Immunostimulatory sequence DNA linked to the Amb a 1 allergen promotes T(H)1 cytokine expression while downregulating T(H)2 cytokine expression in PBMCs from human patients with ragweed allergy.

BACKGROUND: Recent studies have demonstrated that bacterially derived immunostimulatory sequences (ISSs) of DNA can activate the mammalian innate immune system and promote the development of T(H)1 cells. Promotion of T(H)1 immunity by means of immunotherapy in allergic patients has led to the alleviation of symptoms that result from allergen-specific T(H)2 responses. OBJECTIVE: Our purpose was to investigate whether the T(H)1-enhancing properties of ISSs could be used to alter the T(H)2-dominated immune response of allergic PBMCs in vitro. METHODS: Ragweed protein-linked ISS (PLI) was generated from a specific, highly active 22-base ISS and Amb a 1, the immunodominant allergen in ragweed pollen, to combine the T(H)1-enhancing properties of ISSs with allergen selectivity, and its activity was investigated in PBMC cultures from subjects with ragweed allergy. RESULTS: PLI was markedly successful at reversing the dominant allergen-induced T(H)2 profile while greatly enhancing IFN-gamma production. Delivering ISSs in a linked form proved to be much more effective at modulating the resulting cytokine profile than delivering free ISSs in a mixture with unlinked Amb a 1. PLI also demonstrated cytokine-modulating properties, even when used to stimulate cells that had already been primed for 6 days with Amb a 1. The antigen specificity of the action of PLI was confirmed by the observations that PLI enhances Amb a 1--specific T-cell proliferation. CONCLUSION: These data indicate that delivery of ISSs within an antigen-specific context exhibits potent cytokine-modulating activity and, combined with its reduced allergenicity, makes this molecule a strong candidate for use in improved immunotherapy applications.

Case report of venom immunotherapy for a patient with large local reactions.

BACKGROUND: Inadvertent Hymenoptera stings reportedly elicit large local reactions in up to 17% of the general population. Current practice parameters do not recommend venom immunotherapy (IT) for these cases. OBJECTIVE: The goal of this case study was to investigate the clinical and immunologic consequences of venom IT in a newly sensitized individual with large local reactions using an intentional sting challenge before and after treatment to document changes in reaction severity. METHODS: A 47-year-old man became honeybee venom (HBV)-allergic with progressively larger reactions at honeybee sting sites with subsequent stings. Then, a sting on his forefinger produced a large (62 cm) local reaction with swelling throughout the arm that persisted for more than 4 weeks with severe pain. He refused steroid therapy and voluntarily requested venom IT with honeybee-sting challenges to monitor clinical parameters and immunologic changes in his skin and serum before and 7 months post-HBV maintenance IT. RESULTS: A single pre-IT bee sting challenge produced an 11.4-cm wheal with 13-cm erythema at the sting site after 15 minutes, followed by several weeks of edema that involved the entire arm. After rapid escalation of venom IT to maintenance in 7 weeks, a post-maintenance IT sting challenge with two honeybees produced a 3-cm diameter erythema with no wheal at 15 minutes and no late-phase induration. Complete loss of any visible reaction at the field sting site resulted after 13 months of maintenance venom IT. A HBV-specific IgG antibody level >3.5 microg/mL and IgG/IgE antibody molar ratio >500 persisted over the period of venom IT, with venom skin reactivity diminishing 100-fold. CONCLUSIONS: These results support venom IT use in the treatment of Hymenoptera venom-sensitive individuals who experience large local reactions and are at risk for repetitive inadvertent stings.

Recombinant allergens with reduced allergenicity but retaining immunogenicity of the natural allergens: hybrids of yellow jacket and paper wasp venom allergen antigen 5s.

The homologous venom allergen Ag 5s from the yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis) have 59% sequence identity of their respective 204 and 205 amino acid residues, and they have low degrees of antigenic cross-reactivity in insect allergic patients and in animal models. Hybrids containing different segments of these two vespid Ag 5s were expressed in yeast. Circular dichroism spectroscopy suggests the hybrids to have the secondary structure of natural Ag 5. Inhibition ELISA with human and murine Abs suggests the hybrids to have the discontinuous B cell epitopes of the natural Ag 5 but with an altered epitope density. The hybrids were immunogenic in mice for B and T cell responses to both Ag 5s. The N-terminal region of Ag 5 was found to contain its dominant B cell epitope(s). Hybrids containing 10-49 residues of yellow jacket Ag 5 showed 100- to 3000-fold reduction in allergenicity when tested by histamine release assay with basophils of yellow jacket-sensitive patients. Our findings suggest that hybrids represent a useful approach to map the discontinuous B cell epitope-containing regions of proteins. They also suggest that Ag 5 hybrids may be useful immunotherapeutic reagents in man.

Insect sting allergy with negative venom skin test responses.

BACKGROUND: In our 1976 controlled venom immuno rapy trial, 33% of 182 patients with a history of systemic reactions to insect stings were excluded because of negative venom skin test responses. There have been reports of patients with negative skin test responses who have had severe reactions to subsequent stings. OBJECTIVE: Our aim is to increase awareness about the patient with a negative skin test response and insect sting allergy and to determine the frequency and significance of negative skin test responses in patients with a history of systemic reactions to insect stings. METHODS: We prospectively examined the prevalence of negative venom skin test responses in patients with a history of systemic reactions to stings. In patients who gave informed consent, we analyzed the outcome of retesting and sting challenge. RESULTS: Of 307 patients with positive histories screened for our sting challenge study, 208 (68%) had positive venom skin test responses (up to 1 microg/mL concentration), and 99 (32%) had negative venom skin test responses. In 36 (36%) of the 99 patients with negative skin test responses, the venom RAST result was a low positive (1-3 ng/mL), or repeat venom skin test responses were positive; another 7 (7%) patients had high venom-specific IgE antibody levels (4-243 ng/mL). Notably, 56 (57%) of 99 patients with positive histories and negative skin test responses had negative RAST results. In patients with positive skin test responses, sting challenges were performed in 141 of 196 patients, with 30 systemic reactions. Sting challenges were performed on 37 of 43 patients with negative skin test responses and positive venom-specific IgE and in 14 of 56 patients with negative skin test responses and negative RAST results. There were 11 patients with negative skin test responses who had systemic reactions to the challenge sting: 2 had negative RAST results, and 9 had positive RAST results at 1 ng/mL. The frequency of systemic reaction was 21% in patients with positive skin test responses and 22% in patients with negative skin test responses (24% in those with positive RAST results and 14% in those with negative RAST results). CONCLUSIONS: Venom skin test responses can be negative in patients who will subsequently experience another systemic sting reaction. Venom skin test responses are negative in many patients with a history of systemic allergic reactions to insect stings and may be associated with positive serologic test responses for venom-specific IgE antibodies (sometimes strongly positive results). Venom skin test responses should be repeated when negative, along with a serologic IgE antivenom test. Better diagnostic skin test reagents are urgently needed.

Conjugation of immunostimulatory DNA to the short ragweed allergen amb a 1 enhances its immunogenicity and reduces its allergenicity.

BACKGROUND: Allergen immunotherapy is inconvenient and associated with the risk of anaphylaxis. Efforts to improve the safety of immunotherapy by means of chemical modification of allergens have not been successful because it greatly reduced their antigenicity. Recently, immunostimulatory DNA sequences (ISS or CpG motifs) have been shown to act as strong T(H)1 response-inducing adjuvants. OBJECTIVE: We sought to determine whether conjugation of ISS to the major short ragweed allergen Amb a 1 results in enhanced immunotherapeutic potential in mice and decreased allergenicity in human subjects. METHODS: A 22-mer ISS oligodeoxynucleotide (ISS-ODN) was coupled to Amb a 1 and used for immunization of mice, rabbits, and monkeys. RESULTS: In mice the Amb a 1-ISS conjugate induced a T(H)1 response (IFN-gamma secretion), whereas Amb a 1 induced a T(H)2 response (IL-5 secretion). The T(H)1 response was not observed with an Amb a 1-non-ISS conjugate. Coinjection of Amb a 1 with ISS-ODN was much less effective in inducing a T(H)1 response. In mice primed for a T(H)2 response, injection with Amb a 1-ISS conjugate induced a de novo T(H)1 response and suppressed IgE antibody formation after challenge with Amb a 1. Amb a 1-ISS conjugate induced high-titer anti-Amb a 1 IgG antibodies in rabbits and cynomolgus monkeys, whereas Amb a 1 alone or Amb a 1 coinjected with ISS-ODN did not induce a detectable response. Amb a 1-ISS conjugate was less allergenic than Amb a 1 alone, as shown by a 30-fold lower histamine release from human basophils of patients with ragweed allergy, whereas mixing ISS-ODN with Amb a 1 did not reduce histamine release. CONCLUSION: Amb a 1-ISS conjugate has an enhanced T(H)1-biased immunogenicity and reduced allergenicity. It may offer a more effective and safer approach for allergen immunotherapy than currently available methods.

Survey of patients after discontinuing venom immunotherapy.

BACKGROUND: Venom immunotherapy rapidly reduces the risk of a systemic sting reaction in adults from 30% to 70% to less than 2%. When venom immunotherapy is stopped after 5 years or longer, the risk of a systemic sting reaction is 5% to 15% during the first few years after stopping treatment. It is uncertain whether systemic sting reactions will occur more than 5 years after discontinuing venom immunotherapy and whether treatment can be safely stopped in some patients after less than 5 years. OBJECTIVE: The purpose of this study is to estimate the risk of systemic reaction to a sting 10 years after discontinuing treatment and the relative risk after 3 years of treatment compared with that after 5 years or more of treatment. METHODS: Among all patients who had venom immunotherapy at our center, we identified 395 patients who stopped treatment: some had dropped out of therapy early (6-24 months), some stopped after 3 to 4 years, and most completed 5 years or more of venom immunotherapy and were advised to stop by the allergist (many as part of our reported studies of discontinuing venom immunotherapy). RESULTS: Contact was made with 194 patients, including telephone interviews for sting history and requests to visit the office for skin testing and blood sampling. Of these patients, 74 had been included in our original study of patients who had 5 years or more of venom immunotherapy and had sting challenges after 1 to 5 years off venom immunotherapy, as previously reported. Of the 74 in that original study, 61 were reached for this survey, and 30 reported recent stings, with 5 systemic sting reactions. Another 133 patients who had stopped venom immunotherapy were reached: 82 had 5 or more years of venom immunotherapy, 20 had 3 to 4 years of venom immunotherapy, and 31 had less than 2 years of venom immunotherapy. Of 51 patients stung from this group, 27 had 5 or more years of venom immunotherapy (no systemic sting reactions), and 24 had less than 5 years of venom immunotherapy (3 systemic sting reactions). We have now observed a total of 113 patients who had 5 or more years of venom immunotherapy and were stung after stopping. Sixteen (14%) had systemic sting reactions; most were mild, but 4 were severe. Systemic sting reactions occurred in 12 (10.7%) of 112 patients stung in the first 4 years off venom immunotherapy and 5 (10%) of 50 stung more than 5 years off venom immunotherapy. In 4 of 8 patients with current systemic sting reactions, the skin test response was negative, although the venom-IgE response was positive at the previous encounter. All systemic sting reactions were similar in pattern and severity to prevenom immunotherapy reactions in the same patient. CONCLUSIONS: We conclude that the risk of systemic sting reactions when venom immunotherapy is stopped after 5 years or longer remains in the reported range of 5% to 15% in the 5 to 10 years after stopping venom immunotherapy. This risk of systemic sting reactions does not seem to decrease over time, unlike the progressive decline in immunologic markers (skin test and venom-IgE responses). To prospectively assess the risk of recurrent systemic sting reactions, there is a need for sting challenge studies of patients who have been off venom immunotherapy for 5 to 10 years and patients who have stopped venom immunotherapy after just 3 to 4 years treatment.

Relationship between histamine and physiological changes during the early response to nasal antigen provocation.

To investigate the temporal relationships of mediator release and physiological changes during the early response to allergen, we challenged allergic individuals intranasally with antigen and followed their responses. This was done by using small filter paper disks to challenge one nostril and collect secretions from both the challenged and the contralateral nostril, thus enabling us to evaluate the nasonasal reflex. There was a significant increase in sneezing after allergen challenge that peaked within 2 min and returned to baseline. The weights of nasal secretions as well as nasal symptoms increased immediately and remained significantly elevated for 20 min in both nostrils. Nasal airway resistance increased slowly, reaching its peak at approximately 6 min after challenge on the ipsilateral side, but it did not change on the contralateral side. Histamine levels peaked 30 s after removal of the allergen disk on the side of challenge, whereas albumin levels peaked after those of histamine. Lactoferrin paralleled the increase in secretion weights and occurred in both nostrils. Increasing doses of antigen produced dose-dependent increases in all parameters, whereas control challenges produced no response. These studies describe a human model for the evaluation of the allergic response that is capable of simultaneously measuring mediator release and the physiological response, including the nasonasal reflex. This model should prove useful in studying the mechanism of allergic rhinitis in humans.

Daily variations of serum diamine oxidase and the influence of H1 and H2 blockers: a critical approach to routine diamine oxidase assessment.

OBJECTIVE AND DESIGN: Histamine in food has been shown to induce intolerance reactions mimicking food allergy. These reactions seem to be due to impaired histamine metabolism caused by reduced diamine oxidase activity. To validate routine serum diamine oxidase assessment, daily variations of diamine oxidase were evaluated. METHODS: Blood was drawn from each of 20 healthy volunteers (10 female, 10 male; mean age 32.5 years) every 2 h from 9 a.m. to 5 p.m., and diamine oxidase activity was measured using the C14 putrescine method. To assess possible influences of H1 and H2 blockers on diamine oxidase activity, diphenhydramine, ketotifen, cimetidine, and ranitidine were incubated at pharmacologic concentrations with human placental diamine oxidase (identical to neutrophilic and eosinophilic diamine oxidase). Inhibition of diamine oxidase activity was calculated as the percentage of inhibition versus control. In addition, the known diamine oxidase inhibitors, dihydralazine and aminoguanidine, were used as positive controls. RESULTS: Serum diamine oxidase levels showed no significant daily variations (0.041 +/- 0.025; 0.037 +/- 0.022; 0.041 +/- 0.023; 0.040 +/- 0.023; 0.038 +/- 0.025 nKat/l) and no significant sex differences (female 0.040 +/- 0.028 nKat/l versus male 0.039 +/- 0.019 nKat/l). Antihistamines had no influence on diamine oxidase activity except for cimetidine, which caused 25% inhibition at the highest dose tested ( p < 0.0002) (positive control: aminoguanidine 85% inhibition (p< 0.0001), dihydralazine 68% inhibition (p<0.0001)) and diphenhydramine, which caused 19% increase (p<0.0001) of enzyme activity. CONCLUSION: Serum diamine oxidase levels do not show daily variations allowing assessment anytime during office hours. However, diagnostic interpretation of serum diamine oxidase levels may be difficult.

Co-regulation of antigen-specific T lymphocyte responses by type I and type II cyclic AMP-dependent protein kinases (cAK).

While a differential sensitivity to cyclic AMP (cAMP)-mediated signaling between Th1 and Th2 cells has been hypothesized, differential activity of downstream signaling through cAMP-dependent protein kinase (cAK) isoforms remains unexplored. We herein report the effects of type 1- and type 2-specific cAK agonists and antagonists on proliferative responses and cytokine generation from ragweed-driven peripheral blood mononuclear cells (PBMCs) and Amb a 1-specific Th1 and Th2 clones. Rp-8-Cl- and Rp-8-CPT-cAMP were utilized as single agent antagonists of cAKI and cAKII, respectively; 8-AHA-cAMP, with and without 8-PIP-cAMP, and 8-CPT-cAMP, with and without 6-Bnz-cAMP, were used as synergistic agonist pairs specific for the cAKI and cAKII, respectively. Activation of either cAKI or cAKII individually was ineffective in down-regulating proliferative responses of PBMCs or T cell clones; concentration-response curves for the Th1 and Th2 clones were identical. Moreover, inhibition of either cAKI or cAKII individually was ineffective in overcoming the down-regulatory effects of phosphodiesterase inhibition. Activation of either cAKI or cAKII individually was ineffective in down-regulating proinflammatory cytokine generation from T cell clones (interleukin-4 from Th2; interferon-gamma from Th1). However, concurrent activation of both cAKI and cAKII produced down-regulatory effects equivalent to those of the phosphodiesterase inhibitor on both proliferation and cytokine generation. These data suggest a critical role for concurrent activation of cAKI and cAKII in the functional efficacy of antigen-driven downstream signaling due to elevations of intracellular cAMP and argue against differential regulation of Th1 and Th2 responses by cAK subtypes.

Intranasal salmeterol inhibits allergen-induced vascular permeability but not mast cell activation or cellular infiltration.

BACKGROUND: Salmeterol is a long-acting beta2-adrenergic agonist that is widely used in the treatment of asthma. It has been suggested that non-bronchodilator actions of salmeterol may contribute to its efficacy. OBJECTIVE: To further evaluate the potential non-bronchodilator actions of salmeterol in vivo, using a model of nasal challenge with allergen. METHODS: Twelve asymptomatic subjects with seasonal allergic rhinitis participated in a randomized, double-blind, placebo-controlled crossover trial of the effects of a single dose of 100 microg of salmeterol on the response to allergen challenge. Sneezing and symptom scores, and levels of histamine and albumin in nasal lavages, were measured throughout the protocol. Concentrations of tryptase, prostaglandin D2 and lysozyme were measured during the acute allergic response, while levels of IL-3, IL-5 and IL-8 were measured at later time points. Numbers of eosinophils and of total white blood cells were also recorded. RESULTS: Salmeterol did not affect sneezing or symptom scores at any point. During the immediate response to allergen challenge, mast cell activation, reflected by concentrations of histamine, tryptase and prostaglandin D2, and serous glandular secretion, assessed by measurements of lysozyme, were unaffected by salmeterol treatment but vascular permeability, reflected by concentrations of albumin in nasal lavages, was significantly reduced. At later time points, salmeterol had no effect on levels of histamine or albumin and did not affect cellular infiltration. Concentrations of IL-3, IL-5 and IL-8 were not increased by allergen challenge in these subjects, so the effects of salmeterol could not be evaluated. CONCLUSIONS: Treatment with a single dose of salmeterol had no effect on activation of mast cells or cellular infiltration but inhibited vascular permeability. The ability of salmeterol to inhibit antigen-induced vascular permeability may contribute to its therapeutic efficacy in asthma.

IL-4 secretion and histamine release by human basophils are differentially regulated by protein kinase C activation.

The role of protein kinase C (PKC) activation was investigated in the secretion of interleukin-4 (IL-4) protein by human basophils. Phorbol myristate acetate (PMA) induced little to no detectable IL-4 protein in culture supernatants, despite being a potent secretagogue of histamine release by basophils. In fact, the secretion of IL-4 by basophils stimulated with ionomycin alone was down-regulated (30-70%) with the simultaneous addition of PMA. In peripheral blood lymphocytes (PBL), however, the combination of ionomycin and PMA were highly synergistic, resulting in maximum IL-4 release but at a slower rate. PKC inhibitors reversed these effects on IL-4 secretion. In sharp contrast to its inhibitory effect on IL-4 protein secretion, PMA did not block the accumulation of IL-4 mRNA in basophils activated by ionomycin. These data suggest that there are marked differences in the regulatory processes for IL-4 transcription, translation, or secretion between basophils and lymphocytes.

Effects of peptide therapy on ex vivo T-cell responses.

BACKGROUND: Peptide therapy targets T cells directly with short peptides containing multiple T-cell receptor epitopes. Murine studies suggest T-cell anergy as the mechanism of action; however, changes in T-cell cytokine profiles may be more relevant in human beings. OBJECTIVE: We sought to study the effects of peptide therapy on ex vivo antigen-specific T-cell responses. METHODS: Antigen-specific T-cell lines were generated from subjects enrolled in a double-blind, placebo controlled, two-dose study of the ALLERVAX CAT therapeutic, containing Fel d 1 peptides (ImmuLogic Pharmaceutical Corp., Waltham, Mass.) (n = 7, 8, and 7, respectively, for groups receiving placebo, 75 microg, or 750 microg). Each subject had three lines propagated before and after receiving peptide therapy; antigens used were cat hair extract, Fel d 1 peptides, and tetanus toxoid (negative control). Proliferative responses and cytokine generation from each line were assessed after two restimulations with antigen and autologous antigen-presenting cells. RESULTS: The Fel d 1 peptide lines showed a dose-dependent decrease of IL-4 production (p = 0.02 and 0.025, respectively, for the 750 microg group vs both the 75 microg and placebo groups). IL-4 production from the cat hair allergen extract lines and interferon-gamma production from both the Fel d 1 peptide lines and cat hair allergen extract lines showed no statistically significant changes. The control tetanus toxoid lines showed no changes in cytokine production; there were no significant changes in proliferation with any of the antigens in any of the treatment groups. In the clinical arm of the trial, only the 750 microg dose of peptides produced a significant response. CONCLUSIONS: Peptide therapy induces a significant, dose-dependent decrease in peptide-stimulated IL-4 production, consistent with either a shift in T-cell phenotype or peptide-specific T-cell tolerance.

Discontinuing venom immunotherapy: extended observations.

BACKGROUND: Our studies of discontinuing venom immunotherapy after at least 5 years have led to the conclusion that the residual risk of a systemic reaction to a sting was in the range of 5% to 10% in adults, and no severe or life-threatening reaction occurred with 270 challenge stings in 74 patients after 1 to 5 years without venom immunotherapy. OBJECTIVE: The objective of this study was to extend our observation of patients who discontinue venom immunotherapy over 5 to 10 years and to determine which patients are at higher risk for a reaction. METHODS: Patients who discontinued venom immunotherapy were surveyed for 3 consecutive years to determine the frequency of systemic reactions to field stings and the fate of venom sensitivity. The evaluation included the 74 patients previously studied (group 1) and 51 additional patients followed after stopping therapy in our clinical center (group 2). RESULTS: Of the original 74 patients, 11 had field stings again after 3 to 7 years without venom immunotherapy, with one systemic reaction (dyspnea). Of the 51 patients in the other group, 15 were stung, of whom four (26%) had systemic reactions, including respiratory symptoms requiring epinephrine. Review of group 1 and group 2 revealed that half of the patients who had systemic reactions to a sting after stopping venom immunotherapy had a history of a systemic reaction occurring during venom immunotherapy (to an injection or a sting). Systemic reactions occurred in three patients who had negative skin test reactions; all three had very low but detectable venom-specific serum IgE antibody levels as determined by RAST and had a history of systemic reactions during venom immunotherapy. Greater severity of the pretreatment reaction was not associated with higher frequency of reaction to stings after stopping therapy but was associated with greater severity if a reaction did occur. CONCLUSIONS: Venom immunotherapy (yellow jacket/mixed vespid) in adults can be discontinued after 5 to 6 years with a 5% to 10% residual risk of a systemic reaction. Risk factors may include history of a systemic reaction during venom immunotherapy, persistent strongly positive skin test sensitivity, and the severity of the pretreatment reaction.

Differential regulation of IL-4 and IL-13 secretion by human basophils: their relationship to histamine release in mixed leukocyte cultures.

Human basophils are an important source of IL-4, a cytokine that is central to the pathogenesis of allergic inflammation. Recent reports have indicated that these cells also generate IL-13, which shares a number of biologic properties with IL-4. We found basophils to be the major source of IL-13 produced in mixed leukocyte cultures following 20-h activation with a variety of stimuli. While the magnitude of IL-4 protein generated correlated with the percent histamine secreted (r = 0.8; p = 0.007), there was no relationship between the levels of IL-13 detected and the amount of either IL-4 or histamine in cultures activated with IL-3/anti-IgE. The induction of IL-13 secretion also occurred in response to IL-3 alone, without concomitant secretion of either IL-4 or histamine. Although previously shown to inhibit IL-4 secretion, the phorbol ester PMA was a potent stimulus for IL-13 generation from basophils, and this secretion was sensitive to the protein kinase C inhibitor, bisindolylmaleimide. In contrast, bisindolylmaleimide did not prevent cytokine secretion induced by either anti-IgE or IL-3. The immunosuppressant, FK506, while strikingly inhibiting the accumulation of IL-4 mRNA and the secretion of protein in response to IL-3/anti-IgE, had no effect on the generation of IL-13 in these cultures; the resistance was attributed to the IL-3-dependent signaling. Similarly, FK506 had no effect on the secretion of IL-13 in basophil cultures stimulated with PMA. This study suggests that multiple intracellular mechanisms control the generation of IL-13 in basophils, some of which are distinct from those regulating IL-4.

SB 207499 (Ariflo), a potent and selective second-generation phosphodiesterase 4 inhibitor: in vitro anti-inflammatory actions.

First-generation phosphodiesterase 4 (PDE4) inhibitors, such as rolipram, inhibit the activation of immune and inflammatory cells. The clinical use of these compounds is limited by gastrointestinal side effects, such as increased acid secretion and nausea. Consequently, the challenge has been to design novel PDE4 inhibitors that maintain the anti-inflammatory actions of rolipram while achieving an improved side effect profile. Among the first of this new class of PDE4 inhibitors specifically designed to have an improved therapeutic index relative to earlier compounds is SB 207499 (Ariflo) [c-4-cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-r-1-cyclohexanecarboxyl ic acid]. In this study, we compared the anti-inflammatory and gastric secretogogue activities of SB 207499 with those of rolipram. The cellular models used were (1) histamine release from human basophils, (2) tumor necrosis factor-alpha generation in human monocytes, (3) degranulation of human neutrophils, (4) antigen-driven proliferation and cytokine synthesis from human T cells and (5) acid secretion from isolated rabbit gastric glands. SB 207499 inhibited the activation of a variety of immune and inflammatory cells in a concentration-dependent manner: (1) histamine release in basophils [-log IC25 = 6.6 +/- 0.3 vs. 8.0 for (R)-rolipram], (2) lipopolysacchride-induced TNF-alpha formation in monocytes [-log IC50 = 7.0 +/- 0.1 vs. 7.2 +/- 0.1 for (R)-rolipram], (3) fMLP-induced degranulation in neutrophils [-log IC15 = 7.1 +/- 0.2 vs. 6.4 +/- 0.5 for (R)-rolipram], (4) house dust mite induced-proliferation of peripheral blood mononuclear cells [-log IC40 = 6.5 +/- 0.3 vs. 6.4 +/- 0.3 for (R)-rolipram] and (5) ragweed-induced production of interferon-gamma [-log IC50 = 5.4] and interleukin-5 [-log IC50 = 5.0]. Although SB 207499 inhibits the activation of a variety of immune and inflammatory cells with a potency equal to that of rolipram, it is > 100-fold less potent than the latter compound as an acid secretagogue [-log EC50 = 6.1 +/- 0.1 vs. 8.3 +/- 0.2 for (R)-rolipram]. Collectively, these data indicate that SB 207499 retains the anti-inflammatory activity of the prototypical PDE4 inhibitor rolipram but is substantially less likely to stimulate gastric acid secretion.

Nasal provocation with allergen induces a secondary serum IgE antibody response.

The study of the IgE response to seasonal antigen exposure is limited by its occurrence once a year and by the variability of patient exposure to pollens. To overcome these problems, we investigated whether nasal challenge with antigen causes an increase in serum anti-ragweed IgE levels. We challenged individuals with ragweed allergy intranasally with nanogram quantities of ragweed antigen extract and measured their serum anti-ragweed IgE levels before and at weekly intervals after challenge. In a series of studies we found that there was a reproducible rise in antigen-specific serum IgE levels beginning the first week after challenge that plateaued at about 180% of baseline levels during the fourth week and remained elevated for 8 weeks. Not all individuals showed this response. The magnitude of the allergen-specific IgE response to nasal challenge appeared to be greater than the response to seasonal exposure. Treatment with intranasal beclomethasone before challenge did not affect the response. The results demonstrate a human in vivo model for the study of the antigen-specific secondary IgE response to allergen.

A double-blind study of the discontinuation of ragweed immunotherapy.

BACKGROUND: Immunotherapy effectively treats the symptoms of allergic rhinitis and improves its pathophysiology. We studied whether the effects of immunotherapy on the early response to nasal challenge with antigen and seasonal symptoms persist after discontinuation. METHODS: Twenty subjects with ragweed allergy who were receiving immunotherapy and who had nasal challenges performed before initiation of treatment were selected. The patients had been receiving maintenance therapy with aqueous ragweed extract at a dose of 12 microg of Amb a 1 equivalent for a minimum of 3 years, at which point they were randomized to receive either placebo injections or to continue with the maintenance dose. Nasal challenges were performed before and 1 year after randomization. Nasal challenges were monitored by counting the number of sneezes and measuring histamine, N-alpha-tosyl-L-arginine methyl ester-esterase activity, and kinins in recovered nasal lavages. In the same year symptom diaries were collected during the ragweed season. RESULTS: The initial immunotherapy significantly reduced responses to nasal challenge in both groups. The group continuing to receive active treatment showed no significant changes from the response before randomization. In contrast, the group randomized to placebo treatment showed a partial return of histamine, kinins, and N-alpha-tosyl-L-arginine methyl ester-esterase in nasal secretions and the numbers of sneezes. IgG antibodies to ragweed declined only in the group switched to placebo treatment. Seasonal rises of IgE antibodies to ragweed did not return during the first season after treatment was stopped. Symptoms reported during the ragweed season were not different between the groups. CONCLUSIONS: One year after discontinuation of ragweed immunotherapy, nasal challenges showed partial recrudescence of mediator responses even though reports during the season appeared to indicate continued suppression of symptoms.

Corticosteroid modulation of human, antigen-specific Th1 and Th2 responses.

Corticosteroids are potent antiinflammatory agents that modulate human T-lymphocyte responses. Controversy remains as to their possible differential effects on Th1 and Th2 subsets. This study explores the kinetics and efficacy of these agents in human, antigen-driven peripheral blood mononuclear cells (PBMCs) and in nontransformed, antigen-specific Th1 and Th2 clones. Ragweed- and tetanus toxoid-driven proliferative responses of PBMCs from dually sensitized individuals were downregulated equally by dexamethasone (inhibitory concentration of 50% [IC(50)] = 3 x 10(-9) and 2 x 10(-9) mol/L, respectively). The addition of dexamethasone as late as 36 hours after ragweed stimulation still resulted in more than 75% inhibition of the proliferative response, whereas the efficacy of dexamethasone was less than 50% when added 24 hours after tetanus toxoid stimulation. Antigen-induced gene expression for proinflammatory cytokines (IL-4, IL-5, IL-13, and interferon-gamma) from PBMCs was also downregulated by dexamethasone. Proliferation of antigen-specific Th1 and Th2 clones was inhibited by several corticosteroids (hydrocortisone < budesonide < dexamethasone; IC(50) = 10(-6) to 10(-8) mol/L), but no significant differences between Th1 and Th2 clones were evident. IC(50) values in the clones were 10-fold greater than in PBMCs. Gene expression and protein secretion for IL-4, IL-13, and interferon-gamma were downregulated in a concentration-dependent manner by each of the corticosteroids in Th1 and Th2 clones. These data suggest that Th1 and Th2 responses are equally affected by corticosteroids.

Differential regulation of human, antigen-specific Th1 and Th2 responses by the B-7 homologues, CD80 and CD86.

A selectivity of B7.1 (CD80) for promoting Th1 responses and B7.2 (CD86) for promoting Th2 responses in the murine system has recently been suggested. The present study explores this hypothesis, using human PBMCs and antigen-specific Th1 and Th2 clones. Proliferative responses of peripheral blood mononuclear cells (PBMCs) from ragweed-allergic, tetanus toxoid-immunized individuals were downregulated by treatment with anti-CD86 in ragweed- and tetanus toxoid-driven cultures (% Inhibition = 55 +/- 4 and 61 +/- 12, respectively; P < 0.03 relative to untreated cultures). Gene expression in PBMCs for interleukin (IL)-4, IL-5, and interferon gamma (IFNgamma), assessed by reverse-transcriptase polymerase chain reaction, was also downregulated by treatment with anti-CD86 in both the ragweed- and tetanus toxoid-driven systems. Neither independent efficacy nor synergy with anti-CD86 was apparent with anti-CD80 treatment; two different anti-CD80 blocking antibodies yielded identical results. Conversely, antigen-specific Th1 and Th2 clones were insensitive to treatment with either anti-CD80, anti-CD86, or a combination of the two. Unaffected parameters included proliferative response (P < 0.14 and 0.33, respectively, for Th1 and Th2), proinflammatory cytokine gene expression, and cytokine protein secretion into culture supernatants (P < 0.44 and 0.16, respectively, for IL-4 and IFNgamma). We conclude that CD86 is the primary B7 signaling homologue in human PBMC responses, and that second signal pathways through the B7 homologues have no effect on phenotypically differentiated T helper cells in humans.