Perturbation of Mouse Retinal Vascular Morphogenesis by Anthrax Lethal Toxin

Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis

Association of RNA Biosignatures With Bacterial Infections in Febrile Infants Aged 60 Days or Younger

ImportanceYoung febrile infants are at substantial risk of serious bacterial infections; however, the current culture-based diagnosis has limitations. Analysis of host expression patterns ("RNA biosignatures") in response to infections may provide an alternative diagnostic approach.ObjectiveTo assess whether RNA biosignatures can distinguish febrile infants aged 60 days or younger with and without serious bacterial infections.Design, setting, and participantsProspective observational study involving a convenience sample of febrile infants 60 days or younger evaluated for fever (temperature >38° C) in 22 emergency departments from December 2008 to December 2010 who underwent laboratory evaluations including blood cultures. A random sample of infants with and without bacterial infections was selected for RNA biosignature analysis. Afebrile healthy infants served as controls. Blood samples were collected for cultures and RNA biosignatures. Bioinformatics tools were applied to define RNA biosignatures to classify febrile infants by infection type.ExposureRNA biosignatures compared with cultures for discriminating febrile infants with and without bacterial infections and infants with bacteremia from those without bacterial infections.Main outcomes and measuresBacterial infection confirmed by culture. Performance of RNA biosignatures was compared with routine laboratory screening tests and Yale Observation Scale (YOS) scores.ResultsOf 1883 febrile infants (median age, 37 days; 55.7% boys), RNA biosignatures were measured in 279 randomly selected infants (89 with bacterial infections-including 32 with bacteremia and 15 with urinary tract infections-and 190 without bacterial infections), and 19 afebrile healthy infants. Sixty-six classifier genes were identified that distinguished infants with and without bacterial infections in the test set with 87% (95% CI, 73%-95%) sensitivity and 89% (95% CI, 81%-93%) specificity. Ten classifier genes distinguished infants with bacteremia from those without bacterial infections in the test set with 94% (95% CI, 70%-100%) sensitivity and 95% (95% CI, 88%-98%) specificity. The incremental C statistic for the RNA biosignatures over the YOS score was 0.37 (95% CI, 0.30-0.43).Conclusions and relevanceIn this preliminary study, RNA biosignatures were defined to distinguish febrile infants aged 60 days or younger with vs without bacterial infections. Further research with larger populations is needed to refine and validate the estimates of test accuracy and to assess the clinical utility of RNA biosignatures in practice

Choroidal Thickness Profiles in Myopic Eyes of Young Adults in the Correction of Myopia Evaluation Trial Cohort

PurposeTo examine the relationship of choroidal thickness with axial length (AL) and myopia in young adult eyes in the ethnically diverse Correction of Myopia Evaluation Trial (COMET) cohort.DesignCross-sectional, multicenter study.MethodsIn addition to measures of myopia by cycloplegic autorefraction and AL by A-scan ultrasonography, participants underwent optical coherence tomography imaging of the choroid in both eyes at their last visit (14 years after baseline). Using digital calipers, 2 independent readers measured choroidal thickness in the right eye (left eye if poor quality; n = 37) at 7 locations: fovea and 750, 1500, and 2250 μm nasal (N) and temporal (T) to the fovea.ResultsChoroidal thickness measurements were available from 294 of 346 (85%) imaged participants (mean age: 24.3 ± 1.4 years; 44.9% male) with mean myopia of -5.3 ± 2.0 diopters and mean AL of 25.5 ± 1.0 mm. Overall, choroidal thickness varied by location (P < .0001) and was thickest at the fovea (273.8 ± 70.9 μm) and thinnest nasally (N2250, 191.5 ± 69.3 μm). Multivariable analyses showed significantly thinner choroids in eyes with more myopia and longer AL at all locations except T2250 (P ≤ .001) and presence of peripapillary crescent at all locations except T1500 and T2250 (P ≤ .0001). Choroidal thickness varied by ethnicity at N2250 (P < .0001), with Asians having the thinnest and African Americans the thickest choroids.ConclusionChoroids are thinner in longer, more myopic young adult eyes. The thinning was most prominent nasally and in eyes with a crescent. In the furthest nasal location, ethnicity was associated with choroidal thickness. The findings suggest that choroidal thickness should be evaluated, especially in the nasal regions where myopic degenerations are most commonly seen clinically