Laura J. Wozniak

A common peripheral blood gene set for diagnosis of operational tolerance in pediatric and adult liver transplantation.

To identify biomarkers of operational tolerance in pediatric and adult liver transplant recipients, transcriptional profiles were examined from 300 samples by microarrays and Q‐PCR measurements of blood specimens from pediatric and adult liver transplant recipients and normal tissues. Tolerance‐specific genes were validated in independent samples across two different transplant programs and validated by Q‐PCR. A minimal set of 13 unique genes, highly expressed in natural killer cells (p = 0.03), were significantly expressed in both pediatric and adult liver tolerance, irrespective of different clinical and demographic confounders. The performance of this gene set by microarray in independent samples was 100% sensitivity and 83% specificity and the AUC was 0.988 for only three genes by Q‐PCR. 26% of adults and 64% of children with excellent liver allograft function, on minimal or dual immunosuppression, showed high prediction scores for tolerance. Novel peripheral transcriptional profiles can be identified in operational tolerance in pediatric and adult recipients of liver allografts, suggesting a high incidence of a pro‐tolerogenic phenotype in stable patients on chronic immunosuppression. Given the high incidence of viral infections and malignancies in liver transplant recipients, this gene set provides an important monitoring tool that can move the field toward personalized and predictive medicine in organ transplantation.

Donor-specific HLA Antibodies Are Associated with Late Allograft Dysfunction after Pediatric Liver Transplantation

Background The role of donor-specific HLA antibodies (DSA) after pediatric liver transplantation (LTx) is not clearly established. We completed a cross-sectional study to characterize DSA in long-term survivors of pediatric LTx and assess the impact of C1q-binding DSA on allograft outcomes. Methods Serum samples were collected at routine clinic visits from 50 pediatric LTx recipients classified into 3 clinical phenotypes: nontolerant (n = 18) with de novo autoimmune hepatitis (DAIH) and/or late acute cellular rejection (ACR); stable (n = 25) on maintenance tacrolimus; operationally tolerant (n = 7). Samples were blinded, and antibody detection was performed using Luminex single antigen class I and II beads. Patients with positive DSA were tested for C1q-binding DSA. Results DSA were detected in 54% (n = 27) of the patients, with the majority directed at HLA class II antigens (DR, 41%; DQ, 53%). Patients with DSA were younger at the time of LTx (P = 0.016) and time of study (P = 0.024). Mean aspartate aminotransferase, alanine aminotransferase, total bilirubin, and gamma glutamyl transferase were higher in DSA-positive patients, though did not reach statistical significance. Nontolerant patients were significantly more likely to have DQ DSA (61%) compared to stable (20%) and tolerant (29%) patients (P = 0.021). The nontolerant phenotype was associated with DSA and C1q-binding DSA, with odds ratios of 13 (P = 0.015) and 8.6 (P = 0.006), respectively. The presence of DQ DSA was associated with DAIH and late ACR, with odds ratios of 12.5 (P = 0.004) and 10.8 (P = 0.006), respectively. Conclusions Allograft dysfunction is not always evident in patients with DSA, but DQ DSA are strongly associated with DAIH, late ACR, and chronic rejection.

Expression of soluble HLA-G identifies favorable outcomes in liver transplant recipients.

Background. Human leukocyte antigen (HLA)-G displays immunotolerogenic properties toward the main effector cells involved in graft rejection through inhibition of natural killer cell- and cytotoxic T-lymphocyte-mediated cytolysis, and CD4+ T-cell alloproliferation. An increase in serum and graft levels of HLA-G has been noted in transplant patients with improved allograft survival. However, the clinical relevance of soluble serum HLA-G molecules in tolerant pediatric and young adult liver transplant patients remains to be studied. Methods. We examined the serum HLA-G levels in 42 pediatric and young adult liver transplant patients with a mean age of 15 years; 13 patients had operational tolerance (TOL), with complete immunosuppression withdrawal for 2.3 to 13.2 years. Results. Median HLA-G level in patients with acute rejection (AR) was similar to the level in pediatric healthy volunteers (9.9 vs. 4.2 U/mL, P=0.13). HLA-G was higher in patients with stable liver function on immunosuppression (54.6 U/mL) than in patients with AR (P=0.01) and healthy volunteers (P=0.003), but almost 6-fold lower than in TOL patients (325.4 U/mL). HLA-G did not correlate with clinical confounders or a history of posttransplant lymphoproliferative disease or Epstein-Barr virus; although levels in the TOL group were negatively correlated with time after immunosuppression withdrawal (r=−0.75, P=0.003). In rejectors, HLA-G levels trended to negatively correlate with a higher number (r=−0.58) and greater severity of AR episodes (r=−0.56) after 1 year posttransplantation. Conclusions. Increased serum HLA-G levels track with operational tolerance of liver grafts and support favorable outcomes in pediatric and young adult recipients.

Long-term nutrition and predictors of growth and weight gain following pediatric intestinal transplantation.

Background. Advances in intestinal transplantation (ITx) have resulted in improved survival and the opportunity to examine nutritional outcomes. The aim of this study was to describe detailed, long-term nutritional results and identify positive predictors of growth and weight gain following pediatric ITx. Methods. A single-center retrospective, Institutional Review Board-approved review of a prospective database was conducted. Inclusion criteria were ITx recipients 18 years or younger with survival of 6 months or more. Outcomes included anthropometric measurements and biochemical markers at 6, 12, 24, 36, and 48 months post-ITx. More than 25 ITx-related variables were analyzed as potential predictors of growth and weight gain. Statistical analysis was performed using chi-square test, t test, and analysis of variance. Results. Between November 1991 and April 2007, 50 children received 55 ITx; 33 patients met eligibility criteria. Median age at ITx was 2.2 years, follow-up time was 3.8 years, and time from ITx to cessation of total parenteral nutrition was 31 days. The most common micronutrient deficiencies post-ITx were zinc, iron, and copper. Serum protein levels improved significantly over time. Weight gain occurred within 6 months and vertical growth within 12 months, although limited catch-up growth was seen. Early predictors of weight gain and growth included shorter hospitalization and absence of rejection. Long-term predictors were low steroid dosage, infrequent hospitalization, and the use of peptide-based formulas. Conclusions. This represents one of the largest and most comprehensive long-term studies on nutritional outcomes in pediatric ITx. Overall, positive growth and weight gain were seen as were micronutrient deficiencies. Numerous long-term nutritional challenges exist which require a multidisciplinary approach and future prospective studies.

Incidence, timing, and significance of early hypogammaglobulinemia after intestinal transplantation.

Background Despite recent advances in intestinal transplantation (ITx), infection (INF) and acute cellular rejection (ACR) remain major causes of patient and graft loss. Studies in other solid-organ transplantations indicate that low levels of serum immunoglobulin G (IgG) negatively impact outcomes. To date, there have been no studies on IgG after ITx. Methods A retrospective review of an IgG measurement protocol in primary ITx recipients between 2007 and 2011 was undertaken. IgG levels were measured at the time of evaluation, transplantation, and at weekly intervals for 2 months. Hypogammaglobulinemia (HGG) was defined as IgG levels below the lower limit of the 95% confidence interval for age. Associations between HGG, INF, and ACR were tested, and the incidence and timing of INF and ACR were compared. Results Thirty-four patients were transplanted at a mean (SD) age of 12.4 (17.2) years. Most were Latino children with gastroschisis who received multivisceral grafts. Relative to pre-ITx levels, a statistically significant decrease in IgG levels was observed after ITx (P<0.05). Twenty patients (59%) developed HGG during the post-ITx period at a mean (SD) of 9.8 days. No significant associations were identified between HGG and INF or ACR. Conclusions This is the first study to describe serum IgG levels after ITx. A marked decrease in serum IgG levels was observed early on, in most patients. The etiology is potentially related to immunotherapy. HGG was not associated with INF or ACR, possibly related to the sample size and our practice of exogenous intravenous immunoglobulin replacement.

Prevalence and clinical impact of donor-specific alloantibody among intestinal transplant recipients.

Background Rejection remains the leading cause of allograft loss, and a major barrier to improving long-term outcomes after intestinal transplantation. Our aim is to define the prevalence and investigate the role of donor-specific antibody (DSA) on intestinal graft outcomes. Methods The study includes 109 transplants performed in 95 recipients at a single center. Patients were screened for DSA pretransplant, monitored regularly posttransplant and when clinically indicated using the single-antigen bead Luminex assay. Standard induction immunosuppression was with interleukin-2 receptor antagonists, and antithymocyte globulin in high-risk recipients. Maintenance regimens were tacrolimus-based. Results Pretransplant DSA was detected in 12 (11%) recipients with 50% continuing to have circulating antibodies posttransplant. An additional 24 (25%) patients developed de novo DSA, and of these, 71% had persistent antibodies. Recipients with preformed DSA demonstrated elevated risks of early graft failure, whereas those with de novo DSA experienced accelerated graft loss once DSA was detected, reaching a 28% failure rate within 2 years. HLA-DQ mismatch is a significant risk factor for de novo DSA emergence, whereas the persistence of antibodies is predicted by DSA strength and specificity. Although inclusion of the liver in the intestinal allograft imparts an immunological advantage against rejection-related graft loss, this protective effect was lost among recipients with persistent DSA. Conclusions The presence of DSA is associated with inferior graft outcomes among intestinal transplant recipients. An enhanced understanding of the mechanisms by which DSA causes allograft injury, and effective strategies targeting humoral immune reactivity are needed to improve long-term intestinal graft outcomes.

Endoscopy Following Pediatric Intestinal Transplant.

Objectives: Biopsies remain the criterion standard in the diagnosis of intestinal transplant (ITx) rejection, and gastrointestinal endoscopy plays a pivotal role in patient management. Herein, we describe a single-center 23-year endoscopic experience in pediatric ITx recipients. Methods: A retrospective review of endoscopy and pathology reports of all ITx recipients <18 years old transplanted between 1991 and 2013 was performed with the aim of describing the procedural indications, findings, and complications. Results: A total of 1770 endoscopic procedures within 1014 sessions were performed. A combination of esophagogastroduodenoscopy and ileoscopy was the most common procedure (36%). Increased stool output (35%) and surveillance endoscopy (32%) were the most common indications. A total of 162 episodes of biopsy-proven rejection were diagnosed. The first episode of rejection occurred at a median of 1 month after ITx. Of histology-proven rejections, 45% had normal-appearing endoscopies. The rate of procedural complications, including but not limited to bleeding and perforation, was 1.8%. Conclusions: Endoscopy with biopsy plays a significant role in the care of ITx recipients. Multiple procedures are required for graft surveillance, diagnosis of rejection, subsequent treatment, and follow-up of therapy. The gross endoscopic appearance, particularly in mild to moderate acute cellular rejection, does not correlate well with histology. Complex anatomy, complication rates that are higher than patients with non-ITx pediatric endoscopy, and timely histologic interpretation by experienced pathologists are reasons that these procedures should be performed at centers accustomed to caring for ITx recipients. The field would benefit from the development of a noninvasive biomarker to reliably and efficiently detect rejection.

Vitamin D Deficiency in Children With Intestinal Failure Receiving Home Parenteral Nutrition

BACKGROUND Vitamin D plays important roles in both skeletal and nonskeletal health. Limited data suggest that patients with intestinal failure (IF) receiving home parenteral nutrition (PN) are at risk for vitamin D deficiency due to inadequate oral intake, poor absorption, and chronic illness. The purpose of this study was to document vitamin D status in pediatric patients with IF receiving home PN. MATERIALS AND METHODS We performed a 2-year retrospective review of children with IF followed at our center who had been on home PN for ≥6 months and had ≥1 serum 25-hydroxyvitamin D (25-OHD) level checked as part of routine clinical care. Patients were then categorized as deficient (<20 ng/mL), insufficient (20-29 ng/mL), or normal (≥30 ng/mL) based on their lowest vitamin D level. Demographic data and clinical characteristics were also assessed. RESULTS Eleven of 27 children (41%) had ≥1 insufficient 25-OHD level, including one child with vitamin D deficiency. Diagnosis of short bowel syndrome (compared with dysmotility or malabsorption syndromes) was associated with decreased likelihood of suboptimal vitamin D status, with an odds ratio of 0.12 (95% confidence interval, 0.02-0.8, P = .028). Osteopenia was noted in 59% of the cohort. There was a trend toward higher risk for osteopenia in patients with low 25-OHD levels compared with those with normal 25-OHD levels (82% vs 44%, P = .109). CONCLUSION Suboptimal 25-OHD levels are common in children with IF on home PN. This emphasizes the critical importance of routine surveillance of serum vitamin D levels and consideration of enteral supplementation when indicated.

Utility of an immune cell function assay to differentiate rejection from infectious enteritis in pediatric intestinal transplant recipients

The Cylex Immune Cell Function Assay measures cell‐mediated immunity based on ATP production by stimulated CD4 + cells. We hypothesized that this test would discriminate acute cellular rejection (ACR) from infectious enteritis (IE) in pediatric intestinal transplant (ITx) recipients with allograft dysfunction. We retrospectively analyzed 224 Cylex assays drawn in 47 children who received 53 ITx. Samples were classified as stable, ACR, or IE based on clinical status. ATP values were analyzed using Kruskal–Wallis and t‐tests. Overall, there was a statistically significant difference in ATP values based on clinical status (p = 0.03); however, overlap was observed between groups. The median ATP value during ACR was significantly greater than during stable periods (p = 0.02). No difference was seen in IE vs. stability (p = 0.8). The difference in median ATP value in ACR vs. IE approached significance (p = 0.1). Relative to previous levels, ACR episodes were associated with a median ATP increase of 101 ng/mL and IE episodes with a decrease of 3 ng/mL (p = 0.3). These data indicate that the Cylex assay has limited utility in differentiating ACR from IE, largely due to interpatient variability. Following longitudinal intrapatient trends may be an adjunctive tool in discriminating IE from ACR and guiding immunosuppression adjustments in select patients.

Acute antibody‐mediated rejection in ABO‐compatible pediatric liver transplant recipients: case series and review of the literature

Acute AMR is well reported following ABO‐incompatible LTx. However, it remains uncommon in ABO‐compatible LTx. It typically presents with graft dysfunction ≤2 weeks post‐LTx and is often associated with graft loss. We report the clinical presentation, treatment regimen, and outcome of six pediatric LTx recipients diagnosed with early acute AMR based on (i) clinical signs of graft dysfunction, (ii) histopathology indicative of acute injury ± C4d staining, and (iii) presence of HLA DSA. All patients developed elevated ALT and GGT ≤ 45 days post‐LTx. All showed HLA class I (n=4) and/or II (n=6) DSA (peak MFI 6153–11 910). Four had de novo DSA, and two had preformed DSA. Five were initially diagnosed with ACR refractory to steroid therapy. Four exhibited resolution of graft dysfunction with AMR therapy. Two had refractory AMR—one was re‐transplanted; the other was treated with eculizumab and showed improvement in graft function but later died due to a tracheostomy complication. Our case series suggests that AMR following ABO‐compatible LTx may be under‐diagnosed. The presentation can be variable, and treatment should be individualized. Eculizumab may be an option for refractory AMR. Ultimately, future multicenter studies are needed to better define diagnostic criteria, characterize optimal treatment, and assess long‐term outcomes following liver AMR.