ABSTRACTMany pathogens regulate or modify their immune-stimulating ligands to avoid detection by their infected hosts.Listeria monocytogenes, a facultative intracellular bacterial pathogen, interacts with multiple components of mammalian innate immunity during its infection cycle. During replication within the cytosol of infected cells,L. monocytogenesutilizes two multidrug efflux pumps, MdrM and MdrT, to secrete the small nucleic acid second messenger cyclic-di-AMP (c-di-AMP). Host recognition of c-di-AMP triggers the production of type I interferons, including beta interferon (IFN-β), which, surprisingly, promoteL. monocytogenesvirulence. In this study, we have examined the capacity of multiple laboratory and clinical isolates ofL. monocytogenesto stimulate host production of IFN-β. We have identified theL. monocytogenesstrain LO28 as able to hyperinduce IFN-β production in infected cells ∼30-fold more than the common laboratory cloneL. monocytogenesstrain 10403S. Genomic analyses determined that LO28 contains a naturally occurring loss-of-function allele of the transcriptional regulator BrtA and correspondingly derepresses expression of MdrT. Surprisingly, while derepression of MdrT resulted in hyperstimulation of IFN-β, it results in significant attenuation in multiple mouse models of infection. While type I interferons may promoteL. monocytogenesvirulence, this study demonstrates that unregulated expression of the c-di-AMP-secreting efflux pump MdrT significantly restricts virulencein vivoby an unknown mechanism.

Endothelin-A receptor blockade using atrasentan significantly reduces the vascular and neuroretinal complications in diabetic mice. Endothelin-A receptor blockade is a promising therapeutic target in diabetic retinopathy.

Trypsin digest is the gold standard method to analyze the retinal vasculature 1–5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

PurposeTo analyze the topographic correlation between reticular pseudodrusen (RPD) visualized on infrared reflectance (IR) and choroidal vasculature using en-face volumetric spectral-domain optical coherence tomography (SD-OCT).MethodsA masked observer marked individual RPD on IR images using ImageJ (NIH, Bethesda, MD). Using the macular volume scan (Cirrus, Carl Zeiss Meditec Inc, Dublin, CA), the RPE slab function was used to generate a C-scan of the most superficial choroidal vasculature. An independent masked grader created a topographic binary map of the choroidal vasculature by thresholding the en-face image, which was overlaid onto the IR map of RPD. For each IR image, ImageJ was used to generate a random set of dots as “control lesions”.Results17 eyes of 11 patients (78±13.7 years) with RPD were analyzed. The average number of RPD lesions identified on IR images was 414±71.5, of which 49.6±4.3% were located overlying the choroidal vasculature, compared to 45.4±4.0% in controls (p = 0.014). 50.4±4.3% of lesions overlay the choroidal stroma, of which 76.5±3.1% were ≤3 pixels from the choroidal vessels. The percentage of RPD lesions located within ≤3 pixels from the choroidal vasculature was significantly greater than the percentage located ≥7 pixels away. (p<0.0001). Compared to controls (71.6±3.8%), RPD were more likely to be located ≤3 pixels away from choroidal vessels (p = 0.014). In contrast, control lesions were more likely to be ≥7 pixels away from choroidal vessels than RPD (9.1±1.9% vs. 4.8±1.2%, respectively, p = 0.002).ConclusionsOur analysis shows that RPD lesions follow the underlying choroidal vasculature. Approximately half the RPD directly overlay the choroidal vessels and the majority of the remaining lesions were ≤3 pixels (≤30microns) from the vessel edge, supporting the hypothesis that RPD maybe related to pathologic changes at the choroidal level.

Diabetic Retinopathy is one of the hallmark microvascular diseases secondary to diabetes. Endothelial cells and pericytes are key players in the pathogenesis. Interaction between the two cell types is important in the regulation of vascular function and the maintenance of the retinal homeostatic environment. There are currently several approaches to analyze changes in morphology and function of the two cell types. Morphologic approaches include trypsin digest, while functional approaches include studying blood flow. This review explores the advantages and limitations of various methods and summarizes recent experimental studies of EC and pericyte dysfunction in rodent models of DR. An improved understanding of the role played by EC and pericyte dysfunction can lead to enhanced insights into retinal vascular regulation in DR and open new avenues for future treatments that reverse their dysfunction.