Hitomi Inoue

Cytokine release syndrome (CRS) is a life-threatening complication induced by systemic inflammatory responses to infections, including bacteria and chimeric antigen receptor T cell therapy. There are currently no immunotherapies with proven clinical efficacy and understanding of the molecular mechanisms of CRS pathogenesis is limited. Here, we found that patients diagnosed with CRS from sepsis, acute respiratory distress syndrome (ARDS), or burns showed common manifestations: strikingly elevated levels of the four proinflammatory cytokines interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and IL-10 and the coagulation cascade activator plasminogen activator inhibitor-1 (PAI-1). Our in vitro data indicate that endothelial IL-6 trans-signaling formed an inflammation circuit for robust IL-6, IL-8, and MCP-1 production and promoted PAI-1 production; additionally, an IL-6 signaling blockade by the human monoclonal antibody tocilizumab blunted endothelial cell activation. Plasma from severe COVID-19 patients similarly exhibited increased IL-6, IL-10, and MCP-1 levels, but these levels were not as high as those in patients with CRS from other causes. In contrast, the PAI-1 levels in COVID-19 patients were as highly elevated as those in patients with bacterial sepsis or ARDS. Tocilizumab treatment decreased the PAI-1 levels and alleviated critical illness in severe COVID-19 patients. Our findings suggest that distinct levels of cytokine production are associated with CRS induced by bacterial infection and COVID-19, but both CRS types are accompanied by endotheliopathy through IL-6 trans-signaling. Thus, the present study highlights the crucial role of IL-6 signaling in endothelial dysfunction during bacterial infection and COVID-19.

p70 S6 kinase alpha (p70alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70alpha activation in a manner that can be overcome by coexpression of p70alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70alpha phosphorylation in vitro is accompanied by a substantial restoration in p70alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70alpha activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined.

BACKGROUND: The mammalian target of rapamycin (mTOR) belongs to the family of phosphoinositide (PI)-kinase-related kinases that includes the ataxia-telangiectasia gene product (ATM). mTOR plays a critical role in controlling translational effectors such as p70 S6 kinase alpha (p70 alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). RESULTS: We show that the C-terminal region of mTOR, which is highly conserved among the PI-kinase-related kinases, plays a critical role in the mTOR protein kinase activity. Deletion of the C-terminal residues did not adversely affect the expression of mTOR, but caused a nearly complete loss of the mTOR protein kinase activity toward both 4EBP1 and p70 alpha in vitro. These deletions also abolished the ability of a rapamycin-resistant mTOR mutant to rescue the activity of p70 alpha from inhibition induced by rapamycin in vivo. Furthermore, replacement of Trp2545, a conserved residue in the C-terminal region throughout the PI-kinase-related kinase family, abolished the function of the mTOR kinase, both in vivo and in vitro. However, substitution of 32 C-terminal residues of mTOR with those of ATM did not restore the mTOR function. CONCLUSIONS: These findings define an indispensable role for the noncatalytic C-terminal region of mTOR and indicate that, although this highly conserved region may be important throughout the PI-kinase-related kinase family, it is not functionally interchangeable within the family.

The effects of bile salts on the dissolution behavior of indomethacin and phenylbutazone were investigated. Bile salts such as sodium desoxycholate and sodium cholate considerably enhanced the dissolution of both drugs in pH 7.3 buffer at 37°. The results indicated that the enhancement of the dissolution of indomethacin in the presence of bile salts was mainly due to micellar solubilization. On the other hand, the enhanced dissolution of phenylbutazone may be due to the wetting effect, increasing the effective surface area of the powder.