Manufacturing, quality control, and GLP-grade preclinical study of nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles.

BACKGROUND: Human adipose stromal cells-derived extracellular vesicles (haMSC-EVs) have been shown to alleviate inflammation in acute lung injury (ALI) animal models. However, there are few systemic studies on clinical-grade haMSC-EVs. Our study aimed to investigate the manufacturing, quality control (QC) and preclinical safety of clinical-grade haMSC-EVs. METHODS: haMSC-EVs were isolated from the conditioned medium of human adipose MSCs incubated in 2D containers. Purification was performed by PEG precipitation and differential centrifugation. Characterizations were conducted by nanoparticle tracking analysis, transmission electron microscopy (TEM), Western blotting, nanoflow cytometry analysis, and the TNF-α inhibition ratio of macrophage [after stimulated by lipopolysaccharide (LPS)]. RNA-seq and proteomic analysis with liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to inspect the lot-to-lot consistency of the EV products. Repeated toxicity was evaluated in rats after administration using trace liquid endotracheal nebulizers for 28 days, and respiratory toxicity was evaluated 24 h after the first administration. In vivo therapeutic effects were assessed in an LPS-induced ALI/ acute respiratory distress syndrome (ARDS) rat model. RESULTS: The quality criteria have been standardized. In a stability study, haMSC-EVs were found to remain stable after 6 months of storage at - 80°C, 3 months at - 20 °C, and 6 h at room temperature. The microRNA profile and proteome of haMSC-EVs demonstrated suitable lot-to-lot consistency, further suggesting the stability of the production processes. Intratracheally administered 1.5 × 108 particles/rat/day for four weeks elicited no significant toxicity in rats. In LPS-induced ALI/ARDS model rats, intratracheally administered haMSC-EVs alleviated lung injury, possibly by reducing the serum level of inflammatory factors. CONCLUSION: haMSC-EVs, as an off-shelf drug, have suitable stability and lot-to-lot consistency. Intratracheally administered haMSC-EVs demonstrated excellent safety at the tested dosages in systematic preclinical toxicity studies. Intratracheally administered haMSC-EVs improved the lung function and exerted anti-inflammatory effects on LPS-induced ALI/ARDS model rats.

Accuracy of intraoperative aberrometry versus modern preoperative methods in post-myopic laser vision correction eyes undergoing cataract surgery with capsular tension ring placement.

PURPOSE: To assess the accuracy of intraoperative wavefront aberrometry (IWA) versus modern intraocular lens formulas in post-myopic laser vision correction (LVC) patients undergoing cataract surgery with capsular tension ring placement. METHODS: This is a retrospective chart review conducted at an academic outpatient center. All post-myopic LVC eyes undergoing cataract surgery with IWA from a single surgeon from 05/2017 to 12/2019 were included. All patients received a capsular tension ring (CTR). Mean numerical error (MNE), median numerical error (MedNE), and percentages of prediction error within 0.50D, 0.75D, and 1.00D were calculated for the above formulas. RESULTS: Twenty-seven post-myopic LVC eyes from 18 patients were included. In post-myopic LVC, MNE with Optiwave Refractive Analysis (ORA), Barrett True K (BTK), Haigis, Haigis-L, Shammas, SRK/T, Hill-RBF v3.0, and W-K AL-adjusted Holladay 1 were + 0.224, - 0.094, + 0.193, - 0.231, - 0.372, + 1.013, + 0.860, and + 0.630 (F = 8.49, p < 0.001). MedNE were + 0.125, - 0.145, + 0.175, + 0.333, + 0.333, + 1.100, + 0.880, and + 0.765 (F = 7.89, p < 0.001), respectively. BTK provided improved accuracy in both MNE (p < 0.001) and MedNE (p = .033) when compared to ORA in pairwise analysis. If the ORA vs. BTK-suggested IOL power were routinely selected, 30% and 15% of eyes would have projected hyperopic outcomes, respectively (p = 0.09). CONCLUSIONS: Our study suggests that in post-myopic LVC eyes undergoing cataract surgery with CTRs, BTK performed more accurately than ORA with regard to accuracy and yielded a lower percentage of eyes with hyperopic outcomes. Haigis, Haigis-L, and Shammas yielded similar results to ORA with regard to accuracy and percentage of eyes with hyperopic outcomes. On average, Shammas and Haigis-L suggested IOLs that would yield outcomes more myopic than expected when compared to BTK.

Colorectal carcinoma presenting in the orbit: mass effect from an uncommon cause.

An 84-year-old male with previously documented poor medical follow-up presented with progressive painless proptosis of the right eye. Right upper eyelid ptosis, limited motility, proptosis, and inferomedial displacement of the right globe were noted on the exam. Computed tomography (CT) imaging revealed a right retrobulbar extraconal heterogenous mass with ill-defined borders. Biopsy revealed a malignant adenocarcinoma with tumor markers suggestive of a colorectal primary. A rectal mass was identified during a systemic workup. After biopsy, the patient was diagnosed with stage IV metastatic rectal adenocarcinoma. He began palliative radiation therapy shortly following diagnosis.

Inhibition of GCK-IV kinases dissociates cell death and axon regeneration in CNS neurons.

Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.