David Rowe

Epstein-Barr virus BZLF1 trans-activator specifically binds to a consensus AP-1 site and is related to c-fos

Two regions of the Epstein‐Barr virus BZLF1 trans‐activator protein have sequence similarity to the c‐fos protein. Part of the similarity corresponds to the region of c‐fos which is similar to the DNA binding domain of c‐jun and GCN‐4. The structure of the exon which contains this region in c‐fos and BZLF1 is also highly conserved between the two genes. Complete BZLF1 protein and a C terminal fragment were prepared either as purified fusion proteins or by in vitro translation from a BZLF1 cDNA. Gel retardation and DNase footprinting assays using these proteins show that BZLF1 is a sequence specific DNA binding protein capable of binding to a target sequence which contains a consensus AP‐1 site.

Epstein–Barr virus load monitoring: its role in the prevention and management of post-transplant lymphoproliferative disease

Abstract: The Epstein–Barr virus load in the peripheral blood at the time of diagnosis of post‐transplant lymphoproliferative disease (PTLD) is elevated 1000‐ to 10,000‐fold compared to the level detected in normal latency. With the use of quantitative polymerase chain reaction (PCR), changes in the viral load over time can be measured with a two‐ to fourfold accuracy. This has allowed early detection of first‐time infections and reactivations that may lead to PTLD and has provided an opportunity to intervene before symptomatic disease has occured. Viral load monitoring has also been used to follow patients with PTLD and, along with other parameters, provided an assessment of the effectiveness of therapeutic protocols. Viral load monitoring has led to the discovery that at least two‐thirds of transplant recipients become persistent viral load carriers. While the persistent load appears to be largely carried in latently infected memory B cells, more work is needed to clearly define this type of persistent infection and determine the risks associated with it. New diagnostic tests need to be developed to distinguish the persistent latent viral loads from viral loads that are likely to become symptomatic PTLD.

The management of Epstein–Barr virus associated post‐transplant lymphoproliferative disorders in pediatric solid‐organ transplant recipients

Abstract: Despite a growing understanding of the pathogenesis and spectrum of Epstein–Barr virus (EBV) and EBV‐associated post‐transplant lymphoproliferative disease (PTLD) in organ transplant recipients, the optimal management of this complication remains controversial. The absence of comparative data evaluating potential therapeutic strategies explains the lack of uniformly accepted guidelines for the management of PTLD. The purpose of this review is to provide an overview of potential therapies and offer a set of guidelines for the management of EBV‐associated PTLD in children.

Chronic High Epstein-Barr Viral Load State and Risk for Late-Onset Posttransplant Lymphoproliferative Disease/Lymphoma in Children

Increased use of serial EBV‐PCR monitoring after pediatric transplantation has led to the identification of asymptomatic patients who carry very high viral loads over prolonged periods. The significance of this high‐load state is unknown. We speculated that this state may identify patients at high risk for development of late PTLD/lymphoma. We reviewed data on 71 pediatric heart recipients who had serial viral load monitoring since 1997. Chronic high‐load state was defined as the presence of >16 000 genome copies/mL whole blood on ≥50% of samples over at least 6 months. Among 20 high‐load carriers (eight following prior PTLD, seven with prior symptomatic EBV infection, five without previous EBV disease), 9 (45%) developed late‐onset PTLD 2.5–8.4 years posttransplant (including with four Burkitts lymphoma). Among 51 controls with low (n = 39) or absent (n = 12) loads, only 2 (4%; p < 0.001 absent/low vs. high load) developed late PTLD/lymphoma. By multivariable analysis, high‐load carrier state (OR = 12.4, 95% CI 2.1–74.4) and prior history of PTLD (OR = 10.7, 95% CI 1.9–60.6) independently predicted late PTLD. A chronic high EBV‐load state is not benign and is a predictor of de novo or recurrent PTLD.

Predictive negative value of persistent low Epstein-Barr virus viral load after intestinal transplantation in children.

Background. The correlation between an elevated Epstein-Barr virus(EBV) viral load in the peripheral blood and the subsequent development of EBV-associated posttransplant lymphoproliferative disease (PTLD) is the basis for strategies using serial measurements of the EBV viral load to guide preemptive therapy (PT). Neither the frequency, duration of monitoring, nor the predictive negative value of viral load monitoring for asymptomatic patients with persistent low or nondetectable viral loads against the development of PTLD has been established. Methods. Since April 1994, children undergoing intestinal transplantation (ITx) underwent serial monitoring of the EBV viral load in their peripheral blood using a quantitative competitive EBV polymerase chain reaction assay (PCR). Samples were obtained every 2 weeks for the first 3 months and then every 1–3 months depending on the patients clinical condition. EBV viral loads ≥40 (for patients who were EBV seronegative pre-ITx) and≥200 (for those who were seropositive) genome copies/105 peripheral blood lymphocytes were felt to identify patients at increased risk for PTLD and generally prompted PT. Results. A total of 30 ITx recipients were compliant with our monitoring protocol; 23/30 are alive 6–59 months post-ITx. A total of 12/30 never had a viral load >40 and did not receive PT. In contrast, 18/30 had ≥1 high viral load (≥200); the first high viral load was measured a median of 59 days post-ITx (range 1–440). A late rise (>6 months post-ITx) was seen in only 2/18 children. A total of 0/12 patients with persistently low viral loads received PT and none developed PTLD. In contrast, 5/18 with ≥1 one high viral load (including 2/14 who received and 3/4 who did not receive PT) developed PTLD. All five children with PTLD were EBV seropositive pre-ITx and experienced their first high EBV PCR within the first 3 months after ITx. Conclusions. The predictive negative value of persistently low or nondetectable EBV viral loads was 100% in this study. Patients with nondetectable or low viral loads for the first 6 months after ITx did not develop PTLD regardless of their pretransplant EBV serological status. The frequency of viral load monitoring can be safely decreased for patients whose viral loads remain low for the first 6 months ITx.

Measurement of Epstein-Barr Virus DNA Loads in Whole Blood and Plasma by TaqMan PCR and in Peripheral Blood Lymphocytes by Competitive PCR

ABSTRACT Epstein-Barr virus (EBV) DNA load values were measured in samples of whole blood (n = 60) and plasma (n = 59) by TaqMan PCR and in samples of peripheral blood lymphocytes (PBLs) (n = 60) by competitive PCR (cPCR). The samples were obtained from 44 transplant recipients. The whole-blood and PBL loads correlated highly (r2 > 0.900), whereas the plasma and PBL loads correlated poorly (r2 = 0.512). Testing of whole blood by TaqMan PCR is an acceptable alternative to testing of PBLs by cPCR for quantifying EBV DNA load.

CMV‐IVIG for Prevention of Epstein Barr Virus Disease and Posttransplant Lymphoproliferative Disease in Pediatric Liver Transplant Recipients

A randomized controlled trial of CMV‐IVIG (cytomegalovirus‐intravenous immunoglobulin) for prevention of Epstein Barr virus (EBV) posttransplant lymphoproliferative disease (PTLD) in pediatric liver transplantation (PLTx) recipients was begun in Pittsburgh and subsequently expanded to four additional sites. Protocol EB viral loads were obtained in a blinded fashion; additional loads could be obtained for clinical indications. Patients were followed for 2 years post‐LTx. Eighty‐two evaluable patients (39 CMV‐IVIG, 43 placebo) developed 18 episodes of EBV disease (7 CMV‐IVIG, 11 placebo) including nine cases of PTLD (three CMV‐IVIG, six placebo). No significant differences were seen in the adjusted 2‐year EBV disease‐free rate (CMV‐IVIG 79%, placebo 71%) and PTLD‐free rate (CMV‐IVIG 91%, placebo 84%) between treatment and placebo groups at 2 years (p > 0.20). The absence of significant effect of CMV‐IVIG may be explained by a lack of efficacy of the drug or limitations of sample size.

The role of antiviral and immunoglobulin therapy in the prevention of Epstein-Barr virus infection and post-transplant lymphoproliferative disease following solid organ transplantation

Abstract: The recognition of the importance of Epstein–Barr virus (EBV) infection, including EBV‐associated post‐transplant lymphoproliferative disease (PTLD), has led to a new focus on the prevention of this problem. This paper reviews the scientific rationale behind, and clinical experience with, the use of chemoprophylaxis (using acyclovir or ganciclovir) and immunoprophylaxis (using intravenous immunoglobulin) in the prevention of EBV/PTLD. While some centers have already introduced the use of one or both of these agents as standard prophylaxis against the development of this complication, published data in support of these protocols are currently lacking. Well designed clinical trials are necessary to evaluate the potential role of both antiviral and immunoglobulin agents in the prevention of EBV/PTLD in organ transplant recipients.

Epstein-Barr Virus Gene Expression in the Peripheral Blood of Transplant Recipients with Persistent Circulating Virus Loads

Pediatric solid organ transplant recipients are at risk for Epstein-Barr virus (EBV)-driven lymphoproliferative disease. The expression of 5 sentinel EBV genes (EBNA1, EBNA2a, LMP1, LMP2a, and ZEBRA) was examined in solid organ transplant recipients who developed persistent virus loads in their peripheral blood lymphocytes after transplantation. Two distinct groups were identified. LMP2a gene expression alone was detected among 12 of 14 patients carrying EBV loads < or =100 copies/10(5) lymphocytes. The other 2 low-load carriers made LMP2a RNA but also expressed LMP1 RNA. In contrast, LMP2a and LMP1 gene expression was detected among 11 of 13 patients carrying a virus load >100 copies/10(5) lymphocytes. Two high-load carriers made LMP1 RNA but not the RNA for LMP2a or any of the other viral genes. Therefore, persistent low-load carriers appear to maintain an apparently normal state of latent viral infection, whereas high-load carriers display a unique LMP1:LMP2a pattern of viral gene expression that has not been previously described.

Response of elevated epstein-barr virus DNA levels to therapeutic changes in pediatric liver transplant patients: 56-month follow up and outcome

Background. Posttransplant lymphoproliferative disease (PTLD) is a serious complications after liver transplantation. Epstein-Barr virus (EBV) load measured by quantitative competitive polymerase chain reaction (PCR) has been used as an early marker for the development of PTLD and a guide for initiating preemptive therapy. The aim of this study is to report the results of EBV DNA PCR screening in a transplant population and to examine the risk factors for developing elevated EBV DNA PCR and the effect of interventions for reducing EBV DNA levels. Methods. Medical records of 44 children who underwent liver transplantation and EBV DNA PCR screening during a 3-year period were reviewed, and the patients were prospectively followed up for another 2 years. Eleven patients who developed elevated EBV DNA PCR levels, defined as ≥40 genomes/105 peripheral blood lymphocytes (PBL) in pretransplant EBV-seronegative patients and ≥200 genomes/105 PBL in pretransplant-seropositive patients, were treated. The initial intervention was reduction of immunosuppression and initiation of anti-viral therapy in all patients, with administration of cytomegalovirus immunoglobulin (CMV-IgG) in two patients. CMV-IgG was then given to five of the patients who did not respond to the initial intervention. Results. The initial intervention resulted in the reduction of EBV DNA PCR levels to below threshold values in 4 of 11 patients. Five patients who did not respond to the initial interventions were subsequently given intravenous CMV-IgG. The EBV DNA PCR level fell in all five of these patients during the course of treatment with CMV-IgG, with a significant reduction (to threshold levels or by two dilutions) in four of the five patients. No episodes of graft rejection were observed. Conclusion. Eleven patients (25%) developed elevated EBV DNA PCR after liver transplantation. There were no identifiable risk factors for developing elevated EBV DNA PCR. A combination of reducing immunosuppression, adding antiviral agents, and initiating CMV-IgG resulted in a significant reduction of EBV DNA levels in nine (82%) patients during the follow-up period.