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.

Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells.

During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses.IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.

Entry of Classical Swine Fever Virus into PK-15 Cells via a pH-, Dynamin-, and Cholesterol-Dependent, Clathrin-Mediated Endocytic Pathway That Requires Rab5 and Rab7.

UNLABELLED: Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small, enveloped, positive-strand RNA virus. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. However, the mechanisms of CSFV entry into cells are not well characterized. In this study, we used systematic approaches to dissect CSFV cell entry. We first observed that CSFV infection was inhibited by chloroquine and NH4Cl, suggesting that viral entry required a low-pH environment. By using the specific inhibitor dynasore, or by expressing the dominant negative (DN) K44A mutant, we verified that dynamin is required for CSFV entry. CSFV particles were observed to colocalize with clathrin at 5 min postinternalization, and CSFV infection was significantly reduced by chlorpromazine treatment, overexpression of a dominant negative form of the EPS15 protein, or knockdown of the clathrin heavy chain by RNA interference. These results suggested that CSFV entry depends on clathrin. Additionally, we found that endocytosis of CSFV was dependent on membrane cholesterol, while neither the overexpression of a dominant negative caveolin mutant nor the knockdown of caveolin had an effect. These results further suggested that CSFV entry required cholesterol and not caveolae. Importantly, the effect of DN mutants of three Rab proteins that regulate endosomal traffic on CSFV infection was examined. Expression of DN Rab5 and Rab7 mutants, but not the DN Rab11 mutant, significantly inhibited CSFV replication. These results were confirmed by silencing of Rab5 and Rab7. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab7 during the early phase of infection within 45 min after virus entry. These results indicated that after internalization, CSFV moved to early and late endosomes before releasing its RNA. Taken together, our findings demonstrate for the first time that CSFV enters cells through the endocytic pathway, providing new insights into the life cycle of pestiviruses. IMPORTANCE: Bovine viral diarrhea virus (BVDV), a single-stranded, positive-sense pestivirus within the family Flaviviridae, is internalized by clathrin-dependent receptor-mediated endocytosis. However, the detailed mechanism of cell entry is unknown for other pestiviruses, such as classical swine fever (CSF) virus (CSFV). CSFV is the etiological agent of CSF, a highly contagious disease of swine that causes numerous deaths in pigs and enormous economic losses in China. Understanding the entry pathway of CSFV will not only advance our knowledge of CSFV infection and pathogenesis but also provide novel drug targets for antiviral intervention. Based on this objective, we used systematic approaches to dissect the pathway of entry of CSFV into PK-15 cells. This is the first report to show that the entry of CSFV into PK-15 cells requires a low-pH environment and involves dynamin- and cholesterol-dependent, clathrin-mediated endocytosis that requires Rab5 and Rab7.

The potential mechanism of bursal-derived BP8 on B cell developments.

The bursa of Fabricius, the key humoral immune organ unique to birds, is critical for B cell differentiation and antibody production. BP8 (AGHTKKAP) is a novel immunomodulatory peptide that regulates B-cell development. Gene microarray was used to investigate the mechanism of BP8 on B cell development. BP8 regulated expressions of 1,570 genes that were involved in retinol metabolism, the Wnt signaling pathway, MAPK pathway, Jak-Stat pathway, Notch signaling pathway, cytokine-cytokine receptor interaction, and Ca(2+) signals. Finally, BP8 triggered ADH7 and RDH10 expression, interacted with retinol binding protein, and regulated retinol uptake in vitro and vivo. These data reveal a bursal-derived multifunctional factor, BP8, as a novel biomaterial which is essential for the development of the immune system and represents an important linker between the B cell development and retinol metabolism. This study elucidates the mechanisms involved in humoral immune system and has implications in treating human diseases.

BP8, a novel peptide from avian immune system, modulates B cell developments.

The bursa of Fabricius (BF) is the key humoral immune organ unique to birds, and is critical for early B-lymphocyte proliferation and differentiation. However, the molecular basis and mechanisms through which the BF regulates B cell development are not fully understood. In this study, we isolated and identified a new bursal peptide (BP8, AGHTKKAP) by RP-HPLC and MALDI-TOF-MS. BP8 promoted colony-forming pre-B formation, bound B cell precursor, regulated B cell development in vitro as well as in vivo, upstream of the EBF-E2A-Pax5 regulatory complex and increased immunoglobulin secretion. These data revealed a bursal-derived multifunctional factor BP8 as a novel biomaterial which is essential for the development of the immune system. This study elucidates further the mechanisms involved in humoral immune system and has implications in treating human diseases.

BP5 regulated B cell development promoting anti-oxidant defence.

Bursa of Fabricius is the humoral immune system for B cell differentiation and antibody production. Bursopentine (BP5) is a novel immunomodulatory peptide and significantly stimulated an antigen-specific immune response in mice. BP5 was also found to protect LPS-activated murine peritoneal macrophages from oxidative stress. In this study, the effects of BP5 on B cell development were examined. The results suggested that BP5 markedly promoted B cell development by increasing CFU-pre B, and affected the redox homeostasis regulation of B cells. To study the molecular mechanism of effect of bursal-derived BP5, this research utilized 2D-E and MALDI-TOF/TOF to analyze the differentially expressed proteins of BP5-treated WEHI-231 cells. The results showed that BP5 affected the redox homeostasis regulation of WEHI-231 cells and induced alterations in the protein expression profiles related to the oxidoreduction coenzyme metabolic process, precursor metabolites and energy, proteolysis, RNA splicing and translation and cellular process, respectively. BP5 also induced glucose-6-phosphate dehydrogenase (G6PD) activity, an essential anti-oxidant cofactor. We found that the redox homeostasis regulation effect of BP5 was reduced in G6PD-deficient cells. These data suggested that BP5 affected the redox balance toward reducing conditions by promoting the expression of G6PD, which in turn regulated the glutathione redox cycle and other processes.

Identification and characterization of novel immunomodulatory bursal-derived pentapeptide-II (BPP-II).

The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.

Griffithsin inhibits Japanese encephalitis virus infection in vitro and in vivo.

Griffithsin (GRFT) is a broad-spectrum antiviral protein that is effective against several glycosylated viruses. Here, we have evaluated the in vitro and in vivo antiviral activities of GRFT against Japanese encephalitis virus (JEV) infection. In vitro experiments showed that treatment of JEV with GRFT before inoculation of BHK-21 cells inhibited infection in a dose-dependent manner, with 99 % inhibition at 100 µg/ml and a 50 % inhibitory concentration (IC(50)) of 265 ng/ml (20 nM). Binding assays suggested that binding of GRFT to JEV virions inhibited JEV infection. In vivo experiment showed that GRFT (5 mg/kg) administered intraperitoneally before virus infection could completely prevent mortality in mice challenged intraperitoneally with a lethal dose of JEV. Our study also suggested that GRFT prevents JEV infection at the entry phase by targeting the virus. Collectively, our data demonstrate that GRFT is an antiviral agent with potential application in the development of therapeutics against JEV or other flavivirus infections.

The suppressive effects of Bursopentine (BP5) on oxidative stress and NF-ĸB activation in lipopolysaccharide-activated murine peritoneal macrophages.

BACKGROUND/AIM: Bursopentine (BP5) is a novel thiol-containing pentapeptide isolated from chicken bursa of Fabricius, and is reported to exert immunomodulatory effects on B and T lymphocytes. It has been found that some thiol compounds, such as glutathione (GSH) and N-acetylcysteine (NAC) protect living cells from oxidative stress. This led us to investigate whether BP5 had any ability to protect macrophages from oxidative stress as well as any mechanism that might underlie this process. METHODS: Murine peritoneal macrophages activated by lipopolysaccharide (LPS) (2 µg/ml) were treated with single bouts (0, 25, 50, and 100 µM) of BP5. RESULTS: BP5 potently suppressed the markers for oxidative stress, including nitric oxide (NO), reactive oxygen species (ROS), lipid peroxidation, and protein oxidation. It also decreased the expression and activity of inducible nitric oxide synthase (iNOS) and promoted a protective antioxidant state by elevating GSH content and by activating the expression and activity of certain key antioxidant and redox enzymes, including glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT). This suppressive effect on oxidative stress was accompanied by down-regulated expression and activity of nuclear factor kappa B (NF-κB). CONCLUSION: These findings demonstrate that BP5 can protect LPS-activated murine peritoneal macrophages from oxidative stress. BP5 may have applications as an anti-oxidative stress reagent.

The design and recombinant protein expression of a consensus porcine interferon: CoPoIFN-α.

CoPoIFN-α is a recombinant non-naturally occurring porcine interferon-α (IFN-α). It was designed by scanning 17 porcine IFN-α nonallelic subtypes and assigning the most frequently occurring amino acid in each position. We used a porcine IFN-α (PoIFN-α) derived from domestic pig as a control. Both porcine IFN-α genes were introduced into yeast expression vector PpICZα-A and expressed in Pichia pastoris. The antiviral unit of these two IFN-αs were assayed in MDBK, PK-15 and MARC-145 cells against vesicular stomatitis virus (VSV), and their inhibitory abilities on pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) replication were also examined, respectively. We found the antiviral activity (units/mg) of CoPoIFN-α was 46.4, 63.6 and 53.5-fold higher than that of PoIFN-α for VSV inhibition in MDBK, PK-15 and MARC-145 cells, 4.8-fold higher for PRV inhibition in PK-15 cells, and 5-fold higher for PRRSV inhibition in MARC-145 cells. Our results also showed that the PRV and PRRSV-specific cytopathic effect (CPE) could be inhibited in the cells pretreated with CoPoIFN-α and PoIFN-α, and the virus titers in the cells pretreated with CoPoIFN-α were lower than those cells pretreated with PoIFN-α by 10-20-fold. The antiproliferative activity of CoPoIFN-α was significantly higher than that of PoIFN-α on a molar basis. The mRNA level of Mx1 and OAS1 genes in PK-15 cells induced by CoPoIFN-α were enhanced about 4.6-fold and 3.2-fold compared to that induced by PoIFN-α. Based on a homology model of CoPoIFN-α and IFNAR2, all of the different residues between native PoIFN-α and CoPoIFN-α were not involved in IFNAR1 binding site, and there is no direct interaction between these residues and IFNAR2, either. We speculate that the higher activity of CoPoIFN-α was likely due to the electrostatic potential introduced by residue Arg156 around the binding site or a structural perturbation caused by these different residues. This may enhance the overall binding affinity of CoPoIIFN-α and the receptors. Thus, CoPoIFN-α may have the potential to be used in therapy of porcine diseases.

Role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in MARC-145 cells.

Lipid rafts play an important role in the life cycle of many viruses. Cholesterol is a critical structural component of lipid rafts. Although the porcine reproductive and respiratory syndrome virus (PRRSV) has restricted cell tropism for cells of the monocyte/macrophage lineage, a non-macrophage cell MARC-145 was susceptible to PRRSV because of the expression of virus receptor CD163 on the cell surface, therefore MARC-145 cells is used as model cell for PRRSV studies. In order to determine if cholesterol is involved in PRRSV infection in MARC-145 cells, we used three pharmacological agents: methyl-ß cyclodextrin (MßCD), mevinolin, and filipin complex to deplete cholesterol in MARC-145. Although these agents act by different mechanisms, they all significantly inhibited PRRSV infection. The inhibition could be prevented by addition of exogenous cholesterol. Cell membrane cholesterol depletion after virus infection had no effect on PRRSV production and cholesterol depletion pre-infection did not reduce the virus attachment, suggesting cholesterol is involved in virus entry. Further results showed that cholesterol depletion did not change expression levels of the PRRSV receptor CD163 in MARC-145, had no effect on clathrin-mediated endocytosis, but disturbed lipid-raft-dependent endocytosis. Collectively, these studies suggest that cholesterol is critical for PRRSV entry, which is likely to be mediated by a lipid-raft-dependent pathway.

Multiple linear B-cell epitopes of classical swine fever virus glycoprotein E2 expressed in E.coli as multiple epitope vaccine induces a protective immune response.

Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. Epitope-based vaccines is one of the current focuses in the development of new vaccines against classical swine fever virus (CSFV). Two B-cell linear epitopes rE2-ba from the E2 glycoprotein of CSFV, rE2-a (CFRREKPFPHRMDCVTTTVENED, aa844-865) and rE2-b (CKEDYRYAISSTNEIGLLGAGGLT, aa693-716), were constructed and heterologously expressed in Escherichia coli as multiple epitope vaccine. Fifteen 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with epitopes twice at 2-week intervals. All epitope-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:16 to 1:256. At this time, the pigs were subjected to challenge infection with a dose of 1 × 106 TCID50 virulent CSFV strain. After challenge infection, all of the rE2-ba-immunized pigs were alive and without symptoms or signs of CSF. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 5 days post challenge infection. The data from in vivo experiments shown that the multiple epitope rE2-ba shown a greater protection (similar to that of HCLV vaccine) than that of mono-epitope peptide(rE2-a or rE2-b). Therefore, The results demonstrated that this multiple epitope peptide expressed in a prokaryotic system can be used as a potential DIVA (differentiating infected from vaccinated animals) vaccine. The E.coli-expressed E2 multiple B-cell linear epitopes retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.

Design and evaluation of a multi-epitope peptide against Japanese encephalitis virus infection in BALB/c mice.

Epitope-based vaccination is a promising means to achieve protective immunity and to avoid immunopathology in Japanese encephalitis virus (JEV) infection. Several B-cell and T-cell epitopes have been mapped to the E protein of JEV, and they are responsible for the elicitation of the neutralizing antibodies and CTLs that impart protective immunity to the host. In the present study, we optimized a proposed multi-epitope peptide (MEP) using an epitope-based vaccine strategy, which combined six B-cell epitopes (amino acid residues 75-92, 149-163, 258-285, 356-362, 373-399 and 397-403) and two T-cell epitopes (amino acid residues 60-68 and 436-445) from the E protein of JEV. This recombinant protein was expressed in Escherichia coli, named rMEP, and its protective efficacy against JEV infection was assessed in BALB/c mice. The results showed that rMEP was highly immunogenic and could elicit high titer neutralizing antibodies and cell-mediated immune responses. It provided complete protection against lethal challenge with JEV in mice. Our findings indicate that the multi-epitope vaccine rMEP may be an attractive candidate vaccine for the prevention of JEV infection.

The Inducing Role and Molecular Basis of Bursal Hexapeptide (BHP) on Avian Immature B Cell.

BACKGROUND: The Bursa of Fabricius is an acknowledged central humoral immune organ unique to birds, which provides an ideal research model on the immature B cell development. OBJECTIVE: In this article, our motivation is to study the role on sIgM and establish the molecular basis and functional processes of Bursal Hexapeptide (BHP) in avian immature B cells DT40 cell lines. METHODS: In this article, we detected the expressions of sIgM mRNA with qPCR in DT40 cells with BHP treatment, and investigated the gene expression profiles of BHP-treated DT40 cells, employing microarray analyses. Also, to validate the differentially expressed genes, we performed KEGG pathway and Gene Ontology analysis in the BHP-treated DT40 cells. Finally, we comparatively analyzed the similar regulated genes and their involved immune functional processes between DT40 cell and mouse immature B cell line WEHI231 cell with BHP treatment. RESULTS: Following the proposed framework, we proved that the BHP enhanced the mRNA expression levels of IgM in DT40 cells, and induced 460 upregulated genes and 460 downregulated genes in BHP-treated DT40 cells. The pathway analysis showed that the differentially regulated genes in DT40 cell line with BHP treatment were involved in 12 enrichment pathways, in which Toll-like receptor signaling pathway was the vital pathways, and cytokine-cytokine receptor interaction and Jak-STAT signaling pathway were another two important pathways in BHP-treated DT40 cells. Moreover, BHP induced the immune related biological processes in BHP-treated DT40 cells, including T cell related, cytokine related, lymphocyte related, and innate immune response GO terms. Finally, the comparatively analysis showed that there were two downregulated genes GATA3 and IFNG to be found co-existed among the differentially expressed genes in BHP-treated DT40 cell and WEHI231 cells, which shared some same immune related functional processes in both cell lines. CONCLUSION: After the applying the framework, we proved the inducing roles and the gene expression profiles of BHP on avian immature B cells, and verified some molecular basis from the KEGG and GO analysis. These results provided the insight for mechanism on immature B cell differentiation, and offer the essential direction for 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.

Bursin as an adjuvant is a potent enhancer of immune response in mice immunized with the JEV subunit vaccine.

Adjuvants play an important role in the formulation of effective and appropriate vaccines. A series of synthetic bursin and bursin-like peptides was heterologously expressed in Escherichia coli. The use of bursin as an adjuvant enhanced the specific immune responses of mice immunized with a recombinant Japanese encephalitis virus E-binding domain (JEV E-BD) fusion protein. The effect was determined in the form of protective anti-JEV E titers, antibodies (IgG1 and IgG2a), spleen cell lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and the T-lymphocyte sub-type composition. The IgG2a titer and interferon-gamma level suggested that the E-BD protein potentiates the Th1 immune response. These responses were changed when the immunogen was combined with one of the synthetic peptides as adjuvant. JEV-neutralization assay results show that the presence of bursin significantly enhance the JEV-neutralizing titer. We conclude that bursin as an adjuvant is a potent enhancer of immune response in mice immunized with the JEV subunit vaccine, and represents a promising adjuvant for vaccination.

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 immunomodulatory peptide bursopentin (BP5) enhances proliferation and induces sIgM expression in DT40 cells.

BACKGROUND: In the recent past, many studies have been focused on extracts of BF and multiple biologically active factors and their effects on humoral immune system in chickens and birds. However, the mechanism of those immunomodulatory peptides on the B lineage cells proliferation and antibody production in chicken is fairly unknown. DT40 cell line, an avian leucosis virus-induced chicken pre-B cell line, expresses immunoglobulin M (IgM) isotype B cell reporter in the plasma membrane. There are many evidences suggesting that DT40 cells are best characterized as a bursal stem cell line. Because of the unique characteristics of DT40 cell line, it has been widely used to observe biological processes of pre-B lymphocyte cell within living cells. METHODS: The chicken B cell line DT40 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and cytotoxicity was studied. Also, effect of BP5 on cell proliferation and cell cycle distribution of DT40 cells was studied. Also, the effect of BP5 on sIgM mRNA expression was studied by using real-time PCR. OBJECTIVES: To investigat the effects of Bursopentin (Cys-Lys-Arg-Val-Tyr, BP5) on a chicken promyelocyte cell line DT40, assays of cell proliferation, cell cycle distribution, detection of surface immunoglobulin G (sIgM) mRNA expression and gene microarray analysis were performed. RESULTS: The results showed that BP5 displayed concentration-dependent effects on the proliferation, cell cycle, and sIgM mRNA expression in DT40 cells. And the analysis of expression profiles identified a signature set of 3022 genes (1254 up regulated genes, 1762 down regulated genes), which clearly discriminated the BP5-treated DT40 cells from control with high certainty (P≤0.02). The results of microarray analysis were confirmed by quantitative reverse transcription-polymerase chain reaction for 12 of the differentially expressed genes. CONCLUSION: Theses findings showed the immuno-activity effect of BP5 on B lymphocyte and indicated that BP5 treatment regulated eight signaling pathways, in which Toll-like signaling pathway was the most significant enrichment pathway.

[Synthesis of hybrid antimicrobial peptide CecA-mag gene and it's secretion expression in Pichia pastoris].

According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mag gene was synthesized and cloned into pPICZa-A to construct the recombinant expression vector pPICZa-A-CA. The SacI-linearized plasmid pPICZalpha-A-CA was transformed into P. pastoris SMD1168 by electroporation. Under the control of the promoter AOX'(alcoholoxidase') , an approximately 1.9kDa cecA-mag protein was expressed. Antibacterial assays demonstrated that cecA-mag had broad spectrum of antimicrobial property against Gram-positive as well as Gram-negative bacteria especially showed potent antibacterial activity against ampicillin resistant bacteria, such as pathogenic E. coli. In addition, the hybrid antibacterial peptide showed an extreme heat stable and acid stable characteristic. These results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional cecA-mag for both research and industrial purpose. Based on these characteristics, the recombinant antibacterial peptide cecA-mag displays application foreground in the field of prevention of disease, and can be used as additives of animal feedstuff and so on.