Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus.

Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

Tim-3 protects against cisplatin nephrotoxicity by inhibiting NF-κB-mediated inflammation.

The impact of Tim-3 (T cell immunoglobulin and mucin domain-containing protein 3) on cisplatin-induced acute kidney injury was investigated in this study. Cisplatin-induced Tim-3 expression in mice kidney tissues and proximal tubule-derived BUMPT cells in a time-dependent manner. Compared with wild-type mice, Tim-3 knockout mice have higher levels of serum creatinine and urea nitrogen, enhanced TUNEL staining signals, more severe 8-OHdG (8-hydroxy-2' -deoxyguanosine) accumulation, and increased cleavage of caspase 3. The purified soluble Tim-3 (sTim-3) protein was used to intervene in cisplatin-stimulated BUMPT cells by competitively binding to the Tim-3 ligand. sTim-3 obviously increased the cisplatin-induced cell apoptosis. Under cisplatin treatment conditions, Tim-3 knockout or sTim-3 promoted the expression of TNF-α (tumor necrosis factor-alpha) and IL-1ß (Interleukin-1 beta) and inhibited the expression of IL-10 (interleukin-10). NF-κB (nuclear factor kappa light chain enhancer of activated B cells) P65 inhibitor PDTC or TPCA1 lowed the increased levels of creatinine and BUN (blood urea nitrogen) in cisplatin-treated Tim-3 knockout mice serum and the increased cleavage of caspase 3 in sTim-3 and cisplatin-treated BUMPT cells. Moreover, sTim-3 enhanced mitochondrial oxidative stress in cisplatin-induced BUMPT cells, which can be mitigated by PDTC. These data indicate that Tim-3 may protect against renal injury by inhibiting NF-κB-mediated inflammation and oxidative stress.

TRAIL inhibition by soluble death receptor 5 protects against acute myocardial infarction in rats.

Acute myocardial infarction (AMI) is associated with high morbidity and mortality. An effective therapeutic strategy is to rescue cardiomyocytes from death. Apoptosis is a key reason of cardiomyocyte death that can be prevented. In this study, we investigated the role of TNF-related apoptosis-inducing ligand (TRAIL) in initiating apoptosis by binding to death receptor 5 (DR5), and this procession is inhibited by soluble DR5 (sDR5) in rats after AMI. First, we found that the level of TRAIL in serum was down-regulated in AMI patients. Then, TRAIL and DR5 expression was analysed in the myocardium of rats after AMI, and their expression was up-regulated. sDR5 treatment reduced the myocardial infarct size and the levels of CK-MB and cTn-I in serum. The expression of caspase 3 and PARP is decreased, but the anti-apoptotic factor Bcl-2 was increased in sDR5 treatment rats after AMI. DR5 expression was also analysed after sDR5 treatment and it was down-regulated, and a low level of DR5 expression seemed to be beneficial for the myocardium. Overall, our findings indicated that sDR5 decreases myocardial damage by inhibiting apoptosis in rat after AMI. We expect to observe the potential therapeutic effects of sDR5 on AMI in the future.

Blocking the death checkpoint protein TRAIL improves cardiac function after myocardial infarction in monkeys, pigs, and rats.

Myocardial infarction (MI) is a leading cause of death worldwide for which there is no cure. Although cardiac cell death is a well-recognized pathological mechanism of MI, therapeutic blockade of cell death to treat MI is not straightforward. Death receptor 5 (DR5) and its ligand TRAIL [tumor necrosis factor (TNF)-related apoptosis-inducing ligand] are up-regulated in MI, but their roles in pathological remodeling are unknown. Here, we report that blocking TRAIL with a soluble DR5 immunoglobulin fusion protein diminished MI by preventing cardiac cell death and inflammation in rats, pigs, and monkeys. Mechanistically, TRAIL induced the death of cardiomyocytes and recruited and activated leukocytes, directly and indirectly causing cardiac injury. Transcriptome profiling revealed increased expression of inflammatory cytokines in infarcted heart tissue, which was markedly reduced by TRAIL blockade. Together, our findings indicate that TRAIL mediates MI directly by targeting cardiomyocytes and indirectly by affecting myeloid cells, supporting TRAIL blockade as a potential therapeutic strategy for treating MI.

The E3 ubiquitin ligase Itch is required for B-cell development.

The E3 ubiquitin ligase Itch interacts with Foxo1 and targets it for ubiquitination and degradation during follicular helper T-cell differentiation, whereas the transcription factor Foxo1 plays a critical role in B-cell development. Thus, we proposed that Itch mediates B-cell differentiation. Unexpectedly, we found that Itch deficiency downregulated Foxo1 expression in B cells. Itch cKO (conditional knock out in B cells) mice had fewer pro-B cells in the bone marrow, more small resting IgM-IgD-B cells in the periphery, and lower B-cell numbers in the lymph nodes through decreased Foxo1-mediated IL-7Rα, RAG, and CD62L expression, respectively. Importantly, Itch deficiency reduced Foxo1 mRNA expression by up-regulating JunB-mediated miR-182. Finally, Foxo1 negatively regulated JunB expression by up-regulating Itch. Thus, we have identified a novel regulatory axis between Itch and Foxo1 in B cells, suggesting that Itch is essential for B-cell development.

Pre-existing CD19-independent GL7(-) Breg cells are expanded during inflammation and in mice with lupus-like disease.

Interleukin 10 (IL-10)-producing regulatory B-cells (Bregs) suppress inflammatory responses that mediate autoimmune diseases. However, it is unknown whether Bregs derive from a pre-existing dedicated B-cell lineage or if any B-cell can differentiate into Bregs in response to BCR or TLR activation. GL7(+) B-cells are antigen-experienced differentiated B-cells while GL7(-/lo) are at an early stage of B-cell differentiation. While both GL7(-/lo) and GL7(+) B cells can produce IL-10, differentiation of GL7(-) B-cells into Bregs does not require CD19- or Bcl6-induced signals, suggesting that BCR-induced proliferation or Ig class-switching is not necessary for generation of Breg cells. Of particular importance, we show that GL7(-) Breg cells are dramatically expanded in lupus-like mice and GL7(-) Bregs suppressed inflammatory responses in lupus-like mice by inducing expansion of Foxp3(+)Treg cells. Taken together, these results suggest that pre-existing GL7(-)IL-10(+) cells are expanded during inflammation, differentiate into GL7(+) Bregs and contribute to immune-regulation in lupus-like mice.

Activated cytotoxic lymphocytes promote tumor progression by increasing the ability of 3LL tumor cells to mediate MDSC chemoattraction via Fas signaling.

The Fas/FasL system transmits intracellular apoptotic signaling, inducing cell apoptosis. However, Fas signaling also exerts non-apoptotic functions in addition to inducing tumor cell apoptosis. For example, Fas signaling induces lung cancer tumor cells to produce prostaglandin E2 (PGE2) and recruit myeloid-derived suppressor cells (MDSCs). Activated cytotoxic T lymphocytes (CTLs) induce and express high levels of FasL, but the effects of Fas activation initiated by FasL in CTLs on apoptosis-resistant tumor cells remain largely unclear. We purified activated CD8(+) T cells from OT-1 mice, evaluated the regulatory effects of Fas activation on tumor cell escape and investigated the relevant mechanisms. We found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell-mediated chemoattraction of MDSCs via Fas signaling, which was favorable to tumor growth. Our results indicate that CTLs may participate in the tumor immune evasion process. To the best of our knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. Our findings implicate a strategy to enhance the antitumor immune response via reduction of negative immune responses to tumors promoted by CTLs through Fas signaling.

Exosomes with membrane-associated TGF-ß1 from gene-modified dendritic cells inhibit murine EAE independently of MHC restriction.

We have previously demonstrated that exosomes from dendritic cells (DCs) secreting TGF-ß1 (sTGF-ß1-EXOs) delay the development of murine inflammatory bowel disease (IBD). In this study, we isolated exosomes from DCs expressing membrane-associated TGF-ß1 (mTGF-ß1-EXOs) and found mTGF-ß1-EXOs had more potent immunosuppressive activity than sTGF-ß1-EXOs in vitro. Treatment of mice with mTGF-ß1-EXOs inhibited the development and progression of myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE even after disease onset. Treatment of mice with mTGF-ß1-EXOs also impaired Ag-specific Th1 and IL-17 responses, but promoted IL-10 responses ex vivo. Treatment with mTGF-ß1-EXOs decreased the frequency of Th17 cells in EAE mice, which might be associated with the down-regulation of the p38, ERK, Stat3, and NF-κB activation and IL-6 expression in DCs. Treatment with mTGF-ß1-EXOs maintained the regulatory capacity of Treg cells, and adoptive transfer of CD4(+)Foxp3(+)Treg cells from mTGF-ß1-EXO-treated EAE mice dramatically prevented the development of EAE in the recipients. Moreover, treatment with mTGF-ß1-EXOs from C57BL/6 mice effectively prevented and inhibited proteolipid protein (PLP) peptide-induced EAE in BALB/c mice. These results indicate that mTGF-ß1-EXOs possess powerful immunosuppressive ability and can effectively inhibit the development and progression of EAE in different strains of mice.

Pharmacophore-based drug design and biological evaluation of novel ABCB1 inhibitors.

Overexpression of ABCB1 is one of major barriers for multidrug resistance in chemotherapy and limits drug oral bioavailability. Inhibition of ABCB1 would sensitize multidrug resistance in clinical cancer chemotherapy. With this aim, a 3D pharmacophore model was created based on known ABCB1 inhibitors with correlation coefficient of 0.94, comprising three hydrophobic features and one hydrogen bond acceptor. It was further validated and used to search our in-house 3D database for potential ABCB1 inhibitors. The inhibitory activities of the best hits were evaluated by several biological assays, such as rhodamine 123 accumulation assay, chemosensitization assay, multidrug resistance 1-Madin-Darby canine kidney cells/Madin-Darby canine kidney cells permeability assay. Finally, compounds YZ-3 and YZ-16 were identified as potential leads to be developed in the designing of novel potent ABCB1 inhibitors.

New use for an old drug: inhibiting ABCG2 with sorafenib.

Human ABCG2, a member of the ATP-binding cassette transporter superfamily, represents a promising target for sensitizing MDR in cancer chemotherapy. Although lots of ABCG2 inhibitors were identified, none of them has been tested clinically, maybe because of several problems such as toxicity or safety and pharmacokinetic uncertainty of compounds with novel chemical structures. One efficient solution is to rediscover new uses for existing drugs with known pharmacokinetics and safety profiles. Here, we found the new use for sorafenib, which has a dual-mode action by inducing ABCG2 degradation in lysosome in addition to inhibiting its function. Previously, we reported some novel dual-acting ABCG2 inhibitors that showed closer similarity to degradation-induced mechanism of action. On the basis of these ABCG2 inhibitors with diverse chemical structures, we developed a pharmacophore model for identifying the critical pharmacophore features necessary for dual-acting ABCG2 inhibitors. Sorafenib forms impressive alignment with the pharmacophore hypothesis, supporting the argument that sorafenib is a potential ABCG2 inhibitor. This is the first article that sorafenib may be a good candidate for chemosensitizing agent targeting ABCG2-mediated MDR. This study may facilitate the rediscovery of new functions of structurally diverse old drugs and provide a more effective and safe way of sensitizing MDR in cancer chemotherapy.