Selection pressure on the hepatitis B virus pre-S/S and P open reading frames in Tongan subjects with a chronic hepatitis B virus infection.

Identification of the full repertoire of hepatitis B virus (HBV) peptides that are presented to CD8+ T cells by common HLA class I alleles will be useful for designing immunotherapies for chronic hepatitis B. One hundred and seventy five cloned sequences containing the pre-S/S and P open reading frames (ORF) of the HBV were obtained from serum HBV-DNA of HBeAg-positive (n=4) and HBeAg-negative (inactive healthy carriers (IHC), n=16) Tongan subjects with an inactive chronic HBV infection. In addition, 34 and 32 sequences were obtained 5.2±1.4 (mean±SD) years apart from eight subjects. PAML was used to identify codons in the pre-S/S and P ORFs that were under positive selection pressure (ω>1). The number of non-synonymous substitutions in these codons was compared in IHC who were homozygous for either HLA-B∗4001 (n=9) or HLA-B*5602 (n=7), and who were either positive (n=6) or negative (n=10) for HLA-A*02. 34 codons in the pre-S/S and 11 codons in the P ORFs were under positive selection pressure. There was a higher number of non-synonymous substitutions in these codons in HBeAg-negative versus HBeAg-positive subjects in the P (p=0.02) but not the pre-S/S (p=0.64) ORF. There was no association between any HLA class I allele and non-synonymous substitutions in these codons. There was no increase in positive selection pressure on the pre-S/S and P ORFs with time. In conclusion, we could not find HLA class I-restricted selection pressure on any pre-S/S or P ORF amino acid; raising the possibility that peptide-based immunotherapies for chronic hepatitis B may not require peptides from these ORFs.

Biliary complications following orthotopic liver transplantation: a 10-year audit.

BACKGROUND: Biliary complications following liver transplantation result in major morbidity. We undertook a 10-year audit of the incidence, management and outcomes of post-transplant biliary complications at the New Zealand Liver Transplant Unit. METHODS: Prospectively collected data on 348 consecutive liver transplants performed between February 1998 and October 2008 were reviewed. The minimum follow-up was 6 months. RESULTS: A total of 309 adult and 39 paediatric transplants were performed over the study period. Of these, 296 (85%) were whole liver grafts and 52 (15%) were partial liver grafts (24 split-liver, eight reduced-size and 20 live-donor grafts). There were 80 biliary complications, which included 63 (18%) strictures and 17 (5%) bile leaks. Partial graft, a paediatric recipient and a Roux-en-Y biliary anastomosis were independent predictors of biliary strictures. Twenty-five (40%) strictures were successfully managed non-operatively and 38 (60%) required surgery (31 biliary reconstructions, three segmental resections and four retransplants). Seven (41%) bile leaks required surgical revision and 10 (59%) were managed non-operatively. There was no mortality related directly to biliary complications. CONCLUSIONS: Biliary complications affected one in five transplant recipients. Paediatric status, partial graft and Roux-en-Y anastomosis were independently associated with the occurrence of biliary strictures. Over half of the affected patients required surgical revision, but no mortality resulted from biliary complications.

Associations between HLA class I alleles and escape mutations in the hepatitis B virus core gene in New Zealand-resident Tongans.

The full repertoire of hepatitis B virus (HBV) peptides that bind to the common HLA class I molecules found in areas with a high prevalence of chronic HBV infection has not been determined. This information may be useful for designing immunotherapies for chronic hepatitis B. We identified amino acid residues under positive selection pressure in the HBV core gene by phylogenetic analysis of cloned DNA sequences obtained from HBV DNA extracted from the sera of Tongan subjects with inactive, HBeAg-negative chronic HBV infections. The repertoires of positively selected sites in groups of subjects who were homozygous for either HLA-B*4001 (n = 10) or HLA-B*5602 (n = 7) were compared. We identified 13 amino acid sites under positive selection pressure. A significant association between an HLA class I allele and the presence of nonsynonymous mutations was found at five of these sites. HLA-B*4001 was associated with mutations at E77 (P = 0.05) and E113 (P = 0.002), and HLA-B*5602 was associated with mutations at S21 (P = 0.02). In addition, amino acid mutations at V13 (P = 0.03) and E14 (P = 0.01) were more common in the seven subjects with an HLA-A*02 allele. In summary, we have developed an assay that can identify associations between HLA class I alleles and HBV core gene amino acids that mutate in response to selection pressure. This is consistent with published evidence that CD8(+) T cells have a role in suppressing viral replication in inactive, HBeAg-negative chronic HBV infection. This assay may be useful for identifying the clinically significant HBV peptides that bind to common HLA class I molecules.

Pre- and postoperative immunonutrition in patients undergoing liver transplantation: a pilot study of safety and efficacy.

BACKGROUND & AIMS: Malnutrition is common in patients with end-stage liver disease and is a risk factor for post-transplant morbidity. The goal of this study was to assess the safety of an immune-enhancing diet in patients undergoing liver transplantation and to investigate its effects on nutritional status. METHODS: Fifteen consecutive patients received oral Impact (0.6l/d) for a median 54 (range 10-168)d pre-transplant and enteral Impact was started early after transplant. Total body protein was measured prior to commencing supplemental Impact, immediately prior to transplant and 10, 15, 30, 90, and 180 days post-transplant. The results were compared with those from 17 patients who received standard nutritional intervention. RESULTS: All study patients tolerated Impact pre- and postoperatively and there were no safety concerns. Over the preoperative period total body protein increased significantly (P = 0.017). In 7 patients followed for 6 months post-transplant, a significant (P = 0.026) loss of body protein occurred over the first 15 postoperative days which was regained by 6 months. In the patients who did not receive Impact, body protein did not change preoperatively and the loss after surgery was not regained. Infectious complications occurred in 5/15 (33%) Impact patients and 12/17 (71%) non-Impact patients (P = 0.074). CONCLUSIONS: In patients with end-stage liver disease, our results suggest the possibility that Impact may have a role in improving preoperative nutritional status, hastening recovery after transplant, and reducing postoperative infectious complications. These potential benefits need to be confirmed in a randomised controlled trial.

Intragraft gene and protein expression in rat liver allografts treated with costimulatory blockade alone or in combination with CyA.

BACKGROUND: Costimulatory blockade has been shown to prevent acute rejection (AR) and promote long-term graft survival in a number of animal models including nonhuman primates. The effect of concomitant administration of conventional immunosuppressives on long-term liver allograft survival and intragraft expression of immune mediators has not previously been examined. MATERIALS AND METHODS: A high-responding Dark Agouti to Lewis orthotopic liver transplant (LEW OLT) model was used to compare anti-CD154 alone, or in combination with cyclosporin (CyA) on allograft survival. Donor-specific reactivity was assessed by mixed lymphocyte reaction (MLR) and allogeneic skin grafts. Surviving rats were euthanized on day 150 and intragraft gene (CD80, 86, 152, 154, IFN-gamma, IL-2, IL-6, IL-10, IL-13, TNF-alpha, TGF-beta, IL-7, Fas-ligand, Granzyme B, bax, and bcl(2)) and protein (CD4, CD8, ED1, CD154, CD80, CD86) expression was measured. RESULTS: Untreated control recipients had a median survival time of 5 days. Recipients treated with anti-CD154 survived to beyond 150 days with no evidence of AR. Concomitant administration of CyA did not alter the long-term survival. There was no difference in the serum aspartate aminotransferase between treatment groups or a change over time. All treated recipients showed a reduction in donor-specific MLR at day 40 and 60 but had persistence of donor reactivity to skin grafts at day 100. Histologically, liver architecture was well preserved despite the presence of a nondestructive mononuclear cell infiltrate. Analysis of intragraft gene expression revealed an inverse relationship between the duration of anti-CD154 therapy and the gene expression of costimulatory molecules and Th1 cytokine transcripts. The pro-apoptotic gene, bax, was increased in recipients treated with anti-CD154, but not CyA, compared with normal liver. CONCLUSIONS: These data demonstrate that anti-CD154 therapy either alone or in combination with CyA allows for the long-term survival of liver allografts in the rat despite there being a difference in the intragraft gene and protein profile.

Analysis of intragraft gene and protein expression of the costimulatory molecules, CD80, CD86 and CD154, in orthotopic liver transplant recipients.

CD40-CD154 and/or CD28-CD80/86 costimulatory blockade induces long-term allograft survival in numerous animal models. Studies examining the expression of costimulatory molecules during acute cellular rejection (ACR) have been limited to renal and cardiac allografts. The aim of this study was to describe the relationship between intragraft costimulatory molecule expression in OLT recipients and ACR. Forty-five liver biopsies were obtained at reperfusion and day 7. Gene and protein expression of CD80, CD86 and CD154 were analyzed by RT-PCR and immunohistochemistry. CD154 protein expression was present in 13 of 18 patients with a RAI score of 4, but in only two of 14 patients with a RAI score of <4. There was a strong association between the RAI score and the presence of CD80 and CD154 immunoreactivity. CD86 protein expression did not correlate with the severity of ACR. In reperfusion biopsies CD154, but not CD80 or CD86, protein expression correlated with the total ischaemic time. There was no association between expression of costimulatory molecule genes and ACR. In conclusion, we have demonstrated an association between CD154 and CD80 protein expression and ACR in orthotopic liver allografts.

Costimulatory blockade prevents early rejection, promotes lymphocyte apoptosis, and inhibits the upregulation of intragraft interleukin-6 in an orthotopic liver transplant model in the rat.

Costimulatory pathways have a pivotal role in the T-cell response to alloantigen. The role of costimulatory blockade with anti-CD154 in orthotopic liver transplantation (OLT) has not been examined previously. This study aims to investigate effects of anti-CD154 and CTLA4-immunoglobulin (Ig) in the early post-OLT period using a major histocompatibility complex-disparate fully arterialized OLT model in the rat. Lewis rats underwent OLT with Dark Agouti liver allografts. Recipients were randomized to receive (1) isotype control, (2) anti-CD154, (3) CTLA4-Ig, or (4) cyclosporine A (CyA). Rats were killed day 8, and specimens were obtained for histological examination, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, immunohistochemistry, and quantitative reverse-transcriptase polymerase chain reaction. An additional five transplant recipients were treated with anti-CD154 for 14 days postoperatively to assess long-term allograft survival. All isotype control animals died on or before day 6 of acute rejection. Apart from four deaths caused by nonimmunologic causes, all treated recipients survived to day 8. The median survival of rats treated for 14 days with anti-CD154 was greater than 150 days. Serum aspartate aminotransferase and bilirubin levels normalized by day 3 in the CyA group and day 5 in transplant recipients treated with costimulatory blockade. Histologically, there was no difference between isotype controls and CTLA4-Ig-treated animals, whereas anti-CD154-treated transplant recipients had a lower Banff score. CD4+ and CD8+ T-cell infiltrates were prominent in transplant recipients treated with costimulatory blockade. Intragraft analysis showed an increase in lymphocyte apoptosis, Fas ligand messenger RNA expression, and reduction in interleukin-6 gene expression in transplant recipients treated with costimulatory blockade. Costimulatory blockade did not alter intragraft gene expression of other mediators of T-cell priming, differentiation, and effector function compared with isotype control animals. In conclusion, costimulatory blockade prevented acute rejection, enabled long-term survival, and increased intragraft lymphocyte apoptosis in a high-responding rat OLT model.