Antigen-driven basophil activation is indicative of early Necator americanus infection in IgE-seronegative patients.

BACKGROUND: Parasitic worms induce a strong, polarized T(H)2-type immune response. The kinetics of gastrointestinal nematode-induced T(H)2-type responses, especially in the context of primary infection, have been extensively studied in experimental infection models but not in human subjects. OBJECTIVE: We sought to determine the kinetics of basophil sensitization in subjects infected with Necator americanus during the first 12 weeks after infection. METHODS: Thirty nonasthmatic subjects with allergic rhinoconjunctivitis were randomized in a double-blind manner to cutaneous administration of either 10 hookworm infective larvae or histamine placebo. Blood samples were taken at regular intervals for 12 weeks, and basophil activation was determined in whole blood by measuring CD63 and CD203c levels on stimulation with N americanus excretions/secretions. Parasite-specific immunoglobulin responses were assessed by means of ELISA and Western blotting. RESULTS: Median values reflecting basophil activation (CD203c/CD63 double-positive cells) in the excretion/secretion-stimulated infected group steadily increased after week 4, consistently achieving statistical significance compared with the placebo group between 6 and 12 weeks after infection. Only parasite-specific IgM levels increased significantly during this period, whereas total and parasite-specific IgE levels did not differ between groups. CONCLUSION: Basophils are sensitized early in the context of a low-dose primary infection with N americanus in the absence of measurable total and specific IgE serum level increase.

Bacterial N-acylhomoserine lactone-induced apoptosis in breast carcinoma cells correlated with down-modulation of STAT3.

Cell growth is promoted by mitogens and survival factors, which activate intracellular signalling pathways to control cell cycle progression and cellular integrity. Proliferation signals are transmitted through Ras and Rho family small G-proteins coupled to mitogen-activated protein kinase (MAPK) cascades, while survival signals are propagated by lipid-dependent kinases such as phosphatidylinositide 3-kinases (PI3Ks) and protein kinase B (Akt/PKB). Recently, signal transducer and activator of transcription (STAT) proteins were identified as positive regulators of proliferation in a variety of cell types. Persistent activation of these pathways is associated with tumour cell growth, whereas their inhibition can halt proliferation and precipitate apoptotic cell death. The human pathogen Pseudomonas aeruginosa uses quorum-sensing signal molecules (QSSMs) to regulate virulence gene expression. QSSMs also suppress host immune responses although the mechanism of suppression is unknown. Here, we demonstrate that the QSSM N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) from P. aeruginosa blocks proliferation and induces apoptosis in human BC cell lines. Analyses of signalling events reveal that OdDHL has little or no effect on MAPK cascades, partially inhibits the Akt/PKB pathway and ablates STAT3 activity. Pharmacological inhibition of each pathway independently indicates that STAT3 activity is critical for BC cell proliferation and survival, while a constitutively active STAT3 confers resistance to OdDHL. These results support the notion of OdDHL as a bioactive molecule in eukaryotic systems and a paradigm for a novel class of antiproliferative compounds.

A T-cell-dependent humoral immune response is preserved during the administration of the nerve agent pre-treatment pyridostigmine bromide in a murine model.

Immune regulation, either via the autonomic nervous system or by a proposed "non-neuronal" cholinergic system, suggests that the immune system may be susceptible to perturbation by compounds affecting cholinergic function. Here, the current UK and US nerve agent pre-treatment, pyridostigmine bromide (PB) and the related anti-acetylcholinesterase (AChE) compounds physostigmine (PHY) and BW284c51 were tested for their ability to affect mouse splenocyte function in vitro. In addition, PB, at a dose equivalent to that received during pre-treatment for nerve agent poisoning, was tested for its effect on a T-cell-dependent humoral response to antigen in vivo in the mouse. None of the anti-AChEs tested affected concanavalin A (Con A)-, anti-CD3- or lipopolysaccharide LPS-driven splenocyte proliferation, in vitro, at concentrations expected to give effective nerve agent pre-treatment. However, higher concentrations (>100 microM) particularly of PHY caused some inhibition of the proliferative responses. In vivo, PB or saline was administered via 28-day mini-osmotic pumps to give a 25-40% inhibition of whole blood AChE in the PB-treated animals. During PB or saline administration, primary and secondary doses (i.p.) of sheep red blood cells (SRBC) were given and the humoral response determined by monitoring anti-SRBC IgM and IgG levels. Splenocytes isolated from the experimental animals were also examined for their proliferative and cytokine responses to stimulation. No remarkable effects of PB were seen during the period of AChE inhibition on the humoral immune response. However, a modest elevation in IL-2 and IFN(gamma) in Con A-stimulated lymphocytes was seen in PB-treated animals following pump removal. Overall these data suggest that, in vivo, the SRBC stimulated T-cell-dependent immune response is unaffected by the administration of PB at pre-treatment doses.

Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators.

Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone (3O, C(12)-HSL), is described. Twenty-four single or combination systematic alterations of the structural components of 3O, C(12)-HSL were introduced as described. Given the already defined immunological profile of the parent compound, 3O, C(12)-HSL, these compounds were assayed for their ability to inhibit murine and human leucocyte proliferation and TNF-alpha secretion by lipopolysaccharide (LPS) stimulated human leucocytes in order to provide an initial structure-activity profile. From IC(50) values obtained with a murine splenocyte proliferation assay, it is apparent that acylated l-homoserine lactones with an 11-13 C side chain containing either a 3-oxo or a 3-hydroxy group are optimal structures for immune suppressive activity. These derivatives of 3O, C(12)-HSL with monounsaturation and/or a terminal nonpolar substituent on the side chain were also potent immune suppressive agents. However, structures lacking the homoserine lactone ring, structures lacking the l-configuration at the chiral center, and those with polar substituents were essentially devoid of activity. The ability of compounds selected from the optimal activity range to modulate mitogen-driven human peripheral blood mononuclear cell proliferation and LPS-induced TNF-alpha secretion indicates the suitability of these compounds for further investigation in relation to their molecular mechanisms of action in TNF-alpha driven immunological diseases, particularly autoimmune diseases such as psoriasis, rheumatoid arthritis, and type 1 (autoimmune) diabetes.

An in vitro investigation of the effects of the nerve agent pretreatment pyridostigmine bromide on human peripheral blood T-cell function.

The current pretreatment against nerve agent poisoning deployed by the UK and US armed forces is the acetylcholinesterase (EC 3.1.1.7) inhibitor pyridostigmine bromide (PB). At higher doses, PB is also used to treat the autoimmune disease myasthenia gravis. In both cases, the therapeutic effect is mediated by inhibition of acetylcholinesterase (AChE) at cholinergic synapses. However, the location of AChE is not restricted to these sites. AChE, acetylcholine (ACh) receptors and choline acetyltransferase have been reported to be expressed by T cells, suggesting that cholinergic signalling may exert some modulatory influence on T-cell function and consequently on the immune system. The aim of this study was to investigate the role of the T-cell cholinergic system in the immunological activation process and to examine whether inhibitors of AChE such as PB affect immune function. To investigate this, human peripheral blood mononuclear cells (PBMC) were stimulated using either mitogen, cross-linking of the T-cell receptor and co-receptors with antibodies (anti-CD3/CD28) or by antigen presentation in the presence of various AChE inhibitors and ACh receptor agonists or antagonist. Several indices were used to assess T-cell activation, including the secretion of IL-2, cell proliferation and expression of CD69. Treatment with PB had no significant effect on the immunological assays selected. Physostigmine (PHY), a carbamate compound similar to PB, consistently showed inhibition of T-cell activation, but only at concentrations in excess of those required to inhibit AChE. No evidence was found to support previously published findings showing muscarinic enhancement of cell proliferation or IL-2 secretion.

Immunosuppressive but non-LasR-inducing analogues of the Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone.

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (1) is involved not only in bacterial activation but also in subversion of the host immune system, and this compound might thus be used as a template to design immunosuppressive agents, provided derivatives devoid of quorum-sensing activity could be discovered. By use of a leukocyte proliferation assay and a newly developed bioluminescent P. aeruginosa reporter assay, systematic modification of 1 allowed us to delineate the bacterial LasR-induction and host immunosuppressive activities. The main determinant is replacement of the methylene group proximal to the ß-ketoamide in the acyl chain of 1 with functions containing heteroatoms, especially an NH group. This modification can be combined with replacement of the homoserine lactone system in 1 with stable cyclic groups. For example, we found the simple compound N(1)-(5-chloro-2-hydroxyphenyl)-N(3)-octylmalonamide (25d) to be over twice as potent as 1 as an immune suppressor while displaying LasR-induction antagonist activity.

Immunologic profiles of persons recruited for a randomized, placebo-controlled clinical trial of hookworm infection.

Data from epidemiologic studies suggest that hookworm infections, in establishing an immunologic phenotype conducive to parasite survival, may protect against the development of allergic disease. We describe immunologic findings from a clinical study designed to investigate the safety of iatrogenic hookworm infection in participants with allergic rhinitis. The low, relatively safe level of hookworm infection used in this study was immunogenic, inducing eosinophilia and a significant specific IgG response. Importantly, no potentiation of IgE responses to the environmental allergens to which the participants were sensitized was seen. However, no evidence of systemic immune regulation was seen in infected participants. This finding may indicate that the level of infection or the frequency of infection may have to be altered in future trials to induce a therapeutically conducive immunologic phenotype.

Development of a model of hookworm infection exhibiting salient characteristics of human infection.

Patent and pathologic infections of the human hookworm Necator americanus were established in the common marmoset (Callithrix jacchus). In a pilot study, a laboratory strain of N. americanus was compared with a fresh field isolate. Pathology was more severe in animals infected with a fresh isolate. In all animals, infection was associated with increased total plasma IgE and production of IgG specific to adult worm excretory/secretory (ES) products. Histamine was released by basophils in response to IgE, ES products, and a recombinant hookworm allergen, calreticulin. The pilot study indicated the potential of this animal model of hookworm infection and led us to investigate the consequences of infecting a further cohort with the fresh field isolate. This second study confirmed our initial findings, that it is possible to investigate the human hookworm N. americanus in a model exhibiting many of the characteristics of the immunology of hookworm infection in its definitive host.

Basophil competence during hookworm (Necator americanus) infection.

A popular hypothesis to explain parasite survival in the presence of a pronounced T helper 2 phenotype in helminth-parasitized populations has been Fc epsilonRI blockade by parasite-induced polyclonal IgE. To begin to test the hypothesis that Fc epsilonRI-bearing cells would be refractory to activation in parasitized populations, we investigated basophil function in 43 individuals from a hookworm endemic area. Study individuals had high levels of total IgE and eosinophilia and a mean hookworm burden of 2,257 epg. Basophils from all members of this parasitized population were shown to release histamine to a number of agonists, including anti IgE and a hookworm allergen, calreticulin. These data would indicate that Fc epsilonRI blockade at the level of the basophil did not occur in this parasitized population despite the presence of possible immunologic blocking agents. This would suggest that this effector arm of the T helper 2 phenotype remains operative in infected populations.

Differential immune modulatory activity of Pseudomonas aeruginosa quorum-sensing signal molecules.

Pseudomonas aeruginosa releases a spectrum of well-regulated virulence factors, controlled by intercellular communication (quorum sensing) and mediated through the production of small diffusible quorum-sensing signal molecules (QSSM). We hypothesize that QSSM may in fact serve a dual purpose, also allowing bacterial colonization via their intrinsic immune-modulatory capacity. One class of signal molecule, the N-acylhomoserine lactones, has pleiotropic effects on eukaryotic cells, particularly those involved in host immunity. In the present study, we have determined the comparative effects of two chemically distinct and endobronchially detectable QSSM, N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and 2-heptyl-3-hydroxy-4 (1H)-quinolone or the Pseudomonas quinolone signal (PQS), on human leukocytes exposed to a series of stimuli designed to detect differential immunological activity in vitro. 3-Oxo-C12-HSL and PQS displayed differential effects on the release of interleukin-2 (IL-2) when human T cells were activated via the T-cell receptor and CD28 (a costimulatory molecule). 3-Oxo-C12-HSL inhibited cell proliferation and IL-2 release; PQS inhibited cell proliferation without affecting IL-2 release. Both molecules inhibited cell proliferation and the release of IL-2 following mitogen stimulation. Furthermore, in the presence of Escherichia coli lipopolysaccharide, 3-oxo-C12-HSL inhibited tumor necrosis factor alpha release from human monocytes, as reported previously (K. Tateda et al., Infect. Immun. 64:37-43, 1996), whereas PQS did not inhibit in this assay. These data highlight the presence of two differentially active immune modulatory QSSM from P. aeruginosa, which are detectable endobronchially and may be active at the host/pathogen interface during infection with P. aeruginosa, should the bronchial airway lymphoid tissues prove to be accessible to QSSM.