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D I Pritchard

Publications and source records attributed to D I Pritchard.

At least 37 records · Page 2Linked to original sources
PubMed1999

A hookworm allergen which strongly resembles calreticulin.

Immmoglobulin E-rich plasma from patients from Papua New Guinea infected with Necator americanus has been used to probe an adult N. americanus cDNA library for the presence of hookworm allergens. Using this approach, one hookworm allergen has been identified as calreticulin, which was subsequently expressed in Escherichia coli. Little serological cross reactivity was seen between the recombinant calreticulins of this hookworm and its host. Prospective roles for hookworm calreticulin in the host-parasite relationship are discussed in depth.

Allergens↗
PubMed1999

Caenorhabditis elegans as a biomonitor for immunological stress in nematodes.

An experimental system has been developed using Caenorhabditis elegans (Secernentea: Rhabditida), to monitor immunological stress in nematodes. The transgenic C. elegans strain PC72 carries a lacZ reporter gene fused to a C. elegans hsp16-1 gene, which is inducible for beta-galactosidase activity at the heat stress temperature of 26 degrees C. The investigate the possibility of using PC72 to monitor immunological stress, its surface coat was targeted, to mimic immune attack, by raising immune sera against surface coat components selectively removed by the cationic detergent cetyltrimethylammoniunm bromide. Initially, a highly significant induction of beta-galactosidase activity was seen in PC72 incubated in either surface-reactive or naïve rabbit serum. Complement (C3) was detected over the entire surface of adult PC72 and was thought to be responsible for stress-induction with naïve sera. When the immunoglobulin (Ig)G fraction of naïve sera was used in isolation, no stress-induction was seen. In contrast, a two-fold increase in beta-galactosidase activity was seen in the presence of surface-reactive IgG (SR-IgG) which recognised surface components of between 6 and 40 kDa in western blot. The belief that surface reactive IgG could induce a stress response was reinforced by analysis of hsp-16 protein expression. Cationised ferritin was then used to assess whether stress-induction was truly a surface reactive event; binding of cationised ferritin to the nematode surface also resulted in two-fold induction of beta-galactosidase activity. To investigate the downstream biological effects of stress induction, worm growth and fecundity were measured in the presence of IgG preparations. A significant reduction was seen in both worm length and fecundity only when larvae were incubated in surface-reactive IgG, compared to both naïve IgG and K-medium controls. In conclusion, it would appear that C. elegans is a suitable model to monitor the induction of immunological stress at the level of the nematode surface coat. Given the ability of nematode surface antigens to protect the vaccinated host in animal model systems, and the close phylogenetic relationships which exist between C. elegans and nematodes of medical and veterinary importance, it is conceivable that the immunological targets in or on the surface of C. elegans warrant rapid identification.

Animals↗
PubMed1999

Cu/Zn superoxide dismutase in excretory-secretory products of the human hookworm Necator americanus. An electron paramagnetic spectrometry study.

EPR spectrometry was used to investigate the effect of excretory/secretory product from Necator americanus on superoxide radical anions generated by xanthine/xanthine oxidase as a measure of excretory/secretory product superoxide dismutase activity. Using 1,1',5,5'-dimethylpyrollidine-N-oxide (DMPO) as a superoxide spin-trapping agent a 12-line EPR spectrum characteristic of the DMPO-OOH adduct was observed to decrease in the presence of excretory/secretory product. Superoxide dismutase activity was proportional to excretory/secretory protein concentration, was inhibited with cyanide treatment and was progressively destroyed with increasing time of heat denaturation of excretory/secretory product. Using a purpose-built chamber the superoxide dismutase activity of excretory/secretory product from live worms in culture was shown to accumulate with time to a maximum at 4 h. The electron paramagnetic resonance spectrum obtained for the frozen excretory/secretory product of N. americanus recorded at 77 K is typical of Cu(II) in a protein matrix. The results are consistent with the presence of an active Cu/Zn superoxide dismutase in excretory/secretory product from N. americanus and demonstrate a method for the unequivocal determination of the fate of superoxide anions in the presence of live worms.

Animals↗
PubMed1999

Sequence diversity of TT virus in geographically dispersed human populations.

TT virus (TTV) is a newly discovered DNA virus originally classified as a member of the Parvoviridae. TTV is transmitted by blood transfusion where it has been reported to be associated with mild post-transfusion hepatitis. TTV can cause persistent infection, and is widely distributed geographically; we recently reported extremely high prevalences of viraemia in individuals living in tropical countries (e.g. 74% in Papua New Guinea, 83% in Gambia; Prescott & Simmonds, New England Journal of Medicine 339, 776, 1998). In the current study we have compared nucleotide sequences from the N22 region of TTV (222 bases) detected in eight widely dispersed human populations. Some variants of TTV, previously classified as genotypes 1a, 1b and 2, were widely distributed throughout the world, while others, such as a novel subtype of type 1 in Papua New Guinea, were confined to a single geographical area. Five of the 122 sequences obtained in this study (from Gambia, Nigeria, Papua New Guinea, Brazil and Ecuador) could not be classified as types 1, 2 or 3, with the variant from Brazil displaying only 46-50% nucleotide (32-35% amino acid) sequence similarity to other variants. This study provides an indication of the extreme sequence diversity of TTV, a characteristic which is untypical of parvoviruses.

Africa, Western↗
PubMed1999

Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration.

The infective larvae of Necator americanus were shown to secrete all mechanistic classes of proteolytic enzymes with two overall pH optima of 6.5 and 8.5 using fluorescein isothiocyanate-labeled casein as the substrate. Since infective larvae are obligate skin penetrators, the effect of each of these enzyme classes against macromolecules derived from human skin was examined. Larval secretions were shown to degrade collagen types I, III, IV, and V, fibronectin, laminin, and elastin. All the skin macromolecules tested were hydrolyzed by aspartyl proteinase activity, which was inhibitable by pepstatin A. Collagen and elastin was also hydrolyzed by metalloproteinase activity, while the serine proteinase activity hydrolyzed only elastin. As a consequence of these experiments, the effect of proteinase inhibitors on the penetration of live larvae through hamster skin was tested. Larval penetration was significantly inhibited only by pepstatin A, confirming the importance of the aspartyl proteinase activity during the skin penetration process.

Animals↗
PubMed1998

Enzymatically active papain preferentially induces an allergic response in mice.

Human exposure to papain, a cysteine proteinase, is associated with hypersensitivity reactions. We demonstrate in mice that enzymatically active papain preferentially induces an IgG1 response and results in mast cell degranulation, both features typical of an allergic reaction. Inactive papain, blocked with E-64, appears to desensitize mice to subsequent challenge by active enzyme. These results suggest that the enzymatic activity of papain is critical in inducing an allergic response and that the use of inactive allergens may be a possible strategy for desensitizing allergic individuals.

Animals↗
PubMed1998

Heligmosomoides polygyrus immunomodulatory factor (IMF), targets T-lymphocytes.

Experiments were performed to investigate the immunological site of action of an immunomodulatory factor (IMF), isolated from the gastrointestinal nematode Heligmosomoides polygyrus. IMF inhibited antibody production in murine and human 'T-helper (Th-2) driven' immunoassays. The effects were mediated via T lymphocytes as T cell-depleted cultures failed to respond to IMF, a result confirmed by prepulsing discrete cell subsets with the immunomodulant. Although the molecular nature and mode of action of IMF has yet to be determined, it would appear to be a relatively small non-proteinaceous molecule. From this data, we suggest that H. polygyrus secretes a systemically-active IMF from the intestinal lumen, to down-regulate Th-2 cell development in order to promote its survival in a potentially immunologically hostile environment.

Adjuvants, Immunologic↗
PubMed1998

Mite allergens: significance of enzymatic activity.

Ten years ago, the cloning and sequencing of a cDNA encoding the group I allergen of house-dust mites unequivocally determined that protein allergens may have biochemical functions in addition to their ability to bind IgE. Since this discovery, several groups have speculated that the biochemical activities of allergens, or substances associated with allergens, may be involved in their immunogenicity or allergenicity. This paper will focus on just one biochemical function, proteolytic activity, and will be illustrated by examples of our own work that we believe support the hypothesis that this category of molecules are endowed with the properties of proallergic adjuvants.

Allergens↗
PubMed1998

The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone has immunomodulatory activity.

Diverse gram-negative bacterial cells communicate with each other by using diffusible N-acyl homoserine lactone (AHL) signal molecules to coordinate gene expression with cell population density. Accumulation of AHLs above a threshold concentration renders the population "quorate," and the appropriate target gene is activated. In pathogenic bacteria, such as Pseudomonas aeruginosa, AHL-mediated quorum sensing is involved in the regulation of multiple virulence determinants. We therefore sought to determine whether the immune system is capable of responding to these bacterial signal molecules. Consequently the immunomodulatory properties of the AHLs N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) were evaluated in murine and human leukocyte immunoassays in vitro. OdDHL, but not OHHL, inhibited lymphocyte proliferation and tumor necrosis factor alpha production by lipopolysaccharide-stimulated macrophages. Furthermore, OdDHL simultaneously and potently down-regulated the production of IL-12, a Th-1-supportive cytokine. At high concentrations (>7 x 10(-5) M) OdDHL inhibited antibody production by keyhole limpet hemocyanin-stimulated spleen cells, but at lower concentrations (<7 x 10(-5) M), antibody production was stimulated, apparently by increasing the proportion of the immunoglobulin G1 (IgG1) isotype. OdDHL also promoted IgE production by interleukin-4-stimulated human peripheral blood mononuclear cells. These data indicate that OdDHL may influence the Th-1-Th-2 balance in the infected host and suggest that, in addition to regulating the expression of virulence determinants, OdDHL may contribute to the pathogenesis of P. aeruginosa infections by functioning as a virulence determinant per se.

4-Butyrolactone↗
PubMed1997

The relationship between immunological responsiveness controlled by T-helper 2 lymphocytes and infections with parasitic helminths.

It should have been difficult until relatively recently for immunologists to ascribe a sound biological reason for the continued possession of the allergic phenotype in human populations. Nevertheless, for the past 20 years or so textbooks of immunology have routinely exhibited fanciful and perhaps exaggerated diagrams as to how IgE and eosinophils killed all helminth parasites. These diagrams were largely based on perhaps selective in vitro observations, and it is only now that immunoparasitologists, working on human populations under arduous conditions in the field, are able to provide data to corroborate these findings, and perhaps ascribe a useful purpose for a generally pathological immune response termed Type I hypersensitivity. The present paper reviews much of this recent literature, and asks a number of pertinent questions relating to the relationship between what we now know to be T-helper 2 lymphocyte-driven immunological responsiveness and infections with parasitic helminths.

Allergens↗
PubMed1997

Heterogeneous proteolytic specificity and activity of the house dust mite proteinase allergen Der p I.

BACKGROUND: Exposure of the skin or respiratory tract to proteinases is frequently associated with allergic sensitization. This is of particular significance in the domestic indoor environment where the proteolytic activity of Der p I, the group I allergen of the house dust mite Dermatophagoides pteronyssinus, may influence the allergenicity of mites. Using class-specific proteinase inhibitors and active-site affinity chromatography, we have previously shown that Der p I exhibits a mixed cysteine-serine proteinase activity. Measurement of the amount of cleavage, however, did not determine whether the inhibitors used were targeting exactly the same proteolytic mechanism. OBJECTIVE: To resolve this issue, we have examined whether the cleavage specificity of the cysteine and serine proteinase activities of Der p I was the same. METHODS: HPLC and mass spectrometry were used to analyse and identify the products of a Der p I-digested peptide substrate and thus identify the peptide bonds cleaved. RESULTS: Der p I cleaves different peptide bonds, depending upon the class of proteolytic mechanism used. In the model peptide substrate insulin B chain, the cysteine and serine proteinase activities of Der p I showed preference for glutamic acid and arginine respectively in the P1 position. CONCLUSION: These data suggest the existence of more than one mechanistic form of the allergen immunologically identified as Der p I. If proteolytic activity is indeed a function of allergenicity, this information may have important implications for the pathogenicity of Der p I and the ability of innate antiproteinase defences in the respiratory tract to prevent immune sensitization.

Allergens↗
PubMed1997

The pro-allergic influences of helminth parasites.

Parasitic infection is highly allergenic, and the present paper illustrates how parasites might disrupt the regulation of IgE synthesis, resulting in heightened Th-2 responses. The study of parasites, and dysregulation of the IgE network, could in turn provide information relating to the aetiology of allergic diseases such as asthma and atopic dermatitis.

Animals↗
PubMed1996

The anti-haemostatic strategies of the human hookworm Necator americanus.

The human hookworm Necator americanus appears to have evolved a number of complementary strategies to overcome the host's haemostatic processes. These include the inhibition of blood coagulation, platelet aggregation and mediator release, and the secretion of fibrinogenolytic enzymes. These strategies presumably allow the parasite to establish the chronic infections so often documented in human populations.

Animals↗
PubMed1996

Peripheral blood based T cell-containing and T cell-depleted culture systems for human IgE synthesis: the role of T cells.

BACKGROUND: Comparable T cell-containing and T cell-depleted culture systems for human IgE synthesis are currently not available. OBJECTIVE: This has prompted us to develop peripheral blood mononuclear cell (PBMC) based culture systems for human IgE synthesis in the presence and absence of T cells. METHODS: In this paper we describe simplified conditions for in vitro synthesis of high levels of IgE by human peripheral blood B cells, both in T cell-containing cultures and in anti-CD40 stimulated T cell-depleted cultures. RESULTS: T cell-depleted cultures released approximately 20 times more IgE [range 410-2220 ng/mliter (mean 1270 ng/mliter); based on six experiments using cells from three donors] than did T cell-containing cultures [range 23-105 ng/mliter (mean 58 ng/mliter); based on 15 experiments using cells from three donors]. Reconstitution experiments were performed to investigate the role of T cells on IgE synthesis. Adding T cells back to the anti-CD40 stimulated T cell-depleted cultures resulted in a dose-dependent inhibition of IgE production. In the absence of anti-CD40 low numbers of T cells stimulated, while high numbers suppressed, IgE production: the optimal ratio of T cells to non-T cells for maximal IgE production was found to be 1:1. At this ratio, irradiated (non-replicating) T cells supported a much greater IgE synthesis than did non-irradiated T cells. CONCLUSION: The development of these systems provides directly comparable T cell-containing and T cell-depleted cultures for human IgE synthesis from peripheral blood, allowing further study of the role of T cells in IgE regulation. These systems will also be of use for determining whether potential modulators of IgE synthesis act on the T cells or on other cell types.

Cells, Cultured↗
PubMed1996

Isolation and characterization of the tsetse thrombin inhibitor: a potent antithrombotic peptide from the saliva of Glossina morsitans morsitans.

A potent and specific inhibitor of the human coagulation protease thrombin was identified in salivary gland extracts of the tsetse fly, Glossina morsitans morsitans, an important vector of African trypanosomiasis. This low molecular weight peptide (MW = 3,530 Da as determined by laser desorption mass spectrometry) was purified using a combination of size-exclusion chromatography and reverse-phase, high-performance liquid chromatography, respectively. Amino terminal sequencing of the purified protein reveals no homology to any previously identified serine protease inhibitor or naturally occurring anticoagulant. The tsetse thrombin inhibitor (TTI) is a stoichiometric inhibitor of thrombin, with an apparent equilibrium dissociation inhibitory constant (Ki*) [corrected] of 584 x 10(-15)M. In addition, it is also a potent inhibitor of thrombin-induced platelet aggregation. Like other hematophagous arthropods, tsetse flies appear to have evolved a novel protease inhibitor capable of antagonizing host hemostasis and facilitating blood feeding.

Amino Acid Sequence↗
PubMed1995

A major house dust mite allergen disrupts the immunoglobulin E network by selectively cleaving CD23: innate protection by antiproteases.

Asthma is a chronic life-threatening disease of worldwide importance. Although allergic asthma and related atopic conditions correlate strongly with immune sensitization to house dust mites, it is unclear why antigens from mites provoke such powerful allergic immune responses. We have characterized the protease activity of Der p I, the group I protease allergen of the house dust mite Dermatophagoides pteronyssinus, and here report that it cleaves the low-affinity immunoglobulin (Ig) E Fc receptor (CD23) from the surface of human B lymphocytes. Der p I selectively cleaves CD23 and has no effect on the expression of any other B cell surface molecules tested. We speculate that this loss of cell surface CD23 from IgE-secreting B cells may promote and enhance IgE immune responses by ablating an important feedback inhibitory mechanism that normally limits IgE synthesis. Furthermore, since soluble CD23 is reported to promote IgE production, fragments of CD23 released by Der p I may directly enhance the synthesis of IgE. alpha 1-Antiprotease, a pulmonary antiprotease, is also shown to inhibit the cleavage of CD23 by Der p I. This may be significant in the etiopathogenesis of asthma, because other indoor pollutants associated with asthma are known to potently inhibit this antiprotease. These data suggest that the proteolytic activity of Der p I, the group I allergen of the house dust mite D. pteronyssinus, is mechanistically linked to the potent allergenicity of house dust mites. Furthermore, inhibition of Der p I by alpha 1-antiprotease suggests a mechanism by which confounding factors, such as tobacco smoke, may act as a risk factor for allergic asthma.

Animals↗