T helper 2 biased de novo immune response to Keyhole Limpet Hemocyanin in humans.

BACKGROUND: Allergen-specific T helper 2 (Th2) cells play an important role in the pathogenesis of atopic disorders. To date, no model system exists in humans that would allow the monitoring of a developing de novo Th2 immune response in vivo. OBJECTIVE: The aim of the experiment was to establish an immunization protocol inducing a de novo Th2 response in humans using Keyhole Limpet Hemocyanin (KLH) as neo-antigen. METHODS: The double-blind placebo-controlled, parallel-group study was conducted in two groups of subjects (16 healthy volunteers and 16 patients with allergic rhinitis). Subjects received three i.m. injections of 100 microg KLH adsorbed to aluminium hydroxide or matching placebo (alum alone) in intervals of 2 weeks. On day 43, KLH alone (10 microg) was given intra-dermally (i.d.) to all subjects to assess immediate and late-phase skin responses. RESULTS: The immunization protocol was well tolerated, highly specific and efficient. Antigen-specific production of Th2-cytokines (mainly IL-5 and IL-13) by PBMCs suggested a Th2 pattern, as did the presence of KLH-specific IgG4 in sera. Intra-dermal KLH challenge induced an immediate-type of response predominantly in atopic subjects followed by a late-phase skin reaction. The latter was accompanied by the presence of IgE(+) cells, eosinophils and a strong up-regulation of IL-4 and IL-13 along with the absence of Th1 transcripts in biopsies taken from the site of antigen challenge. IL-17 and IL-22 transcripts were detected only in healthy subjects skin following KLH challenge, while IL-1beta and IL-33 expression did not differ between the healthy and the atopics. CONCLUSIONS: The immunization protocol resulted in the elicitation of a local and peripheral Th2 immune response in both healthy and atopic individuals. This may permit the investigation and monitoring of novel immunomodulatory strategies aiming to interfere with Th2 responses in man. The relevance of lack of Th17 cells in atopic skin in this model remains to be determined.

Nitric oxide interacts with oxygen free radicals to evoke the release of adenosine and adenine nucleotides from rat hippocampal slices.

The present study examined some possible mechanisms underlying the previously demonstrated release of adenosine by nitric oxide (NO) donors. Perfusion with the NO-donor S-nitroso-N-acetyl penicillamine (SNAP; 300 microM) led to a significant increase in the release of [3H]purines from both unstimulated and electrically stimulated hippocampal slices prelabeled with [3H]adenine. The NO-donor also evoked the release of endogenous ATP and ADP from unstimulated slices and, when combined with electrical stimulation, the release of ATP, AMP and adenosine. The SNAP-induced [3H]purine release was calcium-dependent, but not affected by the glutamate receptor antagonists MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]-cyclohepten-5,10-imine;100 nM) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 10 microM). Zaprinast (5 microM), an inhibitor of the cyclic GMP-dependent phosphodiesterase and 8-Br-cyclic GMP (0.01-1 mM) failed to evoke the release of purines, whereas generation of oxygen free radicals by xanthine plus xanthine oxidase did evoke purine release. Coperfusion of SNAP with the free radical scavengers superoxide dismutase (SOD; 60 microg/ml) and catalase (50 microg/ml) reduced or eliminated the ability of the NO-donor to enhance [3H]purine release, but the poly (ADP-ribosyl) synthetase (PARS) inhibitor benzamide (500 microM) did not affect it. These data indicate that NO interacts with superoxide, likely forming peroxynitrite, which subsequently acts to release adenosine and adenine nucleotides from hippocampal tissue.

Release of adenosine from rat hippocampal slices by nitric oxide donors.

In the present study we have used perfused hippocampal slices to examine the hypothesis that nitric oxide (NO) can evoke the release of adenosine from nervous tissue. ATP stores were labeled by incubation with [3H]adenine. Electrical field stimulation at 5 Hz for 5 min evoked the release of 3H-purines, and this was enhanced by the NO donor S-nitroso-N-acetylpenicillamine (SNAP). Stimulation at 10 Hz for 15 min evoked a larger release of 3H-purines, which was enhanced in a concentration-dependent manner by both SNAP and sodium nitroprusside (SNP), with SNP being 100-fold less potent than SNAP. N-Acetylpenicillamine (N-AP) was without effect. SNAP had a markedly reduced, although significant, effect when given in the absence of field stimulation. Using HPLC it was found that SNAP enhanced the release of both endogenous and labeled adenosine in a concentration-dependent manner. At the highest concentration used (1 mM), SNAP increased electrically evoked release of endogenous adenosine 100-fold and unstimulated adenosine release eightfold. The ability of SNAP to enhance adenosine release was eliminated in the added presence of hemoglobin (10 microM), further supporting the proposal that the effects of SNAP were due to the liberation of NO. These data provide direct evidence that NO evokes a concentration-dependent release of adenosine from both stimulated and unstimulated nerves of the hippocampus. It is suggested that such NO-stimulated adenosine release may contribute to some of the reported effects of NO donors in the nervous system.