Influenza Inactive Virus Vaccine with the Fusion Peptide (rTα1- BP5) Enhances Protection Against Influenza Through Humoral and Cell-Mediated Immunity

Thymosin α1 (Tα1) and Bursopentin (BP5) are both immunopotentiators. To explore whether the thymosin α1-Bursopentin (rTα1-BP5) is an adjuvant or not, we cloned the gene of Tα1-BP5 and provided evidence that the gene of Tα1-BP5 in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. To evaluate the immune adjuvant properties of rTα1-BP5, chickens were immunized with rTα1-BP5 combined with H9N2 avian influenza whole-inactivated virus (WIV). The titers of HI antibody, antigen-specific antibodies, Avian influenza virus (AIV)-neutralizing antibodies, levels of Th1-type cytokines (gamma interferon (IFN-γ)) and Th2-type cytokines (interleukin 4 (IL-4)), and lymphocyte proliferation responses were determined. We found that rTα1-BP5 enhanced HI antibody and antigen-specific immunoglobulin G (IgG) antibodies titers, increased the level of AIV-neutralizing antibodies, induced the secretion of Th1- and Th2-type cytokines, and promoted the proliferation of T and B lymphocyte. Furthermore, virus challenge experiments confirmed that rTα1-BP5 contributed to the inhibition replication of the virus (H9N2 AIV (A/chicken/Jiangsu/NJ07/05) from chicken lungs. Altogether, these findings suggest that rTα1-BP5 is a novel adjuvant suitable for H9N2 avian influenza vaccine

The Contribution of Antimicrobial Peptides to Immune Cell Function: A Review of Recent Advances

The development of novel antimicrobial agents to replace antibiotics has become urgent due to the emergence of multidrug-resistant microorganisms. Antimicrobial peptides (AMPs), widely distributed in all kingdoms of life, present strong antimicrobial activity against a variety of bacteria, fungi, parasites, and viruses. The potential of AMPs as new alternatives to antibiotics has gradually attracted considerable interest. In addition, AMPs exhibit strong anticancer potential as well as anti-inflammatory and immunomodulatory activity. Many studies have provided evidence that AMPs can recruit and activate immune cells, controlling inflammation. This review highlights the scientific literature focusing on evidence for the anti-inflammatory mechanisms of different AMPs in immune cells, including macrophages, monocytes, lymphocytes, mast cells, dendritic cells, neutrophils, and eosinophils. A variety of immunomodulatory characteristics, including the abilities to activate and differentiate immune cells, change the content and expression of inflammatory mediators, and regulate specific cellular functions and inflammation-related signaling pathways, are summarized and discussed in detail. This comprehensive review contributes to a better understanding of the role of AMPs in the regulation of the immune system and provides a reference for the use of AMPs as novel anti-inflammatory drugs for the treatment of various inflammatory diseases

A Novel Bacillus Velezensis for Efficient Degradation of Zearalenone

Zearalenone (ZEN) is considered one of the most serious mycotoxins contaminating grains and their by-products, causing significant economic losses in the feed and food industries. Biodegradation pathways are currently considered the most efficient solution to remove ZEN contamination from foods. However, low degradation rates and vulnerability to environmental impacts limit the application of biodegradation pathways. Therefore, the main research objective of this article was to screen strains that can efficiently degrade ZEN and survive under harsh conditions. This study successfully isolated a new strain L9 which can efficiently degrade ZEN from 108 food ingredients. The results of sequence alignment showed that L9 is Bacillus velezensis. Meanwhile, we found that the L9 degradation rate reached 91.14% at 24 h and confirmed that the primary degradation mechanism of this strain is biodegradation. The strain exhibits resistance to high temperature, acid, and 0.3% bile salts. The results of whole-genome sequencing analysis showed that, it is possible that the strain encodes the key enzyme, such as chitinase, carboxylesterases, and lactone hydrolase, that work together to degrade ZEN. In addition, 227 unique genes in this strain are primarily involved in its replication, recombination, repair, and protective mechanisms. In summary, we successfully excavated a ZEN-degrading, genetically distinct strain of Bacillus velezensis that provides a solid foundation for the detoxification of feed and food contamination in the natural environment

Probiotic Properties of Chicken-Derived Highly Adherent Lactic Acid Bacteria and Inhibition of Enteropathogenic Bacteria in Caco-2 Cells

Lactic acid bacteria (LAB) as probiotic candidates have various beneficial functions, such as regulating gut microbiota, inhibiting intestinal pathogens, and improving gut immunity. The colonization of the intestine is a prerequisite for probiotic function. Therefore, it is necessary to screen the highly adherent LAB. In this study, the cell surface properties, such as hydrophobicity, auto-aggregation, co-aggregation, and adhesion abilities of the six chicken-derived LAB to Caco-2 cells were investigated. All six strains showed different hydrophobicity (21.18–95.27%), auto-aggregation (13.61–30.17%), co-aggregation with Escherichia coli ATCC 25922 (10.23–36.23%), and Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311 (11.71–39.35%), and adhesion to Caco-2 cells (8.57–26.37%). Pediococcus pentosaceus 2–5 and Lactobacillus reuteri L-3 were identified as the strains with strong adhesion abilities (26.37% and 21.57%, respectively). Moreover, these strains could survive in a gastric acid environment at pH 2, 3, and 4 for 3 h and in a bile salt environment at 0.1%, 0.2%, and 0.3% (w/v) concentration for 6 h. Furthermore, the cell-free supernatant of P. pentosaceus 2–5 and L. reuteri L-3 inhibited the growth of enteropathogenic bacteria and the strains inhibited the adhesion of these pathogens to Caco-2 cells. In this study, these results suggested that P. pentosaceus 2–5 and L. reuteri L-3, isolated from chicken intestines might be good probiotic candidates to be used as feed additives or delivery vehicles of biologically active substances

Chloroquine Inhibition of Autophagy Enhanced the Anticancer Effects of <i>Listeria monocytogenes</i> in Melanoma

Listeria monocytogenes has been shown to exhibit antitumor effects. However, the mechanism remains unclear. Autophagy is a cellular catabolic process that mediates the degradation of unfolded proteins and damaged organelles in the cytosol, which is a double-edged sword in tumorigenesis and treatment outcome. Tumor cells display lower levels of basal autophagic activity than normal cells. This study examined the role and molecular mechanism of autophagy in the antitumor effects induced by LM, as well as the combined antitumor effect of LM and the autophagy inhibitor chloroquine (CQ). We investigated LM-induced autophagy in B16F10 melanoma cells by real-time PCR, immunofluorescence, Western blotting, and transmission electron microscopy and found that autophagic markers were increased following the infection of tumor cells with LM. The autophagy pathway in B16F10 cells was blocked with the pharmacological autophagy inhibitor chloroquine, which led to a significant increase in intracellular bacterial multiplication in tumor cells. The combination of CQ and LM enhanced LM-mediated cancer cell death and apoptosis compared with LM infection alone. Furthermore, the combination of LM and CQ significantly inhibited tumor growth and prolonged the survival time of mice in vivo, which was associated with the increased colonization and accumulation of LM and induced more cell apoptosis in primary tumors. The data indicated that the inhibition of autophagy by CQ enhanced LM-mediated antitumor activity in vitro and in vivo and provided a novel strategy to improving the anticancer efficacy of bacterial treatment

Characterisation of a Plancitoxin-1-Like DNase II Gene in <i>Trichinella spiralis</i>

<div><p>Background</p><p>Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the <i>Trichinella spiralis</i> (<i>T. spiralis</i>) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain.</p><p>Methodology/Principal Findings</p><p>In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from <i>T. spiralis</i> were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (<i>Ts</i>-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (r<i>Ts</i>-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that r<i>Ts</i>-Pt was recognised by serum from <i>T. spiralis</i>-infected mice; the anti-r<i>Ts</i>-Pt serum recognised crude antigens but not ES antigens. The <i>Ts</i>-Pt gene was examined at all <i>T. spiralis</i> developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that <i>Ts</i>-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. r<i>Ts</i>-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C.</p><p>Conclusions</p><p>This study indicated that <i>Ts</i>-Pt was classified as a somatic protein in different <i>T. spiralis</i> developmental stages, and demonstrated for the first time that an expressed DNase II protein from <i>T. spiralis</i> had nuclease activity.</p></div

<i>Escherichia coli</i> and <i>Candida albicans</i> Induced Macrophage Extracellular Trap-Like Structures with Limited Microbicidal Activity

<div><p>The formation of extracellular traps (ETs) has recently been recognized as a novel defense mechanism in several types of innate immune cells. It has been suggested that these structures are toxic to microbes and contribute significantly to killing several pathogens. However, the role of ETs formed by macrophages (METs) in defense against microbes remains little known. In this study, we demonstrated that a subset of murine J774A.1 macrophage cell line (8% to 17%) and peritoneal macrophages (8.5% to 15%) form METs-like structures (METs-LS) in response to <i>Escherichia coli</i> and <i>Candida albicans</i> challenge. We found only a portion of murine METs-LS, which are released by dying macrophages, showed detectable killing effects on trapped <i>E. coli</i> but not <i>C. albicans</i>. Fluorescence and scanning electron microscopy analyses revealed that, <i>in vitro,</i> both microorganisms were entrapped in J774A.1 METs-LS composed of DNA and microbicidal proteins such as histone, myeloperoxidase and lysozyme. DNA components of both nucleus and mitochondrion origins were detectable in these structures. Additionally, METs-LS formation occurred independently of ROS produced by NADPH oxidase, and this process did not result in cell lysis. In summary, our results emphasized that microbes induced METs-LS in murine macrophage cells and that the microbicidal activity of these METs-LS differs greatly. We propose the function of METs-LS is to contain invading microbes at the infection site, thereby preventing the systemic diffusion of them, rather than significantly killing them.</p></div

METs-LS-induced cell death is independent of necrosis.

<p><b>A:</b> The J774A.1 METs-LS induced by <i>C. albicans</i> were stained with SYTOX Green and Hoechst 33342, revealing that the METs-LS are released from either viable (i) or dead (ii) macrophage cells after 120 min incubation. The arrows indicate viable METs-LS formation cells with unchanged nuclei shape, the arrowheads indicate dead MET-LS formation cells with enlarged nuclei. Scale Bars: 10 µm. These experiments were repeated independently 3 times with similar results. <b>C:</b> J774A.1 macrophages were infected with <i>E. coli</i> or <i>C. albicans</i> for 15, 30, 60, 120 and 180 min, respectively. The LDH level in the supernatant of each group was quantified. The data are presented as the means ± SD of three independent experiments.</p

METs-LS show limited killing effect on <i>E. coli</i> but not on <i>C. albicans</i>.

<p>J774A.1 macrophages or murine peritoneal macrophages were infected with <i>E. coli</i> or <i>C. albicans</i> and incubated for the indicated time points. <b>A–B:</b> The survival rates of <i>E. coli</i> (A) and <i>C. albicans</i> (B) incubated with J774A.1 macrophages. <b>C–D:</b> The survival rates of <i>E. coli</i> (C) and <i>C. albicans</i> (D) incubated with peritoneal macrophages. Experiments were performed 3 times with similar results, and a representative experiment is shown as the means ± SD (n = 3). *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 compared with the control groups by two tailed Student’s t-test, respectively. <b>E–F:</b> PI staining was performed to determine the dead macrophages and dead microbes trapped in the METs-LS formed by J774A.1 cells (E) and peritoneal macrophages (F) after 120 min co-incubation. Macrophages were labeled with Hoechst 33342 (Blue). METs-LS, dead macrophages and dead microbes were stained positive by PI (Red). The solid and hollow arrowheads indicate viable and dead microbes in <i>E. coli</i> (i-ii) or <i>C. albicans</i> (iii-iv) infected groups, respectively. Only a portion of GFP- <i>E. coli</i> trapped by METs-LS released from dead macrophages was killed. Scale Bars: 10 µm. <b>G:</b> The quantification of dead <i>E. coli</i> trapped in METs-LS released from viable and dead J774A.1 macrophages and peritoneal macrophages, result is shown as the means ± SD (n = 10), ***P<0.001 compared with control group by two tailed Student’s t-test. These experiments were repeated independently 3 times with similar results.</p

Formation of METs-LS is independent of ROS produced by NADPH oxidase.

<p><b>A:</b> Fluorescence microscope determination of intracellular ROS production in PMA (positive control), <i>E. coli-</i> and <i>C. albicans</i>-stimulated J774A.1 macrophages using DCF ROS probe (Green). DNA was stained with Hoechst 33342 (Blue). The arrows indicate macrophages releasing METs-LS. <b>B:</b> The quantification of ROS production in negative, positive (PMA), <i>E. coli-</i> and <i>C. albicans-</i>infected macrophage groups. The fluorescence intensity of the ROS probe was measured using a fluorescence plate reader. The data are presented as the means ± SD of three independent experiments. *<i>P</i><0.05 and ***<i>P</i><0.001 compared with the medium-only culture control group by ANOVA with Bonferroni’s post-test. <b>C:</b> Determination of <i>E. coli</i> or <i>C. albicans</i> induced J774A.1 METs-LS in the presence of 10 µM DPI. Hoechst 33342 and SYTOX Green were added to assess METs-LS formation. <b>D:</b> Determination of intracellular ROS production in PMA stimulated J774A.1 macrophages in the presence or absence of 10 µM DPI. DNA was stained with Hoechst 33342 and intracellular ROS was determined by DCF ROS probe. Scale Bars: 10 µm. These experiments were repeated independently 3 times with similar results.</p