Presence Of Acute Cellular Rejection Research Articles

Tissue Expression of Programmed Cell Death 1 Ligand1 (PD-L1) in Biopsies of Transplant Livers of Pediatric Patients as a Possible Marker of Acute Cellular Rejection

Introduction: Preclinical models have demonstrated that PD-1 and its ligand programmed death ligand1 (PD-L1) play significant roles in both graft induction and the maintenance of immune tolerance. It has also been suggested that PD-L1 tissue expression may predict graft rejection; however, the available data are sparse and inconclusive. Some studies were conducted on patients with cancer; most of them do not concern the liver, especially within the context of the use of immunohistochemical tests. Therefore, the aim of our study was to assess the relationship between tissue expression of PD-L1 in a unique material, i.e., in the liver biopsies of pediatric patients after transplantation with the presence of acute cellular rejection (ACR). Material and Methods: This retrospective study enrolled 55 biopsies from 55 patients who underwent protocol liver biopsies. The control group consisted of 19 biopsies from 13 patients diagnosed with acute cellular rejection (rejection activity index/RAI/ from 2 to 8). An immunohistochemical (IHC) staining for PD-L1 was performed in all of the liver specimens; its expression was analyzed in different regions of liver tissue (in inflammatory infiltrates and within the endothelium and hepatocytes). The following changes were re-evaluated in each specimen: features of any kind of rejection (acute cellular, antibody-mediated, chronic); the presence and severity of fibrosis (Ishak scale); and the presence of cholestasis and steatosis. Clinical parameters were also evaluated, including tests of liver function (AST, ALT, GGT, bilirubin). Results: The age of patients in the study group ranged from 2.37 to 18.9 years (median 13.87 years), with the time after transplantation being 1–17 years (median 8.36 years). The age of patients in the control group ranged from 1.48 to 17.51 years (median 7.93 years), with their biopsies being taken 0.62–14.39 years (median 1.33 years) after transplantation. We found a statistically significant relationship between PD-L1 expression on inflammatory infiltrates and ACR; however, there was no statistically significant relationship between PD-L1 endothelial expression and ACR. PD-L1 was not positive in the hepatocytes regardless of if it was the study or control group that was under observation. Conclusion: PD-L1 appears to be a promising marker to predict graft rejection.

Is M30 staining a reliable marker predicting graft rejection and fibrosis in protocolar liver biopsies in post-liver transplant pediatric patients?

Liver transplantation is currently a treatment of choice in patients with end-stage liver disease. Acute cellular rejection (ACR), antibody-mediated rejection (AMR), and chronic rejection (ChR) are major causes of graft injury. Therefore, new markers predicting graft rejection are investigating. Apoptosis has been recently proposed as one of the mechanisms contributing to liver fibrosis in liver grafts. Coarse needle liver biopsy is still a gold standard in monitoring post-transplant pathologies. The aim of this study was to assess the utility of immunohistochemical (IHC) staining for M30 (cytokeratin 18), as a prognostic marker of rejection in pediatric recipients of liver transplant and predicting marker of liver fibrosis and worse follow-up. The study enrolled 55 biopsies from 55 patients aged 2.37 to 18.9 years (median 13.87 years) who underwent protocolar liver biopsies taken 1-17 years after liver transplantation (median 8.36 years). The control group (positive control group) consisted of 26 biopsies from 16 patients in whom acute ACR was diagnosed. IHC staining for M30 (cytokeratin 18) and histochemical Azan staining were performed in all liver specimens. The following changes were re-evaluated in each specimen: features of ACR (the severity was assessed using RAI/Rejection Activity Index/Scale, which ranges from 3-9 points and include 3 histopathological changes suggestive of rejection), AMR or ChR; severity of fibrosis (Ishak Scale); presence of cholestasis and steatosis. Clinical parameters including laboratory tests of liver function (AST, ALT, GGTP, bilirubin) were also evaluated. M30 expression correlated with presence of acute cellular rejection. However, no relationship was found between M30 expression and severity of fibrosis. M30 staining, marker of apoptosis, seems to be a promising marker predicting acute cellular rejection.

Low AlloSure® in HeartCare® Associated with Low Risk of Significant Allograft Rejection: An Analysis of SHORE

<h3>Purpose</h3> HeartCare is a non-invasive method of surveillance for cardiac allograft rejection comprised of gene expression profiling (AlloMap, AM) and donor derived cell-free DNA (AlloSure-Heart, AS). AM scores inform immune quiescence whereas AS reflects presence of tissue injury. <h3>Methods</h3> Surveillance HeartCare Outcomes Registry (SHORE) is a multicenter, observational registry assessing the clinical utility of AS/AM. AM score >30 (<6 months) or > 34 (≥6 months) was deemed positive (AM+). AS result >0.15% was defined as positive (AS+). The presence of acute cellular rejection ≥2R or antibody mediated rejection (AMR 1, 2, or 3) was assessed at 14 and 30 days following AS/AM. <h3>Results</h3> Of the 4927 samples analyzed, 2507 (50.9%) were concordant low AS and AM (AS-/AM-), 558 (11.3%) were AS+/AM+. Discordance was noted in 1245 (25.3%) with AS+/AM- and 617 (12.5%) with AS-/AM+. The AS+/AM+ cohort had significantly more rejection events when compared with the AS-/AM- group at 14 days (1.25% vs 0.4%, p = 0.032) with a trend toward more rejection at 30 days (1.61% vs 0.68%, p = 0.055). The AS-/AM+ group had fewer rejection episodes when compared with those with AS+/AM+ at 30 days (0.16% vs 1.61%, p = 0.017) with a trend toward fewer rejection events at 14 days (0.16% vs 1.25%, p = 0.055). The AS-/AM- group had the fewest number of endomyocardial biopsies performed when compared to all other cohorts at 14 days (p < 0.05). <h3>Conclusion</h3> AS/AM provides complementary information regarding probability of clinically significant allograft rejection. In those with discordant AM and AS results, presence of a low AS was associated with lower risk of rejection in the subsequent 14 and 30 days. A concordantly low AS and AM may safely obviate a surveillance endomyocardial biopsy in the appropriately selected.

Peripheral blood eosinophilia as a marker of acute cellular rejection in lung transplant recipients

To assess the association between EOS and the presence of acute cellular rejection in lung transplant recipients. Retrospective study of 583 transbronchial biopsies (TBB) performed in 256 lung transplant patients between 2012 and 2018. We analyzed age, sex, underlying pathology, date of transplant, indications for TBB, presence and degree of ACR, and the simultaneous absolute and relative EOS. ACR were observed in 170 of 583 TBB (29.2%). EOS in patients with ACR were higher than in patients without ACR (203.6 ± 248/mm3 vs 103.1 ± 153/mm3; p < 0.001). High levels of both absolute and relative EOS were associated with the presence of ACR regardless of the underlying disease (odds ratio [OR] 1.003; 95% confidence interval [CI], 1.002-1.004; OR 1.226; 95% CI, 1.120-1.342) and time after transplant (OR 1.003; 95% CI, 1.002-1.004 and OR 1.239; 95% CI, 1.132-1.356). Moreover, both absolute and relative EOS were strongly associated with moderate and severe grades of ACR (OR 3.55; 95% CI, 3.00-4.10 and OR 3.56; 95% CI, 3.00-4.12). EOS are elevated in ACR, especially in moderate or severe ACR. Increased vigilance for ACR is therefore advisable in lung transplant recipients with elevated EOS.

Tissue Doppler velocities for ruling out rejection in heart transplant recipients in the daily routine of the echocardiography laboratory: a feasibility study

Abstract Funding Acknowledgements Type of funding sources: None. Background The sum of systolic and early diastolic myocardial velocities at lateral mitral annulus, evaluated by tissue Doppler (in absolute values, s’+e’) in the apical four chambers view is a parameter whose value for ruling out acute cellular rejection (ACR) in heart transplant (HTx) recipients has been acknowledged by the European and Brazilian recommendations for the use of imaging in HTx. A recent study has shown its independent association with ACR in the context of classical and myocardial deformation variables, with a negative predictive value (NPV) of 98% for treatment requiring ACR (TR-ACR, grade ≥ 2R) for a cut-off point of ≥23 cm/s. This work was performed under experimental conditions (only one expert echocardiographer, only one high-end equipment). Purpose Our objective was to study the diagnostic utility of this parameter for ruling out rejection in HTx recipients in the daily routine of the echocardiography laboratory. Methods From October 2017 to May 2020, serial echocardiograms were performed to 33 consecutive HTx recipients, in the 3 hours after the routine surveillance endomyocardial biopsies (EMB), in the first year after HTx. Three sonographers, seven cardiology residents and six expert echocardiographers participated in the acquisition and interpretation of the images, with seven different echocardiographic machines in only one centre. We analysed the association of s’+e’ with the presence of ACR, and the NPV of s’+e’ ≥23 cm/s for ruling out TR-ACR was investigated. Results A total of 176 pairs of EMB and echocardiogram were obtained. The value of s’+e’ was significantly lower with higher severity of rejection: 25.6 ± 5.5 cm/s, 23.8 ± 5.1 cm/s and 21.6 ± 3.5 cm/s for ACR grade 0R (n = 91, 52%), 1R (n = 67, 38%) and ≥2R (n = 18, 10%), respectively, p = 0.005. It was also lower when comparing studies with (≥1R) and without rejection (23.4 ± 4.9 cm/s versus 25.6 ± 5.5 cm/s, p = 0.005), or TR-ACR versus the rest of series (21.6 ± 3.5 cm/s versus 24.8 ± 5.3 cm/s, p = 0.01). The area under curve for the detection of TR-ACR was 0.67 (CI95% 0.56-0.78), p = 0.02. A cut-off point of ≥23 cm/s, present in 57% of the studies, showed a NPV of 95% for TR-ACR. Conclusion Lateral mitral annulus velocities showed an excellent NPV of 95% for TR-ACR detection in HTx recipients when evaluated in the daily routine of an echocardiography laboratory with a wide variety of operators and echocardiographic equipment. This finding could be useful for reducing the number of EMB in selected cases.

Recipient-Derived Immune Cell Phenotyping of Bronchoalveolar Lavage Identifies Patients with Biopsy Proven Early Acute Cellular Rejection after Lung Transplantation: A Pilot Study

Acute cellular rejection (ACR) is associated with the ultimate development of chronic lung allograft dysfunction. Transbronchial biopsy, the gold standard for the diagnosis of ACR, is associated with potential morbidity. We set out to define the correlation between the recipient-derived immune cell content in bronchoalveolar lavage (BAL) and biopsy proven early ACR (<4 months). We analyzed BAL samples from 8 lung transplant recipients with early ACR and 5 without ACR. Immunophenotyping was performed using spectral flow cytometry; cell origin (donor v recipient) was determined by staining for human leukocyte antigen. T test was used to identify differences in immune cell populations between those with and without ACR. Live CD45+ cells were defined as CD4+ and CD8+ T cells, B cells (CD19+, CD20+), CD56+ lymphocytes, and monocytes (CD14+CD206+/CD64+). BAL was obtained at 1.5 months (range 0.5-3.5) from transplant at time of ACR and at 1.6 months (range 0.5 - 2.8) for non-ACR. The presence of ACR was associated with a statistically significant decrease in the proportion of donor monocytes (Figure 1: Mean 85%, SD 15% v 34%, SD 16%; p <0.001) and donor-derived regulatory T cells (Tregs), defined as CD4+CD25+FOXP3+ (Mean 3%, SD 3% v Mean 9%, SD 3%; p = 0.004). When comparing absolute number of both donor and recipient immune cells found in the BAL, patients with ACR had a statistically significant increase in the number of recipient-derived CD4+ T cells (Mean 7%, SD 5% v 1%, SD 0.05%; p value = 0.02), and a trend towards increased recipient-derived CD8+ T cells (Mean 18%, SD 27% v <1%, SD 1%; p value = 0.17). Biopsy proven, early ACR is associated with a relative decline in donor monocytes and donor Tregs, and increased proportion of recipient-derived CD4+ T cells. These results support further investigation into the role of BAL immune cell phenotyping as a potential less-invasive biomarker for ACR.

P937 Left atrial longitudinal strain as a marker of acute cellular rejection in heart transplant recipients: impact of intervendor variability

Abstract Introduction and purpose Preliminary reports suggests that left atrial longitudinal strain (LALS) variables are a sensitive marker of acute cellular rejection (ACR) in heart transplant recipients (HTxR), discriminating between those studies without rejection and those with any grade of rejection. Intervendor variability is a concern in the widespread use of this technique. Our objective was to compare the LALS evaluated by two different softwares. Methods From September 2014 to October 2016 we performed, in 18 consecutive adult HTxR in their first year posttransplantation, serial echocardiographic exams within 3 hours of the routine surveillance endomyocardial biopsies (EMB), in a single centre. Peak average longitudinal strain, and strain rate were measured in the left atrium in the apical four chambers view in all studies, using both softwares, its association with the presence of ACR was investigated, and intervendor variability was evaluated. Results a total of 147 pairs of EMB and echo exams were performed, 65 with no rejection (grade 0R), 82 with any grade of ejection (grades 1R and 2R). Intraclass correlation coeficients for intervendor reproducibility for LALS and LALSR were 0.4 (95%CI 0.26 - 0.57) and 0.3 (95%CI -0.06 - 0.52) respectively. The number of segments evaluable by each software was significantly different. Association of LALS with rejection is shown in the table. Conclusions In this monocentric prospective study, left atrial longitudinal strain variables were found to be a sensitive marker of acute cellular rejection in heart transplant recipients. Although intervendor reproducibility was poor, these results were consistent between both software. Results Software n/N (%)* Variable No ACR ACR≥1 p value Siemens 114/147 (77.5) Peak atrial LS 19.4 ± 7.4 15.5 ± 6.3 0.006 114/147 (77.5) Peak atrial LSR 1.5 ± 0.4 1.3 ± 0.5 0.005 TomTec 131/147 (89.1) Peak atrial LS 19.1 ± 6.2 14.1 ± 5.4 &amp;lt;0.005 131/147 (89.1) Peal atrial LSR 1.0 ± 0.4 0.8 ± 0.3 &amp;lt;0.005 n: number of exams evaluable by each software. N: total numbers of exams. *p = 0.01 for comparison between the proportion of exams evaluable by each software.

Peripheral Blood Eosinophil Count as a Marker of Pulmonary Allograft Rejection

Purpose Non-invasive methods for early diagnosis of acute cellular rejection (ACR) are needed. Previous studies of other solid organ transplanted recipients have shown a relationship between the eosinophil count in peripheral blood (EOSp) and the presence of ACR. However, the relationship between this biomarker and ACR in lung transplant patients remains unclear.Objective: To asses the relationship between EOSp and the presence of allograft rejection in lung transplant recipients. Methods A retrospective clinical review of 363 transbronchial biopsies (BTB) performed in 194 lung transplant patients between 2014 and 2018. We analyzed: age, date of transplant, the presence and degree of ACR, and the simultaneous absolute number of EOSp. BTB indications were classified as: 1. Protocol BTB in the first month after transplant and after ACR treatment. 2. Decrease of lung function. 3. Other indications The relationship between the EOSp count and ACR was analyzed by the Chi-square test . We calculated the sensitivity, specificity of the EOSp count to diagnose ACR. The precision of the EOS count to predict ACR was analyzed using a receiver operating characteristic ROC curve. Results BTB showed ACR in 117 of 363 cases (32.2%). The most frequent ACR degree was A2 (53%). The EOSp count in patients with ACR was higher than non-ACR patients (197 vs. 92.7; p 197 EOSp was 91% and the area under the ROC curve was 0.724 with 95% confidence interval (CI) 0.636-0.813. Conclusion The monitoring of the EOSp count can be useful to increase the diagnostic yield for ACR of BTB performed by protocol or lung function deterioration.

Tacrolimus Variability: A Cause of Donor-Specific Anti-HLA Antibody Formation in Children.

The most important determinant of long-term graft survival in renal transplantation is adequate immunosuppression. Inadequate immunosuppression may lead to graft loss due to the presence of anti-HLA antibody. The aim of this study was to investigate the effect of variability in tacrolimus blood concentration on anti-HLA antibody development in pediatric recipients of living-donor renal transplants. Pediatric recipients of living-donor renal transplants were retrospectively evaluated. Patients with a minimum of two years of follow-up who were administered tacrolimus were included in the study. Patients who had pretransplant anti-HLA antibody were excluded. Variability in tacrolimus blood concentration was assessed using the coefficient of variation ("tacrolimus CV") method. Tacrolimus CV was calculated separately for the first 6months post-transplant, between 6 and 12months post-transplant, and from the end of the first year post-transplant to the last follow-up. We constructed receiver operating characteristic (ROC) curves of the tacrolimus CV for each group to find the best cutoff value. A total of 67 patients (including 48 males; 72%) with a mean age of 15.16 ± 4.43years were included in the study. Anti-HLA antibody positivity was detected in 12 patients (18%). More than three HLA mismatches and the presence of acute cellular rejection correlated with the development of anti-HLA antibody (p = 0.056, 0.009). Tacrolimus CVs for the three periods were 0.37 ± 0.11, 0.31 ± 0.18, and 0.35 ± 0.12, respectively. The cutoff value of tacrolimus CV for anti-HLA antibody development was calculated as 0.32 with a sensitivity of 90.91% and specificity of 50.94% [AUC (area under the curve) 0.713, p = 0.023]. During the second 6-month period and after a year post-transplant, the percentage of patients with tacrolimus CV >0.32 was significantly higher in the anti-HLA antibody positive group than in the antibody negative group (67% vs 31%, p = 0.027; 83% vs 47%, p = 0.033). The eGFR (estimated glomerular filtration rate) was similar for the anti-HLA antibody negative and positive groups (78.72 ± 2.86 vs 77.45 ± 8.08, p > 0.05). High tacrolimus concentration variability appears to be associated with anti-HLAantibody formation in pediatric recipients of living-donor renal transplants.

Longitudinal Analysis of the Intestinal Microbiota in Liver Transplantation.

BackgroundIncreasing evidence suggests that the intestinal microbiota plays an important role in liver diseases. However, the dynamics of the intestinal microbiota during liver transplantation (LT) and its potential role in clinical course remain unknown.MethodsWe prospectively analyzed the intestinal microbiota of 38 patients who underwent LT in Kyoto University Hospital. We characterized the microbial compositions of fecal specimens from LT patients using a metagenomics approach by an Illumina MiSeq platform. We analyzed the diversity of microbiota sequentially from pretransplantation until 2 months after LT and also compared the microbiota during an episode of acute cellular rejection (ACR) and bloodstream infections (BSI) to the microbial composition of time-matched fecal specimens obtained from patients who did not experience ACR or BSI, respectively.ResultsThree hundred twenty fecal specimens were analyzed. Dynamic changes were observed in the microbial composition of LT recipients during the perioperative period. Over the course of LT, the mean diversity index decreased during the first 3 weeks after LT and gradually increased during our observation period. The loss of intestinal microbiota diversity was associated with high Child-Pugh scores, high model for end-stage liver disease scores, ACR, and BSI. At the family level, Bacteroides, Enterobacteriaceae, Streptococcaceae, and Bifidobacteriaceae were increased whereas Enterococcaceae, Lactobacillaceae, Clostridiaceae, Ruminococcaceae, and Peptostreptococcaceae were decreased in ACR patients.ConclusionsThe microbiota of LT patients was associated with the severity of liver diseases and the presence of ACR and BSI. These results lay the groundwork for more comprehensive investigations of microbiota characteristics to identify diagnostic markers for transplant health and to guide intervention strategies to improve transplant outcomes.

Interstitial Pneumonitis and the Risk of Chronic Allograft Rejection in Lung Transplant Recipients

The presence of interstitial pneumonitis (IP) on surveillance lung biopsy specimens in lung transplant recipients is poorly described, and its impact on posttransplant outcomes is not established. The following study assessed the association of posttransplant IP with the development of bronchiolitis obliterans syndrome (BOS). We examined all recipients of primary cadaveric lung transplants at our institution between January 1, 2000, and December 31, 2007 (N = 145). Patients had bronchoscopies with BAL, and transbronchial biopsies performed for surveillance during posttransplant months 1, 3, 6, and 12 as well as when clinically indicated. Patients were given a diagnosis of IP if, in the absence of active infection and organizing pneumonia, they showed evidence of interstitial inflammation and fibrosis on two or more biopsy specimens. IP was a significant predictor of BOS (OR, 7.84; 95% CI, 2.84-21.67; P &lt; .0001) and was significantly associated with time to development of BOS (hazard ratio, 3.8; 95% CI, 1.93-7.39; P = .0001) within the first 6 years posttransplant. The presence of IP did not correlate with a significantly higher risk of mortality or time to death. There was no association between the presence of IP and the development of or time to acute rejection. The presence of IP on lung transplant biopsy specimens suggests an increased risk for BOS, which is independent of the presence of acute cellular rejection.

Aspiration, Localized Pulmonary Inflammation, and Predictors of Early-Onset Bronchiolitis Obliterans Syndrome after Lung Transplantation

We hypothesized that immune mediator concentrations in the bronchoalveolar fluid (BALF) are predictive of bronchiolitis obliterans syndrome (BOS) and demonstrate specific patterns of dysregulation, depending on the presence of acute cellular rejection, BOS, aspiration, and timing of lung transplantation. We prospectively collected 257 BALF samples from 105 lung transplant recipients. The BALF samples were assessed for absolute and differential white blood cell counts and 34proteins implicated in pulmonary immunity, inflammation, fibrosis, and aspiration. There were elevated BALF concentrations of interleukin (IL)-15, IL-17, basic fibroblast growth factor, tumor necrosis factor-α, and myeloperoxidase, and reduced concentrations of α1-antitrypsin, which were predictive of early-onset BOS. Patients with BOS had an increased percentage of BALF lymphocytes and neutrophils, with a reduced percentage of macrophages (p < 0.05). The BALF concentrations of IL-1β; IL-8; interferon-γ-induced protein 10; regulated upon activation, normal T-cell expressed and secreted; neutrophil elastase; and pepsin were higher in patients with BOS (p < 0.05). Among those with BOS, BALF concentrations of IL-1RA; IL-8; eotaxin; interferon-γ-induced protein 10; regulated upon activation, normal T-cell expressed and secreted; myeloperoxidase; and neutrophil elastase were positively correlated with time since transplantation (p < 0.01). Those with worse grades of acute cellular rejection had an increased percentage of lymphocytes in their BALF (p< 0.0001) and reduced BALF concentrations of IL-1β, IL-7, IL-9, IL-12, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-γ, and vascular endothelial growth factor (p ≤ 0.001). Patients with aspiration based on detectable pepsin had increased percentage of neutrophils (p < 0.001) and reduced BALF concentrations of IL-12 (p < 0.001). The BALF levels of IL-15, IL-17, basic fibroblast growth factor, tumor necrosis factor-α, myeloperoxidase, and α1-antitrypsin at 6 to 12 months after lung transplantation are predictive of early-onset BOS, and those with BOS and aspiration have an augmented chemotactic and inflammatory balance of pulmonary leukocytes and immune mediators. These data justify the surgical prevention of aspiration and argue for the refinement of antirejection regimens.

Co-occurrence of Nonanastomotic Biliary Stricture and Acute Cellular Rejection in Liver Transplant

Nonanastomotic biliary stricture is generally considered the most troublesome biliary complication after liver transplant. Nonanastomotic biliary stricture owing to immunologic cholangiopathy (such as acute cellular rejection) has not been reported. We describe 2 patients with the co-occurrence of nonanastomotic biliary stricture and acute cellular rejection after pediatric live-donor liver transplant. CASE 1: A 13-month-old male infant with liver cirrhosis underwent an ABO-identical live-donor liver transplant using a left lateral segment graft. Eighty days after the live-donor liver transplant, ever with liver dysfunction and dilatation of the intrahepatic bile duct occurred. Percutaneous transhepatic biliary drainage and a liver biopsy were performed. The histopathologic evaluation indicated the presence of acute cellular rejection. After percutaneous transhepatic biliary drainage and steroid pulse treatment, the patient showed good clinical outcome. CASE 2: A 21-month-old female infant with biliary atresia underwent an ABO-identical live-donor liver transplant using a left lateral segment graft. Twenty-six days after the live-donor liver transplant, percutaneous transhepatic biliary drainage for B3 and a liver biopsy were performed, owing to fever, with liver dysfunction, and dilatation of the intrahepatic bile duct. Histopathologic evaluation indicated the presence of acute cellular rejection. After percutaneous transhepatic biliary drainage and steroid pulse treatment, the patient showed good clinical outcome. It is important for patients with nonanastomotic biliary stricture to undergo early liver biopsy because the nonanastomotic biliary stricture may be coincident with, or caused by, acute cellular rejection.

Perfusion Fluid Contamination in Relation to Recipient Survival and Acute Cellular Rejection in Orthotopic Liver Transplantation: Retrospective Analysis

Introduction A perfusion fluid used in the preservation of a grafted liver represents a medium suitable for microorganism growth. This study investigated the prevalence of perfusion fluid contamination, acute cellular rejection (ACR) episodes, and patient survival rate. Method This is a retrospective study, based on an electronic database allocating cases of orthotopic liver transplantation. The exclusion criteria were as follows: having been submitted to multiple organ transplantation, liver retransplantation only, and those whose samples had not been collected or sent on the back table procedure or were unobtainable (usually the samples were sent when there was donor infection suspicion/positivity). Our posttransplantation infection prophylactic protocol consisted of ampicillin/sulbactam for 72 hours. The variables in the study were as follows: fluid contamination, presence of acute cellular rejection (ACR, Banff classification), and recipient survival at the first year. Statistical analysis was performed using descriptive statistics and chi-square with Fisher exact test considering significant P < .05. Results We observed perfusion fluid contamination in 15/121 (12.39%). The agents were as follows: Klebsiella pneumoniae in 6 (4.96%), Staphylococcus epidermidis in 5 (4.13%), and Acinetobacter baumanii in 3 (2.48%) and negative cultures in 106 (87.60%). Only 1 patient had matching for donor infection and positivity hemoculture after the transplantation ( K pneumoniae) and he was the only patient associated with fluid infection and death. The recipients who had their fluid preservation with positive cultures had more ACR and the survival rate was similar among those with or without infection. Conclusion Optimization of microbiological procedures can be performed including fungal and bacterial cultures.

Universal noninvasive detection of solid organ transplant rejection

It is challenging to monitor the health of transplanted organs, particularly with respect to rejection by the host immune system. Because transplanted organs have genomes that are distinct from the recipient's genome, we used high throughput shotgun sequencing to develop a universal noninvasive approach to monitoring organ health. We analyzed cell-free DNA circulating in the blood of heart transplant recipients and observed significantly increased levels of cell-free DNA from the donor genome at times when an endomyocardial biopsy independently established the presence of acute cellular rejection in these heart transplant recipients. Our results demonstrate that cell-free DNA can be used to detect an organ-specific signature that correlates with rejection, and this measurement can be made on any combination of donor and recipient. This noninvasive test holds promise for replacing the endomyocardial biopsy in heart transplant recipients and may be applicable to other solid organ transplants.

Induction Therapy With Antithymocyte Globulin Before Reperfusion

The use of induction immunosuppressive agents in pediatric lung transplantation is not universal. The rationale of induction therapy is to use the strongest immunosuppressive drugs at the time when the risk of acute cellular rejection (ACR) is highest. The timing of induction is not universal. We hypothesize that early treatment with antilymphocyte globulin (ATG) prior to reperfusion of the first donor lung will decrease the incidence of ACR. The initial dose of ATG was given during the operative procedure when the recipient was on cardiopulmonary bypass after removal of the recipient lungs and prior to implantation. Patients received additional doses daily for four days. All children were monitored for ACR during the first 6 months posttransplant with transbronchial biopsies at defined intervals (weeks-months) and when clinically indicated. Presence of ACR was defined by International Society for Heart & Lung Transplantation guidelines. Recipients from two pediatric centers received ATG based on this protocol. A total of 18 patients were treated with this protocol, and the follow-up period was 6 to 45 months. A total of 63 flexible bronchoscopies with transbronchial biopsies were performed during the first 6 months. A single episode of ACR (≥ grade A2) was identified in this patient population for an incidence of 5.2% ACR grade A2 or above in this patient population. Induction therapy with ATG prior to donor lung reperfusion is associated with a low incident of ACR during the first 6 months posttransplant in our patient cohort. Long-term follow-up is needed to ascertain the full effect of this treatment protocol.

Elevated Levels of Serum Interleukin-6 are Associated With Low Grade Cellular Rejection in Patients With Heart Transplantation

Endomyocardial biopsy is the gold-standard procedure to diagnose acute cellular rejection after heart transplantation. This study assessed whether the blood levels of cytokines involved in inflammation and immune activation are useful to detect the presence of acute cellular rejection. Methods Blood specimens collected before 275 endomyocardial biopsies in 66 patients were assayed for levels of TNFα, IL6, IL1β, and IL2 receptor. The biopsies were grouped according to the presence ( n = 41) or absence ( n = 234) of acute cellular rejection grade ≥3A of the International Society for Heart and Lung Transplantation. We compared the levels of cytokines in the two groups. Results Circulating IL6 levels were significantly higher when there was a low grade (0–2) cellular rejection in the biopsy versus the group of biopsies grade ≥3A (19.8 ± 27 versus 12.9 ± 10 pg/mL; P = .001). An IL6 level higher than 30 pg/mL showed a negative predictive value of 95% for the presence of acute rejection grade ≥ 3A. Conclusion In heart transplant patients, high levels of serum IL6 were associated with low grade cellular rejection. Determination of IL6 levels may be useful to reduce the number of endomyocardial biopsies during follow-up in these patients.

Use of C4d as a Diagnostic Adjunct in Lung Allograft Biopsies

Purpose: Humoral allograft rejection is a defined mechanism for cardiac and renal graft dysfunction; C4d deposition, a stable component of complement activation, inversely correlates with graft survival. With the recent recognition of humoral rejection in lung grafts, we examined C4d's role as a prognostic adjunct in lung allografts. Material and Methods: Twenty‐three lung recipients underwent biopsies for deterioration in clinical status or routine surveillance. Clinically unwell patients possessed acute rejection or bronchiolitis obliterans syndrome (BOS). Biopsies attributable to infection were excluded from the study. In addition to routine light microscopy, an attempt was made to correlate the clinical status and morphologic findings with the pattern of C4d deposition and also to compare these clinical and morphologic parameters with the other assessed immunoreactants. Panel reactive antibody testing was also carried out at various points in their post transplantation course whereby in 6 of the cases the samples were procured at exactly the same time as the tissue samples. Results: The patients were segregated into two groups: those patients with recurrent acute rejection and those with BOS. In those patients with symptomatic acute rejection, all biopsies showed light microscopic and immunofluorescent evidence of humoral allograft rejection. The level of C4d was positively correlated with the degree of parenchymal injury, the hallmark being one of septal capillary necrosis. In addition, high and intermediate levels of C4d correlated with a clinical diagnosis of acute rejection. C4d was the strongest predictor of parenchymal injury and of the clinical status (p < .0001) compared to other the immunoreactants C1q, C5b‐9 and immunoglobulin. There was no specific correlation between C4d deposition and the presence of acute cellular rejection. In those patients fulfilling clinical criteria of BOS, deposits of C4d as well as other immunoreactants were found in the bronchial wall as opposed to the rarity of this finding in bon‐BOS patients. However the only statistically significant predictor of BOS was bronchial wall deposition of C1q. In no case were panel reactive antibodies at significant levels discovered post transplantation. Conclusions: In the context of acute rejection, C4d deposition correlates with clinical evidence of rejection and the degree of humoral rejection assessed pathologically; there is no association with the presence of histocompatibility related antibodies. It is a more specific predictor of allograft status compared to other immunoreactants. C4d deposition within the bronchial wall is a feature of BOS and hence may be used as a marker of chronic graft dysfunction. The antigenic target resulting in C4d deposition may not be histocompatibility related.

Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification.

The incidence of acute humoral rejection (AHR) in renal allograft biopsies has been difficult to determine because widely accepted diagnostic criteria have not been established. C4d deposition in peritubular capillaries (PTC) of renal allografts has been proposed as a useful marker for AHR. This study was designed to test the relative value of C4d staining, histology, and serology in the diagnosis of AHR. Of 232 consecutive kidney transplants performed at a single institution from July 1995 to July 1999, all patients (n = 67) who developed acute rejection within the first 3 mo and had a renal biopsy with available frozen tissue at acute rejection onset, as well as posttransplant sera within 30 d of the biopsy, were included in this study. Hematoxylin and eosin and periodic acid-Schiff stained sections were scored for glomerular, vascular, and tubulointerstitial pathology. C4d staining of cryostat sections was done by a sensitive three-layer immunofluorescence method. Donor-specific antibodies (DSA) were detected in posttransplant recipient sera using antihuman-globulin-enhanced T cell and B cell cytotoxicity assays and/or flow cytometry. Widespread C4d staining in PTC was present in 30% (20 of 67) of all acute rejection biopsies. The initial histologic diagnoses of the C4d(+) acute rejection cases were as follows: AHR only, 30%; acute cellular rejection (ACR) and AHR, 45%; ACR (CCTT types 1 or 2) alone, 15%; and acute tubular injury (ATI), 10%. The distinguishing morphologic features in C4d(+) versus C4d(-) acute rejection cases included the following: neutrophils in PTC, 65% versus 9%; neutrophilic glomerulitis, 55% versus 4%; neutrophilic tubulitis, 55% versus 9%; severe ATI, 75% versus 9%; and fibrinoid necrosis in glomeruli, 20% versus 0%, or arteries, 25% versus 0%; all P < 0.01. Mononuclear cell tubulitis was more common in the C4d(-) group (70% versus 100%; P < 0.01). No significant difference between C4d(+) and C4d(-) acute rejection was noted for endarteritis, 25% versus 32%; interstitial inflammation (mean % cortex), 27.2 +/- 27% versus 38 +/- 21%; interstitial hemorrhage, 25% versus 15%; or infarcts, 5% versus 2%. DSA were present in 90% (18 of 20) of the C4d(+) cases compared with 2% (1 of 47) in the C4d(-) acute rejection cases (P < 0.001). The pathology of the C4d(+) but DSA(-) cases was not distinguishable from the C4d(+), DSA(+) cases. The C4d(+) DSA(-) cases may be due to non-HLA antibodies or subthreshold levels of DSA. The sensitivity of C4d staining is 95% in the diagnosis of AHR compared with the donor-specific antibody test (90%). Overall, eight grafts were lost to acute rejection in the first year, of which 75% (6 of 8) had AHR. The 1-yr graft failure rate was 27% (4 of 15) for those AHR cases with only capillary neutrophils versus 40% (2 of 5) for those who also had fibrinoid necrosis of arteries. In comparison, the 1-yr graft failure rates were 3% and 7%, respectively, in ACR 1 (Banff/CCTT type 1) and ACR 2 (Banff/CCTT type 2) C4d(-) groups. A substantial fraction (30%) of biopsy-confirmed acute rejection episodes have a component of AHR as judged by C4d staining; most (90%), but not all, have detectable DSA. AHR may be overlooked in the presence of ACR or ATI by histology or negative serology, arguing for routine C4d staining of renal allograft biopsies. Because AHR has a distinct therapy and prognosis, we propose that it should be classified separately from ACR, with further sub-classification into AHR 1 (neutrophilic capillary involvement) and AHR 2 (arterial fibrinoid necrosis).

Quantitative assessment of cell adhesion molecule gene expression in endomyocardial biopsy specimens from cardiac transplant recipients using competitive polymerase chain reaction.

Adhesion of leukocytes to vascular endothelium is an early step in cardiac allograft rejection leading to migration of lymphocytes into parenchymal tissues. Cell adhesion molecule (CAM) protein expression appears to increase as a result of rejection. The relationship of CAM gene expression to rejection is less well defined. The goal of this study was to define cell adhesion molecule gene expression in relation to the presence of acute cellular rejection in endomyocardial biopsies from cardiac transplant recipients. To quantitatively assess intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin gene expression, we developed a competitive PCR system using nonhomologous DNA fragments (MIMICs) with complementary sequences to CAM gene-specific primers as internal standards. MIMIC fragments with known concentrations were mixed in serial dilutions with constant amounts of cDNA from the biopsy specimens and amplified with common primers under the same polymerase chain reaction conditions. The relative CAM cDNA concentrations were determined by comparing the density of MIMIC to target cDNA bands on agarose gel. ICAM-1, VCAM-1, and E-selectin mRNA concentrations were analyzed from 38 cardiac transplant biopsies divided into 3 groups according to ISHLT rejection grade: group 1-grade 0 (n=13); group 2-grade 1A or 1B (n=13); group 3-grade 3A (n=12). Glyceraldehyde-3-phosphate dehydrogenase (a constitutive gene) was quantified in the same way as CAMs to normalize the relative levels of CAMs. The results expressed as mean (1x10(-3) pM) (+/-SEM) in groups 1, 2, and 3, respectively, were: ICAM-1; 5+/-1; 57+/-4*; 64+/-13*, VCAM-1; 0.8+/-0.1; 6+/-1**; 9+/-1*, E-selectin; 0.4+/-0.2; 0.8+/-0.2; 0.4+/-0.1 (*P<0.001 versus group 1; **P<0.01 versus group 1). ICAM-1 and VCAM-1 gene expression was increased during rejection in endomyocardial biopsy specimens. Competitive polymerase chain reaction can be used to quantitatively assess gene expression in biopsy specimens from patients.