Antibody-mediated Cardiac Allograft Rejection Research Articles

Precise treatment of acute antibody-mediated cardiac allograft rejection in rats using C4d-targeted microbubbles loaded with nitric oxide

Antibody-mediated rejection (AMR) constitutes an important cause of cardiac allograft loss; however, all current therapeutic strategies represent systemic applications with unsatisfactory efficacy. Previously, we successfully non-invasively detected C4d, a specific marker for AMR diagnosis, in allografts using C4d-targeted microbubbles (MBC4d). In this study, we extended this approach by incorporating nitric oxide (NO), as high NO levels manifest immunosuppressive and anti-thrombotic effects. We designed novel MBC4d loaded with NO (NO-MBC4d). A rat model of AMR was established by pre-sensitization with skin transplantation. Contrast-enhanced ultrasound (CEUS) images were obtained and quantitatively analyzed following NO-MBC4d injection. Allograft survival and histologic features were analyzed to evaluate the therapeutic effect and underlying mechanism of NO-MBC4d toward AMR. We successfully obtained CEUS images following NO-MBC4d injection and demonstrated that the ultrasound signal intensity of the myocardial area and clearance time of NO-MBC4d both increased with increased C4d grade, thereby realizing non-invasive diagnosis of AMR. Furthermore, allograft survival was significantly prolonged, and rejection was obviously attenuated following NO-MBC4d injection through significant suppression of thrombosis and reduction of inflammatory cell infiltrates. Overall, the therapeutic efficacy was significantly improved in the NO-MBC4d group compared with the control NO-MB group, demonstrating that precise treatment could significantly improve the therapeutic efficacy compared with that afforded by systemic applications. This study presented a novel tool to provide simultaneous non-invasive diagnosis and precise treatment of AMR using NO-MBC4d CEUS imaging, which may be expected to provide a better option for recipients with AMR in clinic.

Noninvasive and quantitative measurement of C4d deposition for the diagnosis of antibody-mediated cardiac allograft rejection.

BackgroundC4d is a specific biomarker for the diagnosis of antibody-mediated rejection (AMR) after cardiac transplantation. Although strongly recommended, routine C4d surveillance is hindered by the invasive nature of endomyocardial biopsy. Targeted ultrasound (US) has high sensitivity, and C4d is abundantly expressed within the graft of patients experiencing AMR, which makes it possible to visualize C4d deposition in vivo using targeted US.MethodsWe designed a serial dilution of C4d-targeted microbubbles (MBC4d) using a streptavidin-biotin conjugation system. A rat model of AMR with C4d deposition was established by pre-sensitization with skin transplantation before cardiac transplantation. MBC4d were injected into recipients and then qualitatively and quantitatively analyzed using the destruction-replenishment method with a clinical US imaging system and analyzed by software.FindingsWe successfully obtained qualitative images of C4d deposition in a wide cardiac allograft section, which, for the first time, reflected real-time C4d distribution. Moreover, normal intensity difference was used for quantitative analysis and exhibited an almost nearly linear correlation with the grade of C4d deposition according to the pathologic evidence. In addition, MBC4d injection did not affect the survival and aggravate injury, which demonstrates its safety.InterpretationThis study demonstrates a noninvasive, quantitative and safe evaluation method for C4d. As contrast-enhanced US has been widely used in clinical settings, this technology is expected to be applied quickly to clinical practice.FundNational Natural Science Foundation of China and Guangdong Province, Leading Scientific Talents of Guangdong special support program, the Science and Technology Project of Guangdong Province and Guangzhou City.

Polymorphisms in genes related to the complement system and antibody-mediated cardiac allograft rejection

Heart transplantation (HT) is a life-saving treatment for patients with end-stage heart failure. One of the main problems after HT is the humoral response termed antibody-mediated rejection (AMR). Complement activation plays a key role in AMR contributing to graft damage. The aim of this study was to analyze genetic variants in genes related to the complement pathways that could be associated with the development of AMR. Analysis of 51 genes related to the complement pathway was performed by next-generation sequencing in 46 HT recipients, 23 with and 23 without AMR. Statistical analysis was performed with SNPstats and R. We identified 2 single nucleotide polymorphisms, 1 in the mannose-binding lectin 2 gene (p.Gly54Asp-MBL2) and 1 in the complement factor properdin gene (p.Asn428(p=)-CFP), that showed significant association with the absence and development of AMR, respectively. Moreover, the presence of the rare allele in p.Gly54Asp-MBL2 control patients correlated with an immunodeficiency of mannose-binding lectin (6.24 ng/ml vs 207.50 ng/ml, p < 0.01), whereas the presence of the rare allele p.Asn428(p=)-CFP in patients with AMR correlated with higher levels of properdin protein (14.65 μg/ml vs 10.77 μg/ml, p < 0.05). AMR is a complex phenotype affected by many recipient factors. Variants in p.Gly54Asp-MBL2 and p.Asn428(p=)-CFP genes, encoding mannose-binding lectin 2 and properdin, may influence the risk of AMR.

Graft-Derived CCL2 Increases Graft Injury During Antibody-Mediated Rejection of Cardiac Allografts

The pathogenic role of macrophages in antibody-mediated rejection (AMR) remains unclear. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic factor for monocytes and macrophages. The current studies used a murine model of AMR to investigate the role of graft-derived CCL2 in AMR and how macrophages may participate in antibody-mediated allograft injury. B6.CCR5−/−/CD8−/− recipients rejected MHC-mismatched WT A/J allografts with high donor-reactive antibody titers and diffuse C4d deposition in the large vessels and myocardial capillaries, features consistent with AMR. In contrast, A/J.CCL2−/− allografts induced low donor-reactive antibody titers and C4d deposition at Day 7 posttransplant. Decreased donor-reactive CD4 T cells producing interferon gamma were induced in response to A/J.CCL2−/− versus WT allografts. Consequently, A/J.CCL2−/− allograft survival was modestly but significantly longer than A/J allografts. Macrophages purified from WT allografts expressed high levels of IL-1β and IL-12p40 and this expression and the numbers of classically activated macrophages were markedly reduced in CCL2-deficient allografts on Day 7. The results indicate that allograft-derived CCL2 plays an important role in directing classically activated macrophages into allografts during AMR and that macrophages are important contributors to the inflammatory environment mediating graft tissue injury in this pathology, suggesting CCL2 as a therapeutic target for AMR.

Role of Graft-Derived CCL2/MCP-1 in Antibody-Mediated Rejection of Cardiac Allografts.

The role of monocytes/macrophages in antibody-mediated rejection (AMR) remains unclear. We have reported that murine CCR5-/-/CD8-/- recipients produce high titers of donor-specific antibody and reject MHC-mismatched heart allografts. The current studies tested the magnitude of injury and alloimmune responses to grafts deficient in expression of the monocyte chemoattractant MCP-1/CCL2 during AMR. B6.CCR5-/-/CD8-/- recipients rejected MHC-mismatched wild-type A/J allografts with high donor-reactive antibody titers and diffuse C4d deposition in the large vessels and myocardial capillaries, features consistent with AMR. Donor-specific CD4 T cells producing IFN-γ in the recipient spleen were significantly decreased in response to A/J.CCL2-/- vs. A/J allografts and this resulted in decreased donor-reactive antibody production and C4d deposition in allografts in B6. CCR5-/-/CD8-/- recipients at day 7 post-transplant. Consequently, A/J.CCL2-/- allograft survival was slightly but significantly longer than that of A/J allografts (MST: wild-type A/J, day 8 vs. A/J.CCL2-/-, day 10, P < 0.01). Allograft CCL2 deficiency reduced classically activated (M1) macrophage and other inflammatory leukocyte infiltration into the allografts. Strikingly, A/J.CCL2-/- allograft survival was much longer than wild-type A/J allografts in recipients treated with 0.25 mg anti-CD40L mAb at the time of transplant (MST: wild-type A/J, day 21.5 vs. A/J.CCL2-/-, day 58, P < 0.01) with lower titers of donor-reactive antibody and decreased leukocyte infiltration. CCL2 deficiency also reduced pro-fibrotic gene expression and delayed the development of interstitial fibrosis when compared to wild-type A/J allografts. The results indicate that allograft-derived CCL2/MCP-1 plays an important role in directing M1 macrophages and other inflammatory cell infiltration into allografts and in the development of donor-reactive antibody and vascular rejection and suggest CCL2/MCP-1 as a therapeutic target for antibody-mediated vascular rejection and chronic graft injury.

648 Resurgence of Photopheresis for Recalcitrant Cellular and Antibody-Mediated Cardiac Allograft Rejection

648 Resurgence of Photopheresis for Recalcitrant Cellular and Antibody-Mediated Cardiac Allograft Rejection

Antibody-Mediated Rejection in Heart Transplantation: Case Presentation with a Review of Current International Guidelines

Antibody-mediated rejection (AMR) (humoral rejection) of cardiac allografts remains difficult to diagnose and treat. Interest in AMR of cardiac allografts has increased over the last decade as it has become apparent that untreated humoral rejection threatens graft and patient survival. An international and multidisciplinary consensus group has formulated guidelines for the diagnosis and treatment of AMR and established that identification of circulating or donor-specific antibodies is not required and that asymptomatic AMR, that is, biopsy-proven AMR without cardiac dysfunction is a real entity with worsened prognosis. Strict criteria for the diagnosis of cardiac AMR have not been firmly established, although the diagnosis relies heavily on tissue pathological findings. Therapy remains largely empirical. We review an unfortunate experience with one of our patients and summarize recommended criteria for the diagnosis of AMR and potential treatment schemes with a focus on current limitations and the need for future research and innovation.

Luminex-based virtual crossmatching facilitates combined third-time cardiac and de novo renal transplantation in a sensitized patient with sustained antibody-mediated cardiac allograft rejection

Allosensitization represents a major obstacle to successful re-transplantation since circulating antibodies can elicit antibody-mediated rejection episodes with subsequent graft failure. Since sensitization is primarily considered a contraindication to transplantation the duration for patients waiting for a suitable donor organ to become available is considerably prolonged. Herein, we report on the successful application of a Luminex-based virtual crossmatch approach to facilitate combined third-time cardiac and de novo renal transplantation in a sensitized patient with sustained antibody-mediated cardiac allograft rejection.

Detection of C4d deposition in cardiac allografts: a comparative study of immunofluorescence and immunoperoxidase methods.

Abstract Context.—Complement activation, evidenced by deposition of C4d, is important in the diagnosis of antibody-mediated rejection of cardiac allografts. C4d deposition can be assessed by either immunofluorescence (IF)– or immunoperoxidase (IP)–based methods. The use of methods varies considerably among institutions, but there are few data addressing their diagnostic equivalence. Objective.—To compare IF and IP C4d staining on paired endomyocardial biopsy samples from a large number of heart transplant patients. Design.—Retrospectively selected paired frozen and paraffin-embedded samples from the same biopsy were stained for C4d by IF and IP methods. Capillary staining was scored by using a 0, 1+, 2+, 3+ scale. Results.—A total of 296 biopsy pairs from 70 patients were studied. There were two hundred forty-three cases that were scored 0, twenty-four scored 1+, sixteen scored 2+, and thirteen scored 3+ by IF. Two hundred thirty-one cases scored 0, forty scored 1+, ten scored 2+, and fifteen scored 3+ by...

Detection of C4d Deposition in Cardiac Allografts: A Comparative Study of Immunofluorescence and Immunoperoxidase Methods

Complement activation, evidenced by deposition of C4d, is important in the diagnosis of antibody-mediated rejection of cardiac allografts. C4d deposition can be assessed by either immunofluorescence (IF)- or immunoperoxidase (IP)-based methods. The use of methods varies considerably among institutions, but there are few data addressing their diagnostic equivalence. To compare IF and IP C4d staining on paired endomyocardial biopsy samples from a large number of heart transplant patients. Retrospectively selected paired frozen and paraffin-embedded samples from the same biopsy were stained for C4d by IF and IP methods. Capillary staining was scored by using a 0, 1+, 2+, 3+ scale. A total of 296 biopsy pairs from 70 patients were studied. There were two hundred forty-three cases that were scored 0, twenty-four scored 1+, sixteen scored 2+, and thirteen scored 3+ by IF. Two hundred thirty-one cases scored 0, forty scored 1+, ten scored 2+, and fifteen scored 3+ by IP. Complete agreement was seen in 81% of cases. Among discrepant cases, 89% (n = 51) were minor (±1) and 11% (n = 6) were major (±2). Five of the 6 major discrepancy biopsies came from 2 patients, both of whom had concordant (IF and IP) 3+ results on prior biopsies. The weighted κ value for the entire sample set was 0.78 and for the first biopsy only set (to correct for bias introduced by multiple biopsies from the same patient) the weighted κ value was 0.88. Immunofluorescence and IP C4d staining methods are highly comparable and are both viable options for antibody-mediated rejection surveillance in transplant heart biopsies.

92: Detection of C4d Deposition in Cardiac Allografts: A Comparative Study of Immunofluorescence and Immunoperoxidase Methods

Complement activation, evidenced by deposition of C4d, is important in the diagnosis of antibody-mediated rejection of cardiac allografts. C4d deposition can be assessed by either immunofluorescence (IF) or immunoperoxidase (IP) based methods. The use of methods varies considerably among institutions, but there are few data addressing their diagnostic equivalence. The purpose of this study was to compare IF and IP C4d staining on paired endomyocardial biopsy samples from a large number of heart transplant patients.

Serial monitoring of humoral antibody-mediated rejection of cardiac allografts by C4d staining of interstitial capillaries

C4d “staining” of interstitial capillaries of endomyocardial biopsies serves as an indicator of a humoral component of cardiac allograft rejection. In the present study, two cardiac allograft recipients were monitored serially for both cellular and humoral rejection. Cellular rejection, evaluated by light microscopy, and humoral rejection, judged by C4d immunofluorescent “staining”, were treated with appropriate immunosuppressants. Weekly serial biopsies of the first patient revealed maximal humoral rejection (3 +) after 1 week but diminished to 2 + thereafter. Cellular rejection was graded as 3A after 3 weeks but declined steadily to negligible cellular rejection through week 15. Whereas, cellular rejection peaked at 14–21 days, humoral rejection was greatest in the early post-transplant period. Biopsies of the second patient for 12 weeks revealed grade 1A to 1B moderate cellular rejection. Humoral rejection peaked at 3 + “staining” for C4d after 1 week transplant, and then wavered between 2 + (moderate “staining”) and 1 + (weak “staining”). Results revealed the significance not only of traditional light microscopy in evaluating the severity of cellular rejection in endomyocardial biopsies of cardiac allotransplants, but also the value of C4d immunofluorescent “staining” of interstitial capillaries as an indicator of humoral rejection episodes, which may require modified therapy.

Decay-accelerating Factor Expression May Provide Immunoprotection Against Antibody-mediated Cardiac Allograft Rejection

Not all patients with complement deposition of the heart have developed hemodynamic instability at the time of diagnosis, suggesting that immunoprotective mechanisms reside within grafts. We hypothesized that decay-accelerating factor (DAF) could provide a first-line defense against complement injury, thus explaining the discrepancy in hemodynamics. Thus, we examined the role of DAF in the clinical presentation of antibody-mediated cardiac allograft rejection. Endomyocardial biopsies from C4d(+)/C3d(+) patients were immunoflourescently stained for DAF, and its expression was compared in patients who did (n = 5) and did not (n = 4) exhibit allograft dysfunction. Endomyocardial biopsies of patients without allograft dysfunction displayed intense staining of endothelial bound DAF expression at the time of antibody-mediated cardiac allograft rejection. Conversely, biopsies of patients with allograft dysfunction showed no evidence of DAF at that time. Expression of DAF on cardiac allografts may provide an immunoprotective mechanism against the deleterious effects of complement deposition in patients with antibody-mediated cardiac allograft rejection.

Antibody-Mediated Rejection of Cardiac Allografts in CCR5-Deficient Recipients

Rejected MHC-mismatched cardiac allografts in CCR5(-/-) recipients have low T cell infiltration, but intense deposition of C3d in the large vessels and capillaries of the graft, characteristics of Ab-mediated rejection. The roles of donor-specific Ab and CD4 and CD8 T cell responses in the rejection of complete MHC-mismatched heart grafts by CCR5(-/-) recipients were directly investigated. Wild-type C57BL/6 and B6.CCR5(-/-) (H-2(b)) recipients of A/J (H-2(a)) cardiac allografts had equivalent numbers of donor-reactive CD4 T cells producing IFN-gamma, whereas CD4 T cells producing IL-4 were increased in CCR5(-/-) recipients. Numbers of donor-reactive CD8 T cells producing IFN-gamma were reduced 60% in CCR5(-/-) recipients. Day 8 posttransplant serum titers of donor-specific Ab were 15- to 25-fold higher in CCR5(-/-) allograft recipients, and transfer of this serum provoked cardiac allograft rejection in RAG-1(-/-) recipients within 14 days, whereas transfer of either serum from wild-type recipients or immune serum from CCR5-deficient recipients diluted to titers observed in wild-type recipients did not mediate this rejection. Wild-type C57BL/6 and B6.CCR5(-/-) recipients rejected A/J cardiac grafts by day 11, whereas rejection was delayed (day 12-60, mean 21 days) in muMT(-/-)/CCR5(-/-) recipients. These results indicate that the donor-specific Ab produced in CCR5(-/-) heart allograft recipients is sufficient to directly mediate graft rejection, and the absence of recipient CCR5 expression has differential effects on the priming of alloreactive CD4 and CD8 T cells.

A Comparative Analysis Between Survivors and Nonsurvivors with Antibody Mediated Cardiac Allograft Rejection

Antibody mediated rejection (AMR) is an important cause of graft loss in the post heart transplant period. The following study was conducted to determine differences between survivors and nonsurvivors who developed post heart transplant AMR. We retrospectively reviewed the charts of patients who received a heart transplant between January 1993 and December 2002. Patients with biopsy proven AMR were identified. This group was divided into survivors and nonsurvivors. Groups were compared with regards to demographics, T-cell flow panel of reactive antibodies (PRA), flow cross-matches (anti-donor HLA Class I and II), and short- and long-term outcomes. Results of endomyocardial biopsies were collected to allow calculation of sensitivity, specificity, negative- and positive predictive values as well as accuracy of immunoglobulins and complement split products in association to death. A total of 65 patients (8.9%) were diagnosed with AMR. Mean age was 48 y (range: 8-68 y) and 53.8% were males. Episodes of hemodynamic instability associated with AMR were observed in 37% of patients. Only two deaths were directly attributed to acute AMR. Nearly 20% of AMR patients developed transplant coronary artery disease. Univariate analysis identified T-PRA (P < 0.001), mean T-cell molecules of equivalent soluble fluorochrome (MESF) (P < 0.001) and mean B-cell MESF (P < 0.001) as possible factors associated with death. Neither demographics of complement split products were associated to late death. When studying patients with AMR, pretransplant T-PRA, T-cell, and B-cell MESF may identify individuals at risk of late death.

Successful Management of Antibody-Mediated Cardiac Allograft Rejection With Combined Immunoadsorption and Anti-CD20 Monoclonal Antibody Treatment: Case Report and Literature Review

Chronic rejection is still the major limitation of long-term outcome of heart transplant recipients. Several recent studies demonstrated that a not negligible proportion of chronic allograft rejection episodes are not only mediated by T-cell response but also triggered by pre-transplant and de novo post-transplant donor-specific alloantibodies. This points at a fundamental role of humoral immune response mechanisms that contribute to early and late graft failure. This type of rejection is an unsolved problem solid organ transplantation because current immunosuppressive regimens are generally intended to interfere in T-cell signalling pathways. Various options for the removal of circulating alloantibodies and the prevention of alloantibody formation by B-cell depletion have been described. Nevertheless, effective standardized schemes for the treatment of antibody-mediated graft rejection have to be defined. We present a heart transplant recipient with sustained antibody-mediated graft rejection who was successfully managed with a combination treatment consisting of 3 cycles of immunoadsorption and a single-dose administration of the anti-CD20 monoclonal antibody rituximab.