Humanization of an agonistic anti-death receptor 4 single chain variable fragment antibody and avidity-mediated enhancement of its cell death-inducing activity.

Development of agonistic monoclonal antibodies (mAbs) against the pro-apoptotic molecule death receptor 4 (DR4) [or tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor 1] is an attractive anti-cancer strategy because of their potential for inducing tumor-specific cell death. In this study, we humanized an agonistic anti-DR4 AY4 scFv raised in mice (mAY4) by grafting the complementarity-determining regions (CDRs) onto a fixed human framework, while preserving the so-called Vernier zone residues, a group of framework (FR) residues directly underneath the CDRs, with the murine residues in the humanized antibody, hAY4. The humanized hAY4 scFv maintained the antigen binding affinity and epitope specificity of mAY4. To investigate how the valence of hAY4 scFv affects DR4-mediated cell death, bivalent and trivalent forms of hAY4 scFv were generated by linking a hinge region to the coiled-coil domain of a dimerizing leucine zipper and trimerizing isoleucine zipper, respectively. Compared to the monovalent and bivalent forms, the trivalent hAY4 scFv induced more potent caspase-dependent apoptotic cell death as evidenced by increased activation of caspase-8 and downstream pro-apoptotic molecules. Our results suggest that like other TNF family receptors, avidity-mediated oligomerization of DR4 augments the receptor-mediated apoptotic cell death by promoting intracellular cell death signaling.

Cloning and functional characterization of chicken interleukin-17D.

The chicken interleukin-17D was cloned from a testis cDNA library prepared from the Korean native chicken. The full-length chicken IL-17D (chIL-17D) cDNA consisted of a 348 nucleotide sequence encoding an open reading frame of 116 amino acids with a predicted molecular mass of 13.3kDa. Comparison of the deduced amino acid sequence of chIL-17D with homologous proteins from human, mouse and opossum revealed 64%, 53% and 76% identity, respectively, including six conserved cysteine residues present in the mammalian polypeptides. The chIL-17D gene transcript was expressed in a wide range of tissues, and highest levels were in pancreas, thymus and lung. Following Eimeria maxima infection, levels of the chIL-17D mRNA were up-regulated in the intestinal jejunum, bursa, lung, and spleen but decreased in the thymus. Infected chickens also expressed greater levels of chIL-17D mRNA in CD4(+), CD8(+) and TCR1(+) intestinal intraepithelial lymphocytes while decreased expression was seen in TCR2(+) cells. Treatment of CHCC-OU2 fibroblasts with chIL-17D recombinant protein induced the expression of IL-6 and IL-8. Collectively, these results suggest that chL-17D has structural and functional similarities to mammalian IL-17Ds and that it plays an important role in local gut innate immune responses during experimental coccidiosis.

Construction and application of an avian intestinal intraepithelial lymphocyte cDNA microarray (AVIELA) for gene expression profiling during Eimeria maxima infection.

Intestinal intraepithelial lymphocytes (IELs) are the primary immune effector cells in the gut and play a critical role in eliciting protective immunity to enteric pathogens such as Eimeria, the etiologic agent of avian coccidiosis. In this study, a microarray of genes expressed by intestinal IELs from Eimeria-infected chickens was constructed using the expressed sequence tag (EST) strategy. The avian intestinal IEL cDNA microarray (AVIELA) contained duplicates of 9,668 individual ESTs (6,654 known genes and 3,014 unique singletons of unknown identity) and was used to analyze gene expression profiles during primary and secondary Eimeria maxima infections. Following primary inoculation with E. maxima, the expression levels of 74 genes were significantly altered more than two-fold over the 3-day infection period (51 up-regulated, 23 down-regulated). Following secondary infection, the expression levels of 308 genes were significantly altered (62 up-regulated, 246 down-regulated). Pathway gene analysis indicated that many of the modulated genes were related to apoptosis, JAK/STAT, MAPK, interleukin, and TLR signaling pathways, and involving innate and adaptive immune responses. This chicken IEL microarray will provide a valuable resource for future transcriptional profiling of the genes involved in protective immunity to chicken enteric pathogens.

Immunopathology and cytokine responses in broiler chickens coinfected with Eimeria maxima and Clostridium perfringens with the use of an animal model of necrotic enteritis.

The incidence of necrotic enteritis (NE) due to Clostridium perfringens (CP) infection in commercial poultry has been increasing at an alarming rate. Although pre-exposure of chickens to coccidia infections is believed to be one of the major risk factors leading to NE, the underlying mechanisms of CP virulence remain undefined. The objectives of this study were to utilize an experimental model of NE produced by Eimeria maxima (EM) and CP coinfection to investigate the pathologic and immunologic parameters of the disease. Broilers coinfected with EM plus CP exhibited more severe gut pathology compared with animals given EM or CP alone. Additionally, EM/CP coinfection increased the numbers of intestinal CP bacteria compared with chickens exposed to an identical challenge of CP alone. Coinfection with EM and CP repressed nitric oxide synthase gene expression that was induced by EM alone, leading to lower plasma NO levels. Intestinal expression of a panel of cytokine and chemokine genes following EM/CP coinfection showed a mixed response depending on the transcript analyzed and the time following infection. In general, IFN-alpha, IFN-gamma, IL-1beta, IL-2, IL-12, IL-13, IL-17, and TGF-beta4 were repressed, whereas IL-8, IL-10, IL-15, and LITAF were increased during coinfection compared with challenge by EM or CP alone. These results are discussed in the context of EM and CP to act synergistically to create a more severe disease phenotype leading to an altered cytokine/chemokine response than that produced by infection with the individual pathogens.

Immunomodulatory properties of dietary plum on coccidiosis.

The current study was conducted to evaluate the effect of dietary supplementation with a lyophilized powder made from plums (P) on host protective immune responses against avian coccidiosis, the most economically important parasitic disease of poultry. One-day-old White Leghorn chickens were fed from the time of hatch with a standard diet either without P (control and P 0 groups) or supplemented with P at 0.5% (P 0.5) or 1.0% (P 1.0) of the diet. Animals in the P 0, P 0.5, and P 1.0 groups were orally challenged with 5000 sporulated oocysts of Eimeria acervulina at day 12 post-hatch, while control animals were uninfected. Dietary supplementation of P increased body weight gain, reduced fecal oocyst shedding, and increased the levels of mRNAs for interferon-gamma and interleukin-15 in the P 1.0 group at 10 days post-infection compared with the P 0 group. Furthermore, chickens fed either the P 0.5 or P 1.0 diets exhibited significantly greater spleen cell proliferation compared with the non-plum P 0 group. These results indicate that plum possesses immune enhancing properties, and that feeding chickens a plum-supplemented diet augments protective immunity against coccidiosis.

Unique responses of the avian macrophage to different species of Eimeria.

Coccidiosis is recognized as the major parasitic disease of poultry and is caused by the apicomplexan protozoa Eimeria. Increasing evidence shows the complexity of the host immune response to Eimeria and microarray technology presents a powerful tool for the study of such an intricate biological process. Using an avian macrophage microarray containing 4906 unique gene elements, we identified important host genes whose expression changed following infection of macrophages with sporozoites of Eimeria tenella (ET), Eimeria acervulina (EA), and Eimeria maxima (EM). This approach enabled us to identify a common core of 25 genetic elements whose transcriptional expression is induced or repressed by exposure to Eimeria sporozoites and to identify additional transcription patterns unique to each individual Eimeria species. Besides inducing the expression of IL-1beta, IL-6, and IL-18 and repressing the expression of IL-16, Eimeria treated macrophages were commonly found to induce the expression of the CCL chemokine family members macrophage inflammatory protein (MIP)-1beta (CCLi1), K203 (CCLi3), and ah221 (CCLi7). However, the CXCL chemokine K60 (CXCLi1) was found to be induced by macrophage exposure to E. tenella but was repressed upon macrophage exposure to E. maxima and E. acervulina. Fundamental analysis of avian chemokine and cytokine expression patterns offers insight into the unique avian immunological responses to these related but biologically unique pathogens.

Molecular cloning and characterization of chicken lipopolysaccharide-induced TNF-alpha factor (LITAF).

The inflammatory response to parasites, bacteria, and viruses is mediated by multiple host factors. TNF-alpha is one of the most pleiotropic cytokines in mammals, but has yet to be identified in avian species. In the current study, we isolated a full-length cDNA encoding the chicken homologue of LPS-induced TNF-alpha factor (LITAF), transcription factor, with an open reading frame of 148 amino acids and a predicted molecular mass of 16.0kDa. Quantitative RT-PCR analysis showed that chicken LITAF mRNA was predominantly expressed in spleen and intestinal intraepithelial lymphocytes (IELs). LITAF mRNA levels were up-regulated following in vitro stimulation of macrophages for 4 h with Escherichia coli or Salmonella typhimurium endotoxin, and 18-48 h after treatment with Eimeria acervulina, E. maxima, or E. tenella, three causative agents of avian coccidiosis. LITAF mRNA was up-regulated by more than 700-fold in IELs isolated from E. maxima-infected birds. Purified recombinant LITAF protein expressed in E. coli or COS7 cells exhibited cytotoxic activity against chicken tumor cell lines in vitro, presumably through autocrine activation of TNF-alpha or its chicken homologue. This supposition was strengthened by the fact that treatment of chicken macrophages with recombinant LITAF induced the expression of TL1A (TNFSF 15), a member of the TNF ligand super family. We conclude that chicken LITAF may play an important role in the regulation of TNF-alpha gene expression during the course of coccidiosis or tumorigenesis.

Development and characterization of a recombinant chicken single-chain Fv antibody detecting Eimeria acervulina sporozoite antigen.

Chicken monoclonal antibody (mAb), 8C3, which is reactive with a sporozoite antigen of Eimeria acervulina, is a potential therapeutic agent against avian coccidiosis caused by Eimeria spp. However, production of large amounts of 8C3 mAb in cell culture system is labor intensive and not cost-effective. Accordingly, recombinant single chain variable fragment (ScFv) antibody was constructed by amplification of the V(H) and V(L) genes from chicken hybridoma, 8C3 and when expressed in E. coli gave 5 mg l(-1). The expressed protein showed antigen binding activity equivalent to that of the parental mAb. In addition, nucleotide sequence comparison of 8C3 gene to the germline chicken V(L) genes suggested that the gene conversion with (V)lambda pseudogenes might contribute to the diversification of V(L) genes in chickens.

An oligosaccharide fraction from Korean mugwort herb suppresses death of the mouse thymocytes in culture by down-regulating the Fas death receptor gene.

Korean mugwort herb is a preparation of dried leaves from Artemisia species and has been used as a traditional medicine in Asia. An oligosaccharide fraction, AVF3, purified from the preparation promoted survival of the mouse thymocytes in culture. A mouse gene array study suggests that the AVF3 may modulate Fas/FasL dependent apoptotic cell death and thus has influence on the survival of the thymocytes in culture. RT-PCR analysis confirmed the down-regulation of the Fas gene by the AVF3 treatment, supporting that the AVF3 modulated thymocyte death by suppressing the Fas gene expression.

The polysaccharide fraction AIP1 from Artemisia iwayomogi suppresses apoptotic death of the mouse spleen cells in culture.

A polysaccharide fraction, AIP1, purified from Artemisia iwayomogi was shown to have immunomodulating and anti-tumor activities in mice. In order to determine how the AIP1 fraction exhibits the immunomodulating activity, the effect of the fraction on the apoptosis of mouse spleen cells was investigated. Treatment of the mouse spleen cells with the AIP1 fraction resulted in the suppression of apoptotic death and an extension of cell survival in culture, indicating that the fraction might modulate the death of spleen cells. Treatment of the mice with the AIP1 fraction in vivo also resulted in less apoptosis of the spleen cells, which indicates the physiological relevance of the anti-apoptosis effect of the fraction in vitro. A mouse gene array was used to determine the profile of the gene expression change showing a pattern of up- and down-regulated genes by the AIP1 treatment. This study provides preliminary information regarding the immunomodulatory mechanism of the AIP1 fraction.