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R J Tierney

Publications and source records attributed to R J Tierney.

14 recordsLinked to original sources
PubMed2000

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.

B-Lymphocytes↗
PubMed2000

Cytotoxic T-lymphocyte responses to a polymorphic Epstein-Barr virus epitope identify healthy carriers with coresident viral strains.

Cytotoxic T-lymphocyte (CTL) responses to Epstein-Barr virus (EBV) tend to focus on a few immunodominant viral epitopes; where these epitope sequences are polymorphic between EBV strains, host CTL specificities should reflect the identity of the resident strain. In studying responses in HLA-B27-positive virus carriers, we identified 2 of 15 individuals who had strong CTL memory to the pan-B27 epitope RRIYDLIEL (RRIY) from nuclear antigen EBNA3C but whose endogenous EBV strain, isolated in vitro, encoded a variant sequence RKIYDLIEL (RKIY) which did not form stable complexes with B27 molecules and which was poorly recognized by RRIY-specific CTLs. To check if such individuals were also carrying an epitope-positive strain (either related to or distinct from the in vitro isolate), we screened DNA from freshly isolated peripheral blood mononuclear cells for amplifiable virus sequences across the EBNA3C epitope, across a different region of EBNA3C with type 1-type 2 sequence divergence, and across a polymorphic region of EBNA1. This showed that one of the unexplained RRIY responders carried two distinct type 1 strains, one with an RKIY and one with an RRIY epitope sequence. The other responder carried an RKIY-positive type 1 strain and a type 2 virus whose epitope sequence of RRIFDLIEL was antigenically cross-reactive with RRIY. Of 15 EBV-seropositive donors analyzed by such assays, 12 appeared to be carrying a single virus strain, one was coinfected with distinct type 1 strains, and two were carrying both type 1 and type 2 viruses. This implies that a small but significant percentage of healthy virus carriers harbor multiple, perhaps sequentially acquired, EBV strains.

Adult↗
PubMed1998

Epidemiology of infection with Epstein-Barr virus types 1 and 2: lessons from the study of a T-cell-immunocompromised hemophilic cohort.

In apparent contrast to earlier work on Epstein-Barr virus (EBV) carriage in the general Caucasian population, in vitro virus isolations from human immunodeficiency virus (HIV)-positive male homosexual cohorts have shown frequent examples of multiple EBV infection and an overall prevalence of type 2 EBV strains exceeding 30%. Here we ask to what extent these findings might hold true in another T-cell-immunocompromised cohort, HIV-positive hemophilic patients. Resident EBV strains were rescued within lymphoblastoid cell lines derived from the blood and throat washings of 39 such individuals, using the same in vitro protocols of virus isolation as for the homosexual cohort. A mean of 19 independent cell lines was made per patient, and in each case the resident virus was characterized by PCR-based viral genomic analysis and by immunoblotting to reveal the viral "EBNAprint." By these criteria a significant proportion (14 of 39) of the hemophilic cohort carried more than one EBV strain, suggesting that T-cell impairment does indeed sensitize virus carriers to reinfection with new strains of exogenously transmitted virus. However, the overall incidence of type 2 EBV infection was 10%, which is close to that observed in the earlier work with healthy carriers and substantially lower than that seen in HIV-positive homosexuals. We infer that type 2 EBV is relatively rare in the general Caucasian population but has become endemic in the homosexual community.

AIDS-Related Opportunistic Infections↗
PubMed1997

Conserved CTL epitopes within EBV latent membrane protein 2: a potential target for CTL-based tumor therapy.

In healthy virus carriers, EBV is subject to strong CTL responses that principally target the EBV nuclear Ag (EBNA) 3A, 3B, 3C subset of virus proteins. In vitro-reactivated CTLs of this kind have proved very effective in treating EBV-positive immunoblastic lymphoma, a malignancy that expresses the full range of virus proteins. However, targeting other EBV-positive tumors will require CTLs that recognize some of the subdominant viral Ags since in nasopharyngeal carcinoma and EBV-positive Hodgkin's disease, EBNA1, latent membrane protein (LMP) 1, and LMP2 are the only virus proteins present. Studying healthy virus carriers (Caucasian and Chinese), we identified five CTL target epitopes in LMP2 restricted through HLA alleles particularly common in the southern Chinese population, which is most at risk for nasopharyngeal carcinoma (HLA-A2, 50%; A11, 50%; A24, 30%; and B40, 32%). Furthermore, we analyzed the effect of HLA subtype polymorphism, especially in the context of A2 for which four subtypes are present at significant frequency in the Chinese population. As to virus polymorphism, LMP2 epitope sequences (in contrast to EBNA 3A, 3B, and 3C epitopes) were shown to be antigenically conserved among EBV isolates from different world populations, including viruses present in nasopharyngeal carcinoma and Hodgkin's disease biopsy samples. Thus, nasopharyngeal carcinoma and Hodgkin's disease are predicted to express LMP2 proteins that contain conserved CTL target epitopes restricted through common HLA alleles; boosting responses to these epitopes could form the basis of a CTL-based therapy for these malignancies.

Amino Acid Sequence↗
PubMed1996

Frequency of multiple Epstein-Barr virus infections in T-cell-immunocompromised individuals.

The Epstein-Barr virus (EBV) carrier state is characterized by latent infection of the general B-cell pool and by chronic virus replication at oropharyngeal sites. In Caucasian populations, most healthy carriers seem to harbor one dominant transforming virus strain, usually of type I rather than type 2, which persists over time and is detectable both in the blood and in the throat. This finding implies that once the virus carrier state is established, both viral reservoirs are largely if not completely protected from infection with additional strains. However, it is not known which facets of the immune response offer that protection. Here we address this question by a detailed study of EBV carriage in patients T-cell immunocompromised as a result of chronic human immunodeficiency virus (HIV) infection. Resident EBV strains were rescued from blood and from throat washings by using an in vitro transformation assay which aims to minimize bias toward faster-growing transformants; in this way, a mean of 16 independent isolations were made from each of 35 HIV-positive (predominantly male homosexual) patients. These virus isolates were characterized first at the DNA level by PCR amplification across type-specific polymorphisms in the EBNA2 and EBNA3C genes and across the 30-bp deletion and 33-bp repeat loci in the LMP1 gene and then at the protein level by immunoblotting for the strain-specific "EBNAprint" of EBNA1, -2, and -3C molecular weights. By these criteria, 18 of 35 patients harbored only one detectable EBV strain, usually of type 1, as do healthy carriers. However, the other 17 patients showed clear evidence of multiple infection with different EBV strains. In eight cases these strains were of the same type, again usually type 1, and were more often found coresident in throat washings than in the blood. By contrast, a further nine patients gave evidence of coinfection with type 1 and type 2 strains, and in these cases both virus types were detectable in the blood as well as in the throat. Immunological assays on these HIV-positive patients as a group showed a marked impairment of T-cell responses, reflected in reduced levels of EBV-specific cytotoxic T-cell memory, but an elevation of humoral responses, reflected in raised antibody titers to the EBV envelope glycoprotein gp340 and by the maintenance of virus neutralizing antibodies in serum. We infer that selective impairment of the T-cell system predisposes the host to infection with additional exogenously transmitted EBV strains.

AIDS-Related Opportunistic Infections↗
PubMed1996

Isolation of intertypic recombinants of Epstein-Barr virus from T-cell-immunocompromised individuals.

All wild-type isolates of Epstein-Barr virus (EBV) analyzed to date for allelic polymorphisms of the nuclear antigen EBNA2 gene (in the BamHI YH region of the genome) and of the EBNA3A,-3B, -3C genes (tandemly arranged in the BamHI E region) have proved either uniformly type 1 or uniformly type 2 at all four loci. The absence of detectable intertypic recombination in the wild probably reflects the rarity with which individual carriers, and certainly individual target cells, become coinfected with both virus types. Studying a group of human immunodeficiency virus-positive T-cell-immunocompromised patients known to be at enhanced risk of multiple EBV infections, we have isolated intertypic EBV recombinants from 2 of 40 patients analyzed. These recombinants, whose in vitro transforming capacity appeared at least equal to that of type 1 strains, carried a type 1 EBNA2 allele and type 2 EBNA3A,-3B, and -3C alleles. This was clearly demonstrable at the DNA level by PCR amplification using type-specific primer-probe combinations and was confirmed at the protein level (for EBNA2 and EBNA3C) by immunoblotting with type-specific antibodies. In one patient, the recombinant appeared to be the predominant strain, being the virus most commonly rescued by in vitro transformation both from the blood and from the throat washings on two separate occasions 20 months apart. A regular type 1 virus strain was also present in this individual, but this was not related to the recombinant since the two viruses carried type 1 EBNA2 genes with different patterns of variance from the B95.8 prototype sequence. In the other patient, recombinants were isolated on one occasion from the blood and on a separate occasion, 21 months later, from the throat; these recombinants were almost certainly related, being identical at several genomic polymorphisms and differing only in one facet of the "EBNAprint," the size of the EBNA1 protein. Three different type 1 viruses were also isolated from this patient, two of which carried EBNA2 genes with the same pattern of sequence variation from B95.8 as the recombinant; however, since this is a fairly common pattern of variance, the relationship of these viruses to the recombinant remains an open question. We infer that intertypic recombinants of EBV are not uncommon in HIV-positive T-cell-immunocompromised patients, that they arise in such individuals as a consequence of their increased frequency of mixed-type infections, and that they will prove capable of efficient transmission in the human population.

Amino Acid Sequence↗
PubMed1994

Epstein-Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state.

Epstein-Barr virus (EBV) can display different forms of latent infection in B-cell lines in vitro; however, the types of infection normally established by the virus in vivo remain largely unexplored. Here we have approached this question by analyzing the types of viral RNAs present in mononuclear cells freshly isolated from the blood of 14 infectious mononucleosis patients undergoing primary EBV infection and 6 long-term virus carriers. Reverse transcription-PCR amplifications were carried out with a panel of oligonucleotide primers and probes which specifically detect (i) the EBER1 RNA common to all forms of latency, (ii) transcripts either from the Cp and Wp promoters generating all six nuclear antigen (EBNA1, -2, -3A, -3B, -3C, -LP) mRNAs or from the Fp promoter generating a uniquely spliced EBNA1 mRNA, (iii) the latent membrane protein (LMP1 and 2A) mRNAs, and (iv) the BZLF1 mRNA, an immediate-early marker of lytic cycle. Viral transcription in infectious mononucleosis mononuclear cells (and in the B-cell-enriched fraction) regularly included the full spectrum of latent RNAs seen during EBV-induced B-cell growth transformation in vitro, i.e., EBER1, Cp/Wp-initiated EBNA mRNAs, and LMP1/LMP2 mRNAs, in the absence of lytic BZLF1 transcripts. In addition, transcripts with the splice pattern of Fp-initiated EBNA1 mRNA, hitherto seen only in vivo in certain EBV-positive tumors, were frequently detected. In long-term virus carriers, the mononuclear cells were again positive for latent (EBER1) and negative for lytic (BZLF1) markers; Cp/Wp-initiated RNAs were not detected in these samples, but in several individuals it was possible to amplify both Fp-initiated EBNA1 mRNA and LMP2A mRNA signals. We suggest (i) that primary infection is associated with a transient virus-driven expansion of the infected B-cell pool through a program of virus gene expression like that seen in in vitro-transformed cells and (ii) that long-term virus carriage is associated with a switch from Cp/Wp to Fp usage and thus to a more restricted form of latent protein expression that may render the infected cells less susceptible to recognition by the virus-specific cytotoxic T-cell response.

Antigens, Viral↗
PubMed1994

Suicide intervention training evaluation: a preliminary report.

To date, very little work has been done on evaluating training in suicide intervention. This study developed and piloted a comprehensive method for evaluating suicide intervention training by applying three studies of immediate training effects on (a) suicide intervention abilities, (b) attitudes to suicide and suicide intervention, and (c) knowledge about suicide. The focus of the evaluation was a broadly used 2-day suicide intervention training program. Changes in suicide intervention abilities were measured by the Suicide Intervention Response Inventory (SIRI) and by performance in simulated suicide intervention situations, scored with the Suicide Intervention Protocol (SIP). Subjects consisted of 19 workshop participants in a pre-post condition and 17 participants in a post-test only condition. Results indicated significant increases in skills in suicide intervention situations. No significant effects were noted on the SIRI. Results from the attitudes and knowledge studies were very preliminary. They are reported here so that others may become aware of the methodology being used and the status of evaluation of the target program. Implications for further research are discussed.

Adult↗
PubMed1984

Inferential reading abilities of mildly mentally retarded and nonretarded students.

The inferential operations of mildly mentally retarded students reading at the intermediate level were investigated using methods based on discourse comprehension theory. We hypothesized that problems encountered in reading by these students are related to difficulties in generating logical inferences. Mildly retarded junior-high students and nonretarded third-grade students of the same reading comprehension level read and recalled a descriptive expository and a narrative passage. On the expository passage mildly retarded students generated the same quantity of inferences as did nonretarded students, but the inferences were qualitatively inferior. On the narrative passage the differences between the two groups were not significant. These findings were discussed in relation to the cognitive functioning of mildly retarded students.

Adolescent↗