Human gammadelta T cells modulate the mite allergen-specific T-helper type 2-skewed immunity.

BACKGROUND: Gammadelta T cells have been described as one of immune regulators in patients with infection, malignancy, and allergy. OBJECTIVE: To elucidate the ability of gammadelta T cells as an allergen immunotherapy candidate, the effectiveness of human gammadelta T cells in allergen-specific T-helper type 2 (Th2)-type T cells was evaluated in vitro. METHODS: House dust mite-specific Th2-type T cell clones, Bacillus Calmette-Guerin (BCG)-specific Th1-type T cell clones, and gammadelta T cell lines were established from the peripheral blood mononuclear cells of two patients with allergic rhinitis. The effectiveness of gammadelta T cells and BCG-specific Th1-type T cell clones in the modulation of allergen-specific Th2 cells in terms of their cytokine productions was evaluated. RESULTS: In response to cognate antigens, the gammadelta T cell lines demonstrated a proliferation and production of IFN-gamma that exceeded that of BCG-specific Th1-type T cell clones (mean stimulation index: 14.5 vs. 2.8, mean IFN-gamma: 130.5 vs. 10.0 pg/mL). When the gammadelta T cell lines and mite-allergen-specific Th2 clones were co-cultured with each other, only the levels of IL-4 (mean, -87%) decreased, but not the levels of IL-5 and IL-13, with an increasing concentration of gammadelta T cell antigen and IFN-gamma production (mean, +730%). CONCLUSION: These results demonstrated that gammadelta T cells derived from allergic patients might thus have a partial ability to modulate allergen-specific Th2-skewed immunity.

Improved glucose tolerance via enhanced glucose-dependent insulin secretion in dipeptidyl peptidase IV-deficient Fischer rats.

Glucagon-like peptide-1 (GLP-1) is an incretin, which induces glucose-dependent insulin secretion. GLP-1 is rapidly degraded by dipeptidyl peptidase IV (DPPIV) after its release. We investigated whether DPPIV-deficient F344/DuCrj rats show improved glucose tolerance when compared with DPPIV-positive F344/Jcl rats. Oral glucose tolerance test indicated improved glucose tolerance in F344/DuCrj rats, but blood glucose levels of the two strains were almost the same 120 min after the glucose bolus. Valine-pyrrolidide, a DPPIV inhibitor, had no effect on the glucose tolerance of F344/DuCrj rats, but improved that of F344/Jcl rats. Enhanced insulin secretion and high plasma active GLP-1 levels were detected in an intraduodenal glucose tolerance test. Glucose tolerance is improved in DPPIV-deficient F344/DuCrj rats via enhanced insulin release mediated by high active GLP-1 levels. Our results suggest that DPPIV inhibition is a rational strategy to treat diabetic patients by improving glucose tolerance with low risk of hypoglycemia.

Smoking and oxidant stress: assay of isoprostane in human urine by gas chromatography-mass spectrometry.

Isoprostane (8-epi-prostaglandin F2alpha) is synthesized non-enzymatically from arachidonate and active oxygen. We examined the relationship of smoking and excretion of isoprostane in urine with gas chromatography-mass spectrometry selected ion monitoring assay and the stable isotope dilution method. Urine isoprostane concentrations were significantly higher in smokers (n=81, 605.24+/-59.01 ng/mg creatinine) than in non-smokers (n=39, 424.07+/-70.37 ng/mg creatinine), but concentrations in ex-smokers (n=21, 487.27+/-98.48 ng/mg creatinine) did not differ significantly from those in the other groups. In smokers, age, the duration of smoking, and the number of cigarettes per day were not correlated with urine isoprostane concentrations. However, urine isoprostane concentrations were negatively correlated with time since quitting in ex-smokers and with age in non-smokers. These results indicate that smoking increases isoprostane concentration in urine and suggest that smoking causes lipid peroxidation by oxidant stress.

Dietary supplementation with fish oil rich in omega-3 polyunsaturated fatty acids in children with bronchial asthma.

Omega-3 polyunsaturated fatty acids have anti-inflammatory effects in vitro, and high dietary levels are associated with a lower incidence of inflammatory diseases. However, only limited effects have been demonstrated in asthma. The effects of dietary supplementation with fish oil for 10 months in 29 children with bronchial asthma was investigated in a randomized controlled fashion. In order to minimize the effects of environmental inhaled allergens and diet, this study was performed in a long-term treatment hospital. Subjects received fish oil capsules containing 84 mg eicosapentaenoic acid (EPA) and 36 mg docosahexaenoic acid (DHA) or control capsules containing 300 mg olive oil. The daily dosages of EPA and DHA were 17.0-26.8 and 7.3-11.5 mg x kg body weight(-1), respectively. Asthma symptom scores decreased and responsiveness to acetylcholine decreased in the fish oil group but not in the control group. In addition, plasma EPA levels increased significantly only in the fish oil group (p<0.0088). No significant side-effects were observed. The present results suggest that dietary supplementation with fish oil rich in the omega-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid is beneficial for children with bronchial asthma in a strictly controlled environment in terms of inhalant allergens and diet.

Simultaneous assay of prostaglandins and thromboxane in the cerebrospinal fluid by gas chromatography-mass spectrometry-selected ion monitoring.

A method of simultaneous analysis of prostaglandins (PGs) and thromboxane (TX) B2 in cerebrospinal fluid (CSF) with GC-MS-SIM was established. Deuterated PGs and TXB2 were used as internal standards: tetra-deuterated PGE2 (d4-PGE2) for PGE2, PGE1 and PGD2; d5-PGF2alpha for PGF2alpha and 9alpha,11beta-PGF2 and 8-epi PGF2alpha; d4-TXB2 for TXB2; and d4-6-keto PGF1alpha for 6-keto PGF1alpha. The PGs and TXB2 were derivatized to the methyl ester of the methoxim dimethyisopropylsilyl (DMiPSi) ether form or the methyl ester of the DMiPSi ether form with simultaneous preparation. Samples were extracted with octadecyl silica gel and purified in two steps with silisic acid gel chromatography between derivatization steps. The calibration curve of each PG and TXB2 was linear from 10 pg to 10 ng with the isotope dilution method. The levels of the seven types of PG and of TXB2 were assayed simultaneously in the cerebrospinal fluid (CSF) from patients with aseptic meningitis. The CSF pattern of the PG and TXB2 concentrations in mumps meningitis differed from those in other types of aseptic meningitis and in disease controls.

Catalytic and structural importance of Gly-454, Tyr-455, and Leu-456 in the carboxy-terminal region of Escherichia coli F1-ATPase alpha subunit.

Monoclonal antibody alpha110 recognizes Leu-456 in the alpha subunit of the Escherichia coli F1-ATPase. Binding of this antibody to the alpha subunit or mutation of this residue to Pro caused enhancement of the ATPase activity, suggesting that this residue is involved in the catalytic mechanism of this molecule (H. Kanazawa et al. (1995) Arch. Biochem. Biophys. 317, 348-356). Leu-456 together with Gly-454 and Tyr-455 are the only residues in the carboxy-terminal 75 amino acids conserved among various species, suggesting that these three residues play important roles in catalysis by the ATPase. Here, we introduced site-directed mutations into these residues. Not only L456P but also G454L, Y455K, Y455L, and L456N mutations caused enhancement of the ATPase activity. Surprisingly, Y455V, L456H, and L456S caused assembly defects of F1 subunits on the membrane. Reconstitution of the alpha betagamma complex from the wild-type beta and gamma subunits with the mutant alpha subunit (L4gamma6P) exhibited enhanced ATPase activity. Addition of delta or epsilon fused to glutathione S-transferase which are functionally similar to the delta and epsilon subunits, respectively, to the reconstituted F1-ATPase did not cause significant enhancement of its activity. Decreased interaction between alpha and beta subunits with the L456P mutation was detected by the yeast two-hybrid system. According to the deduced three-dimensional structure of the bovine a subunit, Leu-456, Gly-454, and Tyr-455 are included in a small alpha helix. These results suggest that this alpha helix affects interaction of the alpha subunit with the beta subunit but not with delta or epsilon, which may be important for the catalytic mechanism and F1 assembly.

Reconstitution of the F1-ATPase activity from purified alpha, beta, gamma and delta or epsilon subunits with glutathione S-transferase fused at their amino termini.

Systems for overexpression and purification of active alpha, beta and gamma subunits of Escherichia coli H(+)-ATPase were established. The alpha and beta subunits recovered as soluble form were purified by hydroxyapatite column chromatography. Since the gamma subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate. By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active gamma subunit for reconstitution of the F1-ATPase activity with the purified alpha and beta subunit was obtained. The delta and epsilon subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography. These fused proteins (delta-GST and epsilon-GST) were incubated with the purified alpha, beta and gamma subunits and applied to affinity chromatography. The alpha beta gamma delta-GST and alpha beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins. These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the active complex. The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly. The delta-GST bound the gamma subunit significantly, and the alpha and beta subunits very weakly.

Determination of 9 alpha, 11 beta-prostaglandin F2 in human urine and plasma by gas chromatography-mass spectrometry.

Sensitive and specific assay methods for 9 alpha, 11 beta-prostaglandin F2 (9 alpha, 11 beta-PGF2) by gas chromatography-mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9 alpha, 11 beta-PGF2 was based on the use of the methyl ester-dimethylisopropylsilyl ether derivative, and pentadeuterated PGF2 alpha as a convenient internal standard. The calibration graph for 9 alpha, 11 beta-PGF2 was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise ratio = 2.3). The method was applied to the determination of 9 alpha, 11 beta-PGF2 in urine and plasma samples from patients with bronchial asthma.

The effect of platelet-activating factor on IgE binding to, and IgE-dependent biological properties of, human eosinophils.

This study investigated the effect of platelet-activating factor (PAF), leukotriene B4 (LTB4) histamine and formyl-methionyl-leucyl-phenylalanine (FMLP) on immunoglobulin E (IgE) binding and IgE-dependent cytotoxicity of human normal density eosinophils. The binding of a native myeloma IgE to normal human eosinophils was measured by flow cytometry using a fluorescein-conjugated polyclonal anti-IgE antibody. Preincubation with PAF (optimal at 10(-7)M), but not lyso-PAF or FMLP, gave dose-dependent increases in IgE binding. PAF and LTB4 gave significant increases in IgE binding after 5 min preincubation (P less than 0.05); the effect was further enhanced at 30 min (P less than 0.01). This was further confirmed using the rosette assay where PAF and LTB4, but not lyso-PAF or FMLP, gave dose- and time-dependent increases in IgE eosinophil rosettes. Eosinophil cytotoxicity for schistosomula of Schistosoma mansoni, incubated with immune serum, was also significantly enhanced (P less than 0.01) by PAF in a dose-dependent fashion (optimal at 10(-8) M). Schistosomula coated with FPLC-purified IgE fractions were susceptible to killing by normal density eosinophils, and this was enhanced with PAF (10(-8)M), LTB4 (10(-7)M) and histamine (10(-5)M) but not with FMLP (10(-7)M) or lyso-PAF. IgE-dependent cytotoxicity was confirmed by the removal of contaminating IgG from IgE-rich fractions, and by the abolishment of IgE-dependent cytotoxicity after IgE adsorption. These results suggest that PAF (and to a lesser extent LTB4 and histamine) increase IgE binding, IgE-dependent adherence and cytotoxicity of normal human eosinophils. Although IgE receptors have not been identified, the data support current concepts that certain biological properties of eosinophils may be IgE associated.

Enhancement of the expression of eosinophil IgE receptor (Fc epsilon R2) and its function by platelet-activating factor.

Eosinophils possess low-affinity Fc immunoglobulin E (IgE) receptors (Fc epsilon R2). We have compared platelet-activating factor (PAF) with lyso-PAF, leukotriene B4 (LTB4) and histamine for their ability to influence the binding of a native myeloma IgE to normal-density human eosinophils, using flow cytometry and a fluorescein-conjugated anti-IgE polyclonal antibody. Preincubation with PAF gave a dose- and time-dependent increase in IgE binding optimal at 10(-7) M (P less than 0.01) and 30 min respectively. A smaller but significant effect was observed with LTB4 and histamine (optimal at 10(-7) M (P less than 0.01) and 10(-5) M (P less than 0.05) respectively). Diluent alone or lyso-PAF had no effect. These results suggest enhanced expression of Fc epsilon R2 by stimulated eosinophils. Further confirmation of enhancement was obtained using two functional assays, i.e., cytotoxicity and mediator (LTC4) generation. IgE-dependent cytotoxicity against schistosomula of Schistosoma mansoni was enhanced by prior incubation of normal eosinophils with PAF (optimal at 10(-7) M), but not lyso-PAF. No LTC4 was detectable following incubation of unstimulated eosinophils with S. mansoni coated with specific IgE. Preincubation of these cells with PAF (10(-7) M) resulted in the generation of 4.4 pmol of LTC4 per 10(6) cells. Our data demonstrate that normal-density eosinophil Fc epsilon R2 expression can be up-regulated in vitro by PAF and LTB4 and that these changes are accompanied by enhanced functional properties.

High molecular weight-neutrophil chemotactic activity in nasal allergy.

HMW-NCA is involved in the pathogenesis of nasal hypersensitivity reactions in humans. We, however, have little evidence about the role of neutrophils which migrate to the site of allergic reactions by HMW-NCA and other chemotactic mediators. This role of neutrophils in nasal hypersensitivity still remains to be clarified.

Release of high-molecular-weight neutrophil chemotactic activity from resected human nasal turbinate after antigen challenge.

Eleven patients with allergic rhinitis were studied. All subjects were sensitive to house dust mite documented by skin test, RAST score, and nasal provocation test. The patients needed lower turbinectomy because of chronic hypertrophic rhinitis. Tissues of the lower turbinate were obtained at the time of surgery, fragmented, and subsequently challenged with house dust mite in vitro. Diffusates were collected for measurement of neutrophil chemotactic activity (NCA) and histamine. NCA and histamine were released in a dose-dependent manner, and the time course of release of these mediators was identical. Release of NCA and histamine correlated significantly (p less than 0.001). The prior administration of the antiallergic drugs, disodium cromoglycate or tranilast, significantly blocked the release of NCA and histamine from antigen-challenged tissues. NCA released from nasal tissues eluted as a single peak with estimated molecular size of between 669 kd and 440 kd in three subjects and as two or three peaks in two patients. These results provide evidence that NCA might be involved in the pathogenesis of allergic rhinitis.

Release of high molecular weight-neutrophil chemotactic activity from human tissues, cells and secretion.

Release of high molecular weight-neutrophil chemotactic activity from human tissues, cells and secretion was studied in vivo and in vitro. Lung, nasal turbinate, nasal polyps, skin of neurofibromatosis, basophils from chronic myeloid leukemia and cultured basophilic cells from cord blood released this mediator following calcium ionophore, antigen, anti-IgE or homogenization in vitro. Its release was also demonstrated in human nasal secretions from patients with allergic rhinitis following antigen challenge. Regarding mononuclear cells no release of this mediator was observed from normal donors or asthmatic patients having no active attack upon challenge with calcium ionophore, phytohemagglutinin or antigen. Homogenized duodenum released high molecular weight-neutrophil chemotactic activity but less activity in comparison with other tissues or cells mentioned above.

Studies on steroids. CCXXXVIII. Determination of bile acids in liver tissue by gas chromatography-mass spectrometry with negative ion chemical ionization detection.

A method for the determination of bile acids in 2-10 mg of human liver tissue by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Unconjugated, glycine- and taurine-conjugated bile acids labelled with 18O and 2H were used as internal standards. The preparation of these compounds was attained by the exchange reaction of the carbonyl group with H218O, followed by metal hydride reduction. Bile acids in solubilized liver tissue were extracted with a Sep-Pak C18 cartridge, separated into the unconjugated, glycine- and taurine-conjugated fractions by ion-exchange chromatography on piperidinohydroxypropyl-Sephadex LH-20 and then derivatized to the pentafluorobenzyl ester-dimethylethylsilyl ethers. Subsequent resolution of each fraction into lithocholate, deoxycholate, chenodeoxycholate, ursodeoxycholate and cholate was attained by GC on a cross-linked 5% phenylmethyl silicone fused-silica capillary column where bile acids were monitored with a characteristic carboxylate anion [M - 181]-in the NICI mode using isobutane as a reagent gas. The newly developed method was applied to the quantitation of bile acids in liver tissue with satisfactory sensitivity and reliability.

Disodium cromoglycate inhibits activation of human inflammatory cells in vitro.

Recent clinical studies indicate that disodium cromoglycate (DSCG) may have a direct effect on inflammatory cells because the drug reversed various changes in leukocyte function, such as increased membrane-receptor expression and enhanced cytotoxic capacity observed in peripheral white blood cells from subjects with asthma undergoing allergen-inhalation challenge. In the present study, we have demonstrated that DSCG, at low concentrations (a concentration of drug required to produce 50% inhibition, approximately 10(-8) mol/L) and in a time-dependent fashion, directly inhibited the activation in vitro of human neutrophils, eosinophils, and monocytes. Peripheral blood leukocytes were incubated with the synthetic chemoattractant, formyl-methionyl-leucyl-phenylalanine (at an optimal concentration of 10(-8) mol/L), and activation was assessed by measuring increases in the percentages of complement and IgG (Fc) rosettes as well as the enhanced capacity of these cells to kill target organisms (schistosomula of Schistosoma mansoni). DSCG at a concentration of 10(-7) mol/L totally inhibited both the formyl-methionyl-leucyl-phenylalanine-induced enhancement of complement and IgG rosettes, as well as increased schistosomular killing. These observations indicate that DSCG directly inhibits the secretory properties of inflammatory cells and that in turn might have important implications in modulating mechanisms contributing to the inflammatory component of asthma and allergic disease. It may also help to explain why compounds with considerably greater mast cell stabilizing properties than DSCG have been so disappointing when they are evaluated clinically.