Tumor necrosis factor-␣ (TNF-␣), a cytokine secreted by inflammatory cells, has been implicated in several inflammatory disease, is a potent, orally active inhibitor of the TNF-␣ convertase (TACE), an enzyme responsible for proteolytic cleavage of the membrane bound precursor, pro-TNF-␣. Ro 32-7315 inhibited a recombinant form of TACE (IC 50 ϭ 5.2 nM) with selectivity over related matrix metalloproteinases. In a cellular assay system, THP-1 cell line, and in human and rat whole blood, Ro 32-7315 significantly reduced lipopolysaccharide (LPS)-induced TNF-␣ release with IC 50 values of 350 Ϯ 14 nM (n ϭ 5), 2.4 Ϯ 0.5 M (n ϭ 5), and 110 Ϯ 18 nM (n ϭ 5), respectively. Oral administration of Ro 32-7315 to Wistar rats caused a dose-dependent inhibition of LPS-induced release of systemic TNF-␣ with an ED 50 of 25 mg/kg. Treatment (days 0 -14) of Allen and Hamburys hooded rats with Ro 32-7315 (2.5, 5, 10, and 20 mg/kg, i.p., twice daily) significantly reduced adjuvant-induced secondary paw swelling (42, 71, 83, and 93%, respectively) as compared with the vehicle group. In the Ro 32-7315-treated group, the reduced paw swelling was associated with improved lesion score and joint mobility. Furthermore, in a placebocontrolled, single-dose study, Ro 32-7315 given orally (450 mg) significantly suppressed ex vivo, LPS-induced TNF-␣ release in the whole-blood samples taken from healthy male and female volunteers (mean inhibition of 42% over a 4-h duration, n ϭ 6). These data collectively support the potential use of such a compound for the oral treatment of inflammatory disorders.
We report contraction-expansion array (CEA) microchannels for high-yield blood plasma separation utilizing differential inertial migration. An abrupt change of the cross-sectional area curves fluid streams and accelerates the flow velocity, which induces Dean flow. Utilizing the balance between inertial lift force and Dean drag force, we were able to separate blood plasma from human whole blood with a level of 62.2% yield. The CEA microfluidic device is easy to fabricate in a single layer fabrication process and expected to be useful for a simple blood plasma extraction on a chip with high-yield.
The CRISPR/Cas9 system has proved to be a powerful tool for knockout and knock-in in various species. When 2 components—Cas9 and single guide (sg)RNA—are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for gene editing in the CRISPR/Cas9 system, some studies have reported the production of Cas9-expressing animals, such as mice, which could be used to increase gene editing efficiency in subsequent experiments. In previous reports, we successfully produced 4 Cas9-expressing cattle via microinjection (Hahn et al. 2016 Reprod. Fertil. Dev. 29, 211). Primary cells from these calves had Cas9 activity because transfection of only sgRNA resulted in gene deletion. The aim of this study was to analyse the blood of the transgenic cattle to investigate the effect of Cas9 expression on health. Two of 4 transgenic calves died; one had severe ruminant tympany, failed to respond to treatment, and died at 4 months of age, and the other died at 5 months of age due to accidental ingestion of a needle from a feed bunk. Blood samples were obtained from the surviving 2 transgenic cattle (1 male and 1 female) at 7 and 12 months for blood analysis. Five milliliters of whole blood samples was collected from the jugular vein. Portions were used for CBC (Hemavet 950, Drew Scientific, Miami Lakes, FL, USA) and for serum chemistry analysis (BS-400, Mindray, Shenzhen, China). Average values for white blood cells (9600 and 1057/mm3), neutrophils (4590 and 3870/mm3), lymphocytes (4020 and 5910/mm3), red blood cells (732,000 and 798,000/mm3), hemoglobin (9.5 and 10.2 g dL−1), packed cell volume (24.3 and 25.3%), platelet (439,000 and 327,500/mm3), AST (76 and 104 IU), ALP (140 and 133 IU), BUN (7.5 and 10.5 mg dL−1), and creatinine (1.3 and 1.0 mg dL−1) of male and female transgenic calves were within the reference range. Additionally, there was no difference in general health information, including body temperature and feeding. In conclusion, we demonstrated that continuous Cas9 expression in transgenic cattle did not affect health status of the surviving calves in terms of blood analysis. They have grown up without any health issues and are currently 14 (female) and 15 (male) months old. In the near future, we will evaluate their germline transmission by natural breeding or in vitro fertilization. This work was supported by BK21 PLUS Program for Creative Veterinary Science, NRF (NRF-2017R1A2B3004972), and Seoul Milk Coop (SNU 550–20160004).
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