Podocyte depletion precedes progressive glomerular damage in several kidney diseases. However, the current standard of visual detection and quantification of podocyte nuclei from brightfield ...microscopy images is laborious and imprecise.
We have developed PodoSighter, an online cloud-based tool, to automatically identify and quantify podocyte nuclei from giga-pixel brightfield whole-slide images (WSIs) using deep learning. Ground-truth to train the tool used immunohistochemically or immunofluorescence-labeled images from a multi-institutional cohort of 122 histologic sections from mouse, rat, and human kidneys. To demonstrate the generalizability of our tool in investigating podocyte loss in clinically relevant samples, we tested it in rodent models of glomerular diseases, including diabetic kidney disease, crescentic GN, and dose-dependent direct podocyte toxicity and depletion, and in human biopsies from steroid-resistant nephrotic syndrome and from human autopsy tissues.
The optimal model yielded high sensitivity/specificity of 0.80/0.80, 0.81/0.86, and 0.80/0.91, in mouse, rat, and human images, respectively, from periodic acid-Schiff-stained WSIs. Furthermore, the podocyte nuclear morphometrics extracted using PodoSighter were informative in identifying diseased glomeruli. We have made PodoSighter freely available to the general public as turnkey plugins in a cloud-based web application for end users.
Our study demonstrates an automated computational approach to detect and quantify podocyte nuclei in standard histologically stained WSIs, facilitating podocyte research, and enabling possible future clinical applications.
3D bioprinting has become a versatile and powerful method in tissue engineering and regenerative medicine and is increasingly adapted by other disciplines due to its tremendous potential beyond its ...typical applications. However, commercially available 3D bioprinting systems are typically expensive circumventing the broad implementation, including laboratories in low-resource settings. To address the limitations of conventional and commercially available technology, we developed a 3D bioprinter by modification of an off-the-shelf 3D desktop printer, that can be installed within a single day, is of handy size to fit into a standard laminar flow hood, customizable, ultra-low cost and thus, affordable to a broad range of research labs, or educational institutions. We evaluate accuracy and reproducibility of printing results using alginate and alginate/gelatin-hydrogels and demonstrate its potential for biomedical use by printing of various two-and three-dimensional cell-free and mammalian cell-laden objects using recombinant HEK
cells, stably expressing yellow fluorescent protein (YFP) as a model system and high-content imaging. We further provide a parts list and 3D design files in STL and STEP format for reconstructing the device. A time-lapse video of the custom-built device during operation is available at https://vimeo.com/274482794.
OBJECTIVES
The aim of this study is to investigate the clinical characteristics and postoperative outcome of patients with a bicuspid aortic valve (BAV) suffering acute dissection in comparison with ...their tricuspid peers.
METHODS
Between 1995 and 2011, 460 consecutive patients underwent emergency repair for acute type A aortic dissection. In 379 patients without connective tissue disease, the aortic valve morphology could clearly be specified (91.6% tricuspid and 8.4% bicuspid).
RESULTS
At the time of dissection, patients with a bicuspid valve were younger (46.7 ± 13 vs 61.6 ± 12 years, P < 0.001) with the entry tear more often located in the root compared with those with a tricuspid valve (bicuspid: 31.3% vs tricuspid: 6.3%, P < 0.001). Consequently, surgical repair warranted root replacement in 93.8% of bicuspid vs 28.8% of tricuspid valve patients (P < 0.001). The leading pathology was medial necrosis/degeneration in bicuspid and atherosclerosis in tricuspid patients (P = 0.166). Hospital mortality was 20.3% and not significantly different between the two valve morphologies, even despite the younger age of bicuspid patients: 28.1% among bicuspids vs 19.6% among tricuspids (P = 0.255). Survival after discharge was 63.3% at 10 years for all patients. BAV patients had a significantly better survival with 100% at 10 years compared with 60.2% in tricuspid valve patients (P = 0.011). Mean follow-up among survivors was comparable for bicuspid and tricuspid patients (3.7 and 4.1 years, respectively).
CONCLUSIONS
Patients with BAV have a distinctive dissection pattern with the entry tear frequently located in the aortic root and—despite their younger age—are subject to substantial hospital mortality. For bicuspid patients suffering from dissection, composite root replacement yields an excellent outcome equal to an age- and gender-matched normal population.
Bone-marrow endothelial cells in the haematopoietic stem-cell niche form a network of blood vessels that regulates blood-cell traffic as well as the maintenance and function of haematopoietic stem ...and progenitor cells. Here, we report the design and in vivo performance of systemically injected lipid-polymer nanoparticles encapsulating small interfering RNA (siRNA), for the silencing of genes in bone-marrow endothelial cells. In mice, nanoparticles encapsulating siRNA sequences targeting the proteins stromal-derived factor 1 (Sdf1) or monocyte chemotactic protein 1 (Mcp1) enhanced (when silencing Sdf1) or inhibited (when silencing Mcp1) the release of stem and progenitor cells and of leukocytes from the bone marrow. In a mouse model of myocardial infarction, nanoparticle-mediated inhibition of cell release from the haematopoietic niche via Mcp1 silencing reduced leukocytes in the diseased heart, improved healing after infarction and attenuated heart failure. Nanoparticle-mediated RNA interference in the haematopoietic niche could be used to investigate haematopoietic processes for therapeutic applications in cancer, infection and cardiovascular disease.
BACKGROUND—The choice of the best conduit for root/ascending disease and its impact on longevity remain controversial in quinquagenarians.
METHODS AND RESULTS—A total of 205 patients (men=155) ...between 50 and 60 years (mean, 55.7±2.9 years) received either a stentless porcine xenoroot (n=78) or a mechanically valved composite prosthesis (n=127) between February 1998 and July 2011. Of these, 166 patients underwent root replacement for aneurysmal disease (porcine39% n=65; mechanical61% n=101; P=0.5), 25 for acute type A aortic dissection (porcine32% n=8; mechanical68% n=17; P=0.51), and 14 for endocarditis/iatrogenic injury involving the aortic root (6.4% n=5 versus 7.1% n=9; P=1.0). The predominant aortic valve pathology was stenosis in 19% (n=38), regurgitation in 50% (n=102), combined valvular dysfunction in 26% (n=54), and normal aortic valve function in 5% (n=11). Concomitant procedures included coronary artery bypass grafting (13%), mitral valve repair (7%), and partial/complete arch replacement (12%/4%), with no significant differences between porcine and mechanical root replacement. Overall hospital mortality was 7.3%, with no difference between the 2 types of valve prostheses (7.7% for porcine and 7.1% for mechanical root replacement; P=1.0). Follow-up averaged 5.4±3.7 years (1096 patient-years) and was 100% complete. Freedom from aorta-related reoperation at 12 years was not statistically different between the groups (porcine94.9% versus mechanical96.1%; P=0.73). Survival was equivalent between both groups, with a 5-year survival of 86±3% (porcine88±4%; mechanical85±3%; P=0.96) and a 10-year survival of 76% (porcine80±7%; mechanical75±5%; P=0.84). The linearized mortality rate was 3.1%/patient-year (porcine2.9%/patient-year; mechanical3.2%/patient-year).
CONCLUSIONS—In quinquagenerians, long-term survival after stentless porcine xenograft aortic root replacement is equivalent to that after a mechanical Bentall procedure. These results bring into question the predominance of mechanical composite conduits for root replacement in quinquagenerians, particularly in the current era of transcatheter valve-in-valve procedures for structural valve deterioration.
Paraplegia remains the most devastating complication after thoracic and thoracoabdominal aortic aneurysm (TAA/A) repair. The collateral network (CN) concept of spinal cord perfusion suggests ...segmental artery (SA) occlusion to mobilize redundant intraspinal and paraspinal arterial sources and ultimately trigger arteriogenesis, leading to spinal cord blood flow restoration within 96 to 120 hours. This principle is used by the two-staged approach to TAA/A-repair--which has lead to an elimination of paraplegia in an experimental model. However, the clinical implementation of a two-staged surgical procedure is challenging, particularly in the absence of an appropriate vascular segment for a "staged" open anastomosis or an appropriate endovascular landing zone. Selective, transfemoral minimally invasive SA coil embolization (MISACE) could provide the solution for one-stage repair of extensive aortic pathologies by triggering arteriogenic CN preconditioning and thereby allowing for recruitment of otherwise redundant arterial collaterals to the spinal cord.
The feasibility of MISACE was explored in a single animal using an established piglet model. A 6F sheet was introduced via the femoral artery, and a 4F standard Judkins catheter was used for selective angiography and coil insertion. All thoracic and lumbar aortic SAs (15 pairs; Th4-L5) were successfully identified by dye injection. Pediatric platinum endovascular coils (Trufill Pushable Coils, 3 × 20 mm; Cordis, Waterloo, Belgium) were deployed to serially occlude the SA mimicking a CN preconditioning procedure.
All intercostal (thoracic) and lumbar aortic SAs (Th4-L5) were successfully identified and occluded by coil embolization. Successful SA coil embolization was verified intraoperatively by selective dye injection on angiography. No intraoperative coil dislodgement occurred. Autopsy revealed complete occlusion of all embolized SAs enhanced by early local thrombus formation. Thrombotic material was found only distally to the coils. No SA dissection was observed at the aortic SA origins.
The MISACE technique allows for rapid serial endovascular occlusion of all thoracic and lumbar SAs. This new innovative approach bares the potential to CN preconditioning at the respective level of aortic pathology--to allow for adequate perioperative spinal cord blood supply--before conventional open or endovascular surgery. Selective, transarterial MISACE might lead to a dramatic reduction of ischemic spinal cord injury after open and endovascular TAA/A repair in the future.
In the past few years, five different somatostatin (SRIF) receptor subtypes (sst
1–5) have been identified, which form a distinct group in the superfamily of G-protein—coupled receptors. The ...naturally occurring somatostatins SRIF-28, SRIF-25, and SRIF-14 all reveal high-affinity binding for sst
1–5. In contrast, short synthetic analogs that are in clinical use, such as SMS 201–995, RC 160, or BIM 23014, primarily interact with the sst
2 subtype. Some SRIF analogs were previously reported to be selective for one SRIF receptor subtype, eg, the sst
2 (MK 678), the sst
3 (BIM 23056), or the sst
5 (BIM 23052, L362–L855) subtype. However, when we studied the binding affinities of these SRIF analogs for human (h) sst
1–5 expressed in either CHO or COS-1 cells, we were unable to confirm these previously reported selectivities. The absence of sst antagonists is a major drawback for investigating the functional role of each sst subtype. We used site-directed mutagenesis to identify amino acids that determine ligand specificity for sst
2. A single Ser305 to Phe mutation in TM VII increased the affinity of hsst
1 for SMS 201–995 nearly 100-fold, and when Gln291 was also exchanged to Asn in TM VII of hsst
1, almost full sst
2-like binding of SMS 201–995 was obtained. These data may aid in the design and synthesis of new selective type sst ligands. We have identified the expression of sst subtypes in nonclassical SRIF target tissue such as the lung. The pK
i values for SRIF and various SRIF analogs in rat lung tissue preparations were in close correlation with those obtained for CHO cells expressing the sst
4 subtype. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the predominant expression of mRNA specific for sst
4 in mouse, rat, and human lung tissue, confirmed by autoradiographies of rat lung. No specific binding for
125ITyr
3-SMS 201–995 was detected, since SMS 201–995 has low affinity for sst
4. In contrast, specific binding of
125ISRIF-28 to rat lung sections was demonstrated, which could be displaced by unlabelled SRIF-14 and SRIF-28, indicating specific, high affinity binding of this radioligand to sst
4 receptors.
The immunosuppressant everolimus used in organ transplantation is formulated as a conventional tablet for adults and a dispersible tablet that can be administered in water for pediatric use. As part ...of the pediatric clinical development program, the relative bioavailability and food effect for the dispersible tablet were evaluated in healthy adult subjects as a prelude to characterizing the steady‐state pharmacokinetics in pediatric kidney allograft recipients. In a randomized, open‐label, three‐way crossover study, 24 healthy adults received single 1.5‐mg oral doses of everolimus as (1) six 0.25‐mg dispersible tablets in water, (2) two 0.75‐mg conventional tablets, and (3) six 0.25‐mg dispersible tablets in water after a high‐fat breakfast. Cmax and AUC were evaluated by standard bioequivalence testing to determine relative bioavailability and to quantify the effect of food. In a multicenter open‐label efficacy/safety trial, pediatric renal allograft recipients received 0.8 mg/m2 (maximum 1.5 mg) bid everolimus as dispersible tablets in water. Serial trough concentrations over the first week and a steady‐state pharmacokinetic profile on day 7 posttransplant were collected in 19 patients ranging from ages 2 to 16 years old. The bioavailability of everolimus from the dispersible tablet was 10% lower relative to the conventional tablet, with a ratio (90% confidence interval) of 0.90 (0.76–1.07). After a high‐fat meal, tmax was delayed by a median 2.5 hours, and Cmax was reduced by 50%. Overall absorption, however, was not affected by food inasmuch as the fed/fasting AUC ratio was 0.99 (0.83–1.17). In pediatric patients, steady state was reached between days 3 and 5. The corresponding steady‐state parameters were as follows: Cmin, 4.4 ± 1.7 ng/ml; Cmax, 13.6 ± 4.2 ng/ml; and AUC, 87 ± 27 ng•h/ml. Steady‐state concentration‐time profiles in pediatric transplant patients receiving the dispersible tablet were comparable to those of adult patients receiving the conventional tablet when both were dosed to yield similar trough concentrations. If a pediatric patient is converted from the everolimus dispersible tablet to the conventional tablet, this should be based on a 1:1 milligram switch with subsequent therapeutic drug monitoring to further individualize the dose as needed. The dispersible tablet formulation should be taken consistently either with or without food to minimize fluctuations in exposure over time.
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