Ragweed Pollen Extract Research Articles

A Double-Blind Placebo-Controlled Evaluation of Sublingual Immunotherapy with a Standardized Ragweed Extract in Patients with Seasonal Rhinitis

Background: There is a growing consensus on the benefits of sublingual-swallow immunotherapy in the treatment of allergic diseases. Methods: This randomized, double-blind placebo-controlled study was undertaken to assess the efficacy and safety of sublingual immunotherapy with standardized ragweed pollen extract tablets, in patients with an allergic rhinitis. A total of 110 outpatients were randomized (immunotherapy [I]: 55; placebo [P]: 55), of whom 99 were analyzable for efficacy (I: 48; P: 51) and 106 analyzable for safety (I: 53; P: 53). After a 28-day progression phase, the patients received a maintenance treatment during 6.5 months. Efficacy variables included a global assessment of efficacy (patient/ investigator), symptoms and medication scores as well as the frequency of asthma attacks. Results: In the active treatment group, 43 patients completed the study, versus 49 on placebo. During the whole period of pollination, the difference favoring immunotherapy was highly significant for the global assessment by the patient (p = 0.004) and by the investigator (p = 0.005). Adverse reactions were reported more often in the active treatment but mild or moderate, and they abated after dose adjustment. A subgroup analysis of those patients receiving the highest dose of immunotherapy (3 tablets 3 times a week) showed a highly significant response for rhinitis and conjunctivitis total scores by comparison to lower dosages. Conclusion: This study confirms the efficacy and safety of sublingual immunotherapy and strongly suggests a dose-response relationship.

Effect of lysed Enterococcus faecalis FK-23 (LFK) on allergen-induced peritoneal accumulation of eosinophils in mice.

The interest in anti-allergy immunoregulation by lactic acid bacteria has been growing for the last few decades. There is some evidence to suggest that lysed Enterococcus faecalis FK-23 (LFK) could relieve the clinical symptoms of pollinosis. However, the mechanism responsible for this phenomenon remains unknown. To identify the effect of LFK, a lysozyme treated and heat-killed preparation from the lactic acid bacteria Enterococcus faecalis FK-23 strain, on allergen-induced eosinophil accumulation. BALB/c mice were sensitized with ragweed pollen extract, and peritoneal accumulation of eosinophils was induced. A total of 60 mg (0.5 mL) LFK was orally administered to the experimental mouse every day during 21 days of the sensitization period. In addition, LFK 4 mg, 25 mg and 60 mg (each 0.5 mL) were also orally administered to a mouse of each group every day for 21 days. Saline was fed in a dose of 0.5 mL/mouse per day for the same duration as a control. Compared with control mice, LFK-treated mice exhibited decreased ragweed pollen allergen-induced peritoneal accumulation of eosinophils (P = 0.013), which showed a tendency to be in a dose-dependent fashion (P = 0.14). The results provide laboratory evidence of the role for LFK, a lactic acid bacteria preparation, in combating eosinophil accumulation.

Amb a 1–linked CpG oligodeoxynucleotides reverse established airway hyperresponsiveness in a murine model of asthma

Background: Recently, it has been demonstrated that immunostimulatory DNA sequences (ISS) containing CpG motifs prevent the development of allergic airway responses in murine models of disease. However, few studies have addressed the issue of whether these agents will reverse established Tm H2-driven allergic airway responses. Objective: The aim of this study was to determine whether intradermal delivery of an immunogenic protein of ragweed pollen linked to an immunostimulatory DNA sequence could reverse an established allergic response in the mouse lung. Methods: Mice sensitized and challenged with ragweed pollen extract were treated intradermally twice at 1-week intervals with an ISS chemically linked to Amb a 1 (Amb a 1-ISS). One week after the Amb a 1-ISS treatment, mice were rechallenged intratracheally with ragweed extract, and airway responses were assessed. Results: Amb a 1-ISS treatment of ragweed-sensitized and ragweed-challenged mice significantly reversed allergen-induced airway hyperresponsiveness and suppressed the total number of eosinophils in bronchoalveolar lavage fluid. The inhibitory effect of Amb a 1-ISS was associated with a marked increase in IFN-γ levels by Amb a 1-stimulated splenocytes and a shift in the antibody profile from a T H2-directed IgG1 response to a T H1-directed IgG2a response. Interestingly, the inhibitory effect of Amb a 1-ISS on allergen-driven airway hyperresponsiveness was independent of suppression of T H2 cytokine production. Conclusion: These results demonstrate that intradermal delivery of allergen-specific DNA conjugates can reverse established allergic responses in the murine lung, supporting their potential use in the treatment of human asthma. (J Allergy Clin Immunol 2002;109:455-62.)

IL-10 inhibits allergen-specific TH2 cytokine production by human peripheral blood mononuclear cells from patients with ragweed allergies

~ A IL-10 Inhibits Allergen-Specific TH2 Cytokine Production by 1 ~jl-lr Human Peripheral Blood Mononuclear Cells From Patients With Ragweed Allergies J Sun, Susanna Goncharova, M Conway, A Dalrymple, Elizabeth C Cates, Martin R Stampfli, B Wong, Susan Waserman, Manel Jordana McMaster University, Hamilton, ON, Canada Type 2 polarized T cells and their effector cytokines play a central role in allergic diseases. The objective of this study was to investigate whether ILl0, a cytokine with powerful immuno-suppressive activities, inhibits Th2 cytokine production by peripheral blood mononuclear cells (PBMC) from patients with ragweed allergies. The patients were assessed by skin prick test and clinical history, and PBMC were isolated from these ragweed allergic individuals and non-atopic controls prior to pollen season. PBMC were stimulated with a ragweed pollen extract and cytokine synthesis was measured in culture supematants. Spontaneous production of IL-5 and IL-13 varied with each individual and was not significantly different between the atopic and non-atopic group. In ragweed allergic patients, addition of ragweed extract to the cell culture markedly enhanced levels of IL-5 and IL-13. In contrast, PBMC from non-atopic controls did not respond to ragweed stimulation. Both spontaneous and allergen-induced IL-5 and IL13 production were significantly inhibited by recombinant human IL10. IL10 also suppressed Th2 cytokine production induced by PMA, a T cell polyclonal stimulator. Interestingly, IL-4 synthesis could not be detected in any of the samples tested. We are currently assessing the impact of IL-10 on chemokine production. We also extracted RNA from cultured cells at 3h, 6h, 12h, 24 h, 72h, and 5 days and are assessing the expression of chemokine receptors and co-stimulatory molecules using real time quantitative PCR. Our findings in samples collected prior to pollen season will be compared to samples being generated during pollen season. In summary, PBMC from ragweed allergic individuals produce significant levels of IL-5 and IL-13 in response to ragweed stimulation. This response is inhibited by exogenous IL-10. Thus, our results further the notion that IL10 has therapeutic potential in allergic diseases.

Purification and characterization of an arginine-specific peptidase from ragweed (Ambrosia artemisiifolia) pollen.

Ragweed (Ambrosia artemisiifolia) is clinically the most important source of seasonal aeroallergens, as it is responsible for the majority and most severe cases of hay fever (allergic rhinitis). Extracts from pollen grains have been shown to contain numerous proteins with various functions, including a novel serine proteolytic enzyme with chymotrypsin-like specificity that has been previously described (J. Biol. Chem. 1996; 271:26227-26232). We now report the isolation and properties of a second, trypsin-like enzyme with a molecular mass near 80 kD, from ragweed pollen extracts. This enzyme has a blocked N-terminus, a pH optimum near 9.0, and requires Ca2+ for stability and activity, but not reducing agents. The enzyme is inhibited by diisopropyl fluorophosphate, a general serine class proteinase inhibitor, and more specifically by N-p-tosyl-L-lysine chloromethyl ketone. Activity toward protein substrates was not detected, but various synthetic substrates and small biologically active peptides were efficiently cleaved, with a strong preference for Arg in the P1 position and either Arg or Gly in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. Significantly, atrial natriuretic peptide and angiotensin 2, whose degradation would amplify kinin activity and influence inflammatory diseases of the respiratory tract and nasal passages, were also rapidly hydrolyzed. Thus, the ragweed pollen endopeptidase may be involved in the inactivation of regulatory neuropeptides during pollen-initiated allergic reactions.

Identification of common allergenic structures in mugwort and ragweed pollen

Background: Despite the rare occurrence of ragweed in Middle Europe, a surprisingly high number of patients allergic to mugwort, a frequently encountered weed, display IgE reactivity against ragweed pollen allergens. Objective: The aim of this study was to investigate whether the high prevalence of IgE reactivity against ragweed in patients allergic to mugwort is caused by the presence of common allergenic determinants. We also sought to characterize any cross-reactive allergens. Methods: Common allergenic structures in mugwort and ragweed pollen were characterized by qualitative IgE immunoblot inhibition experiments performed with natural allergen extracts and recombinant allergens. The degree of cross-reactivity was estimated by quantitative CAP-FEIA competitions. The clinical significance of cross-reactive IgE antibodies was studied with histamine release experiments and nasal provocation tests. Results: Mugwort and ragweed RAST values were significantly correlated in a population of 82 Austrian patients allergic to mugwort. IgE antibodies cross-reacted with allergens of comparable molecular weight that were present in both extracts. By using recombinant birch profilin and specific antisera for IgE inhibition experiments, profilin was identified as one of the cross-reactive components in mugwort and ragweed pollen. Preincubation of sera from patients allergic to mugwort with mugwort extract inhibited IgE binding to ragweed pollen extract greater than 80%. Mugwort and ragweed pollen extract induced comparable histamine release and reduction of nasal air flow in a patient with IgE reactivity against the major mugwort allergen Art v 1. Conclusion: In addition to profilin, mugwort and ragweed pollen contain a number of cross-reactive allergens, among them the major mugwort allergen Art v 1. Cross-reactive IgE antibodies can lead to clinically significant allergic reactions. (J Allergy Clin Immunol 1998;101:196-206.)

Structural analysis of N-glycans from allergenic grass, ragweed and tree pollens: core alpha1,3-linked fucose and xylose present in all pollens examined.

The N-glycans from soluble extracts of ten pollens were examined. The pyridylaminated oligosaccharides derived from these sources were subject to gel filtration and reverse-phase HPLC, in conjunction with exoglycosidase digests, and in some cases matrix-assisted laser desorption-ionisation mass spectrometry. In comparison to known structures, it was possible to determine the major structures of the N-glycans derived from Kentucky blue grass (Poa pratensis), rye (Secale cerale), ryegrass (Lolium perenne), short ragweed (Ambrosia elatior), giant ragweed (Ambrosia trifida), birch (Betula alba), hornbeam (Carpinus betulus), horse chestnut (Aesculus hippocastanum), olive (Olea europaea) and snake-skin pine (Pinus leucodermis) pollen extracts. For grass pollens the major glycans detected were identical in properties to: [structure in text] Grass pollens also contained some minor structures with one or two non-reducing terminal N-acetylglucosamine residues. In the ragweed pollens, the major structures carried core alpha1,3-linked fucose with or without the presence of xylose. In tree pollen extracts, the major structures were either xylosylated, with or without fucose and terminal N-acetylglucosamine residues, with also significant amounts of oligomannose structures. These results are compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens.

Microencapsulation

Subcutaneous immunotherapy has been used successfully to treat atopic diseases for many years. However, its use is associated with several problems, the most bothersome being systemic allergic reactions. This has led to evaluation of alternative methods of treatment, including oral immunotherapy. For the most part, oral administration of allergens has not proven an effective substitute because of the lack of potency. Extraordinarily large oral doses were required to achieve clinical improvement, but even these doses were ineffective with some allergens. To resolve this difficulty, we have developed a novel system of encapsulating allergens which protects them from the destructive effects of gastric acid and pepsin. Most importantly, encapsulated allergens administered orally to mice elicited vigorous systemic immune responses, with stimulation of predominantly CD4+ T helper type 2 lymphocytes. Three studies of encapsulated short ragweed pollen extracts (eSRW) in short ragweed (SRW)-sensitive humans have been done. Treated patients developed high titres of SRW IgG antibodies, had blunting of the expected anamnestic increase in SRW IgE antibodies, downregulated the expected seasonal increase in nasal SRW IgA antibodies and had a decrease in nasal sensitivity to SRW on provocative testing. Symptoms were significantly decreased in treated patients if they could tolerate a high dose of eSRW. This was accomplished without inducing any systemic reactions, with an oral dose only slightly higher than that used in high dose subcutaneous immunotherapy. These encouraging results suggest that encapsulated allergens have the potential to become important therapeutic agents in immunotherapy.

Evidence of Hop Japanese pollinosis in Korea: IgE sensitization and identification of allergenic components

Background: Hop Japanese (Hop J) pollens are abundant in the air of Korea during the autumn season. Their significance as a source of allergic sensitization is still unclear. Objectives: We sought to detect the sensitization rate to Hop J pollen as an inhalant allergen and to identify its allergenic components. Method: We carried out skin prick tests with Hop J pollen extract in 1287 patients with respiratory allergy who visited our hospital over the course of 1 year. The serum-specific IgE antibody to Hop J pollen antigen was detected by ELISA, and its binding specificity was confirmed by the ELISA inhibition test. To confirm the respiratory sensitization, bronchoprovocation tests were performed in 16 asthmatic patients sensitive to this pollen. To characterize allergenic components, Hop J pollen extract was analyzed by means of sodium dodecylsulfate–polyacrylamide gel electrophoresis followed by IgE immunoblotting. Results: A positive result on the skin prick test (G2+ of the antigen to histamine ratio) was noted in 79 (6.1%) patients. The serum-specific IgE antibody was detected in 18 (41.9%) patients among 43 positive reactors tested. The ELISA inhibition test with the addition of Hop J pollen extract showed a dose-dependent response. Minimal inhibitions were noted with addition of ragweed and mugwort pollen extracts. Nine asthmatic patients showed significant bronchoconstriction after inhalation of the Hop J pollen extract (five early and four dual asthmatic responders), and all of them had high specific IgE binding. Immunoblot analysis revealed 12 IgE-binding components ranging from 13 to 89 kd. Three bands (13 kd, 74 kd, and 80 kd) were bound to the IgE among the sera tested from more than 50% of the patients. Conclusion: We believe that the Hop J pollen should be considered as a relevant allergen during the autumn season and thus included in skin test batteries in Korea. Some patients diagnosed as having “intrinsic” asthma or rhinitis might be sensitized to this pollen and other previously unknown allergens. (J Allergy Clin Immunol 1997;100:475-9.)

Oral immunotherapy with short ragweed extract in a novel encapsulated preparation: A double-blind study

Background: In the past, oral immunotherapy with allergens has had limited clinical effectiveness, presumably because of gastrointestinal destruction of allergens. Objective: We have developed a new technique for microencapsulating protein antigens that permits them, when given orally, to bypass the stomach and be delivered to the small intestine in a highly immunogenic form. This study's purpose was to confirm the immunologic potency of orally administered short ragweed pollen extracts (SRW) microencapsulated (mSRW) by this new technique and to study the effectiveness of mSRW in controlling the symptoms of ragweed-induced hay fever. Methods: Twenty-one SRW-sensitive patients were treated with mSRW in a double-blind placebo-controlled study. Serum SRW IgG and IgE antibodies and nasal secretory IgA antibodies were determined. During the ragweed season, symptoms were quantified by symptom-medication scoring. Results: The treated patients had high titers of serum SRW IgG antibodies (1.15 μg/ml at baseline, increasing to 21.21 μg/ml), experienced regulation of the seasonal increase in serum SRW IgE antibodies (+9% vs +59% in placebo-treated patients), and produced a small amount of nasal SRW IgA antibodies. Despite an insubstantial pollen count, the symptom-medication scores in the treated group were lower than those in the placebo group (4.28 vs 6.18, p = 0.059), but the differences were statistically significant only in the subgroup that tolerated high doses (>20 μg of Amb a 1 in 19 of 21 patients, p = 0.04). These effects were accomplished without inducing any systemic reactions with a dose of mSRW (mean, 23.8 μg of Amb a 1) only slightly higher than that used in high-dose subcutaneous immunotherapy. Conclusion: Oral mSRW seems a safe, easily administered, and immunologically potent treatment for ragweed-induced hay fever, but its ultimate utility requires further study. (J Allergy Clin Immunol 1997;100:30-8.)

Potential therapeutic efficacy of allergen-monomethoxypolyethylene glycol conjugates for in vivo inactivation of sensitized mast cells responsible for common allergies and asthma.

Skin sites of rats, which had been systemically sensitized to ovalbumin (OVA) were injected intradermally with murine anti-DNP IgE mAbs or with murine polyspecific IgE to recombinant Bet v 1. Injection of OVA(mPEG)10-11 conjugates into these skin sites inhibited passive cutaneous anaphylaxis (PCA) on subsequent intravenous challenge with DNP44-BSA and rBet v 1; by contrast, neither OVA nor an unrelated mPEG conjugate affected the PCA reactions. In dogs sensitized to both OVA and ragweed pollen extract (RAG), inhalation of either allergen (AL) caused a dramatic increase in airway resistance (Rrs). By contrast, administration of an aerosol of OVA(mPEG) caused no change in Rrs. Moreover, thereafter, (1) in spite of repeated challenges with aerosolized OVA over many months, the increase in Rrs on inhalation of OVA was blocked and (2) insufflation of RAG resulted in increase in Rrs of only about 50% in relation to that prior to inhalation of the conjugate; this dog's anti-RAG hyperreactivity remained blunted over many months. It is concluded that AL-mPEG conjugates of optimal composition inactivate sensitized mast cells and basophils, as manifested by a significant decrease of cutaneous or airway responses on subsequent challenge with the respective AL(s).

Immunologic effects of encapsulated short ragweed extract: a potent new agent for oral immunotherapy.

Oral allergen immunotherapy with conventional allergens has not been a useful mode of treatment because of the lack of potency of allergens when administered by this route. To study the immunologic potency of short ragweed pollen extracts microencapsulated by a new technique administered orally to short ragweed pollen-sensitive humans and to establish the dose of oral microencapsulated short ragweed pollen extract required for these effects. Nine short ragweed pollen-sensitive patients were treated with a new oral agent for immunotherapy, microencapsulated short ragweed pollen extract, in an open study. The effectiveness of this treatment was determined by comparison to a group of nine matched short ragweed pollen-sensitive patients who received no treatment. Treated patients developed high titers of short ragweed-specific IgG and IgE antibodies and their expected seasonal increase in IgE antibodies was regulated. The dose of microencapsulated short ragweed pollen extract required to achieve these effects was only slightly higher than the dose of short ragweed pollen extract used in high dose subcutaneous immunotherapy. Furthermore, this dose was achieved in 7 weeks. There were no side effects other than mild gastrointestinal ones. The nine treated patients fared clinically better during the ragweed season than the untreated patients in this open study. This study suggests that allergens microencapsulated by this new technique may make oral immunotherapy a practical mode of treatment.

Comparison of efficacy, safety, and skin test inhibition of cetirizine and astemizole

Astemizole, an H1-histamine-receptor antagonist prescribed for seasonal allergic rhinitis, has a slow onset of action and a strong suppressive effect on the wheal and flare reaction, which interferes with skin testing results. The newer antihistamine cetirizine appears to have a rapid onset of action and a low potential to interfere with posttreatment skin testing results. To compare the efficacy, safety, and skin test inhibition of astemizole and cetirizine in the treatment of seasonal allergic rhinitis. In a double-blind, parallel-group study conducted at six sites during ragweed pollination season, 263 subjects were randomized to receive 10 mg of astemizole, 5 mg of cetirizine, or 10 mg of cetirizine daily for 2 weeks. The subjects rated seven allergic rhinitis symptoms daily, the subjects and investigators provided global assessments of the responses to the treatments, and the subjects rated their satisfaction with the treatments. Thirty-nine subjects at one study site underwent quantitative skin testing before and after treatment. As measured by reduction from baseline in total symptom severity score, which was the primary efficacy measure in the study, all three treatments significantly relieved the symptoms of allergic rhinitis (P less than .05). This finding was supported by the global ratings and the subject satisfaction ratings. There were no significant differences among the three treatments for reduction from baseline in total symptom severity score. The mean subject satisfaction score with 10 mg of cetirizine was significantly greater than that with astemizole (P less than .05). In the skin tests performed 3, 7, and 14 days after the end of antihistamine treatment, the subjects who had received the cetirizine doses had significantly greater mean sum of wheal and mean sum of erythema values than those who had received the astemizole dose (P less than .05). Sensitivity to ragweed pollen extract returned to 90% of baseline within three days of the end of cetirizine treatment. Both drugs were well tolerated and their adverse event profiles were similar. Astemizole and cetirizine are effective and well tolerated in alleviating the symptoms of ragweed-induced allergic rhinitis. Cetirizine inhibits skin test results to a much lesser extent than does astemizole. Physicians may wish to consider the potential for skin test inhibition when selecting an antihistamine for patients with allergic rhinitis.

Neurotropin inhibits accumulation of eosinophils induced by allergen through the suppression of sensitized T-cells

A non-protein extract isolated from the inflamed dermis of rabbits inoculated with vaccinia virus (Neurotropin®) has been clinically used in Japan as an analgesic and anti-allergic drug. To clarify its anti-allergic mechanism, the effect of Neurotropin on eosinophil accumulation induced by ragweed pollen extract or plateletactivating factor (PAF) was examined in BALB/c mice. Neurotropin inhibited the T-cell-dependent accumulation of eosinophils induced by allergen in a dose-dependent manner when administered during sensitization. However, Neurotropin was unable to inhibit the T-cell-independent accumulation of eosinophils induced by PAF. A T-cell transfer experiment was performed to address the inhibitory mechanism. A marked accumulation of eosinophils was observed when recipients were injected i.p. with allergen and Nylon wool column-passed splenic T-cells from the sensitized donor mice. However, significant accumulation of eosinophils was not observed when sensitized donor mice were administered with Neurotropin but not saline. Taken together, these results suggest that Neurotropin inhibits the accumulation of eosinophils induced by allergen via the suppression of sensitized T-cell induction, or alternatively by interfering with T-cell function.

Allergen-specific IgG1 and IgG3 through Fc gamma RII induce eosinophil degranulation.

Evidence suggests that eosinophils contribute to inflammation in bronchial asthma by releasing chemical mediators and cytotoxic granule proteins. To investigate the mechanism of eosinophil degranulation in asthma, we established an in vitro model of allergen-induced degranulation. We treated tissue culture plates with short ragweed pollen (SRW) extract and sera from either normal donors or SRW-sensitive patients with asthma. Eosinophils were incubated in the wells and degranulation was assessed by measurement of eosinophil-derived neurotoxin in supernatants. We detected degranulation only when sera from SRW-sensitive patients were reacted with SRW. Anti-IgG and anti-Fc gamma RII mAb, but not anti-IgE or anti-Fc epsilon RII mAb, abolished the degranulation. IgG-depleted serum did not induce degranulation; IgE-depleted serum triggered as much degranulation as untreated serum. Furthermore, serum levels of SRW-specific IgG1 or IgG3 correlated with the amounts of released eosinophil-derived neurotoxin. When eosinophils were cultured in wells coated with purified IgG or IgE, eosinophil degranulation was observed only with IgG. Finally, human IgG1 and IgG3, and less consistently IgG2, but not IgG4, induced degranulation. Thus, sera from patients with SRW-sensitive asthma induce eosinophil degranulation in vitro through antigen-specific IgG1 and IgG3 antibodies. These antibodies may be responsible for degranulation of eosinophils in inflammatory reactions, such as bronchial asthma.

Induction of oral tolerance after feeding of ragweed pollen extract in mice

The induction of oral tolerance following the feeding of ragweed pollen and its extract was investigated in BALB/c mice. Antibody class-specific immune suppression could be observed, and the IgE response was specifically suppressed, depending on the amount of ragweed pollen extract fed when subsequently immunized with ragweed extract together with Al(OH) 3 as an adjuvant. A multiple feeding was more effective than a single feeding of antigen, and the IgE response was completely suppressed when 20 mg of pollen extract was fed for 5 consencutive days. On the other hand, IgG production was not suppressed even though a large amount of ragweed pollen or its extract was fed. Furthermore, no secretion of antigen-specific IgA into saliva was observed in control animals or those fed ragweed pollen extract. Thus, pollen extract feeding may be potentially useful for the prophylaxis or therapy of allergic rhinitis induced by ragweed.

Polymers for delivering peptides and proteins

The use of polymers for delivering peptide and protein drugs is described. Soluble-polymer technology attempts to bind a polymer to all sites on therapeutic protein molecules that cause the body to recognize the molecules as foreign. Goals include a stable linkage, water solubility, low immunogenicity, prolonged half-life, and intact biological activity. Polyethylene glycol (PEG)-adenosine deaminase (ADA), or pegademase bovine, has FDA-approved labeling as replacement therapy for ADA deficiency in patients with severe combined immunodeficiency disease who are not suitable candidates for bone marrow transplantation. Pegademase bovine reverses the toxic accumulation of adenosine and deoxyadenosine in adenosine deaminase-deficient cells, restoring the immune system. PEG-asparaginase (pegaspargase) has shown promise in patients with acute lymphocytic leukemia; allergic reactions have been minimal. Animal studies suggest that superoxide dismutase has potential use in conditions in which the body's ability to remove oxygen free radicals is reduced, such as burns and myocardial infarction; coupling with PEG may greatly increase the protein's half-life. Other PEG-conjugated proteins under investigation include PEG-catalase, PEG-uricase, PEG-honeybee venom, PEG-hemoglobin, and PEG-modified ragweed pollen extract. Dextran, albumin, DL-amino acids, and polyvinyl pyrrolidone have also been studied as protein carriers; most of the products created thus far have not shown much promise. The coupling of polymers to proteins has yielded protein drugs with intact biological activity and reduced immunogenicity, but much remains to be learned about this technology.

Purification and immunochemical characterization of recombinant and native ragweed allergen Amb a II

The complete sequence of a cDNA encoding Amb a II and its relationship to the Amb a I family of allergens has recently been described [Rogers et al. (1991) J. Immun. 147, 2547–2552; Griffith et al. (1991 a), Int. Archs Allergy appl. Immun. 96, 296–304]. In this study, we present results generated with rabbit antipeptide antisera that recognize Amb a II or Amb a I, but not both. The specificity of two anti- Amb a II antipeptide sera, anti-RAE-50.K and anti-RAE-51.K, was verified on Western blots of recombinant Amb a II and Amb a I.1. These two sera, directed against separate regions of the Amb a II molecule, detected three individual 38-kDa Amb a II isoforms on 2D Western blots of aqueous ragweed pollen extract. These Amb a II isoforms have pi in the 5.5–5.85 range and can be easily distinguished from Amb a I isoforms with pI in the 4.5–5.2 range detected by an anti- Amb a I specific peptide antiserum. The Amb a II isoforms have also been individually purified from pollen, positively identified as Amb a II by amino acid sequencing, and visualized as separate bands on IEF gels. An analysis of Amb a II cDNA sequences generated by PCR led to the prediction of three Amb a II isoforms with pI of 5.74, 5.86 and 5.97 that are very similar to the pI deduced from 2D Western blot analysis. Recombinant Amb a I.I and Amb a II have been expressed in E. coli, purified in their denatured form, and examined by ELISA for their capacity to bind pooled allergic human IgE. Purified native Amb a and Amb a II from pollen were shown to have very similar IgE-binding properties. In contrast, Amb a II had a markedly reduced IgE-binding capacity as compared to Amb a I.I. These data suggest that recombinant Amb a I.I and Amb a II, isolated in a denatured form, diner significantly in their IgE-binding properties whereas the native molecules isolated from pollen do not.

Allergenic cross-reactivity between Parthenium and ragweed pollen allergens.

Cross-reactivity of allergens from the pollen of the Compositae weeds, Parthenium hysterophorus (American feverfew) and Ambrosia (ragweed), in 2 groups of patients with different geographic distributions was studied. Parthenium-sensitive Indian patients, who were never exposed to ragweed, elicited positive skin reactions with ragweed pollen extracts. A significant correlation in the RAST scores of Parthenium and ragweed-specific IgE was observed with the sera of Parthenium and ragweed-sensitive Indian and US patients, respectively. RAST inhibition experiments demonstrated that the binding of IgE antibodies in the sera of ragweed-sensitive patients to short (W1) and giant (W3) ragweed allergen discs could be inhibited by up to 94% by Parthenium pollen extracts. Similar inhibition (up to 82%) was obtained when the sera of Parthenium rhinitis patients were incubated with ragweed allergen extracts. A dose-dependent proliferation of lymphocytes from a Parthenium-sensitive rhinitis patient with elevated levels of both Parthenium and ragweed-specific IgE was observed when incubated with Parthenium and ragweed pollen extracts. A 1.6-fold higher proliferation, however, was observed with Parthenium pollen extract at a concentration of 100 micrograms/ml. These results suggest that shared epitopes present on Parthenium and ragweed pollen allergens are recognized by both Indian and US patients sensitized by exposure to Parthenium and ragweed pollen, respectively. The high degree of cross-reactivity between Parthenium and ragweed pollen allergens suggests that individuals sensitized to Parthenium may develop type-I hypersensitivity reactions to ragweed and vice versa when they travel to regions infested with the weed to which they had not been previously exposed.

Role of sodium in antigen-induced contraction of tracheal smooth muscle in dogs

We examined the role of Na + influx in the airway response to antigen (ragweed pollen extract) in sensitized dogs, using amiloride analogs to block Na +-dependent processes. In in vivo studies, respiratory resistance was measured in amiloride treated and untreated groups. The resistance increased by 9.3 cmH 2O·L −1·sec in response to ragweed aerosol in the untreated group, but increased only by 5.2 cmH 2O·L −1·sec in the treated group. In in vitro studies, isometric tension was measured in ragweed pollen sensitized tracheal strips. Tissues were treated with amiloride or its derivatives (50 μM) for specifically blocking Na + channels (phenamil), Na +-H + exchanger [5-( N-methyl N-guanidinocarbonyl methyl)-amiloride] r Na +-Ca 2+ exchanger [5(4-chlorobenzyl)-2′,4′-dimethylbenzamil]. In untreated strips, tension increased in response to ragweed by 1.9 ± 0.5 mN/mg. The increase was reduced by phenamil (95.2 ± 2.5%; P<0.01 and amiloride (41.7 ± 13.1%; P<0.01), but not by the other two agents. Furthermore, phenamil also inhibited histamine-induced tension response and histamine-induced 22Na + uptake of the muscle. We conclude that antigen-induced airway response is attenuated blocking Na + influx in smooth muscle.