Retinal Pigment Epithelium and Müller Progenitor Cell Interaction Increase Müller Progenitor Cell Expression of PDGFRα and Ability to Induce Proliferative Vitreoretinopathy in a Rabbit Model

Purpose. Proliferative vitreoretinopathy (PVR) is a complication of retinal detachment characterized by redetachment of the retina as a result of membrane formation and contraction. A variety of retinal cells, including retinal pigment epithelial (RPE) and Müller glia, and growth factors may be responsible. Platelet-derived growth factor receptor alpha (PDGFRα) is found in large quantities in PVR membranes, and is intrinsic to the development of PVR in rabbit models. This study explores the expression of PDGFR in cocultures of RPE and Müller cells over time to examine how these two cell types may collaborate in the development of PVR. We also examine how changes in PDGFRα expression alter Müller cell pathogenicity. Methods. Human MIO-M1 Müller progenitor (MPC) and ARPE19 cells were studied in a transmembrane coculture system. Immunocytochemistry and Western blot were used to look at PDGFRα, PDGFRβ, and GFAP expression. A transfected MPC line cell line expressing the PDGFRα (MIO-M1α) was generated, and tested in a rabbit model for its ability to induce PVR. Results:. The expression of PDGFRα and PDGFRβ was upregulated in MIO-M1 MPCs cocultured with ARPE19 cells; GFAP was slightly decreased. Increased expression of PDGFRα in the MIO-M1 cell line resulted in increased pathogenicity and enhanced ability to induce PVR in a rabbit model. Conclusions:. Müller and RPE cell interaction can lead to upregulation of PDGFRα and increased Müller cell pathogenicity. Müller cells may play a more active role than previously thought in the development of PVR membranes, particularly when stimulated by an RPE-cell-rich environment. Additional studies of human samples and in animal models are warranted

SU9518 inhibits proliferative vitreoretinopathy in fibroblast and genetically modified Muller cell-induced rabbit models

PURPOSE: Proliferative vitreoretinopathy (PVR) is a complication of retinal detachment that can lead to surgical failure and vision loss. Previous studies suggest that a variety of retinal cells, including RPE and Muller glia, may be responsible. Platelet-derived growth factor receptor alpha (PDGFRalpha) has been strongly implicated in the pathogenesis, and found to be intrinsic to the development of PVR in rabbit models. We examine whether SU9518, a tyrosine kinase inhibitor with PDGFRalpha specificity, can inhibit the development of PVR in fibroblast and Muller cell rabbit models of PVR. METHODS: SU9518 was injected in rabbit eyes along with fibroblasts, Muller cells (MIO-M1), or Muller cells transfected to increase their expression of PDGFRalpha (MIO-M1alpha). Indirect ophthalmoscopy and histopathology were used to assess efficacy and toxicity. RESULTS: SU9518 was an effective inhibitor of PVR in both fibroblast and Muller cell models of PVR. No toxic effects were identified by indirect ophthalmoscopy or histopathology. CONCLUSIONS: SU9518 is an effective and safe inhibitor of PVR in rabbit models, and could potentially be used in humans for the treatment of this and other proliferative diseases of the retina involving fibrosis and gliosis. Further animal studies need to be performed to examine retinal toxicity and sustained delivery mechanisms

Stable engraftment of human microbiota into mice with a single oral gavage following antibiotic conditioning

Abstract Background Human microbiota-associated (HMA) animal models relying on germ-free recipient mice are being used to study the relationship between intestinal microbiota and human disease. However, transfer of microbiota into germ-free animals also triggers global developmental changes in the recipient intestine, which can mask disease-specific attributes of the donor material. Therefore, a simple model of replacing microbiota into a developmentally mature intestinal environment remains highly desirable. Results Here we report on the development of a sequential, three-course antibiotic conditioning regimen that allows sustained engraftment of intestinal microorganisms following a single oral gavage with human donor microbiota. SourceTracker, a Bayesian, OTU-based algorithm, indicated that 59.3 ± 3.0% of the fecal bacterial communities in treated mice were attributable to the donor source. This overall degree of microbiota engraftment was similar in mice conditioned with antibiotics and germ-free mice. Limited surveys of systemic and mucosal immune sites did not show evidence of immune activation following introduction of human microbiota. Conclusions The antibiotic treatment protocol described here followed by a single gavage of human microbiota may provide a useful, complimentary HMA model to that established in germ-free facilities. The model has the potential for further in-depth translational investigations of microbiota in a variety of human disease states

Growth of vegetative <i>C</i>. <i>difficile</i> is inhibited by post-FMT bile acids.

<p>A) Hourly OD₆₀₀ measurements for NAP1 cells in BHIS alone (circle), BHIS with pre-FMT bile acids (triangle), and BHIS with post-FMT bile acids (square). B) OD₆₀₀ at 24 h for 10 isolates. Legend: BA = bile acids; and * = p <0.01. Data represent mean ± SEM.</p

<i>C</i>. <i>difficile</i> spores germinate in response to primary bile acids.

<p>A) (Left Panel) Relative OD₆₀₀ of NAP1 spores exposed to 0.5 mM (square) or 2 mM (triangle) TA versus BHIS alone (circle). (Right Panel) Relative OD₆₀₀ of spores from 10 isolates after 20 min exposure to 0.5 or 2 mM TA vs. BHIS alone. B) (Left Panel) Relative OD₆₀₀ of NAP1 spores exposed to 0.5 mM (square), 1 mM (diamond), or 2 mM (triangle) CA versus BHIS alone (circle). (Right Panel) Relative OD₆₀₀ of spores from 10 isolates after 20 min exposure to 0.5, 1, or 2 mM CA vs. BHIS alone. C) (Left) Relative OD₆₀₀ of NAP1 spores exposed to 0.25 mM (dashed line), 0.5 mM (square), 1 mM (diamond), or 2 mM (triangle) CDCA versus BHIS alone (circle). (Right Panel) Relative OD₆₀₀ of spores from 10 isolates after 20 min exposure to 0.25, 0.5, 1, or 2 mM CDCA vs. BHIS alone. OD₆₀₀(t)/OD₆₀₀(t₀) = OD₆₀₀ normalized to initial OD₆₀₀ (relative OD₆₀₀). Legends: *** = p < 0.001, ** = p < 0.01; * = p < 0.05, n.s. = non-significant. BHIS = BHI with yeast extract and L-cysteine; TA = taurocholate; CA = cholate; and CDCA = chenodeoxycholic acid. Data represent mean ± SEM.</p

Changes in Colonic Bile Acid Composition following Fecal Microbiota Transplantation Are Sufficient to Control <i>Clostridium difficile</i> Germination and Growth

<div><p>Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent <i>Clostridium difficile</i> infection (R-CDI), but its mechanisms remain poorly understood. Emerging evidence suggests that gut bile acids have significant influence on the physiology of <i>C</i>. <i>difficile</i>, and therefore on patient susceptibility to recurrent infection. We analyzed spore germination of 10 clinical <i>C</i>. <i>difficile</i> isolates exposed to combinations of bile acids present in patient feces before and after FMT. Bile acids at concentrations found in patients’ feces prior to FMT induced germination of <i>C</i>. <i>difficile</i>, although with variable potency across different strains. However, bile acids at concentrations found in patients after FMT did not induce germination and inhibited vegetative growth of all <i>C</i>. <i>difficile</i> strains. Sequencing of the newly identified germinant receptor in <i>C</i>. <i>difficile</i>, CspC, revealed a possible correspondence of variation in germination responses across isolates with mutations in this receptor. This may be related to interstrain variability in spore germination and vegetative growth in response to bile acids seen in this and other studies. These results support the idea that intra-colonic bile acids play a key mechanistic role in the success of FMT, and suggests that novel therapeutic alternatives for treatment of R-CDI may be developed by targeted manipulation of bile acid composition in the colon.</p></div

<i>C</i>. <i>difficile</i> spores do not germinate in response to secondary bile acids.

<p>A) (Left Panel) Relative OD₆₀₀ of NAP1 spores exposed to 0.5 mM (square), 1 mM (diamond), or 2 mM (triangle) DCA versus BHIS alone (circle). (Right Panel) Relative OD₆₀₀ of spores from 10 isolates after 20 min exposure to 0.5, 1, or 2 mM DCA vs. BHIS alone. B) (Left Panel) Relative OD₆₀₀ of NAP1 spores exposed to 0.5 mM (square), 1 mM (diamond), or 2 mM (triangle) LCA versus BHIS alone (circle). (Right Panel) Relative OD₆₀₀ of spores from 10 isolates after 20 min exposure to 0.5, 1, or 2 mM LCA vs. BHIS alone. OD₆₀₀(t)/OD₆₀₀(t₀) = OD₆₀₀ normalized to initial OD₆₀₀ (relative OD₆₀₀). Legend: n.s. = non-significant. BHIS = BHI with yeast extract and L-cysteine; DCA = deoxycholate; and LCA = lithocholic acid. Data represent mean ± SEM.</p