De novo donor-specific antibodies in belatacept-treated vs cyclosporine-treated kidney-transplant recipients: Post hoc analyses of the randomized phase III BENEFIT and BENEFIT-EXT studies.

Donor-specific antibodies (DSAs) are associated with an increased risk of antibody-mediated rejection and graft failure. In BENEFIT and BENEFIT-EXT, kidney-transplant recipients were randomized to receive belatacept more intense (MI)-based, belatacept less intense (LI)-based, or cyclosporine-based immunosuppression for up to 7 years (84 months). The presence/absence of HLA-specific antibodies was determined at baseline, at months 6, 12, 24, 36, 48, 60, and 84, and at the time of clinically suspected episodes of acute rejection, using solid-phase flow-cytometry screening. Samples from anti-HLA-positive patients were further tested with a single-antigen bead assay to determine antibody specificities, presence/absence of DSAs, and mean fluorescence intensity (MFI) of any DSAs present. In BENEFIT, de novo DSAs developed in 1.4%, 3.5%, and 12.1% of belatacept MI-treated, belatacept LI-treated, and cyclosporine-treated patients, respectively. The corresponding values in BENEFIT-EXT were 3.8%, 1.1%, and 11.2%. Per Kaplan-Meier analysis, de novo DSA incidence was significantly lower in belatacept-treated vs cyclosporine-treated patients over 7 years in both studies (P < .01). In patients who developed de novo DSAs, belatacept-based immunosuppression was associated with numerically lower MFI vs cyclosporine-based immunosuppression. Although derived post hoc, these data suggest that belatacept-based immunosuppression suppresses de novo DSA development more effectively than cyclosporine-based immunosuppression.

Posttransplant reduction in preexisting donor-specific antibody levels after belatacept- versus cyclosporine-based immunosuppression: Post hoc analyses of BENEFIT and BENEFIT-EXT.

BENEFIT and BENEFIT-EXT were phase III studies of cytotoxic T-cell crossmatch-negative kidney transplant recipients randomized to belatacept more intense (MI)-based, belatacept less intense (LI)-based, or cyclosporine-based immunosuppression. Following study completion, presence/absence of HLA-specific antibodies was determined centrally via solid-phase flow cytometry screening. Stored sera from anti-HLA-positive patients were further tested with a single-antigen bead assay to determine antibody specificities, presence/absence of donor-specific antibodies (DSAs), and mean fluorescent intensity (MFI) of any DSAs present. The effect of belatacept-based and cyclosporine-based immunosuppression on MFI was explored post hoc in patients with preexisting DSAs enrolled to BENEFIT and BENEFIT-EXT. In BENEFIT, preexisting DSAs were detected in 4.6%, 4.9%, and 6.3% of belatacept MI-treated, belatacept LI-treated, and cyclosporine-treated patients, respectively. The corresponding values in BENEFIT-EXT were 6.0%, 5.7%, and 9.2%. In both studies, most preexisting DSAs were of class I specificity. Over the first 24 months posttransplant, a greater proportion of preexisting DSAs in belatacept-treated versus cyclosporine-treated patients exhibited decreases or no change in MFI. MFI decline was more apparent with belatacept MI-based versus belatacept LI-based immunosuppression in both studies and more pronounced in BENEFIT-EXT versus BENEFIT. Although derived post hoc, these data suggest that belatacept-based immunosuppression decreases preexisting DSAs more effectively than cyclosporine-based immunosuppression.

Monorchis lewisi n. sp. (Trematoda: Monorchiidae) from the surf bream, Acanthopagrus australis (Sparidae), in Moreton Bay, Australia.

We describe Monorchis lewisi n. sp. (Monorchiidae) from the surf bream, Acanthopagrus australis (Günther, 1859) (Sparidae), in Moreton Bay, eastern Australia. The new species differs from most existing species of Monorchis Monticelli, 1893 in its possession of an elongate I-shaped excretory vesicle, and from other congeners in the relative configuration of the gut and suckers. Ovipusillus mayu Dove & Cribb, 1998 is re-reported from Gnathanodon speciosus (Forsskål, 1775) (Carangidae) from Moreton Bay. We report new second internal transcribed spacer (ITS2) and 28S rDNA sequence data for both species. Bayesian inference and Maximum Likelihood analyses of the 28S rDNA dataset suggest that existing subfamily and genus concepts within the family require substantial revision.

The Road to HLA Antibody Evaluation: Do Not Rely on MFI.

Technological advances in HLA laboratory testing undoubtedly improved the sensitivity and specificity of HLA antibody assessment but not without introducing a set of challenges regarding data interpretation. In particular, the introduction of solid-phase single-antigen bead (SAB) antibody assessment brought the belief that mean fluorescence intensity (MFI) was a quantifiable value. As such, MFI levels heavily influenced HLA antibody reporting, monitoring, and clinical practice. However, given that SAB testing was neither intended for nor approved to be quantifiable, is the use of MFI in current clinical and laboratory practice valid? What, if anything, does this numerical value actually reveal about the pathogenic potential of the antibody? What are the pitfalls and caveats associated with reporting MFI? Herein, we travel the road to HLA antibody assessment and explore the reliability of MFI values to make clinical decisions.

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.

Apolipoprotein L1 gene variants in deceased organ donors are associated with renal allograft failure.

Apolipoprotein L1 gene (APOL1) nephropathy variants in African American deceased kidney donors were associated with shorter renal allograft survival in a prior single-center report. APOL1 G1 and G2 variants were genotyped in newly accrued DNA samples from African American deceased donors of kidneys recovered and/or transplanted in Alabama and North Carolina. APOL1 genotypes and allograft outcomes in subsequent transplants from 55 U.S. centers were linked, adjusting for age, sex and race/ethnicity of recipients, HLA match, cold ischemia time, panel reactive antibody levels, and donor type. For 221 transplantations from kidneys recovered in Alabama, there was a statistical trend toward shorter allograft survival in recipients of two-APOL1-nephropathy-variant kidneys (hazard ratio [HR] 2.71; p = 0.06). For all 675 kidneys transplanted from donors at both centers, APOL1 genotype (HR 2.26; p = 0.001) and African American recipient race/ethnicity (HR 1.60; p = 0.03) were associated with allograft failure. Kidneys from African American deceased donors with two APOL1 nephropathy variants reproducibly associate with higher risk for allograft failure after transplantation. These findings warrant consideration of rapidly genotyping deceased African American kidney donors for APOL1 risk variants at organ recovery and incorporation of results into allocation and informed-consent processes.

HLA antibody detection with solid phase assays: great expectations or expectations too great?

Alloantibodies directed against HLA antigens, are a barrier to long-term solid organ allograft survival. The clinical impact of preformed, donor-directed HLA alloantibodies range from acceptable risk to unequivocal contraindication for organ transplantation. HLA antibodies are key factors that limit patient access to donor organs. Serological methods were once the only approach to identify HLA antigens and antibodies. Limitations in these technologies led to the development of solid phase approaches. In the early 1990s, the development of the polymerase chain reaction enabled DNA-based HLA antigen testing to be performed. By the mid-1990s, microparticle-based technology that utilized flow cytometry for analysis was developed to detect both classes I and II HLA antibodies. These methodologies revolutionized clinical histocompatibility testing. The strengths and weaknesses of these assays are described in detail in this review.

Solid-phase assays for the detection of alloantibody against human leukocyte antigens: panacea or Pandora?

Serological assessments of antibodies directed against human leucocyte antigens (HLA) formed the basis of early histocompatibility testing (Patel & Terasaki, 1969 N Engl J Med, 280, 735). However, over the past decade, significant advances in HLA antibody detection technologies have emerged. The development and implementation of solid-phase assays has led to safer and more efficient allocation of organs by effectively distinguishing HLA from non-HLA antibodies. Although solid-phase assays are not standardized, they are widely accepted as the new 'gold standard'. However, this technology is not without its challenges. This review is intended to provide a better understanding of solid-phase HLA antibody testing and will focus on important caveats associated with this evolving technology. Examples of the limitations of the technology as well as common data misinterpretations will be shown. Both of which could pose potential harm to transplant recipients (Tait et al., Transplantation, 95, 19).

New digeneans (Opecoelidae) from hydrothermal vent fishes in the south eastern Pacific Ocean, including one new genus and five new species.

A new genus and five new species of digeneans are reported from fishes at hydrothermal vent sites in the South East Pacific Rise region. Biospeedotrema n. gen. (Opecoelidae: Stenakrinae) is distinguished from other stenakrines by the more or less symmetrical testicular configuration, with the uterus passing between the testes, sometimes distinctly into the post-testicular region. Biospeedotrema jolliveti n. gen., n. sp. from Ventichthys biospeedoi (Ophidiidae) is distinguished by the vitelline fields which extend only slightly into the post-testicular region, the intestinal bifurcation is dorsal to the ventral sucker, the genital pore is slightly dextrally submedian or median, the cirrus sac is short and the caeca are broad and overlap the testes, usually reaching into the post-testicular region. Biospeedotrema parajolliveti n. sp. from Thermichthys hollisi differs from Biospeedotrema jolliveti in being squat, always just wider than long, the tegument is wrinkled, the testes are lobate, and the caeca only just reach to the testes. Biospeedotrema biospeedoi n. sp. from T. hollisi differs from its congeners in its body-shape, uterine extent posterior to the testes and the small vitellarium. Caudotestis ventichthysi n. sp. (Opecoelidae: Stenakrinae) from V. biospeedoi is distinguished from its five congeners in various combinations of caecal length, cirrus sac length, internal seminal vesicle shape, vitelline extent and distribution, forebody length and egg-size. Buticulotrema thermichthysi n. sp. (Opecoelidae: Opecoelininae) from T. hollisi (Bythitidae) is distinguished from its only congener by its very long, very strongly muscular oesophagus, bifurcating dorsally to the posterior part of the ventral sucker, the long, narrow pars prostatica and distal male duct and the sinistral genital pore at the level of the pharynx. The phylogenetic position for three of these species, Buticulotrema thermichthysi, Biospeedotrema jolliveti and Biospeedotrema biospeedoi, is assessed based on ssrDNA and lsrDNA sequences, which verify the position of these species in the Opecoelidae. 

Class II alloantibody and mortality in simultaneous liver-kidney transplantation.

Hyperacute kidney rejection is unusual in crossmatch positive recipients of simultaneous liver-kidney transplants (SLKT). However, recent data suggest that these patients remain at risk for antibody-mediated kidney rejection. To further investigate the risk associated with donor-specific alloantibodies (DSA) in SLKT, we studied 86 consecutive SLKT patients with an available pre-SLKT serum sample. Serum samples were analyzed in a blinded fashion for HLA DSA using single antigen beads (median florescence intensity≥2,000=positive). Post-SLKT samples were analyzed when available (76%). Thirty patients had preformed DSA, and nine developed de novo DSA. Preformed class I DSA did not change the risk of rejection, patient or allograft survival. In contrast, preformed class II DSA was associated with a markedly increased risk of renal antibody mediated rejection (AMR) (p=0.006), liver allograft rejection (p=0.002), patient death (p=0.02), liver allograft loss (p=0.02) and renal allograft loss (p=0.045). Multivariable modeling showed class II DSA (preformed or de novo) to be an independent predictor of patient death (HR=2.2; p=0.043) and liver allograft loss (HR=2.2; p=0.044). These data warrant reconsideration of the approach to DSA in SLKT.

Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA.

Solid phase multiplex-bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte-based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R(2) ), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.

Donor-directed MHC class I antibody is preferentially cleared from sensitized recipients of combined liver/kidney transplants.

For patients with chronic renal and liver diseases, simultaneous liver and kidney transplantation (SLKT) is the best therapeutic option. The role of a pretransplant donor-specific antibody (DSA) in SLKT is unclear. We report the results of a retrospective review from 7/08 to 10/09 of SLKT at our institution. Monitoring of DSA was performed using single antigen bead assay. Between 7/08 and 10/09, there were six SLKT who had preformed DSA and positive XM (four class I and II DSA, one class I DSA only, one class II only). One-year patient and renal graft survival was 83%. Death-censored liver allograft survival was 100%. Acute humoral rejection (AHR) of the kidney occurred in 66% (three with both class I and II DSA and one with only class II DSA) of patients. In those with AHR, class I antibodies were rapidly cleared (p < 0.01) while class II antibodies persisted (p = 0.25). All patients who had humoral rejection of their kidney had preformed anticlass II antibodies. Liver allografts may not be fully protective of the renal allograft, especially with pre-existing MHC class II DSA. Long-term and careful follow-up will be critical to determine the impact of DSA on both allografts.

Revisiting traditional risk factors for rejection and graft loss after kidney transplantation.

Single-antigen bead (SAB) testing permits reassessment of immunologic risk for kidney transplantation. Traditionally, high panel reactive antibody (PRA), retransplant and deceased donor (DD) grafts have been associated with increased risk. We hypothesized that this risk was likely mediated by (unrecognized) donor-specific antibody (DSA). We grouped 587 kidney transplants using clinical history and single-antigen bead (SAB) testing of day of transplant serum as (1) unsensitized; PRA = 0 (n = 178), (2) third-party sensitized; no DSA (n = 363) or (3) donor sensitized; with DSA (n = 46), and studied rejection rates, death-censored graft survival (DCGS) and risk factors for rejection. Antibody-mediated rejection (AMR) rates were increased with DSA (p < 0.0001), but not with panel reactive antibody (PRA) in the absence of DSA. Cell-mediated rejection (CMR) rates were increased with DSA (p < 0.005); with a trend to increased rates when PRA>0 in the absence of DSA (p = 0.08). Multivariate analyses showed risk factors for AMR were DSA, worse HLA matching, and female gender; for CMR: DSA, PRA>0 and worse HLA matching. AMR and CMR were associated with decreased DCGS. The presence of DSA is an important predictor of rejection risk, in contrast to traditional risk factors. Further development of immunosuppressive protocols will be facilitated by stratification of rejection risk by donor sensitization.

Taxonomic approaches to and interpretation of host specificity of trematodes of fishes: lessons from the Great Barrier Reef.

The taxonomy of trematodes of Great Barrier Reef (GBR) fishes has been studied in some detail for over 20 years. Understanding of the fauna has been informed iteratively by approaches to sampling, understanding of morphology, the advent of molecular methodology and a feed-back loop from the emergent understanding of host specificity. Here we analyse 658 host-parasite combinations for 290 trematode species, 152 genera and 28 families from GBR fishes. These are reported from 8 orders, 38 families, 117 genera and 243 species of fishes. Of the 290 species, only 4 (1·4%) have been reported from more than one order of fishes and just 23 (7·9%) infect more than one family; 77·9% of species are known from only one genus, and 60% from only one species of fish. Molecular studies have revealed several complexes of cryptic species and others are suspected; we conclude that no euryxenous host distribution should be accepted on the basis of morphology only. The occurrence of individual trematode species in potential hosts is patchy and difficult to predict reliably a priori or explain convincingly a posteriori. These observations point to the need for a vigorous iterative interaction between the accretion of host specificity data and its interpretation.

Sperm ultrastructure of the digenean Siphoderina elongata (Platyhelminthes, Cryptogonimidae) intestinal parasite of Nemipterus furcosus (Pisces, Teleostei).

The ultrastructure of the spermatozoon of Siphoderina elongata was studied by transmission electron microscopy. A description and drawings of the mature spermatozoon are presented in this paper. Several ultrastructural elements of this male gamete have been observed: a nucleus, two mitochondria, two axonemes of 9 + "1" pattern, external ornamentation of the plasma membrane, spine-like bodies and cortical microtubules. The presence, the location or the number of these elements have been compared with other digenean spermatozoa. Moreover, a close attention was paid to the organization of the external ornamentation region. This zone presents a single row of cortical microtubules disposed in a semi-circle around a mitochondrion and associated with external ornamentation and spine-like bodies. The aim of this study is to highlight criteria which can be interesting in Platyhelminthes phylogeny.