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Biomedical subjects

Margaret M Harnett

Publications and source records attributed to Margaret M Harnett.

At least 19 recordsLinked to original sources
PubMed2006

Differential polarization of immune responses by plant 2S seed albumins, Ber e 1, and SFA8.

The plant 2S seed albumins Ber e 1 and SFA8, although structurally very similar, vary with respect to their allergenic properties. Whereas the former represents a major allergen, the latter appears to promote only weak allergenic responses. The aim of this investigation was to determine whether the allergenic properties of Ber e 1 and SFA8 reflected differential polarization of dendritic cell (DC) and Th cell responses. We thus investigated the effect of recombinant forms of both allergens on DC and Th cell responses as indicated by cell surface phenotype and cytokine production. Exposure of murine DCs to SFA8, but not Ber e 1, resulted in production of the cytokines IL-12 p40 and TNF-alpha by a mechanism independent of recognition by TLRs. Furthermore, depending on the mouse strain used, increased expression of MHC class II and costimulatory molecules such as CD40, CD80, and CD86 was associated with exposure to SFA8, but not Ber e 1. In coculture experiments using the DO11.10 transgenic T cell that recognizes OVA peptide, DCs exposed to both allergens induced T cells to produce IFN-gamma, but only Ber e 1 could induce significant production of IL-4 and IL-5. Likewise, analysis of transcription factors shows increased T-bet with respect to both allergens, but also GATA-3 with respect to Ber e 1. Overall, our data are consistent with the idea that the ability of Ber e 1, but not SFA8, to act as a potent allergen may reflect differences in their ability to induce IL-12 production.

2S Albumins, Plant↗
PubMed2006

What causes lymphocyte hyporesponsiveness during filarial nematode infection?

Filarial nematodes persist in the parasitized host by modulating immune responsiveness. A feature of this that has been observed in a multitude of studies dating back several decades is an inability of lymphocytes to respond appropriately to filarial nematode antigens and, in some cases, to other stimuli. The consistency of this observation, allied to the ease of measurement of lymphocyte hyporesponsiveness, has resulted in many attempts to understand its cause.

Animals↗
PubMed2005

ES-62, an immunomodulator secreted by filarial nematodes, suppresses clonal expansion and modifies effector function of heterologous antigen-specific T cells in vivo.

ES-62 is a phosphorylcholine-containing glycoprotein secreted by filarial nematodes, which has previously been shown to possess a range of immunomodulatory capabilities. We now show, using a CD4+ transgenic TCR T cell adoptive transfer system, that ES-62 can modulate heterologous Ag (OVA)-specific responses in vivo. Thus, in contrast to the mixed IgG1-IgG2a response observed in control animals, ES-62-treated mice exhibited a Th2-biased IgG Ab response as evidenced by stable enhancement of anti-OVA IgG1 production and a profound inhibition of anti-OVA IgG2a. Consistent with this, Ag-specific IFN-gamma produced was suppressed by pre-exposure to ES-62 when T cells were rechallenged ex vivo. However, the response observed was not classical Th2, because although Ag-specific IL-5 production was enhanced by pre-exposure to ES-62, IL-13, and IL-4 were inhibited when T cells were rechallenged ex vivo. Moreover, such T cells produced lower levels of IL-2 and proliferated less upon Ag rechallenge ex vivo. Finally, pre-exposure to ES-62 inhibited the clonal expansion of the transferred Ag-specific CD4+ T cells and altered the functional response of such T cells in vivo, by modulating the kinetics and reducing the extent of their migration into B cell follicles.

Animals↗
PubMed2005

Immunomodulation via novel use of TLR4 by the filarial nematode phosphorylcholine-containing secreted product, ES-62.

Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-alpha, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-gamma. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-alpha production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.

Acute-Phase Proteins↗
PubMed2005

Subversion of immunological signalling by a filarial nematode phosphorylcholine-containing secreted product.

Modulation of immune responses is an important strategy employed by pathogens to enable their survival in host organisms. Secreted immunomodulatory molecules are key weapons in the pathogen's battle with the host immune system. In this review, we will discuss the immunomodulatory effects of the phosphorylcholine-containing filarial nematode glycoprotein, ES-62, on the host immune system and summarise the results of our studies to identify the intracellular signalling pathways targeted by ES-62 to achieve these effects.

Animals↗
PubMed2005

Direct quantitation of T cell signaling by laser scanning cytometry.

Current flow cytometric technology allows quantitative assessment of surface and intracellularly expressed molecules on isolated cells. However, the need to disrupt tissues prevents correlation of phenotypic expression with anatomical location. In contrast, immunohistochemistry in conjunction with conventional or confocal microscopy allows localisation of staining, but little in the way of quantitation. The laser scanning cytometer (LSC) allows a combination of both approaches, as it can apply quantitative flow cytometric laser technology to intact tissue. The purpose of this protocol is to describe in vitro and ex vivo methods for quantifying cell signaling molecule expression and activation within antigen-specific T cells by laser scanning cytometry.

Animals↗
PubMed2005

Signalling mechanisms underlying subversion of the immune response by the filarial nematode secreted product ES-62.

Secretion of immunomodulatory molecules is a key strategy employed by pathogens to enable their survival in host organisms. For example, arthropod-transmitted filarial nematodes, which achieve longevity within the infected host by suppressing and modulating the host immune response, produce excretory-secretory (ES) products that have been demonstrated to possess immunomodulatory properties. In this review we discuss the immunomodulatory effects of the phosphorylcholine-containing filarial nematode-secreted glycoprotein ES-62 and describe the intracellular signal transduction pathways it targets to achieve these effects.

Animals↗
PubMed2004

Differential signalling during B-cell maturation.

The molecular mechanism by which the antigen receptors (BCR) on B cells can elicit differential maturation state-specific responses is one of the central problems in B-cell differentiation yet to be resolved. Indeed, many of the early signalling events detected following BCR ligation, such as activation of protein tyrosine kinases (PTK), phospholipase C (PLC), phosphoinositide-3-kinase (PI 3K), protein kinase C (PKC) and the RasMAPK (mitogen activating protein kinase) signalling cascades are observed throughout B-cell maturation. However, it is becoming clear that the differential functional responses of these BCR-coupled signals observed during B-cell maturation are dependent on a number of parameters including signal strength and duration, subcellular localisation of the signal, maturation-restricted expression of downstream signalling effector elements/isoforms and modulation of signal by co-receptors. Thus, the combined signature of BCR signalling is likely to dictate the functional response and act as a developmental checkpoint for B-cell maturation.

Animals↗
PubMed2004

Differences in the kinetics, amplitude, and localization of ERK activation in anergy and priming revealed at the level of individual primary T cells by laser scanning cytometry.

One of the potential mechanisms of peripheral tolerance is the unresponsiveness of T cells to secondary antigenic stimulation as a result of the induction of anergy. It has been widely reported that antigenic unresponsiveness may be due to uncoupling of MAPK signal transduction pathways. However, such signaling defects in anergic T cell populations have been mainly identified using immortalized T cell lines or T cell clones, which do not truly represent primary Ag-specific T cells. We have therefore attempted to quantify signaling events in murine primary Ag-specific T cells on an individual cell basis, using laser-scanning cytometry. We show that there are marked differences in the amplitude and cellular localization of phosphorylated ERK p42/p44 (ERK1/2) signals when naive, primed and anergic T cells are challenged with peptide-pulsed dendritic cells. Primed T cells display more rapid kinetics of phosphorylation and activation of ERK than naive T cells, whereas anergic T cells display a reduced ability to activate ERK1/2 upon challenge. In addition, the low levels of pERK found in anergic T cells are distributed diffusely throughout the cell, whereas in primed T cells, pERK appears to be targeted to the same regions of the cell as the TCR. These data suggest that the different consequences of Ag recognition by T cells are associated with distinctive kinetics, amplitude, and localization of MAPK signaling.

Animals↗
PubMed2004

ES-62, a filarial nematode-derived immunomodulator with anti-inflammatory potential.

Arthropod-transmitted filarial nematodes can survive for in excess of a decade via modulation of the vertebrate host immune system. Although human infection can result in very severe pathology, most infected individuals show remarkably little evidence of this. Analysis of the anti-nematode response indicates that apparently pathology-free individuals have an anti-inflammatory immunological phenotype and it has been suggested that this favours maintenance of host good health. It is considered that parasite-derived molecular secretions contribute to the anti-inflammatory phenotype and we have thus investigated the properties of a filarial nematode glycoprotein secreted in some abundance, ES-62. This molecule shows a plethora of immunomodulatory activities that can be classified as anti-inflammatory. It has been observed in a number of studies that several autoimmune disorders including rheumatoid arthritis (RA) exhibit reduced incidence and severity in geographic regions in which filarial nematodes are transmitted to humans. Furthermore, it has been speculated that these two observations are linked although molecular explanations for such an association have not been forthcoming. Although the aetiology of RA remains unknown a majority of data are consistent with it being mediated via excess pro-inflammatory cytokine production. Given that ES-62 is anti-inflammatory, we hypothesised that it might be able to counter the pathology associated with diseases like RA. Indeed, we found that exposure to ES-62 prevented initiation of collagen-induced arthritis (CIA) in a murine model and also suppressed progression of established disease. Ex vivo analyses demonstrated that these effects correlated with inhibition of TNF-alpha production and inhibition of collagen-specific TH-1 responses. The nematode product was also able to suppress pro-inflammatory cytokine release in vitro in synovial cells derived from RA patients. ES-62 thus constitutes a pathogen-derived immunomodulator with significant therapeutic potential.

Animals↗
PubMed2004

CD40: a growing cytoplasmic tale.

CD40, a member of the tumor necrosis factor (TNF) receptor family that is expressed on B cells, monocytes, dendritic cells, endothelial cells, and epithelial cells, as well as on B cell lymphomas and carcinomas, activates multiple signaling pathways. In B cells, the response to CD40 is complex and depends on the maturation status of the cell. It is well established that CD40 can promote cell survival through up-regulation of the expression of genes encoding antiapoptotic proteins. However, a new role for CD40 signaling is being recognized in promoting progression through the cell cycle. The roles of the phosphoinositide 3-kinase, mitogen-activated protein kinase, and nuclear factor kappaB pathways in mediating CD40 stimulation of the cell cycle are described.

Animals↗
PubMed2004

FcgammaRIIb-mediated negative regulation of BCR signalling is associated with the recruitment of the MAPkinase-phosphatase, Pac-1, and the 3'-inositol phosphatase, PTEN.

The low-affinity receptor for IgG, FcgammaRIIb, negatively regulates B cell antigen receptor (BCR)-mediated proliferative signalling. FcgammaRIIb has been reported to mediate this inhibition by uncoupling the BCR from the RasMAPkinase pathway. We now show that FcgammaRIIb-mediated negative feedback inhibition also correlates with induction of an Erk-associated phosphatase activity that reflects the rapid association of Erk and the MAPkinase phosphatase, Pac-1, and dephosphorylation and inactivation of ErkMAPkinase. This mechanism of abrogating ongoing ErkMAPkinase signalling therefore provides a rationale for rapid immune-complex-mediated feedback inhibition of active antigen-driven B cell responses. In addition, FcgammaRIIb signalling also induces the recruitment and activation of the 3'-inositol phosphatase, PTEN, which by antagonising PI 3kinase activity and inhibiting BCR-coupling to the anti-apoptotic kinase, Akt, provides an additional mechanism for FcgammaRIIb-mediated negative regulation of BCR-coupling to ErkMAPkinase, cell survival and proliferation.

Animals↗
PubMed2004

In vivo exposure of murine dendritic cell and macrophage bone marrow progenitors to the phosphorylcholine-containing filarial nematode glycoprotein ES-62 polarizes their differentiation to an anti-inflammatory phenotype.

We have previously shown in an in vitro study that the filarial nematode phosphorylcholine (PC)-containing glycoprotein ES-62 promotes a murine dendritic cell (DC) phenotype that induces T helper type 2 (Th2) responses. We now show that, in addition to directly priming Th2 responses, ES-62 can act to dampen down the pro-inflammatory DC responses elicited by lipopolysaccharide. Furthermore, we also demonstrate that murine DCs and macrophages derived ex vivo from bone marrow cells exposed in vivo to ES-62 by release from osmotic pumps are hyporesponsive to subsequent stimulation with lipopolysaccharide. These effects can be largely mimicked by exposure to the PC moiety of ES-62 conjugated to an irrelevant protein. The data we provide are, as far as we aware, the first to show that a defined pathogen product can modulate the developmental pathway of bone marrow cells of the immune system in vivo. Such a finding could have important implications for the use of pathogen products or their derivatives for immunotherapy.

Animals↗
PubMed2004

The anti-inflammatory potential of the filarial nematode secreted product, ES-62.

Filarial nematodes achieve long-term infection via modulation of the host immune system. Although human infection can result in severe pathology, the majority of infected individuals exhibit little evidence of this. Analysis of the immune response during infection indicates that the apparently healthy majority have an anti-inflammatory phenotype and it has been speculated that this may contribute to maintenance of host health. Recent data suggest that parasite-derived molecular secretions contribute to the anti-inflammatory phenotype and we have thus characterised a major filarial nematode secreted glycoprotein, ES-62. This molecule has been found to possess broad immunomodulatory activities that are in general, anti-inflammatory. It has long been recognised that several autoimmune disorders including rheumatoid arthritis (RA) exhibit reduced incidence and severity in geographic regions in which filarial nematodes are endemic. Furthermore, it has been speculated that these two observations are causally linked. However, molecular explanations for such an association have not been forthcoming. Although the aetiology of RA is unknown most data suggest that it is mediated via a pro-inflammatory immune response associated with excess cytokine production. Given that ES-62 is anti-inflammatory, we hypothesised that it might possess activity against diseases like RA. Indeed we found that subcutaneous injection of ES-62 prevented initiation of collagen-induced arthritis (CIA) and also suppressed progression of established disease. Ex vivo analyses demonstrated that these effects were due to inhibition of TNF-alpha production and reversal of collagen specific TH-1 responses. The nematode product was also found to inhibit pro-inflammatory cytokine release in vitro in synovial cells derived from RA patients. ES-62 thus represents a parasite-derived immunomodulator with significant therapeutic potential.

Anti-Inflammatory Agents↗
PubMed2003

Bcl-(xL) antagonism of BCR-coupled mitochondrial phospholipase A(2) signaling correlates with protection from apoptosis in WEHI-231 B cells.

Crosslinking of the antigen receptors on the immature B-cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. Commitment to such B-cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A2 activation, disruption of mitochondrial function, and cathepsin B activation. CD40 signaling has been reported to rescue WEHI-231 B cells from BCR-driven apoptosis primarily via up-regulation of the antiapoptotic protein Bcl-xL. Coupling of the BCR to the mitochondrial phospholipase A2-dependent apoptotic pathway can be prevented by rescue signals via CD40. We now show that overexpression of Bcl-xL can prevent mitochondrial phospholipase A2 activation, disruption of mitochondrial potential, and postmitochondrial execution of BCR-mediated apoptosis via cathepsin B activation. Moreover, overexpression of Bcl-xL protects WEHI-231 B cells from mitochondrial disruption and apoptosis resulting from culture with exogenous arachidonic acid, the product of phospholipase A2 action, suggesting that Bcl-xL may act to antagonize arachidonic acid-mediated disruption of mitochondrial integrity. However, although Bcl-xL expression can mimic CD40-mediated rescue of BCR-driven apoptosis, it cannot substitute for CD40 signaling in the reversal of BCR-mediated growth arrest of WEHI-231 B cells. Rather, CD40 signaling additionally induces conversion of arachidonic acid to prostaglandin E2 (PGE2), which promotes WEHI-231 B-cell proliferation by restoring the sustained, cycling extracellular signal-regulated/mitogen-activated protein kinase (ErkMAPkinase) signaling required for cell cycle progression.

Animals↗
PubMed2003

A novel MyD-1 (SIRP-1alpha) signaling pathway that inhibits LPS-induced TNFalpha production by monocytes.

MyD-1 (CD172) is a member of the family of signal regulatory phosphatase (SIRP) binding proteins, which is expressed on human CD14+ monocytes and dendritic cells. We now show a novel role for MyD-1 in the regulation of the innate immune system by pathogen products such as lipopolysaccharide (LPS), purified protein derivative (PPD), and Zymosan. Specifically, we demonstrate that ligation of MyD-1 on peripheral blood mononuclear cells (PBMCs) inhibits tumor necrosis factor alpha (TNFalpha) secretion but has no effect on other cytokines induced in response to each of these products. In an attempt to understand the molecular mechanisms underlying this surprisingly selective effect we investigated signal transduction pathways coupled to MyD-1. Ligation of the SIRP was found to recruit the tyrosine phosphatase SHP-2 and promote sequential activation of phosphatidylinositol (PI) 3-kinase, phospholipase D, and sphingosine kinase. Inhibition of LPS-induced TNFalpha secretion by MyD-1 appears to be mediated by this pathway, as the PI 3-kinase inhibitor wortmannin restores normal LPS-driven TNFalpha secretion. MyD-1-coupling to this PI 3-kinase-dependent signaling pathway may therefore present a novel target for the development of therapeutic strategies for combating TNFalpha production and consequent inflammatory disease.

Antibodies, Monoclonal↗
PubMed2003

19 A solution structure of the filarial nematode immunomodulatory protein, ES-62.

ES-62, a protein secreted by filarial nematodes, parasites of vertebrates including humans, has an unusual posttranslational covalent addition of phosphorylcholine to an N-type glycan. Studies on ES-62 from the rodent parasite Acanthocheilonema viteae ascribe it a dominant role in ensuring parasite survival by modulating the host immune system. Understanding this immunomodulation at the molecular level awaits full elucidation but distinct components of ES-62 may participate: the protein contributes aminopeptidase-like activity whereas the phosphorylcholine is thought to act as a signal transducer. We have used biophysical and bioinformatics-based structure prediction methods to define a low-resolution model of ES-62. Sedimentation equilibrium showed that ES-62 is a tightly bound tetramer. The sedimentation coefficient is consistent with this oligomer and the overall molecular shape revealed by small angle x-ray scattering. A 19 A model for ES-62 was restored from the small-angle x-ray scattering data using the program DAMMIN which uses simulated annealing to find a configuration of densely packed scattering elements consistent with the experimental scattering curve. Analysis of the primary sequence with the position-specific iterated basic local alignment search tool, PSI-BLAST, identified six closely homologous proteins, five of which are peptidases, consistent with observed aminopeptidase activity in ES-62. Differences between the secondary structure content of ES-62 predicted using the consensus output from the secondary structure prediction server JPRED and measured using circular dichroism are discussed in relation to multimeric glycosylated proteins. This study represents the first attempt to understand the multifunctional properties of this important parasite-derived molecule by studying its structure.

Amino Acid Sequence↗