Abstract3,5-bis(benzylidene)-4-piperidones are being advanced as synthetic analogs of curcumin for anticancer and anti-inflammatory properties. We performed structure-activity relationship studies, by testing several synthesized 3,5-bis(benzylidene)-4-piperidones for anti-proliferative activity in lung adenocarcinoma H441 cells. Compared to the lead compound 1, or 3,5-bis(2-fluorobenzylidene)-4-piperidone, five compounds were found to be more potent (IC 50 < 30 μM), and sixteen compounds possessed reduced cell-killing efficacy (IC 50 > 50 μM). Based on the observations, we synthesized 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] (29 or CLEFMA) as a novel analog of 1. CLEFMA was evaluated for antiproliferative activity in H441 cells, and was found to be several folds more potent than compound 1. We did not find apoptotic cell population in flow cytometry, and the absence of apoptosis was confirmed by the lack of caspase cleavage. The electron microscopy of H441cells indicated that CLEFMA and compound 1 induce autophagic cell death that was inhibited by specific autophagy inhibitor 3-methyladenine. The results suggest that the potent and novel curcuminoid, CLEFMA, offers an alternative mode of cell death in apoptosis-resistant cancers.

Synthetic curcuminoid EF24 was studied for its effect on the maturation and inflammatory response in murine bone marrow derived immortalized JAWS II dendritic cells (DCs). EF24 reduced the expression of LPS-induced MHC class II, CD80 and CD86 molecules. It also abrogated the appearance of dendrites, a typical characteristic of mature DCs. These effects were accompanied by the inhibition of LPS-induced activation of transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB). Simultaneous reduction of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, IL-6] both at the mRNA and secreted levels was also observed. To investigate the dependency of LPS effects on MyD88 adaptor protein, we transfected JAWS II DCs with dominant negative MyD88 plasmid construct (MyD88-DN). EF24 reduced NF-κB activity and TNF-α secretion in a MyD88-dependent manner. These results suggest that EF24 modulates DCs by suppressing their maturation and reducing the secretion of inflammatory cytokines. Further, it appears that EF24 acts at or upstream of MyD88 in the LPS-TLR4/MyD88/NF-κB pathway.

The accumulation and deposition of β-amyloid (Aβ) is one postulated cause of Alzheimer’s disease (AD). In addition to its direct toxicity on neurons, Aβ may induce neuroinflammation through the concomitant activation of microglia. Emerging evidence suggests that microglia-mediated neuroinflammation plays an important role in the pathogenesis of AD. As brain macrophages, microglia engulf misfolded-Aβ by phagocytosis. However, the accumulated toxic Aβ may paradoxically “hyper-activate” microglia into a neurotoxic proinflammatory and less phagocytotic phenotype, contributing to neuronal death. This study reports that the known drug furosemide is a potential probe molecule for reducing AD-neuroinflammation. Our data demonstrate that furosemide inhibits the secretion of proinflammatory TNF-α, IL-6, and nitric oxide; downregulates the mRNA level of Cd86 and the protein expression of COX-2, iNOS; promotes phagocytic activity; and enhances the expression of anti-inflammatory IL-1RA and arginase. Our mechanism of action studies further demonstrate that furosemide reduces LPS-induced upregulation of endoplasmic reticulum (ER) stress marker genes, including Grp78, Atf4, Chop, tXbp1, and sXbp1. These data support the observation that furosemide is a known drug with the capacity to downregulate the proinflammatory microglial M1 phenotype and upregulate the anti-inflammatory M2 phenotype, a potentially powerful and beneficial pharmacologic effect for inflammatory diseases such as AD.

The novel coronavirus SARS-CoV-2 has become a global health concern. The morbidity and mortality of the potentially lethal infection caused by this virus arise from the initial viral infection and the subsequent host inflammatory response. The latter may lead to excessive release of pro-inflammatory cytokines, IL-6 and IL-8, as well as TNF-α ultimately culminating in hypercytokinemia (“cytokine storm”). To address this immuno-inflammatory pathogenesis, multiple clinical trials have been proposed to evaluate anti-inflammatory biologic therapies targeting specific cytokines. However, despite the obvious clinical utility of such biologics, their specific applicability to COVID-19 has multiple drawbacks, including they target only one of the multiple cytokines involved in COVID-19’s immunopathy. Therefore, we set out to identify a small molecule with broad-spectrum anti-inflammatory mechanism of action targeting multiple cytokines of innate immunity. In this study, a library of small molecules endogenous to the human body was assembled, subjected to in silico molecular docking simulations and a focused in vitro screen to identify anti-pro-inflammatory activity via interleukin inhibition. This has enabled us to identify the loop diuretic furosemide as a candidate molecule. To pre-clinically evaluate furosemide as a putative COVID-19 therapeutic, we studied its anti-inflammatory activity on RAW264.7, THP-1 and SIM-A9 cell lines stimulated by lipopolysaccharide (LPS). Upon treatment with furosemide, LPS-induced production of pro-inflammatory cytokines was reduced, indicating that furosemide suppresses the M1 polarization, including IL-6 and TNF-α release. In addition, we found that furosemide promotes the production of anti-inflammatory cytokine products (IL-1RA, arginase), indicating M2 polarization. Accordingly, we conclude that furosemide is a reasonably potent inhibitor of IL-6 and TNF-α that is also safe, inexpensive and well-studied. Our pre-clinical data suggest that it may be a candidate for repurposing as an inhaled therapy against COVID-19.

BackgroundCoccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively.ResultsWe found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (TEM) and IFN-γ release.ConclusionsIn conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.

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BackgroundUnresolved and prolonged inflammation is a pathological basis of many disorders such as cancer and multiple organ failure in shock. Interleukin-1 receptor (IL-1R) superfamily consists of IL-1R1 and pathogen pattern recognition receptor toll-like receptor-4 (TLR4) which, upon ligand binding, initiate pro-inflammatory signaling. The study objective was to investigate the effect of a diphenyldifluoroketone EF24 on the expression of IL-1R1 and TLR4 in lipopolysaccharide (LPS)-stimulated dendritic cells (DCs).MethodsImmortalized murine bone marrow-derived JAWS II dendritic cells (DC) were challenged with LPS (100 ng/ml) for 4 h. The LPS-stimulated DCs were treated with 10 μM of EF24 for 1 h. The expression levels of IL-1R1 and TLR4 were monitored by RT-PCR, immunoblotting, and confocal microscopy. The effect of EF24 on the viability and cell cycle of DCs was examined by lactate dehydrogenase assay and flow cytometry, respectively.ResultsEF24 treatment suppressed the LPS-induced TLR4 and IL-1R1 expression in DCs. However, the expression levels of IL-1RA and IL-1R2 were not influenced by either LPS or EF24 treatments. These effects of EF24 were associated with a decrease in LPS-induced expression of phospho-NF-kB p65, indicative of its role in the transcriptional control of IL-1R superfamily members. We did not find any significant effect of EF24 on the proliferation or cell cycle of DCs.ConclusionsThe results suggest that EF24 influences IL-1R superfamily signaling pathway in ways that could have salutary effects in inflammation. The pluripotent anti-inflammatory actions of EF24 warrant further investigation of EF24 in inflammatory conditions of systemic nature.