Should HLA mismatch acceptability for sensitized transplant candidates be determined at the high-resolution rather than the antigen level?

Defining HLA mismatch acceptability of organ transplant donors for sensitized recipients has traditionally been based on serologically defined HLA antigens. Now, however, it is well accepted that HLA antibodies specifically recognize a wide range of epitopes present on HLA antigens and that molecularly defined high resolution alleles corresponding to the same low resolution antigen can possess different epitope repertoires. Hence, determination of HLA compatibility at the allele level represents a more accurate approach to identify suitable donors for sensitized patients. This approach would offer opportunities for increased transplant rates and improved long term graft survivals.

Center-defined unacceptable HLA antigens facilitate transplants for sensitized patients in a multi-center kidney exchange program.

Multi-center kidney paired donation (KPD) is an exciting new transplant option that has not yet approached its full potential. One barrier to progress is accurate virtual crossmatching for KPD waitlists with many highly sensitized patients. Virtual crossmatch results from a large multi-center consortium, the National Kidney Registry (NKR), were analyzed to determine the effectiveness of flexible center-specific criteria for virtual crossmatching. Approximately two-thirds of the patients on the NKR waitlist are highly sensitized (>80% CPRA). These patients have antibodies against HLA-A (63%), HLA-B (66%), HLA-C (41%), HLA-DRB1 (60%), HLA-DRB3/4/5 (18-22%), HLA-DQB1 (54%) and HLA-DPB1 (26%). With donors typed for these loci before activation, 91% of virtual crossmatches accurately predicted an acceptable cell-based donor crossmatch. Failed virtual crossmatches were attributed to equivocal virtual crossmatches (46%), changes in HLA antibodies (21%), antibodies against HLA-DQA (6%), transcription errors (6%), suspected non-HLA antibodies (5%), allele-specific antibodies (1%) and unknown causes (15%). Some failed crossmatches could be prevented by modifiable factors such as more frequent assessment of HLA antibodies, DQA1 typing of donors and auditing data entry. Importantly, when transplant centers have flexibility to define crossmatch criteria, it is currently feasible to use virtual crossmatching for highly sensitized patients to reliably predict acceptable cell-based crossmatches.

Dynamic challenges inhibiting optimal adoption of kidney paired donation: findings of a consensus conference.

While kidney paired donation (KPD) enables the utilization of living donor kidneys from healthy and willing donors incompatible with their intended recipients, the strategy poses complex challenges that have limited its adoption in United States and Canada. A consensus conference was convened March 29-30, 2012 to address the dynamic challenges and complexities of KPD that inhibit optimal implementation. Stakeholders considered donor evaluation and care, histocompatibility testing, allocation algorithms, financing, geographic challenges and implementation strategies with the goal to safely maximize KPD at every transplant center. Best practices, knowledge gaps and research goals were identified and summarized in this document.

Beta-adrenergic receptor polymorphisms and cardiac graft function in potential organ donors.

Prior studies have demonstrated associations between beta-adrenergic receptor (ßAR) polymorphisms and left ventricular dysfunction-an important cause of allograft nonutilization for transplantation. We hypothesized that ßAR polymorphisms predispose donor hearts to LV dysfunction after brain death. A total of 1043 organ donors managed from 2001-2006 were initially studied. The following ßAR single nucleotide polymorphisms were genotyped: ß1AR 1165C/G (Arg389Gly), ß1AR 145A/G (Ser49Gly), ß2AR 46G/A (Gly16Arg) and ß2AR 79C/G (Gln27Glu). In multivariable regression analyses, the ß2AR46 SNP was significantly associated with LV systolic dysfunction, with each minor allele additively decreasing the odds for LV ejection fraction <50%. The ß1AR1165 and ß2AR46 SNPs were associated with higher dopamine requirement during the donor management period: donors with the GG and AA genotypes had ORs of 2.64 (95% CI 1.52-4.57) and 2.70 (1.07-2.74) respectively for requiring >10 µg/kg/min of dopamine compared to those with the CC and GG genotypes. However, no significant associations were found between ßAR SNPs and cardiac dysfunction in 364 donors managed from 2007-2008, perhaps due to changes in donor management, lack of power in this validation cohort, or the absence of a true association. ßAR polymorphisms may be associated with cardiac dysfunction after brain death, but these relationships require further study in independent donor cohorts.

The 4G/4G genotype of the PAI-1 (serpine-1) 4G/5G polymorphism is associated with decreased lung allograft utilization.

Widespread thrombi are found among donor lungs rejected for transplantation. The 4G/5G polymorphism in the plasminogen activator inhibitor (PAI-1) gene impacts transcription and the 4G allele is associated with increased PAI-1 levels. We hypothesized that the 4G/4G genotype would be associated with decreased lung graft utilization, potentially because of worse oxygenation in the donor. We genotyped donors managed by the California Transplant Donor Network from 2001 to 2008 for the 4G/5G polymorphism in the PAI-1 gene. Non-Hispanic donors from 2001 to 2005 defined the discovery cohort (n = 519), whereas donors from 2006 to 2008 defined the validation cohort (n = 369). We found, that the odds of successful lung utilization among Non-Hispanic white donors were lower among donors with the 4G/4G genotype compared to those without this genotype in both the discovery (OR = 0.55, 95% CI = 0.3-0.9, p = 0.02) and validation (OR = 0.5, 95% CI = 0.3-0.9, p = 0.03) cohorts. This relationship was independent of age, gender, cause of death, drug use and history of smoking. Donors with the 4G/4G genotype also had a lower PaO2/FiO2 ratio (p = 0.03) and fewer donors with the 4G/4G genotype achieved the threshold PaO2/FiO2 ratio ≥ 300 (p = 0.05). These findings suggest a role for impaired fibrinolysis resulting in worse gas exchange and decreased donor utilization.

Strategies and technical challenges in allele level Class II typing in 2578 bone marrow transplantation donor-recipient pairs.

Human leukocyte antigen typing of 2578 donor-recipient pairs whose transplantation was facilitated by the National Marrow Donor Program allowed for an in-depth analysis of the accuracy of high-volume allele level testing data. The methods employed provided allele level typing at DRB1/3/5, DQA1, DQB1, DPA1, and DPB1 using sequence-specific oligonucleotide probe hybridization (SSOPH), polymerase chain reaction (PCR) restriction fragment length polymorphism analysis, sequence specific PCR, and direct sequence-based typing (SBT). Each typing was independently tested by two laboratories in Phase 1, and in subsequent phases targeted samples were typed in duplicate by SBT to monitor typing quality. Comparison with prior transplant center typing was also evaluated. SSOPH detected discrepancies ranged from 0.6% at DPB1 to 5.1% at DQB1 in Phase 1. The majority of discrepancies, 62%, resulted from human error such as sample handling, result interpretation, or clerical errors. Alleles that are frequently discrepant have been identified in this predominantly white population.

Increasing mixed chimerism and the risk of graft loss in children undergoing allogeneic hematopoietic stem cell transplantation for non-malignant disorders.

We performed quantitative PCR-based serial chimerism testing of whole blood (WB) and CD3+ cells and retrospectively correlated the results of chimerism tests and the risk of graft loss in children undergoing transplant for non-malignant disorders. Twenty-four children were included in this study. All patients initially engrafted; subsequently, 12% lost the graft, 21% achieved complete donor chimerism and 67% had mixed chimerism (MC). Patients underwent delayed taper of cyclosporine (CsA) if they had MC. Overall survival was 87+/-7% (s.d.) at 5-years post transplant, and it was not affected by chimerism status. Both WB and CD3+ chimerism showed significant fluctuations with a peak in autologous cell signal occurring at a median of 7 months for WB and 2 months for CD3+ cells. Initial post transplant chimerism percentage in either WB or CD3+ lineage was not related to graft loss. Increasing MC to >30% host cells was seen in 33% of patients, and it was related to increased risk of graft loss, as previously published. However, 63% of children with increasing MC did not lose their graft. Additional studies of post transplant chimerism are required to improve our ability to accurately identify children at risk of graft loss following transplant for non-malignant disorders.

HLA gene amplification and hybridization analysis of polymorphism. HLA matching for bone marrow transplantation of a patient with HLA-deficient severe combined immunodeficiency syndrome.

The treatment of choice for certain immunodeficiency syndromes and hematological disorders is bone marrow transplantation (BMT). The success of BMT is influenced by the degree of HLA compatibility between recipient and donor. However, aberrant expression of HLA sometimes makes it difficult, if not impossible, to determine the patient's HLA type by standard serological and cellular techniques. We describe here the application of new molecular biological techniques to perform high resolution HLA typing independent of HLA expression. A patient with HLA-deficient severe combined deficiency was HLA typed using in vitro amplification of the HLA genes and sequence-specific oligonucleotide probe hybridization (SSOPH). Two major advances provided by this technology are:detection of HLA polymorphism at the level of single amino acid differences; and elimination of a requirement for HLA expression. Although the patient's lymphocytes lacked class II HLA proteins, polymorphism associated with DR7,w53;DQw2;DRw11a (a split of DR5), w52b (a split of DRw52);DQw7 were identified. The patient's class I expression was partially defective, and typing was accomplished by a combination of serological (HLA-A and -C) and SSOPH analysis (HLA-B). Complete patient haplotypes were predicted after typing of family members [A2;B35(w6); Cw4; DRw11a(w52b);DQw7 and A2;B13(w4); Cw6;DR7(w53); DQw2]. Potential unrelated donors were typed and a donor was selected for BMT.

Reduced intensity allogeneic umbilical cord blood transplantation in children and adolescent recipients with malignant and non-malignant diseases.

There is a significant amount of morbidity and mortality following myeloablative umbilical cord blood transplantation (UCBT). Reduced intensity (RI) conditioning offers an alternative to myeloablative conditioning before UCBT. We investigated RI-UCBT in 21 children and adolescents with malignant (n=14), and non-malignant diseases (n=7). RI conditioning consisted of fludarabine (150-180 mg/m2) with either busulfan (< or = 8 mg/kg)+rabbit antithymocyte globulin (R-ATG; n=16) or cyclophosphamide+R-ATG+/-etoposide (n=5). Human leukocyte antigen match: 4/6 (n=13), 5/6 (n=5) and 6/6 (n=3). The median total nucleated cell and CD34+ cell dose per kilogram were 3.58 x 10(7) and 2.54 x 10(5), respectively. The median time for neutrophil and platelet engraftment was 17.5 and 52 days, respectively. There were six primary graft failures (chronic myelogenous leukemia (CML), beta-thalassemia, hemophagocytic lymphohistiocytosis (HLH) and myelodysplastic syndrome (MDS)). The probability of developing grade II to grade IV acute graft-versus-host disease (GVHD) and chronic GVHD was 28.6 and 16.7%, respectively. Incidence of transplant-related mortality (TRM) was 14%. The 5 years overall survival (OS) in all patients was 59.8%. The 5 years OS for patients with average versus poor-risk malignancy was 77.8 versus 22.2% (P=0.03). RI-UCBT may result in graft failure in specific high-risk chemo-naïve patients (CML, beta-thalassemia, HLH and MDS), but in more heavily pretreated pediatric and adolescent recipients results in rapid engraftment and may be associated with decreased severe GVHD and TRM.

Reduced intensity conditioning using intravenous busulfan, fludarabine and rabbit ATG for children with nonmalignant disorders and CML.

The major problems with busulfan/cyclophosphamide (Bu/Cy)-containing conditioning regimens are acute toxicities and graft failure. To decrease acute toxicities, we have prospectively evaluated a reduced intensity conditioning (RIC) regimen using targeted dosing of i.v. busulfan, fludarabine, and rabbit ATG (Bu/Flu/rATG) in children with diagnoses that historically would have been conditioned with Bu/Cy regimens. Nineteen pediatric patients were enrolled in the study. The donors included HLA-matched and one antigen-mismatched unrelated volunteers (n = 11), unrelated cord blood (n = 1), and related donors (n = 7). Four patients developed graft failure, which occurred between 1 and 8.5 months post transplant. All four of them underwent a second transplantation and 3/4 are alive without evidence of disease. The mean follow-up of living patients is 29.5 +/- s.d. 11 months. Despite excellent 2-year post-transplant overall survival (89 +/- s.d.7%) and event-free survival (74 +/- s.d.10%), the study was closed prematurely due to high graft failure rate (21%). Receiving a transplant from a mismatched unrelated donor was identified as a risk factor for graft failure. The Bu/Flu/rATG RIC regimen was very well tolerated, resulted in excellent overall survival, and provided sustained engraftment in patients undergoing transplant from matched sibling and unrelated donors. However, it did not provide sustained engraftment in the majority of children with nonmalignancies undergoing mismatched unrelated donor transplants.

In vitro biosynthesis of the beta-subunit of the Na+/K+-ATPase in developing brine shrimp: glycosylation and membrane insertion.

We demonstrate here translation, glycosylation, and membrane insertion of the beta-subunit of the Na+/K+-ATPase of the developing brine shrimp, Artemia, in a reticulocyte lysate translation system. The apparent molecular weight of the primary translation product as determined by SDS-PAGE is 33,000 +/- 1000 (n = 7). When microsomal membranes are present during the entire translation period, a new band with an apparent molecular weight of 37,000 +/- 1000 (n = 7) appears. This change in apparent molecular weight is due to the addition of about two N-linked oligosaccharides. The temporal relationship between protein synthesis and glycosylation have also been examined. Glycosylation and membrane insertion could be achieved if membranes were added after completion of about 70% of the peptide chain. However, glycosylation did not occur if membranes were added after the completion of translation of the beta-subunit. The beta-subunit was synthesized on membrane-bound polysomes, where about two N-linked oligosaccharides were added to the growing polypeptide chain. These studies demonstrate that in vitro translation systems will be useful for studying the biosynthesis of the beta-subunit of the brine shrimp, which is a good model system to examine the developmental regulation of the Na+/K+-ATPase.

Molecular cloning of the Na,K-ATPase alpha-subunit in developing brine shrimp and sequence comparison with higher organisms.

We report here the molecular cloning, nucleotide sequence, and predicted amino acid sequence of an alpha-subunit of the developmentally useful model, Artemia. The amino acid sequence shows divergence from that of mammals, birds, Torpedo, and Drosophila. However, regions in the putative ATP binding and transmembrane domains show absolute or high levels of conservation. Major differences occur in the amino-terminal domain and several other hypervariable regions. These differences are consistent with the suggestion that the brine shrimp is a 'fast clock' organism which diverged from the precursors of vertebrates 0.5-1 billion years ago.

Pseudosymmetry in the structure of luteinizing hormone-releasing hormone. Studies on a series of novel analogs.

Pseudosymmetry in the LH-RH structure is described. Eleven analogs of LH-RH (SMALLER THAN Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) have been synthesized by the fragment condensation method and the repetitive excess mixed anhydride method. Multiple substitutions have been made in the LH-RH sequence, which retain the pseudosymmetry of the LH-RH molecule, while presenting fewer problems of synthesis than the corresponding residues in the natural decapeptide. Thus Trp3, Ser4, Tyr5, Gly6, Leu7, and Arg8 residues were replaced by amino acids having similar properties to the residues that they replace. In all but one of the peptides the Gly10-NH2 residue was replaced by ethylamide, while in the remaining peptide, 1-methyl-5-aminomethyltetrazole (AMT-Me) was substituted at position 10. The compounds were assayed in vitro and in vivo. The following analogs had in vivo and in vitro activities in the range 1-28 percent relative to LH-RH: I, smaller than Glu-His-Phe-Ala-Tyr-Gly-Leu-Arg-Pro-NHEt; II, smaller than Glu-His-Phe-Gly-Tyr-Gly-Leu-Arg-Pro-NHEt; VII, smaller than Glu-His-Phe-Ala-Tyr-Gly-Phe-Arg-Pro-NHEt; IX, smaller than Glu-His-Phe-Ala-Tyr-D-Ala-Leu-Arg-Pro-NHEt; XI, smaller than Glu-His-Phe-Gly-Tyr-Gly-Leu-Arg-Pro-AMT-Me.

Peripherally acting enkephalin analogues. 1. Polar pentapeptides.

The design, synthesis, and biological activity of a series of highly polar enkephalin-related pentapeptides are reported. These analogues incorporate structural features that exclude them from the central nervous system and thereby restrict their action to peripherally located receptors. Hydrophilic analogues were obtained by introduction of polar D-amino acid residues at position 2 and, in certain cases, by conversion of the N-terminal amino group of the Tyr residue to a guanidino function. The peptides were synthesized by classical solution methods. All compounds demonstrated in vitro opioid activity in the GPI and all were shown to possess antinociceptive activity in chemically induced writhing models. The analgesic effects were shown to be predominantly peripherally mediated by antagonism of antinociception with the peripheral antagonist N-methylnalorphine. Comparative data obtained in writhing and hot-plate tests were also supportive of a peripheral mode of action. Compound 13a, L-tyrosyl-D-arginylglycyl-L-4-nitrophenylalanyl-L-prolinamide (BW 443C), was identified as having a favorable pharmacological profile, indicating a high level of peripheral selectivity, and worthy of further investigation.

Peripherally acting enkephalin analogues. 2. Polar tri- and tetrapeptides.

The design, synthesis, and biological activity of a series of D-Arg2-enkephalin-derived tetrapeptide amides and tripeptide aralkylamides are reported. These polar analogues were designed to be excluded from the central nervous system with their action thus limited to peripheral opioid receptors. The effects of the nature of the aromatic ring, aryl ring substitution, and aralkylamine chain length on activity were investigated; in a number of cases the N-terminal amino group of Tyr1 was converted to a guanidino group to further increase hydrophilicity. The peptides were all synthesized by classical solution methodology. The opioid activity of the peptides was assessed in vitro on the guinea pig ileum and their antinociceptive activity was determined in vivo in chemically induced writhing models (peripheral activity) and in the hot-plate test (central activity), in rodents. That the analgesic effects were predominantly mediated in the periphery was demonstrated by antagonism of antinociception by the peripheral opioid antagonist N-methylnalorphine and by comparison of the activities in the writhing and hot-plate tests. As a class, the tetrapeptides were more potent than the tripeptides; N alpha-amidination generally increased activity. A number of compounds exhibited very potent opioid activity and had the desired pharmacological profile, indicating a high degree of peripheral selectivity.

The predictive value of HLA-DR oligotyping for MLC responses.

Comparison of HLA proteins between a patient and potential unrelated marrow donors is difficult because many similar, but not identical, HLA proteins are expressed in the human population. A reliable and practical method to detect these subtle differences is provided by oligotyping, a new technique that identifies polymorphic sequences in the genes encoding the HLA proteins. Oligotyping was used to compare polymorphic HLA-DR sequences in 286 pairs of samples from patients and potential unrelated donors who were serologically matched for HLA-DR specificities. Oligotyping detected HLA-DR differences in 53% of these pairs and all mismatched pairs were reactive in primary mixed lymphocyte cultures. Where HLA-DR disparity was not detected by oligotyping, 37% of the pairs were nonreactive in MLC. The remaining 63% often contained an allele associated with the HLA-DRw11 serological specificity. In the absence of HLA-DRB1*11, oligotyping was predictive of MLC reactivity for samples with HLA-DR2, -DR4, and DRw52. In clinical settings, the ability to predict MLC reactivity on the basis of precise HLA typing provides an alternative to MLC. Further, the relationship between specific polymorphic sequences and reactivity in MLC may lead to more fundamental insights into the mechanisms involved in alloreactive responses.

Influence of pretransplant pregnancy on survival of renal allografts from living donors.

BACKGROUND: The presence of a small number of cells of donor origin in organ transplant recipients (microchimerism) may influence allograft survival and may induce tolerance. Postpartum women may be microchimeric to offspring hematopoietic cells up to 27 years. We hypothesized that mothers receiving renal allografts from offspring would have better graft survival compared with either fathers receiving allografts from offspring, or mothers receiving allografts from nonoffspring donors. METHODS: We analyzed 1803 living related kidney transplants from the UNOS database performed between January 1, 1990, and December 31, 1995, for mothers and fathers who received grafts from offspring with one haplotype match. We also compared these mothers with parous females receiving a kidney from nonoffspring donors (spouse and other biologically related or unrelated family members). A multivariate logistic regression method was used to analyze the effect of donor type, as well as other recipient, donor, and transplant characteristics, on graft and patient survival. RESULTS: Mothers receiving one haplotype-matched offspring renal allografts did not have better graft survival at 1 or 3 years posttransplant compared with fathers receiving similar grafts. There was also no difference in graft or patient survival between mothers receiving kidney grafts from either offspring or nonoffspring donors. Graft survival in mothers with multiple pregnancies was poorer than those with a single pregnancy. CONCLUSIONS: It is possible that persistent microchimerism of fetal cells in maternal circulation may, for some mothers, cause a detectable improvement in graft or patient survival. Comparison of female and male recipients from the UNOS database did not reveal any differences in outcomes. If mothers are tolerant to their offspring, our results indicate that this microchimerism may not improve renal allograft or patient survival in offspring donor to maternal recipient combinations. Lastly, more sensitive pretransplant cross-match assays may need to be implemented in multiparous women, given our results.

Antinociceptive effects of the novel opioid peptide BW443C compared with classical opiates; peripheral versus central actions.

1. To investigate peripherally mediated antinociceptive effects of opioids, the activity of a novel polar enkephalin analogue H-Tyr-D-Arg-Gly-Phe (4-NO2)-Pro-NH2 (BW443C) has been compared with those of classical tertiary opiates against different nociceptive stimuli in the mouse. 2. In chemically-induced writhing models BW443C, administered subcutaneously, demonstrated dose-related antinociceptive effects less potent than morphine and of a similar order to pethidine and D-propoxyphene. In assays using heat as the noxious stimulus BW443C was markedly less potent than any of the opiates tested. 3. In heat-induced assays, but not in chemically-induced writhing assays, BW443C demonstrated a 'U'-shaped dose-time response relationship. Morphine, pethidine and D-propoxyphene showed simple, approximately linear, dose-time effects in all assays. 4. When given subcutaneously, the inhibitory effects of BW443C and morphine in chemically-induced writhing were antagonized by naloxone given intraperitoneally. The inhibitory effects on writhing of BW443C, but not those of morphine, were also antagonized by prior intraperitoneal administration of the quaternary opioid antagonist N-methyl nalorphine. When this antagonist was administered intracerebroventricularly, the antinociceptive effects in writhing of both BW443C and morphine were antagonized. 5. It is concluded that BW443C, being only poorly able to cross the blood brain barrier, demonstrates peripherally mediated opioid antinociceptive effects in chemically-induced writhing models. In heat-induced models, that detect centrally acting opioids, BW443C is effective only at high doses and at time intervals after dosing sufficient to allow slow penetration of drug into the CNS. It is suggested that the peripheral antinociceptive actions of BW443C are mediated by inhibition of sensory neurones.

A conformational analysis for leucine-enkephalin using activity and binding data of synthetic analogues.

1. Leucine-enkephalin and some analogues were assayed for activity in vitro on the mouse vas deferens and for binding to opiate receptors from rat brain. 2. The experimental data were analysed in terms of the stringency for glycine, a D-amino acid or an L-amino acid at each position in the peptide. 3. The observed configurational specificity was compared with the stringency that would be predicted to occur if enkephalin adopted certain hydrogen-bonded conformations at the receptor. 4. A small subset of the conformations examined was found to be compatible with the experimental data.