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.     

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, H₉N₂ 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.     

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.     

Gene expression profiling of hybridoma cells after bursal-derived bioactive factor BP5 treatment

Bursa of Fabricius is the acknowledged vital humoral immune system for B cell differentiation and antibody production. To study the molecular mechanism underlying the effect of bursal-derived BP5, we used gene microarray to analyze the genomic expression profiling of BP5-treated hybridoma cells. BP5 exhibited an immunomodulatory effect on antibody production in hybridoma cells and induced alterations in the gene expression profiles related to the immune-related biological processes, such as T cell activation and proliferation, B cell activation, B cell-mediated immunity, and cytokines cytokine production involved in immune response. In addition, 26 biological pathways associated with immunomodulatory functions were regulated in BP5-treated hybridoma cells, in which p53 signal pathway played an important role in antitumor. Among these regulated genes, 12 differentially expressed genes were verified by qRT-PCR. The activation of p53 activity by BP5 was further confirmed by p53 luciferase reporter assay and p53 expression. Our data revealed that bursal-derived BP5 could regulate various immune-related cellular processes, including antitumor factor p53 signal pathway, perhaps partially accounting for the reported immunomodulatory roles and novel antiproliferation on tumor cells functions of bursal-derived bioactive factor BP5.     

Isolation, modulatory functions on murine B cell development and antigen-specific immune responses of BP11, a novel peptide from the chicken bursa of Fabricius

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds which plays important roles in B lymphocyte differentiation. Here, a new bursal peptide (BP11) with the amino acid sequence DVAGKLPDNRT was identified and characterized from BF. It was proved that BP11 promoted CFU pre-B formation, and regulated B cell differentiation, including increase the percentage of immature and mature B cells in BM cells co-cultured with IL-7. BP11 also exerted immunomodulatory function on antigen-specific immune responses in BALB/c mice immunized with inactivated influence virus (AIV, H9N2 subtype) vaccine, including enhancing AIV-specific antibody and cytokine production. Furthermore, it was noteworthy that BP11 stimulated antibody productions and potentiates the Th1 and Th2-type immune responses in dose-dependent manner in chicken. These results suggested that BP11 might be highly relevant for the development of avian immune system.     

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.     

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.     

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.     

Dysfunction of simian immunodeficiency virus/simian human immunodeficiency virus-induced IL-2 expression by central memory CD4+ T lymphocytes

Production of IL-2 and IFN-gamma by CD4+ T lymphocytes is important for the maintenance of a functional immune system in infected individuals. In the present study, we assessed the cytokine production profiles of functionally distinct subsets of CD4+ T lymphocytes in rhesus monkeys infected with pathogenic or attenuated SIV/simian human immunodeficiency virus (SHIV) isolates, and these responses were compared with those in vaccinated monkeys that were protected from immunodeficiency following pathogenic SHIV challenge. We observed that preserved central memory CD4+ T lymphocyte production of SIV/SHIV-induced IL-2 was associated with disease protection following primate lentivirus infection. Persisting clinical protection in vaccinated and challenged monkeys is thus correlated with a preserved capacity of the peripheral blood central memory CD4+ T cells to express this important immunomodulatory cytokine.     

Apoptosis and other immune biomarkers predict influenza vaccine responsiveness

Despite the importance of the immune system in many diseases, there are currently no objective benchmarks of immunological health. In an effort to identifying such markers, we used influenza vaccination in 30 young (20–30 years) and 59 older subjects (60 to >89 years) as models for strong and weak immune responses, respectively, and assayed their serological responses to influenza strains as well as a wide variety of other parameters, including gene expression, antibodies to hemagglutinin peptides, serum cytokines, cell subset phenotypes and in vitro cytokine stimulation. Using machine learning, we identified nine variables that predict the antibody response with 84% accuracy. Two of these variables are involved in apoptosis, which positively associated with the response to vaccination and was confirmed to be a contributor to vaccine responsiveness in mice. The identification of these biomarkers provides new insights into what immune features may be most important for immune health.     

Absence of in vitro innate immunomodulation by insect‐derived short proline‐rich antimicrobial peptides points to direct antibacterial action in vivo

Some antimicrobial peptides (AMPs) have been described to exert immunomodulatory effects, which may contribute to their in vivo antibacterial activity. Very recently, we could show that novel oncocin and apidaecin derivatives are potently antibacterially active in vivo. Therefore, we studied oncocin and apidaecin derivatives for their effects on murine dendritic cells (DC) and macrophages and compared them with well‐known immunomodulatory activities of murine cathelicidin‐related antimicrobial peptide (CRAMP). To characterize the immunomodulatory activity of the peptides on key cells of the innate immune system, we stimulated murine DC and macrophages with the oncocin and apidaecin derivatives alone, or in combination with lipopolysaccharide (LPS). We analyzed the secretion of pro‐inflammatory cytokines, the expression of surface activation markers, and the chemotactic activity of the AMPs. In contrast to LPS, none of the oncocin and apidaecin derivatives alone has an influence on cytokine or surface marker expression by DC and macrophages. Furthermore, the tested oncocin and apidaecin derivatives do not modulate the immune response after LPS stimulation, whereas CRAMP shows a reduction of the LPS‐mediated immune response as expected. All peptides tested are not chemotactic for DC. Together, lack of in vitro immunomodulatory effects by oncocin and apidaecin derivatives on key cells of the innate murine immune system suggests that their potent in vivo antibacterial activity relies on a direct antibacterial effect. This will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of tumor necrosis factor mRNA translation by a rationally designed immunomodulatory peptide

Based on sequences of immunomodulatory peptides derived from the heavy chain of HLA Class I, novel immunomodulatory peptides with increased potency were developed by computer-aided rational design. Allotrap 1258 was characterized in detail and shown to inhibit cell-mediated immune responses in vitro and in vivo. Immunomodulatory activity was associated with the capability of the peptides to modulate heme oxygenase (HO) activity. In this study we analyzed the effect of Allotrap 1258 on cytokine expression. Allotrap 1258 inhibited concanavalin A- and lipopolysaccharide-induced human and mouse tumor necrosis factor (TNF) production in vitro and in vivo but had no effect on interleukin (IL)-1, IL-2, IL-4, IL-6, or IL-10 expression. Experiments with HO-1/KO and iNOS/KO mice showed that Allotrap 1258-mediated inhibition of TNF was independent of HO-1 and iNOS. Quantitation of TNF protein expression and mRNA steady state levels demonstrated that Allotrap 1258-mediated inhibition occurred at the translational level. Deletion of the AU-rich element in the 3'-untranslated region (UTR) of TNF mRNA, a region known to be involved in TNF mRNA translation, had minimal effect on Allotrap 1258-mediated inhibition. However, replacement of the TNF 3'-UTR with the human globin 3'-UTR rendered the peptide inactive. This demonstrates that besides AU-rich elements, other sequences in the 3'-UTR of TNF mRNA are involved in translational control of TNF expression. Such sequences are necessary for Allotrap 1258-mediated inhibition of TNF production.     

High‐sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients’ sera

The characterization of peptides bound to human leukocyte antigen (HLA) class I is of fundamental importance for understanding CD8+ T cell‐driven immunological processes and for the development of immunomodulatory therapeutic strategies. However, until now, the mass spectrometric analysis of HLA‐bound peptides has typically required billions of cells, still resulting in relatively few high‐confidence peptide identifications. Capitalizing on the recent developments in mass spectrometry and bioinformatics, we have implemented a methodology for the efficient recovery of acid‐eluted HLA peptides after purification with the pan‐reactive antibody W6/32 and have identified a total of 27 862 unique peptides with high confidence (1% false discovery rate) from five human cancer cell lines. More than 93% of the identified peptides were eight to 11 amino acids in length and contained signatures that were in excellent agreement with published HLA binding motifs. Furthermore, by purifying soluble HLA class I complexes (sHLA) from sera of melanoma patients, up to 972 high‐confidence peptides could be identified, including melanoma‐associated antigens already described in the literature. Knowledge of the HLA class I peptidome should facilitate multiplex tetramer technology‐based characterization of T cells, and allow the development of patient selection, stratification and immunomodulatory therapeutic strategies.     

Peptides as regulators of the immune system: emphasis on somatostatin

Study of the communication between nervous and immune systems culminated in the understanding that cytokines, formerly considered exclusively as immune system-derived peptides, are endogenous to the brain and display central actions. More recently, immune cells have been recognized as a peripheral source of “brain-specific” peptides with immunomodulatory actions. This article reviews studies concerning reciprocal effects of selected cytokines and neuropeptides in the nervous and immune systems, respectively. The functional equivalence of these two categories of communicators is discussed with reference to the example of the actions of neuropeptide somatostatin in the immune system.     

Tick modulation of host immunity: an important factor in pathogen transmission.

Immunological interactions at the tick host interface involve innate and specific acquired host immune defenses and immunomodulatory countermeasures by the tick. Tick feeding stimulates host immune response pathways involving antigen-presenting cells, cytokines, B-cells, T-cells, circulating and homocytotropic antibodies, granulocytes, and an array of biologically active molecules. In response to host immune defenses, tick-mediated host immunosuppressive countermeasures inhibit: host antibody responses; complement activation; T-cell proliferation; and cytokine elaboration by macrophages and Th1-lymphocytes. Immunosuppressive proteins identified in tick salivary glands and saliva have been partially characterised. Tick-induced host immunosuppression facilitates blood meal acquisition and is an important factor in the transmission/establishment of the tick-borne disease-causing agent, Borrelia burgdorferi. A novel strategy for control of tick-borne pathogens is proposed.     

A possible role of autogenous IFN-beta for cytokine productions in human fibroblasts

It has been already known that human diploid fibroblasts are able to produce not only high levels of IFN-beta but also various kinds of cytokines by poly rI: poly rC, and some inflammatory cytokines are induced by IFN-beta gene activation. We also obtained similar results. However, in our system, cytokine productions were extremely enhanced by treating the cells with a low dose of type 1 IFN and the priming effects on cytokine productions were blocked by cycloheximide similar to those on IFN-beta productions. Most of cytokines were produced later than IFN-beta and synthesis patterns of their mRNA showed the same phenomena. We made clear that cytokine productions by poly rI: poly rC are mediated by secreted IFN-beta at a protein level using a monoclonal antibody against human IFN-beta. Further, it was shown that intra-cellular IFN-beta which is not secreted might also participate in cytokine productions. Meanwhile, IL-1beta induced various kinds of cytokines in human fibroblasts and production time courses of these cytokines were similar to those of poly rI: poly rC induced cytokines. Although secreted IFN-beta was not detected in IL-1beta stimulated culture, expression of IFN-beta mRNA was augmented. These results showed that priming effects of type 1 IFN on cytokine productions by poly rI: poly rC might not be the direct action, but successive IFN-beta production might be essential in the production processes of other cytokines. Further, it was suggested that inducible IFN-beta might also take part in IL-1beta-induced cytokine productions.     

Differential cytoplasmic and whole cellular expression of histone H1 within the avian bursa of Fabricius

Bursal anti-steroidogenic peptide (BASP), purified from the chicken bursa of Fabricius (BF), has been previously demonstrated to be a potent and efficacious inhibitor of steroid hormone biosynthesis from chicken ovarian, and both mammalian and avian adrenal cells in vitro. Other studies have demonstrated that BASP can markedly reduce avian and mammalian mitogen-stimulated lymphocyte proliferation. Recent studies have indicated that BASP has a structural and functional relationship with histone H1. Immunohistochemical studies using a monoclonal antibody, which is known to recognize a common histone H1 epitope from several plant and animal species identified the protein within the cytoplasm and nucleus of distinct cells within both the cortex and medulla of all BF follicles. Additionally, epithelial cells within the BF expressed the protein strongly in the cytoplasm with reduced nuclear staining. In contrast, the same antibody did not recognize the protein in thymus of the same animals. The differential expression of histone H1 immunoreactivity within selected cells of the BF may support a previous proposed role of histone H1 in extranuclear and extracellular signaling in chickens and possibly other species.     

Isolation: Analysis and Properties of Three Bradykinin–Potentiating Peptides (BPP-II, BPP-III, and BPP-V) From Bothrops Neuwiedi Venom

In the course of systematic investigations on low-molecular-weight compounds from the venom of Crotalidae and Viperidae, we have isolated and characterized at least three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom by gel filtration on Sephadex G-25 M, Sephadex G-10 followed by HPLC. The peptides showed bradykinin-potentiating action on isolated guinea-pig ileum, for which the BPP-V was more active than of BPP-II, and BPP-III, rat arterial blood pressure, and a relevant angiotensin-converting enzyme (ACE) competitive inhibiting activity. The kinetic studies showed a K _i of the order of 9.7 × 10^–3 M to BPP-II, 7 × 10^–3 M to BPP-III, and 3.3 ×10^–3 M to BPP-V. The amino acid sequence of the BPP-III has been determined to be pGlu-Gly-Gly-Trp-Pro-Arg-Pro-Gly-Pro-Glu-Ile-Pro-Pro, and the amino acid compositions of the BPP-II and BPP-V by amino acid analysis were 2Glu-2Gly-1Arg-4Pro-lIle and 2Glu-2Gly-lSer-3Pro-2Val-Ille, with molecular weight of 1372, 1046, and 1078, respectively.     

MHC motif viewer

In vertebrates, the major histocompatibility complex (MHC) presents peptides to the immune system. In humans, MHCs are called human leukocyte antigens (HLAs), and some of the loci encoding them are the most polymorphic in the human genome. Different MHC molecules present different subsets of peptides, and knowledge of their binding specificities is important for understanding the differences in the immune response between individuals. Knowledge of motifs may be used to identify epitopes, to understand the MHC restriction of epitopes, and to compare the specificities of different MHC molecules. Algorithms that predict which peptides MHC molecules bind have recently been developed and cover many different alleles, but the utility of these algorithms is hampered by the lack of tools for browsing and comparing the specificity of these molecules. We have, therefore, developed a web server, MHC motif viewer, that allows the display of the likely binding motif for all human class I proteins of the loci HLA A, B, C, and E and for MHC class I molecules from chimpanzee (Pan troglodytes), rhesus monkey (Macaca mulatta), and mouse (Mus musculus). Furthermore, it covers all HLA-DR protein sequences. A special viewing feature, MHC fight, allows for display of the specificity of two different MHC molecules side by side. We show how the web server can be used to discover and display surprising similarities as well as differences between MHC molecules within and between different species. The MHC motif viewer is available at .