Tailored environments for directed mesenchymal stromal cell proliferation and differentiation using decellularized extracellular matrices in conjunction with substrate modulus.

Decellularised extracellular matrix (dECM) produced by mesenchymal stromal cells (MSCs) is a promising biomaterial for improving the ex vivo expansion of MSCs. The dECMs are often deposited on high modulus surfaces such as tissue culture plastic or glass, and subsequent differentiation assays often bias towards osteogenesis. We tested the hypothesis that dECM deposited on substrates of varying modulus will produce cell culture environments that are tailored to promote the proliferation and/or lineage-specific differentiation of MSCs. dECM was produced on type I collagen-functionalised polyacrylamide hydrogels with discrete moduli (∼4, 10, and 40 kPa) or in a linear gradient of modulus that spans the same range, and the substrates were used as culture surfaces for MSCs. Fluorescence spectroscopy and mass spectrometry characterization revealed structural compositional changes in the dECM as a function of substrate modulus. Softer substrates (4 kPa) with dECM supported the largest number of MSCs after 7 days (∼1.6-fold increase compared to glass). Additionally, osteogenic differentiation was greatest on high modulus substrates (40 kPa and glass) with dECM. Nuclear translocation of YAP1 was observed on all surfaces with a modulus of 10 kPa or greater and may be a driver for the increased osteogenesis on the high modulus surfaces. These data demonstrate that dECM technology can be integrated with environmental parameters such as substrate modulus to improve/tailor MSC proliferation and differentiation during ex vivo culture. These results have potential impact in the improved expansion of MSCs for tailored therapeutic applications and in the development of advanced tissue engineering scaffolds. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are extensively used in tissue engineering and regenerative medicine due to their ability to proliferate, differentiate, and modulate the immune environment. Controlling MSC behavior is critical for advances in the field. Decellularised extracellular matrix (dECM) can maintain MSC properties in culture, increase their proliferation rate and capacity, and enhance their stimulated differentiation. Substrate stiffness is another key driver of cell function, and previous reports have primarily looked at dECM deposition and function on stiff substrates such as glass. Herein, we produce dECM on substrates of varying stiffness to create tailored environments that enhance desired MSC properties such as proliferation and differentiation. Additionally, we complete mechanistic studies including quantitative mass spec of the ECM to understand the biological function.

Amniotic membrane graft to patch an overfiltering trabeculectomy flap.

Purpose: To describe the use of an amniotic membrane graft (AMG) with fibrin sealant to address an overfiltering trabeculectomy flap encountered intraoperatively. Observations: A 35-year-old female with severe primary open angle glaucoma underwent trabeculectomy with mitomycin C due to uncontrolled intraocular pressure (IOP). Intraoperatively, the elastic nature of the scleral flap led to overfiltration, causing persistent anterior chamber shallowing despite numerous sutures. To decrease but not completely shut down aqueous outflow through the trabeculectomy flap, we utilized AMG and fibrin sealant to stabilize the flap. Postoperatively, the patient had a formed anterior chamber, elevated bleb and significantly reduced IOP, without the need for additional glaucoma medications. Conclusions and importance: Amniotic membrane grafts (AMG) with fibrin sealant may help regulate aqueous flow efflux, maintain anterior chamber stability, and mitigate the risk of postoperative hypotony in trabeculectomy surgery. AMG was chosen in this setting given its anti-inflammatory, anti-fibrotic properties, as well as its optically clear nature to allow for post-operative visualization of the flap. AMG allows for early postoperative stabilization of the scleral flap without complete obstruction, and may be useful in patients at risk of early postoperative hypotony.

Durable responses to alectinib in murine models of EML4-ALK lung cancer requires adaptive immunity.

Lung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g., alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). However, factors that control this response are not well understood. While the contribution of the immune system in mediating the response to immunotherapy has been extensively investigated, less is known regarding the contribution of immunity to TKI therapeutic responses. We previously demonstrated a positive association of a TKI-induced interferon gamma (IFNγ) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used three murine models of EML4-ALK lung cancer to test the role for host immunity in the alectinib therapeutic response. The cell lines (EA1, EA2, EA3) were propagated orthotopically in the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by µCT and immune cell content was measured by flow cytometry and multispectral immunofluorescence. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA. All cell lines were similarly sensitive to alectinib in vitro and as orthotopic tumors in immunocompetent mice, exhibited durable shrinkage. However, in immunodeficient mice, all tumor models rapidly progressed on TKI therapy. In immunocompetent mice, EA2 tumors exhibited a complete response, whereas EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of TKI treatment. Prior to treatment, EA2 tumors had greater numbers of CD8+ T cells and fewer neutrophils compared to EA1 tumors. Also, RNAseq of cancer cells recovered from untreated tumors revealed elevated levels of CXCL9 and 10 in EA2 tumors, and higher levels of CXCL1 and 2 in EA1 tumors. Analysis of pre-treatment patient biopsies from ALK+ tumors revealed an association of neutrophil content with shorter time to progression. Combined, these data support a role for adaptive immunity in durability of TKI responses and demonstrate that the immune cell composition of the tumor microenvironment is predictive of response to alectinib therapy.

Improvement of Mesenchymal Stromal Cell Proliferation and Differentiation via Decellularized Extracellular Matrix on Substrates With a Range of Surface Chemistries.

Decellularized extracellular matrix (dECM) deposited by mesenchymal stromal cells (MSCs) has emerged as a promising substrate for improved expansion of MSCs. To date, essentially all studies that have produced dECM for MSC expansion have done so on tissue culture plastic or glass. However, substrate surface chemistry has a profound impact on the adsorption of proteins that mediate cell-material interactions, and different surface chemistries can cause changes in cell behavior, ECM deposition, and the in vivo response to a material. This study tested the hypothesis that substrate surface chemistry impacts the deposition of ECM and its subsequent bioactivity. This hypothesis was tested by producing glass surfaces with various surface chemistries (amine, carboxylic acid, propyl, and octyl groups) using silane chemistry. ECM was deposited by an immortalized MSC line, decellularized, and characterized through SDS-PAGE and immunofluorescence microscopy. No significant difference was observed in dECM composition or microarchitecture on the different surfaces. The decellularized surfaces were seeded with primary MSCs and their proliferation and differentiation were assessed. The presence of dECM improved the proliferation of primary MSCs by ~100% in comparison to surface chemistry controls. Additionally, the adipogenesis increased by 50-90% on all dECM surfaces in comparison to surface chemistry controls, and the osteogenesis increased by ~50% on the octyl-modified surfaces when dECM was present. However, no statistically significant differences were observed within the set of dECM surfaces or control surfaces. These results support the null hypothesis, meaning surface chemistry (over the range tested in this work) is not a key regulator of the composition or bioactivity of MSC-derived dECM. These results are significant because they provide an important insight into regenerative engineering technologies. Specifically, the utilization of dECM in stem cell manufacturing and tissue engineering applications would require the dECM to be produced on a wide variety of substrates. This work indicates that it can be produced on materials with a range of surface chemistries without undesired changes in the bioactivity of the dECM.

A Case of Refractory Open-angle Glaucoma with Failed Baerveldt Glaucoma Implant and Trabeculectomy Treated with Ab Externo XEN Gel Stent Placement.

Aim: We report a case of successful intraocular pressure (IOP) management in a patient with refractory primary open-angle glaucoma (POAG) following implantation of XEN gel implant in the same hemisphere as prior failed filtering surgeries [i.e., Baerveldt glaucoma implant (BGI) and trabeculectomy bleb)]. Background: Glaucoma is a major cause of blindness worldwide and is typically associated with elevated IOP and retinal ganglion cell loss. Treatment centers around decreasing IOP with eye drops and surgical interventions. The advent of minimally invasive glaucoma surgeries (MIGS) has expanded therapeutic options for patients who have failed traditional treatments. The XEN gel implant creates a shunt between the anterior chamber and the subconjunctival or sub-tenon's space, allowing for drainage of aqueous humor without significant tissue disruption. Given that the XEN gel implant also results in bleb formation, it is generally recommended to avoid placement in the same quadrant of prior filtering surgeries. Case description: A 77-year-old man with a 15-year history of severe POAG of OU presents with persistently elevated IOP despite multiple filtering surgeries and maximal eye drop regimen. The patient had a superotemporal BGI in OU and a scarred trabeculectomy bleb superiorly in the right eye (OD). He underwent an open conjunctiva ab externo XEN gel implant placement in the OD in the same hemisphere as previous filtering surgeries. At 12 months postoperatively, the IOP range continues to be maintained within goal without complications. Conclusion: The XEN gel implant can be successfully placed in the same hemisphere as prior filtering surgeries and can achieve goal IOP without any surgical complications at 12 months postoperatively. Clinical significance: A XEN gel implant can effectively lower patients' IOP and can be a unique surgical option in refractory cases of POAG with multiple failed filtering surgeries, even when inserted in close proximity to prior filtering surgeries. How to cite this article: Amoozadeh SA, Yang MC, Lin KY. A Case of Refractory Open-angle Glaucoma with Failed Baerveldt Glaucoma Implant and Trabeculectomy Treated with Ab Externo XEN Gel Stent Placement. J Curr Glaucoma Pract 2022;16(3):192-194.

Accurate Identification of the Trabecular Meshwork under Gonioscopic View in Real Time Using Deep Learning.

PURPOSE: Accurate identification of iridocorneal structures on gonioscopy is difficult to master, and errors can lead to grave surgical complications. This study aimed to develop and train convolutional neural networks (CNNs) to accurately identify the trabecular meshwork (TM) in gonioscopic videos in real time for eventual clinical integrations. DESIGN: Cross-sectional study. PARTICIPANTS: Adult patients with open angle were identified in academic glaucoma clinics in both Taipei, Taiwan, and Irvine, California. METHODS: Neural Encoder-Decoder CNNs (U-nets) were trained to predict a curve marking the TM using an expert-annotated data set of 378 gonioscopy images. The model was trained and evaluated with stratified cross-validation grouped by patients to ensure uncorrelated training and testing sets, as well as on a separate test set and 3 intraoperative gonioscopic videos of ab interno trabeculotomy with Trabectome (totaling 90 seconds long, 30 frames per second). We also evaluated our model's performance by comparing its accuracy against ophthalmologists. MAIN OUTCOME MEASURES: Successful development of real-time-capable CNNs that are accurate in predicting and marking the TM's position in video frames of gonioscopic views. Models were evaluated in comparison with human expert annotations of static images and video data. RESULTS: The best CNN model produced test set predictions with a median deviation of 0.8% of the video frame's height (15.25 µm) from the human experts' annotations. This error is less than the average vertical height of the TM. The worst test frame prediction of this model had an average deviation of 4% of the frame height (76.28 µm), which is still considered a successful prediction. When challenged with unseen images, the CNN model scored greater than 2 standard deviations above the mean performance of the surveyed general ophthalmologists. CONCLUSIONS: Our CNN model can identify the TM in gonioscopy videos in real time with remarkable accuracy, allowing it to be used in connection with a video camera intraoperatively. This model can have applications in surgical training, automated screenings, and intraoperative guidance. The dataset developed in this study is one of the first publicly available gonioscopy image banks (https://lin.hs.uci.edu/research), which may encourage future investigations in this topic.

Capnocytophaga canimorsus blebitis: case report and review of literature.

BACKGROUND: Capnocytophaga canimorsus is a facultative anaerobic, slow-growing, capnophilic, Gram-negative bacillus, that is commonly found in the microflora of canine and feline oral cavities. Capnocytophaga infections are an emerging zoonotic disease that can cause fatal systemic infections in immunocompromised individuals. Localized ocular Capnocytophaga infections, including keratitis, blepharitis, and endophthalmitis, can lead to severe eye threatening situations. To our knowledge, there is currently no documented case of Capnocytophaga canimorsus blebitis with bleb perforation after trabeculectomy. CASE PRESENTATION: Our case report and literature review features a novel case of Capnocytophaga blebitis that occurred after trabeculectomy, associated with close dog contact (i.e. face licking). The patient had underwent trabeculectomy 10 years prior and presented with conjunctival injection, perforated bleb, and hypotony. Overall, patient was medically treated subconjunctival vancomycin, gentamicin and moxifloxacin drops. Trabeculectomy revision was performed with good visual outcome. Bacterial cultures grew Capnocytophaga canimorsus. CONCLUSIONS: We discuss the strategies for diagnosis, treatment, and common risk factors for ocular Capnocytophaga infections. At-risk patients with ocular infections should be asked about close contact with dogs and cats; and treated promptly with the proper antibiotic regimen.

Surge of Miller Fisher variant and Guillain-Barré syndrome in two downtown Los Angeles community teaching hospitals.

Guillain-Barré syndrome (GBS) and Miller Fisher variant (MFv) cases spiked threefold in Los Angeles, with a high proportion of MFv cases. MFv is underdiagnosed when accompanying neurological symptoms are mild. This report emphasizes the seasonality of GBS and its relation to ganglioside antibodies.

Faster Recovery of Internal Ophthalmoplegia than External Ophthalmoplegia in a Miller Fisher Variant of Guillain-Barre Syndrome.

We present a case of classic Miller Fisher Syndrome (MFS) variant of Guillain-Barre Syndrome (GBS) with detailed description in the difference between the internal and external ophthalmoplegia. They are different in their onset, duration, and recovery.

Salzmann's nodular degeneration of cornea associated with thyroid eye disease.

Salzmann's nodular degeneration (SND) typically occurs in patients who are female, 50-60 years old, and have a history of corneal inflammation and irritation. Multiple case reports have documented associations between SND and trachoma, viral infections, trauma, contact lens wear, corneal surgeries and corneal exposure. The authors describe a patient with bilateral SND confirmed by anterior segment optical coherence tomography (OCT) imaging in the context of thyroid eye disease (TED) and history of LASIK. Treatment involved propylthiouracil (PTU), artificial tear use, loteprednol etabonate ophthalmic gel, eyelid taping and selenium supplementation and prospective superficial keratectomy with diamond burr polish.

Drug-induced Acute Angle-closure Glaucoma: A Review.

AIM: Our goal is to review current literature regarding drug-induced acute angle-closure glaucoma (AACG) and provide ophthalmologists and general practitioners with a thorough understanding of inciting medications and treatment pitfalls to be avoided. BACKGROUND: Drug-induced AACG is an ophthalmological emergency that ophthalmologists and general practitioners should be familiar with, given its potentially blinding consequences. Common anatomical risk factors for AACG include a shallow anterior chamber depth, short axial length, plateau iris configuration, thick lens, anteriorly positioned lens, and rarely, intraocular tumor. Demographic risk factors include female sex, Asian ethnicity, family history, and advanced age. In patients with predisposing factors, acute angle closure can be triggered by various classes of medications including adrenergic agonists, anticholinergics, cholinergics, sulfonamides, supplements, and serotonergic medications. Physicians prescribing such inciting medications should be aware of their potentially sight-threatening adverse effects and to inform patients of the warning symptoms. Patients typically present with elevated intraocular pressure (IOP), headache, nausea, blurry vision, and halos around lights. REVIEW RESULTS: There are two main mechanisms of drug-induced AACG, both with different treatment strategies. The first mechanism of drug-induced AACG is pupillary block and iridocorneal angle closure secondary to thickening of iris base with mydriasis. The second mechanism of drug-induced AACG is anterior displacement of the lens-iris diaphragm due to mass effect (e.g., blood, misdirected aqueous humor, and tumors), uveal effusion, or weakened zonules. CONCLUSION: This paper reviews drug-induced AACG, high-risk anatomical features, underlying mechanisms, inciting medications, and options for treatment and prevention. CLINICAL SIGNIFICANCE: With proper understanding of the underlying mechanism of drug-induced AACG, physicians can respond promptly to save their patients' vision by employing the correct treatment strategy. HOW TO CITE THIS ARTICLE: Yang MC, Lin KY. Drug-induced Acute Angle-closure Glaucoma: A Review. J Curr Glaucoma Pract 2019;13(3):104-109.

Transferable Matrixes Produced from Decellularized Extracellular Matrix Promote Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells and Facilitate Scale-Up.

Decellularized extracellular matrixes (dECM) derived from mesenchymal stem cell (MSC) cultures have recently emerged as cell culture substrates that improve the proliferation, differentiation, and maintenance of MSC phenotype during ex vivo expansion. These biomaterials have considerable potential in the fields of stem cell biology, tissue engineering, and regenerative medicine. Processing the dECMs into concentrated solutions of biomolecules that enable the useful properties of the native dECM to be transferred to a new surface via a simple adsorption step would greatly increase the usefulness and impact of this technology. The development of such solutions, hereafter referred to as transferable matrixes, is the focus of this article. In this work, we produced transferable matrixes from dECM derived from two human placental MSC cell lines (DMSC23 and CMSC29) using pepsin digestion (P-ECM), urea extraction (U-ECM), and mechanical homogenization in acetic acid (AA-ECM). Native dECMs improved primary DMSC proliferation as well as osteogenic and adipogenic differentiation, compared with traditional expansion procedures. Interestingly, tissue culture plastic coated with P-ECM was able to replicate the proliferative effects of native dECM, while U-ECM was able to replicate osteogenic differentiation. These data illustrate the feasibility of producing dECM-derived transferable matrixes that replicate key features of the native matrixes and show that different processing techniques produce transferable matrixes with varying bioactivities. Additionally, these transferable matrixes are able to coat 1.3-5.2 times the surface area covered by the native dECM, facilitating scale-up of this technology.

Inhibitory Neuron Transplantation into Adult Visual Cortex Creates a New Critical Period that Rescues Impaired Vision.

The maturation of inhibitory circuits in juvenile visual cortex triggers a critical period in the development of the visual system. Although several manipulations of inhibition can alter the timing of the critical period, none have demonstrated the creation of a new critical period in adulthood. We developed a transplantation method to reactivate critical period plasticity in the adult visual cortex. Transplanted embryonic inhibitory neurons from the medial ganglionic eminence reinstate ocular dominance plasticity in adult recipients. Transplanted inhibitory cells develop cell-type-appropriate molecular characteristics and visually evoked responses. In adult mice impaired by deprivation during the juvenile critical period, transplantation also recovers both visual cortical responses and performance on a behavioral test of visual acuity. Plasticity and recovery are induced when the critical period would have occurred in the donor animal. These results reveal that the focal reactivation of visual cortical plasticity using inhibitory cell transplantation creates a new critical period that restores visual perception after childhood deprivation.

The utility of 'low-grade intraepithelial lesion, cannot exclude high grade' diagnosis: a single institution's experience.

OBJECTIVE: Squamous intraepithelial lesions (SILs) are classified as low-grade SIL (LGSIL) and high-grade SIL (HGSIL). 'LGSIL cannot exclude high grade' (LGSIL-H) interpretive category has been used in cases where findings exceed criteria for LGSIL, but do not fulfill the criteria for HGSIL. This study analyzed follow-up histology of LGSIL-H cases and compared the follow-up results of LGSIL-H with LGSIL to determine the utility of LGSIL-H category using a single institution's experience. STUDY DESIGN: Pap smears with LGSIL-H interpretation from 2005 to 2008 were retrieved. Histological follow-up results for LGSIL-H cases were analyzed and compared to the follow-up results of LGSIL cases. RESULTS: Cases with LGSIL-H interpretation (311) comprised 0.18% of all cases (170,307). Follow-up was available for 144 patients and 13.2% had benign findings, 51.4% had cervical intraepithelial neoplasia (CIN) 1, and 35.4% had CIN 2 or higher. In comparison, of 425 patients with LGSIL, 22.6% had benign findings, 71% had CIN 1 and 6.4% had CIN 2 or higher. CONCLUSION: A significantly greater number of patients with LGSIL-H interpretation had a CIN 2 or higher lesion on follow-up compared to patients with LGSIL. This suggests LGSIL-H may be a useful diagnostic category.

Nodular pulmonary amyloidosis with an unusual protein composition diagnosed by fine-needle aspiration biopsy: a case report.

Nodular pulmonary amyloidosis is rare, but a number of individual cases have been reported in the literature. Radiologically, pulmonary amyloid nodules often mimic cancer and hence require biopsy to obtain a specimen for definitive diagnosis. We describe a case of isolated nodular pulmonary amyloidosis with an unusual amyloid composition that given the patient's clinical history has not been described in the literature to the best of our knowledge, making this case unique. A 58-year-old woman with a history of mixed connective tissue disease was found to have multiple lung nodules on chest computed tomography (CT) that were radiologically suspicious for cancer. A CT-guided percutaneous fine-needle aspiration (FNA) biopsy of one of the nodules was performed. A diagnosis of amyloid was rendered using special stains and polarized light microscopic examination. Immunohistochemical stains revealed that the amyloid comprised beta-2 microglobulin as well as both kappa and lambda light chains. Further work up showed no evidence of systemic amyloidosis. We were successful in rendering an accurate diagnosis of a pulmonary amyloidoma on material procured by CT-guided FNA thus avoiding more invasive procedures. In addition, immunohistochemical staining revealed an unexpected and highly unusual protein composition of the amyloid.

Tissue-specific and glucose-responsive expression of the pancreatic derived factor (PANDER) promoter.

Pancreatic derived factor (PANDER) is a recently identified cytokine-like protein that is dominantly expressed in the islets of Langerhans of the pancreas. To investigate the mechanism of tissue-specific regulation of PANDER, we identified and characterized the promoter region. The transcriptional start site was identified 520 bp upstream of the translational start codon by 5'-RLM-RACE. Computer algorithms identified several islet-associated and glucose-responsive binding motifs that included A and E boxes, hepatocyte nuclear factors 1 and 4, Oct-1, and signal transducer and activator of transcription 3, and 5. Reporter gene analysis revealed cell type-specific PANDER promoter expression in islet and liver-derived cell lines. Levels of PANDER mRNA were directly concordant to the observed cell type-specific PANDER promoter gene expression. The minimal element was mapped to the 5'-UTR and located between +200 and +491 relative to the transcriptional start site and imparted maximal gene expression. In addition, several putative glucose-responsive binding sites were further functionally characterized to reveal critical regulatory elements of PANDER. The PANDER promoter was demonstrated to be glucose-responsive in a dose-dependent manner in murine insulinoma beta-TC3 cells and primary murine islets, but unresponsive in glucagon-secreting alpha-TC3 cells. Our findings revealed that the 5'-UTR of PANDER contains the minimal element for gene expression and imparts both tissue-specificity and glucose-responsiveness. The regulation of PANDER gene expression mimics that of insulin and suggests a potential biological function of PANDER involved in metabolic homeostasis.