Background Physicians treating COVID-19 patients increasingly believe that the hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating biomarkers seen across the spectrum of COVID-19 have not been characterized compared to healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. Objective To identify and characterize the host inflammatory response to SARS-CoV-2 infection, we assessed levels of proteins related to immune responses and cardiovascular disease, in patients stratified as mild, moderate, and severe, versus matched healthy controls. Methods Blood samples from adult patients hospitalized with COVID-19 were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of 185 markers. Results Results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within COVID-19 patients, and strong biomarker association with patient response as measured by Ordinal scale. As patients progress, we observe statistically significant dysregulation of IFNγ, IL-1RA, IL-6, IL-10, IL-19, MCP-1, -2, -3, CXCL9, CXCL10, CXCL5, ENRAGE and PARP-1. Furthermore, in a limited series of patients who were sampled frequently confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm. Conclusion These wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. They also provide insights into mechanisms of SARS-CoV-2 pathogenesis and the host response.

Mutant Abl kinases (such as BCR-Abl) drive the development of leukemia; however little is known regarding whether Abl kinases contribute to the development or progression of solid tumors. We recently demonstrated that endogenous Abl kinases (c-Abl, Arg) are activated by deregulated ErbB receptors and Src kinases, and drive invasion of aggressive breast cancer cells. In this study, we examined whether activation of endogenous Abl kinases affects transformation, proliferation and survival, which are major contributors to breast cancer development and metastatic progression. Using a pharmacological inhibitor and RNAi, we demonstrate that activation of endogenous Abl kinases dramatically promotes breast cancer cell proliferation and anchorage-independent growth in serum, as well as survival following nutrient deprivation. Activation of Abl kinases mediates phosphorylation of STAT3, and promotes proliferation by accelerating G 1 -S progression. Moreover, we identify IGF-1R as a novel upstream activator of endogenous Abl kinases, and demonstrate that Abl kinase activation is required for IGF-1-stimulated cell cycle progression in breast cancer cells. Since activation of Abl kinases affects multiple steps of breast cancer development and progression, Abl kinase inhibitors are likely to be effective agents for the treatment of breast cancers containing highly active Abl kinases.

Despite 35 years of clinical trials, there is little improvement in one-year survival rates for patients with metastatic melanoma, and the disease is essentially untreatable if not cured surgically. The paucity of chemotherapeutic agents that are effective for treating metastatic melanoma indicates a dire need to develop new therapies. Here, we found a previously unrecognized role for c-Abl and Arg in melanoma progression. We demonstrate that the kinase activities of c-Abl and Arg (c-Abl, Arg) are elevated in primary melanomas (60%), in a subset of benign nevi (33%), and in some human melanoma cell lines. Using siRNA and pharmacological approaches, we show that c-Abl/Arg activation is functionally relevant because it is required for melanoma cell proliferation, survival, and invasion. Significantly, we identify the mechanism by which activated c-Abl promotes melanoma invasion by showing that it transcriptionally upregulates MMP-1, and using rescue approaches we demonstrate that c-Abl promotes invasion via a STAT3→MMP-1 pathway. Additionally, we show that c-Abl and Arg are not merely redundant, as active Arg drives invasion in a STAT3-independent manner, and upregulates MMP-3 and MT1-MMP, in addition to MMP-1. Most importantly, c-Abl and Arg not only promote in vitro processes important for melanoma progression, but also promote metastasis in vivo, as inhibition of c-Abl/Arg kinase activity with the c-Abl/Arg inhibitor, nilotinib, dramatically inhibits metastasis in a mouse model. Taken together, these data identify c-Abl and Arg as critical, novel, drug targets in metastatic melanoma, and indicate that nilotinib may useful in preventing metastasis in patients with melanomas harboring active c-Abl and Arg.

Anaplasma phagocytophilum infects neutrophils and myeloid, endothelial, and tick cell lines to reside within a host cell-derived vacuole that is indispensible for its survival. Here, we identify APH_0032 as an Anaplasma-derived protein that associates with the A. phagocytophilumoccupied vacuolar membrane (AVM). APH_0032 is a 66.1 kDa acidic protein that electrophoretically migrates with an apparent molecular weight of 130 kDa. It contains a predicted transmembrane domain and tandemly arranged direct repeats that comprise 46% of the protein. APH_0032 is undetectable on Anaplasma organisms bound to the surfaces of HL-60 cells, but is detected on the AVM and surfaces of intravacuolar bacteria beginning 24 h post-infection. APH_0032 localizes to the AVM in HL-60, THP-1, HMEC-1, and ISE6 cells. APH_0032, along with APH_1387, which encodes a confirmed AVM protein, is transcribed during A. phagocytophilum infection of tick salivary glands and murine neutrophils. APH_0032 localizes to the AVM in neutrophils recovered from infected mice. The Legionella pneumophila Dot/IcM type IV secretion system (T4SS) can heterologously secrete a CyaA-tagged version of the A. phagocytophilum VirB/D T4SS effector, AnkA, but fails to secrete CyaA-tagged APH_0032 or -APH_1387. These data confirm APH_0032 as an Anaplasma-derived AVM protein and hint that neither it nor APH_1387 are T4SS effectors.

Obligate vacuolar pathogens produce proteins that localize to the host cell-derived membranes of the vacuoles in which they reside, yielding unique organelles that are optimally suited for pathogen survival. Anaplasma phagocytophilum is an obligate vacuolar bacterium that infects neutrophils and causes the emerging and potentially fatal disease human granulocytic anaplasmosis. Here we identified APH_1387 as the first A. phagocytophilum-derived protein that associates with the A. phagocytophilum-occupied vacuolar membrane (AVM). APH_1387, also referred to as P100, is a 61.4-kDa acidic protein that migrates with an apparent molecular weight of 115 kDa on SDS-PAGE gels. It carries 3 tandem direct repeats that comprise 58% of the protein. Each APH_1387 repeat carries a bilobed hydrophobic alpha-helix domain, which is a structural characteristic that is consistent with the structure of chlamydia-derived proteins that traverse inclusion membranes. APH_1387 is not detectable on the surfaces of A. phagocytophilum dense core organisms bound at the HL-60 cell surface, but abundant APH_1387 is detected on the surfaces of intravacuolar reticulate cell and dense core organisms. APH_1387 accumulates on the AVM throughout infection. It associates with the AVM in human HL-60, THP-1, and HMEC-1 cells and tick ISE6 cells. APH_1387 is expressed and localizes to the AVM in neutrophils recovered from A. phagocytophilum-infected mice. This paper presents the first direct evidence that A. phagocytophilum actively modifies its host cell-derived vacuole.