Kidney transplant outcomes in patients with antibodies to human neutrophil antigen 3a.

BACKGROUND: Antibody mediated rejection (ABMR) of kidney transplants has been shown to occur in the absence of a known donor specific antibody to human leucocyte antigen (HLA). Antibodies to the human neutrophil antigen (HNA) system have been detected in kidney transplant recipients and linked to ABMR in the absence of an HLA donor specific antibody (DSA), but there remains limited literature regarding this. METHODS: Case series analysis was carried out examining three cases of HNA-3a antibody positive flow cytometry cross match (FC-XM) from two transplant centres in Scotland. RESULTS: All patients included were female and had been sensitised as a result of pregnancy. One live donor recipient with HNA-3a antibodies identified prior to transplant received ATG induction and has had a good outcome. The remaining two patients received deceased donor transplants. HNA-3a antibodies were indicated following a retrospective flow cytometry crossmatch. Both patients received Basiliximab induction and both have experienced ABMR requiring supplementary immunosuppression. CONCLUSIONS: The predicted rate of HNA-3a antibodies amongst patients awaiting kidney transplant in the UK is <1%. However, with increasing evidence to support a role for HNA-3a antibodies in the development of ABMR there may be value in screening at risk groups to allow for augmented immunosuppression to be considered at the time of kidney transplant.

High grade urothelial carcinoma in kidney transplant patients with a history of BK viremia - Just a coincidence?

INTRODUCTION: Kidney transplant recipients (KTR) have a three-to-four-fold increased risk of developing urothelial carcinoma (UC) compared to the general population. BK polyoma virus (BKV) infection is known to affect approximately 15% of KTR. In vitro models support a potential pathogenic role for BKV in the development of UC. We describe a series of UC in kidney transplant recipients. METHODS: Electronic patient records were searched to identify KTR with UC who had undergone kidney only or simultaneous kidney and pancreas transplantation in a single UK center between 2009 and 2015. Where available, stored pathological samples were retrieved, re-examined and stained for SV40 as a marker of BKV using standard staining protocols for kidney biopsy samples. RESULTS: Fourteen KTR had developed UC post-transplant. Of these, 10 KTR had a history of BKV infection post-transplant. Six of these 10 KTR developed a rare micropapillary tumor subtype of UC which is typically only found in <1% of UC cases. All six micropapillary tumor samples stained positive for SV40, including samples from metastases. Three tumor samples were available from the four KTR with no history of BKV infection and were not micropapillary subtype and were negative for SV40. Three micropapillary tumors from immunocompetent patients were examined as controls and were negative for SV40. CONCLUSIONS: These findings would support a pathogenic role for BK virus in the development of rare micropapillary subtype urothelial tumors in the kidney transplant population.

A case of membranous nephropathy with microspherular deposits progressing to collapsing nephropathy and dialysis dependency, but with full recovery following immunosuppression.

Membranous nephropathy with microspherular deposits is a rare renal condition associated with sub-nephrotic or nephrotic-range proteinuria. We report a case presenting with severe nephrotic syndrome and pathological features of collapsing glomerulopathy. This is the first case we are aware of that progressed to requiring dialysis. The patient received rituximab and corticosteroids. She has now been off dialysis for over a year with both serum creatinine and urine protein-creatinine ratio returning to baseline.

Comparison of kidney transplant outcomes in HLA compatible and incompatible transplantation: A national cohort study.

BACKGROUND: Reports of HLA incompatible (HLAi) kidney transplant outcomes are inconclusive, especially in the context of lower level Donor Specific Antibodies (DSA). METHODS: Multi-centre national cohort study of HLAi kidney transplant recipients matched in 1:2 ratio with HLA compatible (HLAc) kidney transplant recipients. HLAi defined as DSA identified by Luminex. Antibody mediated rejection (AMR) and transplant-survival were analysed using Kaplan-Meier plots. Propensity score (PS) matching was used to compare recipient and transplant survival between groups. RESULTS: We included 61 HLAi and 122 HLAc recipients; mean age 46 years; 60% female. MFIT0 : 3327 (IQR 1352-6458), 23 (38%) were Flow cytometry crossmatch positive (FC-XMPOS ). DSAPOS /FC-XMPOS transplantation carried an increased risk of AMR at 1 year (52%) compared to DSAPOS /FC-XMNEG (27%) and HLAc (0%). Unadjusted death censored graft loss at 3 years was 13% (HLAi) and 8% (HLAc). Three-year patient survival was 95% in HLAc, 84% in DSAPOS /FC-XMNEG and 69% in DSAPOS /FC-XMPOS recipients; 58% of HLAi deaths were infection-related. HLA incompatibility was associated with a decreased 3-year survival in our PS-matched cohort. CONCLUSION: In kidney transplantation, DSA and positive FC-XM carries an increased risk of AMR. Despite inferior transplant and survival outcomes compared to HLAc transplantation, it remains a realistic option for highly sensitized patients facing prolonged waiting times and reduced survival on dialysis.

Highly sensitised patients awaiting deceased donor renal transplants are disadvantaged by the presence of denatured HLA antibody detected in routine HLA antibody testing.

Luminex single antigen bead (SAB) assays used to detect HLA antibodies may artificially increase sensitisation in highly sensitised patients (HSP). The presence of denatured HLA (dHLA) within the assay enables antibodies specific to cryptic HLA epitopes to bind, such antibodies are not clinically relevant. We sought to exclude dHLA reactivity in a cohort of very HSP, calculated reaction frequency (cRF) 95%-100% and determine the effect upon sensitisation. Such patients have limited access to suitable donors and small changes in their HLA antibody profile, particularly where their cRF is 100%, can increase their opportunity of a transplant. We determined the presence of dHLA by aligning antibody reactivity which did not correspond to known HLA class I epitope mismatches with the results of assays modified to detect class I dHLA. 130 class I dHLA reactions were identified within 11 HSP, all of whom had clear sensitising events. cRF was corrected for dHLA, mean cRF 98.2% (93-100) pre and 95.5% (87-100) post correction (p = 0.0156). An increase in the number of predicted compatible donors (p = 0.0078) after dHLA correction was demonstrated. Two manufacturers SAB assays were used. A reduction of patients with 100% cRF was observed for both manufactures. dHLA is contributing to sensitisation in HSP and is detrimental to their chances of receiving a compatible transplant. The observed dHLA reactivity varied according to kit manufacturers (p = 0.0001), this is potentially a useful finding for laboratories wishing to discriminate between nHLA and dHLA, but without the resources required to regularly perform dHLA assay and epitope analyses.

Monoallelic IFT140 pathogenic variants are an important cause of the autosomal dominant polycystic kidney-spectrum phenotype.

Autosomal dominant polycystic kidney disease (ADPKD), characterized by progressive cyst formation/expansion, results in enlarged kidneys and often end stage kidney disease. ADPKD is genetically heterogeneous; PKD1 and PKD2 are the common loci (∼78% and ∼15% of families) and GANAB, DNAJB11, and ALG9 are minor genes. PKD is a ciliary-associated disease, a ciliopathy, and many syndromic ciliopathies have a PKD phenotype. In a multi-cohort/-site collaboration, we screened ADPKD-diagnosed families that were naive to genetic testing (n = 834) or for whom no PKD1 and PKD2 pathogenic variants had been identified (n = 381) with a PKD targeted next-generation sequencing panel (tNGS; n = 1,186) or whole-exome sequencing (WES; n = 29). We identified monoallelic IFT140 loss-of-function (LoF) variants in 12 multiplex families and 26 singletons (1.9% of naive families). IFT140 is a core component of the intraflagellar transport-complex A, responsible for retrograde ciliary trafficking and ciliary entry of membrane proteins; bi-allelic IFT140 variants cause the syndromic ciliopathy, short-rib thoracic dysplasia (SRTD9). The distinctive monoallelic phenotype is mild PKD with large cysts, limited kidney insufficiency, and few liver cysts. Analyses of the cystic kidney disease probands of Genomics England 100K showed that 2.1% had IFT140 LoF variants. Analysis of the UK Biobank cystic kidney disease group showed probands with IFT140 LoF variants as the third most common group, after PKD1 and PKD2. The proximity of IFT140 to PKD1 (∼0.5 Mb) in 16p13.3 can cause diagnostic confusion, and PKD1 variants could modify the IFT140 phenotype. Importantly, our studies link a ciliary structural protein to the ADPKD spectrum.

Characterization of a single chain variable fragment of nivolumab that targets PD-1 and blocks PD-L1 binding.

Activated T-cells express Programmed cell Death protein 1 (PD-1), a key immune checkpoint receptor. PD-1 functions primarily in peripheral tissues, where T cells may encounter tumor-derived immunosuppressive ligands. Monoclonal antibodies that disrupt the interaction between T-cell derived PD-1 and immunosuppressive ligands, such as PD-L1, have revolutionized approaches to cancer therapy. For instance, Nivolumab is a monoclonal Ab that targets human PD-1 and has played an important role in immune checkpoint therapy. Herein we report the purification and initial characterization of a ~27 kDa single chain variable fragment (scFv) of Nivolumab that targets human PD-1 and blocks binding by PD-L1. The possibility that the anti-PD-1 scFv can serve as both an anti-tumor agent and as an anti-viral agent is discussed. IMPORTANCE: The clinical significance of anti-PD-1 antibodies for treatment of a range of solid tumors is well documented (reviewed in [1-4]). In this report, we describe the results of studies that establish that an anti-PD-1 scFv purified from E. coli binds tightly to human PD-1. Furthermore, we demonstrate that upon binding, the anti-PD-1 scFv disrupts the interaction between PD-1 and PD-L1. Thus, the properties of this scFv, including its small size, stability and affinity for human PD-1, suggest that it has the potential to be a useful reagent in subsequent immunotherapeutic, diagnostic and anti-viral applications.

The impact of age on patient tolerance of mycophenolate following kidney transplantation.

BACKGROUND: In Scotland, standard maintenance immunosuppression following kidney transplantation consists of mycophenolate (MPA), tacrolimus and prednisolone irrespective of recipient age. We analyzed the tolerability of this immunosuppression regimen and the association with transplant outcomes. METHODS: A national, multicentre retrospective analysis of patients transplanted in 2015 and 2016, comparing graft function, acute rejection, significant infection rates and immunosuppression dosing between patients aged 18 and 59 years (Group 1) and ≥60 years (Group 2). RESULTS: Of the 490 patients, 26% were aged ≥60 years. Acute rejection (AR) rates at 1 year were 15% and 11% in Groups 1 and 2, respectively. Full-dose MPA was poorly tolerated with 53% in Group 1 and 77% in Group 2 requiring dose reduction or cessation. Female gender and age ≥60 years were independent predictors for MPA dose changes. One year following MPA dose reduction, AR risk was low (5%) in Group 2, however, those remaining on full dose MPA had a 79% increased rate of serious infections. CONCLUSION: The majority of renal transplant recipients aged ≥60 fail to tolerate full-dose MPA. In this group, MPA dose reduction is associated with low rejection rates, but full-dose MPA is associated with high infection rates. We suggest that a tailored approach to immunosuppression in elderly recipients incorporating lower doses of MPA may be appropriate.

A case report-Two manufacturers SAB testing kits can reveal different HLA antibody profiles-Identifying prozone and denatured antigen.

Recent reports have identified that the presence of non-native conformation HLA to which antibody can bind upon Luminex HLA Class I single antigen beads, can vary in levels between different manufacturers kits and that the prozone effect may also be specific to particular products. We present a case in which both prozone and non-native HLA reactive antibodies were observed, which raises important questions on how SAB assays are utilised, especially in the post-transplant monitoring setting. A 56-year old, highly sensitised female patient awaiting a regraft received a HLAi renal transplant. Post-transplant monitoring showed discordant results between two SAB manufacturers assays, with one assay identifying a potential de novo HLA DSA. HLA Class I antibody reactivity was observed which was directed towards the Bw6 public epitope, which is present upon HLA molecules encoded for by numerous HLA-B alleles. However in the day 19 post-transplant sample reactivity spread beyond the Bw6 epitope. To investigate the possibility of a prozone type effect influencing the testing kit the day 19 post-transplant sample was diluted 1:10 with PBS and reanalysed. After dilution the Bw6 reactivity was observed again, however the suspect de novo DSA still persisted. An analysis of the mismatched epitopes identified one manufacturer's assay as being confounded by the presence of denatured reactivity as well as prozone.

The impact of donor and recipient common clinical and genetic variation on estimated glomerular filtration rate in a European renal transplant population.

Genetic variation across the human leukocyte antigen loci is known to influence renal-transplant outcome. However, the impact of genetic variation beyond the human leukocyte antigen loci is less clear. We tested the association of common genetic variation and clinical characteristics, from both the donor and recipient, with posttransplant eGFR at different time-points, out to 5 years posttransplantation. We conducted GWAS meta-analyses across 10 844 donors and recipients from five European ancestry cohorts. We also analyzed the impact of polygenic risk scores (PRS), calculated using genetic variants associated with nontransplant eGFR, on posttransplant eGFR. PRS calculated using the recipient genotype alone, as well as combined donor and recipient genotypes were significantly associated with eGFR at 1-year posttransplant. Thirty-two percent of the variability in eGFR at 1-year posttransplant was explained by our model containing clinical covariates (including weights for death/graft-failure), principal components and combined donor-recipient PRS, with 0.3% contributed by the PRS. No individual genetic variant was significantly associated with eGFR posttransplant in the GWAS. This is the first study to examine PRS, composed of variants that impact kidney function in the general population, in a posttransplant context. Despite PRS being a significant predictor of eGFR posttransplant, the effect size of common genetic factors is limited compared to clinical variables.

Polygenic risk score as a determinant of risk of non-melanoma skin cancer in a European-descent renal transplant cohort.

Renal transplant recipients have an increased risk of non-melanoma skin cancer (NMSC) compared to in the general population. Here, we show polygenic risk scores (PRS) calculated from genome-wide association studies (GWAS) of NMSC in a general, nontransplant setting, can predict risk of, and time to posttransplant skin cancer. Genetic variants, reaching predefined P-value thresholds were chosen from published squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) nontransplant GWAS. Using these GWAS, BCC and SCC PRS were calculated for each sample across three European ancestry renal transplant cohorts (n = 889) and tested as predictors of case:control status and time to NMSC posttransplant. BCC PRS calculated at P-value threshold 1 × 10-5 was the most significant predictor of case:control status of NMSC posttransplant (OR = 1.61; adjusted P = .0022; AUC [full model adjusted for clinical predictors and PRS] = 0.81). SCC PRS at P-value threshold 1 × 10-5 was the most significant predictor of time to posttransplant NMSC (adjusted P = 9.39 × 10-7 ; HR = 1.41, concordance [full model] = 0.74). PRS of nontransplant NMSC is predictive of case:control status and time to NMSC posttransplant. These results are relevant to how genomics can risk stratify patients to help develop personalized treatment regimens.

Using omics to explore complications of kidney transplantation.

The importance of genetic and biochemical variation in renal transplant outcomes has been clear since the discovery of the HLA in the 1950s. Since that time, there have been huge advancements in both transplantation and omics. In recent years, there has seen an increased number of genome-, proteome- and transcriptome-wide studies in the field of transplantation moving away from the earlier candidate gene/protein approaches. These areas have the potential to lead to the development of personalized treatment depending on individual molecular risk profiles. Here, we discuss recent progress and the current literature surrounding omics and renal transplant complications.

Hearing loss and renal syndromes.

The association between ear and kidney abnormalities has long been recognized; however, the connection between these two disparate organs is not always straightforward. Although Alport syndrome is the most well-known, there are over 20 disorders that need to be considered in the differential diagnosis of patients with both ear and kidney abnormalities. Commonalities are present between the kidney and ear in a number of structural proteins, developmentally important transcription factors, ciliary proteins, and channel proteins, and mutations in these pathways can lead to disease in both organ systems. This manuscript reviews the congenital disorders with both hearing and kidney manifestations.

Replication of JC Virus DNA in the G144 Oligodendrocyte Cell Line Is Dependent Upon Akt.

Progressive multifocal leukoencephalopathy (PML) is an often-fatal demyelinating disease of the central nervous system. PML results when oligodendrocytes within immunocompromised individuals are infected with the human JC virus (JCV). We have identified an oligodendrocyte precursor cell line, termed G144, that supports robust levels of JCV DNA replication, a central part of the JCV life cycle. In addition, we have determined that JC virus readily infects G144 cells. Furthermore, we have determined that JCV DNA replication in G144 cells is stimulated by myristoylated (i.e., constitutively active) Akt and reduced by the Akt-specific inhibitor MK2206. Thus, this oligodendrocyte-based model system will be useful for a number of purposes, such as studies of JCV infection, establishing key pathways needed for the regulation of JCV DNA replication, and identifying inhibitors of this process.IMPORTANCE The disease progressive multifocal leukoencephalopathy (PML) is caused by the infection of particular brain cells, termed oligodendrocytes, by the JC virus. Studies of PML, however, have been hampered by the lack of an immortalized human cell line derived from oligodendrocytes. Here, we report that the G144 oligodendrocyte cell line supports both infection by JC virus and robust levels of JCV DNA replication. Moreover, we have established that the Akt pathway regulates JCV DNA replication and that JCV DNA replication can be inhibited by MK2206, a compound that is specific for Akt. These and related findings suggest that we have established a powerful oligodendrocyte-based model system for studies of JCV-dependent PML.

The Evolution of the Journal Club: From Osler to Twitter.

Journal clubs have typically been held within the walls of academic institutions and in medicine have served the dual purpose of fostering critical appraisal of literature and disseminating new findings. In the last decade and especially the last few years, online and virtual journal clubs have been started and are flourishing, especially those harnessing the advantages of social media tools and customs. This article reviews the history and recent innovations of journal clubs. In addition, the authors describe their experience developing and implementing NephJC, an online nephrology journal club conducted on Twitter.

Isolation of a monoclonal antibody that recognizes the origin binding domain of JCV, but not SV40, large T-antigen.

Within immunocompromised populations, the JC polyomavirus is the cause of the often-fatal disease Progressive Multifocal Leukoencephalopathy (PML). JC virus encodes a protein, termed T-antigen (T-ag), which is essential for its replication and pathogenicity. Previous studies of JCV T-ag have, in general, used antibodies raised against SV40 T-ag. Unfortunately, SV40 T-ag is also detected in humans and therefore there have been concerns about cross-reactivity. To address this issue, we have isolated a monoclonal antibody that binds to the JCV, but not the SV40, T-ag origin-binding domain (OBD). Furthermore, the region on the surface of the JCV T-ag OBD that is recognized by the "anti-JCV OBD mAb" has been mapped. We also demonstrate that the "anti-JCV OBD mAb" will be a useful reagent for standard techniques (e.g., Westerns blots and ELISAs). Finally, we note that additional monoclonal Abs that are specific for the T-ags encoded by the other human polyomaviruses could be generated by adopting the approach described herein.

Structural Based Analyses of the JC Virus T-Antigen F258L Mutant Provides Evidence for DNA Dependent Conformational Changes in the C-Termini of Polyomavirus Origin Binding Domains.

The replication of human polyomavirus JCV, which causes Progressive Multifocal Leukoencephalopathy, is initiated by the virally encoded T-antigen (T-ag). The structure of the JC virus T-ag origin-binding domain (OBD) was recently solved by X-ray crystallography. This structure revealed that the OBD contains a C-terminal pocket, and that residues from the multifunctional A1 and B2 motifs situated on a neighboring OBD molecule dock into the pocket. Related studies established that a mutation in a pocket residue (F258L) rendered JCV T-ag unable to support JCV DNA replication. To establish why this mutation inactivated JCV T-ag, we have solved the structure of the F258L JCV T-ag OBD mutant. Based on this structure, it is concluded that the structural consequences of the F258L mutation are limited to the pocket region. Further analyses, utilizing the available polyomavirus OBD structures, indicate that the F258 region is highly dynamic and that the relative positions of F258 are governed by DNA binding. The possible functional consequences of the DNA dependent rearrangements, including promotion of OBD cycling at the replication fork, are discussed.