Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.

Prostanoids modulate inflammation and alloimmune responses during graft rejection

Acute rejection of a transplanted organ is characterized by intense inflammation within the graft. Yet, for many years transplant researchers have overlooked the role of classic mediators of inflammation such as prostaglandins and thromboxane (prostanoids) in alloimmune responses. It has been demonstrated that local production of prostanoids within the allograft is increased during an episode of acute rejection and that these molecules are able to interfere with graft function by modulating vascular tone, capillary permeability, and platelet aggregation. Experimental data also suggest that prostanoids may participate in alloimmune responses by directly modulating T lymphocyte and antigen-presenting cell function. In the present paper, we provide a brief overview of the alloimmune response, of prostanoid biology, and discuss the available evidence for the role of prostaglandin E2 and thromboxane A2 in graft rejection.

Anti-tumor necrosis factor-a for the treatment of steroid-refractory acute graft-versus-host disease

Allogeneic stem cell transplantation has been increasingly performed for a variety of hematologic diseases. Clinically significant acute graft-versus-host disease (GVHD) occurs in 9 to 50% of patients who receive allogeneic grafts, resulting in high morbidity and mortality. There is no standard therapy for patients with acute GVHD who do not respond to steroids. Studies have shown a possible benefit of anti-TNF-a (infliximab)for the treatment of acute GVHD. We report here on the outcomes of 10 recipients of related or unrelated stem cell transplants who received 10 mg/kg infliximab, iv, once weekly for a median of 3.5 doses (range: 1-6) for the treatment of severe acute GVHD and who were not responsive to standard therapy. All patients had acute GVHD grades II to IV (II = 2, III = 3, IV = 5). Overall, 9 patients responded and 1 patient had progressive disease. Among the responders, 3 had complete responses and 6 partial responses. All patients with cutaneous or gastrointestinal involvement responded, while only 2 of 6 patients with liver disease showed any response. None of the 10 patients had any kind of immediate toxicity. Four patients died, all of them with sepsis. Six patients are still alive after a median follow-up time of 544 days (92-600) after transplantation. Considering the severity of the cases and the bad prognosis associated with advanced acute GVHD, we find our results encouraging. Anti-TNF-a seems to be a useful agent for the treatment of acute GVHD.

Heterotopic transplantation of glycerin-preserved trachea: effect of respiratory epithelium desquamation on acute rejection

An effective preservation method and decreased rejection are essential for tracheal transplantation in the reconstruction of large airway defects. Our objective in the present study was to evaluate the antigenic properties of glycerin-preserved tracheal segments. Sixty-one tracheal segments (2.4 to 3.1 cm) were divided into three groups: autograft (N = 21), fresh allograft (N = 18) and glycerin-preserved allograft (N = 22). Two segments from different groups were implanted into the greater omentum of dogs (N = 31). After 28 days, the segments were harvested and analyzed for mononuclear infiltration score and for the presence of respiratory epithelium. The fresh allograft group presented the highest score for mononuclear infiltration (1.78 ± 0.43, P <= 0.001) when compared to the autograft and glycerin-preserved allograft groups. In contrast to the regenerated epithelium observed in autograft segments, all fresh allografts and glycerin-preserved allografts had desquamation of the respiratory mucosa. The low antigenicity observed in glycerin segments was probably the result of denudation of the respiratory epithelium and perhaps due to the decrease of major histocompatibility complex class II antigens.

Human polyclonal anti-hepatitis B surface antigen immunoglobulin reduces the frequency of acute rejection after liver transplantation for chronic hepatitis B

BACKGROUND: Use of polyclonal anti-hepatitis B surface antigen immunoglobulin (HBIg) has been shown to reduce hepatitis B virus (HBV) recurrence after liver transplantation (LT) and to decrease the frequency of acute cellular rejection (ACR). However, the protective role of HBIg against ACR remains controversial, since HBV infection has been also associated with a lower incidence of ACR. AIM: To assess the relationship between HBIg immunoprophylaxis and the incidence of rejection after LT. METHODS: 260 patients (158 males, 43 ± 14 years old) submitted to LT were retrospectively evaluated and divided into three groups, according to the presence of HBsAg and the use of HBIg. Group I was comprised of HBsAg-positive patients (n = 12) that received HBIg for more than 6 months. Group II was comprised of HBsAg-positive patients that historically have not received HBIg or have been treated irregularly for less than 3 months (n = 10). Group III was composed of 238 HBsAg-negative subjects that have not received HBIg. RESULTS: HBIg-treated patients (group I) had significantly less ACR episodes, when compared to group II and III. No differences between groups II and III were observed. CONCLUSIONS: Long-term HBIg administration contributes independently to reduce the number of ACR episodes after LT.

Diagnosis of Rejection by Analyzing Ventricular Late Potentials in Heart Transplant Patients

Background: Heart transplant rejection originates slow and fragmented conduction. Signal-averaged ECG (SAECG) is a stratification method in the risk of rejection. Objective: To develop a risk score for rejection, using SAECG variables. Methods: We studied 28 transplant patients. First, we divided the sample into two groups based on the occurrence of acute rejection (5 with rejection and 23 without). In a second phase, we divided the sample considering the existence or not of rejection in at least one biopsy performed on the follow-up period (rejection pm1: 18 with rejection and 10 without). Results: On conventional ECG, the presence of fibrosis was the only criterion associated with acute rejection (OR = 19; 95% CI = 1.65-218.47; p = 0.02). Considering the rejection pm1, an association was found with the SAECG variables, mainly with RMS40 (OR = 0.97; 95% CI = 0.87-0.99; p = 0.03) and LAS40 (OR = 1.06; 95% IC = 1.01-1.11; p = 0.03). We formulated a risk score including those variables, and evaluated its discriminative performance in our sample. The presence of fibrosis with increasing of LAS40 and decreasing of RMS40 showed a good ability to distinguish between patients with and without rejection (AUC = 0.82; p < 0.01), assuming a cutoff point of sensitivity = 83.3% and specificity = 60%. Conclusion: The SAECG distinguished between patients with and without rejection. The usefulness of the proposed risk score must be demonstrated in larger follow-up studies.

Histopathological analysis of pre-implantation donor kidney biopsies: association with graft survival and function in one year post-transplantation

Introduction: Pre-implantation kidney biopsy is a decision-making tool when considering the use of grafts from deceased donors with expanded criteria, implanting one or two kidneys and comparing this to post-transplantation biopsies. The role of histopathological alterations in kidney compartments as a prognostic factor in graft survival and function has had conflicting results. Objective: This study evaluated the prevalence of chronic alterations in pre-implant biopsies of kidney grafts and the association of findings with graft function and survival in one year post-transplant. Methods: 110 biopsies were analyzed between 2006 and 2009 at Santa Casa de Porto Alegre, including live donors, ideal deceased donors and those with expanded criteria. The score was computed according to criteria suggested by Remuzzi. The glomerular filtration rate (GFR) was calculated using the abbreviated MDRD formula. Results: No statistical difference was found in the survival of donors stratified according to Remuzzi criteria. The GFR was significantly associated with the total scores in the groups with mild and moderate alterations, and in the kidney compartments alone, by univariate analysis. The multivariate model found an association with the presence of arteriosclerosis, glomerulosclerosis, acute rejection and delayed graft function. Conclusion: Pre-transplant chronic kidney alterations did not influence the post-transplantation one-year graft survival, but arteriosclerosis and glomerulosclerosis is predictive of a worse GFR. Delayed graft function and acute rejection are independent prognostic factors.

Prevention of bone marrow graft failure by an anti LFA-1 monoclonal antibody

Graft rejection is the major cause of failure of HLA mismatched bone marrow transplantation because of residual host immunity. we have proposed to use a monoclonal murine antibody specific for the LFA-1 molecule (25-3) to prevent graft failure in HLA mismatched bone marrow transplantation (BMT). The rationale for this approach is three fold: LFA-1 deficient patients (3/3) do not reject HLA mismatched BMT; anti LFA-1 blocka in vitro the induction of T cell responses and T/ non T cytotoxic functions; LFA-1 is not expressed by other cells than leucocytes. We have accordingly treated twenty two patients with inherited diseases and 8 with leikemia. The bone marrow was T cells depled by E rosetting of Campath antibody. The antibody was given at days -3, -1, +1, +3, +5 at dose of .1 mg/kg/d for the first 9 and then .2mg/kg/d from day -3 to +6. Engraftment occured in 23/30 patients as shown by at least HLA typing. Hematological recovery was rapid, GVH was limited. Side effects of antibody infusion included fever and possibly an increased incidence of early bacteral infection (sepsis, 1 death). Immunological reconstitution occured slowly leading in six cases to EBV-induced B cell poliferation (1 death and in two others to transient auto immune hemolytic anemia. There has been only one secondary graft rejection. Sisteen patients are alive 3 to 26 months post transplant with functional grafts. Although the number of patients treated is still low the absence of late rejection so far, gives hope for long term maintenance of the graft using anti LFA-1. Since the antibody is an IgG 1 unable to bind human complement, and since it is known to inhibit phagocytosis, there is a good suggestion that 25-3 act through functional blocking of host T and non T luymphocytes at both induction and effector levels.

Clinical and pathological correlations of C4d immunostaining and its infl uence on the outcome of kidney transplant recipients

INTRODUCTION: C4d is a marker of antibody-mediated rejection (ABMR) in kidney allografts, although cellular rejection also have C4d deposits. OBJECTIVE: To correlate C4d expression with clinico-pathological parameters and graft outcomes at three years. METHODS: One hundred forty six renal transplantation recipients with graft biopsies by indication were included. C4d staining was performed by paraffin-immunohistochemistry. Graft function and survival were measured, and predictive variables of the outcome were determined by multivariate Cox regression. RESULTS: C4d staining was detected in 48 (31%) biopsies, of which 23 (14.7%) had diffuse and 25 (16%) focal distribution. Pre-transplantation panel reactive antibodies (%PRA) class I and II were significantly higher in C4d positive patients as compared to those C4d negative. Both glomerulitis and pericapillaritis were associated to C4d (p = 0.002 and p < 0.001, respectively). The presence of C4d in biopsies diagnosed as no rejection (NR), acute cellular rejection (ACR) or interstitial fibrosis/ tubular atrophy (IF/TA) did not impact graft function or survival. Compared to NR, ACR and IF/TA C4d-, patients with ABMR C4d+ had the worst graft survival over 3 years (p = 0.034), but there was no difference between ABMR versus NR, ACR and IF/TA that were C4d positive (p = 0.10). In Cox regression, graft function at biopsy and high %PRA levels were predictors of graft loss. CONCLUSIONS: This study confirmed that C4d staining in kidney graft biopsies is a clinically useful marker of ABMR, with well defined clinical and pathological correlations. The impact of C4d deposition in other histologic diagnoses deserves further investigation.

Tuberculosis in renal transplant patients: The experience of a single center in Medellín-Colombia, 2005-2013

Introduction: Tuberculosis is a common opportunistic infection in renal transplant patients. Objective: To obtain a clinical and laboratory description of transplant patients diagnosed with tuberculosis and their response to treatment during a period ranging from 2005 to 2013 at the Pablo Tobón Uribe Hospital. Methods: Retrospective and descriptive study. Results: In 641 renal transplants, tuberculosis was confirmed in 12 cases. Of these, 25% had a history of acute rejection, and 50% had creatinine levels greater than 1.5 mg/dl prior to infection. The disease typically presented as pulmonary (50%) and disseminated (33.3%). The first phase of treatment consisted of 3 months of HZRE (isoniazid, pyrazinamide, rifampicin and ethambutol) in 75% of the cases and HZME (isoniazid, pyrazinamide, moxifloxacin and ethambutol) in 25% of the cases. During the second phase of the treatment, 75% of the cases received isoniazid and rifampicin, and 25% of the cases received isoniazid and ethambutol. The length of treatment varied between 6 and 18 months. In 41.7% of patients, hepatotoxicity was associated with the beginning of anti-tuberculosis therapy. During a year-long follow-up, renal function remained stable, and the mortality rate was 16.7%. Conclusion: Tuberculosis in the renal transplant population studied caused diverse nonspecific symptoms. Pulmonary and disseminated tuberculosis were the most frequent forms and required prolonged treatment. Antituberculosis medications had a high toxicity and mortality. This infection must be considered when patients present with a febrile syndrome of unknown origin, especially during the first year after renal transplant.

Vascular endothelial growth factor-A enhances indoleamine 2,3-dioxygenase expression by dendritic cells and subsequently impacts lymphocyte proliferation

Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.

Cytomegalovirus infection in renal transplant recipients diagnosed by nested-PCR

A prospective study of cytomegalovirus (CMV) infection was carried out on 34 renal transplant recipients managed at a General Hospital in Ribeirão Preto, SP, Brazil. Serologic tests showed that all patients were infected with CMV before renal transplantation. Two nested-PCR techniques with primers that recognize sequences of the glycoprotein B (gB) and H (gH) genes were used for CMV detection in blood and urine samples during the post-transplantation period. CMV was detected more frequently in blood samples than in urine samples (P<0.001). Thirty-three patients had CMV detected at least once in blood and/or urine samples. Seven of these patients (21.2%) were diagnosed as having symptomatic CMV infection and showed a worse clinical outcome, with a higher death rate (P = 0.03). No association between CMV viremia and graft rejection was observed. Nested-PCR was not useful to identify patients at risk for symptomatic CMV infection since only 21.2% of the patients with CMV infection were symptomatic.

Pharmacokinetic/pharmacodynamic relationships of FTY720 in kidney transplant recipients

FTY720 is a new and effective immunosuppressive agent, which produces peripheral blood lymphopenia through a lymphocyte homing effect. We investigated the relationship between the dose of FTY720 or blood concentration (pharmacokinetics, PK) and peripheral lymphopenia (pharmacodynamics, PD) in 23 kidney transplant recipients randomized to receive FTY720 (0.25-2.5 mg/day) or mofetil mycophenolate (2 mg/day) in combination with cyclosporine and steroids. FTY720 dose, blood concentrations and lymphocyte counts were determined weekly before and 4 to 12 weeks after transplantation. The effect of PD was calculated as the absolute lymphocyte count or its reductions. PK/PD modeling was used to find the best-fit model. Mean FTY720 concentrations were 0.36 ± 0.05 (0.25 mg), 0.73 ± 0.12 (0.5 mg), 3.26 ± 0.51 (1 mg), and 7.15 ± 1.41 ng/ml (2.5 mg) between 4 and 12 weeks after transplantation. FTY720 PK was linear with dose (r² = 0.98) and showed low inter- and intra-individual variability. FTY720 produced a dose-dependent increase in mean percent reduction of peripheral lymphocyte counts (38 vs 42 vs 56 vs 77, P < 0.01, respectively). The simple Emax model [E = (Emax * C)/(C + EC50)] was the best-fit PK/PD modeling for FTY720 dose (Emax = 87.8 ± 5.3% and ED50 = 0.48 ± 0.08 mg, r² = 0.94) or concentration (Emax = 78.3 ± 2.9% and EC50 = 0.59 ± 0.09 ng/ml, r² = 0.89) vs effect (% reduction in peripheral lymphocytes). FTY720 PK/PD is dose dependent and follows an Emax model (EC50 = 0.5 mg or 0.6 ng/ml). Using lymphopenia as an FTY720 PD surrogate marker, high % reductions (~80%) in peripheral lymphocytes are required to achieve best efficacy to prevent acute allograft rejection.

Allogeneic hematopoietic stem cell transplantation from an alternative stem cell source in Fanconi anemia patients: analysis of 47 patients from a single institution

We transplanted 47 patients with Fanconi anemia using an alternative source of hematopoietic cells. The patients were assigned to the following groups: group 1, unrelated bone marrow (N = 15); group 2, unrelated cord blood (N = 17), and group 3, related non-sibling bone marrow (N = 15). Twenty-four patients (51%) had complete engraftment, which was not influenced by gender (P = 0.87), age (P = 0.45), dose of cyclophosphamide (P = 0.80), nucleated cell dose infused (P = 0.60), or use of anti-T serotherapy (P = 0.20). Favorable factors for superior engraftment were full HLA compatibility (independent of the source of cells; P = 0.007) and use of a fludarabine-based conditioning regimen (P = 0.046). Unfavorable factors were > or = 25 transfusions pre-transplant (P = 0.011) and degree of HLA disparity (P = 0.007). Intensity of mucositis (P = 0.50) and use of androgen prior to transplant had no influence on survival (P = 0.80). Acute graft-versus-host disease (GVHD) grade II-IV and chronic GVHD were diagnosed in 47 and 23% of available patients, respectively, and infections prevailed as the main cause of death, associated or not with GVHD. Eighteen patients are alive, the Kaplan-Meyer overall survival is 38% at ~8 years, and the best results were obtained with related non-sibling bone marrow patients. Three recommendations emerged from the present study: fludarabine as part of conditioning, transplant in patients with <25 transfusions and avoidance of HLA disparity. In addition, an extended family search (even when consanguinity is not present) seeking for a related non-sibling donor is highly recommended.

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Studentt-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.