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.

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.

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.

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.

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.

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.

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.

Decreased donor-specific cytotoxic T cell precursor frequencies one year after clinical lung transplantation do not reflect transplantation tolerance: a comparison of lung transplant recipients with or without bronchiolitis obliterans syndrome.

BACKGROUND: Decreased in vitro T cell alloreactivity, demonstrated by decreased frequencies of peripheral blood donor-specific T cell precursors, may reflect a tolerant state after transplantation and lower the risk for development of chronic graft dysfunction. It is unknown whether a decrease in donor-specific T cell frequencies also occurs after clinical lung transplantation and if such a decrease lowers the risk for bronchiolitis obliterans syndrome (BOS), a hallmark of chronic graft dysfunction. Therefore, we compared changes in posttransplant donor-specific cytotoxic T lymphocyte (CTLp) and helper T lymphocyte precursor (HTLp) frequencies in lung allograft recipients with good graft function and in recipients with BOS. METHODS: Donor and third party specific CTLp and HTLp frequencies were determined by limiting dilution assay in pre- and posttransplant (1 year) peripheral blood samples of lung allograft recipients with good graft function (n = 13) and BOS (n = 10). RESULTS: In recipients with good graft function, mean donor-specific CTLp frequencies decreased after transplantation (183 vs. 16 precursors before and after transplantation, respectively). Additionally, HTLp frequencies decreased but this was not specific for donor alloantigens because third party-specific HTLp frequencies decreased also. Surprisingly, recipients with BOS also showed a decrease in mean donor-specific CTLp frequencies after transplantation (332 vs. 49 precursors before and after transplantation, respectively). Again, HTLp frequencies decreased nonspecifically. CONCLUSIONS: We conclude that donor-specific CTLp frequencies decrease after lung transplantation, but that this does not result in transplantation tolerance protecting the lung against the development of chronic graft dysfunction.

Activation of alloreactive T cells by allogeneic nonprofessional antigen-presenting cells and interleukin-12 from bystander autologous professional antigen-presenting cells.

BACKGROUND: After solid organ transplantation most alloantigens are presented to the recipient's immune system by normal tissue cells, which can be considered to act as nonprofessional antigen-presenting cells (APC). It is well accepted that such nonprofessional APC fail to activate recipient resting T cells due to their inability to deliver costimulatory activity. In our study, we tested a hypothesis that such costimulatory activity may be provided by "bystander" recipient professional APC. METHODS: We set up mixed lymphocyte cultures (MLC) of purified T cell responders and T cell stimulator cells, the latter as nonprofessional APC carrying allogeneic MHC class I and II, and tested if responder-type autologous APC could facilitate responder T cell proliferation. In this assay also the effects of anti-CD28 antibody and interleukin- (IL) 1beta, IL-6, or IL-12 mediated costimulation on responder T cell proliferation and IL-2 production were investigated. RESULTS: Autologous APC, i.e., monocytes, were found to facilitate the proliferative response of resting T cells stimulated by allogeneic nonprofessional APC. IL-12 was identified as the most important costimulatory factor for induction of proliferation. IL-1beta enhanced IL-2 production and proliferation of allostimulated resting T cells but its presence was not essential. Although CD28 triggering alone was ineffective, this costimulatory pathway enhanced IL-2 production and proliferation when combined with IL-12 or IL-1beta. CONCLUSIONS: We conclude that costimulatory activity for activation of resting human T cells by nonprofessional donor APC can be delivered through activity of bystander recipient-type autologous APC. This mechanism of allostimulation may contribute to the induction and perpetuation of alloreactivity "in vivo" in a time frame when intragraft professional donor-type APC have been replaced with professional recipient-type APC.

Donor-specific hyporeactivity after liver transplantation: prominent decreases in donor-specific cytotoxic T lymphocyte precursor frequencies independent of changes in helper T lymphocyte precursor frequencies or suppressor cell activity.

BACKGROUND: The development of immunological donor-specific hyporeactivity may account for the low incidence of chronic rejection after clinical liver transplantation. We investigated whether hyporeactivity commonly develops after liver transplantation by analyzing precursor frequencies of donor-reactive cytotoxic (CTLp) and helper (HTLp) T lymphocytes and mixed lymphocyte culture (MLC) reactivity in liver allograft recipients. We further studied whether CTLp hyporeactivity correlated with changes in donor-specific HTLp frequencies or suppressor cell activity. METHODS: CTLp and HTLp frequencies and MLC reactivity against donor and third-party spleen cells were determined in pre- and posttransplantation peripheral blood samples from 18 recipients with good graft function 2 years after transplantation. By mixing posttransplantation samples (with "putative" suppressor cell activity) with pretransplantation samples (in which normal CTL activity with no suppressor cell activity is expected), the presence of suppressor cell activity in peripheral blood was analyzed. RESULTS: Two years after transplantation, all but one (94%) of the recipients had developed CTLp hyporeactivity as evidenced by reduced donor-specific CTLp frequencies. The development of hyporeactivity was not specific for any particular underlying disease. The occurrence of HTL hyporeactivity, however, was less frequent: 38% and 20% of recipients were HTLp and MLC hyporeactive, respectively. Decreases in CTLp frequencies did not correlate with decreased donor-specific HTL function or suppressor cell activity in peripheral blood samples. CONCLUSIONS: Donor-specific CTLp hyporeactivity can develop in the majority of liver allograft recipients, irrespective of underlying disease. Donor-specific HTL hyporeactivity, however, occurs infrequently. A reduction in donor-specific CTLp frequencies was found to be independent of changes in donor-specific HTLp or suppressor cell activity, suggesting that other mechanisms (e.g., clonal deletion) are operative in the reduction of donor-specific CTLp after liver transplantation.

Long-term outcome of lung transplantation is predicted by the number of HLA-DR mismatches.

BACKGROUND: The importance of HLA mismatch in determining long-term outcome in lung transplantation remains largely uncertain. METHODS: A retrospective analysis of 102 consecutive primary lung transplants was performed to identify risk factors for poor long-term outcome after lung transplantation defined as graft survival and bronchiolitis obliterans syndrome (BOS) stage I and II. Variables included were patient characteristics (age, sex, prior diagnosis), the number of HLA mismatches between donor and recipient, cold ischemic time, cytomegalovirus serologic concordance, number of acute rejections, and time to first rejection. Variables carrying significance in a univariate analysis were subjected to a proportional hazard regression analysis. RESULTS: In the multivariate analysis, an increased number of acute rejections correlated positively with decreased graft survival (risk ratio [RR] = 1.25; 95% confidence interval [CI], 1.05-1.5; P = 0.011), development of BOS stage I (RR = 1.36/episode; 95% CI, 1.16-1.58;P < 0.001), and BOS stage II (RR = 1.42/episode; 95% CI, 1.2-1.67; P < 0.001). An increased time to rejection correlated positively with reduced graft survival (RR = 1.03/day; 95% CI, 1.01-1.06; P = 0.02), and BOS stage I and II (both RR = 1.04/day; 95% CI, 1.01-1.07; P < 0.005). Compared with 2 HLA-DR mismatches, 0 or 1 mismatch was associated with improved graft survival (RR = 0.43; 95% CI, 0.19-0.98; P = 0.045) and protected against development of BOS stage I (RR = 0.47; 95% CI, 0.23-0.98; P = 0.044) and BOS stage II (RR = 0.35; 95% CI, 0.15-0.83; P = 0.017). CONCLUSIONS: HLA-DR mismatching appears to be a risk factor for the development of BOS and graft loss. Improved outcome after lung transplantation might be achieved with prospective matching for HLA-DR. Alternatively, the amount and type of immunosuppressive drugs may be guided by the degree of HLA-DR (mis)matching.

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.

Mutual tolerance after liver and not after heart transplantation? Evaluation of patient-anti-donor and donor-anti-patient responses by mixed lymphocyte culture.

The ultimate goal in organ transplantation is the induction of donor-specific transplantation tolerance. The fact that in some patients it is possible to withdraw immunosuppressive therapy completely, suggests that immunological adaptation or donor-specific nonresponsiveness can occur following transplantation. In earlier studies we have shown that after blood transfusion, the mixed lymphocyte reactivity of the donor against patient peripheral blood mononuclear lymphocytes taken after blood transfusion gradually decreased with time. This may reflect the induction of an immunoregulatory mechanism, which protects the recipient against an immune reaction of the donor, enhancing a state of mixed chimerism. A similar phenomenon might also play a role in the immunological mechanism leading to transplantation tolerance. Therefore, we studied responses in patients with a well-functioning liver and heart transplant using a primed lymphocyte test (PLT) and a mixed lymphocyte culture (MLC). Two years after liver transplantation the PLT and MLC responses of patient against donor were decreased significantly compared to the situation before transplantation. The response of donor against patient was also lower two years after transplantation. The decreased responses were donor-specific since responses to third-party cells generally remained unchanged. In heart transplant recipients we could not detect a donor-specific downregulation. The reversed response, of donor against patient, was not different from responses of third-party against patient cells. Therefore, we conclude that donor-specific nonresponsiveness is not induced in patients with well-functioning heart transplants. In contrast, after a successful liver transplantation the response of patient against donor is decreased, as is the reversed response. It may be valuable to test whether in liver transplant patients withdrawing or reducing of maintenance immunosuppression is permitted for patients who appear to have developed two-way donor-specific hyporeactivity.

Biochemical and serological identification of major histocompatibility complex antigens in outbred chickens.

Serology and biochemistry were used to identify major histocompatibility complex (MHC) types in chicken lines selected for high and low antibody response to sheep red blood cells. Serological typing was performed by direct haemagglutination, using antisera obtained by erythrocyte alloimmunization within the lines. Four serotypes were identified, called preliminarily B114, B119, B121 and B124. Subsequently, these types were characterized for their B-G and B-F products by biochemical analysis, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and one-dimensional IEF (isoelectric focusing) respectively. The B114, B119 and B121 serotypes displayed each characteristic banding patterns for both B-G and B-F. No additional types or subtypes were identified by biochemistry within these serotypes. The B124 serotype however could be subtyped into three different haplotypes with specific banding patterns for B-G and B-F. None of the haplotypes in the selection lines were identical for both B-G and B-F with the tested reference B haplotypes. Comparison of B-G alleles revealed similar, but not identical B-G patterns for B114 and B-G14, whereas B124C and B-G23, as well as B119 and B-G19 displayed indistinguishable patterns. For B-F, only B121 and B-F21 banding patterns were indistinguishable by IEF. All other B-F types differed from the reference types.

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.

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.

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.

Biochemical identification of B-F and B-G allelic variants of the chicken major histocompatibility complex.

Biochemical methods were used to analyse B-F and B-G antigens of the chicken major histocompatibility complex (MHC). In a panel of 12 inbred or partially inbred chicken lines the MHC haplotypes, originally defined by serological and histogenetical methods, were compared. Using monoclonal 18-6G2, allele-specific B-G patterns were obtained by immunoblotting. Comparison of B-G12 and B-G2 revealed a shared banding pattern, but additional products were detected for B-G12. The B-F products of B2 and B12 had identical IEF patterns. The identical B-F products and partially shared B-G products might explain the serological cross-reaction between these haplotypes. In addition, the IEF pattern of B-F21 appeared similar to B-F2 and B-F12, but the partial proteolysis map showed a clear difference. Although two B-F bands could be detected per haplotype, no evidence for the expression of more than one B-F locus was found. The biochemical methods enabled a precise definition of expressed MHC products and can be a useful tool for the identification of B-alleles in other chicken lines or outbred chickens for their MHC antigens.

Glycolytic activity in human red cell populations separated by a combination of density and counterflow centrifugation. Evidence for an improved separation of red cells according to age.

Human red blood cells were separated by a discontinuous Percoll density gradient into fractions of increasing density. Red cells comprising the lowest and highest density fractions, respectively, were subsequently separated according to mean cell volume (MCV) by means of counterflow centrifugation. The activities of 4 red cell age-dependent enzymes (hexokinase (HK), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD) and aspartate aminotransferase (ASAT) were highest in the red cell fraction with low density/large MCV, although the difference from red cell enzyme activities in the total low density fraction was not significant. These 4 enzyme activities were lowest in the fraction of red cells with high density/small MCV. The relative activities of the enzymes in the high density/small MCV fraction, as compared to the unseparated cell population, were: HK (58%), PK (49%), G6PD (53%) and ASAT (28%). These activities were all significantly lower than those measured in the total high density red cell fraction. The rates of lactate production in the low density/large MCV cells (0.89 +/- 0.15 mumol X min-1 X 10(-11) cells) is approximately 3-fold higher than in high density/small MCV cells (0.33 +/- 0.03 mumol X min-1 X 10(-11) cells). This latter value is 1.8-fold lower than the rate of lactate production in the total high density red cell fraction (0.59 +/- 0.14 mumol X min-1 X 10(-11) cells) and is, in contrast to lactate production in other density/size fractions, insensitive to phosphate as a metabolic stimulus. It is argued that the combination of density gradient and counter-flow centrifugation offers a greater potential for obtaining an old red cell population than classical red cell density centrifugation alone.