PURPOSE: This study aimed to evaluate early-phase safety of subretinal application of AAVanc80.CAG.USH1Ca1 (OT_USH_101) in wild-type (WT) pigs, examining the effects of a vehicle control, low dose, and high dose. METHODS: Twelve WT pigs (24 eyes) were divided into three groups: four pigs each received bilateral subretinal injections of either vehicle, low dose (3.3 × 1010 vector genomes [vg] per eye), or high dose (1.0 × 1011 vg per eye). Total retinal thickness (TRT) was evaluated using optical coherence tomography and retinal function was assessed with full-field electroretinography (ff-ERG) at baseline and two months post-surgery. After necropsy, retinal changes were examined through histopathology, and human USH1C_a1/harmonin expression was assessed by quantitative PCR (qPCR) and Western blotting. RESULTS: OT_USH_101 led to high USH1C_a1 expression in WT pig retinas without significant TRT changes two months after subretinal injection. The qPCR revealed expression of the human USH1C_a1 transgene delivered by the adeno-associated virus vector. TRT changes were minimal across groups: vehicle (256 ± 21 to 243 ± 18 μm; P = 0.108), low dose (251 ± 32 to 258 ± 30 μm; P = 0.076), and high dose (242 ± 24 to 259 ± 28 μm; P = 0.590). The ff-ERG showed no significant changes in rod or cone responses. Histopathology indicated no severe retinal adverse effects in the vehicle and low dose groups. CONCLUSIONS: Early-phase clinical imaging, electrophysiology, and histopathological assessments indicated that subretinal administration of OT_USH_101 was well tolerated in the low-dose treatment arm. OT_USH_101 treatment resulted in high expression of human USH1C_a1. Although histopathological changes were not severe, more frequent changes were observed in the high-dose group.

• MeSH
• Cytoskeletal Proteins genetics   MeSH
• Dependovirus genetics   MeSH
• Electroretinography * MeSH
• Genetic Therapy methods   MeSH
• Genetic Vectors * MeSH
• Injections, Intraocular * MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Tomography, Optical Coherence * MeSH
• Swine MeSH
• Cell Cycle Proteins genetics   MeSH
• Gene Expression Regulation MeSH
• Retina * metabolism   pathology   MeSH
• Transgenes * MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Data-driven cell tracking and segmentation methods in biomedical imaging require diverse and information-rich training data. In cases where the number of training samples is limited, synthetic computer-generated data sets can be used to improve these methods. This requires the synthesis of cell shapes as well as corresponding microscopy images using generative models. To synthesize realistic living cell shapes, the shape representation used by the generative model should be able to accurately represent fine details and changes in topology, which are common in cells. These requirements are not met by 3D voxel masks, which are restricted in resolution, and polygon meshes, which do not easily model processes like cell growth and mitosis. In this work, we propose to represent living cell shapes as level sets of signed distance functions (SDFs) which are estimated by neural networks. We optimize a fully-connected neural network to provide an implicit representation of the SDF value at any point in a 3D+time domain, conditioned on a learned latent code that is disentangled from the rotation of the cell shape. We demonstrate the effectiveness of this approach on cells that exhibit rapid deformations (Platynereis dumerilii), cells that grow and divide (C. elegans), and cells that have growing and branching filopodial protrusions (A549 human lung carcinoma cells). A quantitative evaluation using shape features and Dice similarity coefficients of real and synthetic cell shapes shows that our model can generate topologically plausible complex cell shapes in 3D+time with high similarity to real living cell shapes. Finally, we show how microscopy images of living cells that correspond to our generated cell shapes can be synthesized using an image-to-image model.

• MeSH
• Caenorhabditis elegans * MeSH
• Humans MeSH
• Mitosis MeSH
• Lung Neoplasms * MeSH
• Neural Networks, Computer MeSH
• Image Processing, Computer-Assisted methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Live cells act as biological lenses and can be employed as real-world optical components in bio-hybrid systems. Imaging at nanoscale, optical tweezers, lithography and also photonic waveguiding are some of the already proven functionalities, boosted by the advantage that cells are fully biocompatible for intra-body applications. So far, various cell types have been studied for this purpose, such as red blood cells, bacterial cells, stem cells and yeast cells. White Blood Cells (WBCs) play a very important role in the regulation of the human body activities and are usually monitored for assessing its health. WBCs can be considered bio-lenses but, to the best of our knowledge, characterization of their optical properties have not been investigated yet. Here, we report for the first time an accurate study of two model classes of WBCs (i.e., monocytes and lymphocytes) by means of a digital holographic microscope coupled with a microfluidic system, assuming WBCs bio-lens characteristics. Thus, quantitative phase maps for many WBCs have been retrieved in flow-cytometry (FC) by achieving a significant statistical analysis to prove the enhancement in differentiation among sphere-like bio-lenses according to their sizes (i.e., diameter d) exploiting intensity parameters of the modulated light in proximity of the cell optical axis. We show that the measure of the low intensity area (S: Iz

• MeSH
• Holography * methods   MeSH
• Humans MeSH
• Lymphocytes MeSH
• Microscopy * methods   MeSH
• Monocytes MeSH
• Optics and Photonics MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The 255th ENMC workshop on Muscle Imaging in Idiopathic Inflammatory myopathies (IIM) aimed at defining recommendations concerning the applicability of muscle imaging in IIM. The workshop comprised of clinicians, researchers and people living with myositis. We aimed to achieve consensus on the following topics: a standardized protocol for the evaluation of muscle images in various types of IIMs; the exact parameters, anatomical localizations and magnetic resonance imaging (MRI) techniques; ultrasound as assessment tool in IIM; assessment methods; the pattern of muscle involvement in IIM subtypes; the application of MRI as biomarker in follow-up studies and clinical trials, and the place of MRI in the evaluation of swallowing difficulty and cardiac manifestations. The following recommendations were formulated: In patients with suspected IIM, muscle imaging is highly recommended to be part of the initial diagnostic workup and baseline assessment. MRI is the preferred imaging modality due to its sensitivity to both oedema and fat accumulation. Ultrasound may be used for suspected IBM. Repeat imaging should be considered if patients do not respond to treatment, if there is ongoing diagnostic uncertainty or there is clinical or laboratory evidence of disease relapse. Quantitative MRI is established as a sensitive biomarker in IBM and could be included as a primary or secondary outcome measure in early phase clinical trials, or as a secondary outcome measure in late phase clinical trials. Finally, a research agenda was drawn up.

• MeSH
• Biomarkers MeSH
• Muscle, Skeletal pathology   MeSH
• Humans MeSH
• Magnetic Resonance Imaging methods   MeSH
• Myositis * diagnosis   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Clinical Conference MeSH
• Geographicals
• Netherlands MeSH

Solid tumor metastases cause most cancer-related deaths. The prevention of their occurrence misses suitable anti-metastases medicines newly labeled as migrastatics. The first indication of migrastatics potential is based on an inhibition of in vitro enhanced migration of tumor cell lines. Therefore, we decided to develop a rapid test for qualifying the expected migrastatic potential of some drugs for repurposing. The chosen Q-PHASE holographic microscope provides reliable multifield time-lapse recording and simultaneous analysis of the cell morphology, migration, and growth. The results of the pilot assessment of the migrastatic potential exerted by the chosen medicines on selected cell lines are presented.

• Publication type
• Journal Article MeSH

Signal transducer and activator of transcription 3 (Stat3) is responsible for many aspects of normal development and contributes to the development and progression of cancer through regulating epithelial cell identity and cancer stem cells. In breast cancer, Stat3 is associated with triple-negative breast cancers (TNBC) and its function has been related to the activation of p63, itself a marker of basal-like TNBC and a master regulator of stem cell activities. Stat3 activation is controlled by dual phosphorylation at tyrosine 705 (pTyr705) and serine 727 (pSer727), although it is unclear whether these have equivalent effects, and whether they are related or independent events. To address these issues, we investigated Stat3 phosphorylation at the two sites by immunohistochemistry in 173 patients with TNBC. Stat3 phosphorylation was assessed by automated quantitative measurements of digitized scanned images and classified into four categories based on histoscore. The results were analyzed for associations with multiple markers of tumor phenotype, proliferation, BRCA status, and clinicopathological characteristics. We show that the levels of pTyr705- and pSer727-Stat3 were independent in 34% of tumors. High pTyr705-Stat3 levels were associated with the luminal differentiation markers ERβ/AR and MUC1, whereas tumors with high levels of pSer727-Stat3 were more likely to be positive for the basal marker CK5/6, but were independent of p63 and were EGFR negative. Combined high pSer727- and low Tyr705-Stat3 phosphorylation associated with basal-like cancer. Although high Stat3 phosphorylation levels were associated with less aggressive tumor characteristics, they did not associate with improved survival, indicating that Stat3 phosphorylation is an unfavorable indicator for tumors with an otherwise good prognosis according to clinicopathological characteristics. These findings also show that pTyr705-Stat3 and pSer727-Stat3 associate with specific breast tumor phenotypes, implying that they exert distinct functional activities in breast cancer.

• MeSH
• Phenotype MeSH
• Phosphorylation MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Serine genetics   MeSH
• STAT3 Transcription Factor metabolism   MeSH
• Triple Negative Breast Neoplasms * pathology   MeSH
• Tyrosine genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Oxidative stress has been implied in cellular injury even in the early phases of multiple sclerosis (MS). In this study, we quantified levels of biomarkers of oxidative stress and antioxidant capacity in cerebrospinal fluid (CSF) in newly diagnosed MS patients and their associations with brain atrophy and iron deposits in the brain tissue. Consecutive treatment-naive adult MS patients (n = 103) underwent brain MRI and CSF sampling. Healthy controls (HC, n = 99) had brain MRI. CSF controls (n = 45) consisted of patients with non-neuroinflammatory conditions. 3T MR included isotropic T1 weighted (MPRAGE) and gradient echo (GRE) images that were processed to quantitative susceptibility maps. The volume and magnetic susceptibility of deep gray matter (DGM) structures were calculated. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-iso prostaglandin F2α (8-isoPG), neutrophil gelatinase-associated lipocalin (NGAL), peroxiredoxin-2 (PRDX2), and malondialdehyde and hydroxyalkenals (MDA + HAE) were measured in CSF. Compared to controls, MS patients had lower volumes of thalamus, pulvinar, and putamen, higher susceptibility in caudate nucleus and globus pallidus, and higher levels of 8-OHdG, PRDX2, and MDA + HAE. In MS patients, the level of NGAL correlated negatively with volume and susceptibility in the dentate nucleus. The level of 8-OHdG correlated negatively with susceptibility in the caudate, putamen, and the red nucleus. The level of PRDX2 correlated negatively with the volume of the thalamus and both with volume and susceptibility of the dentate nucleus. From MRI parameters with significant differences between MS and HC groups, only caudate susceptibility and thalamic volume were significantly associated with CSF parameters. Our study shows that increased oxidative stress in CSF detected in newly diagnosed MS patients suggests its role in the pathogenesis of MS.

• Publication type
• Journal Article MeSH

OBJECTIVE: Surgery is the only curative treatment for primary hyperparathyroidism. Parathyroid scintigraphy is one method used to preoperatively localize the lesion. We examined time-related changes in radiopharmaceutical uptake in parathyroid adenomas (PTAs) and thyroid gland by quantitative single-photon-emission computed tomography (SPECT) imaging to assess differences between rapid and delayed washout patterns. PATIENTS AND METHODS: The study group consisted of 35 histologically verified PTAs after radio-guided surgery extirpation in 33 patients with primary hyperparathyroidism. Patients underwent a three-phase SPECT/CT study of the neck and upper thorax post 99mTc-methoxyisobutylisonitrile (MIBI) injection. Images were reconstructed using a proprietary ordered-subset-conjugate-gradient-maximization algorithm (Siemens xSPECT Quant). PTAs were divided into those with a rapid (group A) and those with a slow (group B) washout pattern. SUVmax values of PTAs and thyroid gland tissue at 10, 90 and 180 min post 99mTc-MIBI injection were recorded and statistically assessed. Retention indexes related to the early examination were calculated for PTA and thyroid gland (RI-PTA and RI-TG). RESULTS: There were 11 PTAs in group A and 24 in group B. Significant between-group differences in PTA SUVmax and PTA/thyroid gland ratios were observed only at 180 min postinjection (P = 0.0297, P = 0.0222, respectively). RI-PTAs differed significantly at 90 and 180 min postinjection (P = 0.0298, P = 0.0431). No differences in PTA volumes, thyroid gland SUVmax values or RI-TG were observed between the groups. CONCLUSION: PTAs with rapid and slow washout patterns have different characteristics on quantitative analysis in later phases. No significant differences in directly measurable quantitative values (SUVmax, PTA/thyroid gland ratio) at the early stages of multi-phase examination were observed.

• MeSH
• Parathyroid Neoplasms * MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: The rate of reparative osteogenesis controls when an implant is sufficiently stable as to allow functional loading. Using a mini pig model, the rate of reparative osteogenesis in two types of implant sites for example, an osteotomy versus a fresh extraction socket were compared. METHODS: Eight adult mini pigs were used for the study. In phase I, three premolars were extracted on one side of the oral cavity; 12 weeks later, in phase II, osteotomies were produced in healed extraction sites, and contralateral premolars were extracted. Animals were sacrificed 1, 5, and 12 weeks after phase II. Bone repair and remodeling were evaluated using quantitative micro-computed tomographic imaging, histology, and histochemical assays coupled with quantitative dynamic histomorphometry. RESULTS: One week after surgery, extraction sockets and osteotomy sites exhibited similar patterns of new bone deposition. Five weeks after surgery, mineral apposition rates (MARs) were elevated at the injury sites relative to intact bone. Twelve weeks after surgery, the density of new bone in both injury sites was equivalent to intact bone but quantitative dynamic histomorphometry and cellular activity assays demonstrated bone remodeling was still underway. CONCLUSIONS: The mechanisms and rates of reparative osteogenesis were equivalent between fresh extraction sockets and osteotomies. The volume of new bone required to fill a socket, however, was significantly greater than the volume required to fill an osteotomy. These data provide a framework for estimating the rate of reparative osteogenesis and the time to loading of implants placed in healed sites versus fresh extraction sockets.

• MeSH
• Dental Implantation, Endosseous methods   MeSH
• Tooth Extraction methods   MeSH
• Swine, Miniature MeSH
• Swine MeSH
• Bicuspid surgery   MeSH
• Bone Remodeling MeSH
• Dental Implants * MeSH
• Tooth Socket * diagnostic imaging   surgery   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In this paper, a novel U-Net-based method for robust adherent cell segmentation for quantitative phase microscopy image is designed and optimised. We designed and evaluated four specific post-processing pipelines. To increase the transferability to different cell types, non-deep learning transfer with adjustable parameters is used in the post-processing step. Additionally, we proposed a self-supervised pretraining technique using nonlabelled data, which is trained to reconstruct multiple image distortions and improved the segmentation performance from 0.67 to 0.70 of object-wise intersection over union. Moreover, we publish a new dataset of manually labelled images suitable for this task together with the unlabelled data for self-supervised pretraining.

• Publication type
• Journal Article MeSH