Molecular events contributing to successful pediatric cardiac transplantation in HLA sensitized recipients.

Antibodies to HLA resulting in positive cytotoxicity crossmatch are generally considered a contraindication for cardiac transplantation. However, cardiac transplantations have been performed in children by reducing the Abs and modifying immunosuppression. To identify mechanisms leading to allograft acceptance in the presence of Abs to donor HLA, we analyzed priming events in endothelial cells (EC) by incubating with sera containing low levels of anti-HLA followed by saturating concentration of anti-HLA. Pre-transplant sera were obtained from children with low levels of Abs to HLA who underwent transplantation. EC were selected for donor HLA and exposed to sera for 72 h (priming), followed by saturating concentrations of anti-HLA (challenge). Priming of EC with sera induced the phosphatidylinositol 3-kinase/Akt mediated by the BMP4/WNT pathway and subsequent challenge with panel reactive antibody sera increased survival genes Bcl2 and Heme oxygenase-1, decreased adhesion molecules, induced complement inhibitory proteins and reduced pro-inflammatory cytokines. In contrast, EC which did not express donor HLA showed decreased anti-apoptotic genes. Primed EC, upon challenge with anti-HLA, results in increased survival genes, decreased adhesion molecules, induction of complement inhibitory proteins, and downregulation of pro-inflammatory cytokines which may result in accommodation of pediatric cardiac allografts despite HLA sensitization.

Isolated vascular "v" lesions in liver allografts: How to approach this unusual finding.

According to the Banff criteria for kidney allografts, isolated vascular or "v" lesions are defined as intimal inflammation, age-inappropriate fibro-intimal hyperplasia, or both, without the presence of associated interstitial T cell-mediated rejection (TCMR). In general, these lesions portend a worse outcome for kidney allografts, particularly in those where the "v" lesions are identified in patients with coexistent donor specific antibodies (DSA) or later after transplantation. Although affected arteries are rarely sampled in liver allograft biopsies, we identified nine patients at a mean of 1805 days posttransplantation and compared these to matched controls. Almost half (4 of 9) of the study patient biopsies showed inflammatory arteritis associated with focal or diffuse C4d positivity, which was not observed in matched controls. One "v" lesion patient progressed to rejection-related graft failure and two developed moderate/severe TCMR in subsequent biopsies, whereas only one rejection episode occurred in follow-up biopsies, and no rejection-related deaths or graft failures were detected in controls. In conclusion, patients with liver allograft isolated "v" lesions should undergo further evaluation and closer follow-up for impending TCMR and/or underlying co-existent chronic antibody-mediated rejection (AMR).

Elevated ST2 Distinguishes Incidences of Pediatric Heart and Small Bowel Transplant Rejection.

Elevated serum soluble (s) suppressor of tumorigenicity-2 is observed during cardiovascular and inflammatory bowel diseases. To ascertain whether modulated ST2 levels signify heart (HTx) or small bowel transplant (SBTx) rejection, we quantified sST2 in serially obtained pediatric HTx (n = 41) and SBTx recipient (n = 18) sera. At times of biopsy-diagnosed HTx rejection (cellular and/or antibody-mediated), serum sST2 was elevated compared to rejection-free time points (1714 ± 329 vs. 546.5 ± 141.6 pg/mL; p = 0.0002). SBTx recipients also displayed increased serum sST2 during incidences of rejection (7536 ± 1561 vs. 2662 ± 543.8 pg/mL; p = 0.0347). Receiver operator characteristic (ROC) analysis showed that serum sST2 > 600 pg/mL could discriminate time points of HTx rejection and nonrejection (area under the curve [AUC] = 0.724 ± 0.053; p = 0.0003). ROC analysis of SBTx measures revealed a similar discriminative capacity (AUC = 0.6921 ± 0.0820; p = 0.0349). Quantitative evaluation of both HTx and SBTx biopsies revealed that rejection significantly increased allograft ST2 expression. Pathway and Network Analysis of biopsy data pinpointed ST2 in the dominant pathway modulated by rejection and predicted tumor necrosis factor-α and IL-1ß as upstream activators. In total, our data indicate that alloimmune-associated pro-inflammatory cytokines increase ST2 during rejection. They also demonstrate that routine serum sST2 quantification, potentially combined with other biomarkers, should be investigated further to aid in the noninvasive diagnosis of rejection.

Preformed and de novo donor specific antibodies in visceral transplantation: long-term outcome with special reference to the liver.

Despite improvement in early outcome, rejection particularly chronic allograft enteropathy continues to be a major barrier to long-term visceral engraftment. The potential role of donor specific antibodies (DSA) was examined in 194 primary adult recipients. All underwent complement-dependent lymphocytotoxic crossmatch (CDC-XM) with pre- and posttransplant solid phase HLA-DSA assay in 156 (80%). Grafts were ABO-identical with random HLA-match. Liver was included in 71 (37%) allografts. Immunosuppression was tacrolimus-based with antilymphocyte recipient pretreatment in 150 (77%). CDC-XM was positive in 55 (28%). HLA-DSA was detectable before transplant in 49 (31%) recipients with 19 continuing to have circulating antibodies. Another 19 (18%) developed de novo DSA. Ninety percent of patients with preformed DSA harbored HLA Class-I whereas 74% of recipients with de novo antibodies had Class-II. Gender, age, ABO blood-type, cold ischemia, splenectomy and allograft type were significant DSA predictors. Preformed DSA significantly (p < 0.05) increased risk of acute rejection. Persistent and de novo HLA-DSA significantly (p < 0.001) increased risk of chronic rejection and associated graft loss. Inclusion of the liver was a significant predictor of better outcome (p = 0.004, HR = 0.347) with significant clearance of preformed antibodies (p = 0.04, OR = 56) and lower induction of de novo DSA (p = 0.07, OR = 24). Innovative multifaceted anti-DSA strategies are required to further improve long-term survival particularly of liver-free allografts.

A critical appraisal of methods to grade transplant glomerulitis in renal allograft biopsies.

Transplant glomerulitis is an increasingly recognized lesion in renal transplant biopsies. To develop a refined grading system, we defined glomerulitis by the presence of ≥5 leukocytes/glomerulus and evaluated 111 biopsies using three different grading systems: (i) percentage of glomerular involvement, (ii) peak inflammation in the most severely affected glomerulus and (iii) presence/absence of endocapillary occlusion by inflammatory cells. Endocapillary occlusion had no impact on graft survival, but was associated with increased serum creatinine, proteinuria and subsequent transplant glomerulopathy. Grading based on either percent or peak glomerular involvement correlated with graft failure and peritubular capillaritis. However, the percent glomerular involvement method had the additional advantage of displaying associations with: concurrent proteinuria, focal or diffuse immunoperoxidase peritubular capillary C4d staining, 1-year postbiopsy serum creatinine, subsequent detection of donor-specific antibody and development of transplant glomerulopathy. Patients with >75% glomerular involvement also revealed persistent high-grade glomerulitis on follow-up biopsies despite antirejection treatment. In conclusion, grading of glomerulitis is a meaningful exercise, and a quantification system based on percentage of glomerular involvement shows the most robust associations with clinical parameters and prognosis.

Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups.

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

1.7 year follow-up after bortezomib therapy for refractory antibody mediated rejection.

In summary, 1.7 years after transplantation, bortezomib rescue has been durably effective in salvaging our patient with refractory antibody mediated rejection. The only price has been persistently high levels of BK viruria. The presence of ongoing and even recurrent donor specific antibody has made it difficult to reduce immunosuppression further, and the concern that the high levels of BK viruria will eventually progress to viremia and nephropathy necessitates continued therapy with very low dose cidofovir and leflunomide. The absence of C1q binding DSA with stable renal function may provide some reserved optimism that the DSA that is detectable by convention Luminex assay may have reduced pathological implications. However, more data and prolonged follow-up are needed to determine whether or not non-complement binding DSA has an adverse pathological role.

Two hundred living donor kidney transplantations under alemtuzumab induction and tacrolimus monotherapy: 3-year follow-up.

Alemtuzumab has been used in off-label studies of solid organ transplantation. We extend our report of the first 200 consecutive living donor solitary kidney transplantations under alemtuzumab pretreatment with tacrolimus monotherapy and subsequent spaced weaning to 3 years of follow-up. We focused especially on the causes of recipient death and graft loss, and the characteristics of rejection. The actuarial 1-, 2- and 3-year patient and graft survivals were 99.0% and 98.0%, 96.4% and 90.8% and 93.3% and 86.3%, respectively. The cumulative incidence of acute cellular rejection (ACR) at the following months was 2%

Allospecific CD154+ T cells associate with rejection risk after pediatric liver transplantation.

Antigen-specific T cells, which express CD154 rapidly, but remain untested in alloimmunity, were measured with flow cytometry in 16-h MLR of 58 identically-immunosuppressed children with liver transplantation (LTx), to identify Rejectors (who had experienced biopsy-proven rejection within 60 days posttransplantation). Thirty-one children were sampled once, cross-sectionally. Twenty-seven children were sampled longitudinally, pre-LTx, and at 1-60 and 61-200 days after LTx. Results were correlated with proliferative alloresponses measured by CFSE-dye dilution (n = 23), and CTLA4, a negative T-cell costimulator, which antagonizes CD154-mediated effects (n = 31). In cross-sectional observations, logistic regression and leave-one-out cross-validation identified donor-specific, CD154 + T-cytotoxic (Tc)-memory cells as best associated with rejection outcomes. In the longitudinal cohort, (1) the association between CD154 + Tc-memory cells and rejection outcomes was replicated with sensitivity/specificity 92.3%/84.6% for observations at 1-60 days, and (2) elevated pre-LTx CD154 + Tc-memory cell responses were associated with significantly increased incidence (p = 0.02) and hazard (HR = 7.355) of rejection in survival/proportional hazard analysis. CD154 expression correlated with proliferative alloresponses (r = 0.835, p = 7.1e-07), and inversely with CTLA4 expression of allospecific CD154 + Tc-memory cells (r =-0.706, p = 3.0e-05). Allospecific CD154 + T-helper-memory cells, not CD154 + Tc-memory, were inhibited by increasing Tacrolimus concentrations (p = 0.026). Collectively, allospecific CD154 + T cells provide an estimate of rejection risk in children with LTx.

IMPDH1 gene polymorphisms and association with acute rejection in renal transplant patients.

Inosine 5'-monophosphate dehydrogenase 1 (IMPDH1) catalyzes the rate-limiting step of the de novo pathway for purine synthesis and is a major target of the immunosuppressive drug mycophenolic acid (MPA). Few variants of the IMPDH1 gene have been reported. The objective of this study was to identify and characterize IMPDH1 variants to determine whether genetic variation contributes to differences in MPA response and toxicity in transplant patients. Seventeen genetic variants were identified in the IMPDH1 gene with allele frequencies ranging from 0.2 to 42.7%. In this study, 191 kidney transplant patients who received mycophenolate mofetil were genotyped for IMPDH1. Two single-nucleotide polymorphisms, rs2278293 and rs2278294, were significantly associated with the incidence of biopsy-proven acute rejection in the first year post-transplantation. Future studies of the multifactorial nature of acute rejection must consider IMPDH1 polymorphisms in MPA-treated patients.

Alemtuzumab pre-conditioning with tacrolimus monotherapy in pediatric renal transplantation.

We employed antibody pre-conditioning with alemtuzumab and posttransplant immunosuppression with low-dose tacrolimus monotherapy in 26 consecutive pediatric kidney transplant recipients between January 2004 and December 2005. Mean recipient age was 10.7 +/- 5.8 years, 7.7% were undergoing retransplantation, and 3.8% were sensitized, with a PRA >20%. Mean donor age was 32.8 +/- 9.2 years. Living donors were utilized in 65% of the transplants. Mean cold ischemia time was 27.6 +/- 6.4 h. The mean number of HLA mismatches was 3.3 +/- 1.3. Mean follow-up was 25 +/- 8 months. One and 2 year patient survival was 100% and 96%. One and 2 year graft survival was 96% and 88%. Mean serum creatinine was 1.1 +/- 0.6 mg/dL, and calculated creatinine clearance was 82.3 +/- 29.4 mL/min/1.73 m(2). The incidence of pre-weaning acute rejection was 11.5%; the incidence of delayed graft function was 7.7%. Eighteen (69%) of the children were tapered to spaced tacrolimus monotherapy, 10.5 +/- 2.2 months after transplantation. The incidence of CMV, PTLD and BK virus was 0%; the incidence of posttransplant diabetes was 7.7%. Although more follow-up is clearly needed, antibody pre-conditioning with alemtuzumab and tacrolimus monotherapy may be a safe and effective regimen in pediatric renal transplantation.

Recovery of functional memory T cells in lung transplant recipients following induction therapy with alemtuzumab.

Profound T-cell depletion with the monoclonal antibody alemtuzumab facilitates reduced maintenance immunosuppression in abdominal and lung transplantation. While the phenotype of the post-depletional T cells has been characterized, little is known about their function. In the present study, global and CMV-specific T-cell function was assessed longitudinally in 23 lung transplant (LTx) recipients using T-cell assays (ImmuKnow and T Cell Memory, Cylex, Columbia, MD) during the first year posttransplant after induction therapy. Recovery of mitogen responses were seen at 2 weeks posttransplantation (65%PHA; 58% Con A), despite the low number of circulating T cells (<2%). These responses declined at 4-5 months (24%PHA; 54% Con A) and were partially reconstituted by 9 months (46% PHA; 73% Con A). CMV-specific responses recovered in 80% of R+ patients as early as 2 weeks posttransplant (n = 5) and 72% of patients had a memory response by 3 months (n = 11). In contrast, only 2 of 5 patients who did not exhibit memory responses pre-transplant (R-) developed transient CMV-specific T-cell responses. Our results show that profound depletion of T cells induced by alemtuzumab spares the functional subset of CMV-specific memory T cells. Conversely, CMV R- patients predepletion may require a prolonged period of prophylaxis.

Voriconazole prophylaxis in lung transplant recipients.

Lung transplant recipients have one of the highest rates of invasive aspergillosis (IA) in solid organ transplantation. We used a single center, nonrandomized, retrospective, sequential study design to evaluate fungal infection rates in lung transplant recipients who were managed with either universal prophylaxis with voriconazole (n = 65) or targeted prophylaxis (n = 30) with itraconazole +/- inhaled amphotericin in patients at high risk (pre- or posttransplant Aspergillus colonization [except Aspergillus niger]). The rate of IA at 1 year was better in lung transplant recipients receiving voriconazole prophylaxis as compared to the cohort managed with targeted prophylaxis (1.5% vs. 23%; p = 0.001). Twenty-nine percent of cases in the targeted prophylaxis group were in patients colonized with A. niger who did not receive itraconazole. A three-fold or higher increase in liver enzymes was noted in 37-60% of patients receiving voriconazole prophylaxis as compared to 15-41% of patients in the targeted prophylaxis cohort. Fourteen percent in the voriconazole group as compared to 8% in the targeted prophylaxis group had to discontinue antifungal medications due to side effects. Voriconazole prophylaxis can be used in preventing IA in lung transplant recipients. Regular monitoring of liver enzymes and serum concentrations of calcineurin inhibitors are required to avoid hepatotoxicity and nephrotoxicity.

Steroid-free tacrolimus monotherapy after pretransplantation thymoglobulin or Campath and laparoscopy in living donor renal transplantation.

Living donor renal transplantation was performed under a regimen of recipient pretreatment and low-dose postoperative immunosuppression with subsequent weaning. From October 9, 2002, to December 31, 2004, 196 consecutive, unselected laparoscopic live donor nephrectomies resulting in 196 living donor renal transplantations were performed. Recipients were pretreated with rabbit antithymocyte globulin (thymoglobulin; 24 patients or [12%]) or Campath 1H (alemtuzumab; 166 patients [85%]), or were not in protocol (6 patients [3%]), and were given postoperative steroid-free low-dose tacrolimus immunosuppressive monotherapy with subsequent weaning. There was no donor mortality. Major and minor donor morbidities were 2.6% and 4.2%, respectively. Laparoscopic live donor nephrectomy recipient outcomes with a mean follow-up of 401 days included (1) recipient and graft survival of 99.0% and 97.4%, respectively; (2) no ureteral stenosis; (3) 0.5% delayed graft function, from recurrent focal segmental glomerulosclinosis; and (4) no vascular thrombosis. The incidence of acute rejection at 30, 90, and 401 days was 1.5%, 3.8%, and 11.2% (all 196 recipients), 0%, 25%, and 29.2% (thymoglobulin recipients), and 1.8%, 3.9%, and 8.4% (Campath 1H recipients), respectively. Sixty-six patients (33.7%) are receiving spaced-dose immunosuppressive monotherapy. The mean creatinine concentration in all recipients was 1.5 +/- 1.1 mg/dL. There were no instances of cytomegalovirus tissue invasive disease or posttransplantation lymphoproliferative disease. The incidence of new-onset posttransplantation insulin-dependent diabetes was 0.5%. At current follow-up, the use of Campath 1H rather than thymoglobulin for pretreatment seems to have significantly improved the efficacy of our regimen.

Preservation of post-transplant lung function with aerosol cyclosporin.

Post-lung transplant use of aerosol cyclosporin (ACsA) is considered by examining the relationship between deposited aerosol dose and effect. In a sub-study of placebo controlled trials of ACsA as a rejection prophylaxis, 15 drug subjects received aerosol dose quantification tests to gage their ability to effectively deposit the nebulised drug in their transplanted lung(s). A total of seven placebo subjects received mock deposition tests. The deposited doses and mock doses were compared to changes in the forced expiratory volume in one second, at six time points during the 2-yr trial period (ACsA was started within 6 weeks post-transplant). Linear relationships were demonstrated between deposited dose and improvement in lung function in the drug subjects at all intervals. Mock dose data from placebo subjects did not demonstrate similar correlation. Based on these results, subjects were grouped by dose and compared. Subjects depositing > or = 5 mg of the drug in the periphery of their transplant(s) had improving pulmonary function on average. Low-dose and placebo subjects demonstrated declines, more A2-A4 rejection events in the latter portion of the trial, and more chronic rejection beyond the end of the trial. A dose-to-effect relationship is demonstrated for aerosol cyclosporin in terms of pulmonary function and biopsy proven rejection.

Aerosol cyclosporin therapy in lung transplant recipients with bronchiolitis obliterans.

The majority of patients who develop bronchiolitis obliterans, after lung transplantation, die within 2-3 yrs after onset since treatment with conventional immunosuppression is typically ineffective. A case/control study was conducted in lung transplant recipients with biopsy-documented bronchiolitis obliterans to determine whether aerosol cyclosporin use contributed to increased survival. The cases comprised 39 transplant recipients who received open-label aerosol cyclosporin treatment in addition to conventional immunosuppression. The controls were transplant recipients treated with conventional immunosuppression alone. There were 51 controls from the University of Pittsburgh Medical Center and 100 from a large multicentric database (Novartis Lung Transplant Database). Forced expiratory volume in one second expressed as a percentage of the predicted value was an independent predictor of survival in all patients with bronchiolitis obliterans. Cox proportional-hazards analysis revealed a survival advantage for aerosol cyclosporin cases compared to the Pittsburgh control group. A survival advantage was also seen when comparing study cases to multicentric controls. Aerosol cyclosporin, given with conventional immunosuppression to lung transplant recipients with bronchiolitis obliterans, provides a survival advantage over conventional therapy alone.

Real-time monitoring of acute liver-allograft rejection using the Banff schema.

BACKGROUND: The Banff schema is the internationally accepted standard for grading acute liver-allograft rejection, but it has not been prospectively tested. METHODS: Complete Banff grading was prospectively applied to 2,038 liver-allograft biopsies from 901 adult tacrolimus-treated primary hepatic allograft recipients between August 1995 and September 2001. Histopathologic data was melded with demographic, clinical, and laboratory data into a database on an ongoing basis using locally developed software. RESULTS: Acute rejection developed in 575 of 901 (64%) patients and the worst grade was mild in 422 of 575 (73%). At least one episode of moderate or severe acute rejection developed in 153 of 901 (17%) patients and most episodes, irrespective of severity, occurred within the first year after transplantation. Patients with moderate or severe acute rejection showed higher alanine aminotransferase (P =0.007) and aspartate aminotransferase ( P=0.07) levels and were more likely to develop perivenular fibrosis on follow-up biopsies (P =0.001) and graft failure from acute or chronic rejection ( P=0.004) than those with mild rejection. Regardless of severity, 80% of patients with acute rejection did not develop significant fibrosis in follow-up biopsies, and graft failure from acute or chronic rejection occurred in only 11 of 901 (1%) allografts. CONCLUSIONS: Most acute-rejection episodes are mild and do not lead to clinically significant architectural sequelae. When tested prospectively under real-life and -time conditions, the Banff schema can be used to identify those few patients who are potentially at risk for more significant problems. Creation, capture, and integration of non-free text, or "digital," pathology data can be used to prospectively conduct outcomes-based research in transplantation.

The epidemiology of parainfluenza virus infection in lung transplant recipients.

Human parainfluenza virus (HPIV) is a common cause of seasonal respiratory tract infections. However, little is known about the clinical presentation and impact of HPIV infections in lung transplant recipients. We reviewed HPIV infections at the University of Pittsburgh Medical Center. From January 1990 through May 2000, 32 cases of HPIV infection were identified. HPIV infection was found in 24 lung transplant recipients (75%), all of whom were included in the study group. Diagnosis was established at a median of 2.1 years after transplantation (range, 0.6-5 years). Presenting symptoms included cough (17 patients), shortness of breath (16), and temperature elevation (4). Respiratory failure occurred in 5 patients (21%). The HPIV serotypes were HPIV-1 (7 patients), HPIV-2 (2), and HPIV-3 (15 [63%]). Twenty-two patients underwent transbronchial biopsy, and 18 (82%) showed signs of acute allograft rejection. Seven patients (32%) subsequently were found to have bronchiolitis obliterans.

Long-term survival of rat cardiac allografts by intrathymic plus portal venous injections of donor bone marrow cells and short-term tacrolimus immunosuppression.

Intrathymic (IT) or portal venous (PV) injection of donor antigens has been shown to prolong organ acceptance in low responder rat strain combinations. We determined whether a combination of these strategies would prolong cardiac allograft survival in high responder combinations. Wistar Furth rats received 1 x 10(8) ACI rat bone marrow cells (BMCs) via IT, intravenous (IV), PV, IV + PV, IT + IV or IT + PV route at the time of ACI cardiac transplantation. Without tacrolimus (FK), all grafts were acutely rejected. With FK immunosuppression (1.5 mg/kg per day, I. M., days 0-4), single BMC injection did not increase graft survival beyond 93 days, whereas 70% of grafts survived indefinitely ( > 150 days) when IT and PV BMCs were combined. Animals receiving IT and PV BMCs also had less allograft vasculopathy. Thus, IT and PV injections of donor BMCs under a brief course of FK synergistically improve cardiac allograft survival.