Comparative Safety, Immunogenicity, and Efficacy of CEF Cell-Based and DF-1 Cell Line Adapted Infectious Bursal Disease Vaccines in Specific-Pathogen-Free Chickens.

Infectious bursal disease (IBD) is an immunosuppressive and economically important disease of young chickens caused by infectious bursal disease virus (IBDV). The National Veterinary Institute (Bishoftu, Ethiopia) produces intermediate IBDV vaccine using primary chicken embryo fibroblast (CEF) cells, a method with technical and economical cumbersome. This study assessed the safety, immunogenicity, and efficacy of DF-1 cell line-adapted IBDV LC-75 vaccine strain in reference to the CEF-based vaccine. Confluent monolayer of DF-1 cells was infected with IBDV and cells with cytopathic effects were passaged until 3rd passage. Viral growth was confirmed using a one-step RT-PCR targeting IBDV VP2 gene. Viral titer increased from 1st passage through 3rd passage. Safety was assessed in 30 specific-pathogen-free chickens (15 chickens/group) injected with 10-fold field dose of each vaccine intraocularly and monitored for 21 days. For immunogenicity and efficacy, 60 specific-pathogen-free chickens were grouped into 3 (20 chickens/group). First and 2nd group received DF-1 cell and CEF-based IBDV vaccines, respectively. The 3rd group served as unvaccinated control. Antibody response was measured using iELISA. Chickens were challenged 4 weeks postvaccination with very virulent IBDV (vvIBDV) intraocularly and followed-up for 10 days. Vaccination did not cause any adverse reactions during the 21 days of follow-up. In addition, both vaccines induced higher antibody titer 14 and 24 days-post-vaccination as compared to unvaccinated controls (p < 0.05). Moreover, DF-1 and CEF-based IBDV LC-75 vaccines rendered a complete protection against vvIBDV. Contrarily, morbidity and mortality in unvaccinated chickens was 50% and 30%, respectively. The results indicated that DF-1 and CEF cell-based IBDV vaccines are comparably immunogenic and efficacious. Therefore, DF-1 cell-line can be considered an affordable and convenient alternative to the CEF-based approach. The suitability of DF-1 cells to grow other IBDV strains and safety of these vaccines on bursa of Fabricius should further be investigated.

The Roles of Bursal Nonapeptide (BP9) on AIV Vaccine Immune Response in Chick Immunization and on Avian Immature B Cell.

BACKGROUND: Bursa of Fabricius plays the vital functions on B cell development and antibody production in poultry. The bursal-derived peptide plays the essential roles on avian immature B cell development. OBJECTIVES: Here we explored the functions of the recently reported bursal nonapeptide (BP9) on the antibody production and the molecular basis of BP9 on avian immature B cell. METHODS: Chicken were twice immunized with Avian Influenza Virus (AIV) inactivated vaccine plus with BP9 at three dosages, respectively. On two weeks after the second immunization, sera samples were collected from all experimental groups to measure AIV-specific Agglutination Inhibition (HI) antibody titers. Also, on 7th day after the second immunization, spleen lymphocytes were isolated from the immunized chicken to detect the lymphocyte viabilities. DT40 cells were treated with BP9 from 0.02 to 2 µg/mL for 4 and 20h to detect sIgM mRNA levels, and total RNAs from BP9-treated DT40 cells were collected to investigate the gene expression profiles of DT40 cells, and to analyze the enriched pathways and functional biological processes. Finally, nine gene expressions were validated with quantitative PCR (qPCR). RESULTS: Our investigation proved the strong regulatory roles of BP9 on AIV-specific HI antibody titers and lymphocyte viabilities. BP9 promoted sIgM mRNA levels in DT40 cells, and upregulated 598 gene expressions and downregulated 395 gene expressions in DT40 cells with 0.2µg/mL BP9 treatment. Moreover, our findings verified the significantly enriched six pathways and various the biological functional processes of BP9 on avian immature B cell. Also, we found eight signaling pathways in the enriched biological processes of BP9-treated DT40 cells, and the expressions of nine selected genes with qPCR were identical to that of microarray data. CONCLUSION: BP9 promoted the antibody production in the 21-old-day chicken immunization, and stimulated the sIgM expression in DT40 cells. Furthermore, we analyzed the gene expression profile and immune-related biological processes of DT40 cells treated with BP9, which provided some new insights into the mechanism on immature B cell development, and provided important references for adjuvant development on vaccine improvement and clinical application.

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.

Bursal Hexapeptide, A Potential Immunomodulator, Inhibits Tumor Cells Proliferation via p53 Signaling Pathway.

BACKGROUND: The Bursa of Fabricius (BF) is acknowledged as the central humoral immune organ unique to birds. Bursal Hexapeptide (BHP, AGCCNG) is a recently reported bursal-derived bioactive peptide. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of BHP. METHOD: In this paper, Gene microarray analyses demonstrated that BHP regulated expression of 1347 genes, of which 832 were up-regulated and 515 were down-regulated. Differentially expressed genes involved in various pathways were identified, of which 16 pathways were associated with immune responses and tumorigenic processes. RESULT: Specifically, we found that BHP selectively inhibited tumor cell proliferation. Furthermore, BHP enhanced antitumor factor p53 luciferase activity and stimulated expression of p53, p21, and p130 protein. Moreover, we observed that the inhibitory effect of BHP on cell proliferation and premature senescence in a p53-dependent manner. CONCLUSION: Taken together, we uncovered that BHP may be involved in antitumor suppressor via p53 signaling pathway.

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.

Dietary L-arginine modulates immunosuppression in broilers inoculated with an intermediate strain of infectious bursa disease virus.

BACKGROUND: The effects of dietary l-arginine (Arg) on immunosuppression following infectious bursal disease virus (IBDV) inoculation in broiler chickens were evaluated. The design of this study was a 5 × 2 factorial arrangement (n = 5) with five Arg concentrations (starter: 9.9, 13.9, 17.6, 21.3 and 25.3 g kg(-1) ; grower-finisher: 9.5, 13.5, 17.1, 20.1 and 23.6 g kg(-1) ) with or without IBDV inoculation (IBDV or saline inoculation at 14 days). Chickens were sampled at 2, 4 and 6 days post-inoculation (DPI) and 42 days of age. RESULTS: The IBDV inoculation decreased (P = 0.05) CD3(+) , CD4(+) , and CD8(+) T cell counts at 2 days post-inoculation (DPI) and monocyte counts at 6 DPI; and reduced (P < 0.05) bursal interleukin-1ß (IL-1ß) mRNA expression at 2 DPI and serum IL-6 concentration at 4 DPI. Increasing Arg concentration increased (P < 0.05) CD4(+) and CD8(+) T cell counts at 2 DPI, linearly increased (P = 0.05) CD3(+) T cell counts in IBDV-inoculated groups and monocyte counts in control groups at 4 DPI; increased (P < 0.05) serum IL-6 concentration in IBDV-inoculated groups at 2 DPI; and increased (P < 0.05) serum anti-IBDV antibody titres at 42 days of age. CONCLUSION: Varying concentrations of Arg supplementation attenuated IBDV inoculation induced immunosuppression via modulating circulating T cell sub-populations.

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.

Bursopentin (BP5) from chicken bursa of fabricius attenuates the immune function of dendritic cells.

Bursopentine (BP5), a novel pentapeptide isolated from chicken bursa of fabricius, has been proved to have immunomodulatory effects on B and T lymphocytes, anti-oxidative stress on macrophages, and antiproliferation on tumor cells. However, the effects of BP5 on the immune function exhibited by dendritic cells (DCs), which are regarded as a major target for immunomodulators, remain unknown. In this study, we examined the effects of BP5 on the activation and maturation of murine bone marrow-derived DCs. Our results showed that BP5 significantly suppressed the secretion of lipopolysaccharide (LPS)-induced pro-inflammatory (TNF-α, IL-1ß, IL-6 and IL-12p70) and anti-inflammatory (IL-10) cytokines by DCs, and this impact was not due to its cytotoxicity. Besides, BP5 reversed the morphological changes and attenuated the expression of phenotypic markers (MHC II, CD40, CD80 and CD86 molecules) in LPS-induced DCs. Furthermore, BP5 restored the decreased FITC-dextran uptake in LPS-treated DCs, arrested the LPS-induced migration of DCs and abrogated the promoting ability of LPS-induced DCs for allogeneic T cell proliferation. These findings show a new immunopharmacological capability of BP5 and provide a novel approach in the prevention and therapy of chronic inflammation and autoimmunity via abolishing the immune function of DCs.

Melatonin receptor subtypes Mel1a and Mel1c but not Mel1b are associated with monochromatic light-induced B-lymphocyte proliferation in broilers.

This study determined the effects of melatonin (MEL) and its receptors on monochromatic light-induced bursal B-lymphocyte proliferation in broiler chickens. In vivo, green light (GL) enhanced the proliferation of B lymphocytes in bursas by 16.49% to 30.83% and the expression of MEL receptor subtypes 1a (Mel1a), Mel1b, and Mel1c receptors in bursas by 6.91% to 366.98% than other light colors. However, pinealectomy reduced these parameters and eliminated the differences between GL and other light groups. In vitro, the MEL-induced bursal B-lymphocyte proliferation was most suppressed by prazosin (P = 0.001, selective Mel1c antagonist), followed by luzindole (P = 0.022, nonselective Mel1a/Mel1b antagonist), but not by 4-phenyl-2-propionamideotetralin (P = 0.144, selective Mel1b antagonist). Similarly, dibutyryl-cyclic adenosine monophosphate (cAMP; analog of cAMP; P = 0.017) but not 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (P = 0.736; activator of exchange protein directly activated by cAMP) significantly inhibited bursal B-lymphocyte proliferation. These results suggest that MEL mediates GL-induced bursal B-lymphocyte proliferation through Mel1c and Mel1a receptors but not Mel1b receptors by activating the cAMP/protein kinase A pathway.

Immunomodulatory roles and functional analysis of pre-B lymphocyte DT40 cells with the bursal-derived BSP-II treatment.

The bursa of Fabricius, the acknowledged central humoral immune organ, is vital to B cell differentiation. However, the regulatory function of the bursal-derived peptide on avian B cell proliferation has not been reported. BSP-II is a recently reported bursal-derived bioactive peptide. In this paper, 75 days-old chicks were twice subcutaneously immunized with BSP-II and inactivated avian influenza virus (AIV, H(9)N(2) strain). It was proved that BSP-II induced a strongly AIV-specific HI antibody production in the immunized chicks. Also, BSP-II could enhance avian pre-B lymphocyte DT40 cell viability. To investigate the global patterns of gene expression in DT40 cells after BSP-II treatment, gene microarray was carried out. It was identified that the differentially expressed genes were involved in various pathways, of which six pathways were associated with signaling transductions, including ErbB signaling, MAPK signaling, Toll-like receptor signaling, Notch signaling, mTOR signaling, and Wnt signaling. Finally, RT-qPCR was used to confirm the microarray expression data. These results indicated the molecular basis of pre-B lymphocyte viability with BSP-II treatment, which provided a potential mechanism of the bursa of Fabricius on pre-B lymphocyte viability, differentiation, and development. These results are valid for the mechanism of the bursa of Fabricius on B lymphocytes development.

Characterization and immunomodulatory function comparison of various bursal-derived peptides isolated from the humoral central immune organ.

The bursa of Fabricius (BF) is the acknowledged central immune organ, which is important to the B cell differentiation and antibody production. However, due to difficult purification, the immunomodulatory peptides from BF were little reported. In this study, the extract samples of BF were taken to a chromatographic analysis by RP-HPLC. Five novel low molecular weight peptides were isolated from BF, with amino acid sequences of YEYAY, RMYEE, GPPAT, AGCCNG, and RRL, and named as Bursal pentapeptide (BPP)-III, -IV, -V, and Bursal hexapeptide (BHP), and Bursal tripeptide (BTP), respectively. BSP-I, BSP-II, BPP-I and BPP-II are recently reported to be the bursal-derived bioactive peptides. In this paper, we analyzed the chemical formula and characteristics of these nine bursal-derived peptides. The immunization comparative experiment verified the different immunomodulatory activity of these nine bursal peptides on antibody and cytokine productions. Furthermore, the results showed that at reachable concentrations, BPP-II and BPP-I induced antibody productions, lymphocyte viabilities and cytokine responses in different dose-dependent manner in the immunized mice model, respectively. These results provided important orientations for the comprehensively understanding and study of the humoral central immune system of human, and provided a novel insight on the treatment of serious disease and immune improvement of human.

Interleukin 4 increases CCR9 expression and homing of lymphocytes to gut-associated lymphoid tissue in chickens.

The effects of in vitro and in vivo IL-4 supplementation on thymocyte and splenocyte CCR9 mRNA amount and migration were studied. Thymocytes, splenocytes, splenocytes+thymocytes (2:1), and splenocytes+bursocyte cells (2:1) were supplemented with either 0 or 5 ng/ml IL-4 for 5d. CCR9 mRNA was undetectable in all experimental groups supplemented with 0 ng/ml IL-4. IL-4 treatment (5 ng/ml) upregulated (P=0.01) CCR9 mRNA only in the splenocyte+thymocyte cell culture. IL-4-mediated CCR9 mRNA induction in the splenocyte+thymocyte cell culture was dependent on the in vitro dose of IL-4 supplementation. IL-4-treated splenocyte+thymocyte cells when injected in vivo preferentially migrated to cecal tonsils. In vivo supplementation of IL-4 was achieved through in ovo injection of recombinant chicken IL-4 plasmid. Cecal tonsils in chicks hatched from IL-4-plasmid-injected eggs weighed more, had a higher amount of CCR9 mRNA, and had a higher percentage of CD8(+) cells than cecal tonsils from chicks hatched from PBS-injected eggs. It could be concluded that IL-4 induces CCR9 mRNA in thymocytes and splenocytes and directs the migration of cells to gut-associated lymphoid tissue.

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.

A bursal pentapeptide (BPP-I), a novel bursal-derived peptide, exhibits antiproliferation of tumor cell and immunomodulator activity.

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds. Here, we isolated a novel bursal pentapeptide I (BPP-I), LGPGP, from BF. BPP-I could play inhibition effect on MCF-7 but not on CEF or Vero cell proliferation in vitro, and enhance antitumor factor p53 protein expression. Also, BPP-I stimulated antibody production in a dose-dependent manner in hybridoma cell. Furthermore, BPP-I could induce various immune responses in mice immunization experiments, including increase antibody production and cytokines IL-4 and IFN-γ level, and induce T-cell immunophenotyping. These results suggest that BPP-I is a potential immunomodulator of antitumor and immunity. The study could provide some novel insights on the probable candidate reagent for the antitumor and immune improvement.

Bursopentine as a novel immunoadjuvant enhances both humoral and cell-mediated immune responses to inactivated H9N2 Avian Influenza virus in chickens.

There is an urgent need for identification of a new adjuvant capable of selectively promoting an efficient immune response for use with vaccines and especially subunit vaccines. Our pervious study showed that Bursopentine (BP5) is a novel immunomodulatory peptide and has the ability to significantly stimulate an antigen-specific immune response in mice. In this study, the potential adjuvant activities of BP5 were examined in chickens by coinjection of BP5 and an inactivated avian influenza virus (AIV) (A/Duck/Jiangsu/NJ08/05 [AIV H9N2 subtype]). The results suggested that BP5 markedly elevated serum hemagglutination inhibition (HI) titers and antigen-specific antihemagglutinin (anti-HA) antibody (IgG) levels, induced both Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ])- and Th2 (IL-4)-type cytokines, promoted the proliferation of peripheral blood lymphocytes, and increased populations of CD3(+) T cells and their subsets CD4(+) (CD3(+) CD4(+)) T cells and CD8(+) (CD3(+) CD8(+)) T cells. Furthermore, a virus challenge experiment revealed that BP5 contributes to protection against homologous avian influenza virus challenge by reducing viral replication in chicken lungs. This study indicates that the combination of inactivated AIVs and BP5 gives a strong immune response at both the humoral and cellular levels and implies that BP5 is a novel immunoadjuvant suitable for vaccine design.

Isolation, antiproliferation on tumor cell and immunomodulatory activity of BSP-I, a novel bursal peptide from chicken humoral immune system.

The bursa of Fabricius (BF) is acknowledged as central humoral immune organ unique to birds. Our purpose was to identify the potential function of a novel bursal-derived bioactive peptide. A bursal septpeptide (BSP-I), EPASGMM, first isolated from BF, reduced MCF and Hela tumor cells proliferation, and enhanced antitumor factor p53 luciferase activity and protein expression. Further, we found the significantly immune inducing function of BSP-I on antigen-specific immune response in BALB/c mice intraperitoneally immunized with inactivated avian influence virus (AIV, H(9)N(2) subtype) vaccine, including of enhancing the antibody (IgG, the isotypes IgG1 and IgG2a) production, and stimulating cytokines IL-4 and IFN-γ level, and inducing T cell immunophenotyping and lymphocyte proliferation. These results suggested that as the bioactive peptide from avian humoral immune system, various biological function of BSP-I may have far-reaching implication on immune system significance, which might provide novel insight on linking between humoral immune system and development of effective immunotherapeutic strategies for treating human cancers diseases.

Immunomodulatory activities of a new pentapeptide (Bursopentin) from the chicken bursa of Fabricius.

The bursa of Fabricius (BF) is a central immune organ in birds, and some peptides from chicken BF have demonstrated important immune functions. Here, a new 626.27 Da pentapeptide, Bursopentin (BP5, Cys-Lys-Arg-Val-Tyr) was isolated and purified by reverse-phase high-performance liquid chromatography. In this study, we examined the effects of BP5 on antigen-specific immune response in BALB/c mice sensitized with inactivated avian influenza virus (AIV) [A/Duck/Jiangsu/NJ08/05 (AIV H9N2 subtype)]. The results suggested that BP5 enhanced anti-hemagglutinin antibody (IgG, the isotypes IgG1 and IgG2a) production, induced both of Th1- (IL-2 and IFN-γ) and Th2-type (IL-4 and -10) cytokines, increased proliferations of splenic lymphocyte subsets CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+) and B cells, and enhanced cytotoxic T-lymphocyte activity of the activated splenocytes against NIH3T3 cells. The effects of BP5 on the proliferation of isolated T- and/or B-cell populations of BALB/c mice were assessed, and the data suggested that BP5 promoted spleen lymphocyte proliferation by activating B cells directly and T cells indirectly. Further analysis revealed that B-lymphocyte proliferation induced by BP5 is mediated by reactive oxygen species generated from thiol auto-oxidation of BP5. Furthermore, our data indicated that protein kinase C, mitogen-activated protein kinase, and nuclear factor kappa B are involved in the signal transductions during the BP5-induced B lymphocyte proliferation. This study indicates that BP5 could be a potential immunomodulator for future immuno-pharmacological use.

Isolation and potential immunological characterization of TPSGLVY, a novel bursal septpeptide isolated from the bursa of Fabricius.

The bursa of Fabricius is central immune organ unique to birds, and the extract is immunocompetent in stimulating B cell differentiation and enhancing antibody production. However, except for bursin, the active peptides from the bursa of Fabricius are little reported. In the paper, a novel bursal septpeptide (BSP-II) with the amino acids sequence of TPSGLVY was identified and similar to the MGC53864 protein of Gallus gallus. We investigated the effects of BSP-II on the immune response in terms of the antibodies titers (IgG1 and IgG2alpha), the levels of interferon-gamma and interleukin-4 cytokines, spleen cell lymphocyte proliferation, and the T-lymphocyte subtype composition. It was noteworthy that BSP-II potentiates the Th1 and Th2-type immune responses in dose-dependent manner. BSP-II had specific enhancing effects on the hybridoma SP2/0 cell proliferation at two different serum concentrations (20% and 5%), but had no connection with the dose of BSP-II. The antibody secreting level of hybridoma SP2/0 cells rose in 5% and 20% serum when the concentrations of BSP-II increased. Also, BSP-II had effect on the viabilities of tumor cells (Hela and SP2/0). All the results indicated that BSP-II was able to significantly induce various immune responses and involved in the cell viability of different tumor cell lines. Our observations implied that BSP-II might be a novel biological active factor from the bursa of Fabricius with immunomodulatory activities.

Expression of pituitary adenylate cyclase-activating polypeptide in the primary lymphoid organs of the duck Anas platyrhynchos.

The expression of pituitary adenylate cyclase-activating polypeptide (PACAP) was studied in the thymus and bursa of Fabricius of the duck Anas platyrhynchos, at different ages, using immunohistochemistry, Western blotting, RT-PCR and sequencing. In the thymus, PACAP immunoreactivity (-ir) was found in lymphoid cells. CD68/ and PGP 9.5/PACAP38 double labelling showed that PACAP was not expressed either in macrophages or in epithelial cells, suggesting that the PACAP-positive cells observed were lymphoid cells. Immunoreactive lymphocytes were observed in the interlobular septa. They increased in number with ageing. In the bursa, PACAP-ir was found in nerve fibres and in a few lymphoid cells. RT-PCR revealed PACAP mRNA expression in the thymus but not in the bursa. These results suggest that PACAP plays a role in the functions of the immune system in birds.