The molecular evolutionary characteristics of new isolated H9N2 AIV from East China and the function of vimentin on virus replication in MDCK cells.

BACKGROUND: The low pathogenic H9N2 AIV caused the serious impact on the poultry industry and public safety. Our purpose was to investigate the molecular evolutionary characteristics of the new isolated H9N2 virus and investigate the intracellular target protein of H9N2 AIV replication in sensitive cells. METHODS: AIV A/chicken/Shandong/LY1/2017 (H9N2) was isolated from the cloaca of the healthy chicken in Shandong, and the full-length eight gene segments of this isolated H9N2 AIV were amplified by RT-PCR and analyzed. MDCK cells were used as the target cell model, and VOPBA assay and LC-MS/MS were carried out to identify the virus-binding protein of H9N2 AIV. MDCK cells were pre-treated with the special antibody and siRNA, and treated with H9N2 AIV to detect the virus replication. Additionally, Vimentin-pcDNA3.0 was successfully constructed, and transinfected into MDCK cells, and then H9N2 AIV mRNA was detected with RT-PCR. RESULTS: Phylogenetic analysis revealed that HA, NA, PB2, PB1, PA, NP and M seven genes of the isolated H9N2 AIV were derived from A/Chicken/Shanghai/F/98, while NS gene was derived from A/Duck/Hong Kong/Y439/97. The cleavage site sequence of HA gene of the isolated H9N2 AIV was a PARSSR G pattern, and the left side sequence (224 ~ 229) of receptor binding site was NGQQGR pattern, which were similar to that of A/Chicken/Shanghai/F/98. Following VOPBA assay, we found one protein of about 50KDa binding to H9N2 AIV, and the results of LC-MS/MS analysis proved that vimentin was the vital protein binding to H9N2 AIV. The pre-incubation of the specific antibody and siRNA decreased the viral RNA level in MDCK cells treated with H9N2 AIV. Furthermore, we found that over-expressed vimentin increased H9N2 AIV replication in MDCK cells. CONCLUSIONS: These findings suggested that the isolated H9N2 AIV might be a recent clinical common H9N2 strain, and vimentin protein might be one vital factor for H9N2 AIV replication in MDCK cells, which might be a novel target for design and development of antiviral drug.

Disruption of Striatal-Enriched Protein Tyrosine Phosphatase Signaling Might Contribute to Memory Impairment in a Mouse Model of Sepsis-Associated Encephalopathy.

Sepsis-associated encephalopathy (SAE) is a potentially irreversible acute cognitive dysfunction with unclear mechanism. Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase which normally opposes synaptic strengthening by regulating key signaling molecules involved in synaptic plasticity and neuronal function. Thus, we hypothesized that abnormal STEP signaling pathway was involved in sepsis-induced cognitive impairment evoked by lipopolysaccharides (LPS) injection. The levels of STEP, phosphorylation of GluN2B (pGluN2B), the kinases extracellular signal-regulated kinase 1/2 (pERK), cAMP-response element binding protein (CREB), synaptophysin, brain derived neurotrophic factor (BDNF), and post-synaptic density protein 95 (PSD95) in the hippocampus, prefrontal cortex, and striatum were determined at the indicated time points. In the present study, we found that STEP levels were significantly increased in the hippocampus, prefrontal cortex, and striatum following LPS injection, which might resulted from the disruption of the ubiquitin-proteasome system. Notably, a STEP inhibitor TC-2153 treatment alleviated sepsis-induced memory impairment by increasing phosphorylation of GluN2B and ERK1/2, CREB/BDNF, and PSD95. In summary, our results support the key role of STEP in sepsis-induced memory impairment in a mouse model of SAE, whereas inhibition of STEP may provide a novel therapeutic approach for this disorder and possible other neurodegenerative diseases.

The immunomodulatory functions and molecular mechanism of a new bursal heptapeptide (BP7) in immune responses and immature B cells.

The bursa of Fabricius (BF) is the acknowledged central humoural immune organ unique to birds and plays a vital role in B lymphocyte development. In addition, the unique molecular immune features of bursal-derived biological peptides involved in B cell development are rarely reported. In this paper, a novel bursal heptapeptide (BP7) with the sequence GGCDGAA was isolated from the BF and was shown to enhance the monoclonal antibody production of a hybridoma. A mouse immunization experiment showed that mice immunized with an AIV antigen and BP7 produced strong antibody responses and cell-mediated immune responses. Additionally, BP7 stimulated increased mRNA levels of sIgM in immature mouse WEHI-231 B cells. Gene microarray results confirmed that BP7 regulated 2465 differentially expressed genes in BP7-treated WEHI-231 cells and induced 13 signalling pathways and various immune-related functional processes. Furthermore, we found that BP7 stimulated WEHI-231 cell autophagy and AMPK-ULK1 phosphorylation and regulated Bcl-2 protein expression. Finally, chicken immunization showed that BP7 enhanced the potential antibody and cytokine responses to the AIV antigen. These results suggested that BP7 might be an active biological factor that functions as a potential immunopotentiator, which provided some novel insights into the molecular mechanisms of the effects of bursal peptides on immune functions and B cell differentiation.

Activation of ß2-Adrenoceptor Attenuates Sepsis-Induced Hippocampus-Dependent Cognitive Impairments by Reversing Neuroinflammation and Synaptic Abnormalities.

Sepsis-associated encephalopathy induces cognitive dysfunction via mechanisms that commonly involve neuroinflammation and synaptic plasticity impairment of the hippocampus. The ß2-adrenoceptor (ß2-AR) is a G-protein coupled receptor that regulates immune response and synaptic plasticity, whereas its dysfunction has been implicated in various neurodegenerative diseases. Thus, we hypothesized abnormal ß2-AR signaling is involved in sepsis-induced cognitive impairment. In the present study, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) to mimic the clinical human sepsis-associated encephalopathy. The levels of hippocampal ß2-AR, proinflammatory cytokines tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), IL-6, cAMP-response element binding protein (CREB), brain derived neurotrophic factor (BDNF), post-synaptic density protein 95 (PSD95), and NMDA receptor 2 B subtypes (GluN2B) were determined at 6, 12, 24 h and 7 and 16 days after CLP. For the interventional study, mice were treated with ß2-AR agonist clenbuterol in two ways: early treatment (immediately following CLP) and delayed treatment (on the 8th day following CLP). Neurobehavioral performances were assessed by open field and fear conditioning tests. Here, we found that hippocampal ß2-AR expression was significantly decreased starting from 12 h and persisted until 16 days following CLP. Besides, sepsis mice also exhibited increasing neuroinflammation, down-regulated CREB/BDNF, decreasing PSD95 and GluN2B expression, and displayed hippocampus-dependent cognitive impairments. Notably, early clenbuterol treatment alleviated sepsis-induced cognitive deficits by polarizing microglia toward an anti-inflammatory phenotype, reducing proinflammatory cytokines including IL-1ß, TNF-α, and up-regulating CREB/BDNF, PSD95, and GluN2B. Intriguingly, delayed clenbuterol treatment also improved cognitive impairments by normalization of hippocampal CREB/BDNF, PSD95, and GluN2B. In summary, our results support the beneficial effects of both early and delayed clenbuterol treatment, which suggests that activation of ß2-AR has a translational value in sepsis-associated organ dysfunction including cognitive impairments.

The Inducing Roles of the New Isolated Bursal Hexapeptide and Pentapeptide on the Immune Response of AIV Vaccine in Mice.

BACKGROUND: Bursa of Fabricius is the acknowledged central humoral immune organ. The bursal-derived peptides play the important roles on the immature B cell development and antibody production. OBJECTIVES: Here we explored the functions of the new isolated bursal hexapeptide and pentapeptide on the humoral, cellular immune response and antigen presentation to Avian Influenza Virus (AIV) vaccine in mice immunization. METHODS: The bursa extract samples were purified following RP HPLC method, and were analyzed with MS/MS to identify the amino acid sequences. Mice were twice subcutaneously injected with AIV inactivated vaccine plus with two new isolated bursal peptides at three dosages, respectively. On two weeks after the second immunization, sera samples were collected from the immunized mice to measure AIV-specific IgG antibody levels and HI antibody titers. Also, on 7th day after the second immunization, lymphocytes were isolated from the immunized mice to detect T cell subtype and lymphocyte viabilities, and the expressions of co-stimulatory molecule on dendritic cells in the immunized mice. RESULTS: Two new bursal hexapeptide and pentapeptide with amino acid sequences KGNRVY and MPPTH were isolated, respectively. Our investigation proved the strong regulatory roles of bursal hexapeptide on AIV-specific IgG levels and HI antibody titers, and lymphocyte viabilities, and the significant increased T cells subpopulation and expressions of MHCII molecule on dendritic cells in the immunized mice. Moreover, our findings verified the significantly enhanced AIV-specific IgG antibody and HI titers, and the strong increased T cell subpopulation and expressions of CD40 molecule on dendritic cells in the mice immunized with AIV vaccine and bursal pentapeptide. CONCLUSION: We isolated and identified two new hexapeptide and pentapeptide from bursa, and proved that these two bursal peptides effectively induced the AIV-specific antibody, T cell and antigen presentation immune responses, which provided an experimental basis for the further clinical application of the bursal derived active peptide on the vaccine improvement.

The Functions and Mechanism of a New Oligopeptide BP9 from Avian Bursa on Antibody Responses, Immature B Cell, and Autophagy.

The bursa of Fabricius is an acknowledged central humoral immune organ unique to birds, which is vital to B cell differentiation and antibody production. However, the function and mechanism of the biological active peptide isolated from bursa on B cell development and autophagy were less reported. In this study, we isolated a new oligopeptide with nine amino acids Leu-Met-Thr-Phe-Arg-Asn-Glu-Gly-Thr from avian bursa following RP-HPLC, MODIL-TOP-MS, and MS/MS, which was named after BP9. The results of immunization experiments showed that mice injected with 0.01 and 0.05 mg/mL BP9 plus JEV vaccine generated the significant increased antibody levels, compared to those injected with JEV vaccine only. The microarray analysis on the molecular basis of BP9-treated immature B cell showed that vast genes were involved in various immune-related biological processes in BP9-treated WEHI-231 cells, among which the regulation of cytokine production and T cell activation were both major immune-related processes in WEHI-231 cells with BP9 treatment following network analysis. Also, the differentially regulated genes were found to be involved in four significantly enriched pathways in BP9-treated WEHI-231 cells. Finally, we proved that BP9 induced the autophagy formation, regulated the gene and protein expressions related to autophagy in immature B cell, and stimulated AMPK-ULK1 phosphorylation expression. These results suggested that BP9 might be a strong bursal-derived active peptide on antibody response, B cell differentiation, and autophagy in immature B cells, which provided the linking among humoral immunity, B cell differentiation, and autophagy and offered the important reference for the effective immunotherapeutic strategies and immune improvement.

NLRP3/Caspase-1 Pathway-Induced Pyroptosis Mediated Cognitive Deficits in a Mouse Model of Sepsis-Associated Encephalopathy.

Sepsis-associated encephalopathy (SAE) is a common complication that leads to long-term cognitive impairments and increased mortality in sepsis survivors. The mechanisms underlying this complication remain unclear and an effective intervention is lacking. Accumulating evidence suggests the nucleotide-binding domain-like receptor protein3 (NLRP3)/caspase-1 pathway is involved in several neurodegenerative diseases. Thus, we hypothesized that the NLRP3/caspase-1 pathway is involved in NLRP3-mediated pyroptosis, maturation and release of inflammatory cytokines, and cognitive deficits in SAE. We used the NLRP3 inhibitor MCC950 and the caspase-1 inhibitor Ac-YVAD-CMK to study the role of the NLRP3/caspase-1 pathway in pyroptosis and cognitive deficits in a mouse model of SAE. Mice were randomly assigned to one of six groups: sham+saline, sham+MCC950, sham+Ac-YVAD-CMK, cecal ligation and puncture (CLP)+saline, CLP+MCC950, and CLP+Ac-YVAD-CMK. Surviving mice underwent behavioral tests or had hippocampal tissues collected for histochemical analysis and biochemical assays. Our results show that CLP-induced hippocampus-dependent memory deficits are accompanied by increased NLRP3 and caspase-1 positive cells, and augmented protein levels of NLRP3, caspase-1, gasdermin-D, and pro-inflammatory cytokines in the hippocampus. In addition, administration of MCC950 or Ac-YVAD-CMK rescues cognitive deficits and ameliorates increased hippocampal NLRP3-mediated neuronal pyroptosis and pro-inflammatory cytokines. Our results suggest that the NLRP3/caspase-1 pathway-induced pyroptosis mediates cognitive deficits in a mouse model of SAE.

The Functions of Bursal Hexapeptide (BHP) on Immune Response and the Molecular Mechanism on Immature B Cell.

BACKGROUND: The bursa of Fabricius (BF) is an acknowledged central immune organ, and is important to B cell differentiation. Bursal hexapeptide (BHP) is the recently reported bursalderived peptide, while its inducing function on immune response is uncertain. OBJECTIVES: The main objective of this study was to analyze the immune responses to JEV vaccine in mice induced by BHP plus JEV vaccine, and to detect the signal and biological functions of BHP on immature B cells. METHODS: Mice were immunized with Japanese encephalitis virus (JEV) vaccine and BHP from 0.01 mg/mL to 0.25 mg/mL to detect antibody response and cellular immune response, respectively. The production of IgG, IgG1 and IgG2a specific to JEV in serum from immunized mice were measured by ELISA, and T cell subpopulation from immunized mice were detected with using fluorochrome conjugated mAbs of the corresponding PE-Cys/FITC/PE by flow cytometry. Spleen cells from all immunized mice were harvested after one week of second immunization for lymphocyte proliferation assay. Mouse immature B cell WEHI-231 cell was treated with 0.01µg/mL BHP for 4h, and analyzed the involved biological function and pathway of differentially expressed genes with gene microarray. RESULTS: BHP co-immunization with JEV vaccine generated significant increased antibody levels, neutralizing antibody titers and spleen lymphocyte viability, compared to that of vaccine control. The subpopulations of T cells in spleen lymphocytes were significantly modified in the mice coimmunized with JEV vaccine and BHP. The analysis results of gene expression profiles of WEHI- 231 mouse immature B cells with BHP treatment showed that the regulated genes with BHP treatment were involved various immune related biological functions, including proliferation and activation of lymphocyte and T cell, T cell mediated immunity and regulation of adaptive immune response. Furthermore, BHP stimulated three significant enriched pathways, including amphetamine addiction, long-term potentiation, and RIG-I-like receptor signaling pathway. CONCLUSION: Our results indicated BHP induced significant humoral and cellular immunity to JEV vaccine, and regulated various biological processes and signalling related to immune activation in immature B cells. These results proposed the immunomodulatory function and mechanism of BHP on immune induction, which provided the novel insight on the candidate reagent for immune improvement.

The Regulatory Functions of a New Tetrapeptide from the Bursa of Fabricius on AIV Vaccine Immunization and Antibody Production.

BACKGROUND: Understanding the regulatory functions of the biological peptide from the humoral central immune organ bursa of Fabricius on vaccine immune responses and antibody production is of vital importance. OBJECTIVES: Here we thoroughly verified the immunomodulatory functions of the new tetrapeptide BP4 from the bursa of Fabricius on vaccine immune responses in mice and chicken immunizaiton model, and on potential intracellular signaling during antibody production. METHOD: BP4 was isolated and identified by Reverse Phase High Performance Liquid Chromatography and matrix-assisted laser desorption ionization time of flight mass spectrometry. immunomodulatory functions of BP4 was verified by AIV vaccine immunization on mice and chickens regarding roles in vivo, by monitoring the impact of signalling inhibitors in hybridoma cells on antibody production in vitro. RESULTS: Our investigation revealed the strong inducing roles of new isolated BP4 on immune responses in mice immunization, the immunomodulatory effects in the immunized chicken, four potential key intracellular signaling during antibody production in hybrdoma cells. CONCLUSION: The new bursal-derived peptide BP4 was isolated and identified, and the immunomodulatory effects on antigen-specific immune responses in vivo and in vitro were verified, suggesting BP4 might be highly relevant to the humoral immune responses, and PI3K/Akt, p38 MAPK, NF-κB and tyrosine phosphorylation signaling might be the key activated intracellular signaling during antibody production during BP4 stimulation, which provided a novel potential adjuvant candidate for vaccine immunization improvement and precaution on animal epidemic disease.