Evaluation of Antibodies Directed Against Two GPCRs, Anti-AT1R and Anti-ETAR, on Kidney Transplant Outcome.

BACKGROUND: The role of an alloimmune response against non-self-antigens is established in organ transplantation. HLA incompatibilities are mainly responsible for this recognition between donor and recipient, but they may also be involved in the reactivity against other alloantigens expressed on the allograft resulting from an autoimmune response developed against selfantigens. OBJECTIVE: Our study aimed to determine the presence of non-anti-HLA antibodies (anti-AT1R and anti-ETAR) in sera from patients with end-stage renal disease, who underwent kidney transplantation in pre- and post-transplantation samples to study their influence on the development and evolution of acute humoral rejections and DSAs. METHODS: Antibodies (Abs) against two G protein-coupled receptors (GPCRs), angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR), have been detected in the sera of transplant recipients, who experience allograft dysfunction, patients with coronary heart disease, marginal hypertension and refractory, vascular lesions, myocardial hypertrophy and chronic inflammatory diseases, such as atherosclerosis or sclerosis. RESULTS: Kidney graft recipients were monitored for anti-ETAR, -AT1R, and -HLA Abs in pre-and post-transplant evolution, and anti-AT1R and/or -ETAR Abs were detected in 24% of recipients (22.4% with anti-AT1R Abs and 9.8% with anti-ETAR Abs). Due to acute humoral rejection, Graft loss was detected in 6.4% of patients with anti-GPCRs non-HLA Abs, and 3.2% had DSA anti-HLA Abs. In this research, we have described how the function of the anti-GPCRs autoAbs and how these Abs that activate GPCRs could influence graft outcome. CONCLUSION: In conclusion, there is a high association of non-HLA anti-GPCRs Abs levels with reduced kidney function after transplantation, especially in the presence of DSA anti-HLA Abs. Although more studies are needed, anti-AT1R and anti-ETAR antibodies may be helpful biomarkers that allow the risk of graft loss to be assessed.

Influence of Preformed Antibodies in Liver Transplantation.

The significance of human leukocyte antigen (HLA) matching and preformed donor-specific antibodies (DSAs) in liver transplantation remains unclear. The aim of this study was to analyze the presence of DSAs in a large cohort of 810 liver recipients undergoing liver transplant to determine the influence on acute (AR) or chronic liver rejection (CR), graft loss and allograft survival. DSAs were identified using complement dependent cytotoxicity crossmatch (CDC-CM) and multiplexed solid-phase-based flow cytometry assay (Luminex). CDC-CM showed that a 3.2% of liver transplants were positive (+CDC-CM) with an AR frequency of 19.2% which was not different from that observed in negative patients (-CDC-CM, 22.3%). Only two patients transplanted with +CDC-CM (7.6%) developed CR and suffered re-transplant. +CDC-CM patients showed a significantly lower survival rate compared to -CDC-CM patients (23.1% vs. 59.1%, p = 0.0003), developing allograft failure within the first three months (p < 0.00001). In conclusion, we have demonstrated a relationship between the presence of preformed DSAs and the low graft liver survival, indicating the important role and the potential interest of performing this analysis before liver transplantation. Our results could help to detect patients with an increased risk of graft loss, a better choice of liver receptors as well as the establishment of individualized immunosuppressive regimens.

In vitro intracellular IFNγ, IL-17 and IL-10 producing T cells correlates with the occurrence of post-transplant opportunistic infection in liver and kidney recipients.

AIM: To validate intracellular cytokine production functional assay as means of cell-mediated immunity monitoring of post-transplant patients with opportunistic infection (OI). METHODS: Intracellular cytokine-producing CD4+ and CD8+ T-cell monitoring was carried out in 30 liver transplant (LTr) and 31 kidney transplant (KTr) recipients from 2010 to 2012. Patients were assessed in our Department of Immunology at the Clinical University 'Hospital Virgen de la Arrixaca-IMIB' in Murcia, Spain for one year following transplantation. FACS Canto II flow cytometer was employed to quantify the intracellular production of IL-17, IFNγ and IL-10 cytokines on stimulated CD4+CD69+ and CD8+CD69+ T cells and BD FACS DIVA v.6 software was used to analysed the data. Statistical analysis was carried out using SPSS 22.0. RESULTS: LTr with OI had significantly lower % of CD8+CD69+IFNγ+ T cells at 60 (7.95 ± 0.77 vs 26.25 ± 2.09, P < 0.001), 90 (7.47 ± 1.05 vs 30.34 ± 3.52, P < 0.001) and 180 (15.31 ± 3.24 vs 24.59 ± 3.28, P = 0.01) d post-transplantation. Higher % of CD4+CD69+IL-10+ as well as CD4+CD69+IL-17+ T cells were yet reported at 30 (14.06 ± 1.65 vs 6.09 ± 0.53, P = 0.0007 and 4.23 ± 0.56 vs 0.81 ± 0.14, P = 0.005; respectively), 60 (11.46 ± 1.42 vs 4.54 ± 0.91, P = 0.001 and 4.21 ± 0.59 vs 1.43 ± 0.42, P = 0.03; respectively) and 90 d (16.85 ± 1.60 vs 4.07 ± 0.63, P < 0.001 and 3.97 ± 0.43 vs 0.96 ± 0.17, P = 0.001). Yet, KTr with OI had significantly lower percentage of CD4+CD69+IFNγ+ at 30 (11.80 ± 1.59 vs 20.64 ± 3.26, P = 0.035), 60 (11.19 ± 1.35 vs 15.85 ± 1.58, P = 0.02), 90 (11.37 ± 1.42 vs 22.99 ± 4.12, P = 0.028) and 180 (13.63 ± 2.21 vs 21.93 ± 3.88, P = 0.008) d post-transplantation as opposed to CD4+CD69+IL-10+ and CD8+CD69+IL-10+ T cells which percentages were higher at 30 (25.21 ± 2.74 vs 8.54 ± 1.64, P < 0.001 and 22.37 ± 1.35 vs 17.18 ± 3.54, P = 0.032; respectively), 90 (16.85 ± 1.60 vs 4.07 ± 0.63, P < 0.001 and 23.06 ± 2.89 vs 10.19 ± 1.98, P = 0.002) and 180 (21.81 ± 1.72 vs 6.07 ± 0.98, P < 0.001 and 19.68 ± 2.27 vs 10.59 ± 3.17, P = 0.016) d post-transplantation. The auROC curve model determined the most accurate cut-off values to stratify LTr and KTr at high risk of OI and Cox Regression model confirmed these biomarkers as the most significant risk factors to opportunistic infection. CONCLUSION: Post-transplant percentages of T-cell subsets differed significantly amongst infected- and non-infected-LTr and -KTr and yet this imbalance was found to contribute towards a worst clinical outcome.

CD28 biomarker quantification and expression level profiles in CD4+ T-lymphocytes in solid organ transplantation.

The introduction of anti-calcineurin-based therapies has led to an increase in the one-year survival as well as graft function rates in patients undergoing solid organ transplantation (SOT). Nonetheless, early cellular acute rejection (EAR) incidence still remains a major challenge that irrevocably heads to poor outcomes. The mechanisms underlying CD4 T cell activation in SOT are still under research. In this sense, CD28 co-stimulatory molecule plays a pivotal role triggering CD4 T cell activation as well as survival maintenance. Previous own studies stated the role that CD4+CD28+ circulating T lymphocytes plays before and during EAR episodes. We assessed the percentage as well as the absolute number of CD28 molecules on CD4+ T cells as predictive surrogate biomarker of EAR in a prospective cohort of liver and kidney transplant recipients. Quantitative analysis of CD28 was carried out on whole peripheral blood samples by flow cytometry. Decreased pre-transplant expression of CD28 was associated with EAR in both study groups. Furthermore, the expression of CD28 within the rejected group, experimented an up-regulation upon transplantation. These preliminary results suggest that patients undergoing liver or kidney transplant can be stratified at high risk of EAR according to their CD28 molecule expression on peripheral CD4+ T lymphocytes.

Molecular targets on B-cells to prevent and treat antibody-mediated rejection in organ transplantation. Present and Future.

INTRODUCTION: Waiting lists for transplant are increasing day after day since transplantation is still the best option for the treatment of end-stage organ failure. Likewise, our increasing knowledge about how immune system mounts the response against allograft is at the point that up to 95% of acute T-cell-mediated rejections are effectively controlled by current immunosuppressive therapy. Nevertheless, this is not the case for acute and chronic antibody-mediated rejections (ABMR), where treatments to reduce the level of donor specific antibodies (DSAs) remain suboptimal, causing the majority of acute and chronic graft losses. AREAS COVERED: T-cell immunosuppressant agents are usually ineffective to treat humoral rejection and current strategies to prevent ABMR include plasmapheresis; high doses of intravenous immunoglobulin; anti-CD20 monoclonal antibodies to treat B-cells; and in some cases Bortezomib, which mainly affects plasmoblast and plasma cells, or antibodies against components of complement cascade. EXPERT OPINION: We reassess the B-cell ontogeny in order to propose new molecular targets that interrupt the antibody production pathways and their eventual pathological injury. We also take a look at the pharmaceutical industry to find agents that are currently being assayed for B-cell pathologies and that could be used in the future to prevent and treat ABMR.

HLA Epitopes and Shared Molecular Eplet Characterization and Their Implication on Transplant Outcome: The Experience of One Center.

We report three interesting cases concerning antibody-mediated rejection (AMR), associated or not with anti-donor-specific antibodies, and detection of implicated molecular epitopes. The first report presents a case of intra-allele sensitization. The second case presents an interesting case concerning Luminex mean fluorescence intensity (MFI) levels considered to be low risk antibodies (<1000), but producing AMR. The third case occurred after a second kidney transplantation mediated by antibodies directed against HLA-C antigens (MFI<1000) in the previous transplantation (which was considered to be an indicator of low-risk of AMR).

Evolutionary site-number changes of ribosomal DNA loci during speciation: complex scenarios of ancestral and more recent polyploid events.

Several genome duplications have been identified in the evolution of seed plants, providing unique systems for studying karyological processes promoting diversification and speciation. Knowledge about the number of ribosomal DNA (rDNA) loci, together with their chromosomal distribution and structure, provides clues about organismal and molecular evolution at various phylogenetic levels. In this work, we aim to elucidate the evolutionary dynamics of karyological and rDNA site-number variation in all known taxa of subtribe Vellinae, showing a complex scenario of ancestral and more recent polyploid events. Specifically, we aim to infer the ancestral chromosome numbers and patterns of chromosome number variation, assess patterns of variation of both 45S and 5S rDNA families, trends in site-number change of rDNA loci within homoploid and polyploid series, and reconstruct the evolutionary history of rDNA site number using a phylogenetic hypothesis as a framework. The best-fitting model of chromosome number evolution with a high likelihood score suggests that the Vellinae core showing x = 17 chromosomes arose by duplication events from a recent x = 8 ancestor. Our survey suggests more complex patterns of polyploid evolution than previously noted for Vellinae. High polyploidization events (6x, 8x) arose independently in the basal clade Vella castrilensis-V. lucentina, where extant diploid species are unknown. Reconstruction of ancestral rDNA states in Vellinae supports the inference that the ancestral number of loci in the subtribe was two for each multigene family, suggesting that an overall tendency towards a net loss of 5S rDNA loci occurred during the splitting of Vellinae ancestors from the remaining Brassiceae lineages. A contrasting pattern for rDNA site change in both paleopolyploid and neopolyploid species was linked to diversification of Vellinae lineages. This suggests dynamic and independent changes in rDNA site number during speciation processes and a significant lack of correlation between 45S and 5S rDNA evolutionary pathways.

Incomplete sequence homogenization in 45S rDNA multigene families: intermixed IGS heterogeneity within the single NOR locus of the polyploid species Medicago arborea (Fabaceae).

BACKGROUND AND AIMS: Ribosomal sequences have become the classical example of the genomic homogenization of nuclear multigene families. Despite theoretical advantages and modelling predictions that support concerted evolution of the 45S rDNA, several reports have found intragenomic polymorphisms. However, the origins and causes of these rDNA polymorphisms are difficult to assess because seed plants show a wide range of 45S rDNA loci number variation, especially in polyploids. Medicago arborea is a tetraploid species that has a single 45S rDNA locus. This feature makes this species a suitable case study to assess the fate of ribosomal IGS homogenization in polyploid species showing nucleolus organizer region (NOR) reduction. METHODS: The intergenic spacer (IGS) region was amplified by long PCR and the fragments were cloned and sequenced by a primer-walking strategy. The physical mapping of the whole and partial IGS variants was assessed by fluorescent in situ hybridization (FISH) and fibre-FISH methods on mitotic chromosomes and extended DNA fibres, respectively. KEY RESULTS: Two IGS fragments of 4·8 and 3·5 kb were obtained showing structural features of functional sequences. The shorter variant appears to be a truncated copy of the 4·8 kb fragment that lacks the duplication of the transcription initiation site region and the entire D region. The physical localization of the two IGS variants on metaphase chromosomes and extended DNA fibres using FISH corroborated their joint presence within the same locus. In addition, no spatial structure of the two variants was detected within the NOR. CONCLUSIONS: The results suggest that full sequence homogenization is not operating within the NOR locus of M. arborea. The structure of the NOR locus reported here departs from the models of IGS heterogeneity present in plants and caution against assuming the widespread belief that intragenomic ribosomal heterogeneity is mainly due to sequence variation between paralogous loci.

Partial sequence homogenization in the 5S multigene families may generate sequence chimeras and spurious results in phylogenetic reconstructions.

Multigene families have provided opportunities for evolutionary biologists to assess molecular evolution processes and phylogenetic reconstructions at deep and shallow systematic levels. However, the use of these markers is not free of technical and analytical challenges. Many evolutionary studies that used the nuclear 5S rDNA gene family rarely used contiguous 5S coding sequences due to the routine use of head-to-tail polymerase chain reaction primers that are anchored to the coding region. Moreover, the 5S coding sequences have been concatenated with independent, adjacent gene units in many studies, creating simulated chimeric genes as the raw data for evolutionary analysis. This practice is based on the tacitly assumed, but rarely tested, hypothesis that strict intra-locus concerted evolution processes are operating in 5S rDNA genes, without any empirical evidence as to whether it holds for the recovered data. The potential pitfalls of analysing the patterns of molecular evolution and reconstructing phylogenies based on these chimeric genes have not been assessed to date. Here, we compared the sequence integrity and phylogenetic behavior of entire versus concatenated 5S coding regions from a real data set obtained from closely related plant species (Medicago, Fabaceae). Our results suggest that within arrays sequence homogenization is partially operating in the 5S coding region, which is traditionally assumed to be highly conserved. Consequently, concatenating 5S genes increases haplotype diversity, generating novel chimeric genotypes that most likely do not exist within the genome. In addition, the patterns of gene evolution are distorted, leading to incorrect haplotype relationships in some evolutionary reconstructions.

Amplification, contraction and genomic spread of a satellite DNA family (E180) in Medicago (Fabaceae) and allied genera.

BACKGROUND AND AIMS: Satellite DNA is a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNA is an important element in genome organization and evolution in plants. Here we assess the presence and physical distribution of the repetitive DNA E180 family in Medicago and allied genera. Our goals were to gain insight into the karyotype evolution of Medicago using satellite DNA markers, and to evaluate the taxonomic and phylogenetic signal of a satellite DNA family in a genus hypothesized to have a complex evolutionary history. METHODS: Seventy accessions from Medicago, Trigonella, Melilotus and Trifolium were analysed by PCR to assess the presence of the repetitive E180 family, and fluorescence in situ hybridization (FISH) was used for physical mapping in somatic chromosomes. KEY RESULTS: The E180 repeat unit was PCR-amplified in 37 of 40 taxa in Medicago, eight of 12 species of Trigonella, six of seven species of Melilotus and in two of 11 Trifolium species. Examination of the mitotic chromosomes revealed that only 13 Medicago and two Trigonella species showed FISH signals using the E180 probe. Stronger hybridization signals were observed in subtelomeric and interstitial loci than in the pericentromeric loci, suggesting this satellite family has a preferential genomic location. Not all 13 Medicago species that showed FISH localization of the E180 repeat were phylogenetically related. However, nine of these species belong to the phylogenetically derived clade including the M. sativa and M. arborea complexes. CONCLUSIONS: The use of the E180 family as a phylogenetic marker in Medicago should be viewed with caution. Its amplification appears to have been produced through recurrent and independent evolutionary episodes in both annual and perennial Medicago species as well as in basal and derived clades.