Citations

INTRODUCTION: We examined autoimmunity markers (AIM) and autonomic dysfunction in patients with chronic neurogastroenterological symptoms and their relationship to joint hypermobility/hypermobility spectrum disorder (JH/HSD). METHODS: AIM positivity was defined as a diagnosis of known autoimmune/autoinflammatory disorder with at least 1 positive seromarker of autoimmunity or at least 2 positive seromarkers by themselves. Three cohorts were studied: (i) retrospective (n = 300), (ii) prospective validation cohort (n = 133), and (iii) treatment cohort (n = 40), administered open-label intravenous immunoglobulin (IVIG). RESULTS: AIM positivity was found in 40% and 29% of the retrospective and prospective cohorts, the majority of whom (71% and 69%, respectively) had autoinflammatory disorder. Significantly more patients with AIM had elevations of C-reactive protein (31% vs 15%, P < 0.001) along with an increased proportion of cardiovascular autonomic dysfunction (48% vs 29%; P < 0.001), small fiber neuropathy (20% vs 9%; P = 0.002), and HLADQ8 positivity (24% vs 13%, P = 0.01). Patients with JH/HSD were more likely to have AIM (43% vs 15%, P = 0.001) along with more severe autonomic and gastrointestinal (GI) symptom scores. IVIG treatment was associated with robust improvement in pain, GI, and autonomic symptoms, but adverse events were experienced by 62% of patients. DISCUSSION: Autoimmune markers and autonomic dysfunction are common in patients with unexplained GI symptoms, especially in those with JH/HSD. Many patients seem to respond to IVIG treatment, but this needs to be confirmed by controlled trials. These results highlight the need for vigilance for autoimmune and autonomic factors and JH/HSD in patients with neurogastroenterological disorders. Clinicaltrials.gov, NCT04859829.

Mesoporous silica nanoparticles coated with a lipid bilayer (protocells) are a stable, versatile and biocompatible delivery platform that allows surface engineering to target specific cells and intracellular cargo delivery. The endo‐lysosomal escape of this type of drug carriers is important to enhance the therapeutic efficacy of their cargo. Motoneurons can be specifically targeted by surface‐coating protocells with the subunit B of cholera toxin (CTB‐protocells). The central hypothesis was that CTB‐protocells escape endo‐lysosomal compartments after internalization by motoneurons. In a first study, fixed NSC‐34 motoneuron‐like cells were treated with fluorescently labeled CTB‐protocells (for 1, 3, 6 and 24 hours), immunostained for lysosomes (with anti‐LAMP1) or early endosomes (with anti‐EEA1) and imaged using multicolor confocal microscopy. In a second study, NSC‐34 cells treated with CTB‐protocells and labeled with the acidic vesicle tracker LysoTracker were visualized using time lapse confocal imaging. In these two studies, colocalization of CTB‐protocells with lysosomes or endosomes was analyzed using ImageJ and found to remain less than 20% up to 24 hours. The fraction of CTB‐protocells in endo‐lysosomes increased only slightly between 1 and 24 hours. In a third study, transmission electron microscopy was used to confirm the intracellular localization of CTB‐protocells in NSC‐34 motoneuron‐like cells. CTB‐protocells were visualized in the cytosol and were not detected within larger vacuole compartments after 24‐hour incubation. In conclusion, CTB‐protocells escape lysosomal degradation in motoneurons making them a suitable vehicle to target drug delivery to motoneurons.

Cholera toxin B modified protocells (CTB‐protocells) are a promising, customizable approach for targeting and delivering drugs to motoneurons. Understanding the endocytic mechanism and intracellular fate of CTB‐protocells is essential for safe therapeutic application and cargo delivery efficiency, and also to elucidate characteristics of motoneuron physiology that are not well understood. To assess the energy dependent uptake of CTB‐protocells by motoneurons, intracellular protocell fluorescence was measured at 4°C and 37°C. Confocal analysis demonstrated that after 3 h at 4°C, there is a 40% reduction in CTB‐protocell uptake compared to the uptake observed at 37°C (p<0.001). Pharmacological endocytic pathway inhibitors were used to elucidate the preferential endocytotic mechanism. Results showed that amiloride (macropinocytosis inhibitor) and methyl‐β‐cyclodextrin (inhibitor of cholesterol dependent endocytic processes) significantly reduced the uptake of CTB‐protocells (p<0.001), consistent with endocytosis being dependent on the integrity of lipid rafts. To track intracellular fate, immunofluorescence techniques and live confocal microscopy were used to demonstrate colocalization of CTB‐protocells with lysosomes, endoplasmic reticulum (ER) and Golgi apparatus at 1 h and 3 h. Colocalization of CTB‐protocells with lysosomes was less than 20%. Both, ER and Golgi colocalization increased from ~20 to ~40% in a 2‐h period (from 1 h to 3 h). To evaluate retrograde transport in motoneurons in vivo , CTB‐protocells were intrapleurally injected in rats. Evidence of protocells in phrenic nerve axons measured by protocell fluorescence intensity supported retrograde transport. In conclusion, uptake of CTB‐protocells is energy‐dependent and mediated principally by macropinocytosis. Additionally, CTB‐protocells are able to escape lysosomal degradation, and are trafficked to relevant subcellular compartments after endocytosis.

At present, there are limited delivery systems to target neurons in the central nervous system. Neurological diseases are commonly hindered by treatments lacking drug selectivity to the neurons of interest. In particular, motoneuron diseases will likely require selective targeting of therapeutic interventions, which can be delivered via the peripheral nervous system. Nanoparticles are novel drug delivery systems with exceptional therapeutic potential. Specifically, mesoporous silica nanoparticles coated with a lipid bilayer (protocells) are a suitable treatment vehicle to target the peripheral and central nervous system because they offer advantageous fusion characteristics including 1) increased surface area with larger loading capacity, 2) stability and 3) improved biocompatibility. A number of natural toxins exist that already target the nervous system and could be used as a targeting strategy. Cholera toxin produced by the bacterium Vibrio cholerae has an atoxic subunit (CTB) that binds to gangliosides present on neuronal membranes and is a reliable motoneuron tracer. We hypothesized that CTB‐coated protocells specifically target motoneurons. In culture, CTB uptake is evident in motoneuron‐like NSC‐34 cells but not in L6 muscle cells, consistent with the suitability of CTB as a motoneuron targeting strategy. Fluorescently labeled protocells (130 nm in diameter) were bound to CTB using biotin‐neutravidin. CTB‐protocells showed significantly greater motoneuron uptake compared to non‐CTB coated protocells when quantified by measurements of intracellular protocell fluorescence intensity. Confocal images were used for co‐localization of fluorescently labeled protocells and lysosomes. Protocells effectively escape endo‐lysosomal degradation delivering their cargo intracellularly. Biocompatibility was evidenced by lack of cytotoxicity in cultured motoneurons and unimpaired cellular proliferation. To evaluate the targeting efficacy of CTB‐protocells in tissue, we utilized isolated diaphragm muscle‐phrenic nerve preparations. Evidence of protocell uptake at diaphragm neuromuscular junctions measured by protocell fluorescence intensity supports uptake into phrenic motoneuron axon terminals. In conclusion, CTB‐protocells are a promising, customizable approach for targeting and delivering drugs to motoneurons in vivo.

Diseases of the central nervous system commonly affect motoneurons. In particular, spinal cord injury, aging and neurological disorders can cause motoneuron dysfunction or loss. There are no effective diagnostic tools or treatments that specifically target motoneurons. We hypothesized that nanoparticles can be used to target drug delivery to motoneurons. Specifically, mesoporous silica nanoparticles (MSNPs) may be ideal since they can be independently modified allowing loading of various cargos and surface‐engineered for bio‐functionality. The aim of this study was to characterize the role of nanoparticle size, concentration, time of incubation and surface coating on uptake by cultured motoneurons. Four different fluorescently‐labeled MSNPs were evaluated: PEG‐PEI‐Ace MSNPs of 150 nm and 50 nm, lipid coated MSNPs (protocell ‐ 140 nm) and PEG‐PEI MSNPs (50nm). PEG‐PEI‐Ace showed significant greater motoneuron uptake (p<0.05) after longer periods of incubation (12h for 50 nm & 24h for 150 nm). Protocells and PEG‐PEI achieved maximal uptake by 3h. Differentiated motoneurons displayed significant uptake at cell soma and dendrites. Regardless of cell density and duration of treatment, there was minimal evidence of cytotoxicity by any MSNP. In conclusion, MSNPs can be used to target delivery to motoneurons. Specifically, coating, size and concentration affect motoneuron uptake. Smaller and coated PEG‐PEI‐Ace MSNPs showed substantial uptake by motoneurons. These results may help inform the appropriate characteristics of MSNPs for specific in vivo application.

BackgroundThe ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non‐EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation.MethodsWe compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non‐target EVs in the final EV preparation.ResultsTo determine enrichment of prostate derived EVs relative to non‐target MPs, anti‐PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody‐based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein.ConclusionsWhile attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.

Background —The Telectronics Accufix pacing leads were recalled in November 1994 after 2 deaths and 2 nonfatal injuries were reported. This multicenter clinical study (MCS) of patients with Accufix leads was designed to determine the rate of spontaneous injury related to the J retention wire and results of lead extraction. Methods and Results —The MCS included 2589 patients with Accufix atrial pacing leads that were implanted at or who were followed up at 12 medical centers. Patients underwent cinefluoroscopic imaging of their lead every 6 months. The risk of J retention wire fracture was ≈5.6%/y at 5 years and 4.7%/y at 10 years after implantation. The annual risk of protrusion was 1.5%. A total of 40 spontaneous injuries were reported to a worldwide registry (WWR) that included data from 34 672 patients (34 892 Accufix leads), including pericardial tamponade (n=19), pericardial effusion (n=5), atrial perforation (n=3), J retention wire embolization (n=4), and death (n=6). The risk of injury was 0.02%/y (95% CI, 0.0025 to 0.072) in the MCS and 0.048%/y (95% CI, 0.035 to 0.067) in the WWR. A total of 5299 leads (13%) have been extracted worldwide. After recall in the WWR, fatal extraction complications occurred in 0.4% of intravascular procedures (16 of 4023), with life-threatening complications in 0.5% (n=21). Extraction complications increased with implant duration, female sex, and J retention wire protrusion. Conclusions —Accufix pacing leads pose a low, ongoing risk of injury. Extraction is associated with substantially higher risks, and a conservative management approach is indicated for most patients.

Background— Restenosis after percutaneous coronary intervention (PCI) is a major problem affecting 15% to 30% of patients after stent placement. No oral agent has shown a beneficial effect on restenosis or on associated major adverse cardiovascular events. In limited trials, the oral agent tranilast has been shown to decrease the frequency of angiographic restenosis after PCI. Methods and Results— In this double-blind, randomized, placebo-controlled trial of tranilast (300 and 450 mg BID for 1 or 3 months), 11 484 patients were enrolled. Enrollment and drug were initiated within 4 hours after successful PCI of at least 1 vessel. The primary end point was the first occurrence of death, myocardial infarction, or ischemia-driven target vessel revascularization within 9 months and was 15.8% in the placebo group and 15.5% to 16.1% in the tranilast groups ( P =0.77 to 0.81). Myocardial infarction was the only component of major adverse cardiovascular events to show some evidence of a reduction with tranilast (450 mg BID for 3 months): 1.1% versus 1.8% with placebo ( P =0.061 for intent-to-treat population). The primary reason for not completing treatment was ≥1 hepatic laboratory test abnormality (11.4% versus 0.2% with placebo, P <0.01). In the angiographic substudy composed of 2018 patients, minimal lumen diameter (MLD) was measured by quantitative coronary angiography. At follow-up, MLD was 1.76±0.77 mm in the placebo group, which was not different from MLD in the tranilast groups (1.72 to 1.78±0.76 to 80 mm, P =0.49 to 0.89). In a subset of these patients (n=1107), intravascular ultrasound was performed at follow-up. Plaque volume was not different between the placebo and tranilast groups (39.3 versus 37.5 to 46.1 mm 3 , respectively; P =0.16 to 0.72). Conclusions— Tranilast does not improve the quantitative measures of restenosis (angiographic and intravascular ultrasound) or its clinical sequelae.