Greatly reduced risk of EBV reactivation in rituximab-experienced recipients of alemtuzumab-conditioned allogeneic HSCT.

EBV-associated post-transplant lymphoproliferative disease (PTLD) remains an important complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). We retrospectively analysed the incidence and risk factors for EBV reactivation in 186 adult patients undergoing consecutive allo-HSCT with alemtuzumab T-cell depletion at a single centre. The cumulative incidence of EBV reactivation was 48% (confidence interval (CI) 41-55%) by 1 year, with an incidence of high-level EBV reactivation of 18% (CI 13-24%); 8 patients were concurrently diagnosed with PTLD. Amongst patients with high-level reactivation 31/38 (82%) developed this within only 2 weeks of first EBV qPCR positivity. In univariate analysis age⩾50 years was associated with significantly increased risk of EBV reactivation (hazard ratio (HR) 1.54, CI 1.02-2.31; P=0.039). Furthermore, a diagnosis of non-Hodgkin lymphoma (NHL) was associated with greatly reduced risk of reactivation (HR 0.10, CI 0.03-0.33; P=0.0001) and this was confirmed in multivariate testing. Importantly, rituximab therapy within 6 months prior to allo-HSCT was also highly predictive for lack of EBV reactivation (HR 0.18, CI 0.07-0.48; P=0.001) although confounding with NHL was apparent. Our data emphasise the risk of PTLD associated with alemtuzumab. Furthermore, we report the clinically important observation that rituximab, administered in the peri-transplant period, may provide effective prophylaxis for PTLD.

HLA-A11-restricted epitope polymorphism among Epstein-Barr virus strains in the highly HLA-A11-positive Chinese population: incidence and immunogenicity of variant epitope sequences.

An individual's CD8(+)-cytotoxic-T-lymphocyte (CTL) response to Epstein-Barr virus (EBV) latent cycle antigens focuses on a small number of immunodominant epitopes often presented by just one of the available HLA class I alleles; for example, HLA-A11-positive Caucasians frequently respond to two immunodominant HLA A11 epitopes, IVTDFSVIK (IVT) and AVFDRKSDAK (AVF), within the nuclear antigen EBNA3B. Here, we reexamine the spectrum of EBV strains present in the highly HLA-A11-positive Chinese population for sequence changes in these epitopes relative to the Caucasian type 1 prototype strain B95.8. The IVT epitope was altered in 61 of 64 Chinese type 1 viruses, with four different sequence variants being observed, and the AVF epitope was altered in 46 cases with six different sequence variants; by contrast, all 10 Chinese type 2 viruses retained the prototype 2 epitope sequences. All but one of the type 1 epitope variants were poorly recognized by IVT- or AVF-specific CTLs in pulse-chase assays of peptide-mediated target cell lysis. More importantly, we screened HLA-A11-positive Chinese donors carrying viruses with known epitope mutations for evidence of epitope-specific CTL memory by enzyme-linked immunospot assays: none of the type 1 variants tested, nor the type 2 prototype, appeared to be immunogenic in vivo. The data remain consistent with the possibility that, during virus-host coevolution, pressure from the host CTL-mediated immune response has given A11 epitope-loss viruses a selective advantage.

Latent gene sequencing reveals familial relationships among Chinese Epstein-Barr virus strains and evidence for positive selection of A11 epitope changes.

Epstein-Barr virus (EBV) strains from the highly HLA-A11-positive Chinese population are predominantly type 1 and show a variety of sequence changes (relative to the contemporary Caucasian prototype strain B95.8) in the nuclear antigen EBNA3B sequences encoding two immunodominant HLA-A11 epitopes, here called IVT and AVF. This has been interpreted by some as evidence of immune selection and by others as random genetic drift. To study epitope variation in a broader genomic context, we sequenced the whole of EBNA3B and parts of the EBNA2, 3A, and 3C genes from each of 31 Chinese EBV isolates. At each locus, type 1 viruses showed <2% nucleotide divergence from the B95.8 prototype while type 2 sequences remained even closer to the contemporary African prototype Ag876. However, type 1 isolates could clearly be divided into families based on linked patterns of sequence divergence from B95.8 across all four EBNA loci. Different patterns of IVT and AVF variation were associated with the different type 1 families, and there was additional epitope diversity within families. When the EBNA3 gene sequences of type 1 Chinese strains were subject to computer-based analysis, particular codons within the A11-epitope-coding region were among the few identified as being under positive or diversifying selection pressure. From these results, and the observation that mutant epitopes are consistently nonimmunogenic in vivo, we conclude that the immune selection hypothesis remains viable and worthy of further investigation.

Methylation of transcription factor binding sites in the Epstein-Barr virus latent cycle promoter Wp coincides with promoter down-regulation during virus-induced B-cell transformation.

Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites.

Epstein-barr virus nuclear antigen 1 sequences in endemic and sporadic Burkitt's lymphoma reflect virus strains prevalent in different geographic areas.

The Epstein-Barr virus (EBV) nuclear antigen EBNA1 is the only viral protein detectably expressed in virus genome-positive Burkitt's lymphoma (BL); recent work has suggested that viral strains with particular EBNA1 sequence changes are preferentially associated with this tumor and that, within a patient, the tumor-associated variant may have arisen de novo as a rare mutant of the dominant preexisting EBV strain (K. Bhatia, A. Raj, M. J. Gutierrez, J. G. Judde, G. Spangler, H. Venkatesh, and I. T. Magrath, Oncogene 13:177-181, 1996). In the present work we first study 12 BL patients and show that the virus strain in the tumor is identical in EBNA1 sequence and that it is matched at several other polymorphic loci to the dominant strain rescued in vitro from the patient's normal circulating B cells. We then analyze BL-associated virus strains from three different geographic areas (East Africa, Europe, and New Guinea) alongside virus isolates from geographically matched control donors by using sequence changes in two separate regions of the EBNA1 gene (N-terminal codons 1 to 60 and C-terminal codons 460 to 510) to identify the EBNA1 subtype of each virus. Different geographic areas displayed different spectra of EBNA1 subtypes, with only limited overlap between them; even type 2 virus strains, which tended to be more homogeneous than their type 1 counterparts, showed geographic differences at the EBNA1 locus. Most importantly, within any one area the EBNA1 subtypes associated with BL were also found to be prevalent in the general population. We therefore find no evidence that Burkitt lymphomagenesis involves a selection for EBV strains with particular EBNA1 sequence changes.

Orientation of functional activating regions in the Escherichia coli CRP protein during transcription activation at class II promoters.

At class II CRP-dependent promoters the DNA site for CRP overlaps the DNA site for RNA polymerase, covering the -35 region. Transcription activation at class II CRP- dependent promoters requires a contact between an activating region in the upstream subunit of the bound CRP dimer and a contact site in the C-terminal domain of the alpha-subunit of RNA polymerase. Transcription activation is suppressed by amino acid substitutions in the activating region, but activation can be restored by second site substitutions at K52 or E96. These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters. Using the method of 'oriented heterodimers' we show that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer.

Definition of nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL.

Transcription initiation at the Escherichia coli nirB promoter is induced by anaerobic growth and further increased by the presence of nitrite or nitrate in the growth medium. Expression from this promoter is totally dependent on the transcription factor, FNR, which binds between positions -52 and -30 upstream of the transcription startsite. The 20 base pairs from position -79 to -60 contain an inverted repeat of two 10-base sequence elements that are related to sequences at the NarL-binding site at the E. coli narG promoter. Comparison of these, and sequence elements at other promoters regulated by NarL, suggests a consensus NarL-binding sequence. Mutations in the putative NarL-binding site at the nirB promoter decrease FNR-dependent anaerobic induction, suggesting that NarL acts as a helper to FNR during transcription activation. These mutations also suppress induction by nitrite: single mutations at symmetry-related positions have similar effects, whilst double mutations have more severe effects, probably because two NarL subunits bind to the inverted repeat. Disruption of narL decreases nitrite induction of the nirB promoter whilst not suppressing induction by nitrate, suggesting that there may be a second nitrate-responsive factor. Nitrate induction was, however, suppressed by double mutations at symmetry-related positions in the NarL-binding site, suggesting that this putative second factor may bind to sequences similar to those recognized by NarL.

Interconversion of the DNA-binding specificities of two related transcription regulators, CRP and FNR.

In Escherichia coli, FNR and CRP are homologous transcriptional regulators which recognize similar nucleotide sequences via DNA-binding domains containing analogous helix-turn-helix motifs. The molecular basis for recognition and discrimination of their target sites has been investigated by directed amino acid substitutions in the corresponding DNA-recognition helices. In FNR, Glu-209 and Ser-212 are essential residues for the recognition of FNR sites. A V208R substitution confers CRP-site specificity without loss of FNR specificity, but this has adverse effects on anaerobic growth. In contrast, changes at two (V208R and E209D) or three (V208R, S212G and G216K) positions in FNR endow a single CRP-site binding specificity. In reciprocal experiments, two substitutions (R180V and G184S) were required to convert the binding specificity of CRP to that of FNR. Altering Asp-199 in FNR failed to produce a positive control phenotype, unlike substitutions at the comparable site in CRP. Implications for the mechanism of sequence discrimination by FNR and CRP are discussed.

Molecular genetic analysis of an FNR-dependent anaerobically inducible Escherichia coli promoter.

From the effects of 13 deletions and three linker-scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR-dependent induction of activity by anaerobiosis and further nitrite-dependent stimulation of expression. We describe a nirB promoter derivative that allows the cloning of 'cassettes' carrying different FNR-binding sequences and experiments in which a number of point mutations were introduced into these sequences. FNR-dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR-binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function. We have transferred a number of cassette FNR-binding sequences from the nirB promoter to the unrelated melR promoter. The insertion of FNR-binding sequences at the melR promoter is sufficient to confer fnr-dependency on expression. However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR-binding sequence.

Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP.

Expression from the E.coli melR promoter (pmelR) is normally totally dependent on the transcription activator protein, CRP. We describe experiments with a genetically engineered DNA fragment carrying pmelR in which the wild type CRP binding site was replaced with synthetic oligonucleotides containing either FNR or CRP binding sequences. When the synthetic oligonucleotide contains the 22 bp consensus for FNR binding sites, expression from pmelR is dependent on FNR but not CRP. Single changes at either of two symmetrically-related positions create sites that are recognised by both FNR and CRP. Changes at both positions result in a site that is not recognised by FNR but which binds CRP tightly.

Performance of United States citizens with foreign medical education on standardized medical examinations.

To enter the mainstream of American medicine, United States students who enroll in foreign medical schools must either transfer to a United States medical school (usually after passing Part I of the NMBE examination) or complete their studies abroad and then pass FLEX and usually ECFMG examinations. Half those applying for transfer fail Part I. Although virtually all who succeed in transferring subsequently pass Parts II and III, their scores are below the mean of the reference group, and their relative performance progressively deteriorates. Sixty per cent of USFMG's fail the medical portion of the ECFMG examination as compared to an expected failure rate for United States medical students of only 2 to 3 per cent. Thirty-eight per cent of USFMG's who take both ECFMG and FLEX examinations fail the latter. The mean weighted score for USFMG's on FLEX is 75.8, while that for United States graduates is 80.1 and for FMG's 74.7. These preliminary data suggest that United States students who go abroad, regardless of where they complete their studies, do not perform as well on standard tests after they return home as do those who have had their entire education in United States schools. This question needs more systematic study.