Lymph node blood vessels play important roles in the support and trafficking of immune cells. The blood vasculature is a component of the vascular-stromal compartment that also includes the lymphatic vasculature and fibroblastic reticular cells (FRCs). During immune responses, as lymph nodes swell, the blood vasculature undergoes a rapid proliferative growth that is initially dependent on CD11c+ cells and VEGF but is independent of lymphocytes. The lymphatic vasculature grows with similar kinetics and VEGF dependence, suggesting co-regulation of blood and lymphatic vascular growth, but lymphatic growth has been shown to be B cell-dependent. Here we show that blood vascular, lymphatic, and FRC growth are coordinately regulated and identify two distinct phases of vascular-stromal growth—an initiation phase, characterized by upregulated vascular-stromal proliferation, and a subsequent expansion phase. The initiation phase is CD11c+ cell-dependent and T/B cell-independent while the expansion phase is dependent on B and T cells together. Using CCR7−/− mice and selective depletion of migratory skin dendritic cells, we show that endogenous skin-derived dendritic cells are not important during the initiation phase and uncover a modest regulatory role for CCR7. Finally, we show that FRC VEGF expression is upregulated during initiation and that dendritic cells can stimulate increased fibroblastic VEGF, suggesting the scenario that lymph node-resident CD11c+ cells orchestrate the initiation of blood and lymphatic vascular growth in part by stimulating FRCs to upregulate VEGF. These results illustrate how the lymph node microenvironment is shaped by the cells it supports.

The novel coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic, but the factors influencing viral RNA shedding, which would help inform optimal control strategies, remain unclear. Methods: The clinical course and viral RNA shedding pattern of 267 consecutive symptomatic COVID-19 patients admitted to the hospital from January 20, 2020 to March 15, 2020 were evaluated retrospectively. Results: The median duration of viral RNA shedding was 12 days (interquartile range 8-16 days) after the onset of illness. Of the 267 patients included in this study, 65.2% had viral RNA clearance within 14 days, 88.8% within 21 days, and 94.4% within 28 days. Older age (hazard ratio (HR) 0.99, 95% confidence interval (CI) 0.98-1.00; p = 0.04), time lag from illness onset to hospital admission (HR 0.91, 95% CI 0.88-0.94; p < 0.001), diarrhea (HR 0.59, 95% CI 0.36-0.96; p = 0.036), corticosteroid treatment (HR 0.60, 95% CI 0.39-0.94; p = 0.024), and lopinavir/ritonavir use (HR 0.70, 95% CI 0.52-0.94; p = 0.014) were significantly and independently associated with prolonged viral RNA shedding. Conclusions: Early detection and timely hospital admission may be warranted for symptomatic COVID-19 patients, especially for older patients and patients with diarrhea. Corticosteroid treatment is associated with prolonged viral RNA shedding and should be used with caution. Lopinavir/ritonavir use may be associated with prolonged viral RNA shedding in non-severe patients; further randomized controlled trials are needed to confirm this finding.

Complex I is a critical site of O2•− production and the major host of reactive protein thiols in mitochondria. In response to oxidative stress, Complex I protein thiols at the 51 kDa and 75 kDa subunits are reversibly S-glutathionylated. The mechanism of Complex I S-glutathionylation is mainly obtained from insight into GSSG-mediated thiol-disulfide exchange, which would require a dramatic decline in the GSH/GSSG ratio. Intrinsic Complex I S-glutathionylation can be detected in the rat heart at a relatively high GSH/GSSG ratio (Chen, J., et al. J. Biol. Chem 285: 3168–3180, 2010). Thus, we hypothesized that reactive thiyl radical is more likely to mediate protein S-glutathionylation of Complex I. Here we employed immuno-spin trapping and tandem mass spectrometry (LC/MS/MS) to test the hypothesis in the 75 kDa subunit from S-glutathionylated Complex I. Under the conditions of O2•− production in the presence of GSH, we detected Complex I S-glutathionylation at the Cys-226, Cys-367, and Cys-727 of the 75 kDa subunit. Addition of a radical trap, 5, 5-dimethyl-1-Pyroline-N-Oxide (DMPO), significantly decreased Complex I S-glutathionylation, and subsequently increased the protein radical adduct of Complex I-DMPO as detected by immunoblotting using an anti-DMPO antibody. LC/MS/MS analysis indicated that Cys-226, Cys-554, and Cys-727 were involved in DMPO-binding, confirming that formation of the Complex I thiyl radical mediates S-glutathionylation. LC/MS/MS analysis also showed that Cys-554 and Cys-727 were S-sulfonated under conditions of O2•− generation in the absence of DMPO. In myocytes (HL-1 cell line) treated with menadione to trigger mitochondrial O2•− generation, Complex I protein radical and S-glutathionylation were increased. Thus mediation of Complex I S-glutathionylation by the protein thiyl radical provides a unique pathway for the redox regulation of mitochondrial function.

Increased O 2. and NO production is a key mechanism of mito- To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90 and probed with antibodies against 3-nitrotyrosine. Enhancement of protein tyrosine nitration was detected in the post-ischemic myocardium. Isolated SQR was subjected to in vitro protein nitration with peroxynitrite, leading to site-specific nitration at the 70-kDa polypeptide and impairment of SQR electron transfer activity. Protein nitration of SQR further impaired its protein-protein interaction with Complex III. Liquid chromatography/tandem mass spectrometry analysis indicated that Tyr-56 and Tyr-142 were involved in protein tyrosine nitration. When the isolated SQR was subjected to in vitro S-glutathionylation, oxidative modification and impairment mediated by peroxynitrite were significantly decreased, thus confirming the protective effect of S-glutathionylation from the oxidative damage of nitration.