Alternative 2-keto acid oxidoreductase activities in Trichomonas vaginalis.

We have induced high levels of resistance to metronidazole (1 mM or 170 microg ml(-1)) in two different strains of Trichomonas vaginalis (BRIS/92/STDL/F1623 and BRIS/92/STDL/B7708) and have used one strain to identify two alternative T. vaginalis 2-keto acid oxidoreductases (KOR) both of which are distinct from the already characterised pyruvate:ferredoxin oxidoreductase (PFOR). Unlike the characterised PFOR which is severely down-regulated in metronidazole-resistant parasites, both of the alternative KORs are fully active in metronidazole-resistant T.

Identification of a cytotoxic T-lymphocyte response to the novel BARF0 protein of Epstein-Barr virus: a critical role for antigen expression.

The Epstein-Barr virus (EBV)-encoded BARF0 open reading frame gene products are consistently expressed in EBV-positive Burkitt's lymphoma (BL) cell lines, nasopharyngeal carcinoma cell lines, and lymphoblastoid cell lines (LCLs). Here we show that the BARF0 sequence includes an HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope. By using theoretically predicted HLA A2 binding motifs and peptide-loaded antigen presentation-deficient T2 cells, polyclonal BARF0-specific CD8(+) CTLs were isolated from four different healthy EBV-seropositive donors but not from two seronegative donors.

Detection of Blastocystis sp. in domestic dogs and cats.

Blastocystis sp. was detected in faecal samples from domestic dogs and cats in Brisbane, Australia. The prevalence rates were high, with 70.

Anaerobic bacterial metabolism in the ancient eukaryote Giardia duodenalis.

The protozoan parasite, Giardia duodenalis, shares many metabolic and genetic attributes of the bacteria, including fermentative energy metabolism which relies heavily on pyrophosphate rather than adenosine triphosphate and as a result contains two typically bacterial glycolytic enzymes which are pyrophosphate dependent. Pyruvate decarboxylation and subsequent electron transport to as yet unidentified anaerobic electron acceptors relies on a eubacterial-like pyruvate:ferredoxin oxidoreductase and an archaebacterial/eubacterial-like ferredoxin. The presence of another 2-ketoacid oxidoreductase (with a preference for alpha-ketobutyrate) and multiple ferredoxins in Giardia is also a trait shared with the anaerobic bacteria.

Targeting a polyepitope protein incorporating multiple class II-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by CD4+ cytotoxic T lymphocytes: a novel approach to vaccine design.

The role of CD4+ and CD8+ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4+- and CD8+-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8+- or CD4+-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4+ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4+ CTLs.

Involvement of superoxide dismutase and pyruvate:ferredoxin oxidoreductase in mechanisms of metronidazole resistance in Entamoeba histolytica.

Metronidazole resistance has been induced in an axenic strain of Entamoeba histolytica (HTH-56:MUTM) following continuous exposure to steadily increasing drug concentrations. The drug-resistant line is routinely maintained in normally lethal levels of metronidazole (10 microM). Resistance to this concentration of drug was developed over 177 days.

The radiosensitive cell line 180BR is not defective in the major DNA damage-sensing proteins.

The fibroblast culture 180BR, established from a patient showing an adverse response to radiotherapy, has been shown previously to be hypersensitive to ionizing radiation and to be defective in the repair of DNA double-strand breaks. We demonstrate here that the products of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and its regulatory subunits (Ku 70 and Ku 80) are present at normal levels and possess functional activity. The product of the gene mutated in the human genetic disorder ataxia-telangiectasia was also detected in these cells.

Hierarchy of Epstein-Barr virus-specific cytotoxic T-cell responses in individuals carrying different subtypes of an HLA allele: implications for epitope-based antiviral vaccines.

Major histocompatibility complex class I-restricted Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) in healthy virus carriers constitute a primary effector arm of the immune system in controlling the proliferation of virus-infected B cells in vivo. These CTLs generally recognize target epitopes included within the latent antigens of the virus. For example, CTLs from HLA B44+ healthy virus carriers often recognize peptide EENLLDFVRF [corrected] from EBV nuclear antigen 6.

Isolation of full-length ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype.

A gene mutated in the human genetic disorder ataxia-telangiectasia (A-T), ATM, was recently identified by positional cloning. ATM is a member of the phosphatidylinositol-3-kinase superfamily, some of which are protein kinases and appear to have important roles in cell cycle control and radiation signal transduction. We describe herein, to our knowledge, for the first time, the cloning of a full-length cDNA for ATM and correction of multiple aspects of the radio-sensitive phenotype of A-T cells by transfection with this cDNA.

Class I processing-defective Burkitt's lymphoma cells are recognized efficiently by CD4+ EBV-specific CTLs.

In the present study, we have isolated CD4+ CTLs that recognize an epitope from EBV nuclear Ag 2 in association with two different HLA-DQ Ags, DQA1*0501/DQB1*0201 (DQ2) or DQA1*0501/DQB1*0301 (DQ7). Both the HLA-DQ2 and HLA-DQ7 alleles displayed a similar efficiency in the endogenous and exogenous presentation of this epitope. Since earlier studies have shown that the EBV-associated malignancy, Burkitt's lymphoma (BL), escapes class I-restricted immune recognition by down-regulating the expression of peptide transporter genes, we have explored the possibility that these tumor cells can process class II-restricted CTL epitopes.

Human leukocyte antigen phenotype imposes complex constraints on the antigen-specific cytotoxic T lymphocyte repertoire.

The memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, is exceptionally restricted, being dominated by cytotoxic T lymphocytes (CTL) with a single, public T cell receptor (TCR). CTL clones that express this receptor fortuitously cross-react with the alloantigen HLA B44. However, of the two major subtypes of this HLA, B*4402 and B*4403, that differ by a single amino acid, only the former is recognized by these mature CTL clones.

Identification of type B-specific and cross-reactive cytotoxic T-lymphocyte responses to Epstein-Barr virus.

Persistent Epstein-Barr virus (EBV) infection is primarily controlled by HLA class I-restricted memory cytotoxic T-cell (CTL) responses which can be reactivated in vitro by stimulation of peripheral blood lymphocytes with autologous lymphoblastoid cell lines. During an investigation of a donor infected by both type A and type B EBV, CTL specific for type B EBV were isolated. The CTL were found to recognize an epitope encoded by the EBNA-6B gene.

Functional evidence for a colorectal cancer tumor suppressor gene at chromosome 8p22-23 by monochromosome transfer.

Chromosome 8p is considered, from loss of heterozygosity analysis, to be a strong candidate for the location of a tumor suppressor gene inactivated in colorectal cancer. We have found a 53% (27 of 51) rate of allelic loss at the LPL locus on 8p22, with the smallest region of overlap of deletions including the region D8S258 to D8S277. Using microcell-mediated monochromosome 8 transfer into three colorectal cancer cell lines, SW480, SW620 and HT29, we have demonstrated a reduction of tumorigenicity in SW620 hybrids.

Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis.

The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM).

Cytotoxic T-lymphocyte clones specific for an immunodominant epitope display discerning antagonistic response to naturally occurring Epstein-Barr virus variants.

In the present study we have identified Epstein-Barr virus isolates which encode variant sequences within an HLA B35-restricted immunodominant cytotoxic T-lymphocyte (CTL) epitope that act as natural antagonists and can inhibit CTL activity on the wild-type epitope. This effect can be demonstrated if the wild-type epitope is presented as a synthetic peptide or when processed from a full-length Epstein-Barr virus protein expressed by recombinant vaccinia constructs. However, this antagonistic effect was only selectively seen with some CTL clones, while a strong agonistic effect was evident for other clones in the presence of the same variant peptide.

Ataxia-telangiectasia: a multifaceted genetic disorder associated with defective signal transduction.

The gene responsible for the defect in the human genetic disorder ataxia-telangiectasia, ATM, was cloned recently. The part of the gene coding for a phosphatidylinositol 3-kinase domain showed it to be related to a family of genes involved in signal transduction, cell cycle control and the response to DNA damage. The elucidation of the role of the ATM gene product will provide valuable insight into the radiosensitivity, cancer predisposition, immunodeficiency and neuropathology that characterize this syndrome.

Peptide transporter (TAP-1 and TAP-2)-independent endogenous processing of Epstein-Barr virus (EBV) latent membrane protein 2A: implications for cytotoxic T-lymphocyte control of EBV-associated malignancies.

Major histocompatibility [correction of histocampatability] complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) recognizing Epstein-Barr virus (EBV) latent antigens play a pivotal role in restricting the proliferation of EBV-infected normal B cells. However, it is now well established that most of the EBV-associated malignancies escape this potent CTL response in vivo. This resistance to immune surveillance is not due to an obvious CTL dysfunction but has been partly attributed to the down-regulation of the peptide transporters, TAP-1 and TAP-2, thus restricting the endogenous loading of MHC class I molecules with peptides derived from viral nuclear antigens.

Identification of a naturally occurring recombinant Epstein-Barr virus isolate from New Guinea that encodes both type 1 and type 2 nuclear antigen sequences.

In this report we describe an Epstein-Barr virus isolate, derived from the peripheral blood lymphocytes of a healthy adult from Papua New Guinea, that is a recombinant of the two major Epstein-Barr virus types, encoding type 1 Epstein-Barr nuclear antigen 2 (EBNA2) sequences, and type 2 EBNA3, EBNA4, and EBNA6 sequences.

The effects of bistratene A on the development of Plasmodium falciparum in culture.

The effects of the marine ascidian compound bistratene A on in vitro cultures of Plasmodium falciparum were assessed. Concentrations from 0 to 1.5 micrograms ml(-1) of the compound were tested.

The ecology and pathology of Epstein-Barr virus.

Epstein-Barr virus achieves its ubiquitous and uniform epidemiological distribution by a dual strategy of latency to guarantee lifelong persistence and intermittent replication to guarantee transmission. These two functions appear to dictate residence in different cell types: latency in B lymphocytes and replication in epithelial cells. Both of these cell compartments are potential sites for EBV-associated malignancies.

Drug resistance and Giardia.

Giardiasis is a worldwide disease that can cause serious morbidity. Metronidozole is the current recommended drug for treatment, and is mostly still effective. However, Giardia duodenalis, the causative agent, is capable of developing resistance to high levels of metronidozole and other drugs, in vitro, via a number of mechanisms.