Severe acute respiratory syndrome (SARS) is an acute infectious disease of the respiratory system. Although a novel coronavirus has been identified as the causative agent of SARS, the pathogenic mechanisms of SARS are not understood. In this study, sera were collected from healthy donors, patients with SARS, patients with severe SARS, and patients with SARS in convalescence. The serum concentrations of interleukin-1 (IL-1), IL-4, IL-6, IL-8, IL-10, tumor growth factor beta (TGF-␤), tumor necrosis factor alpha (TNF-␣), and gamma interferon (IFN-␥) were measured by enzyme-linked immunosorbent assays (ELISA). The concentrations of IL-1 and TNF-␣ were not significantly different in different groups. The IL-6 concentration was increased in SARS patients and was significantly elevated in severe SARS patients, but the IL-6 concentrations were similar in convalescent patients and control subjects, suggesting that there was a positive relationship between the serum IL-6 concentration and SARS severity. The concentrations of IL-8 and TGF-␤ were decreased in SARS patients and significantly reduced in severe SARS patients, but they were comparable in convalescent SARS patients and control subjects, suggesting that there was a negative relationship between the IL-8 and TGF-␤ concentrations and SARS severity. The concentrations of IFN-␥, IL-4, and IL-10 showed significant changes only in convalescent SARS patients. The IFN-␥ and IL-4 levels were decreased, while the levels of IL-10 were increased, and the differences between convalescent SARS patients and other patient groups were statistically significant. These results suggest that there are different immunoregulatory events during and after SARS and may contribute to our understanding of the pathogenesis of this syndrome.

Increased pulmonary vascular permeability due to endothelial cell (EC) barrier dysfunction is a major pathological feature of acute respiratory distress syndrome/acute lung injury (ARDS/ALI), which is a devastating critical illness with high incidence and excessive mortality. Activation of NADPH oxidase (NOX) induces EC dysfunction via production of reactive oxygen species (ROS). However, the role(s) of NOX isoform(s), and their downstream signaling events, in the development of ARDS/ALI have remained unclear. Cecal Ligation Puncture (CLP) was used to induce preclinical septic ALI in wild-type mice and mice deficient in NOX2 or p47phox, or mice transfected of control siRNA, NOX1 or NOX4 siRNA in vivo. The survival rate of the CLP group at 24 h (26.6%, control siRNA treated) was substantially improved by NOX4 knockdown (52.9%). Mice lacking NOX2 or p47phox, however, had worse outcomes after CLP (survival rates at 0% and 8.3% respectively), whereas NOX1-silenced mice had similar survival rate (30%). NOX4 knockdown attenuated lung ROS production in septic mice, whereas NOX1 knockdown, NOX2 knockout, or p47phox knockout in mice had no effects. In addition, NOX4 knockdown attenuated redox-sensitive activation of the CaMKII/ERK1/2/MLCK pathway, and restored expression of EC tight junction proteins ZO-1 and Occludin to maintain EC barrier integrity. Correspondingly, NOX4 knockdown in cultured human lung microvascular ECs also reduced LPS-induced ROS production, CaMKII/ERK1/2/MLCK activation and EC barrier dysfunction. Scavenging superoxide in vitro and in vivo with TEMPO, or inhibiting CaMKII activation with KN93, had similar effects as NOX4 knockdown in preserving EC barrier dysfunction. In summary, we have identified a novel, selective and causal role of NOX4 (versus other NOX isoforms) in inducing lung EC barrier dysfunction and injury/mortality in a preclinical CLP-induced septic model, which involves redox-sensitive activation of CaMKII/ERK1/2/MLCK pathway. Targeting NOX4 may therefore prove to an innovative therapeutic option that is markedly effective in treating ALI/ARDS.

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.

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