Optic neuritis (ON) is an inflammatory optic neuropathy that is often a harbinger of central nervous system (CNS) demyelinating disorders. ON is frequently misdiagnosed in the clinical arena, leading ...to either inappropriate management or diagnostic delays. As a result, patients may fail to achieve optimal recovery. The treatment response to corticosteroids and long term risk of multiple sclerosis was established in the first clinical trials conducted roughly 30 years ago. Spontaneous resolution was observed in the vast majority of patients and intravenous high-dose corticosteroids hastened recovery; half of the patients eventually developed multiple sclerosis. Over the ensuing decades, the number of inflammatory conditions associated with ON has significantly expanded exposing substantial variability in the prognosis, treatment, and management of ON patients. ON subtypes can frequently be distinguished by distinct clinical, serological, and radiological profiles allowing expedited and specialized treatment. Guided by an increased understanding of the immunopathology underlying optic nerve and associated CNS injuries, novel disease management strategies are emerging to minimize vision loss, improve long-term surveillance strategies, and minimize CNS injury and disability. Knowledge regarding the clinical signs and symptoms of different ON subtypes is essential to guide acute therapy, prognosticate recovery, accurately identify underlying CNS inflammatory disorders, and facilitate study design for the next generation of clinical and translational trials.
We present a γ-ray photon flux and spectral variability study of the flat-spectrum radio quasar 3C 273 over a rapid flaring activity period between September 2009 to April 2010. Five major flares ...were observed in the source during this period. The most rapid flare observed in the source has a flux doubling time of 1.1 hr. The rapid γ-ray flares allow us to constrain the location and size of the γ-ray emission region in the source. The γγ-opacity constrains the Doppler factor δγ ≥ 10 for the highest energy (15 GeV) photon observed by the Fermi-Large Area Telescope (LAT). Causality arguments constrain the size of the emission region to 1.6 × 1015 cm. The γ-ray spectra measured over this period show clear deviations from a simple power law with a break in the 1−2 GeV energy range. We discuss possible explanations for the origin of the γ-ray spectral breaks. Our study suggests that the γ-ray emission region in 3C 273 is located within the broad line region (< 1.6 pc). The spectral behavior and temporal characteristics of the individual flares indicate the presence of multiple shock scenarios at the base of the jet.
Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their ...redifferentiation and restoration of the chondrogenic phenotype. Typically 1×10(5)-5×10(5) chondrocytes are aggregated, resulting in "macro" pellets having diameters ranging from 1-2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1-2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×10(5) human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.
While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An ...extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.
We present the results of a series of radio, optical, X-ray, and γ-ray observations of the BL Lac object S50716+714 carried out between April 2007 and January 2011. The multifrequency observations ...were obtained using several ground- and space-based facilities. The intense optical monitoring of the source reveals faster repetitive variations superimposed on a long-term variability trend on a time scale of ~350 days. Episodes of fast variability recur on time scales of ~60−70 days. The intense and simultaneous activity at optical and γ-ray frequencies favors the synchrotron self-Compton mechanism for the production of the high-energy emission. Two major low-peaking radio flares were observed during this high optical/γ-ray activity period. The radio flares are characterized by a rising and a decaying stage and agrees with the formation of a shock and its evolution. We found that the evolution of the radio flares requires a geometrical variation in addition to intrinsic variations of the source. Different estimates yield robust and self-consistent lower limits of δ ≥ 20 and equipartition magnetic field Beq ≥ 0.36 G. Causality arguments constrain the size of emission region θ ≤ 0.004 mas. We found a significant correlation between flux variations at radio frequencies with those at optical and γ-rays. Theoptical/GeV flux variations lead the radio variability by ~65 days. The longer time delays between low-peaking radio outbursts and optical flares imply that optical flares are the precursors of radio ones. An orphan X-ray flare challenges the simple, one-zone emission models, rendering them too simple. Here we also describe the spectral energy distribution modeling of the source from simultaneous data taken through different activity periods.
Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone ...through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.
The sensitivity of acetylcholine receptor (AChR) antibody testing is thought to be lower in ocular myasthenia gravis (OMG) compared with generalized disease, although estimates in small-scale studies ...vary. There is little information in the literature about the implications of AChR antibody levels and progression from OMG to generalized myasthenia gravis.
To test the hypothesis that serum AChR antibody testing is more sensitive in OMG than previously reported and to examine the association between AChR antibody levels and progression from OMG to generalized myasthenia gravis.
A retrospective, observational cohort study was conducted of 223 patients (mean SD age, 59.2 16.4 years; 139 62.3% male) diagnosed with OMG between July 1, 1986, and May 31, 2013, at 2 large, academic medical centers.
Baseline characteristics, OMG symptoms, results of AChR antibody testing, and progression time to generalized myasthenia gravis (if this occurred) were recorded for each patient. Multiple logistic regression was used to measure the association between all clinical variables and antibody result. Kaplan-Meier survival analysis was performed to examine time to generalization.
Among the 223 participants, AChR antibody testing results were positive in 158 participants (70.9%). In an adjusted model, increased age at diagnosis (odds ratio OR, 1.03; 95% CI, 1.01-1.04; P = .007) and progression to generalized myasthenia gravis (OR, 2.92; 95% CI, 1.18-7.26; P = .02) were significantly associated with positive antibody test results. Women were less likely to have a positive antibody test result (OR, 0.36; 95% CI, 0.19-0.68; P = .002). Patients who developed symptoms of generalized myasthenia gravis had a significantly higher mean (SD) antibody level than those who did not develop symptoms of generalized myasthenia gravis (12.7 16.5 nmol/L vs 4.2 7.9 nmol/L; P = .002).
We demonstrate a higher sensitivity of AChR antibody testing than previously reported in the largest cohort of patients with OMG available to date. Older age, male sex, and progression to generalized myasthenia gravis were significantly associated with a positive antibody test result. In addition, to our knowledge, this is the first report of an association between high AChR antibody levels and progression from OMG to generalized disease.
A mathematical model was developed for mesenchymal stromal cell (MSC) growth in a packed bed bioreactor that improves oxygen availability by allowing oxygen diffusion through a gas-permeable wall. ...The governing equations for oxygen, glucose and lactate, the inhibitory waste product, were developed assuming Michaelis-Menten kinetics, together with an equation for the medium flow based on Darcy's Law. The conservation law for the cells includes the effects of inhibition as the cells reach confluence, nutrient and waste product concentrations, and the assumption that the cells can migrate on the scaffold. The equations were solved using the finite element package, COMSOL. Previous experimental results collected using a packed bed bioreactor with gas permeable walls to expand MSCs produced a lower cell yield than was obtained using a traditional cell culture flask. This mathematical model suggests that the main contributors to the observed low cell yield were a non-uniform initial cell seeding profile and a potential lag phase as cells recovered from the initial seeding procedure. Lactate build-up was predicted to have only a small effect at lower flow rates. Thus, the most important parameters to optimise cell expansion in the proliferation of MSCs in a bioreactor with gas permeable wall are the initial cell seeding protocol and the handling of the cells during the seeding process. The mathematical model was then used to identify and characterise potential enhancements to the bioreactor design, including incorporating a central gas permeable capillary to further enhance oxygen availability to the cells. Finally, to evaluate the issues and limitations that might be encountered scale-up of the bioreactor, the mathematical model was used to investigate modifications to the bioreactor design geometry and packing density.
Validated methods to identify neuro-ophthalmologists in administrative data do not exist. The development of such method will facilitate research on the quality of neuro-ophthalmic care and health ...care utilization for patients with neuro-ophthalmic conditions in the United States.
Using nationally representative, 20% sample from Medicare carrier files from 2018, we identified all neurologists and ophthalmologists billing at least 1 office-based evaluation and management (E/M) outpatient visit claim in 2018. To isolate neuro-ophthalmologists, the National Provider Identifier numbers of neuro-ophthalmologists in the North American Neuro-Ophthalmology Society (NANOS) directory were collected and linked to Medicare files. The proportion of E/M visits with International Classification of Diseases-10 diagnosis codes that best distinguished neuro-ophthalmic care ("neuro-ophthalmology-specific codes" or NSC) was calculated for each physician. Multiple logistic regression models assessed predictors of neuro-ophthalmology specialty designation after accounting for proportion of ophthalmology, neurology, and NSC claims and primary specialty designation. Sensitivity, specificity, and positive predictive value (PPV) for varying proportions of E/M visits with NSC were calculated.
We identified 32,293 neurologists and ophthalmologists who billed at least 1 outpatient E/M visit claim in 2018 in Medicare. Of the 472 NANOS members with a valid individual National Provider Identifier, 399 (84.5%) had a Medicare outpatient E/M visit in 2018. The model containing only the proportion of E/M visits with NSC best predicted neuro-ophthalmology specialty designation (odds ratio 1.05 95% confidence interval 1.04, 1.05; P < 0.001; area under the receiver operating characteristic AUROC = 0.91). Model predictiveness for neuro-ophthalmology designation was maximized when 6% of all billed claims were for NSC (AUROC = 0.89; sensitivity: 84.0%; specificity: 93.9%), but PPV was low (14.9%). The threshold was unchanged when limited only to neurologists billing ≥1% ophthalmology claims or ophthalmologists billing ≥1% neurology claims, but PPV increased (33.3%).
Our study provides a validated method to identify neuro-ophthalmologists who can be further adapted for use in other administrative databases to facilitate future research of neuro-ophthalmic care delivery in the United States.
Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of "pieces" of tumour in mice, but the cellular composition of pieces is not standardised. ...Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ
(NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 μm achieving superior microtissue vascularisation relative to mesh with 36×36 μm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation.
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