ABSTRACT Tumor‐associated monocytes and macrophages are abundant in the tumor microenvironment (TME) and can influence the therapeutic accessibility of antibody drugs. However, the impact of macrophage‐mediated antibody sequestration on the therapeutic efficacy of anti‐epidermal growth factor receptor (EGFR) therapy remains unclear. This study aimed to assess whether monocytes and macrophages attenuate anti‐EGFR therapeutic efficacy by sequestering antibodies. We established a transwell‐based co‐culture platform using human THP‐1 monocytes or macrophages and EGFR‐positive DiFi colorectal cancer cells to evaluate macrophage‐associated antibody sequestration. In vivo xenograft assays, flow cytometric uptake analyses and inhibitor‐based functional studies were performed using the anti‐EGFR monoclonal antibodies cetuximab and panitumumab. Xenograft models co‐implanted with THP‐1 cells demonstrated attenuated responses to anti‐EGFR antibodies compared to tumor‐only models. Monocytes and macrophages significantly reduced the growth‐inhibitory effects of cetuximab and panitumumab in co‐culture. Antibody uptake was increased in THP‐1 cells and THP‐1‐derived macrophages. Fc receptor blockade and uptake‐associated inhibitors partially suppressed antibody uptake and restored antibody‐mediated anti‐tumor activity, suggesting the involvement of FcγR‐dependent and FcγR‐independent pathways. Monocyte‐ and macrophage‐mediated antibody sequestration attenuates anti‐EGFR therapeutic efficacy by reducing antibody accessibility to tumor cells. The established co‐culture platform provides a useful bioanalytical assay system to screen and evaluate antibody accessibility and macrophage‐associated sequestration during the preclinical development of antibody therapeutics.
ABSTRACT Chlorine dioxide (ClO 2 ) is used as a disinfectant in food and environmental sanitation. However, information regarding its efficacy against enterohemorrhagic Escherichia coli (EHEC) under organic load conditions remains limited. This study evaluated the bactericidal activity of aqueous ClO 2 against clinically important EHEC serogroups and compared its efficacy with that of sodium hypochlorite (NaClO). Additionally, other bacterial strains, including enteropathogenic E. coli (EPEC), Escherichia albertii , and E. coli type strains, were also examined. Under organic load conditions, treatment with 100 ppm NaClO resulted in only minor reductions (< 1 log 10 CFU/mL) within 60 s for all strains tested. In contrast, 10 ppm ClO 2 reduced the E. coli type strain and E. albertii by > 3 log 10 CFU/mL, while 100 ppm ClO 2 reduced all tested strains to below the detection limit (> 6 log 10 CFU/mL reduction). EHEC and EPEC strains showed lower susceptibility to 10 ppm ClO 2 (0.2–1.3 log 10 CFU/mL reduction) but were reduced to below the detection limit by 100 ppm ClO 2 . Time‐course assay further demonstrated that 100 and 30 ppm ClO 2 inactivated > 6 log 10 CFU/mL of EHEC within 15 and 30 s, respectively, whereas NaClO showed only limited bactericidal activity over the same period. These results demonstrate that ClO 2 provides substantially greater and more rapid bactericidal activity than NaClO against EHEC in the presence of organic matter. The findings provide quantitative evidence of the concentration‐ and time‐dependent bactericidal efficacy of ClO 2 and provide fundamental information for future studies evaluating its potential application in food hygiene and environmental sanitation.
ABSTRACT Bacteria belonging to the family Enterobacteriaceae occur as commensals or pathogens in humans. The increasing prevalence of multidrug‐resistant bacteria has raised global concern. Following the reclassification of Enterobacteriaceae species into the order Enterobacterales in 2016. However, no quantification method has been reported that is designed and validated in the context of the current order‐level taxonomy of Enterobacterales . Therefore, we aimed to develop a novel reverse transcription quantitative PCR (RT‐qPCR) system for the quantification of Enterobacterales based on 23S rRNA. The system was used to quantify Enterobacterales species, and its applicability was evaluated using fecal samples from 99 patients with gastrointestinal cancer and 10 healthy volunteers. Furthermore, we performed a proof‐of‐concept evaluation of its utility for rapid assessment of antimicrobial susceptibility in carbapenem‐resistant Enterobacterales (CRE). The system exhibited high analytical sensitivity and enabled quantification of diverse Enterobacterales spp., including newly classified species, with theoretical detection limits of 10 3 cells/g feces and 1 cell/mL blood. In healthy volunteers, intestinal Enterobacterales were dominated by Escherichia coli and Enterobacter , whereas preoperative patients with gastrointestinal cancer exhibited greater diversity. In vitro, viable CRE cell counts determined by the RT‐qPCR system 2 h after antibiotic treatment correlated well with culture‐based susceptibility results obtained after 24 h, suggesting the feasibility of rapid antimicrobial susceptibility assessment within 6 h. The developed 23S rRNA RT‐qPCR system provides a sensitive approach for quantifying Enterobacterales within the current taxonomic framework and may facilitate the rapid assessment of antimicrobial activity against CRE. Clinical Trial Registration There is no clinical trial registration.
Staphylococcus aureus is responsible for a wide range of pyogenic infections, food poisoning, and allergic inflammation. S. aureus produces a very wide variety of proteinaceous exotoxins, which are classified by structure as β-barrel pore-forming toxin, oligonucleotide/oligosaccharide-binding (OB)-fold/β-grasp proteins, and triple-helix bundle-containing proteins. We previously reported that three staphylococcal exotoxins belonging to the first two groups activate basophils and mast cells, which are innate immune cells that play a central role in allergic inflammation. In this study, we focused on a group of triple-helix bundle-containing toxins and examined their ability to activate murine bone marrow-derived mast cells and basophils. Extracellular complement-binding protein (Ecb), previously known as a complement inhibitor, was found to induce IL-4 and IL-6 production in basophils but not that of IL-6 and IL-13 in mast cells. Ecb did not induce IL-4 production in murine splenocytes or CD4+ T cells, suggesting that it is a basophil-specific activator. Ecb-induced IL-4 expression in basophils in the absence of serum. The C3-binding-deficient mutant of Ecb also activated basophils, indicating that Ecb activates basophils independently of its complement-inhibiting activity. Dasatinib, a Src kinase inhibitor, prevented Ecb-induced IL-4 production in basophils, suggesting that the activation required a Src family kinase. These findings reveal a novel function of Ecb, that is, the activation of basophils to produce IL-4 in an IgE-independent manner, and suggest a dual Ecb contribution to immune evasion-by interfering with complement activation and skewing Th2 immunity-and a role in the development of allergic inflammation-by inducing IL-4 production.
Many mammalian cells restrict viral replication by utilizing various host restriction factors. We recently demonstrated that CCHC-type zinc-finger-containing protein 3 (ZCCHC3) suppresses human immunodeficiency virus type 1 (HIV-1) replication through multiple mechanisms. We also revealed that single-nucleotide polymorphisms in human ZCCHC3 affect its antiviral function; however, whether similar genetic and functional diversity is present in other species remains unknown. In this study, we investigated the genetic and functional diversity of ZCCHC3 in cynomolgus macaques, a critical animal model for HIV-1-related research. Sequencing analysis of eight independent ZCCHC3 clones per animal revealed substantial amino acid diversity among cynomolgus macaques. We selected 12 representative variants and examined their antiviral activity against several retroviral vectors derived from HIV-1, simian immunodeficiency virus, feline immunodeficiency virus, and murine leukemia virus. Moreover, using replication-competent HIV-1, we showed that selected cynomolgus macaque ZCCHC3 variants can affect both viral production and viral infectivity. These results suggest that the genetic and functional diversity of ZCCHC3 is not limited to humans and underscore the importance of considering ZCCHC3 variation in cynomolgus macaques when using them as animal models for HIV-1-related research.
