Adriana Colovai

The prevalence and clinical significance of C1q-binding donor-specific anti-HLA antibodies early and late after kidney transplantation.

We aimed to determine the prevalence and clinical significance of complement-binding donor-specific antibodies (DSA) detected up to 30 years after kidney transplantation. Group 1 patients included 284 consecutive DSA negative patients who underwent kidney transplantation after 1 May 2009. Group 2 included 405 patients transplanted before this date and followed at our center with functioning allografts. DSA were tested using Luminex Single Antigen and the C1q assay. In Group 1 patients, who were monitored prospectively, 31 (11%) developed de novo DSA during a median follow-up of 2.5 (1.9, 3.6) years. Of these, 11 (4%) had C1q+ and 20 (7%) had C1q negative DSA. In Group 2 patients, 77 (19%) displayed DSA. Among these, 33 (8%) had C1q+ and 44 (11%) had C1q negative DSA. The incidence of acute antibody-mediated rejection (AMR) was significantly higher in C1q+DSA patients in both Group 1 (45%) and Group 2 (15%) compared with C1q negative DSA (5% and 2%) and DSA negative patients (1% and 3%; P < 0.001 and P = 0.001). The incidence of chronic AMR was 36% (Group 1) and 51% (Group 2) in patients with C1q+DSA. In contrast, chronic AMR occurred in 5% and 25% of C1q negative DSA, and 2% and 6% of DSA negative Group 1 and 2 patients, respectively (P < 0.001). Although the graft survival was lower in Group 1 C1q+DSA patients (73%) compared with C1q negative DSA (95%) and DSA negative (94%) patients, the difference was not statistically significant by Kaplan-Meier survival analysis (P = 0.21). Our results indicated that the presence of C1q+ DSA was associated with acute and chronic AMR.

Increased intragraft rejection-associated gene transcripts in patients with donor-specific antibodies and normal biopsies

We investigated why some donor-specific antibody-positive patients do not develop antibody-mediated rejection. Of 71 donor-specific antibody-positive patients, 46 had diagnosis of antibody-mediated rejection and 25 had normal biopsies. Fifty donor-specific antibody-negative patients with normal biopsies were used as a control group. A subgroup of 61 patients with available biopsy and 64 with blood samples were analyzed by microarrays. Both donor-specific antibody-positive/antibody-mediated rejection-positive and negative biopsies showed increased expression of gene transcripts associated with cytotoxic T cells, natural killer cells, macrophages, interferon-gamma, and rejection compared to donor-specific antibody-negative biopsies. Regulatory T-cell transcripts were upregulated in donor-specific antibody-positive/antibody-mediated rejection-positive and B-cell transcripts in donor-specific antibody-positive/antibody-mediated rejection-negative biopsies. Whole-blood gene expression analysis showed increased immune activity in only donor-specific antibody-positive/antibody-mediated rejection-positive but not negative patients. During a median follow-up of 36 months, 4 donor-specific antibody-positive/antibody-mediated rejection-negative patients developed antibody-mediated rejection, 12 continued to have donor-specific antibody, but 9 lost their donor-specific antibody. Gene expression profiles did not predict the development of antibody-mediated rejection or the persistence of donor-specific antibody. Thus, donor-specific antibody-positive/antibody-mediated rejection-negative patients had increased rejection-associated gene transcripts in their allografts despite no histologic findings of rejection but not in their blood. This was found in both biopsy and blood samples of donor-specific antibody-positive/antibody-mediated rejection-positive patients.

Pretransplant immunologic risk assessment of kidney transplant recipients with donor-specific anti-human leukocyte antigen antibodies.

Background Patients with pretransplantation strong donor-specific anti–human leukocyte antigen (HLA) antibodies (DSA) are at higher risk for rejection. We aimed to study the safety of kidney transplantation in patients with lower strength DSAs in a prospective cohort study. Methods Three hundred and seventy-three consecutive adult kidney transplant recipients with (DSA+; n=66) and without (DSA−; n=307) DSA were evaluated. Anti-HLA antibodies with mean fluorescence intensity values over 5,000 for HLA-A, HLA-B, and HLA-DR and more than 10,000 for HLA-DQ were reported as unacceptable antigens. Patients received transplant if flow cytometry T-cell and B-cell cross-match channel shift values were less than 150 and 250, respectively, with antithymocyte globulin and intravenous immunoglobulin induction treatment. Results Patients had a mean number of 1.6±0.8 DSAs with a mean fluorescence intensity value of 2,815±2,550. Twenty-seven percent were flow cytometry cross-match positive with T-cell and B-cell channel shift values of 129±49 and 159±52, respectively. During a median follow-up of 24 months (range, 6–50), there were no statistically significant differences in patient (99% vs. 95%) and graft survival (88% vs. 90%) rates between DSA+ and DSA− groups, respectively. Cumulative acute rejection rates of 11% in the DSA+ group and 12% in the DSA− group were similar. Two DSA+ (3%) and five DSA− (2%) patients developed chronic antibody-mediated rejection (3%). The mean serum creatinine levels were identical between the two groups (1.4±0.6 mg/dL). Conclusion Similar patient and graft survival, and acute rejection rates can be achieved in DSA+ patients compared to DSA− patients with pretransplantation immunologic risk assessment.

Clinical and molecular significance of microvascular inflammation in transplant kidney biopsies.

The diagnostic criteria for antibody-mediated rejection (AMR) are continuously evolving. Here we investigated the clinical and molecular significance of different Banff microvascular inflammation (MVI) scores in transplant kidney biopsies. A total of 356 patients with clinically indicated kidney transplant biopsies were classified into three groups based on MVI scores of 0, 1, 2, or more for Groups 1-3, respectively. Gene expression profiles were assessed using arrays on a representative subset of 93 patients. The incidence of donor-specific anti-HLA antibodies was increased from 25% in Group 1 to 36% in Group 2 and to 54% in Group 3. Acute and chronic AMR were significantly more frequent in Group 3 (15% and 35%) compared with the Group 2 (3% and 15%) and Group 1 (0% and 5%), respectively. Gene expression profiles showed increased interferon-γ and rejection-induced, cytotoxic and regulatory T-cell, natural killer cell-associated and donor-specific antibody (DSA)-selective transcripts in Group 3 compared with Groups 1 and 2. There was no significant difference in gene expression profiles between the Groups 1 and 2. Increased intragraft expression of DSA-selective transcripts was found in the biopsies of C4d- Group 3 patients. Thus, an MVI score of 2 or more was significantly associated with a histological diagnosis of acute and chronic antibody-mediated rejection. Hence, increased intragraft DSA-selective gene transcripts may be used as molecular markers for AMR, especially in C4d- biopsies.

A pathogenesis-based transcript signature in donor-specific antibody-positive kidney transplant patients with normal biopsies

Affymetrix Human Gene 1.0-ST arrays were used to assess the gene expression profiles of kidney transplant patients who presented with donor-specific antibodies (DSAs) but showed normal biopsy histopathology and did not develop antibody-mediated rejection (AMR). Biopsy and whole-blood profiles for these DSA-positive, AMR-negative (DSA +/AMR−) patients were compared to both DSA-positive, AMR-positive (DSA +/AMR +) patients as well as DSA-negative (DSA −) controls. While individual gene expression changes across sample groups were relatively subtle, gene-set enrichment analysis using previously identified pathogenesis-based transcripts (PBTs) identified a clear molecular signature involving increased rejection-associated transcripts in AMR − patients. Results from this study have been published in Kidney International (Hayde et al., 2014 [1]) and the associated data have been deposited in the GEO archive and are accessible via the following link: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50084

Increased access to transplantation of highly sensitized patients under the new kidney allocation system. A single center experience

We aimed to investigate the impact of the new kidney allocation system (KAS) on the rate of transplantation of sensitized patients at our center. Pre-KAS and post-KAS intervals were Jan 1st to Dec 3rd 2014 and Jan 1st 2015 to Dec 3rd 2015, respectively. The number of deceased-donor crossmatches performed by flow cytometry increased from 715 pre-KAS to 1188 post-KAS. The percent of crossmatches performed for sensitized patients with calculated panel reactive antibody (cPRA)>0% increased from 19% pre-KAS to 26% post-KAS (p<0.0001). The number of deceased-donor kidney transplants performed at our center increased from 115 pre-KAS to 125 post-KAS (9% increase). There was a significant increase in the percentage of deceased-donor kidney transplants received by sensitized candidates (from 14% to 26% pre- and post-KAS, respectively; p<0.0001). The highest increase was seen in the patients with cPRA>98%, from 0% to 9%, followed by the group with cPRA 50-79%, from 5% to 8%. This increase was balanced by a decrease of 12% in the percentage of non-sensitized recipients, and a modest decrease of 1% in the group with cPRA 1-49%. In conclusion, transplant rate has increased in sensitized patients after KAS. The highest increase was observed among highly sensitized patients (cPRA>98%).

Pregnancy in sensitized kidney transplant recipients: a single-center experience

We aimed to examine the clinical outcomes of sensitized pregnant kidney transplant recipients.

False positive hepatitis C antibody test results in left ventricular assist device recipients: increased risk with age and transfusions

Left ventricular assist devices (LVADs) have been successfully used in patients with heart failure. However, LVADs may trigger immune activation, leading to higher frequencies of autoantibodies. We describe the clinical, epidemiological, and laboratory characteristics of LVAD recipients with false positive hepatitis C (FPHC) serology among 39 consecutive adult LVAD recipients who bridged to heart transplantation from January 2007 to January 2013 at Montefiore Medical Center. FPHC patients were identified as those with post-LVAD positive hepatitis C ELISA antibody tests and negative confirmatory testing with hepatitis C RNA PCR and/or radioimmunoblot assay. Ten (26%) patients previously seronegative for hepatitis C were found to have FPHC after device placement. Of the 39 patients, 32 had HeartMate II devices. The mean age at LVAD placement was 55 years. FPHC correlated with older age at the time of LVAD implantation and with receipt of packed red blood cell transfusions, but not with gender, fresh frozen plasma transfusions, panel reactive antibodies, globulin fraction, rheumatoid factor, or anticardiolipin antibodies. Clinicians should be aware of this increased risk of FPHC in older LVAD patients and those more heavily transfused in order to avoid unnecessary apprehension and possible delay in transplantation. Further studies should be done to evaluate the possible relationship between transfused blood products and immunomodulation.

The ZFX Target Gene, FAM92A1, Is a Marker of AML Aggressiveness

The propagation of leukemia requires activation of stem cell transcriptional programs that support long term self-renewal. Previously we showed that the stem cell transcription factor ZFX is critically important for propagation of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). We have identified ZFX target genes in mouse and human leukemia cells using gene expression profiling in conjunction with chromatin immunoprecipitation and massively parallel sequencing (ChIP-seq). Some ZFX target genes have a well established role in controlling mitochondrial function and cell metabolism, suggesting that ZFX is required, in part, to maintain metabolic homeostasis in leukemia propagating cells. The biological functions of other ZFX target genes have not been well characterized. One such gene, FAM92A1, is heavily dependent on ZFX for its expression in mouse and human AML cells. Genetic deletion of ZFX in mouse AML and lentiviral RNAi mediated knockdown of ZFX in human AML cell lines markedly decreased FAM92A1 expression. ChIP-seq showed a ZFX binding site in a dense CpG island between the first and second exons, which was confirmed by ChIP-PCR and suggests that ZFX directly controls FAM92A1 transcription at the proximal promoter. Our studies in sorted murine AML cells and human AML patient samples demonstrate that FAM92A1 is preferentially expressed in the leukemia initiating cell-enriched fractions. Analysis of large-scale gene expression studies of human AML patients revealed that high FAM92A1 expression is associated with the Flt3-ITD mutation, high-risk cytogenetics, early AML recurrence, and patient death. Multivariable analysis showed that it was an independent risk factor for shortened progression-free survival. In addition, FAM92A1 expression is increased in patients with myodysplastic syndrome-refractory anemia with excess blasts. These findings identify FAM92A1 as a marker for ZFX transcriptional activity and AML aggressiveness. The increased expression of FAM92A1 in AML with Flt3-ITD and high-risk cytogenetics suggests that it may be a functionally important component of the ZFX-driven self-renewal transcriptional program in high-risk AML. Definitive functional studies are warranted to establish the role of FAM92A1 in AML propagation.