Identification of Heterophilic Epitopes of H1N1 Influenza Virus Hemagglutinin.

Our previous studies found that the H1-50 monoclonal antibody (mAb) of influenza A virus hemagglutinin (HA) cross-reacted with pancreatic tissue and islet ß-cells, and further studies showed that H1-50 mAb binds to prohibitin (PHB) protein of islet ß-cells. These suggest that there are heterophilic epitopes between influenza virus HA and pancreatic tissue, which may be involved in the pathogenesis of type 1 diabetes. To further investigate these heterophilic epitopes, we screened binding epitopes of H1-50 mAb using a phage 12-peptide library. DNA sequencing and comparative analysis were performed on specific positive phage clones, and the sequence of 12-peptide binding to H1-50 mAb was obtained. The binding epitopes of H1-50 mAb in influenza virus HA were determined by sequence analysis and experimental verification, and their distribution within the three-dimensional structure was assessed by PyMOL. The results showed that H1-50 mAb specifically binds to polypeptides (306-SLPFQNIHPITIGK-319) of influenza A virus HA, located in the stem of the HA protein. However, there is no specific binding sequence between H1-50 mAb and the PHB protein of islet ß-cells in the primary structure, and we speculate that the binding of H1-50 mAb to islet ß-cells may depend on the spatial conformation. The identification of the heterophilic epitopes of H1N1 influenza virus hemagglutinin provides a new perspective on type 1 diabetes that may be caused by influenza virus infection, which may contribute to the prevention and control of influenza.

[Inhibition of Paeoniflorin on TNF-α-induced TNF-α Receptor Type I /Nuclear Factor-κB Signal Transduction in Endothelial Cells].

OBJECTIVE: To study the inhibitory effect of paeoniflorin (PAE) on TNF-α-induced TNF receptor type I (TNFR1)-mediated signaling pathway in mouse renal arterial endothelial cells (AECs) and to explore its underlying molecular mechanisms. METHODS: Mouse AECs were cultured in vitro and then they were treated by different concentrations PAE or TNF-α for various time periods. Expression levels of intercellular cell adhesion molecule-1 (ICAM-1) were detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 6-h TNF-α 30 ng/mL), the low dose PAE group (cultured by 2-h PAE 0.8 µmo/L plus 6-h TNF-α 30 ng/mL), the middle dose PAE group (cultured by 2-h PAE 8 µmol/L plus 6-h TNF-α 30 ng/mL), the high dose PAE group (cultured by 2-h PAE 80 µmol/L plus 6-h TNF-α 30 ng/mL) with Western blot analysis. Nuclear translocation of transcription factor NF-κB (NE-κB) was detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 45-mm TNF-α 30 ng/mL), and the high dose PAE group (cultured by 2-h PAE 80 µmol/L plus 45-min TNF-α 30 ng/mL) by immunofluorescent staining. Expression levels of the phosphorylation of extracellular signal-regulated (protein) kinase (ph-ERK) and p38 (ph- p38) were detected in the normal group (cultured by serum-free culture media) and the high dose PAE group (2-h PAE 80 µmol/L culture) by Western blot. NF-κB inhibitor-α (IκBα) protein expressions were detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 30-min TNF-α 30 ng/mL), the high dose PAE group (cultured by 2-h PAE 80 µmol/L plus 30-min TNF-α 30 ng/mL), the p38 inhibitor group (SB group, pretreatment with SB238025 25 µmol/L for 30 min, then treated by PAE 80 µmol/L for 2 h, and finally treated by TNF-α 30 ng/mL for 30 min), the ERK inhibitor group (PD group, treated by PD98059 50 µmol/L for 30 min, then treated by PAE 80 µmol/L for 2 h, and finally treated by TNF-α 30 ng/mL for 30 min) by Western blot. RESULTS: Compared with the normal group, ICAM-1 protein expression levels obviously increased (P < 0.01). Compared with the TNFα group, ICAM-1 protein expression levels were obviously inhibited in the high dose PAE group (P < 0.05). Protein expression levels of ph-p38 and ph-ERK were obviously higher in the hIgh dose PAE group (P < 0.05). Compared with the normal group, IκBα protein expression levels obviously decreased in the TNF-α group (P < 0.01). Compared with the TNFα group, TNF-α-induced IκBα degradation could be significantly inhibited in the high dose PAE group (P < 0.01); the inhibition of PAE on IκBα degradation could be significantly inhibited in the SB group (P < 0.05). NF-κB/p65 signal was mainly located in cytoplasm in the normal group. NF-κB/p65 was translocated from cytoplasm to nucleus after stimulated by 45 min TNF-α in the TNF-α group, while it could be significantly inhibited in the high dose PAE group. CONCLUSIONS: PAE inhibited TNF-α-induced expression of lCAM-1. Its action might be associated with inhibiting TNFR1/NF-κB signaling pathway. p38 participated and mediated these actions.

Development and characterization of a panel of cross-reactive monoclonal antibodies generated using H1N1 influenza virus.

To characterize the antigenic epitopes of the hemagglutinin (HA) protein of H1N1 influenza virus, a panel consisting of 84 clones of murine monoclonal antibodies (mAbs) were generated using the HA proteins from the 2009 pandemic H1N1 vaccine lysate and the seasonal influenza H1N1(A1) vaccines. Thirty-three (39%) of the 84 mAbs were found to be strain-specific, and 6 (7%) of the 84 mAbs were subtype-specific. Twenty (24%) of the 84 mAbs recognized the common HA epitopes shared by 2009 pandemic H1N1, seasonal A1 (H1N1), and A3 (H3N2) influenza viruses. Twenty-five of the 84 clones recognized the common HA epitopes shared by the 2009 pandemic H1N1, seasonal A1 (H1N1) and A3 (H3N2) human influenza viruses, and H5N1 and H9N2 avian influenza viruses. We found that of the 16 (19%) clones of the 84 mAbs panel that were cross-reactive with human respiratory pathogens, 15 were made using the HA of the seasonal A1 (H1N1) virus and 1 was made using the HA of the 2009 pandemic H1N1 influenza virus. Immunohistochemical analysis of the tissue microarray (TMA) showed that 4 of the 84 mAb clones cross-reacted with human tissue (brain and pancreas). Our results indicated that the influenza virus HA antigenic epitopes not only induce type-, subtype-, and strain-specific monoclonal antibodies against influenza A virus but also cross-reactive monoclonal antibodies against human tissues. Further investigations of these cross-reactive (heterophilic) epitopes may significantly improve our understanding of viral antigenic variation, epidemics, pathophysiologic mechanisms, and adverse effects of influenza vaccines.

A comparative study on inhibition of total astragalus saponins and astragaloside IV on TNFR1-mediated signaling pathways in arterial endothelial cells.

BACKGROUND: Both total astragalus saponins (AST) and it's main component astragaloside IV (ASIV) have been used in China as cardiovascular protective medicines. However, the anti-inflammatory activities that are beneficial for cardiovascular health have never been compared directly and the molecular mechanisms remain unresolved. This study was conducted to compare the inhibitory effects of these drugs on TNFα-induced cell responses, related signaling pathways, and the underlying mechanisms in mouse arterial endothelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Real-time qRT-PCR was performed to determine the expression of cell adhesion molecule (CAM) genes. Immunofluorescent staining was used to detect the nuclear translocation of transcription factor NF-κB-p65. Western Blot analysis was used to identify TNFα-induced NF-κB-p65 phosphorylation, IκBα degradation, and caspase-3 cleavage. Cell surface proteins were isolated and TNFα receptor-1(TNFR1) expression was determined. The results suggest that both AST and ASIV attenuate TNFα-induced up-regulation of CAMs mRNA and upstream nuclear translocation and phosphorylation of NF-κB-p65. However, TNFR1-mediated IκBα degradation, cleavage of caspase-3 and apoptosis were inhibited only by AST. These differences in the actions of AST and ASIV could be explained by the presence of other components in AST, such as ASII and ASIII, which also had an inhibitory effect on TNFR1-induced IκBα degradation. Moreover, AST, but not ASIV, was able to reduce TNFR1 protein level on the cell surface. Furthermore, mechanistic investigation demonstrated that TNFR1-mediated IκBα degradation was reversed by the use of TAPI-0, an inhibitor of TNFα converting enzyme (TACE), suggesting the involvement of TACE in the modulation of surface TNFR1 level by AST. CONCLUSION: ASIV was not a better inhibitor than AST, at least on the inhibition of TNFα-induced inflammatory responses and TNFR1-mediated signaling pathways in AECs. The inhibitory effect of AST was caused by the reduction of cell surface TNFR1 level, and TACE could be involved in this action.