Molecular cloning, expression and characterization of Pekin duck programmed death-1.

Programmed death-1 (PD-1) has a pivotal role in the attenuation of adaptive immune responses and peripheral tolerance. Here we describe the identification of the Pekin duck programmed death-1 orthologue (duPD-1). The duPD-1 cDNA encodes a 283-amino acid polypeptide that has an amino acid identity of 70%, 32% and 31% with chicken, murine and human PD-1, respectively. The duck PD-1 gene shares five conserved exons with chicken, murine and human PD-1 genes. A cluster of putative regulatory elements within the conserved region B (CR-B) of the basal promotor is conserved. Homology modeling was most compatible with the two ß-sheet IgV domain structure of murine PD-1. Contact residues, shown to be critical for binding of the respective human and murine PD-1 ligands are mostly conserved between avian and mammalian species, whereas residues that define the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) are highly conserved across higher vertebrates and frog. Constitutive expression of duPD-1 transcripts was predominantly found in lymphocyte-rich tissues, and mitogen-stimulation of duck peripheral blood mononuclear cells transiently increased duPD-1 mRNA expression. A soluble duPD-1 protein was expressed and shown to engage the identified duck PD-1 ligands. Our observations show considerable evolutionary conservation between mammalian and avian PD-1 orthologues. This work will facilitate further investigation of the role of PD-1 signaling in adaptive immunity in the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.

The Pekin duck programmed death ligand-2: cDNA cloning, genomic structure, molecular characterization and expression analysis.

Programmed death-1 (PD-1), upon engagement by its ligands, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), provides signals that attenuate adaptive immune responses. Here we describe the identification of the Pekin duck PD-L2 (duPD-L2) and its gene structure. The duPD-L2 cDNA encodes a 321 amino acid protein that has an amino acid identity of 76% and 35% with chicken and human PD-L2, respectively. Mapping of the duPD-L2 cDNA with duck genomic sequences revealed an exonic structure similar to that of the human Pdcd1lg2 gene. Homology modelling of the duPD-L2 protein was compatible with the murine PD-L2 ectodomain structure. Residues known to be important for PD-1 receptor binding of murine PD-L2 were mostly conserved in duPD-L2 within sheets A and G and partially conserved within sheets C and F. DuPD-L2 mRNA was constitutively expressed in all tissues examined with highest expression levels in lung, spleen, cloaca, bursa, cecal tonsil, duodenum and very low levels of expression in muscle, kidney and brain. Lipopolysaccharide treatment of adherent duck PBMC upregulated duPD-L2 mRNA expression. Our work shows evolutionary conservation of the PD-L2 ectodomain structure and residues important for PD-1 binding in vertebrates including fish. The information provided will be useful for further investigation of the role of duPD-L2 in the regulation of duck adaptive immunity and exploration of PD-1-targeted immunotherapies in the duck hepatitis B infection model.

Identification and characterization of functional CD154 (CD40 ligand) in the Pekin duck.

Binding of CD154, a member of the TNF ligand superfamily, to its receptor CD40 is essential for the development and regulation of adaptive immune responses in mammals. The duck CD154 (DuCD154) encoding gene was isolated from activated splenocytes using RT-PCR. Sequence analysis of the cloned DuCD154 gene revealed an open reading frame of 819 base pairs encoding a 272 amino acid protein. The extracellular domain of DuCD154 was identified and expressed for characterization and generation of antibodies. DuCD154 mRNA was predominantly expressed in spleen, thymus and duodenum. DuCD154 protein generated in cell culture was secreted and formed dimers. DuCD154 markedly enhanced proliferative responses in duck splenocytes when used alone or in conjunction with LPS or PHA. These observations suggest that DuCD154 has functional equivalence with mammalian CD154 and that the central role of CD154 as an immunoregulatory protein had already evolved before the divergence of birds and mammals.

Molecular cloning, expression and characterization of Pekin duck interferon-λ.

Interferons (IFNs) are the first line of defense against viral infections in vertebrates. Type III interferon (IFN-λ) is recognized for its key role in innate immunity of tissues of epithelial origin. Here we describe the identification of the Pekin duck IFN-λ ortholog (duIFN-λ). The predicted duIFN-λ protein has an amino acid identity of 63%, 38%, 37% and 33% with chicken IFN-λ and human IFN-λ3, IFN-λ2 and IFN-λ1, respectively. The duck genome contains a single IFN-λ gene that is comprised of five exons and four introns. Recombinant duIFN-λ up-regulated OASL and Mx-1 mRNA in primary duck hepatocytes. Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding and antiviral activity. The identification and expression of duIFN-λ will facilitate further study of the role of type III IFN in antiviral defense and inflammatory responses of the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.

Genomic structure, molecular characterization and functional analysis of Pekin duck interleukin-10.

Here we describe the cloning and expression of Pekin duck IL-10 (duIL-10) and a six exon-5 intron structure of an IL-10 gene. Two transcripts encoding duIL-10 with an alternatively spliced 3'UTR, and a transcript lacking exon 5 with a novel coding sequence for its C-terminus (duIL-10ΔE5) were isolated from splenocytes. The duIL-10 protein has an amino acid identity of 79% and 47% with chicken and human IL-10, respectively. The duck IL-10 gene shares a similar structure of the respective exons 1-5 with the IL-10 genes of other vertebrates but has an alternative exon. The duIL-10 3D structure by homology modeling was similar to that of the human IL-10 monomer, whereas the predicted duIL-10ΔE5 protein lacks helix F. DuIL-10 and duIL-10ΔE5 transcripts were most abundant in primary and secondary immune organs and lung. Recombinant duIL-10 suppressed duck IL-2 transcripts in mitogen-activated PBMCs. Our observation suggests evolutionary conservation of structure and function of the duIL-10 protein but the roles of the novel IL-10 splice variants in the regulation of duck immune responses and evolution of vertebrate immunity remain to be elucidated.

Immunogenicity and protective efficacy of a DNA vaccine encoding a chimeric protein of avian influenza hemagglutinin subtype H5 fused to CD154 (CD40L) in Pekin ducks.

The potential of CD154 (CD40L) as a powerful immunological adjuvant has been shown in various strategies. In this study we examine the immunogenicity and protective efficacy of a CD40-targeting avian influenza hemagglutinin (HA) subunit DNA vaccine in ducks. DNA constructs encoded the ectodomain of the HA protein of LPAI A/mallard/BC/373/2005 (H5N2) with or without fusion to the ectodomain of duck CD154. CD40-targeting significantly accelerated and enhanced humoral responses to the vector-encoded HA protein. In viral challenge experiments with A/chicken/Vietnam/14/2005 (H5N1), DNA immunization conferred partial protection against the genetically distant HPAI. The observed improved kinetics and magnitude of immune induction suggest that CD40-targeting holds promise for influenza A vaccine development.

Identification of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) isoforms in the Pekin duck.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152) is an inhibitory T cell receptor predominately expressed on activated T cells. The duck CTLA-4 (DuCTLA-4) cDNA and a transcript lacking the predicted transmembrane encoding region (DuCTLA-4DeltaTM) were isolated from splenocytes using RT-PCR. The predicted DuCTLA-4 protein showed an identity of 92%, 49% and 47% with chicken, human and mouse homologues, respectively. Sequence comparison revealed conservation of residues implicated in the B7 ligand binding, disulfide linkages, glycosylation and intracellular signaling. DuCTLA-4 mRNA was predominately expressed in primary and secondary immune organs. DuCTLA-4 and DuCTLA-4DeltaTM transcripts were differentially regulated in PBMCs. Flow cytometric analysis showed constitutive expression of DuCTLA-4 protein on freshly isolated PBMCs and a modest increase upon mitogen stimulation. Our observations suggest that DuCTLA-4 and its isoform DuCTLA-4DeltaTM evolved before the divergence of birds and mammals. Both DuCTLA-4 isoforms have significant structural homology to mammalian CTLA-4 proteins but their individual roles in the regulation of duck immune responses remains to be elucidated.

Plasmapheresis for hemolytic crisis and impending acute liver failure in Wilson disease.

Wilsonian crisis is fatal unless copper removal is initiated early and liver transplantation is performed for patients that fulfill criteria for a poor outcome. We report a patient presenting with severe hemolysis and impending acute liver failure that made a rapid recovery with prompt initiation of plasmapheresis and chelation therapy. Rapid copper removal by plasmapheresis alleviated hemolysis and liver injury. A review of the literature was performed examining the use of plasmapheresis and albumin dialysis with continuous veno-venous hemodialysis or molecular adsorbents and recirculating system.

Immunotargeting with CD154 (CD40 ligand) enhances DNA vaccine responses in ducks.

Engagement of CD154 on activated T cells with CD40 on antigen-presenting cells (APCs) potentiates adaptive immune responses in mammals. Soluble multimeric forms of CD154 have been used as an adjuvant or in immunotargeting strategies to enhance vaccine responses. The objective of our study was to examine the ability of duck CD154 (DuCD154) to enhance DNA vaccine responses in the duck hepatitis B model. Constructs were generated to express the functional domain of DuCD154 (tCD154), truncated duck hepatitis B virus (DHBV) core antigen (tcore) and chimera of tcore fused to tCD154 (tcore-tCD154). Expression in LMH cells demonstrated that all proteins were secreted and that tCD154 and tcore-tCD154 formed multimers. Ducks immunized with the plasmid ptcore-tCD154 developed accelerated and enhanced core-specific antibody responses compared to ducks immunized with ptcore or ptcore plus ptCD154. Antibody responses were better sustained in both ptcore-tCD154- and ptcore plus ptCD154-immunized ducks. Core-specific proliferative responses of duck peripheral blood mononuclear cells were enhanced in ducks immunized with ptcore-tCD154 or ptcore alone. This study suggests that the role of CD154 in the regulation of adaptive immune responses had already evolved before the divergence of birds and mammals. Thus, targeting of antigens to APCs with CD154 is an effective strategy to enhance DNA vaccine responses not only in mammalian species but also in avian species.

Value of an enzymatic assay for the determination of serum ceruloplasmin.

The serum concentration of the copper protein ceruloplasmin has been an important diagnostic indicator of Wilson's disease (WD). It is widely quoted that 95% of people with WD have low serum ceruloplasmin concentrations. Current evidence suggests that a normal serum ceruloplasmin concentration is more common in patients with WD, particularly those with liver disease, perhaps in part because of the routine use of an immunologic assay. This assay might indicate a normal level of ceruloplasmin when the enzymatic activity is lower. Enzymatic activity is the biologically relevant parameter. We compared the immunologic measurement with the enzymatic assessment of oxidase activity in patients with liver or neurologic symptoms of unknown origin in whom WD was considered in the differential diagnosis. Although a strong correlation of ceruloplasmin protein concentration with oxidase activity was observed in controls, this was not the case for these patients. Twelve patients, presenting with various types of hepatic disease, demonstrated a weak correlation between ceruloplasmin protein concentration and oxidase activity. Ten percent of patients with neurologic symptoms ( n = 41) had low ceruloplasmin concentrations and oxidase activity, and another 8% had normal ceruloplasmin concentrations associated with low oxidase activity. Although the enzymatic method is preferred for its biologic relevance, ceruloplasmin analysis is not a reliable diagnostic parameter for the diagnosis of WD in patients with liver disease. An important use of the ceruloplasmin oxidase assay is in the follow-up of patients with WD. Ceruloplasmin oxidase activity was undetectable in sera from patients with WD who were undergoing long-term chelation therapy, suggesting an early sign of copper depletion and a need for subsequent monitoring for symptoms of copper deficiency.

Late acute rejection after liver transplantation: the Western Canada experience.

Acute rejection usually occurs within 3 months posttransplantation. Most centers reduce immunosuppression over 6 to 12 months to minimize opportunistic infection, malignancy, and drug toxicity. Pretransplant disease and low immunosuppression have been reported in association with late acute rejection (LAR). The objective of this study was to determine the incidence, predictive factors, and outcomes of LAR via retrospective review of adult liver transplant recipients in Western Canada from 1989 to 2000. LAR was defined as biopsy-proven acute rejection occurring more than 180 days posttransplantation. Patient characteristics, immunosuppression, and outcome were determined. Both a univariate and multiple logistic regression analysis were performed. LAR occurred in 97 (23%) of 415 patients more than 180 days posttransplantation. Median follow-up was 402 days (range, 180 to 3137 days); 79% of LAR episodes were graded mild. At the time of LAR, 33% were on a steroid taper. A total of 73% of LAR episodes were treated with pulse intravenous steroids, and 5% were steroid-resistant. In the univariate analysis, patients undergoing transplantation for viral etiologies and older age were associated with less LAR. Immunosuppression was significant in a multiple logistic regression model, but not with a proportional hazards model. On multivariate analysis, only patients undergoing transplantation for viral etiologies remained resistant to LAR (hazard ratio, 0.52; range, 0.34 to 0.93, P = .02). There was a trend toward increased chronic rejection in patients who developed LAR (P = .04). LAR is common and occurs after more than 1 year posttransplantation. Patients undergoing transplantation for viral etiologies seem to have a lower risk of LAR. There may be an increased risk of chronic rejection in those developing LAR.