Biomedical subjects

D T Rowe

Publications and source records attributed to D T Rowe.

At least 19 recordsLinked to original sources

PubMed2001

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

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 occurred. 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.

DNA, Viral↗

PubMed1999

Epstein-Barr virus immortalization and latency.

Epstein-Barr virus infects human B lymphocytes. The interaction between the virus and these cells has been the subject of investigation for over three decades. Recent in vitro and in vivo studies, reviewed here, are revealing the mechanisms by which EBV induces and controls proliferation through the expression of six viral nuclear proteins and two plasma membrane proteins. This genetic program is referred to as immortalization and it is suggested that the purpose of immortalization is to use the innate proliferative potential of B cells to inflate the numbers of infected cells prior to virus production and cell lysis. Latency, on the other hand, has only been detected in situ in latently infected humans. It is characterized by the presence of very low numbers of viral episomes that appear to express the RNA for only one protein (Latent Membrane Protein 2) in B cells that bear the markers of a non-activated resting memory subset. Two models are proposed for the mechanism that establishes this state. The differences between immortalization and latency are highlighted in this review and it is suggested that many of the functions currently attributed to latency are actually features of immortalization. An appreciation of this distinction may assist the discussion of the nature of the interaction between the virus and the host in EBV-associated lymphoproliferative diseases and tumors.

B-Lymphocytes↗

PubMed1999

Serum sCD23 level in patients with AIDS-related non-Hodgkin's lymphoma is associated with absence of Epstein-Barr virus in tumor tissue.

The cytokine soluble CD23 (sCD23) acts as a B cell growth factor and is associated with Epstein-Barr virus (EBV) infection. To elucidate the role sCD23 might play in the pathogenesis of AIDS-related non-Hodgkin's lymphoma (AIDS NHL), 101 AIDS NHL patients from the Multicenter AIDS Cohort Study were studied. Serum sCD23 within 18 months prior to lymphoma diagnosis was measured for all patients and EBV in tumor tissue was ascertained for 49 patients. Tumor morphology and primary site were verified from pathology reports and tumor specimens. Bivariate tests and multivariate regression were employed to determine whether serum sCD23 correlated with tumor EBV, morphology, and primary site. Higher levels of serum sCD23 were associated with the absence of tumor EBV and with small noncleaved cell morphology. Thus, the serum sCD23 level does not appear to be mediated by EBV in these patients, but could be related to a pathogenetic mechanism of small noncleaved cell lymphoma.

Adult↗

PubMed1999

Serum soluble CD23 level correlates with subsequent development of AIDS-related non-Hodgkin's lymphoma.

The cytokine soluble CD23 (sCD23) has been shown to act as a B cell growth factor and to be elevated in serum prior to development of AIDS-related non-Hodgkin's lymphoma (AIDS NHL). To further characterize the elevation of serum sCD23 in AIDS NHL patients and investigate its potential as a diagnostic test, a matched case-control study of AIDS NHL (n = 101) was nested within the Multicenter AIDS Cohort Study. Serum sCD23 was measured in cases' and controls' serum specimens at three different time periods (0-6, 6-12, and 12-18 months) and CD4+ thresholds (0-99, 100-199, and 200-299 cells/microl) prior to the case's NHL diagnosis. Changes in serum sCD23 over time were examined in AIDS NHL cases relative to controls, and t tests were performed to determine whether cases' serum sCD23 exceeded that of controls at each time period and CD4+ threshold. Overall, cases' median serum sCD23 levels were approximately double those of controls. Serum sCD23 concentration was positively correlated with lymphocyte counts for both cases and controls. The difference in cases' and controls' serum sCD23 levels became greater as AIDS NHL diagnosis date approached: in the 18 months preceding the case's NHL diagnosis, serum sCD23 was stable in cases but dropped in controls. Although this difference was statistically significant (P < 0.05), it was not clinically significant. It is unlikely that serum sCD23 would make a useful test for AIDS NHL because the magnitude of the difference between cases and controls was small and there was no change in serum sCD23 in cases that would indicate disease.

Adult↗

PubMed1998

Serial measurement of Epstein-Barr viral load in peripheral blood in pediatric liver transplant recipients during treatment for posttransplant lymphoproliferative disease.

BACKGROUND: Few data are available describing the natural history of the Epstein-Barr virus (EBV) viral load after the diagnosis of EBV-associated posttransplant lymphoproliferative disease (PTLD). Accordingly, we prospectively followed the EBV viral load after the diagnosis of EBV/PTLD in seven pediatric orthotopic liver transplant recipients. METHODS: EBV viral loads were serially measured by quantitative competitive polymerase chain reaction of the peripheral blood from pediatric patients with PTLD and correlated with the clinical course of these children. Viral loads >200 genome copies/10(5) peripheral blood lymphocytes were considered consistent with an increased risk of PTLD. Viral loads <200 obtained during treatment for PTLD were considered evidence of "clearance" of EBV; subsequent loads >200 were considered evidence of virologic "rebound." RESULTS: The mean EBV viral load at the time of diagnosis of PTLD was 1029. All patients "cleared" their EBV viral load during the treatment of PTLD; patient and graft survival in this series was 100%. The mean time to clearance of EBV from the peripheral blood (18.8 days) was similar to the mean time to onset of first rejection (13.8 days). EBV viral load at the time of diagnosis of rejection after PTLD was always <100. A rebound in the EBV viral load to >200 was noted in five of seven patients a median of 3.5 months (range 2.3-13 months) after the diagnosis of EBV/PTLD. However, none of these children has had any evidence of PTLD recurrence. CONCLUSIONS: Clearance of the EBV viral load from the peripheral blood seems to correlate with restoration of the host's immune response as noted both by the regression of the PTLD and the onset of rejection. Late rebound of the EBV viral load is common and does not seem to predict disease recurrence.

Child↗

PubMed1997

Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders.

A quantitative competitive PCR (QC-PCR) assay for Epstein-Barr virus (EBV) has been developed to provide accurate measurement of EBV genome load in pediatric transplant recipients at risk for developing posttransplant lymphoproliferative disorder (PTLD). The assay quantifies between 8 and 5,000 copies of the EBV genome in 10(5) lymphocytes after a 30-cycle amplification reaction. For 14 pediatric patients diagnosed with PTLD, the median EBV genome load was 4,000, and 13 of the 14 patients had values of >500 copies per 10(5) lymphocytes. Only 3 of 12 control transplant recipients not diagnosed with PTLD had detectable viral genome loads (median value, 40). This median was calculated by using the highest value obtained by PCR testing on each of these patients posttransplantation. PCR values of >500 copies per 10(5) lymphocytes appear to correlate with a diagnosis of PTLD. By a modified protocol, the EBV genome copy number in latently infected adults was estimated to be <0.1 copy per 10(5) lymphocytes.

Adolescent↗

PubMed1997

Epstein-Barr virus latent membrane protein 2 associates with and is a substrate for mitogen-activated protein kinase.

The latent membrane protein 2 (LMP2) of Epstein-Barr virus interferes with B-lymphocyte signal transduction through the immunoglobulin (Ig) receptor. Two isoforms of LMP2 exist and differ only in that one isoform (LMP2a) contains an N-terminal cytoplasmic domain that the other isoform does not. LMP2a is a phosphoprotein that is phosphorylated on tyrosines and serines in the cytoplasmic domain. GST1-119, a glutathione S-transferase (GST) fusion protein containing the 119 amino acids of the cytoplasmic domain, affinity precipitated serine kinase activity from BJAB cell extracts. The affinity-precipitated kinase phosphorylated LMP2a sequences, and kinase activity was increased following induction. Probing of Western immunoblots of affinity-precipitated proteins showed that the Erk1 form of mitogen-activated protein kinase (MAPK) was present. Purified MAPK phosphorylated GST fusion proteins containing the cytoplasmic domain of LMP2a and mutational analyses were used to identify S15 and S102 as the sites of in vitro phosphorylation. A polyclonal rabbit antiserum was prepared against a maltose binding protein-LMP2a cytoplasmic domain fusion protein (MBP1-119) and used to immunoprecipitate LMP2a from the in vitro-immortalized lymphoblastoid B-cell line B95-8CR. LMP2a immunoprecipitates from B95-8CR contained MAPK as a coprecipitated protein. Cross-linking surface Ig on B95-8CR cells failed to induce MAPK activity within the cells. Treatment of B95-8CR with phorbol myristate acetate (PMA) was able to bypass the Ig receptor block and activate MAPK activity. Phosphorylation of LMP2a on serine residues increased after PMA induction. The possible role for LMP2a serine phosphorylation by MAPK in the control of latency is discussed.

Amino Acid Sequence↗

PubMed1996

Protein-protein interactions between Epstein-Barr virus nuclear antigen-LP and cellular gene products: binding of 70-kilodalton heat shock proteins.

The EBNA-LP protein encoded by the open reading frame in the leader exons of the Epstein-Barr nuclear antigen messages is essential for efficient immortalization of B lymphocytes. Protein-protein interaction studies using affinity precipitation of proteins from [35S]methionine-labeled cell lysates and bacterially expressed maltose binding protein EBNA-LP fusions were performed. A cellular 68/72-kDa doublet protein was detected. This banding pattern was shown to be identical to that obtained in affinity precipitations with fusions of glutathione-S-transferase and Sp1 (a basal transcription factor). For both EBNA-LP and Sp1 the specific interacting cellular proteins have been identified as heat shock proteins (HSP) 72/73. Affinity precipitation of HSP 72/73 with deletion mutants of EBNA-LP maps the interaction domain on EBNA-LP to exon Y2 which is required for immortalization. Immunoprecipitation of EBNA-LP from EBV-positive lymphoblastoid cell lines coprecipitated the HSP 72/73 proteins, indicating that the interaction occurs in vivo as well as in vitro. The association of HSPs with a widening range of nuclear proteins involved in gene expression and proliferation control now includes Sp1 and EBNA-LP and suggests that there is a central role for molecular chaperones in these processes.

Antigens, Viral↗

PubMed1996

Cell cycle stage-specific phosphorylation of the Epstein-Barr virus immortalization protein EBNA-LP.

EBNA-LP is a viral nuclear oncoprotein implicated in the immortalization of B lymphocytes by Epstein-Barr virus. An analysis of EBNA-LP migration on polyacrylamide gels was performed with protein derived from the X50-7 lymphoblastoid cell line blocked by hydroxyurea or aphidicolin at the G1/S phase of the cell cycle or by nocodazole at the G2/M phase. More slowly migrating species of EBNA-LP were detected in G2/M phase-arrested cell extracts. Release from nocodazole G2/M block or treatment with phosphatase caused the more slowly migrating species of EBNA-LP to disappear. Analyses of 32PO(4)(3-)-labeled EBNA-LP protein immunoprecipitated from the drug-synchronized cells showed that phosphorylated EBNA-LP was present throughout the cell cycle but that phosphorylation increased in G2 and was maximal at G2/M. Phosphoamino acid analysis revealed that all phosphorylation was on serine residues only. The ability of EBNA-LP to be phosphorylated by p34(cdc2) kinase and casein kinase II exclusively on serines implicates these enzymes as being potentially involved in EBNA-LP phosphorylation.

Binding Sites↗

PubMed1995

Epstein-Barr virus latent messages with shuffled leader exons: remnants of circumgenomic transcription?

The EBNA transcription unit which is active in Epstein-Barr virus-immortalized latently infected B lymphocytes covers approximately 60% of the 172-kb genome. Since the genome exists as a circular double-stranded DNA molecule in latently infected cells, it is conceivable that complete copies are made during transcription. Rather than attempt to detect gigantic RNA molecules directly, we used RNA-PCR to detect incorporation of leader exons into mRNA in a shuffled order. The downstream U leader exon was detected spliced upstream of the internal repeat leader exons W1 and W2 in the polyadenylated RNA fraction of spontaneous lymphoblastoid cell lines, restricted phenotype BL cell lines Wanyanyi and Wewak2, and in B95-8, Raji, and Akata cells. Quantitative competitive RNA-PCR showed that the ratio of U exon-containing EBNA1 messages to U exon-shuffled leader messages was approximately 10:1, with large variation from cell line to cell line, and was not affected by induction of the lytic cycle in B95-8, Raji, or Akata cells. Messages with shuffled exons contained only the C2W1 alternative splice, which does not produce an initiator AUG methionine codon for EBNA4 gene expression. The results provide evidence for long-range exon skipping and imply that genome-length transcripts may occur and participate in viral gene expression in latency.

Adult↗

PubMed1992

Cell growth effects of Epstein-Barr virus leader protein.

B lymphoblastoid cell lines immortalized with P3HR1/633 Epstein-Barr virus (EBV), which has a deletion in the EBV nuclear antigen leader protein (EBNA-LP) gene, were transfected with a vector expressing wild-type EBNA-LP. The EBNA-LP transfectants grew out faster under G418 selection than control cells but expression of EBNA-LP made no significant difference to growth rate or saturation density of the resulting established cell lines. When the cells expressing EBNA-LP were allowed to grow to saturation and then diluted in fresh medium they underwent DNA synthesis more rapidly than control cultures.

Antigens, Viral↗

PubMed1992

Epstein-Barr virus latent gene expression in uncultured peripheral blood lymphocytes.

In this study of Epstein-Barr virus (EBV) latency, the polymerase chain reaction was used in modified form for amplification and detection of viral mRNA sequences in peripheral blood lymphocytes from healthy seropositive adults. Six known promoters for latent gene expression and eight known gene products were identified in in vitro-immortalized lymphocytes and in the cell lines established spontaneously from seropositive adults. We examined whether mRNA expression in uncultured B cells from four seropositive adults was the same as that which occurred in spontaneously established EBV-positive B-cell lines from the same individuals. A minimum of 17 polymerase chain reaction targets was required to circumscribe the known latent mRNA structures. Expression of the C promoter for the EBNA genes was detected in B-cell RNA from three of the four subjects. Transcripts initiated from the alternative W promoter for EBNA expression were not detected. The spliced transcripts detected in the B cells contained only the C2-to-W1 alternative splice, which was nonproductive for EBNA4 gene expression. None of the other EBNA open reading frames were detected spliced onto the 3' ends of the C promoter-initiated RNAs. Spliced RNA from the TP gene was detected in all four subjects. Expression of the TP gene was restricted to TP1 promoter-initiated RNAs, as no TP2 promoter-initiated transcripts were detected. Expression of RNA from the LMP gene was not detected. The F promoter which is active in the restricted expression latency that occurs in Burkitt's lymphoma cells was not detected being expressed in peripheral blood B cells. This pattern of latent gene expression is unique to uncultured B cells, indicating that there are profound differences between viral latent states in vitro and in situ and suggesting a central role for the TP gene in the latency of EBV.

Adult↗

PubMed1991

Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia.

The BZLF1 protein of Epstein-Barr virus (EBV) is a key immediate-early protein which has been shown to disrupt virus latency in EBV-infected B cells. We have generated a monoclonal antibody, BZ1, to BZLF1 which reacts in immunohistology, immunoblotting, and immunoprecipitation and which recognizes both the active, dimeric form and the inactive, monomeric form of the protein. Biopsies of oral hairy leukoplakia, an AIDS-associated lesion characterized by high-level EBV replication, were examined by immunohistochemistry using the BZ1 monoclonal antibody. A differentiation-associated pattern of BZLF1 expression was observed, BZ1 reacting with nuclei of the upper spinous layer of the lesion. This finding suggests that the BZLF1 promoter may be regulated by the degree of squamous differentiation. A comparison of in situ hybridization to EBV DNA and viral capsid antigen staining with BZ1 reactivity suggested that BZLF1 expression precedes rampant virus replication. The inability to detect EBV in the lower epithelial layers of oral hairy leukoplakia raises questions concerning the nature of EBV latency and persistence in stratified squamous epithelium.

Antibodies, Monoclonal↗

PubMed1990

Structure and function of the Epstein-Barr virus BZLF1 protein.

Five DNA-binding sites for the Epstein-Barr virus BZLF1 protein have been identified within three of the early viral promoters, and four of these binding sites contain a consensus AP-1 site. The part of the BZLF1 protein required for sequence-specific DNA binding to one of these AP-1-like sites was identified by deletion mapping. Site-directed mutagenesis of this DNA target suggests that BZLF1 may work partly by overcoming a cellular repressor of viral transcription.

Amino Acid Sequence↗

PubMed1990

Identification of the Epstein-Barr virus terminal protein gene products in latently infected lymphocytes.

The terminal protein (TP) gene produces two overlapping mRNAs in latently infected lymphocytes that are predicted to encode the similar polypeptides TP1 (497 amino acids) and TP2 (378 amino acids), with TP1 exon 1 providing 119 extra unique residues at the N terminus. Rabbit antisera were raised to procaryotic fusion proteins and used to detect expression of a predicted 53-kilodalton (kDa) TP product in transfected 293 cells and latently infected lymphocytes. Fractionation of transfected 293 cells showed this protein to be localized to an integral membrane preparation. The same fraction of latently infected lymphocytes contained proteins of 53 and 27 to 39 kDa as determined by Western immunoblotting with the TP-specific rabbit antisera. Immunoprecipitation of TP products from 35S-labeled human lymphoblastoid cells (CR/B95-8) was used in pulse-chase experiments and showed that TP1 was a labile protein with a half-life of approximately 2 to 4 h. The anti-fusion protein serum detected a 53-kDa TP1 and degradation products in the range of 25 to 35 kDa. A panel of Burkitt's lymphoma cell lines and cell lines established with virus recovered from the BL cells were analyzed by Western immunoblotting and found to contain the 53-kDa TP1 product, its degradation products, or both. Only two EBV-positive BL cell lines (BL72 and Wewak II) were negative in this assay. The results suggest that a labile TP1 protein may be expressed by most, if not all, EBV-infected cell lines.

Antibodies, Monoclonal↗

PubMed1989

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.

Amino Acid Sequence↗

PubMed1989

Size and stability of the Epstein-Barr virus major internal repeat (IR-1) in Burkitt's lymphoma and lymphoblastoid cell lines.

We have used field inversion gel electrophoresis to survey EBV strains for the size of the major internal repeat, IR-1, and estimate the number of 3.1-kb repeat units present. The B95-8 strain of EBV was estimated to contain 8.6 repeats. The repeat number varies considerably among naturally occurring isolates around a mean of six repeats. Some cell lines harbored multiple viral genomes with differing numbers of repeats and our results suggest that the repeat number in IR-1 is more likely to change during lytic replication than during latency. The Jijoye strain had 6.6 repeats and the Jijoye deletion mutant clone P3HR-1 retained 5.9 repeats setting the size of the P3HR-1 deletion at 6.8 kb. Thus, the nonimmortalizing mutant has retained all of the W1 and W2 exons of the immortalizing parent and has lost only the 3' unique exons of EBNA4 and all of EBNA2.

Antigens, Viral↗

PubMed1989

The type-specific epitopes of the Epstein-Barr virus nuclear antigen 2 are near the carboxy terminus of the protein.

The Epstein-Barr virus nuclear antigen 2 (EBNA 2) shows serotype variation and two serologically distinct groups of viruses have been identified. These correspond to the two hybridization groups of viruses (A and B) that are distinguished by a highly substituted nucleic acid sequence in the middle of the open reading frame of the EBNA 2 gene. An epitope survey of the EBNA 2-coding region was carried out using a new prokaryotic expression vector tailored to express DNA fragments from the M13 sequencing libraries of the B95-8 (type A) and Jijoye (type B) prototype virus strains. Short overlapping stretches of EBNA 2 sequence were expressed as fusion proteins and used in Western blotting with human sera that contained serotype-specific antibodies. The type A-specific epitope was located between residues 378 and 435 of the B95-8 EBNA 2 polypeptide and the type B-specific epitope mapped between residues 390 and 454, at the carboxy terminus of the Jijoye polypeptide chain. All of the type-specific anti-EBNA 2 sera tested reacted with fusion proteins containing one or other of these epitopes. Despite the direct correlation between the hybridization and serological phenotypes, the type-specific epitopes appear to lie in the relatively conserved carboxy-terminal region of EBNA 2. There was no indication that the residues of the non-homologous region contributed to the formation of antibody-combining sites.

Amino Acid Sequence↗