Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells in a rapid manner and with single-cell resolution. Impedance measurements are particularly well-suited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in the presence of both cell types.

Effects of antimicrobial peptides on membrane dynamics: A comparison of fluorescence and NMR experiments.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight resistant infections. They are commonly thought to kill bacteria by perturbing the permeability of their cell membranes. However, bacterial killing requires a high coverage of the cell surface by bound peptides, at least in the case of cationic and amphipathic AMPs. Therefore, it is conceivable that peptide accumulation on the bacterial membranes might interfere with vital cellular functions also by perturbing bilayer dynamics, a hypothesis that has been termed "sand in the gearbox". Here we performed a systematic study of such possible effects, for two representative peptides (the cationic cathelicidin PMAP-23 and the peptaibol alamethicin), employing fluorescence and NMR spectroscopies. These approaches are commonly applied to characterize lipid order and dynamics, but sample different time-scales and could thus report on different membrane properties. In our case, fluorescence anisotropy measurements on liposomes labelled with probes localized at different depths in the bilayer showed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptide-induced reduction in lipid lateral mobility. Finally, laurdan fluorescence indicated that peptide binding reduces water penetration below the headgroups region. Comparable effects were observed also in fluorescence experiments performed directly on live bacterial cells. By contrast, the fatty acyl chain order parameters detected by deuterium NMR spectroscopy remained virtually unaffected by addition of the peptides. The apparent discrepancy between the two techniques confirms previous sporadic observations and is discussed in terms of the different characteristic times of the two approaches. The perturbation of membrane dynamics in the ns timescale, indicated by the multiple fluorescence approaches reported here, could contribute to the antimicrobial activity of AMPs, by affecting the function of membrane proteins, which is strongly dependent on the physicochemical properties of the bilayer.

Toward a theranostic device for gliomas.

BACKGROUND: In the surgical management of glioblastoma, a highly aggressive and incurable type of brain cancer, identification and treatment of residual tissue is the most common site of disease recurrence. Monitoring and localized treatment are achieved with engineered microbubbles (MBs) by combining ultrasound and fluorescence imaging with actively targeted temozolomide (TMZ) delivery. METHODS: The MBs were conjugated with a near-infrared fluorescence probe CF790, cyclic pentapeptide bearing the RGD sequence and a carboxyl-temozolomide, TMZA. The efficiency of adhesion to HUVEC cells was assessed in vitro in realistic physiological conditions of shear rate and vascular dimensions. Cytotoxicity of TMZA-loaded MBs on U87 MG cells and IC50 were assessed by MTT tests. RESULTS: We report on the design of injectable poly(vinyl alcohol) echogenic MBs designed as a platform with active targeting ability to tumor tissues, by tethering on the surface a ligand having the tripeptide sequence, RGD. The biorecognition of RGD-MBs onto HUVEC cells is quantitatively proved. Efficient NIR emission from the CF790-decorated MBs was successfully detected. The conjugation on the MBs surface of a specific drug as TMZ is achieved. The pharmacological activity of the coupled-to-surface drug is preserved by controlling the reaction conditions. CONCLUSIONS: We present an improved formulation of PVA-MBs to achieve a multifunctional device with adhesion ability, cytotoxicity on glioblastoma cells and supporting imaging.

Association Between Psoas Muscle Sarcopenia and Long-Term Survival Following Elective Endovascular Aortic Repair.

INTRODUCTION: The aims of this study were as follows: i) to identify the prevalence of sarcopenia in patients undergoing elective endovascular aortic repair (EVAR) for intact abdominal aortic aneurysm; ii) to assess its association with perioperative morbidity and long-term survival; and iii) to estimate its diagnostic accuracy for prediction of 5-year mortality following the intervention. METHODS: We performed a retrospective review of all patients who underwent elective EVAR from January 1, 2010 through December 31, 2019. The lean psoas muscle area (LPMA; cm2 × HU) was calculated on computed tomography angiography by multiplying psoas muscle area and psoas muscle density. The main exposure variable for this study was the presence of preoperative sarcopenia (LPMA value < 350), and the study cohort was divided in two groups for all subsequent analyses. The primary endpoint was all-cause mortality. RESULTS: The study cohort eventually comprised 338 patients who underwent elective EVAR for intact abdominal aortic aneurysm. In the overall population, 154 patients (45.5%) were classified as sarcopenic. At baseline, patients with sarcopenia were older (mean age: 78 ± 5 versus 75 ± 7 y, P < 0.001) and had lower proportion of males (73.5% versus 93.5%, P < 0.001). At 5 y, the estimated survival rates were 52% versus 74% in sarcopenic and nonsarcopenic patients, respectively (P < 0.001). Using multivariate Cox proportional hazard regression, an independent association was identified between sarcopenia and all-cause mortality in the whole cohort (hazard ratio: 2.63, 95% confidence interval: 1.43-3.36, P = 0.009). CONCLUSIONS: Sarcopenia, defined as LPMA <350 as measured on preoperative computed tomography angiography, can be highly prevalent in patients undergoing elective EVAR. Although the intervention remains safe in the short term, presence of sarcopenia was significantly associated to lower long-term survival irrespective of patients' age or gender.

Prognostic interaction between age and sex on outcomes following carotid endarterectomy.

Background: The aim of this study was to assess the prognostic interaction between age and sex on peri-operative and follow-up outcomes following elective carotid endarterectomy (CEA) for asymptomatic and symptomatic carotid stenosis. Patients and methods: A retrospective review of all patients admitted to a single vascular unit who underwent elective CEA between January, 2015 and December, 2019 was performed. The primary endpoints of the study were overall survival (from index operation) and cumulative stroke rate at thirty days. Results: A total of 383 consecutive patients were included in this study; of these 254 (66.4%) were males. At baseline, males were younger (mean age 73.4±11 vs. 76.3±10 years, p=.01) and with lower proportion of octogenarians (20.4% vs. 28.7%, p=.05). The rate of stroke in symptomatic and asymptomatic patients (males vs. females) were as follows: a) whole cohort 1.9% vs. 2% (p=1.00) and 2.7% vs. 1.3% (p=.66), respectively; b) ≥80 years old 3.7% vs. 0% (p=1.00) and 4% vs. 5.9% (p=1.00), respectively; c) <80 years old 1.2% vs. 3.3% (p=.47) and 2.5% vs. 0% (p=.55), respectively. The 3-year survival estimates were significantly lower for males (84% vs. 92%, p=.03). After stratification by age groups, males maintained inferior survival rates in the strata aged <80 years (85% vs. 97%, p=.005), while no differences were seen in the strata aged ≥80 years (82% vs. 79%, p=.92). Using multivariate Cox proportional hazards, age (HR: 2.1, 95% CI: 1.29-3.3, p=.002) and male gender (HR: 2.5, 95% CI: 1.16-5.5, p=.02) were associated with increased hazards of all-cause mortality. Conclusions: In this study of elective CEA for asymptomatic and symptomatic carotid stenosis, similar peri-operative neurologic outcomes were found in both males and females irrespective of age. Despite being usually older, females have superior long-term survival rates.

Secondary Relining With Focal Flaring of Novel-Generation Balloon-Expandable Covered Stents for Endovascular Treatment of Significant Diameter Mismatch in the Aorto-Iliac Territory.

OBJECTIVES: The aim of this study was to report on the safety and feasibility of secondary relining with focal flaring of novel-generation balloon-expandable covered stents for endovascular treatment of significant diameter mismatch in the aorto-iliac territory. Significant diameter mismatch was defined as >20% difference in the nominal diameter between the intended proximal and distal landing zones. METHODS: Patient A was an 84-year-old man with prior abdominal aortic aneurysm open repair with a straight 20 mm Dacron tube. He presented with a right common iliac artery aneurysm (Ø88 mm) with contained rupture. The Gore Viabahn endoprosthesis (9 mm × 5 cm) was inserted proximally about 15 mm above the occluded ostium of the internal iliac artery. Subsequently, the BeGraft Aortic® (16 mm × 48 mm) was inserted proximally up to the common iliac artery origin; its proximal portion was flared to 22 mm. Patient B was a 77-year-old man with prior endovascular abdominal aortic aneurysm repair with a Medtronic Endurant stent-graft. He presented with occlusion of the right limb of the aortic endoprosthesis and thrombosis that extended down to the level of the superficial femoral artery. After mechanical thrombectomy, two Gore Viabahn endoprosthesis (first one, 8 mm × 10 cm; second one, 10 mm × 15 cm) were inserted into the right iliac limb. Subsequently, the BeGraft Aortic® (12mm × 39mm) was inserted proximally up to the gate of the aortic stent-graft; its proximal portion was flared to 16 mm. RESULTS: Technical success and clinical success were achieved in both patients. Imaging follow-up (6 months for Patient A, 12 months for Patient B) showed correct placement of all stent-grafts without any graft-related adverse event. The patients remained free from new reinterventions or recurrent symptoms. Patient A died 8 months after the index procedure from acute respiratory failure after community acquired pneumonia. CONCLUSION: Secondary relining with focal flaring of novel-generation balloon-expandable covered stents for endovascular treatment of significant diameter mismatch in the aorto-iliac territory is safe and feasible. Although mid-term results seem to be effective, longer follow-up is warranted to establish durability of the technique.

Novel Application of Custom-Made Stent Grafts with Inner Branches for Secondary Treatment After Stent Graft Migration of Previous Infrarenal Endovascular Aortic Repair.

PURPOSE: We present a novel application of custom-made stent grafts (CMSGs) with inner branches to incorporate target vessels (TVs) as an alternative to fenestrations or directional branches for secondary treatment after stent graft migration of previous infrarenal endovascular aortic repair (EVAR). CASE REPORT: Two consecutive patients with stent graft migration of previous EVAR were electively treated at our institution from January 1, 2018 through December 31, 2018. Stent graft migration was defined as radiologic evidence of stent graft displacement >10 mm. In both cases, a proximal type I endoleak was noted, and the residual infrarenal aorta above the previous endograft was unsuitable as the proximal landing zone for a nonfenestrated cuff. Repair was planned by means of a CMSG with 4 inner branches. The procedures were conducted in two-stage fashion to minimize the risk of spinal cord ischemia. The procedures were technically successful with a total of 8 TVs stented. Both patients did not suffer from any early (i.e., up to 30 days) major adverse events, and no access-site complications were noted. At one-year follow-up, computed tomography angiography showed regular placement of the CMSGs, widely patent TVs, absence of any type I or III endoleak, and stable sac size. No late reinterventions were recorded. CONCLUSIONS: Secondary treatment of stent graft migration after previous EVAR is safe and feasible using CSMGs with 4 inner branches. This technique is effective as showed by stable sac size and 100% freedom from TVI at mid-term imaging follow-up. Larger cohorts and longer follow-up are needed to confirm the preliminary results.

Secondary Endovascular Repair of Recurring Lesions and Perioperative Complications after Open Aortic Repair: The Complementary Role of Different Technical Solutions.

BACKGROUND: The aim of our article was to describe the complementary role of different technical solutions for secondary endovascular repair of recurring lesions and perioperative complications after open aortic repair (OAR). METHODS: We describe our clinical experience of secondary endovascular repair after OAR. We included in the analysis all consecutive patients who presented recurring lesions and perioperative complications after OAR and underwent secondary endovascular repair between January 1, 2015 and June 31, 2018. Both elective and nonelective cases were captured. Early end points were technical success, 30-day mortality, 30-day major adverse events, and 30-day vascular access complications. Late end points were survival and freedom from secondary interventions. RESULTS: Three different techniques were used in 6 patients: 2 cases of fenestrated-branched endovascular aortic repair (F-BEVAR), 2 cases of parallel-graft EVAR (pg-EVAR), and 2 cases of off-label use of standard devices. Technical success was 100%. One patient died within 30 days from acute pulmonary embolism. One patient developed acute kidney injury not requiring renal replacement therapy, whereas the remaining 4 patients were free from 30-day major adverse events. The cumulative rate of 30-day vascular access complications was 0%. All the 5 patients who survived the index hospitalization had ≥12 months of clinical and imaging follow-up. At the longest individual follow-up, they all were alive and free from secondary interventions. Computed tomography angiography showed in all cases sustained clinical success. CONCLUSIONS: Secondary endovascular repair of recurring lesions and perioperative complications after OAR is safe and feasible and offers a minimally invasive effective treatment option when a redo surgical operation would be associated with a considerable risk to the patient. Different solutions are available (including F-BEVAR, pg-EVAR, and off-label use of standard devices) and are complementary rather than competitive. Careful preoperative assessment and familiarity with advanced techniques are essential to achieve satisfactory outcomes.

Custom-Made Unibody Conical Endografts for Elective Endovascular Repair of Saccular Infrarenal Abdominal Aortic Aneurysms with Narrow Aortic Bifurcations-Novel Implementation of the Aortoaortic Concept.

The aortoaortic concept for endovascular repair (EVAR) of infrarenal abdominal aortic aneurysms (AAAs) is nearly forgotten but may constitute a valuable option for focal pathologies. Herein, we describe our experience using custom-made (CM) unibody conical endografts for saccular AAAs with narrow (≤20 mm wide) aortic bifurcation (AB) in three patients. Given the narrow AB, the option for a bifurcated stent graft was reputed not optimal. Therefore, we decided to further suggest the construction of a CM unibody conical stent graft with respect to unique anatomical characteristics of the patients. Technical success rate was 100%, and computed tomography angiography at first-month follow-up showed complete sealing proximally and distally with excellent conformability of the endografts in all the cases. All the patients were free from any-type endoleak (EL) and had no evidence of any endograft-related complication (i.e., fracture, thrombosis, or migration) or required any reintervention at their longest follow-up. We conclude that in the proper anatomic setting, the use of CM unibody conical endografts for elective EVAR of saccular AAAs with narrow AB is technically feasible with excellent short-term safety regarding ELs or migration.

Successful Off-Label Use of an Iliac Branch Device to Rescue an Occluded Aortofemoral Bypass Graft.

PURPOSE: To report an alternative approach for rescue of an occluded aortofemoral bypass using the Gore Excluder Iliac Branch Endoprosthesis (IBE). CASE REPORT: A 52-year-old man presented with acute right limb ischemia because of displaced and occluded iliac stents and was treated with aortofemoral bypass. On the third postoperative day, there was early bypass failure due to distal embolization from aortic thrombus. After fluoroscopy-guided balloon thrombectomy of the bypass, an endovascular bailout strategy was used. The Gore Excluder IBE was deployed below the renal arteries (with the external iliac limb opening in the surgical prosthesis and the gate opening within the aortic lumen). After antegrade catheterization of the gate, a Gore Viabahn endoprosthesis was inserted as the bridging endograft and deployed so that it landed just above the preimplanted aortoiliac kissing stents without overlapping them. Completion angiography showed technical success without complications; results were sustained at 1-year follow-up. CONCLUSION: The Gore Excluder IBE may represent a versatile solution for the rescue of complex cases when open surgery would be associated with a considerable risk. This off-label application of a well-recognized endovascular device is safe and feasible and may prove useful as a valuable alternative in properly selected patients.

Hybrid Treatment of a True Right Subclavian Artery Aneurysm Involving the Vertebral Artery using a Covered Stent.

Subclavian artery aneurysms (SAAs) are rare but potentially life- and limb-threatening. We present the case of a 69-year-old man with a true right SAA; the vertebral artery branched off the aneurysm and was the dominant one. A hybrid (combined open surgical and endovascular) repair was performed; the vertebral artery was anastomosed end to side to the common carotid artery through a right supraclavicular incision, then using a percutaneous high brachial artery access, a covered stent was deployed to exclude the SSA. The procedure was technically successful, and computed tomography angiography at 24 months showed regular placement of the endograft with blood flow within it and absence of any endograft-related complication (i.e., stent fracture/thrombosis/displacement or any-type detectable endoleak). This hybrid treatment is safe and feasible with good midterm results and may represent a valuable, less invasive alternative to conventional open surgical approaches.

Urgent Use of Gore Excluder Iliac Branch Endoprosthesis with Left Transaxillary Approach for Preservation of the Residual Hypogastric Artery: A Case Series.

BACKGROUND: Preservation of the residual hypogastric artery (HGA) in patients with previous endovascular aortic aneurysm repair (EVAR) may require complex operative strategies. We report an alternative technique to preserve the residual HGA with the Gore Excluder Iliac Branch endoprosthesis (IBE) in urgent situations. METHODS: We report the case of 2 high-risk patients (unfit for open surgery), with previous EVAR and exclusion of 1 HGA, treated in emergency setting. Both patients met the anatomical requirements for Gore IBE use. Due to lack of the native aortic bifurcation, we used a transaxillary approach to deploy a covered stent (Gore Viabahn) in the target HGA. RESULTS: Technical success was 100%. Computed tomography angiography at 30 days, 6 months, and 1 year showed regular placement of all endografts and patency of all residual HGAs without evidence of any endograft-related complication (i.e., stent fracture, stent thrombosis or stent displacement). There was not any detectable type 1, 2, or 3 endoleak at longest follow-up. CONCLUSIONS: Our case series shows the technical feasibility and the good results of this approach, which may prove useful when the native aortoiliac carrefour is no longer available. The procedure seems to be safe and effective, with optimal primary patency of the stent grafts, freedom from type 1, 2, and 3 endoleaks, and absence of pelvic ischemic complications.