or by short term in vitro exposure of effector cells to recombinant Interleukin-2. It is hoped that insights gained from this research may help in the future development of NK cell specific perioperative immunotherapy relevant to the solid tumor patients undergoing cancer resection. ^

Avian area vasculosa and CAM as rapid in vivo pro-angiogenic and antiangiogenic models.

Angiogenesis, the development of new blood vessels from preexisting ones, is driven by coordinated signaling pathways governed by specific molecules, hemodynamic forces, and endothelial and periendothelial cells. The processes involve adhesion, migration, and survival machinery within the target endothelial and periendothelial cells. Factors that interfere with any of these processes may therefore influence angiogenesis either positively (pro-angiogenesis) or negatively (antiangiogenesis). The avian area vasculosa (AV) and the avian chorioallantoic membrane (CAM) are two useful tools for studying both angiogenesis and antiangiogenesis since they are amenable to both intravascular and topical administration of target, agents, are relatively rapid assays, and can be adapted very easily to study angiogenesis-dependent processes, such as tumor growth. Both models provide a physiological setting that permits investigation of pro-angiogenic and antiangiogenic agent interactions in vivo.

Comparison of a Novel Biodegradable Polymer Sirolimus-Eluting Stent With a Durable Polymer Everolimus-Eluting Stent: Results of the Randomized BIOFLOW-II Trial.

BACKGROUND Biodegradable polymers for release of antiproliferative drugs from drug-eluting stents aim to improve vascular healing. We assessed noninferiority of a novel ultrathin strut drug-eluting stent releasing sirolimus from a biodegradable polymer (Orsiro, O-SES) compared with the durable polymer Xience Prime everolimus-eluting stent (X-EES) in terms of the primary end point in-stent late lumen loss at 9 months. METHODS AND RESULTS A total of 452 patients were randomly assigned 2:1 to treatment with O-SES (298 patients, 332 lesions) or X-EES (154 patients, 173 lesions) in a multicenter, noninferiority trial. The primary end point was in-stent late loss at 9 months. O-SES was noninferior to X-EES for the primary end point (0.10±0.32 versus 0.11±0.29 mm; difference=0.00063 mm; 95% confidence interval, -0.06 to 0.07; Pnoninferiority<0.0001). Clinical outcome showed similar rates of target-lesion failure at 1 year (O-SES 6.5% versus X-EES 8.0%; hazard ratio=0.82; 95% confidence interval, 0.40-1.68; log-rank test: P=0.58) without cases of stent thrombosis. A subgroup of patients (n=55) underwent serial optical coherence tomography at 9 months, which demonstrated similar neointimal thickness among lesions allocated to O-SES and X-EES (0.10±0.04 mm versus 0.11±0.04 mm; -0.01 [-0.04, -0.01]; P=0.37). Another subgroup of patients (n=56) underwent serial intravascular ultrasound at baseline and 9 months indicating a potential difference in neointimal area at follow-up (O-SES, 0.16±0.33 mm(2) versus X-EES, 0.43±0.56 mm(2); P=0.04). CONCLUSIONS Compared with durable polymer X-EES, novel biodegradable polymer-based O-SES was found noninferior for the primary end point in-stent late lumen loss at 9 months. Clinical event rates were comparable without cases of stent thrombosis throughout 1 year of follow-up. CLINICAL TRIAL REGISTRATION URL http://www.clinicaltrials.gov. Unique identifier: NCT01356888.

Ultrasound-assisted versus conventional catheter-directed thrombolysis for acute iliofemoral deep vein thrombosis

BACKGROUND For patients with acute iliofemoral deep vein thrombosis, it remains unclear whether the addition of intravascular high-frequency, low-power ultrasound energy facilitates the resolution of thrombosis during catheter-directed thrombolysis. METHODS AND RESULTS In a controlled clinical trial, 48 patients (mean age 50±21 years, 52% women) with acute iliofemoral deep vein thrombosis were randomized to receive ultrasound-assisted catheter-directed thrombolysis (N=24) or conventional catheter-directed thrombolysis (N=24). Thrombolysis regimen (20 mg r-tPA over 15 hours) was identical in all patients. The primary efficacy end point was the percentage of thrombus load reduction from baseline to 15 hours according to the length-adjusted thrombus score, obtained from standardized venograms and evaluated by a core laboratory blinded to group assignment. The percentage of thrombus load reduction was 55%±27% in the ultrasound-assisted catheter-directed thrombolysis group and 54%±27% in the conventional catheter-directed thrombolysis group (P=0.91). Adjunctive angioplasty and stenting was performed in 19 (80%) patients and in 20 (83%) patients, respectively (P>0.99). Treatment-related complications occurred in 3 (12%) and 2 (8%) patients, respectively (P>0.99). At 3-month follow-up, primary venous patency was 100% in the ultrasound-assisted catheter-directed thrombolysis group and 96% in the conventional catheter-directed thrombolysis group (P=0.33), and there was no difference in the severity of the post-thrombotic syndrome (mean Villalta score: 3.0±3.9 [range 0-15] versus 1.9±1.9 [range 0-7]; P=0.21), respectively. CONCLUSIONS In this randomized controlled clinical trial of patients with acute iliofemoral deep vein thrombosis treated with a fixed-dose catheter thrombolysis regimen, the addition of intravascular ultrasound did not facilitate thrombus resolution. CLINICAL TRIAL REGISTRATION URL http://www.clinicaltrials.gov. Unique identifier: NCT01482273.

Ophthalmic complications and local anesthesia. Pathophysiology and types of eye complications after intraoral dental anesthesia, and clinical recommendations

The present article reviews the different types of ophthalmologic complications following administration of intraoral local anesthesia. Since the first report by Brain in 1936, case reports about that topic have been published regularly in the literature. However, clinical studies evaluating the incidence of ophthalmologic complications after intraoral local anesthesia are rarely available. Previous data point to a frequency ranging from 0.03% to 0.13%. The most frequently described ophthalmologic complications include diplopia (double vision), ptosis (drooping of upper eyelid), and mydriasis (dilatation of pupil). Disorders that rather affect periorbital structures than the eye directly include facial paralysis and periorbital blanching (angiospasm). Diverse pathophysiologic mechanisms and causes have been reported in the literature, with the inadvertent intravascular administration of the local anesthetic considered the primary reason. The agent as well as the vasopressor is transported retrogradely via arteries or veins to the orbit or to periorbital structures (such as the cavernous sinus) with subsequent anesthesia of nerves and paralysis of muscles distant from the oral cavity. In general the ophthalmologic complications begin shortly after administration of the local anesthesia, and disappear once the local anesthesia has subsided.

Systemic Scuticociliatosis (Philasterides dicentrarchi) in sharks.

Scuticociliatosis is an economically important, frequently fatal disease of marine fish in aquaculture, caused by histophagous ciliated protozoa in the subclass Scuticociliatida of the phylum Ciliophora. A rapidly lethal systemic scuticociliate infection is described that affected aquarium-captive zebra sharks (Stegostoma fasciatum), Port Jackson sharks (Heterodontus portusjacksoni), and a Japanese horn shark (Heterodontus japonicus). Animals died unexpectedly or after a brief period of lethargy or behavioral abnormality. Gross findings included necrohemorrhagic hepatitis and increased volumes of celomic fluid. Histologically, 1 or more of a triad of necrotizing hepatitis, necrotizing meningoencephalitis, and thrombosing branchitis were seen in all cases, with necrotizing vasculitis or intravascular fibrinocellular thrombi. Lesions contained variably abundant invading ciliated protozoa. Molecular identification by polymerase chain reaction from formalin-fixed tissues identified these as the scuticociliate Philasterides dicentrarchi (syn. Miamiensis avidus), a novel and potentially emergent pathogen in sharks.

Plasma T-cadherin negatively associates with coronary lesion severity and acute coronary syndrome.

AIMS This study evaluated associations between plasma T-cadherin levels and severity of atherosclerotic disease. METHODS AND RESULTS Three hundred and ninety patients undergoing coronary angiography were divided into three groups based on clinical and angiographic presentation: a group (n=40) with normal coronary arteries, a group (n=250) with chronic coronary artery disease and a group (n=100) with acute coronary syndrome. Plasma T-cadherin levels were measured by double sandwich ELISA. Intravascular ultrasound data of the left-anterior descending artery were acquired in a subgroup of 284 patients. T-cadherin levels were lower in patients with acute coronary syndrome than in normal patients (p=0.007) and patients with chronic coronary artery disease (p=0.002). Levels were lower in males (p=0.002), in patients with hypertension (p=0.002) and inpatients with diabetes (p=0.008), and negatively correlated with systolic blood pressure (p=0.014), body mass index (p=0.001) and total number of risk factors (p=0.001). T-cadherin negatively associated with angiographic severity of disease (p=0.001) and with quantitative intravascular ultrasound measures of lesion severity (p<0.001 for plaque, necrotic core and dense calcium volumes). Significant associations between T-cadherin and intravascular ultrasound measurements persisted even if the regression model was adjusted for the presence of acute coronary syndrome. Multivariate analysis identified a strong (p=0.002) negative association of T-cadherin with acute coronary syndrome, and lower T-cadherin levels significantly (p=0.002) associated with a higher risk of acute coronary syndrome independently of age, gender and cardiovascular risk factors. CONCLUSIONS A reduction in plasma T-cadherin levels is associated with increasing severity of coronary artery disease and a higher risk for acute coronary syndrome.

Impact of local endothelial shear stress on neointima and plaque following stent implantation in patients with ST-elevation myocardial infarction: A subgroup-analysis of the COMFORTABLE AMI-IBIS 4 trial.

BACKGROUND Numerous studies have demonstrated an association between endothelial shear stress (ESS) and neointimal formation after stent implantation. However, the role of ESS on the composition of neointima and underlying plaque remains unclear. METHODS Patients recruited in the Comfortable AMI-IBIS 4 study implanted with bare metal stents (BMS) or biolimus eluting stents (BES) that had biplane coronary angiography at 13month follow-up were included in the analysis. The intravascular ultrasound virtual-histology (IVUS-VH) and the angiographic data were used to reconstruct the luminal surface, and the stent in the stented segments. Blood flow simulation was performed in the stent surface, which was assumed to represent the luminal surface at baseline, to assess the association between ESS and neointima thickness. The predominant ESS was estimated in 3-mm segments and was correlated with the amount of neointima, neointimal tissue composition, and with the changes in the underlying plaque burden and composition. RESULTS Forty three patients (18 implanted with BMS and 25 with BES) were studied. In both stent groups negative correlations were noted between ESS and neointima thickness in BMS (P<0.001) and BES (P=0.002). In BMS there was a negative correlation between predominant ESS and the percentage of the neointimal necrotic core component (P=0.015). In BES group, the limited neointima formation did not allow evaluation of the effect of ESS on its tissue characteristics. ESS did not affect vessel wall remodeling and the plaque burden and composition behind BMS (P>0.10) and BES (P>0.45). CONCLUSIONS ESS determines neointimal formation in both BMS and BES and affects the composition of the neointima in BMS. Conversely, ESS does not impact the plaque behind struts irrespective of stent type throughout 13months of follow-up.

Visualization and quantification of air embolism structure by processing postmortem MSCT data.

Venous air embolism (VAE) is an often occurring forensic finding in cases of injury to the head and neck. Whenever found, it has to be appraised in its relation to the cause of death. While visualization and quantification is difficult at traditional autopsy, Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) offer a new potential in the diagnosis of VAE. This paper reports the findings of VAE in four cases of massive head injury examined postmortem by Multislice Computed Tomography (MSCT) prior to autopsy. MSCT data of the thorax were processed using 3D air structure reconstruction software to visualize air embolism within the vascular system. Quantification of VAE was done by multiplying air containing areas on axial 2D images by their reconstruction intervals and then by summarizing the air volumes. Excellent 3D visualization of the air within the vascular system was obtained in all cases, and the intravascular gas volume was quantified.

Stable coronary artery disease: revascularisation and invasive strategies

Stable coronary artery disease is the most common clinical manifestation of ischaemic heart disease and a leading cause of mortality worldwide. Myocardial revascularisation is a mainstay in the treatment of symptomatic patients or those with ischaemia-producing coronary lesions, and reduces ischaemia to a greater extent than medical treatment. Documentation of ischaemia and plaque burden is fundamental in the risk stratification of patients with stable coronary artery disease, and several invasive and non-invasive techniques are available (eg, fractional flow reserve or intravascular ultrasound) or being validated (eg, instantaneous wave-free ratio and optical coherence tomography). The use of new-generation drug-eluting stents and arterial conduits greatly improve clinical outcome in patients undergoing percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG). PCI is feasible, safe, and effective in many patients with stable coronary artery disease who remain symptomatic despite medical treatment. In patients with multivessel and left main coronary artery disease, the decision between PCI or CABG is guided by the local Heart Team (team of different cardiovascular specialists, including non-invasive and invasive cardiologists, and cardiac surgeons), who carefully judge the possible benefits and risks inherent to PCI and CABG. In specific subsets, such as patients with diabetes and advanced, multivessel coronary artery disease, CABG remains the standard of care in view of improved protection against recurrent ischaemic adverse events.

The Effect of Everolimus Initiation and Calcineurin Inhibitor Elimination on Cardiac Allograft Vasculopathy in De Novo Recipients: One-Year Results of a Scandinavian Randomized Trial.

Early initiation of everolimus with calcineurin inhibitor therapy has been shown to reduce the progression of cardiac allograft vasculopathy (CAV) in de novo heart transplant recipients. The effect of de novo everolimus therapy and early total elimination of calcineurin inhibitor therapy has, however, not been investigated and is relevant given the morbidity and lack of efficacy of current protocols in preventing CAV. This 12-month multicenter Scandinavian trial randomized 115 de novo heart transplant recipients to everolimus with complete calcineurin inhibitor elimination 7-11 weeks after HTx or standard cyclosporine immunosuppression. Ninety-five (83%) patients had matched intravascular ultrasound examinations at baseline and 12 months. Mean (± SD) recipient age was 49.9 ± 13.1 years. The everolimus group (n = 47) demonstrated significantly reduced CAV progression as compared to the calcineurin inhibitor group (n = 48) (ΔMaximal Intimal Thickness 0.03 ± 0.06 and 0.08 ± 0.12 mm, ΔPercent Atheroma Volume 1.3 ± 2.3 and 4.2 ± 5.0%, ΔTotal Atheroma Volume 1.1 ± 19.2 mm(3) and 13.8 ± 28.0 mm(3) [all p-values ≤ 0.01]). Everolimus patients also had a significantly greater decline in levels of soluble tumor necrosis factor receptor-1 as compared to the calcineurin inhibitor group (p = 0.02). These preliminary results suggest that an everolimus-based CNI-free can potentially be considered in suitable de novo HTx recipients.

Everolimus initiation and early calcineurin inhibitor withdrawal in heart transplant recipients: a randomized trial.

In a randomized, open-label trial, everolimus was compared to cyclosporine in 115 de novo heart transplant recipients. Patients were assigned within 5 days posttransplant to low-exposure everolimus (3–6 ng/mL) with reduced-exposure cyclosporine (n = 56), or standard-exposure cyclosporine (n = 59), with both mycophenolate mofetil and corticosteroids. In the everolimus group, cyclosporine was withdrawn after 7–11 weeks and everolimus exposure increased (6–10 ng/mL). The primary efficacy end point, measured GFR at 12 months posttransplant, was significantly higher with everolimus versus cyclosporine (mean ± SD: 79.8 ± 17.7 mL/min/1.73 m2 vs. 61.5 ± 19.6 mL/min/1.73 m2; p < 0.001). Coronary intravascular ultrasound showed that the mean increase in maximal intimal thickness was smaller (0.03 mm [95% CI 0.01, 0.05 mm] vs. 0.08 mm [95% CI 0.05, 0.12 mm], p = 0.03), and the incidence of cardiac allograft vasculopathy (CAV) was lower (50.0% vs. 64.6%, p = 0.003), with everolimus versus cyclosporine at month 12. Biopsy-proven acute rejection after weeks 7–11 was more frequent with everolimus (p = 0.03). Left ventricular function was not inferior with everolimus versus cyclosporine. Cytomegalovirus infection was less common with everolimus (5.4% vs. 30.5%, p < 0.001); the incidence of bacterial infection was similar. In conclusion, everolimus-based immunosuppression with early elimination of cyclosporine markedly improved renal function after heart transplantation. Since postoperative safety was not jeopardized and development of CAV was attenuated, this strategy may benefit long-term outcome.

Effect of everolimus introduction on cardiac allograft vasculopathy--results of a randomized, multicenter trial.

BACKGROUND Everolimus reduces the progression of cardiac allograft vasculopathy (CAV) in de novo heart transplant (HTx) recipients, but the influence on established CAV is unknown. METHODS In this Nordic Certican Trial in Heart and lung Transplantation substudy, 111 maintenance HTx recipients (time post-HTx 5.8 ± 4.3 years) randomized to everolimus+reduced calcineurin inhibitor (CNI) or standard CNI had matching (intravascular ultrasound) examinations at baseline and 12 months allowing accurate assessment of CAV progression. RESULTS No significant difference in CAV progression was evident between the treatment groups (P = 0.30). When considering patients receiving concomitant azathioprine (AZA) therapy (n = 39), CAV progression was attenuated with everolimus versus standard CNI (Δmaximal intimal thickness 0.00 ± 0.04 and 0.04 ± 0.04 mm, Δpercent atheroma volume 0.2% ± 3.0% and 2.6% ± 2.5%, and Δtotal atheroma volume 0.25 ± 14.1 and 19.8 ± 20.4 mm(3), respectively [P < 0.05]). When considering patients receiving mycophenolate mofetil (MMF), accelerated CAV progression occurred with everolimus versus standard CNI (Δmaximal intimal thickness 0.06 ± 0.12 vs. 0.02 ± 0.06 mm and Δpercent atheroma volume 4.0% ± 6.3% vs. 1.4% ± 3.1%, respectively; P < 0.05). The levels of C-reactive protein and vascular cell adhesion molecule-1 declined significantly with AZA+everolimus, whereas MMF+everolimus patients demonstrated a significant increase in levels of C-reactive protein, vascular cell adhesion molecule-1, and von Willebrand factor. CONCLUSIONS Conversion to everolimus and reduced CNI does not influence CAV progression among maintenance HTx recipients. However, background immunosuppressive therapy is important as AZA+everolimus patients demonstrated attenuated CAV progression and a decline in inflammatory markers, whereas the opposite pattern was seen with everolimus+MMF. The different effect of everolimus when combined with AZA versus MMF could potentially reflect hitherto unknown interactions.

Changes of coronary plaque composition correlate with C-reactive protein levels in patients with ST-elevation myocardial infarction following high-intensity statin therapy.

OBJECTIVES Levels of inflammatory biomarkers associate with changes of coronary atheroma burden in statin-treated patients with stable coronary artery disease. This study sought to determine changes of plaque composition in vivo in relation to high-sensitivity C-reactive protein (hs-CRP) levels in patients with ST-elevation myocardial infarction (STEMI) receiving high-intensity statin therapy. METHODS The IBIS-4 study performed serial (baseline and 13-month), 2-vessel intravascular ultrasound (IVUS) and radiofrequency-IVUS of the non-infarct-related arteries in patients with STEMI treated with high-intensity statin therapy. The present analysis included 44 patients (80 arteries) with serial measurements of hs-CRP. RESULTS At follow-up, median low-density lipoprotein cholesterol (LDL-C) levels decreased from 126 to 77 mg/dl, HDL-C increased from 44 to 47 mg/dl, and hs-CRP decreased from 1.6 to 0.7 mg/L. Regression of percent atheroma volume (-0.99%, 95% CI -1.84 to -0.14, p = 0.024) was accompanied by reduction of percent fibro-fatty (p = 0.04) and fibrous tissue (p < 0.001), and increase in percent necrotic core (p = 0.006) and dense calcium (p < 0.001). Follow-up levels of hs-CRP, but not LDL-C, correlated with changes in percent necrotic core (p = 0.001) and inversely with percent fibrous tissue volume (p = 0.008). Similarly, baseline-to-follow-up change of hs-CRP correlated with the change in percent necrotic core volume (p = 0.02). CONCLUSIONS In STEMI patients receiving high-intensity statin therapy, stabilization of VH-IVUS-defined necrotic core was confined to patients with lowest on-treatment levels and greatest reduction of hs-CRP. Elevated CRP levels at follow-up may identify progression of high-risk coronary plaque composition despite intensive statin therapy and overall regression of atheroma volume.