Distinct Gene Expression Profiles Define Anaplastic Grade in Retinoblastoma.

Morbidity and mortality associated with retinoblastoma have decreased drastically in recent decades, in large part owing to better prediction of high-risk disease and appropriate treatment stratification. High-risk histopathologic features and severe anaplasia both predict the need for more aggressive treatment; however, not all centers are able to assess tumor samples easily for the degree of anaplasia. Instead, identification of genetic signatures that are able to distinguish among anaplastic grades and thus predict high- versus low-risk retinoblastoma would facilitate appropriate risk stratification in a wider patient population. A better understanding of genes dysregulated in anaplasia also would yield valuable insights into pathways underlying the development of more severe retinoblastoma. Here, we present the histopathologic and gene expression analysis of 28 retinoblastoma cases using microarray analysis. Tumors of differing anaplastic grade show clear differential gene expression, with significant dysregulation of unique genes and pathways in severe anaplasia. Photoreceptor and nucleoporin expression in particular are identified as highly dysregulated in severe anaplasia and suggest particular cellular processes contributing to the development of increased retinoblastoma severity. A limited set of highly differentially expressed genes also are able to predict severe anaplasia accurately in our data set. Together, these data contribute to the understanding of the development of anaplasia and facilitate the identification of genetic markers of high-risk retinoblastoma.

Reactive Retinal Astrocytic Tumor (Focal Nodular Gliosis): A Case Report.

PURPOSE: To report the clinical and histopathological findings of a reactive retinal astrocytic tumor (RRAT) that progressed to massive retinal gliosis. OBSERVATIONS: The patient presented with an elevated, white-yellow retinal mass and extensive retinal exudation in the left eye. Progressive enlargement of the mass and proliferative vitreoretinopathy eventually led to phthisis bulbi and enucleation. Histologically, the mass showed a predominant astrocytic component with intense glial fibrillary acidic protein staining, hyperplasia, fibrous metaplasia, and osseous metaplasia of the retinal pigment epithelium. The Ki-67 proliferative index was <5%, and few scattered vascular channels were observed. CONCLUSIONS AND IMPORTANCE: These findings show that this tumor is the result of a reactive glial process rather than of neoplastic vascular proliferation. Massive retinal gliosis probably represents the advanced stage of RRAT.

Gleaning Insights from Fecal Microbiota Transplantation and Probiotic Studies for the Rational Design of Combination Microbial Therapies.

Beneficial microorganisms hold promise for the treatment of numerous gastrointestinal diseases. The transfer of whole microbiota via fecal transplantation has already been shown to ameliorate the severity of diseases such as Clostridium difficile infection, inflammatory bowel disease, and others. However, the exact mechanisms of fecal microbiota transplant efficacy and the particular strains conferring this benefit are still unclear. Rationally designed combinations of microbial preparations may enable more efficient and effective treatment approaches tailored to particular diseases. Here we use an infectious disease, C. difficile infection, and an inflammatory disorder, the inflammatory bowel disease ulcerative colitis, as examples to facilitate the discussion of how microbial therapy might be rationally designed for specific gastrointestinal diseases. Fecal microbiota transplantation has already shown some efficacy in the treatment of both these disorders; detailed comparisons of studies evaluating commensal and probiotic organisms in the context of these disparate gastrointestinal diseases may shed light on potential protective mechanisms and elucidate how future microbial therapies can be tailored to particular diseases.

Giant Chondroid Syringoma of the Lower Eyelid.

Chondroid syringoma is a benign mixed tumor characterized by sweat gland elements in a cartilaginous stroma. This rare tumor accounts for only 0.01% of all primary skin tumors and occurs only rarely in the periorbital region. Usually between 0.5 cm and 3.0 cm, risk of malignancy increases in chondroid syringomas greater than 3.0 cm in size. Here, the authors report a rare case of giant chondroid syringoma arising in the lower eyelid, characterized by keratinized stratified epithelium in a cartilaginous stroma. This case illustrates the importance of considering a possible diagnosis of chondroid syringoma in the evaluation of eyelid masses.

Characterization of the Probiotic Yeast Saccharomyces boulardii in the Healthy Mucosal Immune System.

The probiotic yeast Saccharomyces boulardii has been shown to ameliorate disease severity in the context of many infectious and inflammatory conditions. However, use of S. boulardii as a prophylactic agent or therapeutic delivery vector would require delivery of S. boulardii to a healthy, uninflamed intestine. In contrast to inflamed mucosal tissue, the diverse microbiota, intact epithelial barrier, and fewer inflammatory immune cells within the healthy intestine may all limit the degree to which S. boulardii contacts and influences the host mucosal immune system. Understanding the nature of these interactions is crucial for application of S. boulardii as a prophylactic agent or therapeutic delivery vehicle. In this study, we explore both intrinsic and immunomodulatory properties of S. boulardii in the healthy mucosal immune system. Genomic sequencing and morphological analysis of S. boulardii reveals changes in cell wall components compared to non-probiotic S. cerevisiae that may partially account for probiotic functions of S. boulardii. Flow cytometry and immunohistochemistry demonstrate limited S. boulardii association with murine Peyer's patches. We also show that although S. boulardii induces a systemic humoral immune response, this response is small in magnitude and not directed against S. boulardii itself. RNA-seq of the draining mesenteric lymph nodes indicates that even repeated administration of S. boulardii induces few transcriptional changes in the healthy intestine. Together these data strongly suggest that interaction between S. boulardii and the mucosal immune system in the healthy intestine is limited, with important implications for future work examining S. boulardii as a prophylactic agent and therapeutic delivery vehicle.

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice.

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation.

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.

Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.