Is the Success of Cefazolin plus Ertapenem in Methicillin-Susceptible Staphylococcus aureus Bacteremia Based on Release of Interleukin 1-beta?

Cefazolin and ertapenem has been shown to be an effective salvage regimen for refractory methicillin-susceptible Staphylococcus aureus bacteremia. Our findings suggest cefazolin plus ertapenem in vitro stimulates interleukin-1β release from peripheral blood monocytes both with and without S. aureus presence. This IL-1β augmentation was primarily driven by ertapenem. These findings support further exploration of cefazolin plus ertapenem in MSSA bacteremia and may partially explain its marked potency in vivo despite modest synergy in vitro .

The Lipopolysaccharide Mutant Re-LPS Is a Useful Tool for Detecting LPS Contamination in Rheumatoid Synovial Cell Cultures

<b><i>Introduction:</i></b> Lipopolysaccharide (LPS) contamination of commercially available proteins has seriously impeded research on citrullinated fibrinogen (cit-Fb) in rheumatoid synovial cells (RSCs). <b><i>Methods:</i></b> RSCs obtained from 4 rheumatoid arthritis patients who underwent full knee arthroplasty were cultured, stimulated with cit-Fb, and cytokine expression levels were measured. We then evaluated polymyxin-B (PMB), heat inactivation, and rough (R)-type LPS mutants for rapid detection of LPS contamination. <b><i>Results:</i></b> cit-Fb induced expression of <i>CXCL10</i> and <i>IFNB</i> in RSCs via the toll-like receptor. PMB inhibited cit-Fb-mediated CXCL10 gene expression but not protein expression induced by 20 μg/mL cit-Fb. Heat inactivation did not affect LPS-mediated <i>CXCL10</i> or <i>IL-6</i> induction; however, cit-Fb-mediated <i>CXCL10</i>expression was inhibited. Wild-type LPS from <i>Escherichia coli</i> (WT-LPS) strongly induces <i>CXCL10</i> expression, but induction by Ra-LPS was weak, and induction by Rc- and Re-LPS was minimal. Re-LPS suppression of WT-LPS-mediated <i>CXCL10</i> induction in RSCs and peripheral blood monocytes (PBMs) was dose dependent. Furthermore, Re-LPS completely suppressed cit-Fb-mediated <i>CXCL10</i> induction in RSCs and PBMs. <b><i>Conclusion:</i></b> To easily identify LPS contamination during routine experiments, our results suggest that Re-LPS is a better tool for rapid detection of LPS contamination compared to PMB and heat treatment.

649 Efferocytosis drives myeloid inflammasome signaling and Gasdermin D independent secretion of IL-1β to promote tumor growth

BackgroundInflammation has long been associated with different stages of tumorigenesis as well as response to therapy. A key signaling pathway in this context is the casp-1 inflammasome. However, to date, its role in cancer has been contradictory and context dependent. We previously reported myeloid casp-1 can promote tumor growth in T cell independent manner. However, the regulatory mechanism that drives the myeloid intrinsic inflammasome signaling in the context of tumor growth remains largely unknown.MethodsIn order to gain finer details about the inflammasome pathway components in the different myeloid clusters, we analyzed tumor and blood samples from head and neck cancer patients using bulk as well as 10X single cell sequencing platforms. For in vivo tumor studies, genetically engineered preclinical mice models were used. For in vitro functional studies, cells were isolated from mice or human tumors/blood and differentiated to either MDSC or macrophages and subjected to various assays.ResultsOur bulk sequencing of myeloid cells isolated from treatment naïve head and neck tumors revealed an enrichment for inflammasome genes. Unbiased pathway analysis of tumor infiltrating myeloid cells compared to matched peripheral blood monocytes revealed IL-1β signaling to be significantly altered in the tumor myeloids. In our single cell transcriptomic sequencing dataset on human head & neck carcinoma with matched peripheral blood monocytes, we observed similar elevated inflammasome transcriptomic activity within specific clusters of tumor-infiltrating macrophages and myeloid derived suppressor cells. Interestingly, distinct inflammasome sensor genes, specifically NLRP3, had distinct co-expressions with IL-1β in specific myeloid subsets within the TME. Our data also indicates that myeloid-intrinsic caspase-1 signaling paradoxically increased tumor infiltrating myeloid cell survival without significant intratumoral trafficking into the tumor. When we explored the TME regulatory factors that regulate intratumoral myeloid inflammasome signaling, we found that NLRP3 dependent inflammasome signaling and IL-1β production promotes tumor growth in a Gasdermin D independent mechanism. Mechanistically, we show that efferocytosis of dying tumor cells by myeloid cells in the TME directly activates NLRP3 dependent inflammasome signaling and IL-1 β production in myeloid cells to promote tumor growth rate.ConclusionsTo our knowledge, we are the first to attribute the tumor supporting role of myeloid inflammasome signaling to efferocytic clearance of apoptotic debris in the tumor microenvironment. Our study thus opens an enticing option of novel therapeutic modality for treatment of solid tumors in future.Ethics ApprovalAll experimental procedures were approved by the Institutional Review Board of Vanderbilt University Medical Center (IRB: 170172).

Changes in Phenotypic Patterns of Blood Monocytes After Kidney Transplantation and During Acute Rejection

Peripheral blood monocytes, which serve as precursors for tissue macrophages and dendritic cells (DC), play a key role in the immune response to kidney allograft, reparation processes and homeostasis regulation. In this prospective study, we used multicolor flow cytometry to monitor the phenotypic patterns of peripheral monocytes in subjects with uncomplicated outcomes and those with acute rejection. We found a reciprocal increase in the proportion of “classical monocytes” (CD14+CD16-) along with a decline in pro-inflammatory “intermediary” (CD14+CD16+) and “non-classical” (CD14lowCD16+) monocytes in subjects with normal outcomes. In subjects with acute rejection, we observed no reduction in “intermediary” monocytes and no increase in “classical” monocytes. Patients with uncomplicated outcomes exhibited downregulated HLA-DR in all three monocyte subpopulations. However, non-classical monocytes were unaffected in subjects with acute rejection. Expression of CD47 was downregulated after transplantation, while patients with antibody-mediated rejection and donor-specific antibodies showed higher pre-transplant values. In monocytes isolated at the time of biopsy, CD47 expression was higher in individuals with acute rejection compared to patients with normal outcomes one year post-transplant. Expression of CD209 (DC-SIGN) and the proportion of CD163+CD206+ subpopulations were upregulated during the first week after kidney transplantation. CD209 was also upregulated in samples taken on the day of biopsy confirming acute rejection. Our data demonstrate that kidney allograft transplantation is associated with phenotypic changes in peripheral blood monocytes during acute rejection.

Evaluation of the Safety and Feasibility of Apheresis in Dogs: For Application in Metastatic Cancer Research

In patients with solid tumors, circulating tumor cells (CTCs) spread in their blood and function as a seed for metastases. However, the study of CTCs has been limited by their rarity, low frequency, and heterogeneity. The efficient collection of CTCs will contribute to further research of metastatic cancers. Apheresis is a process in which the whole blood of an individual is passed through a machine that isolates a particular constituent and returns the remainder to the circulation. In the present study, we investigated the safety and feasibility of apheresis to separate peripheral blood monocytes (PBMCs), whose density is closely similar to that of CTCs, and to capture intravenously administered human breast cancer cells, MCF7s, from the dogs. No life-threatening events were observed in dogs during the apheresis process. The changes in the hemogram were transient and recovered gradually within a few days after apheresis. During apheresis, 50 mL of PBMCs could be collected from each dog. Notably, a thrombus was formed along the circuit wall during apheresis, which decreased the blood collection pressure. MCF7 cells were successfully captured by the apheresis machine. The captured cells were regrown in vitro and characterized compared with the original cells. In conclusion, apheresis could be safely performed in dogs to isolate CTCs with precautions to maintain hemodynamic stability.

Monocyte gene expression distinguishes enhancing brain parenchymal cysticercal granulomas from tuberculomas

Background In patients with enhancing brain parenchymal lesions, parenchymal neurocysticercosis (pNCC) is often difficult to distinguish from tuberculoma, necessitating biopsy or empirical therapy. Materials and methods In a prospective study, peripheral blood monocytes were isolated from patients with definitive pNCC (n=39) and brain tuberculomas (n=20). Patients with tuberculomas were diagnosed by the presence of concurrent systemic tuberculosis (n=7), pathological or bacteriological confirmation (n=5), and resolution of typical brain lesions following a therapeutic trial of anti-tuberculous therapy (n=8). Expressions of 14 NCC associated monocyte genes were determined by qPCR and analyzed for diagnostic usefulness between the two groups. Results Expression of seven genes (TAX1BP1, RAP1A, PLCG2, TOR3A, GBP1P1, LRRFIP2 and FEZ2) was significantly higher in pNCC patients than in tuberculoma patients with TAX1BP1 and RAP1A expressions greater than 22- and 5-fold higher in pNCC patients. TAX1BP1 had the highest sensitivity of 66.7% at a specificity of 100% in discriminating pNCC from tuberculoma. A combination of TAX1BP1 and RAP1A increased the sensitivity to 84.6% and including GBP1P1 with TAX1BP1 and RAP1A further increased sensitivity to 87.2% while maintaining specificity of 100%. Conclusion Expression of a panel of genes in blood monocytes distinguishes pNCC from brain tuberculomas in patients with enhancing brain lesions.