Outbreak Linked to Morel Mushroom Exposure - Montana, 2023.

During March-April 2023, a total of 51 persons reported mild to severe gastrointestinal illness after eating at restaurant A in Bozeman, Montana. The outbreak resulted in multiple severe outcomes, including three hospitalizations and two deaths. After an inspection and temporary restaurant closure, the Montana Department of Public Health and Human Services and Montana's Gallatin City-County Health Department collaborated with CDC to conduct a matched case-control study among restaurant patrons to help identify the source of the outbreak. Consumption of morel mushrooms, which are generally considered edible, was strongly associated with gastrointestinal illness. A dose-response relationship was identified, and consumption of raw morel mushrooms was more strongly associated with illness than was consumption of those that were at least partially cooked. In response to the outbreak, educational public messaging regarding morel mushroom preparation and safety was shared through multiple media sources. The investigation highlights the importance of prompt cross-agency communication and collaboration, the utility of epidemiologic studies in foodborne disease outbreak investigations, and the need for additional research about the impact of morel mushroom consumption on human health. Although the toxins in morel mushrooms that might cause illness are not fully understood, proper preparation procedures, including thorough cooking, might help to limit adverse health effects.

Characterization of Human iPSC-RPE on a Prosthetic Bruch's Membrane Manufactured From Silk Fibroin.

Purpose: RPE cell transplantation as a potential treatment for AMD has been extensively investigated; however, in AMD, ultrastructural damage affects both the RPE and its underlying matrix support, the Bruch's membrane (BrM). An RPE monolayer supported by a surrogate scaffold could thus provide a more effective approach to cell-based therapy for AMD. Toward this goal, we aimed to establish a functional human induced pluripotent stem cell-derived (hiPSC)-RPE monolayer on a Bombyx mori silk fibroin (BMSF) scaffold. Methods: RPE differentiated from five distinct hiPSC lines were cultured on BMSF membrane coated with extracellular matrix (ECM, COL1), and either regular tissue culture plastic or Transwell coated with ECM (LAM-TCP). Morphologic, gene and protein expression, and functional characteristics of the hiPSC-RPE cultured on different membranes were compared in longitudinal experiments spanning 1 day to ≥3 months. Results: The hiPSC-RPE monolayers on ECM-coated BMSF and TCP could be maintained in culture for ≥3 months and displayed RPE-characteristic morphology, pigmentation, polarity, and expression of RPE signature genes and proteins. Furthermore, hiPSC-RPE on both ECM-coated BMSF and TCP displayed robust expression and secretion of several basement membrane proteins. Importantly, hiPSC-RPE cells on COL1-BMSF and LAM-TCP showed similar efficacy in the phagocytosis and degradation of photoreceptor outer segments. Conclusions: A biomaterial scaffold manufactured from silk fibroin supports the maturation and long-term survival of a functional hiPSC-RPE monolayer. This has significant implications for both in vitro disease modeling and in vivo cell replacement therapy.

Pharmacological Modulation of Photoreceptor Outer Segment Degradation in a Human iPS Cell Model of Inherited Macular Degeneration.

Degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is essential for vision, and studies have implicated altered POS processing in the pathogenesis of some retinal degenerative diseases. Consistent with this concept, a recently established hiPSC-RPE model of inherited macular degeneration, Best disease (BD), displayed reduced rates of POS breakdown. Herein we utilized this model to determine (i) if disturbances in protein degradation pathways are associated with delayed POS digestion and (ii) whether such defect(s) can be pharmacologically targeted. We found that BD hiPSC-RPE cultures possessed increased protein oxidation, decreased free-ubiquitin levels, and altered rates of exosome secretion, consistent with altered POS processing. Application of valproic acid (VPA) with or without rapamycin increased rates of POS degradation in our model, whereas application of bafilomycin-A1 decreased such rates. Importantly, the negative effect of bafilomycin-A1 could be fully reversed by VPA. The utility of hiPSC-RPE for VPA testing was further evident following examination of its efficacy and metabolism in a complementary canine disease model. Our findings suggest that disturbances in protein degradation pathways contribute to the POS processing defect observed in BD hiPSC-RPE, which can be manipulated pharmacologically. These results have therapeutic implications for BD and perhaps other maculopathies.

Functional analysis of serially expanded human iPS cell-derived RPE cultures.

PURPOSE: To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures. METHODS: Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE). RESULTS: RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels. CONCLUSIONS: Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.

iPS cell modeling of Best disease: insights into the pathophysiology of an inherited macular degeneration.

Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.

Integrating photoacoustic ophthalmoscopy with scanning laser ophthalmoscopy, optical coherence tomography, and fluorescein angiography for a multimodal retinal imaging platform.

Photoacoustic ophthalmoscopy (PAOM) is a newly developed retinal imaging technology that holds promise for both fundamental investigation and clinical diagnosis of several blinding diseases. Hence, integrating PAOM with other existing ophthalmic imaging modalities is important to identify and verify the strengths of PAOM compared with the established technologies and to provide the foundation for more comprehensive multimodal imaging. To this end, we developed a retinal imaging platform integrating PAOM with scanning laser ophthalmoscopy (SLO), spectral-domain optical coherence tomography (SD-OCT), and fluorescein angiography (FA). In the system, all the imaging modalities shared the same optical scanning and delivery mechanisms, which enabled registered retinal imaging from all the modalities. High-resolution PAOM, SD-OCT, SLO, and FA images were acquired in both albino and pigmented rat eyes. The reported in vivo results demonstrate the capability of the integrated system to provide comprehensive anatomic imaging based on multiple optical contrasts.