Increased natural killer cell subsets with inhibitory cytokines and inhibitory surface receptors in patients with recurrent miscarriage and decreased or normal subsets in kidney transplant recipients late post-transplant.

Patients with recurrent miscarriage (RM) show up-regulated cytotoxic natural killer (NK) cells that are suspected to play a causal role in abortion. In the present study, we investigated counter-regulating inhibitory mechanisms and compared the results in RM patients with those of healthy controls (HC), patients with end-stage renal disease (ESRD) and kidney transplant recipients late post-transplant (TX). NK, NK T and T cell subsets were analysed in the peripheral blood of 31 RM, 14 female ESRD and nine female TX patients as well as 21 female HC using eight-colour fluorescence flow cytometry. Compared with HC, RM patients showed significantly higher absolute numbers of CD56+ NK cells co-expressing the phenotype interferon (IFN)-γR+ , IL-4+ , transforming growth factor (TGF)-ß+ , IL-4+ human leucocyte antigen D-related (HLA-DR)+ , TGF-ß+ HLA-DR+ , IL-4+ TGF-ß+ , IL-4+ TGF-ß- , IFN-γ+ and/or IL-10- IFN-γ+ (all P ≤ 0·01), more IL-17+ CD56bright (P = 0·028) NK cells and more CD56dim CD16+ NK cells co-expressing IFN-γR, IFN-γ, IL-4 and/or TGF-ß (all P ≤ 0·01). When the same cell subsets were analysed in ESRD or TX patients, cytokine-producing NK cell subsets were not significantly different from those of HC. RM patients showed significantly higher absolute numbers of CD158a+ , CD158b+ , CD158a- CD158e+ (all P < 0·05), NKG2D+ NKG2A+ , NKG2D + NKG2A- , NKG2D+ and/or NKG2A+ (all P ≤ 0·01) CD56+ NK cells and higher CD158a+ , CD158b+ (all P < 0·05), NKG2D+ and/or NKG2A+ (all P < 0·01) CD56dim+ CD16+ NK cells than HC. In contrast, ESRD patients had normal and TX recipients had lower CD158a+ and NKG2D+ NKG2A- CD56+ NK cells and lower CD158a+ CD56dim+ CD16+ NK cells (all P < 0·05) than HC. RM patients have abnormally high circulating NK cells expressing inhibitory cytokines and inhibitory surface receptors which might contribute to the pathogenesis of RM.

Association of peripheral NK cell counts with Helios+ IFN-γ- Tregs in patients with good long-term renal allograft function.

Little is known about a possible interaction of natural killer (NK) cells with regulatory T cells (Treg ) in long-term stable kidney transplant recipients. Absolute counts of lymphocyte and Treg subsets were studied in whole blood samples of 136 long-term stable renal transplant recipients and 52 healthy controls using eight-colour fluorescence flow cytometry. Patients were 1946 ± 2201 days (153-10 268 days) post-transplant and showed a serum creatinine of 1·7 ± 0·7 mg/dl. Renal transplant recipients investigated > 1·5 years post-transplant showed higher total NK cell counts than recipients studied < 1·5 years after transplantation (P = 0·006). High NK cells were associated with high glomerular filtration rate (P = 0·002) and low serum creatinine (P = 0·005). Interestingly, high NK cells were associated with high CD4+ CD25+ CD127- forkhead box protein 3 (FoxP3+ ) Treg that co-express the phenotype Helios+ interferon (IFN)-γ- and appear to have stable FoxP3 expression and originate from the thymus. Furthermore, high total NK cells were associated with Treg that co-express the phenotypes interleukin (IL)-10- transforming growth factor (TGF)-ß+ (P = 0·013), CD183+ CD62L- (P = 0·003), CD183+ CD62+ (P = 0·001), CD183- CD62L+ (P = 0·002), CD252- CD152+ (P < 0·001), CD28+ human leucocyte antigen D-related (HLA-DR- ) (P = 0·002), CD28+ HLA-DR+ (P < 0·001), CD95+ CD178- (P < 0·001) and CD279- CD152+ (P < 0·001), suggesting that these activated Treg home in peripheral tissues and suppress effector cells via TGF-ß and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). The higher numbers of NK and Treg cell counts in patients with long-term good allograft function and the statistical association of these two lymphocyte subsets with each other suggest a direct or indirect (via DC) interaction of these cell subpopulations that contributes to good long-term allograft acceptance. Moreover, we speculate that regulatory NK cells are formed late post-transplant that are able to inhibit graft-reactive effector cells.

Serum levels of chemokines CCL4 and CCL5 in cirrhotic patients indicate the presence of hepatocellular carcinoma.

BACKGROUND: Most hepatocellular carcinomas (HCCs) are diagnosed at an advanced stage. The prognostic value of serum tumour markers alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) is limited. The aim of our study is to evaluate the diagnostic value of serum growth factors, apoptotic and inflammatory mediators of cirrhotic patients with and without HCC. METHODS: Serum samples were collected from cirrhotic potential liver transplant patients (LTx) with (n=61) and without HCC (n=78) as well as from healthy controls (HCs; n=39). Serum concentrations of CRP, neopterin and IL-6 as markers of inflammation and thrombopoietin (TPO), GCSF, FGF basic and VEGF, HMGB1, CK-18 (M65) and CK18 fragment (M30) and a panel of proinflammatory chemokines (CCL2, CCL3, CCL4, CCL5, CXCL5 and IL-8) were measured. Chi square, Fisher exact, Mann-Whitney U-tests, ROC curve analysis and forward stepwise logistic regression analyses were applied. RESULTS: Patients with HCC had higher serum TPO and chemokines (P<0.001 for TPO, CCL4, CCL5 and CXCL5) and lower CCL2 (P=0.008) levels than cirrhotic patients without HCC. Multivariate forward stepwise regression analysis for significant parameters showed that among the studied parameters CCL4 and CCL5 (P=0.001) are diagnostic markers of HCC. Serum levels of TPO and chemokines were lower, whereas M30 was significantly higher in cirrhotic patients than in HCs. CONCLUSIONS: High serum levels of inflammatory chemokines such as CCL4 and CCL5 in the serum of cirrhotic patients indicate the presence of HCC.

Complement C5a receptor gene 450 C/T polymorphism in renal transplant recipients: association of the CT genotype with graft outcome.

Complement-mediated humoral rejection has become the main focus of research in organ transplantation. The aim of this study was to investigate the possible association of the complement C5aR gene 450 C/T polymorphism in antibody-mediated renal allograft rejection. This polymorphism was investigated in 290 first deceased donor kidney graft recipients with well functioning grafts and no rejection treatment during the first transplant year (WFG), 265 recipients with graft failure within the first transplant year (F), and 187 healthy controls. Frequency of the 450 CT genotype was lower in the total population of 555 kidney recipients (4.7%) than in 187 healthy controls (8.6%), but the difference was not statistically significant (P = 0.065). A significantly higher frequency of CT genotype was found in F patients (CT: 6.8%) when compared to WFG patients (CT: 2.8%, P = 0.027). The CT genotype was also significantly lower in WFG patients than in healthy controls (P = 0.009). Low frequency of the C5aR 450 CT genotype, which apparently is a feature of certain kidney diseases, appears to be associated with good graft outcome in kidney transplantation and might be helpful for identifying recipients who are at low risk for graft rejection.

Association between steroid dosage and death with a functioning graft after kidney transplantation.

Death with a functioning graft remains a major challenge following kidney transplantation. Steroid dosing may be a modifiable risk factor. Collaborative Transplant Study (CTS) data were analyzed to assess the relationship between long-term steroid dose and death with function during years 2-5 posttransplant in 41 953 adult recipients of a deceased-donor kidney transplant during 1995-2010. Steroid dose at year 1 correlated significantly with death with function overall, and with death due to cardiovascular disease or infection (all p < 0.001). In patients with optimal graft function (serum creatinine <130 µmol/L) and no anti-rejection treatment during (a) year 1 (b) years 1 and 2, these significant associations remained (all p < 0.001). The center-specific incidence of steroid withdrawal during year 2 showed a significant inverse association with death due to cardiovascular disease (p < 0.001) or infection (p < 0.001) overall, and within the subpopulation with good graft function and no rejection during year 1 (p = 0.002 and p < 0.001, respectively). Maintenance steroid dose shows a highly significant association with death with a functioning graft caused by cardiovascular disease or infection during years 2-5 after kidney transplantation, even in patients with good graft outcomes in whom steroid treatment would appear to be unnecessary.

Influence of test technique on sensitization status of patients on the kidney transplant waiting list.

The exquisitely sensitive single antigen bead (SAB) technique was shown to detect human leukocyte antigen (HLA) antibodies in sera of healthy male blood donors. Such false reactions can have an impact on critical decisions, especially with respect to the determination of unacceptable HLA-antigen mismatches in patients awaiting a kidney transplant. We tested pretransplant sera of 534 patients on the kidney waiting list using complement-dependent cytotoxicity (CDC), enzyme-linked immunosorbent assay (ELISA) and SAB in parallel. Evidence of HLA antibodies was obtained in 5% of patients using CDC, 14% using ELISA, and 81% using SAB. Among patients without history of an immunizing event, 77% showed evidence of HLA antibodies in SAB. In contrast 98% of these patients were negative in ELISA and CDC. In patients without an immunizing event, SAB-detected antibodies reacted not always weakly but with mean fluorescence intensity (MFI) values as high as 14 440. High-MFI-value antibodies were found in some of these patients with HLA specificities that are rather common in general population, consideration of which would lead to unjustified exclusion of potential kidney donors. False SAB reactions can be unveiled by testing with additional antibody assays. Denial of donor kidneys to recipients based on HLA-antibody specificities detected exclusively in the SAB assay is not advisable.

No impact of KIR-ligand mismatch on allograft outcome in HLA-compatible kidney transplantation.

Natural killer (NK) cell function can be modulated by the killer cell immunoglobulin-like receptors (KIR) which interact with human leukocyte antigen (HLA) class I molecules on target cells. KIR-ligand mismatching has recently been shown by van Bergen et al. (American Journal of Transplantation 2011; 11(9): 1959-1964) to be a significant risk factor for long-term graft loss in HLA-A, -B and -DR compatible kidney transplants. To verify this potentially important finding, we performed genotyping of 608 deceased-donor kidney graft recipients and their HLA-A, -B and -DR compatible donors for KIR and HLA, using samples and clinical data provided by the Collaborative Transplant Study. Graft survival of KIR-ligand-matched and -mismatched transplants was compared. We found no impact of KIR-ligand mismatching on 10-year graft survival in HLA-A, -B, -DR compatible kidney transplants. Further analysis did not reveal a significant effect of recipient activating/inhibitory KIR or KIR genotypes on graft survival. Our data do not support the concept that KIR-HLA matching might serve as a tool to improve long-term renal allograft survival.

Ceppellini Lecture 2012: collateral damage from HLA mismatching in kidney transplantation.

Inclusion of human leukocyte antigen (HLA) matching in donor kidney allocation schemes has been based solely on its association with graft survival. Other long-term effects associated with HLA incompatibility are largely unexplored. Data from deceased donor kidney transplants reported to the Collaborative Transplant Study have been analyzed to assess the relation between HLA mismatching and clinical events to 3 years post-transplant, and an overview of these analyses is presented. A significant correlation was observed between the number of mismatches and the need for anti-rejection therapy during the first year post-transplant, which was maintained for HLA-DR and HLA-A + B mismatching separately and at years 2 and 3 post-transplant. The number of HLA-DR mismatches and the number of HLA-A + B mismatches as well as rejection treatment showed significant associations with the dose of maintenance steroids. The cumulative incidences of death with a functioning graft from infection or cardiovascular causes, but not from cancer, were also significantly associated with HLA mismatching. The number of HLA-DR mismatches showed a significant association with the incidence of non-Hodgkin lymphoma and hip fractures. These findings show that the adverse consequences of HLA mismatching on kidney transplants extend beyond an effect on graft survival, and include an increased risk of death with a functioning graft, non-Hodgkin lymphoma and hip fracture.

Characterization of mannose-binding lectin (MBL) variants by allele-specific sequencing of MBL2 and determination of serum MBL protein levels.

Mannose-binding lectin (MBL) is a major component of the lectin pathway of complement activation. High and low MBL levels have been associated with susceptibility and severity of a variety of infectious and autoimmune diseases. Several single-nucleotide polymorphisms (SNPs) in the promoter region and exon 1 of the MBL2 gene are responsible for variations in serum MBL levels. We developed a sequence-based typing method for allele-specific MBL2 genotyping and measured serum MBL protein levels in 24 German blood donors. We identified the common MBL2 haplotypes including five promoter polymorphisms in linkage with the Q allele and correlated serum MBL levels with the respective genotypes. The genotyping method presented here could provide a basis for confirmatory studies in larger cohorts.

Minor H antigen matches and mismatches are equally distributed among recipients with or without complications after HLA identical sibling renal transplantation.

Studies of the effect of minor H antigen mismatching on the outcome of renal transplantation are scarce and concern mainly single center studies. The International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 16 laboratories of the IHIW, the role of 15 autosomal, 10 Y-chromosome encoded minor H antigens and 3 CD31 polymorphisms, was investigated in relation to the incidence of renal graft rejection and graft loss in 444 human leukocyte antigens (HLA)-identical sibling renal transplantations. Recipient and donor DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, C19Orf48, LB-ECGF-1, CTSH, LRH-1, LB-ADIR and HY. The correlation between minor H antigen mismatch and the primary outcome graft rejection or graft loss was statistically analyzed. The incidence of rejection was very low and no correlation was observed between one or more minor H antigen mismatch(es) and a rejection episode (n = 36), of which only eight resulted in graft loss. In summary, in our study cohort of 444 renal transplants, mismatching for neither autosomal nor HY minor H antigens correlate with rejection episodes or with graft loss.

Association of HLA mismatch with death with a functioning graft after kidney transplantation: a collaborative transplant study report.

HLA mismatches may correlate with risk of death with a functioning graft (DWFG) because of requirement for higher immunosuppression doses and more antirejection therapy. Deceased-donor kidney transplants (n = 177 584) performed 1990-2009 and reported to the Collaborative Transplant Study were analyzed. The incidence of DWFG was found to be 4.8% during year 1 posttransplant and 7.7% during years 2-5 (Kaplan-Meier estimates). Most frequent causes of DWFG were infection, cardiovascular disease and malignancy (32.2%, 30.9% and 3.6% in year 1; 16.4%, 29.6% and 15.9% in years 2-5). HLA-A + B + DR mismatches were significantly associated with DWFG during year 1 (p < 0.001), a correlation that diminished but persisted during years 2-5 (p < 0.001). HLA mismatch was associated with DWFG because of infection (p < 0.001 during year 1, p = 0.043 during years 2-5) or cardiovascular disease (p < 0.001 during year 1, p = 0.030 during years 2-5) but not malignancy. There was also a significant association between HLA mismatch and hospitalization for viral (p < 0.001) or bacterial (p = 0.002) infection. Multivariable analysis showed that mismatches for HLA class II were more strongly associated with both hospitalization and DWFG than mismatches for HLA class I.

Characterization of four new HLA alleles: HLA-B*15:01:18, HLA-B*44:110, HLA-C*04:01:22 and HLA-DQB 1*05:14.

We describe four novel HLA alleles, HLA-B*15:01:18, HLA-B*44:110, HLA-C*04:01:22 and HLA-DQB1*05:14.

Treatment of kidney transplant recipients with ACEi/ARB and risk of respiratory tract cancer: a collaborative transplant study report.

Whether treatment with angiotensin-converting enzyme inhibitors (ACEi) and/or angiotensin receptor blockers (ARB) increases the risk of cancer is controversial. Collaborative transplant study data were analyzed according to whether kidney transplant recipients were treated with ACEi/ARB at year 1. Twenty-four thousand and ninety patients were studied of whom 9079 (38%) patients received ACEi/ARB. There were 872 nonskin malignancies during years 2-8 posttransplant, including 107 respiratory/intrathoracic tumors. The standardized incidence ratio (SIR) for all nonskin malignancies was similar between the ACEi/ARB (1.91) and no ACEi/ARB (1.81) groups (p = 0.42). For respiratory/intrathoracic tumors, however, SIR was significantly higher with ACEi/ARB (1.65 vs. 1.09 for no ACEi/ARB, p = 0.033). Multivariate Cox regression analysis showed that ACEi/ARB treatment was not associated with an increased risk of respiratory/intrathoracic tumors in nonsmokers. In patients with a history of smoking, however, the risk of respiratory/intrathoracic tumors was 2.77 (95% CI 1.19-6.43, p = 0.018) in patients without ACEi/ARB treatment as compared to 7.10 (95% CI 3.27-15.4, p < 0.001) in patients treated with ACEi/ARB. Our data indicate that in kidney transplant recipients, ACEi/ARB treatment is associated with a significant increase in the rate of respiratory/intrathoracic tumors in the subpopulation of patients with a history of smoking.

Characterization of three new alleles HLA-A*02:241, HLA-A*02:242 and HLA-A*30:04:02.

We describe three novel alleles HLA-A*02:241, HLA-A*02:242 and HLA-A*30:04:02.

Autologous stimulation of human lymphocyte subpopulation.

The background stimulation universally seen when lymphocytes are cultured in vitro has been shown to be markedly lowered by reducing the proportion of B lymphocytes. B-rich fractions of lymphocytes had extremely high background stimulation. It is concluded that stimulation of T cells, probably by autologous B cells, provides the most probable explanation for the findings described.

QuickMap: a public tool for large-scale gene therapy vector insertion site mapping and analysis.

Several events of insertional mutagenesis in pre-clinical and clinical gene therapy studies have created intense interest in assessing the genomic insertion profiles of gene therapy vectors. For the construction of such profiles, vector-flanking sequences detected by inverse PCR, linear amplification-mediated-PCR or ligation-mediated-PCR need to be mapped to the host cell's genome and compared to a reference set. Although remarkable progress has been achieved in mapping gene therapy vector insertion sites, public reference sets are lacking, as are the possibilities to quickly detect non-random patterns in experimental data. We developed a tool termed QuickMap, which uniformly maps and analyzes human and murine vector-flanking sequences within seconds (available at www.gtsg.org). Besides information about hits in chromosomes and fragile sites, QuickMap automatically determines insertion frequencies in +/- 250 kb adjacency to genes, cancer genes, pseudogenes, transcription factor and (post-transcriptional) miRNA binding sites, CpG islands and repetitive elements (short interspersed nuclear elements (SINE), long interspersed nuclear elements (LINE), Type II elements and LTR elements). Additionally, all experimental frequencies are compared with the data obtained from a reference set, containing 1 000 000 random integrations ('random set'). Thus, for the first time a tool allowing high-throughput profiling of gene therapy vector insertion sites is available. It provides a basis for large-scale insertion site analyses, which is now urgently needed to discover novel gene therapy vectors with 'safe' insertion profiles.

Reassessing the impact of donor HLA-C genotype on long-term liver transplant survival.

HLA-C is the major inhibitory ligand for killer immunoglobulin-like receptors (KIRs) that are expressed on natural killer (NK) cells. Based on their KIR specificity, HLA-C alleles can be divided into two groups, termed HLA-C1 and HLA-C2. Donor HLA-C group has recently been identified by Hanvesakul et al. (Am J Transplant 2008) as a critical determinant of clinical outcome following liver transplantation: Possession of at least one HLA-C group 2 allele by the donor was associated with significantly improved long-term graft and patient survival, presumably due to an inhibition of host NK cell function. To verify this study, we performed genotyping of 913 deceased liver donors for the relevant KIR epitopes of HLA-C and correlated the presence or absence of donor HLA-C2 genotype with graft and patient survival. In our study, donor HLA-C2 genotype had no impact on 10-year graft or patient survival. We cannot confirm a major role of donor HLA-C2 genotype on long-term allograft survival after liver transplantation.

Lymphocyte antigenicity loss with retention of responsiveness.

During culturing at 22 degrees C for more than 4 days, human peripheral blood lymphocytes lose their ability to stimulate allogeneic lymphocytes in mixed lymphocyte cultures. The cells retain their ability to respond to allogeneic lymphocytes or phytohemagglutinin for up to 10 days of culturing. The findings are relevant to reports on successful transplantation of cultured skin.

Prolonged survival of second human kidney transplants.

Rejection of kidney transplants in 264 patients, followed by retransplantation from cadaver donors, resulted in a 1-year survival rate of 51 +/- 3 percent (rate +/- standard error) as compared to 51 +/- 1 percent for first transplants. If the first transplant immunizes the patient or is rejected by immunologically responsive patients, second grafts into the same patients would be expected to be rejected at a higher rate. Only those reject who reject first grafts hyperacutely or between 1 to 3 months were found to have low second graft survival rates. Patients who rejected transplants after 3 months tended to have second transplant survival rates which were higher than their first graft survival rates.