The Barrier Disruption and Pyroptosis of Intestinal Epithelial Cells Caused by Perfringolysin O (PFO) from Clostridium perfringens.

Clostridium perfringens (C. perfringens), a Gram-positive bacterium, produces a variety of toxins and extracellular enzymes that can lead to disease in both humans and animals. Common symptoms include abdominal swelling, diarrhea, and intestinal inflammation. Severe cases can result in complications like intestinal hemorrhage, edema, and even death. The primary toxins contributing to morbidity in C. perfringens-infected intestines are CPA, CPB, CPB2, CPE, and PFO. Amongst these, CPB, CPB2, and CPE are implicated in apoptosis development, while CPA is associated with cell death, increased intracellular ROS levels, and the release of the inflammatory factor IL-18. However, the exact mechanism by which PFO toxins exert their effects in the infected gut is still unidentified. This study demonstrates that a C. perfringens PFO toxin infection disrupts the intestinal epithelial barrier function through in vitro and in vivo models. This study emphasizes the notable influence of PFO toxins on intestinal barrier integrity in the context of C. perfringens infections. It reveals that PFO toxins increase ROS production by causing mitochondrial damage, triggering pyroptosis in IPEC-J2 cells, and consequently resulting in compromised intestinal barrier function. These results offer a scientific foundation for developing preventive and therapeutic approaches against C. perfringens infections.

Characterization of the monoclonal antibody and the immunodominant B-cell epitope of African swine fever virus pA104R by using mouse model.

The African swine fever virus (ASFV) structural protein pA104R is the only histone-like protein encoded by eukaryotic viruses. pA104R is an essential DNA-binding protein required for DNA replication and genome packaging of ASFV, which are vital for pathogen survival and proliferation. pA104R is an important target molecule for diagnosing, treating, and immune prevention of ASFV. This study characterized monoclonal antibodies (mAbs) against pA104R and found them to recognize natural pA104R in ASFV strains with different genotypes, showing high conservation. Confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Furthermore, the pA104R protein functions through the polar interactions between the binding amino acid sites; however, these interactions may be blocked by the recognition of generated mAbs. Characterizing the immunodominant B-cell epitope of the ASFV critical proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and vaccine candidate targets.IMPORTANCEAfrican swine fever (ASF) is a highly pathogenic, lethal, and contagious viral disease affecting domestic pigs and wild boars. As no effective vaccine or other treatments have been developed, the control of African swine fever virus (ASFV) relies heavily on virus detection and diagnosis. A potential serological target is the structural protein pA104R. However, the molecular basis of pA104R antigenicity remains unclear, and a specific monoclonal antibody (mAb) against this protein is still unavailable. In this study, mAbs against pA104R were characterized and found to recognize natural pA104R in ASFV strains with different genotypes. In addition, confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Characteristics of the immunodominant B-cell epitope of ASFV proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and identifying vaccine candidate targets.

African Swine Fever Virus pF778R Attenuates Type I Interferon Response by Impeding STAT1 Nuclear Translocation.

African swine fever virus (ASFV) is an extensive and intricate double-stranded DNA virus with approximately 100% lethality in domestic swine. There is no effective vaccine to combat this virus, and this has led to substantial economic losses in the swine industry. ASFV encodes various proteins that impede interferon-based immune defenses in the host by employing diverse mechanisms. However, the roles of most of these proteins remain unknown. Therefore, understanding the immune evasion mechanisms employed by ASFV may facilitate the development of effective measures against the virus. In this study, we discovered a negative regulation of the type I interferon (IFN) response by the ASFV ribonuclease reductase large subunit pF778R. This novel type Ⅰ IFN response antagonist significantly inhibits IFN-α-induced interferon-stimulated response element promoter activation, precludes the upregulation of various interferon-stimulated genes, and prevents STAT1 nuclear translocation. Mechanistically, pF778R did not affect the protein levels of crucial molecules in the JAK/STAT signaling pathway or engage in direct interactions. However, pF778R expression impedes type I IFN responses mediated by the JAK/STAT signaling pathway. Further investigations revealed that pF778R did not interfere with STAT1 phosphorylation or dimerization, but it inhibited IFN signaling by weakening the nuclear accumulation of activated STAT1. The critical role of the ASFV protein pF778R in evading IFN-I-mediated innate immunity highlights a unique mode of ASFV evasion and provides insights into the pathogenic mechanism of the virus.

Brincidofovir is a robust replication inhibitor against African swine fever virus in vivo and in vitro.

African swine fever virus (ASFV) infection is a major public and socioeconomic concern that has a serious impact on the global swine industry. Unfortunately, there are currently no commercially available vaccines or antiviral agents that are both safe and effective against ASFV. In the study, we use primary porcine alveolar macrophages to screen a kinase inhibitor library for anti-ASFV compounds. Six candidate compounds that inhibited ASFV infection with inhibition of > 90% were identified, among which brincidofovir exhibited optimal inhibitory effects on ASFV. Brincidofovir reduces ASFV replication in a dose-dependent manner (IC50 = 2.76 nM) without cytotoxicity (CC50 = 58 µM). It possesses the ability to reduce viral titres and inhibit viral structural protein expression. Time-of-addition assays suggest that the compound interferes with the post-invasion stage of the viral infection cycle. In pig challenge experiments, brincidofovir was indicated to protect pigs against ASFV-induced lethality by decreasing the viral load in organs and peripheral blood, while it alleviated the histopathological changes associated with ASFV infection. Furthermore, brincidofovir also decreased viral shedding in pigs with ASFV infection. Our data together demonstrate that brincidofovir may serve as a potentially effective agent for the prevention and control of ASFV infection, whereas further investigations are still required.

African swine fever virus pA104R protein acts as a suppressor of type I interferon signaling.

This study evaluates the role of the late viral protein, pA104R, in African swine fever virus immunosuppression. ASFV-encoded pA104R is a putative histone-like protein that is highly conserved throughout different virulent and non-virulent isolates. Previous studies have demonstrated that pA104R plays a vital role in the ASFV replication cycle and is a potential target for antiviral therapy. Here, we demonstrated that pA104R is a potent antagonist of type I interferon signaling. IFN-stimulated response element activity and subsequent transcription of co-transfected and endogenous interferon-stimulated genes were attenuated by pA104R treatment in HEK-293 T cells. Immunoprecipitation assay and reciprocal pull-down showed that pA104R does not interact directly with STAT1, STAT2, or IRF9. However, pA104R could inhibit IFN signaling by attenuating STAT1 phosphorylation, and we identified the critical amino acid residues (R/H69,72 and K/R92,94,97) involved through the targeted mutation functional assays. Although pA104R is a histone-like protein localized to the nucleus, it did not inhibit IFN signaling through its DNA-binding capacity. In addition, activation of the ISRE promoter by IRF9-Stat2(TA), a STAT1-independent pathway, was inhibited by pA104R. Further results revealed that both the transcriptional activation and recruitment of transcriptional stimulators by interferon-stimulated gene factor 3 were not impaired. Although we failed to determine a mechanism for pA104R-mediated IFN signaling inhibition other than attenuating the phosphorylation of STAT1, these results might imply a possible involvement of epigenetic modification by ASFV pA104R. Taken together, these findings support that pA104R is an antagonist of type I interferon signaling, which may interfere with multiple signaling pathways.

African Swine Fever Virus pI215L Inhibits Type I Interferon Signaling by Targeting Interferon Regulatory Factor 9 for Autophagic Degradation.

African swine fever virus (ASFV) is the etiological agent of a highly lethal hemorrhagic disease in domestic pigs and wild boars that has significant economic consequences for the pig industry. The type I interferon (IFN) signaling pathway is a pivotal component of the innate antiviral response, and ASFV has evolved multiple mechanisms to antagonize this pathway and facilitate infection. Here, we reported a novel function of ASFV pI215L in inhibiting type I IFN signaling. Our results showed that ASFV pI215L inhibited IFN-stimulated response element (ISRE) promoter activity and subsequent transcription of IFN-stimulated genes (ISGs) by triggering interferon regulatory factor 9 (IRF9) degradation. Additionally, we found that catalytically inactive pI215L mutations retained the ability to block type I IFN signaling, indicating that this only known viral E2 ubiquitin-conjugating enzyme mediates IFR9 degradation in a ubiquitin-conjugating activity-independent manner. By coimmunoprecipitation, confocal immunofluorescence, and subcellular fractionation approaches, we demonstrated that pI215L interacted with IRF9 and impaired the formation and nuclear translocation of IFN-stimulated gene factor 3 (ISGF3). Moreover, further mechanism studies supported that pI215L induced IRF9 degradation through the autophagy-lysosome pathway in both pI215L-overexpressed and ASFV-infected cells. These findings reveal a new immune evasion strategy evolved by ASFV in which pI215L acts to degrade host IRF9 via the autophagic pathway, thus inhibiting the type I IFN signaling and counteracting the host innate immune response. IMPORTANCE African swine fever virus (ASFV) causes a highly contagious and lethal disease in pigs and wild boars that is currently present in many countries, severely affecting the global pig industry. Despite extensive research, effective vaccines and antiviral strategies are still lacking, and many fundamental questions regarding the molecular mechanisms underlying host innate immunity escape remain unclear. In this study, we identified ASFV pI215L, the only known viral E2 ubiquitin-conjugating enzyme, which is involved in antagonizing the type I interferon signaling. Mechanistically, pI215L interacted with interferon regulatory factor 9 for autophagic degradation, and this degradation was independent of its ubiquitin-conjugating activity. These results increase the current knowledge regarding ASFV evasion of innate immunity, which may instruct future research on antiviral strategies and dissection of ASFV pathogenesis.

Lobetyolin suppressed lung cancer in a mouse model by inhibiting epithelial-mesenchymal transition.

Traditional Chinese medicines are gaining more attention as promising adjuvant agents for conventional chemotherapy. Recent studies have shown that lobetyolin (LBT) is one of the main bioactive compounds of traditional Chinese medicines and it exhibits anticancer activity in several types of cancer. Therefore, this study aimed to investigate the mechanism by which LBT inhibits lung cancer. A549 human lung cancer cells were treated with LBT. In addition, A549 cells were injected into Balc/b nude mice to establish model of lung cancer. The mice were treated with cisplatin (DDP) or LBT alone or in combination, and tumor growth was monitored. Protein levels of E-cadherin, vimentin and matrix metalloproteinase 9 (MMP9) were detected. We found that the combination of LBT and DDP showed stronger effect to inhibit the proliferation of A549 cells compared to LBT or DDP treatment alone. Wound healing assay showed that the ratio of wound healing was significantly lower in LBT group and DDP group and was the lowest in LBT+DDP group. Transwell invasion assay showed that the invasion ability of A549 cells was the weakest in LBT+DDP group. Protein levels of E-cadherin were the highest while those of vimentin and MMP9 were the lowest in A549 cells treated with LBT+DDP. Nude mouse xenograft tumor model showed that the combination of LBT with DDP had the highest efficacy to inhibit the growth of lung cancer, and tumor tissues of mice treated with LBT+DDP had the lowest expression of vimentin and MMP9 and the highest expression of E-cadherin. In conclusion, LBT significantly enhances the efficacy of chemotherapy on lung cancer, and the mechanism may be related to the inhibition of epithelial-mesenchymal transition.

Characterization of a universal neutralizing monoclonal antibody against Glaesserella parasuis CdtB.

Glaesserella parasuis is the etiological agent of Glässer's disease. Although present as a symbiotic bacterium in the respiratory tract of healthy pigs, G. parasuis invades piglets under stress conditions and causes severe systemic infection characterized by fibrinous polyserositis, polyarthritis, and meningitis, which caused high mortality in weaned and nursery herds. Further, the lack of cross-protection against the various serotypes of G. parasuis is a serious concern for the swine industry. Cytolethal distending toxin (CDT) is essential for the pathogenicity of G. parasuis and is a conserved virulence factor. CdtB is the active subunit of CDT, causing DNA double-strand breaks in eukaryotic cells, leading to irreversible cell cycle arrest and apoptosis. However, the immunogenicity and immunogenic domain of G. parasuis CdtB have not been investigated. In this study, monoclonal antibodies (mAbs) against G. parasuis CdtB were screened. One mAb, 4F10, was characterized and found to specifically recognize G. parasuis strains of all serotypes, including non-typeable strains, without showing any reactivity with other swine bacterial pathogens. Additionally, 4F10 exhibited neutralizing activity that restrained the cytotoxicity caused by CdtB. Moreover, the core unit of the epitope 84GVGFPIDEYVWNLGTRSRPN103 recognized by 4F10 was identified. The mAb-4F10 characterized herein provides a candidate for the further investigation of the pathogenic and immunogenic functions of CdtB in G. parasuis and could facilitate future serological diagnosis, prevention, and control of this disease.

Development of a nanoparticle-assisted PCR assay for detection of bovine respiratory syncytial virus.

BACKGROUND: Bovine respiratory syncytial virus (BRSV) is a common pathogen causing respiratory disease in cattle and a significant contributor to the bovine respiratory disease (BRD) complex. BRSV is widely distributed around the world, causing severe economic losses. This study we established a new molecular detection method of BRSV pathogen NanoPCR attributed to the combination of nano-particles in traditional PCR (Polymerase chain reaction) technology. RESULTS: In this study, the BRSV NanoPCR assay was developed, and its specificity and sensitivity were investigated. The results showed that no cross-reactivity was observed for the NanoPCR assay for related viruses, including the infectious bovine rhinotracheitis virus (IBRV), bovine viral diarrhea virus (BVDV), and bovine parainfluenza virus type 3 (BPIV3), and the assay was more sensitive than the conventional PCR assay, with a detection limit of 1.43 × 102 copies recombinant plasmids per reaction, compared with 1.43 × 103 copies for conventional PCR analysis. Moreover, thirty-nine clinical bovine samples collected from two provinces in North-Eastern China, 46.15% were determined BRSV positive by our NanoPCR assay, compared with 23.07% for conventional PCR. CONCLUSIONS: This is the first report to demonstrate the application of a NanoPCR assay for the detection of BRSV. The sensitive and specific NanoPCR assay developed in this study can be applied widely in clinical diagnosis and field surveillance of BRSV infection.

Effects of bile reflux and intragastric microflora changes on lesions of remnant gastric mucosa after gastric operation.

AIM: To investigate the effects of bile reflux and intragastric microflora changes on lesions of remnant gastric mucosa after gastric operation. METHODS: Concentration of bile acid and total bacterial counts (TBC) in gastric juice were measured in 49 patients with peptic ulcer before and after gastrectomy. One year after the operation, sample of gastric mucosa taken from all the patients were used for histological examination. RESULTS: The concentration of gastric bile acid was significantly increased in group B-I, or B-II and SV+A than that in group HSV (P<0.05-0.01). The abnormal histological changes in the remnant gastric mucosa were more common in the first 2 groups than in the last group. CONCLUSION: The type of gastrectomy can affect bile reflux. The abnormal histological changes in the remnant gastric mucosa are closely related to the elevation of bile acid concentration and increase of TBC in gastric juice. HSV can effectively prevent bile reflux and keep the gastric physiological functions stable.

Gene therapy for gastric cancer: a review.

Gastric cancer is common in China, and its early diagnosis and treatment are difficult. In recent years great progress has been achieved in gene therapy, and a wide array of gene therapy systems for gastric cancer has been investigated. The present article deals with the general principles of gene therapy and then focuses on how these principles may be applied to gastric cancer.