The Pekin duck programmed death-ligand 1: cDNA cloning, genomic structure, molecular characterization and mRNA expression analysis.

Programmed death ligand-1 (PD-L1) plays an important role in the attenuation of adaptive immune responses in higher vertebrates. Here, we describe the identification of the Pekin duck PD-L1 orthologue (duPD-L1) and its gene structure. The duPD-L1 cDNA encodes a 311-amino acid protein that has an amino acid identity of 78% and 42% with chicken and human PD-L1, respectively. Mapping of the duPD-L1 cDNA with duck genomic sequences revealed an exonic structure of its coding sequence similar to those of other vertebrates but lacked a noncoding exon 1. Homology modelling of the duPD-L1 extracellular domain was compatible with the tandem IgV-like and IgC-like IgSF domain structure of human PD-L1 (PDB ID: 3BIS). Residues known to be important for receptor binding of human PD-L1 were mostly conserved in duPD-L1 within the N-terminus and the G sheet, and partially conserved within the F sheet but not within sheets C and C'. DuPD-L1 mRNA was constitutively expressed in all tissues examined with highest expression levels in lung and spleen and very low levels of expression in muscle, kidney and brain. Mitogen stimulation of duck peripheral blood mononuclear cells transiently increased duPD-L1 mRNA expression. Our observations demonstrate evolutionary conservation of the exonic structure of its coding sequence, the extracellular domain structure and residues implicated in receptor binding, but the role of the longer cytoplasmic tail in avian PD-L1 proteins remains to be determined.

The Pekin duck IL-10R2 common chain: cDNA cloning, genomic structure, molecular characterization and mRNA expression analysis.

The interleukin-10 receptor 2 (IL-10R2, IL-10Rß) is required for the signalling of the class 2 cytokines IL-10, IL-22, IL-26 and IFN-λ1-3 . Here, we describe the identification of the Pekin duck IL-10R2 (duIL-10R2) common chain and its gene structure. The duIL-10R2 cDNA encodes a 343 amino acid protein that has an amino acid identity of 76% and 42% with chicken and human IL-10R2, respectively. Binding residues of human IL-10R2 for IL-10 and IL-22 were mostly conserved in the avian IL-10R2 proteins within loops L3 and L5, but not within loops L2 and L6. Homology modelling of the duIL-10R2 extracellular domain structure using soluble human IL-10R2 (shIL-10R2, PDB ID: 3LQM) as a template revealed a protruding loop L5 and two distinct clefts between loops L2/L3 and L3/L5, similar to shIL-10R2. However, in contrast to the three amino acid ß-hairpin loop L2 of shIL-10R2, loop L2 of duIL-10R2 is five residues longer. Residues within a putative Tyk2 binding site were highly conserved across all vertebrate IL-10R2 proteins examined. The duIL-10R2 gene shares a seven exon-six intron structure with chicken and human IL-10R2 genes, but avian genes are more compact. DuIL-10R2 mRNA was constitutively expressed in all tissues. Mitogen stimulation of duck peripheral blood mononuclear cells (PBMC) did not alter transcript levels. Our observations suggest that genomic organization and structural features implicated in multiple cytokine-binding properties of human IL-10R2 are conserved in duck IL-10R2, but the evolutionary changes that appear to have lead to low-affinity cytokine interaction within loop L2 are distinct to mammalian species.

cDNA cloning, genomic structure, molecular characterization and mRNA expression analysis of the Pekin duck interleukin-10 receptor 1.

Interleukin-10 (IL-10) mediates its broad anti-inflammatory and immunoregulatory effects through two cell surface receptors by which binding to the IL-10 receptor 1 (IL-10R1) is the initial step that leads to recruitment of IL-10R2 and initiation of the ternary complex signal transduction cascade. The duck IL-10R1 (duIL-10R1) cDNA was obtained by using RT-PCR and 5'RACE. The deduced 574 amino acid protein has an amino acid identity of 62%, 27% and 28% with chicken, mouse and human IL-10R1, respectively. Comparison of the duIL-10R1 cDNA with duck genomic sequences revealed a seven exon-six intron structure of the duck IL-10R1 gene that shares a similar size with the respective exons 1-7 of the chicken and human IL-10R1 genes, but the avian genes are more compact. Promoter analysis identified putative binding sites for regulatory elements such as CCAAT enhancer binding protein-α, specificity protein 1 (Sp1), nuclear factor 1 (NF1), transcriptional regulatory protein Oct-1, nuclear factor (NF) κB and interferon-stimulated gene factor-3 (ISGF-3). A canonical TATA box was absent in proximity of the transcription initiation site, but a CpG island was present. Sequence analysis of the predicted duIL-10R1 protein revealed characteristic features of class-II cytokine receptors (CFR2) family members and a considerable degree of conservation of residues implicated in ligand binding across higher vertebrates. The predicted secondary structure of the duIL-10R1 extracellular domain is compatible with the two-subdomain structure of the human IL-10R1 protein established by its crystal structure. The 3D model structure shows conservation of the positions of conserved contact residues within four of the five ligand-binding loops. Within the cytoplasmic domain, residues implicated in signal transduction were conserved including two redundant peptide motifs GYXXQ essential for recruitment and activation of STAT3. DuIL-10R1 mRNA expression was most abundant in spleen, thymus, peripheral blood mononuclear cells (PBMCs) and lung. Mitogen stimulation of PBMCs transiently increased duIL-10R1 mRNA expression. Our observations suggest significant evolutionary conservation of the IL-10R1 genomic organization, protein structure and receptor function through the JAK/STAT signalling pathway across higher vertebrates.

Efficacy of lamivudine in chronic hepatitis B patients with active viral replication and decompensated cirrhosis undergoing liver transplantation.

Liver transplantation for endstage hepatitis B virus (HBV) infection has been associated with survival inferior to that of liver transplantation in other chronic liver diseases due to HBV reinfection of the graft. Lamivudine is a new nucleoside analog with potent antiviral effects against hepatitis B. Our aim was to test its efficacy when used pre- and posttransplantation in HBV-DNA positive patients with endstage liver disease. Patients received oral lamivudine 100 mg daily both pretransplant and posttransplant. Viral serology, serum and tissue HBV-DNA and liver histology were assessed sequentially. Five consecutive patients with endstage hepatitis B were entered into the trial. Serum HBV-DNA was cleared pretransplant in all patients. Three of four transplanted patients cleared HBeAg and HBsAg postoperatively, whereas all four became negative for serum HBV-DNA (dot-blot and PCR). Liver biopsies were negative for HBV-DNA by PCR in 3 of 4 cases. Lymphocytes were negative for HBV-DNA by PCR in all cases. With follow-up of 3, 14, 16, and 26 months, two patients have normal liver enzymes and normal liver histology and two have developed recurrent hepatitis B. No significant side effects were seen. This pilot study shows that lamivudine can effectively inhibit hepatitis B virus in cirrhotic patients pretransplant and posttransplant. A lamivudine resistant mutant developed in two patients. Transplant recipients with actively replicating HBV related cirrhosis may achieve a good outcome after liver transplantation using lamivudine, but viral resistance is likely to be a significant problem.

Chronic viral hepatitis C: management update.

The management of chronic viral hepatitis C is evolving rapidly. Monotherapy with interferon, the accepted standard of treatment until recently, achieves only a modest sustained virological response rate of 15%. Combination treatment with alpha-2b interferon and ribavirin has been shown to increase sustained response rates to 40% in patients who have never been treated with interferon and to 50% in those who have relapsed following monotherapy with interferon. However, side effects, which have led to the discontinuation of combination treatment in a significant proportion of patients, must be carefully monitored. Treatment with interferon alpha-2b and ribavirin has now been approved in Canada, but the selection and monitoring of patients suitable for combination treatment requires special expertise. Although improvements in current therapeutic options may be possible with more frequent, higher doses or long-acting forms of interferon together with ribavirin, low sustained response rates (i.e., below 30%) for patients with hepatitis C virus genotype 1 emphasize the need for novel antiviral medications that will target the functional sites of the HCV genome.

Lamivudine resistance in hepatitis B: mechanisms and clinical implications.

Lamivudine (beta-L-(-)-2',3'-dideoxy-3'-thiacytidine) has been a major breakthrough in the care of patients with hepatitis B. With prolonged monotherapy the development of resistance is an increasingly recognized problem that limits the long term efficacy of this nucleoside analogue. The most common mutations associated with lamivudine resistance occur within the highly conserved YMDD motif in the C domain of the viral polymerase and are often associated with a compensatory mutation in the proximal B domain. The structural and functional relationship of resistance mutations is reflected in different in vitro sensitivities to lamivudine and changes in replication capacities. During prolonged lamivudine treatment there can be successive changes of different resistant mutants (genotypic succession) or a single mutant can remain the dominant viral species. In patients treated for chronic hepatitis B infection the cumulative incidence of viral resistance reaches over 50% after 3 years. Most patients will have lower serum HBV DNA levels after the emergence of resistance which is ascribed to the decreased replication capacity of these mutants. Although severe flares and ongoing HBe antigen seroconversion can occur in these patients with lamivudine-resistant HBV, the impact of continued therapy on the long-term outcome is still insufficiently studied. In the setting of liver transplantation for HBV-associated disease the clinical course after the emergence of viral resistance is variable but still may lead to disease progression and graft failure. Analogous to the success of combination therapies to delay the emergence of antiviral-resistant HIV, it will be important to combine anti-HBV agents with additive or synergistic antiviral properties and different resistance profiles for future de novo combination therapies for hepatitis B infection.

Adefovir dipivoxil for the treatment of lamivudine-resistant hepatitis B mutants.

Lamivudine has been shown to be an effective therapy for chronic hepatitis B, but resistance to this nucleoside agent is common after prolonged use. Five patients with chronic hepatitis B virus (HBV) infection developed resistance to lamivudine after 9 to 19 months of treatment. In 4 patients this occurred after liver transplantation and the remaining individual had stable cirrhosis. In each case, resistance was confirmed to be caused by one or more mutations in the HBV-DNA polymerase gene and was associated with active underlying liver disease. The patients were treated with adefovir dipivoxil in a dose of 5 to 30 mg daily. Two to 4 log(10) reductions in HBV-DNA levels were observed in 4 cases, and the fifth patient became negative by quantitative polymerase chain reaction (PCR) after retransplantation in conjunction with hepatitis B immunoglobulin (HBIg). Virologic improvement was associated with stable or declining serum alanine transaminase levels in 4 patients. HBV-DNA suppression has been sustained during a mean treatment period of 13 months (range 11 to 15 months), including 1 patient in whom lamivudine has been discontinued. Mild changes in renal function were observed during treatment in most cases but did not require early discontinuation of the drug. This study provides evidence that adefovir dipivoxil can be an effective treatment for lamivudine-resistant HBV mutants as well as wild-type HBV.

Peritonitis and massive granulocytic infiltration of the spleen in adult Still's disease.

A case of adult Still's disease is described which, in addition to the more common manifestations, also included abdominal discomfort. Upon laparoscopy, peritonitis was disclosed; a biopsy showed massive granulocytic infiltration of the spleen which could not be attributed to an infectious disease. The patient did not improve on conventional therapeutic modalities but required intensive combination therapy consisting of high dose acetylsalicylic acid, prednisone, and slow-reacting substances before entering remission.

Genotypic succession of mutations of the hepatitis B virus polymerase associated with lamivudine resistance.

BACKGROUND/AIMS: Hepatitis B mutant strains of virus emerging during treatment with the nucleoside analog lamivudine are being increasingly recognized. In the majority of lamivudine-resistant isolates the mutations have been reported to occur within the YMDD motif of the viral polymerase, either as a single mutation M552I or as M552V concomitant with L528M. We analyzed the time course and genetic succession pattern during the emergence of lamivudine resistance. METHODS: Seven patients with breakthrough viremia in the setting of chronic hepatitis (n=5) or recurrent HBV after liver transplantation (n=2) were investigated. Pre- and post-breakthrough serum samples were evaluated by single- or second-round PCR amplification and sequencing analysis. RESULTS: Genotypic succession of the virus populations was observed to occur from M552I to M552I/L528M (n=2) and from L528M to M552V/L528M (n=1). The double mutations M552I/L528M (n=4) or M552V/L528M (n=2) were found in six out of seven patients, and represented the stable virus populations throughout the follow-up period. Breakthrough viremia was not associated with the single L528M mutation. The mean duration of uninterrupted treatment with lamivudine until breakthrough was 422 days (range 182-642) and was longer in the setting of chronic hepatitis B than in recurrent hepatitis B after liver transplantation. HBV DNA levels after breakthrough were lower than pretreatment levels in the majority of patients with chronic hepatitis but higher after liver transplantation. CONCLUSION: Our observations show that the virus populations conferring resistance to lamivudine can evolve from single to double mutations at amino acid 552 and 528 of the HBV polymerase, and that M552I/ L528M or M552V/L528M seem to be the predominant mutations arising during long-term antiviral therapy with lamivudine.

De novo acute hepatitis B infection in a previously vaccinated liver transplant recipient due to a strain of HBV with a Met 133 Thr mutation in the "a" determinant.

UNLABELLED: De novo HBV infection post-liver transplantation (LT) from an anti-HBc seropositive donor rarely presents as acute failure. We report a 42-year-old Caucasian female, HBsAg and anti-HBc seronegative, with primary biliary cirrhosis who received an allograft from a HBsAg negative, anti-HBc seropositive donor. The patient, previously vaccinated years pre-LT, was re-vaccinated against HBV and 1 year post-LT had an anti-HBs titre of 256 IU/l. Two years post-LT, elevated serum aminotransferases and worsening liver function with an INR of 2.0 developed. The HBsAg became positive, anti-HBs undetectable and serum HBV-DNA >2000 pg/ml by hybridisation assay. Liver biopsy revealed significant ballooning degeneration, piecemeal necrosis and positive immunostaining for HBsAg. Progressive liver failure developed followed by sepsis and terminal multi-organ failure. Subsequent analysis of the predominant HBV strain revealed mutations in the "a" determinant: Met 133 Thr (codon change ATG to ACG) and Asn 131 Thr. CONCLUSION: ' Acute de novo HBV infection from an anti-HBc sero-positive donor may occur long after LT despite protective anti-HBs titres post-vaccination secondary to the emergence of "a" determinant mutated strains of HBV.