Donor Chimera Model for Tolerance Induction in Transplantation.

Tolerance induction is the basis of a successful transplantation with the goal being the re-establishment of homeostasis after transplantation. Non-autograft transplantation disrupts this maintenance drastically which could be avoided by the selection of a novel procedure. At present, the blood group antigens and the genotypes of the donor and recipient are matched before transplantation, combined with a drug regimen that confers general immunosuppression. But the "specific" unresponsiveness of the recipient to the donor organ, implied by "Tolerance", is not achieved in this process. This article introduces the 'Donor Chimera Model' for tolerance induction via the concept of the "Closed Transplantation Loop" which seeks to limit the use of immunosuppressive therapy after transplantation through the induction of tolerance in both the recipient as well as donor (read more)

A simplified method for screening siblings for HLA identity using short tandem repeat (STR) polymorphisms.

Identifying an HLA-matched sibling donor for hematopoietic stem cell transplantation (HSCT) is time-consuming and expensive, and often limited by reimbursement caps imposed by insurance providers. To improve the effectiveness and efficiency of screening for HLA-matched siblings, we developed an assay for determining HLA identity using a panel of nine informative short tandem repeat (STR) loci located throughout the HLA complex. The STR panel was assessed for accuracy in identifying HLA-matched siblings in 88 family workups comprising a total of 132 related donor and recipient typing comparisons. All sibling pairs with identical STR alleles were also HLA identical. Of the 48 pairs mismatched at one or more STR alleles, all were genotypically HLA non-identical at one or more loci. The sensitivity and specificity of STR analysis for identifying HLA-matched siblings were 91% and 100%, respectively. Three false negatives occurred due to an STR mutation or possible HLA-DPB1/DQB1 recombination. Additionally, STR genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, due to extensive homozygosity or shared parental haplotypes. The HLA STR assay is a reliable and rapid test that can be used to inexpensively screen potential sibling donors for HLA identity (read more)

Donor Exposures in Recipients of Pooled Platelet Concentrates

The overall relative increase in donor exposures with the use of pooled platelet concentrates was similar in the two institutions (43% in Germany and 45% in Canada). The highest relative increase in donor exposures was 88% and 60%, respectively, and was observed in patients receiving fewer than 10 transfusions. Both percentages are far less than the currently anticipated 300% to 500% increase in donor exposures, which may be four to six times as high among patients who receive pooled platelet concentrates as among patients who receive apheresis platelet concentrates (read more)

Low mannose-binding lectin serum levels are associated with reduced kidney graft survival

Activation of the complement system is initiated by the alternative, the classical, or the lectin pathway. As the complement system is involved in the pathophysiology of graft rejection after kidney transplantation, we investigated the possible role of mannose-binding lectin in kidney transplantation and the influence of human leukocyte antigen (HLA) immunization on this process. In a prospective study of 544 kidney transplant patients over a follow-up period of 5 years, low serum levels of this lectin at the time of transplantation were found to be significantly associated with decreased 5-year death-censored graft survival (hazard ratio 1.68). Subanalysis showed that this association was confined to non-HLA-immunized patients (hazard ratio 1.93). The strongest association was seen in non-HLA-immunized patients receiving a kidney from a deceased donor (hazard ratio 2.93). No significant association with mannose-binding lectin levels and graft survival were found in HLA-immunized patients. Variant MBL2 genotypes causing low mannose-binding lectin serum concentrations showed the same association pattern. Our findings demonstrate a clear protective role of mannose-binding lectin and thus innate immunity in maintaining kidney graft survival, but these are probably overruled by HLA immunization (read more).

Partially Mismatched Transplantation and HLA Donor Specific Antibodies.

The presence of donor HLA-specific antibodies (DSA) increases engraftment failure risk in partially-HLA mismatched, or HLA-haploidentical, allogeneic marrow (alloBMT) transplantation. As pre-existing sensitization to HLA antigens is not well characterized among candidates for HLA-haploidentical alloBMT, we retrospectively evaluated both the incidence and relative strength of DSA in this patient population. Based on correlations of solid phase antibody assays on the Luminex™ platform with actual crossmatch tests, DSA were characterized as weak for results that were consistent with negative flow cytometric crossmatch results or as moderate to strong for results consistent with positive flow cytometric or cytotoxicity crossmatches. 296 alloBMT candidates (111 [37.5%] female) were evaluated. DSA were detected in 43 (14.5%) candidates, mostly among female candidates (42.9% female vs. 12.5% male). Moderate to strong DSA strength was more frequently encountered when directed against haploidentical donors as compared to mismatched unrelated donors. DSA were most commonly detected in female patients directed against their children. Because the presence of DSA has been considered prohibitive for HLA mismatched alloBMT, we additionally report a desensitization methodology used to reduce DSA to negative or weak levels. i.e., levels well below those detectable in a flow cytometric crossmatch. Nine patients without other available donors underwent desensitization. Eight reduced their DSA to negative or weak levels proceeded to alloBMT and achieved full donor engraftment. These data support routine DSA evaluation in all patients considered for mismatched alloBMT; however, for patients with no other viable options, desensitization to weak or negative DSA levels may afford the opportunity for successful transplantation (read more)

In Search of Immunodominant Minor Histocompatibility Antigens

Major histocompatibility complex (MHC)-associated peptides (MAPs) displayed at the cell surface define our immune self . Under normal circumstances, MAPs presented by MHC class I and II molecules originate from proteolytic degradation of self proteins. Importantly, some MAPs are polymorphic; they are present in some persons, but in other MHC-matched subjects they are absent or present a slightly different amino acid sequence. For historical reasons, these polymorphic MAPs are referred to as minor histocompatibility antigens (MiHAs). They are a consequence of any form of accumulated genetic variation that hinders MAP generation (eg, gene deletion) or the structure of a MAP (eg, single nucleotide polymorphisms) . Thus, MiHAs are essentially genetic polymorphisms viewed from a T cell perspective (read more)

The Paradoxical Functions of B Cells and Antibodies in Transplantation

Scarcely anyone would dispute that donor-specific B cells and the Abs that they produce can cause rejection of transplants. Less clear and more controversial, however, is the possibility that donor-specific B cells and the Abs that they produce are one or more means by which transplants can be protected from injury. In this article, we review and discuss this possibility and consider how less well-known functions of B cells and Abs might impact on the design of therapeutics and the management of transplant recipients (read more)

Kidney Graft Survival in Europe and the United States: Strikingly Different Long-Term Outcomes

Background: Kidney graft survival has never been systematically compared between Europe and the United States.
Methods: Applying period analysis to first deceased-donor (DD) and living-donor kidney grafts from the United Network for Organ Sharing/Organ Procurement and Transplantation Network for the United States and the Collaborative Transplant Study for Europe, we compared overall and age-specific 1-, 5-, and 10-year graft survival for Europeans and white, African, and Hispanic Americans for the 2005 to 2008 period. A Cox regression model was used to adjust for differences in patient characteristics.
Results: For the 2005 to 2008 period, 1-year survival for DD grafts was equal (91%) between Europeans and white and Hispanic Americans, whereas it was slightly lower for African Americans (89%). In contrast, overall 5- and 10-year graft survival rates were considerably higher for Europe (77 and 56%, respectively) than for any of the three U.S. populations (whites, 71 and 46%, Hispanic, 73 and 48%, and African American, 62 and 34%). Differences were largest for recipient ages 0 to 17 and 18 to 29 and generally increased beyond 3 to 4 years after transplantation. Survival patterns for living-donor grafts were similar as those seen for DD grafts. Adjusted hazard ratios for graft failure in United Network for Organ Sharing white Americans ranged between 1.5 and 2.3 (all P<0.001) for 2 to 5 years after transplantation, indicating that lower graft survival is not explained by differences in baseline patient characteristics.
Conclusions: Long-term kidney graft survival rates are markedly lower in the United States compared with Europe. Identifying actionable factors explaining long-term graft survival differences between Europe and the United States is a high priority for improving long-term graft survival (read more)

Preformed Complement-Activating Low-Level Donor-Specific Antibody Predicts Early Antibody-Mediated Rejection in Renal Allografts

Background. Donor-specific anti-HLA antibodies (DSA) are a major cause of alloimmune injury. Transplant recipients with negative complement-dependent cytotoxic crossmatch (CDC-XM) and donor cell-based flow cytometric crossmatch (flow-XM) but low level DSA (i.e., by Luminex) have worse outcomes compared with nonsensitized patients. The aim of this study was to establish whether complement-activating ability in this low-level DSA, present before transplantation, as determined by this technique is important in dictating pathogenicity.
Methods. We retrospectively studied 52 patients with preformed DSA detected by single-antigen flow cytometric fluorescent beads (SAFBs). Patients were transplanted using a steroid-sparing regimen consisting of alemtuzumab induction, 1 week of corticosteroids and tacrolimus monotherapy.Fifteen (29%) of 52 patients experienced antibody-mediated rejection (AMR), whereas 37 (71%) patients did not. There were no demographic differences between patients with AMR and those without. Pretransplant sera were retested using a modified (SAFB) assay, which detects the presence of the complement fragment C4d as a result of DSA-induced complement activation.
Results. C4d+DSA were detected in 10 (19%) of 52 patients. Biopsy-proven AMR occurred in 7 (70%) of the 10 patients with C4d+DSA and in 8 (19%) of 42 patients with C4d-DSA. AMR-free survival was worse in patients with C4d+DSA (P<0.001).
Conclusions. The ability of preformed, low-level, DSA to trigger C4d fixation in vitro in patients with negative conventional crossmatch tests is predictive for AMR. C4d SAFB is potentially a powerful tool for risk stratification prior to transplantation and may allow identification of unacceptable donor antigens, or patients who may require enhanced immunosuppression (read more)

Pancreas Retransplantation: A Second Chance for Diabetic Patients?

Background: If pancreas transplantation is a validated alternative for type 1 diabetic patients with end-stage renal disease, the management of patients who have lost their primary graft is poorly defined. This study aims at evaluating pancreas retransplantation outcome.
Methods: Between 1976 and 2008, 569 pancreas transplantations were performed in Lyon and Geneva, including 37 second transplantations. Second graft survival was compared with primary graft survival of the same patients and the whole population. Predictive factors of second graft survival were sought. Patient survival and impact on kidney graft function and survival were evaluated.
Results: Second pancreas survival of the 17 patients transplanted from 1995 was close to primary graft survival of the whole population (71% vs. 79% at 1 year and 59% vs. 69% at 5 years; P=0.5075) and significantly better than their first pancreas survival (71% vs. 29% at 1 year and 59% vs. 7% at 5 years; P=0.0008) regardless of the cause of first pancreas loss. The same results were observed with all 37 retransplantations. Survival of second simultaneous pancreas and kidney transplantations was better than survival of second pancreas after kidney. Patient survival was excellent (89% at 5 years). Pancreas retransplantation had no impact on kidney graft function and survival (100% at 5 years).
Conclusion: Pancreas retransplantation is a safe procedure with acceptable graft survival that should be proposed to diabetic patients who have lost their primary graft (read more)

Subclinical Epstein–Barr Virus Viremia Among Adult Renal Transplant Recipients: Incidence and Consequences

The natural history and clinical significance of posttransplant Epstein-Barr virus (EBV) infection remain largely unknown. The aims of this study are to describe the incidence, risk factors and consequences of EBV infection after kidney transplantation. A total of 383 consecutive patients having received a kidney transplant between January 2002 and December 2010 were included. EBV polymerase chain reaction (PCR) was performed every 2 weeks for 3 months, and every 4 weeks for the next 9 months. A total of 155 of the 383 patients (40%) had at least one positive viremia during the first year posttransplant. The median time to viremia was day 31 posttransplant (14–329). A total of 73 (47%) had EBV viremia > 10³ log and 23 (15%) had positive viremia for more than 6 months. EBV D+/R− patients (12/18 (67%) versus 143/365 (39%), p = 0.02) and those having received antithymocyte globulins (ATG) (54% vs. 35%; p<0.001) were more likely to develop EBV infection. EBV infection (hazard ratio [HR], 3.03; 95% confidence interval [CI], 1.72–8.29; p = 0.01) was associated with the occurrence of opportunistic infections. A positive EBV PCR during the first 6 months posttransplant was associated with graft loss (HR, 3.04; 95% CI, 1.36–6.79; p = 0.014). EBV reactivation is frequent after transplantation and reflects overimmunosuppression. Prospective studies should examine the association between EBV and graft loss (read more)

Duodenal Infusion of Donor Feces for Recurrent Clostridium difficile

Background : Recurrent Clostridium difficile infection is difficult to treat, and failure rates for antibiotic therapy are high. We studied the effect of duodenal infusion of donor feces in patients with recurrent C. difficile infection. Methods : We randomly assigned patients to receive one of three therapies: an initial vancomycin regimen (500 mg orally four times per day for 4 days), followed by bowel lavage and subsequent infusion of a solution of donor feces through a nasoduodenal tube; a standard vancomycin regimen (500 mg orally four times per day for 14 days); or a standard vancomycin regimen with bowel lavage. The primary end point was the resolution of diarrhea associated with C. difficile infection without relapse after 10 weeks. Results : The study was stopped after an interim analysis. Of 16 patients in the infusion group, 13 (81%) had resolution of C. difficile–associated diarrhea after the first infusion. The 3 remaining patients received a second infusion with feces from a different donor, with resolution in 2 patients. Resolution of C. difficile infection occurred in 4 of 13 patients (31%) receiving vancomycin alone and in 3 of 13 patients (23%) receiving vancomycin with bowel lavage (P<0.001 for both comparisons with the infusion group). No significant differences in adverse events among the three study groups were observed except for mild diarrhea and abdominal cramping in the infusion group on the infusion day. After donor-feces infusion, patients showed increased fecal bacterial diversity, similar to that in healthy donors, with an increase in Bacteroidetes species and clostridium clusters IV and XIVa and a decrease in Proteobacteria species. Conclusions : The infusion of donor feces was significantly more effective for the treatment of recurrent C. difficile infection than the use of vancomycin. (Funded by the Netherlands Organization for Health Research and Development and the Netherlands Organization for Scientific Research; Netherlands Trial Register number, NTR1177) (read more)

Frequency and Determinants of Pregnancy-Induced Child-Specific Sensitization

The aim of this study was to define the frequency and determinants of pregnancy-induced child-specific sensitization shortly after full-term delivery using sensitive single HLA-antigen beads (SAB) and high resolution HLA-typing of the mothers and their children (n = 301). A positive SAB result was defined by a background normalized ratio >1 or a mean fluorescence intensity (MFI) >300, >500 and >1000, respectively. The overall frequency of pregnancy-induced sensitization determined by SAB shortly after full-term delivery was between 45% (MFI > 1000 cut-off) and 76% (ratio cut-off). The rate of child-specific sensitization at the HLA-A/B/C/DRB1 loci was between 28% (MFI > 1000 cut-off) and 38% (ratio cut-off). The number of live birth was associated with a higher frequency of sensitization, which was driven by child-specific, but not third party HLA-antibodies. There was a clear hierarchy of sensitization among the investigated loci (B-locus: 31%; A-locus: 26%; DRB1-locus: 20%; C-locus: 15%; p < 0.0001). Some mismatched paternal HLA-antigens led to a significantly higher rate of sensitization than the average (e.g. HLA-A2, HLA-B49, HLA-B51, HLA-C*15). Furthermore, the mother's own HLA-phenotype—especially HLA-A/B homozygosity—was associated with a higher rate and broadness of sensitization. The number of mismatched HLA-A/B/C eplets strongly correlated with the rate of child-specific class I sensitization (read more)

Pretransplant Interferon-γ Secretion by CMV-Specific CD8+ T Cells Informs the Risk of CMV Replication After Transplantation

In this prospective study we analyzed pretransplant interferon-γ secretion by cytomegalovirus (CMV)-specific CD8+ T cells to assess its possible utility in determining the risk of CMV replication after solid organ transplantation. A total of 113 lung and kidney transplant patients were enrolled in the study but only 55 were evaluable. All CMV-seronegative recipients were pretransplant “nonreactive” (IFNγ <0.2 IU/mL) (11/11), whereas 30/44 (68.2%) CMV-seropositive (R+) recipients were “reactive” (IFNγ ≥0.2 IU/mL) and 14/44 (31.8%) were “nonreactive”. In the R(+) “nonreactive” group, 7/14 (50%) developed posttransplant CMV replication, whereas the virus replicated only in 4/30 (13.3%) of the R(+) “reactive” patients (p = 0.021). According to the best multivariate model, pretransplant “nonreactive” recipients receiving an organ from a CMV-seropositive donor had a 10-fold increased risk of CMV replication compared to pretransplant “reactive” recipients (adjusted OR 10.49, 95% CI 1.88–58.46). This model displayed good discrimination ability (AUC 0.80) and calibration (Hosmer–Lemeshow test, p = 0.92). Negative and positive predictive values were 83.7% and 75%, respectively. The accuracy of the model was 82%. Therefore, assessment of interferon-γ secretion by cytomegalovirus (CMV)-specific CD8+ T cells prior to transplantation is useful in informing the risk of posttransplant CMV replication in solid organ transplant patients (read more)

Correlation of Infused CD3+CD8+ Cells with Single-Donor Dominance after Double-Unit Cord Blood Transplantation

Single-donor dominance is observed in the majority of patients following double-unit cord blood transplantation (dCBT); however, the biological basis for this outcome is poorly understood. To investigate the possible influence of specific cell lineages on dominance in dCBT, flow cytometry assessment for CD34+, CD14+, CD20+, CD3−CD56+, CD3+CD56+ (natural killer), and T cell subsets (CD4+, CD8+, memory, naïve, and regulatory) was performed on individual units. Subsets were calculated as infused viable cells per kilogram of recipient actual weight. Sixty patients who underwent dCBT were included in the final analysis. Higher CD3+ cell dose was statistically concordant with the dominant unit in 72% of cases (P = .0006). Further T cell subset analyses showed that dominance was correlated more with the naive CD8+ cell subset (71% concordance; P = .009) than with the naive CD4+ cell subset (61% concordance; P = .19). These data indicate that a greater total CD3+ cell dose, particularly of naïve CD3+CD8+ T cells, may play an important role in determining single-donor dominance after dCBT (read more)

Allogeneic Transplantation: When is a Mismatch Better than a Match?

An accepted fact in allogeneic transplantation from unrelated donors, supported by multiple publications, including large numbers of transplantation patients, is that matching for human leukocyte antigen (HLA) is critical to ensure the best outcomes for patients. The current gold standard is an unrelated donor matched for 10/10 (or 8/8) HLA alleles (at HLA-A, -B, -C, -DRB1 +/− DQB1). Graft-versus-host disease (GVHD) is increased in the HLA-mismatched setting, and overall survival becomes significantly worse as the number of mismatched alleles at these loci increases  (read more)

Donor-Derived Mesenchymal Stem Cells Combined With Low-Dose Tacrolimus Prevent Acute Rejection After Renal Transplantation: A Clinical Pilot Study

 Background: The deleterious side effects of calcineurin inhibitors have impaired long-term survival after renal allograft. New immunotherapy regimens that minimize or even eliminate calcineurin inhibitors are required to improve transplantation outcome. Mesenchymal stem cells (MSCs) represent a unique cell population with immunosuppressive function and prolong allograft survival in experimental organ transplant models.
Methods: In this pilot study, donor-derived bone marrow MSCs combined with a sparing dose of tacrolimus (50% of standard dose) were administered to six de novo living-related kidney transplant recipients. Six other patients who received a standard dose of tacrolimus were enrolled as a control. The safety of MSC infusion, acute rejection, graft function, and patient and graft survival within 12 months after kidney transplantation were observed. The immune profiles were analyzed at different time points after transplantation.
Results: None of the MSC recipients experienced immediate or long-term toxic side effects associated with MSC infusion. The tacrolimus dose (0.045±0.002 mg/kg) in the MSC group was significantly reduced compared with the control group (0.077±0.005 mg/kg). One acute rejection occurred only in the control group. All patients survived with stable renal function at month 12 and no chimerism was detectable at month 3. Patients in the MSC group showed significantly higher B-cell levels than the control group at month 3.
Conclusion: These preliminary data suggest that the use of MSCs could provide potential benefits in renal transplantation by reducing the dosage of conventional immunosuppressive drug that is required to maintain long-term graft survival and function (read more)

Consensus Guidelines on the Testing and Clinical Management Issues Associated With HLA and Non-HLA Antibodies in Transplantation

Background: The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require resolution and careful consideration when interpreting antibody results.
Methods: With this background, The Transplantation Society convened a group of laboratory and clinical experts in the field of transplantation to prepare a consensus report and make recommendations on the use of this new technology based on both published evidence and expert opinion. Three working groups were formed to address (a) the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups were established in November 2011 and convened for a “Consensus Conference on Antibodies in Transplantation” in Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases for this report.
Results: A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if single-antigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results.
Conclusions: A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods (read more)

HLA Class II Molecules Influence Susceptibility versus Protection in Inflammatory Diseases by Determining the Cytokine Profile [BRIEF REVIEWS]

The MHC in humans encodes the most polymorphic genes, the HLA genes, which are critical for the immune system to clear infection. This can be attributed to strong selection pressure as populations moved to different parts of the world and encountered new kinds of infections, leading to new HLA class II alleles. HLA genes also have the highest relative risk for autoimmune diseases. Three haplotypes, that is, HLA-DR2DQ6, DR4DQ8, and DR3DQ2, account for HLA association with most autoimmune diseases. We hypothesize that these haplotypes, along with their multiple subtypes, have survived bottlenecks of infectious episodes in human history because of their ability to present pathogenic peptides to activate T cells that secrete cytokines to clear infections. Unfortunately, they also present self-peptides/mimics to activate autoreactive T cells secreting proinflammatory cytokines that cause autoimmune diseases (read more)