Differential effects of donor-specific HLA antibodies in living versus deceased donor transplant.

The presence of donor-specific anti-HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long-term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement-dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10-year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10-year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10-year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

Questionable expression of unstable DQ heterodimer containing HLA-DQA1*01:07.

Human leukocyte antigens (HLA)-DQA1*01:07 was identified as an HLA-DQ blank specificity that segregated with the serological HLA-A2, -B7, -DR14, -DR52 haplotype, which carried DQB1*05:03. The blank specificity of DQA1*01:07-DQB1*05:03 may be because of lack of reactivity of available typing sera, or disruption of proper assembly of DQ heterodimer. The cDNA sequence of DQA1*01:07 is nearly identical to DQA1*01:04 except for a variant at position 304, which results in the replacement of an arginine with a cysteine at 79α. To determine whether the DQA1*01:07 product can be expressed on cell-surface, we co-expressed DQA1*01:07 with various DQB1*05 or *06 alleles in fibroblast cells. Cell-surface expression of DQ was detectable when DQA1*01:07 was co-expressed with DQB1*06:04 but undetectable with other DQB1*05 and DQB1*06 alleles, including DQB1*05:03, to which DQA1*01:07 was encoded in cis. These data suggest that DQA1*01:07 may act as a phenotypically null allele in the DQA1*01:07-DQB1*05:03 haplotype, while it can be expressed at a low level in the presences of certain DQB1*06 alleles, such as DQB1*06:04, in trans. Based on the null or low expression of DQA1*01:07 as shown in the previous and present studies, DQA1*01:07 has recently been renamed to DQA1*01:07Q, indicating its questionable expression.

The PROCARE consortium: toward an improved allocation strategy for kidney allografts.

Kidney transplantation is the best treatment option for patients with end-stage renal failure. At present, approximately 800 Dutch patients are registered on the active waiting list of Eurotransplant. The waiting time in the Netherlands for a kidney from a deceased donor is on average between 3 and 4 years. During this period, patients are fully dependent on dialysis, which replaces only partly the renal function, whereas the quality of life is limited. Mortality among patients on the waiting list is high. In order to increase the number of kidney donors, several initiatives have been undertaken by the Dutch Kidney Foundation including national calls for donor registration and providing information on organ donation and kidney transplantation. The aim of the national PROCARE consortium is to develop improved matching algorithms that will lead to a prolonged survival of transplanted donor kidneys and a reduced HLA immunization. The latter will positively affect the waiting time for a retransplantation. The present algorithm for allocation is among others based on matching for HLA antigens, which were originally defined by antibodies using serological typing techniques. However, several studies suggest that this algorithm needs adaptation and that other immune parameters which are currently not included may assist in improving graft survival rates. We will employ a multicenter-based evaluation on 5429 patients transplanted between 1995 and 2005 in the Netherlands. The association between key clinical endpoints and selected laboratory defined parameters will be examined, including Luminex-defined HLA antibody specificities, T and B cell epitopes recognized on the mismatched HLA antigens, non-HLA antibodies, and also polymorphisms in complement and Fc receptors functionally associated with effector functions of anti-graft antibodies. From these data, key parameters determining the success of kidney transplantation will be identified which will lead to the identification of additional parameters to be included in future matching algorithms aiming to extend survival of transplanted kidneys and to diminish HLA immunization. Computer simulation studies will reveal the number of patients having a direct benefit from improved matching, the effect on shortening of the waiting list, and the decrease in waiting time.

Pretransplant HLA mistyping in diagnostic samples of acute myeloid leukemia patients due to acquired uniparental disomy.

Although acquired uniparental disomy (aUPD) has been reported in relapse acute myeloid leukemia (AML), pretransplant aUPD involving chromosome 6 is poorly documented. Such events could be of interest because loss of heterozygosity (LOH) resulting from aUPD in leukemic cells may lead to erroneous results if HLA typing for hematopoietic stem cell donor searches is performed on blood samples drawn during blastic crisis. We report here six AML patients whose HLA typing was performed on DNA extracted from peripheral blood obtained at diagnosis. We observed LOH involving the entire HLA region (three patients), HLA-A, B, C (two patients) and HLA-A only (one patient). An array-comparative genomic hybridization showed that copy number was neutral for all loci, thus revealing partial aUPD of chromosome 6p21. When HLA typing was performed on remission blood samples both haplotypes were detected. A 3-4% LOH incidence was estimated in AML patients with high blast counts. Based on DNA mixing experiments, we determined by PCR sequence-specific oligonucleotide hybridization on microbeads arrays a detection threshold for HLA-A, B, DRB1 heterozygosity in blood samples with <80% blasts. Because aUPD may be partial, any homozygous HLA result should be confirmed by a second typing performed on buccal swabs or on blood samples from the patient in remission.

Variable EBV DNA load distributions and heterogeneous EBV mRNA expression patterns in the circulation of solid organ versus stem cell transplant recipients.

UNLABELLED: Epstein-Barr virus (EBV) driven post-transplant lymphoproliferative disease (PTLD) is a heterogeneous and potentially life-threatening condition. Early identification of aberrant EBV activity may prevent progression to B-cell lymphoma. We measured EBV DNA load and RNA profiles in plasma and cellular blood compartments of stem cell transplant (SCT; n = 5), solid organ transplant recipients (SOT; n = 15), and SOT having chronic elevated EBV-DNA load (n = 12). In SCT, EBV DNA was heterogeneously distributed, either in plasma or leukocytes or both. In SOT, EBV DNA load was always cell associated, predominantly in B cells, but occasionally in T cells (CD4 and CD8) or monocytes. All SCT with cell-associated EBV DNA showed BARTs and EBNA1 expression, while LMP1 and LMP2 mRNA was found in 1 and 3 cases, respectively. In SOT, expression of BARTs was detected in all leukocyte samples. LMP2 and EBNA1 mRNA was found in 5/15 and 2/15, respectively, but LMP1 mRNA in only 1, coinciding with severe PTLD and high EBV DNA. CONCLUSION: EBV DNA is differently distributed between white cells and plasma in SOT versus SCT. EBV RNA profiling in blood is feasible and may have added value for understanding pathogenic virus activity in patients with elevated EBV-DNA.

Association of complement C3 gene variants with renal transplant outcome of deceased cardiac dead donor kidneys.

Local renal complement activation by the donor kidney plays an important role in the pathogenesis of renal injury inherent to kidney transplantation. Contradictory results were reported about the protective effects of the donor C3F allotype on renal allograft outcome. We investigated the influence of the donor C3F allotype on renal transplant outcome, taking all different donor types into account. C3 allotypes of 1265 donor-recipient pairs were determined and divided into four genotypic groups according to the C3F allotype of the donor and the recipient. The four genotypic groups were analyzed for association with primary nonfunction (PNF), delayed graft function, acute rejection, death-censored graft survival and patient survival. Considering all donor types, multivariable analysis found no association of the donor C3F allotype with renal allograft outcome. Also, for living and deceased brain-dead donors, no association with allograft outcome was found. Post hoc subgroup analysis within deceased cardiac dead (DCD) donors revealed an independent protective association of donor C3F allotype with PNF. This study shows that the donor C3F allotype is not associated with renal allograft outcome after kidney transplantation. Subgroup analysis within DCD donors revealed an independent protective association of the donor C3F allotype with PNF, which is preliminary and warrants further validation.

Donor kidney adapts to body dimensions of recipient: no influence of donor gender on renal function after transplantation.

Female kidneys and kidneys from small donors have been suggested to perform worse after kidney transplantation. Here, we evaluate the impact of gender and body dimensions on posttransplantation GFR in living donor transplantation. Two hundred and ninety-three donor-recipient pairs, who were transplanted at our center were evaluated. All pairs had detailed renal function measurement ((125) I-iothalamate and (131) I-hippuran) 4 months predonation in the donor and 2.5 months posttransplantation in donor and recipient. For 88 pairs, 5 years of recipient follow-up was available. Delta GFR was calculated as (recipient GFR-donor single kidney GFR). Recipients of both male and female kidneys had similar renal function at early and long term after transplantation. Male recipients had higher ERPF, ΔGFR and ΔERPF at both time points. Kidneys of donors smaller than their recipient had higher ΔGFR and ΔERPF than kidneys of larger donors at both time points (p < 0.05). In multivariate analysis, ΔGFR was predicted by donor/recipient BSA-ratio together with transplantation related factors (R(2) 0.19), irrespective of donor and recipient gender. In conclusion, in living donor transplantation, female kidneys perform as well as male donor kidneys. Kidneys adapt to the recipient's body size and demands, independent of gender, without detrimental effects in renal function and outcome up to mid-long term.

Rapid quantitative PCR assays for the simultaneous detection of herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6 DNA in blood and other clinical specimens.

Rapid diagnosis of human herpesvirus primary infections or reactivations is facilitated by quantitative PCRs. Quantitative PCR assays with a standard thermal cycling profile permitting simultaneous detection of herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV6) DNA were developed and validated for diagnostic use. High specificity and sensitivity were achieved and the new PCR assays correlated well with commercial PCR assays. Twenty two thousand eight hundred sixty eight PCR tests were undertaken on specimens obtained from immunosuppressed patients. DNAemia was frequent with EBV (43.5%), HHV6 (32.4%), CMV (12.8%), and VZV (12.9%). As already described for EBV and CMV, high virus loads of HHV6 and VZV were associated with clinical symptoms and poor clinical outcome, for example, three of four patients with VZV virus loads >10(5) copies/ml died. A high proportion of lower respiratory specimens was positive for EBV- (38.8%), HHV6- (29.4%), and CMV-DNA (18.2%). For CMV, infection was confirmed in 66.7% of patients by virus isolation or positive pp65 antigenaemia. Differentiation of HHV6A, -B and HSV-1, -2 by melting curve analysis revealed that HHV6A and HSV-2 represented only 1.8% and 3.3% of all positive specimens, respectively. In conclusion, these results indicate significant improvements for the early diagnosis of primary infections or reactivations of five human herpesviruses especially in immunosuppressed patients. Detection of coinfections with multiple herpesviruses is facilitated. Quantitative results enable monitoring of virus load during antiviral therapy. A standard thermal cycling profile permits time and cost effective use in a routine diagnostic setting.

Heme oxygenase-1 genotype of the donor is associated with graft survival after liver transplantation.

Heme oxygenase-1 (HO-1) has been suggested as a cytoprotective gene during liver transplantation. Inducibility of HO-1 is modulated by a (GT)(n) polymorphism and a single nucleotide polymorphism (SNP) A(-413)T in the promoter. Both a short (GT)(n) allele and the A-allele have been associated with increased HO-1 promoter activity. In 308 liver transplantations, we assessed donor HO-1 genotype and correlated this with outcome variables. For (GT)(n) genotype, livers were divided into two classes: short alleles (<25 repeats; class S) and long alleles (> or =25 repeats; class L). In a subset, hepatic messenger ribonucleic acid (mRNA) expression was correlated with genotypes. Graft survival at 1 year was significantly better for A-allele genotype compared to TT-genotype (84% vs. 63%, p = 0.004). Graft loss due to primary dysfunction (PDF) occurred more frequently in TT-genotype compared to A-receivers (p = 0.03). Recipients of a liver with TT-genotype had significantly higher serum transaminases after transplantation and hepatic HO-1 mRNA levels were significantly lower compared to the A-allele livers (p = 0.03). No differences were found for any outcome variable between class S and LL-variant of the (GT)(n) polymorphism. Haplotype analysis confirmed dominance of the A(-413)T SNP over the (GT)(n) polymorphism. In conclusion, HO-1 genotype is associated with outcome after liver transplantation. These findings suggest that HO-1 mediates graft survival after liver transplantation.

Curaçao patients with coronary artery disease have a higher prevalence of the HFE C282Y mutation.

Epidemiological studies indicate a positive relation between iron status and coronary artery disease (CAD) risk The HFE C282Y allele is associated with increased iron status and higher CAD risk. We investigated whether HFE C282Ymight be a CAD risk factor in Curaçao in a case-control study design. The patient group comprised 42 men and 10 women. Fifty-four men and 30 women without history of CAD served as age and gender matched controls. HFE C282Y genotypes were established using sequence-specific priming polymerase chain reaction. None of the investigated subjects were homozygous for HFE C282Y, whereas 5/52 (9.6%) CAD patients and 1/84 controls (1.2%) were heterozygous for HFE C282Y (p = 0.03). The HFE C282Y mutation was 8.8 fold (95% CI 1.001, 77.8; p = 0.049) more prevalent in CAD patients than in controls. The HFE C282Y allele frequency in Curaçao is higher than that of African populations, but comparable with that of Jamaica. We conclude that Curaçao CAD patients have somewhat higher frequency of HFE C282Y heterozygosity than controls, and that the HFE C282Y allele frequency in the Curaçao population is higher than might be expected in persons of African descent. The consequences of HFE C282Y heterozygosity as CAD risk factor are as yet uncertain, since there is no proof that iron lowering reduces CAD risk.

Curaçao patients with coronary artery disease have a higher prevalence of the HFE C282Y mutation

Epidemiological studies indicate a positive relation between iron status and coronary artery disease (CAD) risk The HFE C282Y allele is associated with increased iron status and higher CAD risk. We investigated whether HFE C282Ymight be a CAD risk factor in Curaçao in a case-control study design. The patient group comprised 42 men and 10 women. Fifty-four men and 30 women without history of CAD served as age and gender matched controls. HFE C282Y genotypes were established using sequence-specific priming polymerase chain reaction. None of the investigated subjects were homozygous for HFE C282Y, whereas 5/52 (9.6) CAD patients and 1/84 controls (1.2) were heterozygous for HFE C282Y (p = 0.03). The HFE C282Y mutation was 8.8 fold (95 CI 1.001, 77.8; p = 0.049) more prevalent in CAD patients than in controls. The HFE C282Y allele frequency in Curaçao is higher than that of African populations, but comparable with that of Jamaica. We conclude that Curaçao CAD patients have somewhat higher frequency of HFE C282Y heterozygosity than controls, and that the HFE C282Y allele frequency in the Curaçao population is higher than might be expected in persons of African descent. The consequences of HFE C282Y heterozygosity as CAD risk factor are as yet uncertain, since there is no proof that iron lowering reduces CAD risk

Identification of three new DRB1 alleles, DRB1*0107, *0425 and *13012 and confirmation of DRB4*01033.

The characterization of three novel DRB1 alleles is described, DRB1*0107, DRB1*0425 and DRB1*13012 as well as confirmation of DRB4*01033. Two alleles, DRB1*0107 and *0425, showed amino acid differences with previously identified HLA molecules. In DRB1*0107, the glutamine at position 10 was substituted by a glutamic acid. DRB1*0425 showed one amino acid difference with DRB1*0418 (I to F) at position 67, and five amino acid differences with DRB1*04011 at positions 67 (L to F), 70 (Q to D), 71 (K to R), 74 (A to L) and 86 (G to V). The alleles DRB1*13012 and DRB4*01033 had protein sequences identical to DRB1*13011 and DRB4*01031/01032, respectively. Nucleotide differences were present at position 306 for DRB1*13012 and at position 321 for DRB4*01033.

Identification of a new HLA-B*40 variant, B*4035.

In this report, the novel allele B*40351 is presented. The allele was identified in a Caucasian individual by sequence-based typing. B*4035 is identical to B*4002 in exon 2, but differs in exon 3 at position 463, where it has an A in stead of a C. This results in an amino acid change from arginine to serine at codon 131 of the mature protein. The haplotype carrying the B*4035 was A3 B*4035 Cw2 DR11 DQ3.

Molecular and serological identification of the HLA-A*3404 allele.

In this report we describe a novel HLA-A*34 allele, A*3404, which was initially detected by an unusual serological pattern in two unrelated individuals. Sequencing revealed that the new allele was identical to A*3402 in exons 2 and 3, except for a single nucleotide difference at position 238, changing codon 56 from glycine to arginine. The codon change resulted in positive serological reactions with several sera recognizing A30 and/or A31, implicating an important role for this position in epitope recognition. The allele was identified twice on the haplotype A*3404, B*1402, Cw*0802, DRB1*14, DQB1*05.

Identification of two new HLA-B22 variants, HLA-B*5509 and B*5606.

In our recent study using high-resolution HLA-B locus typing by sequence-based typing (SBT) we identified 9 new alleles in a total of 355 unrelated individuals (4). Three of them concerned an allele belonging to the B22 group. One of them, B*5607, showed the unusual presence of a Bw4 sequence motif, as described previously (5). In this report the other two B22 variants are described; one belonging to the B55 specificity and named B*5509; the other one being a B*56 allele and assigned B*5606, which brings the total number of alleles belonging to the B22 group to 18.

The influence of tumor necrosis factor-alpha and interleukin-10 gene promoter polymorphism on the inflammatory response in experimental human endotoxemia.

In this study, we show that there is no correlation between tumor necrosis factor-alpha gene promoter polymorphism at position -308, interleukin-10 gene promoter polymorphism at position -1082, and the cytokine levels they produce in the human endotoxemia model.

Rapid decreases in donor-specific cytotoxic T lymphocyte precursor frequencies and graft outcome after liver and lung transplantation.

BACKGROUND: A decrease in donor-specific T cell precursor frequencies as seen late, one or more years, after transplantation is assumed to reflect transplantation tolerance, a condition important for long term acceptance of the allograft. However, such late decreases also occur in recipients that developed chronic transplant dysfunction questioning its relevance in transplantation tolerance. We investigated whether early, i.e., the first 6 months, decreases in donor-specific T cell precursor frequencies reflect transplantation tolerance and predict graft outcome after liver and lung transplantation. METHODS: Donor and third party specific cytotoxic (CTLp) and helper T lymphocyte precursor (HTLp) frequencies were analyzed in pretransplant and 1 (or 2) and 6-month blood samples taken from liver and lung recipients and were correlated with graft outcome. RESULTS: In liver allograft recipients with good graft function (n=7), mean donor-specific CTLp frequencies decreased as early as 1 month after transplantation and remained low thereafter. In contrast, mean CTLp frequencies did not decrease in liver allograft recipients with chronic transplant dysfunction (n=6). In lung allograft recipients, donor-specific CTLp frequencies remained relatively high and frequencies were not different between recipients without (n=6) or with (n=6) chronic transplant dysfunction. Donor-specific HTLp frequencies did not change significantly after liver or lung transplantation and did not differ between recipients without or with chronic transplant dysfunction. CONCLUSIONS: An early decrease in donor-specific CTLp correlates with good graft outcome after liver transplantation. Such rapid decreases in alloreactivity do not occur after lung transplantation illustrating the unique capacity of liver allografts to induce transplantation tolerance.