Acaricide resistance and novel photosensitizing approach as alternative acaricides against the camel tick, Hyalomma dromedarii.

The control of the camel tick, Hyalomma dromedarii is very crucial. This study evaluated the novel toxicity of photosensitizers and Phoxim insecticide against H. dromedarii males using the adult immersion tests. Ticks were subjected to sunlight for 10 min post-treatment (PT). The optical characters of the applied materials were determined by UV-Vis spectroscopy (250-900 nm wavelengths). The intensity of spectra decreased as dye concentration decreased. The optical bandgap energies of the dyes at different concentrations were not changed as the concentration changed and decreased as the absorption peak of individual dyes red-shifted. The mortalities 72 h PT reached 42.2%, 44.4%, 51.1%, 71.1%, 46.7%, 48.9%, 44.4%, and 55.6% for chlorophyllin, echinochrome, field stain, methylene blue, phthalocyanine, rhodamine 6G, riboflavin, and safranin, respectively. Methylene blue recorded the highest median lethal concentration (LC50 = 127 ppm) followed by safranin, field stain, rhodamine 6G, phthalocyanine, echinochrome riboflavin, and chlorophyllin (LC50 = 209, 251, 271, 303, 324, 332, and 362 ppm, respectively, 72 h PT). Their median lethal time, LT50, values PT with 240 ppm were 45, 87, 96, 72, 129, 115, 131, and 137 h, respectively. The relative toxicities of the LC50 values 72 h PT showed that chlorophyllin, echinochrome, field stain, methylene blue, phthalocyanine, rhodamine 6G, riboflavin, and safranin were 3.2, 3.6, 4.6, 9.1, 3.8, 4.3, 3.5, and 5.6 times, respectively, more effective than Phoxim. Methylene blue, safranin, and field stain showed a broad absorbance area indicating a large photoactivity and better phototoxicity and could be used as alternative agents to synthetic acaricides.

Influence of Aquatain™, a monomolecular surface film on surface tension for controlling the filarial vector Culex pipiens (Diptera: Culicidae).

Surface tension is a phenomenon in the liquid media and plays an important part in the development and survival of aquatic animals. Influence of Aquatain™ monomolecular film on surface tension was determined against mosquito larvae and pupae at different temperatures (10, 15, 20, 25, 30 and 35 °C) and Aquatain™ doses (0.5, 1.0 and 2.0 ml/m2). In the laboratory, Aquatain™ showed larvicidal and pupicidal effects against the filarial vector Culex pipiens. Higher mortality was observed in late and more weighted instars/stages than young ones as well as in the pupal stage. The pupal mortality reached 76.2%, 86% and 93.3% after 12 h post-treatment at 0.5, 1.0 and 2.0 ml/m2, respectively, and it was completely eliminated (100%) within 24 h compared to 15.1%, 26.9% and 38.2% for 1st larval instar, respectively. Also, results showed at 0.5 ml/m2 with temperature range: 10, 15, 20, 25, 30 and 35 °C, the mortality reached 4.0, 6.7, 10.8, 17.3, 22.7, 29.3% and 32, 44, 54, 72, 84, 97.3% for 1st and 4th larval instar, respectively, where the surface tension (γ) was 65.6, 62.4, 58.0, 57.0, 54.2 and, 49.6 dyn/cm, while the Aquatain™ was more effective on mosquito larvae and pupae at high doses with the temperature range. On the other hand, without Aquatain™ dose, the mortality value ranged between 0.0 - 1.2%, and the surface tension (γ) was 74.5 dyne/cm, which is considered as an accidental death. Aquatain™ was effective against all aquatic phases of mosquitoes, especially against the last and weighted ones. Not only was the efficacy of Aquatain™ increased by increasing the dose, but it also increased with the increased temperature of the environment. This efficiency of Aquatain™ is due to its ability to reduce the surface tension of the water medium, preventing different stages of mosquitoes from reaching the surface for breathing thereby leading to suffocation and death. Therefore, we recommended Aquatain™ in programmes for mosquito control and other aquatic insects as a safe, cost-effective control agent.

Antibody to very late activation antigen 4 prevents interleukin-5-induced airway hyperresponsiveness and eosinophil infiltration in the airways of guinea pigs.

This study examines the effect of monoclonal antibody to very late activation antigen-4 (VLA-4) on IL5-induced airway hyperresponsiveness in vivo and eosinophil accumulation into guinea pig airways. IL5 has been shown to be important in the development of airway hyperresponsiveness and eosinophil accumulation in the guinea pig. Eosinophils, unlike neutrophils, express VLA-4 which mediates the adhesion to vascular cell adhesion molecule-1 on endothelial cells. Thus VLA-4 seems to be an important adhesion molecule in the infiltration of eosinophils from the vasculature into the airway tissue. In addition, it has been shown that IL5 activates VLA-4 on eosinophils to facilitate their adhesion. In the present study, IL5 (1 microg, twice on one day) or vehicle were administered intranasally. Monoclonal antibody (mAb) to VLA-4 (HP1/2) or the isotype-matched control mAb (1E6) were injected 1 hour before each IL5 or vehicle treatment at a dose of 2.5 mg/kg body weight. The next day in vivo bronchial reactivity, eosinophil number in bronchoalveolar lavage (BAL) fluid, and eosinophil peroxidase (EPO) activity in cell-free BAL fluid were determined. IL5 induces an increase in bronchial reactivity to histamine, which is associated with an accumulation of eosinophils into BAL fluid (control: 12 (5 to 42) x 10(5) cells and IL5: 69 (11 to 99) x 10(5) cells, p < 0.05) and an increase of 35% +/- 14% in EPO activity in cell-free BAL fluid. Intravenous administration of anti-VLA-4 mAb, but not of the control antibody, completely inhibits the bronchial hyperresponsiveness as well as the airway eosinophilia found after intraairway application of IL5. HP1/2 also suppresses the IL5-induced increase in EPO activity in cell-free BAL fluid. In conclusion, for the development of IL5-induced airway hyperresponsiveness in the guinea pig, the VLA-4-dependent infiltration and activation of eosinophils in the bronchial tissue seems to be essential.

An interleukin 5 mutant distinguishes between two functional responses in human eosinophils.

Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.

Lung inflammation and epithelial changes in a murine model of atopic asthma.

A murine model of allergen-induced airway inflammation and epithelial phenotypic change, and the time-courses of these events, are described. Mice were sensitized to ovalbumin using an adjuvant-free protocol, and challenged by multiple intratracheal instillations of ovalbumin by a non-surgical technique. Many of the characteristic features of human atopic asthma were seen in the mice. A marked eosinophilic infiltration of lung tissue and airways followed allergen challenge, and its severity increased with each challenge, as did the number of eosinophils in the blood. Lymphocytes, neutrophils, and monocytes also invaded the lungs. Airway macrophages showed signs of activation, their appearance resembling those recovered from antigen-challenged human asthmatic airways. The airway epithelium was thickened and displayed a marked goblet cell hyperplasia in terminal bronchioles and larger airways. After repeated challenges, the reticular layer beneath the basement membrane of the airway epithelium showed fibrosis, reproducing a commonly observed histologic feature of human asthma. Goblet cell hyperplasia began to appear before eosinophils or lymphocytes had migrated across the airway epithelium, and persisted for at least 11 days after the third intratracheal challenge with ovalbumin, despite the number of inflammatory cells in the lungs and airways having decreased to near-normal levels by 4 days. Plugs of mucus occluded some of the airways. These results indicate that some of the phenotypic changes in airway epithelium that follow an allergic response in the lung can be initiated before the migration of eosinophils or lymphocytes across the epithelial layer.

A rapid activation assay for human eosinophils based on adhesion to immobilized ICAM-1, VCAM-1 and IgG.

Interleukin 5 (IL-5) is a T-cell derived cytokine that induces eosinophil growth and differentiation in both mouse and human bone marrow cultures. Elevated levels of IL-5 as well as eosinophils have been detected in the sputum and Bronchoalveolar lavage (BAL) fluids of asthmatics. Since the recruitment of inflammatory cells to tissues requires the participation of adhesion molecules, we have developed a rapid and sensitive assay to examine the effect of IL-5 and other activation stimuli on eosinophil adhesion to recombinant intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Human recombinant IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), tumour necrosis factor alpha (TNF-alpha), RANTES, MCP-3, C5a, PAF, fMLP, PMA and ConA all induced adhesion of purified eosinophils obtained from normal donors to ICAM-1 and VCAM-1 in a dose and time dependent manner. Adhesion was rapid, within 15 minutes of culture at 37 degrees C, and plateaued within 30 minutes. Activated eosinophils also adhered rapidly to immobilized IgG via the type II Fc gamma receptor (CD32). Analysis of the effect of IL-5 on surface molecule expression by FACS analysis revealed increased expression of CD11b molecules and decreased expression of L-selectin, but no change in the expression of CD11a, CD18, CD29, CD49d and CD32. We also show that Mac-i plays an important role in the regulation of eosinophil activation, since antibodies to CD11b can block IL-5 induced adhesion to IgG and IL-5 induced degranulation.

Role of interleukin-5 and substance P in development of airway hyperreactivity to histamine in guinea-pigs.

In this study, we examined the mechanism by which bronchoalveolar lavage (BAL) cells induced hyperreactivity of the trachea in vitro. As both interleukin-5 (IL-5) and substance P (SP) appeared to be involved, the effect of these mediators was examined in vivo. Tracheae were incubated with BAL cells from ovalbumin or saline challenged animals, and from naive animals, in the absence or presence of either IL-5, SP, or both. In addition, the effect of intra-airway application of IL-5, SP, both, or vehicle on tracheal hyperreactivity was examined. Incubation of tracheae with BAL cells from ovalbumin challenged animals induced an increase (30 +/- 10%) in the maximal response to histamine. The hyperreactivity could be completely inhibited by co-incubation with 5-lipoxygenase inhibitor, AA861. The hyperreactivity could be mimicked by incubation of tracheae with BAL cells from naive animals in the presence of IL-5 and SP. After in vivo administration of either IL-5 or SP, maximal responses to histamine were increased and amounted to 105 +/- 35 and 101 +/- 37%, respectively. Administration of IL-5 but not SP induced a significant increase in the number of eosinophils (67 +/- 22%) and eosinophil peroxidase (EPO) activity (94 +/- 33%) in BAL cells. The simultaneous administration of IL-5 and SP did not potentiate the hyperreactivity and eosinophilia observed with IL-5 alone. These data suggest that IL-5 is important in the recruitment of eosinophils, whereas both IL-5 and substance P are involved in the induction of airway hyperreactivity.

Eosinophil infiltration precedes development of airway hyperreactivity and mucosal exudation after intranasal administration of interleukin-5 to mice.

Recently, we demonstrated that antibody to interleukin-5 (IL-5) prevents the infiltration of eosinophils in the respiratory airways and the development of bronchial hyperreactivity in an animal model of allergic asthma. In this study we investigated the influence of long-term intranasal administration of IL-5 on airway responsiveness in vitro, the infiltration of inflammatory leukocytes, and mucosal exudation. Mice (BALB/c) received 1 microgram of recombinant human IL-5 in 30 microliters of saline solution or vehicle alone twice a day for 1, 3, and 7 days. At 3 and 7 days after IL-5 administration, the number of bronchoalveolar lavage eosinophils increased approximately fourfold and sixfold, respectively. Blood eosinophil numbers showed a similar increase. In addition, 7 days after IL-5 treatment, total lung eosinophil peroxidase activity was significantly increased by 170% as compared with controls. The maximal responsiveness of the trachea in vitro to methacholine was significantly increased by 34%, as compared with controls, only at 7 days after IL-5 administration. Furthermore, mucosal exudation was also only increased significantly at 7 days after IL-5 administration. It can be concluded that the IL-5-induced eosinophil infiltration precedes the development of airway hyperreactivity and mucosal exudation.

Identification of key charged residues of human interleukin-5 in receptor binding and cellular activation.

Interleukin-5 (IL-5) is a cytokine that plays a major role in the differentiation and activation of eosinophils. In order to identify which charged residues of human IL-5 are important in binding to its receptor and subsequent cellular activation, we have systematically replaced all of the clusters of charged amino acids with alanine residues. The mutants have been expressed in Escherichia coli, renatured, and purified. They were assayed for ability to cause proliferation of the erythroleukaemic cell line TF-1 and the up-regulation of eosinophil adhesion to ICAM-1. In addition, we studied receptor binding using either immobilized recombinant IL-5 receptor alpha-chain or the alpha/beta-receptor complex expressed on TF-1 cells. The key charged residue involved in binding to the beta-chain of the receptor is Glu-12. This residue is in an identical position to those previously identified in IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) involved in binding to the receptor beta-chain. The alpha-chain binding site is shown to involve the side chains Arg-90 and Glu-109, located in the second beta sheet and after the end of the fourth helix, respectively. It is unique to IL-5 and does not occur in IL-3 or GM-CSF. Understanding the topology of the interaction of IL-5 with its receptor chains will help in the search for rationally designed antagonists of IL-5 function.

CD40 ligand is functionally expressed on human eosinophils.

CD40 ligand (CD40L), a surface molecule which can be expressed by T cells, mast cells and basophils, has been shown to be involved in the control of B cell proliferation, immunoglobulin class switching as well as in the activation of monocytes and T cells. We demonstrate that CD40L can also be expressed constitutively by eosinophils from an hypereosinophilic patient or, upon activation, by the eosinophilic cell line EOL-3 and normal blood eosinophils. Eosinophils were able to induce, in conjunction with IL-4, CD40L-dependent B cell proliferation in vitro. These results suggest that CD40L could play a role in the inflammatory processes during which eosinophil infiltration and activation are observed.

Purification, characterisation and crystallisation of selenomethionyl recombinant human interleukin-5 from Escherichia coli.

Interleukin-5 (IL-5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three-dimensional structure, we have expressed large quantities of recombinant human IL-5 (hIL-5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno-DL-methionine-containing medium. Cell densities of A650 = 10 have been achieved. The selenomethionyl-labelled hIL-5 (Se-hIL-5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B-cell growth factor assay, the Se-hIL-5 is significantly more active than the non-labelled hIL-5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26,326 +/- 6 and 26,280 +/- 8 corresponding to the 4Se and 3Se/1S forms of hIL-5. Unlike the methionine-containing hIL-5, the N-terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se-hIL-5 crystallises in the same space group and unit cell as hIL-5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N-terminal is disordered in the crystal, and the N-terminal selenomethionines are not resolved in the difference Fourier.

IL-5 responsive subsets among normal and lymphomatous murine B cells.

Two normal murine B-cell subpopulations, germinal center and coelomic B cells, and at least some of the lymphomas derived from them, respond to IL-5. In the case of normal B cells, a comitogen (DxS) is required. IFN-gamma is strongly inhibitory to proliferation of the coelomic B-cell subset but not for germinal center cells or the SJL lymphomas derived from them.

Production, characterisation and use of monoclonal antibodies to human interleukin-5 in an enzyme-linked immunosorbent assay.

Two mouse monoclonal antibodies (Mabs) against recombinant human interleukin-5(rhIL-5) have been produced, characterised and purified. Both are IgG1 antibodies and neutralised the activity of rhIL-5 in the B13 assay. Neither Mab cross-reacted with mouse IL-5. A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was developed with different combinations of the mouse Mabs and also a rat anti-mouse IL-5 Mab, TRFK5, which also has activity against rhIL-5. The most sensitive assay, with a lower detection limit of 0.5 ng/ml IL-5, used TRFK5 as the capture antibody and the mouse anti-human IL-5 Mab as second antibody. The sensitivity of this assay was increased by an enhanced chemiluminescent reagent and resulted in a lower limit of detection around 40 pg/ml IL-5.

Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli.

The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines.

In vitro and in vivo studies with purified recombinant human interleukin 5.

The functional activities of highly purified recombinant human IL 5 (hIL 5) have been characterized on a number of cell types in vitro and in BALB/c mice in vivo. In vitro, hIL 5 could induce the differentiation of eosinophils from precursors in both human and mouse bone marrow with approximately the same efficiency. A mouse IL 5/3-dependent B cell line, LyH7.B13, was found to proliferate in response to hIL 5 but not human interleukin 1 (IL 1), interleukin 2 (IL 2), interleukin 3 (IL 3), interleukin 4 (IL 4), interleukin 6 (IL 6), interferon-gamma (IFN-gamma), or granulocyte macrophage-colony stimulating factor (GM-CSF) and was at least 10-fold more sensitive than BCL1 mouse lymphoma cells. We have successfully used this cell line to demonstrate the production of IL 5 by human T cell clones. In marked contrast to its effects on murine B cell lines, hIL 5 had no demonstrable activity on CD23 expression, anti-mu costimulated proliferation or IgM, IgG, or IgE production by tonsillar B cells and did not influence such responses triggered by IL 4. BALB/c mice injected with hIL 5 for 7 consecutive days were shown to develop an eosinophilia comparable to that induced by infection with the parasite Mesocestoid corti.

Toxocara canis: interaction of human blood eosinophils with the infective larvae.

This study was carried out to investigate the nature of the immunological responses which took place in a child who had recently recovered from toxocariasis. She had developed a marked eosinophilia and had high titers of toxocara antibodies. Experiments were performed to examine whether Toxocara canis infective larvae could be killed in the presence of her serum and human eosinophils. Eosinophils with human complement, or this patient's serum, adhered to the surface of the larvae within 10 min. By 40 min, using both light and electron microscopy, it was shown that the cells had flattened against the cuticle and degranulated. However, by 3 hr, eosinophils had begun to detach, and the larvae remained alive for at least 1 week afterward. Further addition of serum or of eosinophils, which were shown to be able to immobilize T. spiralis infective larvae, failed to kill the T. canis larvae. It was concluded that, in this patient, the development of an inflammatory response to a T. canis infection was not associated with the appearance of antibodies capable of inducing eosinophil dependent toxicity to the larvae in vitro. Eosinophil dependent killing mechanisms may be less important than other components of the immune response, in immunity to this parasite in humans.

Simplified method for measuring sex-hormone binding globulin.

We describe a simple, rapid method for measurement of sex-hormone binding globulin. Serial dilutions of pregnancy serum are prepared in serum from males that has been pre-treated by heating to 60 degrees C for 1 h to destroy endogenous binding globulin, which is then determined by a long-used technique to yield a set of "standards." In the assay itself, a fixed amount of [3H]-labeled and unlabeled dihydrotestosterone is incubated with standard or unknown, and the bound fraction precipitated with saturated ammonium sulfate. A plot of percent of the steroid bound vs standard dilution yields a sigmoid curve, from which the results in unknowns can be read by simple extrapolation. Within-assay CVs for pools of serum from men, women, and women in late pregnancy were 6.56, 9.59, and 8.4%, respectively. Between-assay CVs for the same pools were 8.05, 9.5, and 11.5%, respectively. The correlation between results obtained by this method and those of the older technique was 0.95 for samples from non-pregnant subjects and 0.73 for those from pregnant women. Our procedure is simpler and faster than previous methods and accurately measures the differences in the globulin in sera from men, women, and pregnant women. Forty to 50 samples can be assayed in a working day.