Biomedical subjects

Scott M Ward

Publications and source records attributed to Scott M Ward.

8 recordsLinked to original sources

PubMed2006

Mutationally altered signal output in the Nart (NarX-Tar) hybrid chemoreceptor.

Signal-transducing proteins that span the cytoplasmic membrane transmit information about the environment to the interior of the cell. In bacteria, these signal transducers include sensor kinases, which typically control gene expression via response regulators, and methyl-accepting chemoreceptor proteins, which control flagellar rotation via the CheA kinase and CheY response regulator. We previously reported that a chimeric protein (Nart) that joins the ligand-binding, transmembrane, and linker regions of the NarX sensor kinase to the signaling and adaptation domains of the Tar chemoreceptor elicits a repellent response to nitrate and nitrite. As with NarX, nitrate evokes a stronger response than nitrite. Here we show that mutations targeting a highly conserved sequence (the P box) in the periplasmic domain alter chemoreception by Nart and signaling by NarX similarly. In particular, the G51R substitution converts Nart from a repellent receptor into an attractant receptor for nitrate. Our results underscore the conclusion that the fundamental mechanism of transmembrane signaling is conserved between homodimeric sensor kinases and chemoreceptors. They also highlight the plasticity of the coupling between ligand binding and signal output in these systems.

Bacterial Proteins↗

PubMed2005

Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection.

Hepatitis C virus (HCV) becomes persistent in the majority of infected individuals. In doing so, the virus evades host adaptive immune responses, although the mechanisms responsible in this evasion are not clear. Several groups have demonstrated weak or absent HCV-specific CD4+ T cell responses during chronic HCV infection using proliferation assays and, more recently, class II tetramers. However, the functional status of HCV-specific CD4+ T cells in resolved and persistent infection is poorly understood. Using interferon gamma (IFN-gamma) and interleukin 2 (IL-2) enzyme-linked immunospot assays, we analyzed cytokine secretion patterns in chronically infected patients and compared them with those with resolved infection. In the spontaneous resolver group, strong IL-2 secretion in relation to IFN-gamma secretion was observed. However, in the persistently infected group, a consistent and significant loss of IL-2-secreting cells, compared with IFN-gamma-secreting cells, was identified. In vitro addition of IL-2 had a substantial effect in restoring CD4+ T cell activity. In conclusion, failure of IL-2 secretion, as opposed to physical deletion or complete functional unresponsiveness, appears to be an important determinant of the status of CD4+ T cell populations in chronic HCV infection. Loss of IL-2 secretory capacity may lead to disruption of IFN-gamma and proliferative function in vivo-a status that characterizes the cellular immune response in both CD4+ and CD8+ compartments in chronic disease.

Adult↗

PubMed2005

Nitrate-dependent activation of the Dif signaling pathway of Myxococcus xanthus mediated by a NarX-DifA interspecies chimera.

Myxococcus xanthus fibril exopolysaccharide (EPS), essential for the social gliding motility and development of this bacterium, is regulated by the Dif chemotaxis-like pathway. DifA, an MCP homolog, is proposed to mediate signal input to the Dif pathway. However, DifA lacks a prominent periplasmic domain, which in classical chemoreceptors is responsible for signal perception and for initiating transmembrane signaling. To investigate the signaling properties of DifA, we constructed a NarX-DifA (NafA) chimera from the sensory module of Escherichia coli NarX and the signaling module of M. xanthus DifA. We report here the first functional chimeric signal transducer constructed using genes from organisms in two different phylogenetic subdivisions. When expressed in M. xanthus, NafA restored fruiting body formation, EPS production, and S-motility to difA mutants in the presence of nitrate. Studies with various double mutants indicate that NafA requires the downstream Dif proteins to function. We propose that signal inputs to the Dif pathway and transmembrane signaling by DifA are essential for the regulation of EPS production in M. xanthus. Despite the apparent structural differences, DifA appears to share similar transmembrane signaling mechanisms with enteric sensor kinases and chemoreceptors.

Bacterial Proteins↗

PubMed2005

Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection.

The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8(+) T cells. The role of CD4(+)CD25(+) T regulatory (T(reg)) cells in priming and expanding virus-specific CD8(+) T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8(+) T-cell proliferation and gamma interferon (IFN-gamma) frequency were analyzed with/without depletion of T(reg) cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4(+)CD25(+) T(reg) cells inhibited anti-CD3/CD28 CD8(+) T-cell proliferation and perforin expression. Depletion of CD4(+)CD25(+) T(reg) cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-gamma-expressing CD8(+) T cells. Although stimulated CD8(+) T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4(+)CD25(+) regulatory T cells on CD8(+) T-cell proliferation. In conclusion, marked CD4(+)CD25(+) regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8(+) T-cell responses and viral persistence.

CD4 Antigens↗

PubMed2004

Virus-specific CD8+ T lymphocytes within the normal human liver.

The frequency and phenotype of human antiviral memory CD8(+) T cells in blood are well studied, yet little is known about their distribution within tissues. Analysis of antiviral CD8(+) T cell populations derived from a unique set of normal liver and blood samples identified a consistent population of virus-specific cells within the liver. In comparison to the circulating T cells, the liver-derived T cells were present at frequencies which were variably enriched compared to that in the blood, and showed significant differences with regard to the expression of CD45RA, CD45RO, CD95, CCR7, CD27 and CD28. The differences in these cell surface markers are consistent with a mature 'effector memory' phenotype of antigen-specific CD8(+) T cells within the liver. An enrichment of an activated subset of NKT cells (V alpha 24/V beta 11) was also observed, a finding which may be relevant to the regulation of the antiviral populations.

Animals↗

PubMed2004

Ultra-sensitive class I tetramer analysis reveals previously undetectable populations of antiviral CD8+ T cells.

A major breakthrough in cellular immunology has been the development of HLA class I tetramers to analyze CD8(+) T cell responses. However, in many situations, including persistent virus infection, specific T cell responses are rarely detected using this technology. This raises the question of whether such responses are 'deleted' (or 'exhausted') or present below the conventional detection limit for class I tetramer staining. In particular, persistent hepatitis C virus (HCV) infection is characterized by very weak or apparently absent specific CD8(+) T cell responses, even though they are readily detectable in acute disease. Therefore, we assessed the use of anti-PE-labeled magnetic beads to enrich tetramer-positive HCV-specific T cells and identify previously undetectable populations. Using the enrichment technique, HCV-specific T cells could be detected in the majority of infected individuals, whereas these responses were not detected using conventional tetramer staining (8/15 vs. 1/15; p=0.01). Magnetic enrichment could reliably detect very rare HCV-specific responses at frequencies of >0.0011% of CD8(+) T cells (approximately 1/million PBMC), and phenotypic analysis of these rare populations was possible. Therefore, this direct ex vivo technique revealed the persistence of very low frequencies of virus-specific CD8(+) T cells during chronic virus infection and is readily transferable to the study of other viral, self- or tumor-specific T cells.

Antigens, Neoplasm↗

PubMed2003

Generation of CTL responses using Kunjin replicon RNA.

The Kunjin replicon was used to express a polytope that consisted of seven hepatitis C virus cytotoxic T lymphocyte epitopes and one influenza cytotoxic T lymphocyte epitope for vaccination studies. The self-replicating nature of, and expression from, the ribonucleic acid was confirmed in vitro. Initial vaccinations with one dose of Kun-Poly ribonucleic acid showed that an influenza-specific cytotoxic T lymphocyte response was elicited more efficiently by intradermal inoculation compared with intramuscular delivery. Two micrograms of ribonucleic acid delivered in the ear pinnae of mice was sufficient to elicit a detectable cytotoxic T lymphocyte response 10 days post-vaccination. Further vaccination studies showed that four of the seven hepatitis C virus cytotoxic T lymphocyte epitopes were able to elicit weak cytotoxic T lymphocyte responses whereas the influenza epitope was able to elicit strong, specific cytotoxic T lymphocyte responses following three doses of Kun-Poly ribonucleic acid. These studies vindicate the use of the Kunjin replicon as a vector to deliver encoded proteins for the development of cell-mediated immune responses.

Animals↗

PubMed2002

A NarX-Tar chimera mediates repellent chemotaxis to nitrate and nitrite.

Membrane receptors communicate between the external world and the cell interior. In bacteria, these receptors include the transmembrane sensor kinases, which control gene expression via their cognate response regulators, and chemoreceptors, which control the direction of flagellar rotation via the CheA kinase and CheY response regulator. Here, we show that a chimeric protein that joins the ligand-binding, transmembrane and linker domains of the NarX sensor kinase to the signalling and adaptation domains of the Tar chemoreceptor of Escherichia coli mediates repellent responses to nitrate and nitrite. Nitrate induces a stronger response than nitrite and is effective at lower concentrations, mirroring the relative sensitivity to these ligands exhibited by NarX itself. We conclude that the NarX-Tar hybrid functions as a bona fide chemoreceptor whose activity can be predicted from its component parts. This observation implies that ligand-dependent activation of a sensor kinase and repellent-initiated activation of receptor-coupled CheA kinase involve a similar transmembrane signal.

Bacterial Proteins↗