Up-regulated cytokine-inducible SH2-containing protein expression in allergen-stimulated T cells from hen's egg-allergic patients.

BACKGROUND: Although changes in the fine balance of allergen-specific T cells are crucial in the pathogenesis of allergic diseases, their roles in the allergic reaction to hen's eggs (HE) have not yet been fully analysed. OBJECTIVE: Using microarray technology, allergen-stimulated T cells from HE-allergic children were analysed to identify genes that are specifically up-regulated in these cells. METHODS: RNA from CD4(+) CD14(-) cells, fractionated from allergen-stimulated peripheral mononuclear cells, was analysed using a whole-genome microarray and real-time RT-PCR. The protein expression of selected genes was ascertained by flow cytometry. RESULTS: In microarray analyses of allergen-stimulated T cells, 43 genes were up-regulated in HE-allergic children but not in non-HE-allergic children. Among these, up-regulation of three genes, cytokine -inducible SH2-containing protein (CISH), nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor Z (NFKBIZ) and B-cell CLL/lymphoma 2 (BCL2), was confirmed by real-time quantitative RT-PCR. CISH, but not NFKBIZ or BCL2, showed a significantly higher ratio of antigen-stimulated cell transcription over unstimulated cells in HE-allergic than in non-HE-allergic children (P<0.01). Flow-cytometric analysis revealed that the percentage of CD25(+)CISH(+) cells in CD4(+) cells from patients with HE allergy was significantly higher than that in controls (P<0.01). The expression level of CISH was significantly higher in IL-4(+) Th2 cells than in IFN-gamma(+) Th1 cells. CONCLUSION: We noted that CISH expression in allergen-stimulated CD4(+) T cells from HE-allergic patients was significantly increased in both mRNA and protein levels compared with that from non-HE-allergic children.

Allergen-specific helper T cell response in patients with cow's milk allergy: Simultaneous analysis of proliferation and cytokine production by carboxyfluorescein succinimidyl ester dilution assay.

BACKGROUND: The role of antigen-specific T cells in the allergic reaction to cow's milk or in tolerance induction is not yet fully understood. OBJECTIVE: This study was designed to analyse both cow's milk protein (CMP)-specific T cell proliferation and cytokine production simultaneously in children with cow's milk allergy (CMA) in comparison with subjects with various allergic backgrounds. METHODS: Carboxyfluorescein succinimidyl ester was used to detect cow's milk-specific T cells by flow cytometry. The intra-cytoplasmic cytokine production of these antigen-specific T cells was also analysed. RESULTS: Significant differences of both CMP-specific CD4+ cell proliferation and cytokine production between CMA and non-allergic children were observed. While the proliferative responses of children who recently outgrew CMA were not significantly different from those of patients, the patterns of cytokine production were similar to those of non-allergic children. CONCLUSION: These results suggest that the presence of CMP-specific T cell clones per se does not produce CMA, but that the T-helper type 2-skewed pattern of those T cells is associated with adverse reactions. Although it is not possible to distinguish between individual patients with and without CMA on the basis of CFSE assays, these results contribute to the understanding of the pathogenesis and tolerance induction of CMA.

IgE-binding activity to enzyme-digested ovomucoid distinguishes between patients with contact urticaria to egg with and without overt symptoms on ingestion.

BACKGROUND: We occasionally see egg-allergic children who develop contact urticaria to hen's egg despite the absence of the overt symptoms on ingestion. The mechanisms remain to be elucidated. METHODS: Twenty-one subjects with positive reactions to 20-min patch tests for egg-white antigens were divided into subgroups with positive (n = 10) and negative (n = 11) results to oral challenge tests by the same antigens. We measured IgE antibody for egg white and its components, and IgE-binding activities to digestive enzyme-treated ovomucoid by RAST inhibition. RESULTS: There were no significant differences in IgE antibody titers to egg white (positive vs negative: 30.3% vs 15.3%, P=0.130), ovomucoid (21.5% vs 10.2%, P= 0.078), ovotransferrin (9.9% vs 3.7%, P = 0.105), and lysozyme (3.4% vs 2.9%, P=0.944), except ovalbumin (16.8% vs 5.6%, P=0.024), between the positive and negative subjects in the provocation tests. In contrast, the concentration (1.93 microg/ml) of pepsin-treated ovomucoid needed for 50% RAST inhibition in the challenge-positive subjects was significantly (P=0.0003) lower than that (114.9 microg/ml) of negative subjects. Similar but less significant differences were obtained when ovomucoid fragments treated with chymotrypsin (0.91 microg/ml vs 6.86 microg/ml, P=0.014) and trypsin (0.75 microg/ml vs 4.67 microg/ml, P= 0.041) were used as inhibitors. CONCLUSIONS: We suggest that IgE antibodies from subjects showing contact urticaria despite the absence of reactions to the ingestion of egg white recognize the epitope(s) unstable to digestive enzymes.

Losartan and captopril follow different mechanisms to decrease pressor responses in the pithed rat.

1. We investigated the effects of losartan and captopril on noradrenaline (NA) release and vascular reactivity to NA in the pithed rat. 2. The pressor responses to sympathetic nerve stimulation (SNS) before and after i.v. administration of captopril (1 mg/kg), losartan (1 and 10 mg/kg), sodium nitroprusside (SNP: 5 micrograms/kg per min), losartan (1 mg/kg)+captopril (1 mg/kg), captopril (1 mg/kg) + losartan (1 mg/kg) or the bradykinin B2 receptor antagonist HOE 140 (1 mg/kg)+captopril (1 mg/kg) were measured. Plasma NA concentrations were measured during 60 s SNS before and after losartan (1 mg/kg), captopril (1 mg/kg), SNP (5 micrograms/kg per min) or HOE 140 (1 mg/kg)+captopril (1 mg/kg). Pressor responses to exogenous NA were measured before and after administration of losartan (1 mg/kg), captopril (1 mg/kg), HOE 140 (1 mg/kg) + captopril (1 mg/kg) or the nitric oxide synthase (NO) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) + captopril (1 mg/kg). 3. Captopril, losartan and SNP decreased frequency-response curves to a similar extent. The captopril-induced decrease in pressor responses to SNS was restored by pretreatment with HOE140. Adding captopril to losartan decreased the curve more than did adding losartan to captoprill. Both losartan, captopril and HOE 140 + captopril significantly decreased the plasma NA concentration after SNS (34.1 +/- 5.0, 27.4 +/- 2.6 and 41.4 +/- 8.1%, respectively). Sodium nitroprusside did not change the plasma NA concentration after SNS (3.8 +/- 28.2%). The dose-response curves to i.v. NA were not affected by losartan, but were significantly decreased by captopril. However, responses to NA that were reduced by captopril were restored to control values by pretreatment with HOE 140 or L-NAME. 4. We suggest that both losartan and captopril decrease pressor responses to SNS by inhibiting NA release from sympathetic nerve endings; however, captopril also decreases 'vascular reactivity' to NA, which is mediated by nitric oxide produced by activation of the bradykinin B2 receptors.

Effects of adrenaline infusion on plasma lipids and noradrenaline levels in rabbits with adriamycin-induced cardiomyopathy.

1. We investigated the acute effects of adrenaline infusion on plasma lipid levels in vehicle- and adriamycin-treated rabbits. Lipids were measured before and 30 and 60 min after the commencement of continuous intravenous administration of adrenaline (0.06 microgram/kg per min) or saline in pentobarbital-anaesthetized rabbits. 2. Adrenaline infusion significantly increased plasma free fatty acid (P < 0.05) and noradrenaline (NA) levels (P < 0.05) in vehicle-treated control rabbits, but not in adriamycin-treated rabbits. However, adrenaline had no effect on plasma total cholesterol, free cholesterol, high-density lipoprotein-cholesterol, triglyceride or phospholipid levels. 3. Pretreatment with propranolol almost completely inhibited increased plasma free fatty acid and NA levels associated with adrenaline infusion, suggesting that adrenaline increases plasma free fatty acid and NA levels via the stimulation of beta-adrenoceptors in vehicle-treated rabbits. 4. It is suggested that both the production of plasma free fatty acids and the release of NA via the activation of beta-adrenoceptors is reduced in rabbits with adriamycin-induced cardiomyopathy. This may be related to the down-regulation of beta-adrenoceptors caused by elevated plasma NA levels induced by cardiac failure.

Simultaneous evaluation of left- and right-sided heart pumping function during dynamic leg exercise in patients with mild chronic congestive heart failure, with special reference to afterload and plasma noradrenaline.

We simultaneously measured increases in mean pulmonary capillary wedge pressure (delta PCW), mean right atrial pressure (delta RA), and cardiac index (delta CI) in response to dynamic leg exercise in 81 patients with mild congestive heart failure to clarify the relationship between the left-sided and right-sided pumping function of the heart. The ratio of delta CI to delta PCW was used as an index of left-sided heart performance and the delta CI/delta RA as an index of right-sided heart performance. We also determined systemic vascular resistance, as an index of afterload on the left heart; pulmonary vascular resistance, as an index of afterload on the right heart; and the plasma level of noradrenaline before and during dynamic leg exercise. Patients with delta CI/delta PCW > 0.181/ min/m2 per mmHg were regarded as having a well functioning left heart, and the patients with delta CI/delta PCW < or = 0.181/min/m2 per mmHg as having a poorly functioning left heart. Patients with delta CI/delta RA > 0.3111/min/m2 per mmHg were regarded as having a well functioning right heart, and those with delta CI/delta RA < or = 0.311/l/min/m2 per mmHg as having a poorly functioning right heart. Patients were classified into three groups: well functioning left and right heart (normal group; n = 40), poorly functioning left and right heart (bilateral group; n = 34), and poorly functioning left heart and well functioning right heart (left-sided group; n = 7). The systemic vascular resistance index decreased during leg exercise in all patients. The decrease was smaller in the bilateral group and the left-sided group than in the normal group. The pulmonary vascular resistance index increased during exercise in the bilateral group but was unchanged in the normal group and the left-sided group. The plasma level of noradrenaline increased during exercise in all patients, but the increase was greater in the bilateral and left-sided groups than in the normal group. Pretreatment with phentolamine, an alpha-adrenoceptor antagonist, inhibited the increase in the pulmonary vascular resistance index and restored the decrease in the systemic vascular resistance index during exercise in the bilateral group. Our results showed that systemic vascular resistance, which represents afterload on the left heart, increased in the presence of impaired left-sided heart pumping function and pulmonary vascular resistance, which represents afterload on the right heart, increased in the presence of impaired right-sided heart pumping function. The inhibited decrease in systemic vascular resistance and the increase in pulmonary vascular resistance during exercise were associated with alpha-adrenoceptor-mediated vasoconstriction caused by the increase in the plasma level of noradrenaline.

Modulation of noradrenaline release via activation of presynaptic beta-adrenoceptors in rabbits with adriamycin-induced cardiomyopathy.

We investigated the role of beta-adrenoceptors at postganglionic sympathetic nerve endings in noradrenaline release in rabbits with cardiomyopathic congestive heart failure produced by adriamycin (1 mg/kg, I.V., twice a week for 8 weeks). Plasma noradrenaline levels were measured before, 30 min after, and 60 min after the start of continuous intravenous administration of adrenaline (0.06 micrograms/kg/min) in adriamycin-treated and vehicle-treated rabbits in anesthetized condition and pithed condition with electrically stimulated sympathetic outflow (3 Hz, 1 ms square wave pulse, 90 V). In both the anesthetized and pithed conditions, adrenaline increased plasma noradrenaline levels in vehicle-treated rabbits. However, in the adriamycin-treated rabbits, adrenaline had no effect on the plasma noradrenaline level. Pretreatment with propranolol (0.2 mg/kg, bolus I.V. + 0.1 mg/kg/hr, continuous infusion) almost completely abolished the rise in plasma noradrenaline associated with adrenaline infusion in vehicle-treated rabbits. These results suggest that in rabbits with adriamycin-induced cardiomyopathy, the noradrenaline release from the sympathetic nerve endings via the activation of presynaptic beta-adrenoceptors is reduced. This might be due to down-regulation of presynaptic beta-adrenoceptors caused by the elevated plasma noradrenaline due to cardiac failure. However, other possibilities such as reduced affinity or impaired signal transduction cannot be excluded.

The effects of phentolamine and nitroglycerin on right-sided hemodynamics in cardiac patients can be explained by a shift of the systemic venous return curve and right-ventricular output curve.

The present study investigated the effects of phentolamine (PH) and nitroglycerin (NG) on the hemodynamics of the right heart in patients with cardiac disease. The patients were divided into a well-functioning left heart group (W group, n = 15) and a poorly-functioning left heart group (P group, n = 15). Right cardiac hemodynamic parameters and plasma noradrenaline (NA) and adrenaline (A) concentrations were measured before and after administering PH (0.1 mg/kg, i.v.) or NG (0.6 mg, sublingual). In a parallel animal study we obtained a systemic venous return curve by measuring mean circulatory pressure (MCP), mean right atrial pressure (RAP) and cardiac output, before and after administering PH (0.1 mg/kg, i.v.) or NG (12.5 micrograms/kg, i.v.) to anesthetized open-chest dogs (n = 14). We used MCP data (W group: 7.5 mmHg, P group: 10 mmHg) obtained in a separate series of human studies in our laboratory. We constructed the venous return curve by connecting the MCP point on abscissa with the cardiac index (CI)-RAP plot obtained in the clinical study. We also constructed the right ventricular output curve by connecting the point of -2 mmHg on the abscissa with the CI-RAP plot. We obtained the following results: (1) PH shifted the CI-RAP plot to the left and upwards, while NG shifted the CI-RAP plot to the left almost horizontally on the CI-RAP plane, where CI was plotted on ordinate and RAP on abscissa. The length [formula: see text] C = control point, PH = point after PH) of the shift of CI-RAP plot due to PH was greater in the P group than in W group, while there was no difference in the length [formula: see text] C = control point, NG = point after NG) of the shift of CI-RAP plot due to NG between P and W groups. (2) Both PH and NG significantly elevated plasma NA and A concentrations in both the W and P groups. In the P group, PH increased the plasma NA concentration significantly more than did NG, but both drugs increased plasma A concentration to a similar extent. (3) Both PH and NG significantly decreased the mean pulmonary arterial pressure with NG doing so significantly more than PH.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of guanfacine, bunazosin, atenolol and nadolol on blood pressure and plasma noradrenaline responses to cold pressor testing.

1. The role of the presynaptic adrenoceptor subtypes in man was investigated based on observation of the changes in blood pressure (delta BP) and plasma noradrenaline concentration (delta NA) with the cold pressor test (CPT). 2. The CPT was well reproducible for BP and NA when performed at a 2 week interval in patients with mild hypertension. 3. After administration for 4 weeks, guanfacine (Gf; alpha 2-adrenoceptor agonist) decreased the delta NA response to CPT. 4. After administration for 2 or 4 weeks, bunazosin (Bu; alpha 1-adrenoceptor antagonist) atenolol (At; beta 1-adrenoceptor antagonist) and nadolol (Nd; non-selective beta-adrenoceptor antagonist) did not affect the delta NA response to CPT. 5. Both Gf and Bu decreased the systolic blood pressure response (delta SBP) to CPT after 4 weeks of the administration. Neither At nor Nd significantly changed the delta SBP response to CPT. 6. It is likely that Gf stimulated the presynaptic alpha 2 adrenoceptors at the sympathetic nerve endings as well as the central alpha 2 adrenoceptors, inhibiting the release of noradrenaline. It is unlikely that Bu, At and Nd exerted any clearly defined action on the presynaptic adrenoceptors in human hypertensive subjects.

Cellular mechanisms of nitrate action.

It is now generally accepted that organic nitrates generate their vasodilator action via production of nitric oxide. However, the cellular location of the metabolic enzyme(s) responsible for such conversion has not been defined. We examined the production of nitric oxide, via chemiluminescence detection, by various cellular fractions of the bovine coronary artery. We were able to show that the highest activity resides in the plasma membrane. Future isolation and characterization of such metabolic systems will greatly assist our understanding of nitrate action and tolerance. Several cellular mechanisms for nitrate tolerance have been proposed. Among the most popular theories is the "intracellular sulfhydryl depletion hypothesis" originally proposed by Needleman et al. The primary supportive data for this mechanism are that exogeneously added thiols (such as N-acetylcysteine) can potentiate the in vivo activity of nitroglycerin and can partially reverse nitrate tolerance. We showed that a cellular-impermeant thiol, viz: glutathione, can also potentiate the hemodynamic effect of nitroglycerin in rats. We subsequently showed that exogenously administered thiols can promote the formation of vasoactive S-nitrosothiols in blood. Thus, the beneficial effects of thiols on nitrate action might be mediated through an extracellular pathway. Another cellular mechanism for nitrate tolerance suggested that tolerance is caused by an alteration of the enzyme, guanylate cyclase. We showed, however, that blood vessels made tolerant to nitroglycerin remain fully responsive (in terms of in vitro relaxation) toward nitric oxide and S-nitrosothiols. These data showed that, as far as relaxation is concerned, nitrate tolerance did not cause a significant alteration of guanylate cyclase activity toward nitric oxide and S-nitrosothiols.