Reemergence of Reston ebolavirus in Cynomolgus Monkeys, the Philippines, 2015.

In August 2015, a nonhuman primate facility south of Manila, the Philippines, noted unusual deaths of 6 cynomolgus monkeys (Macaca fascicularis), characterized by generalized rashes, inappetence, or sudden death. We identified Reston ebolavirus (RESTV) infection in monkeys by using serologic and molecular assays. We isolated viruses in tissues from infected monkeys and determined viral genome sequences. RESTV found in the 2015 outbreak is genetically closer to 1 of the 4 RESTVs that caused the 2008 outbreak among swine. Eight macaques, including 2 also infected with RESTV, tested positive for measles. Concurrently, the measles virus was circulating throughout the Philippines, indicating that the infection of the macaques may be a reverse zoonosis. Improved biosecurity measures will minimize the public health risk, as well as limit the introduction of disease and vectors.

Quantitative intravital imaging for real-time monitoring of pancreatic tumor cell hypoxia and stroma in an orthotopic mouse model.

Pancreatic cancer is a lethal disease with few successful treatment options. Recent evidence demonstrates that tumor hypoxia promotes pancreatic tumor invasion, metastasis, and therapy resistance. However, little is known about the complex relationship between hypoxia and the pancreatic tumor microenvironment (TME). In this study, we developed a novel intravital fluorescence microscopy platform with an orthotopic mouse model of pancreatic cancer to study tumor cell hypoxia within the TME in vivo, at cellular resolution, over time. Using a fluorescent BxPC3-DsRed tumor cell line with a hypoxia-response element (HRE)/green fluorescent protein (GFP) reporter, we showed that HRE/GFP is a reliable biomarker of pancreatic tumor hypoxia, responding dynamically and reversibly to changing oxygen concentrations within the TME. We also characterized the spatial relationships between tumor hypoxia, microvasculature, and tumor-associated collagen structures using in vivo second harmonic generation microscopy. This quantitative multimodal imaging platform enables the unprecedented study of hypoxia within the pancreatic TME in vivo.

Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction.

Aberrant ceramide build-up in preeclampsia, a serious disorder of pregnancy, causes exuberant autophagy-mediated trophoblast cell death. The significance of ceramide accumulation for lysosomal biogenesis in preeclampsia is unknown. Here we report that lysosome formation is markedly increased in trophoblast cells of early-onset preeclamptic placentae, in particular in syncytiotrophoblasts. This is accompanied by augmented levels of transcription factor EB (TFEB). In vitro and in vivo experiments demonstrate that ceramide increases TFEB expression and nuclear translocation and induces lysosomal formation and exocytosis. Further, we show that TFEB directly regulates the expression of lysosomal sphingomyelin phosphodiesterase (L-SMPD1) that degrades sphingomyelin to ceramide. In early-onset preeclampsia, ceramide-induced lysosomal exocytosis carries L-SMPD1 to the apical membrane of the syncytial epithelium, resulting in ceramide accumulation in lipid rafts and release of active L-SMPD1 via ceramide-enriched exosomes into the maternal circulation. The SMPD1-containing exosomes promote endothelial activation and impair endothelial tubule formation in vitro. Both exosome-induced processes are attenuated by SMPD1 inhibitors. These findings suggest that ceramide-induced lysosomal biogenesis and exocytosis in preeclamptic placentae contributes to maternal endothelial dysfunction, characteristic of this pathology.

Differential effects of the 1,25D3-MARRS receptor (ERp57/PDIA3) on murine mammary gland development depend on the vitamin D3 dose.

1,25 dihydroxyvitamin D3 (1,25D3) is the most potent biologically active form of vitamin D3. Its actions on the mammary gland include cell growth inhibition and anti-cancer effects. This study's purpose was to explore the role of the 1,25D3-membrane associated rapid response steroid (MARRS) receptor in the mammary gland using a tissue-specific knockout mouse model and a vitamin D3 dietary intervention. Three genotype groups were created using the Cre/loxp system to knock-down (+/-) and knockout (-/-) the MARRS receptor in epithelial cells of mammary glands (MG). Abdominal MGs were collected from 6-week old female mice (n = 94) on diets of 10,000 IU/kg (excess), 1,000 IU/kg (sufficient) or 0 IU/kg (deficient) of D3. There was a significant interaction between genotype and diet regarding number of terminal end buds (TEBs) (p = 0.001) and ductal coverage of the fat pad (p = 0.03). MARRS -/- mice on the sufficient diet had significantly fewer TEBs (p = 0.001) compared to MARRS +/+ on the same diet, but the opposite effect was seen in mice on the excess diet. There were no effects of genotype on TEBs when animals were vitamin D3 deficient. These results suggest that there is an effect of MARRS on mammary gland development that is dependent on 25(OH)D status, specifically, altering the number of highly proliferative TEBs. Increased numbers of TEBs have been correlated with increased breast cancer risk later in life. Therefore the results of this study warrant further examination of 25(OH)D status and recommendations in adolescent humans to reduce dietary effects on future breast cancer risk.

Multi-site evaluation of the LN34 pan-lyssavirus real-time RT-PCR assay for post-mortem rabies diagnostics.

Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse Lyssavirus genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance.