Anti-TLR4 (aa 420-435) polyclonal antibody [Biotin]

Owing to the challenges faced by conventional therapeutics, novel peptide antibiotics against multidrug-resistant (MDR) gram-negative bacteria need to be urgently developed. We had previously designed Pro9-3 and Pro9-3D from the defensin of beetleProtaetia brevitarsis; they showed high antimicrobial activity with cytotoxicity. Here, we aimed to develop peptide antibiotics with bacterial cell selectivity and potent antibacterial activity against gram-negative bacteria. We designed 10-meric peptides with increased cationicity by adding Arg to the N-terminus of Pro9-3 (Pro10-1) and its D-enantiomeric alteration (Pro10-1D). Among all tested peptides, the newly designed Pro10-1D showed the strongest antibacterial activity againstEscherichia coli,Acinetobacter baumannii, and MDR strains with resistance against protease digestion. Pro10-1D can act as a novel potent peptide antibiotic owing to its outstanding inhibitory activities against bacterial film formation with high bacterial cell selectivity. Dye leakage and scanning electron microscopy revealed that Pro10-1D targets the bacterial membrane. Pro10-1D inhibited inflammation via Toll Like Receptor 4 (TLR4)/Nuclear factor-kappa B (NF-kappa B) signaling pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Furthermore, Pro10-1D ameliorated multiple-organ damage and attenuated systemic infection-associated inflammation in anE. coliK1-induced sepsis mouse model. Overall, our results suggest that Pro10-1D can potentially serve as a novel peptide antibiotic for the treatment of gram-negative sepsis.

The immunoregulatory effect and immunologic response mechanism of Craterellus cornucopioides (L.) Pers. polysaccharide (CCP) with a triple-helix structure on peritoneal macrophages was investigated in vitro for the first time. These studies demonstrated that treatment of peritoneal macrophages with 80 mu g/mL CCP for 48 h significantly strengthened their phagocytic function as well as increases the activities of lysozyme (LZM), acid phosphatase (ACP) and succinodehydrogenase (SDH) when compared with the untreated group. Furthermore, Western Blot and quantitative real-time polymerase chain reaction (qRT-PCR) assays demonstrated that 80 mu g/mL CCP activated macrophages, significantly increased mRNA expression of cytokines (IL-8, IL-1 beta, IFN-alpha and TNF-alpha) and upregulated the protein expression of cell membrane receptor TLR4, as well as its downstream protein kinase products (MyD88, TAK1, P-IKK alpha/beta and P-MEK) through activation of the TLR4-NF kappa B pathway in peritoneal macrophages. In conclusion, these results showed that the immunomodulatory mechanism of CCP in peritoneal macrophages was associated with the release of NO, related enzymes and cytokines by stimulating the NF-kappa B p50 pathway via TLR4-MyD88-TAK1 signaling. (C) 2020 Published by Elsevier B.V.