Double-blind, placebo-controlled immunotherapy with mixed grass-pollen allergoids

Double-blind, placebo-controlled immunotherapy with mixed grass-pollen allergoids IV. Comparison of the safety and efficacy of two dosages of a high-molecular-weight allergoid J. Bousquet, MD, PhD, A. Hejjaoui, F-B. Michel, MD Montpellier, France

MD, M. Soussana,

MD, and

Specific immunotherapy is still widely used in grass-pollen allergy, but its side effects may limit its use. We tested the safety and efficacy of a formalinized high-molecular-weight allergoid prepared from a mired grass-pollen extract with two injection schedules in a double-blind, placebo-controlled study. Eighteen patients received placebo, 19 received the low-dose schedule (maximal dose: Zoo0 PNU) and 20 received the high-dose schedule (maximal dose: 10,000 PNU). Only one patient presented a systemic reaction of moderate severity for a dose of 1200 PNU. Before the onset of the pollen season, patients had a nasal challenge with orchard grass-pollen grains, a skin test titration. and the titration of serum-specific IgG. Both groups of patients presented a significant reduction in nasal and skin sensitivities and a significant increase in IgG compared to placebo. Symptoms and medications for rhinitis and asthma were studied during the season, and both groups receiving allergoids had a significant reduction of symptom-medication scores for nasal and bronchial symptoms. There was a highly significant correlation between nasal symptom-medication scores during the season and the results of nasal CLJNIMWJNOL challenges. High-molecular-weight allergoids are safe and effective. (J ALLERGY 1990:85:490-7.)

Double-blind, placebo-controlled studies found that specific immunotherapy with formalinized allergoids was effective in controlling symptoms of grass pollen-induced rhinitis, conjunctivitis, and asthma.I-3Two different preparationswere used. The classical allergoid preparedby the two-step procedure according to the method of Marsh et al .4.’ was found to elicit more SRs than an HMW GOID fractionated from the sameallergoid.-’ Methods evaluating the efficacy of immunotherapy include symptom-medicationscores,‘-3.6.7 provocative challengeswith soluble allergenP or pollen grains,’ skin prick tests,‘-3.” and in vitro assays, such as the evolution of allergen-specific IgG.‘-‘, 6. ’ However,

From the Clinique des Maladies Respiratoires,CHU, Hopital I’Aiguelongue, Montpellier, France. Received for publication June 20, 1989. Revised Sept. 20. 1989. Accepted for publication Sept. 25. 1989. Reprint requests: J. Bousquet, MD, Clinique des Maladies Respiratoires, CHU, Hopital I’Aiguelongue, Avenue du Major Flandre, 34059 Montpellier Cedex, France. l/1/17123

490

1 Abbreviations used GOID: Unfractionated allergoid HMW: High-molecular weight HPLC: High-pressure liquid chromatography PNU: Protein nitrogen unit SR: Systemic reaction

there are very few studies comparing objective parameterswith symptom-medicationscoresduring the pollen season, and the relevance of different tests is still a matter of debate. The doseof allergen to be administeredduring specific immunotherapy has not been as yet completely characterized. It has been demonstratedthat low allergen doses,such as thoseadministeredwith the Rinkel method, are ineffective,13 and thus, it was proposedthat the largest tolerated dosebe administered.” However, very large dosesof allergens have induced an unacceptablerate of SR,14leading to the concept of an “optimal dose”lssI6 in which there is a balance between efficacy and safety.

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TABLE 1. Protocol ----

of specific

immunotherapy High-dose

Injection

Day

I

AM

I

9

4 5

8

IO II 9

6 7 x 9 IO

I4 21 28 35 42

2 3

with aller:(o~os 491

10

Dose (PNU)

schedule

Lowdose

Vial

Volume

lcn)

(ml)

100 200 500 1.ooo

200 2.000

0,s 0. I

2,ooo 2.ooo

2.ooo

20.000 20,OfJO 2o.OOQ 20.000 20.000 20,OtHJ

4,000 6.ooO 10,000 10,000 10.000

Dose

schedule - .___ -_. _--_._

Vial (en)

Volume (ml1

loo 200

‘th J 2ooo

il i I) I

0.25 0.5

SO0 I000

‘OOIJ ‘(MM)

(I.3 II i

0. I 0.2 0.3 0,s 0.S 0.5

400 X(HJ I200 2ooo 2000 2000

3CiWJ 3OMl 4tJOoc 4004I 4o(HJ ‘MJO~ I ---.----._.

(PNU)

II I I1 2 Ii : II s \I 5 Il.5 - -. ____

cn. Concentration in PFr’L.

A double-blind, placebo-controlled study was performed in 57 grass pollen-allergic individuals to (1) confirm previous results on the safety and efficacy of an HMW GOID, (2) compare different objective in vivo (nasal challenge) and in vitro (serum grass with pollen-specific IgG and IgE) parameters symptom-medication scores during the pollen season, and (3) study whether large allergoid doses elicit better protection than smaller doses.

MATERIAL Pstients

AND METHODS

Fifty-seven patients (24 men, 11 to 45 years of age; mean, 26.8 f 10.4 years) volunteered to enter the study after informed consent. All patients had had symptoms of rhinitis for 2 to 35 years during the grass-pollen season, and more than half the patients also had symptoms of asthma and/or conjunctivitis. All the patients had a positive prick test to a I / 100 (wt/vol) standardized orchard-grass pollen extract (Stallergenes Laboratories, Fresnes, France). Allergeninduced skin tests are positive when the wheaI size is 75% of the wheal size induced by a 9% codeine-phosphate positive control solution. ” All the patients had a positive RAST to orchard-grass pollen. None of the patients had received any form of specific immunotherapy to pollen extract.

Immunotherapy HMW mired grass-pollen allergoid (HMW-GOID). A six grass-pollen ahergoid (GOID) was prepared by the two-step procedure described by Marsh et al.5 from pollens of six different grass species (Dactylis glomerata, Festuca elatior. Holcus lanatus, Lolium perenne, Phleum pratense. and Poa pratensis). The methods of preparation and control of the lyophilized allergen extract and the lyophilized GOID have been previously published in detail. I. ’ The fractionation of the HMW GOID was done according to a method previous published in detail.’ Briefly, the lyophilized GOID was dissolved in 0.004 mol/L of NH,HCO, and passed through a

preparative Sephadex G-100 column (Pharmacia. Freiburg. West Germany). A 0.05 mol/L of phosphate buffer (pH 7.5) was used as an eluent. All the molecules with a molecular weight of >85 kd were pooled and lyophilized. The yield of HMW GOID was 19.7% of GOID on weight basis. For immunotherapy, HMW GOID was dissolved in 0.9% saline solution containing 0.4% phenol and standardized in PNU. Both preparations (GOID and HMW GOID) were tested in parallel to assess their molecular weight distribution and relative allergenicity by means of high-pressure liquid chromatography, RAST inhibition, and skin testmg. ~The molecular weight distributions of these two preparations were compared by high-pressure liquid chromatography. according to a method previously described in detail.” and confirmed first, that GOID consisted mainly of molecules <44 kd and second, that HMW GOID had a molecular weight of >85 kd. The in vitro allergenic activities of both allergoids in comparison with the native allergen were examined by RAST inhibition as described previously. “’ It was observed that the in vitro allergenic activities of HMW GOID were similar to that of GOID and reduced by approximately lOOO-fold compared to the native allergen. In vivo allergenicity was assessed by SPTs with a parallelline bioassay and a modification of the technique described by Dreborg and Grimme? and Turkeltaub et al.” In vivo allergenic activities of GOID and HMW GOID wcrc reduced by 130-fold and 8500-fold, respectively. compared to the native allergen. Placebo. A placebo was prepared with 0.9% saline containing 0.4% phenol as a preservative and increasing concentrations of histamine dihydrochloride !O.oO to 0.5 mgiml). Immunotherapy. A double-blind. placebo-controlled immunotherapy was performed according to the protocol indicated in Table I. Because we wished to investigate the safety of the extract. we initially used a rapid increase of doses with both schedules, and we later adapted the dosage to large and small dosages. When a systemic or a large local

492

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J. ALLERGY

RHINITIS p
150

-

140

-

130

-

120

-

110

-

100

-

3 5

90 -

ti4

80 -

5

70-

:z

60 -

m

50

005

-

40 30 20

-

10 oGROUPS

OF

PATIENTS

FIG. 1. Mean symptom scores for rhinitis experienced by patients placed in the placebo (P), high-dose IHI, or lowdose (f) groups. Statistical analysis by Mann-Whitney U test.

reaction of >I0 cm occurred, the dosage increment was but we tried to reach the maintenancedose in all patients. The maintenancedosewas then administeredthree times at weekly intervals before the investigations were begun. After that time, the maintenancedose was reduced by one half and administered every 2 weeks during the season. reduced,

Investigations Symptom scores. When the patients attended the clinic in April 1987, they were taught the possible symptomsthat might occur and the medication that they should take.‘-‘. ” The daily symptom-scoreforms had been developed previously in our clinic? I’ and included symptomsof rhinitis (rhinorrhea, sneezing, and nasal obstruction), conjunctivitis (watery eyes, redness of the eyes, and pruritus), and asthma (wheezing and shortnessof breath). The severity of symptoms was scoredfrom 0 to 5. Patientswere allowed to take drugs only if they had symptoms.In caseof nasalsymptoms, they were first, to take nasal cromoglycate, and then, if symptomswere still present, to add nasal beclomethasone, then H, blocker (terfenadine, 60 mg twice daily), and finally, oral corticosteroids. For conjunctivitis, ocular cromoglycate was taken first and terfenadine was added if symptomspersisted. For asthma, inhaled salbutamol was administeredat a dose of 200 p.g, and then the dose was increasedto 600 )*g; if it were necessary,long-acting, sustained-releasetheophylline was added.In the latter case,the patient was asked

CLIN. IMMUNOL. FEBRUARY 1990

to return to the clinic. Patientscompleteddaily forms during the months of May and June. Reportedon the forms were their nasal, ocular, and bronchial symptoms for 12 hours along with all the possible medicationsthat might be used. The symptoms and medications were scored, and average symptom and medication scores were calculated for each patient for rhinitis, conjunctivitis, and asthma. SPT. SPTs were performed according to the modified method of Pepy? with a hypodermic needle on the volar surface of the forearm. The method has previously been described in detail.*’ Briefly, none of the patients had any factor that might have modified the performance of skin tests. Threefold dilutions (8 to 50,OOOBU/ml) of a standardized orchard grass-pollen extract standardized by in vivo and in vitro methods” were tested in parallel with a 9% codeine-phosphatesolution, and the end point titer was calculated as the threshold concentration eliciting a wheal size similar to the wheal size elicited by the 9% codeine-phosphatesolution, as previously described.I-‘. I’. ?’ Nasal provocative challenge. Nasal provocation tests were performed as previously published” according to a technique modified from Naclerio et al.” All patients were testedbetween 8 AM and 12 AM to avoid possible circadian variations by the same investigator. Patients were tested within 3 weeks of the end of immunotherapy (March 15 to April 7) at a time when no grass pollen is found in the atmosphere.None of the patients was receiving treatment that may have affected the performanceof nasalchallenges. None of the patients had been suffering from any form of allergic or infectious rhinitis for the 2 weeks before the challenge. Capsulescontaining lactoseor increasing concentrations of orchard-grasspollen from 50 to 156,250grains (fivefold increment) were preparedby the Stallergtnes Laboratories. Contents were insufflated into nostrils according to a technique modified from Rosenberget a1.26 with a nasalspinhaler (Fisons Laboratories,Loughborough, U .K .) , the patient refraining from breathing during the insufflation. Lactosewas initially insufflated, and eventual symptomswere recorded for 15 minutes. Then, increasing numbers of grains were deposited on the nasal mucosa every 15 minutes until a symptomscoreof 5 was obtained: five consecutivesneezes, 3; a rhinorrhea, 1-3; nasal blockage, 1-3; or itching of the nose, 1. This scoring system was defined according to Idsterballe,’ and we subsequentlyconfirmed the validity of symptom scoresin a previous study.” It was observedthat the releaseof prostaglandin D, in nasal secretionswas significantly (p < OOO5)associatedwith a symptom scoreof 5. The reproducibility of this test was examined, and it was found to be high.‘,‘” Moreover, we observedthat there was a significant con-elationbetweenthe threshold doseinducing a positive nasalchallengeand the severity of nasalsymptoms during the pollen season.’ Serum mired grass pollen-specific IgG. Serum-specific JgC was titrated according to the method of Puttonen and Maasch.*’ RAST disks were prepared according to Ceska et al.= with the mixed grass-pollenextract. IgG coupled on the disk was revealedby radiolabeledStaphylococcus aureus protein A. The results are listed as percent binding of total addedcounts and percent of IgG values from baseline, the

VOLUME NUMBER

Specific immunotherapy

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value of the IgG determination before immunotherapybeing taken as 100%. Serum mixed grass pollen-specific

IgE. Serum-specific IgE was titrated by PhadebasRAST (PhannaciaDiagnostics AB. Uppsala. Sweden) with the sameRAST disks as disks prepared for the titration of specific IgG. The results arc listed as percent binding of total added counts and percent of IgE values from baseline, the value of the IgE determination before immunotherapy being taken as I owi

Design of the study All patients were treated the same year and randomly assignedto one of the three treatmentgroups by rank order: placebo, I8 patients; HMW GOID high dose, 20 patients; and low dose, lo patients. Two patients did not complete the study. Before immunotherapy and between April I and April 21 (i.e.. before the pollen season), all the patients had a skin test titration and the titration of serum grass-pollen IgG and IgE. During the season, all the patients had follow-up by a symptom-medicationscore that they themselvesfilled in cvcry day from May I to June 30, but because of pollen counts. we only analyzed the period ranging from May I to June 15. Statistical analysis was done by nonparametric tests.-“’

RESULTS Immunotherapy

wiih alleronids

493

TABLE II. Comparison of the three grouos of patients

No. of patients

Low

High dosage

dosage

Placebo

20

I(!

:x

28.’

23.7

‘I.5

IO.6 6.3

IO 7 5.x

i 3.1 f). I

6.3

5x

h. I

I .6

I .J

7.0

I I.0 8.1

1h.X 13.2

i 5.6 IYb

7.6 4.6

4.0 7.1

4.6 0.b

Age iyr) Mean SD SFT end point Mean

SD Specific IgE (8 binding) Mean SD Rhinitis duration (yr) Mean SD Severity Mean SD o/cpatients with asthma Conjunctivitis: 9 patients Severity

3.6

3.h

3.5

0.8

0.0

I).!4

55

5.1

SO

85

wi

ho

Mean

3.3’

?.4

2.6

SD

0.8

0.8

0.0

p a!c c 0.03; p b/c < 0.03.

schedule

and side effects

All the patients were able to reach the maintenance dose, but because of some local and/or SRs, some patients reached the maintenance dose later than other patients. Patients placed in the high-dose group received a mean dose of 45,433 2 14,001 PNU. and patients who were in the low-dose group had a mean dose of 10,570 ? 2808 PNU. Large local reactions of >lO cm in diameter and lasting for more than 24 hours were observed in nine patients (four in the low-dose group and five reactions in the high-dose group). SRs of minor importance were noticed in eight patients (three in the placebotreated group, two in the low-dose group, and three SRs in the high-dose group). These reactions appeared between 15 minutes to 6 hours after the last injection, were rn the form of flushing of the face, rhinitis, or urticaria. and subsided without any treatment. These reactions occurred for doses ranging between 1200 and 10,ooO PNU. One patient had a more severe reaction characterized by urticaria, rhinitis, and asthma without hypotension but requiring treatment by 400 t.q of salbutamol (metered-dose inhaler). antihistamines, and methylprednisolone ( 120 mg intramuscularly). However, this reaction might have been due to an intravascular passage of the extract, since it appeared in the low-dose group for a dose of 1200 PNU. whereas the former injection of 1000 PNU performed I week earlier did not elicit any SR.

Symptom

scores

Symptom and medication scores were analyzed for rhinitis and asthma. All the patients had had rhinitis in previous years and all scores were analyzed. For nasal symptoms, patients in the placebo-treated group had a significantly higher mean score of symptoms and medications than patients placed in both HMW GOID-treated groups ( Fig. 1; Table II). Only a subset of patients were first observed with grass polleninduced asthma in the preceding year(s), and data were calculated for these patients only. Only patients who had complained of asthma in previous years had bronchial symptoms during the period of evaluation in the treated groups, whereas two patients who were placed in the placebo-treated group were observed with asthma for the first time during the year 1987. The results of the symptom and medication scores for asthma are similar to the scores observed for rhinitis (Fig. 2; Table III). There was no significant difference between the two treated groups for rhinitis or asthma. Nasal challenge None of the subjects tested had significant symptoms with lactose (score always <2). Patients receiving placebo had a nasal response for a significantly lower mean number of orchard grass-pollen gmins

494

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TABLE IV. Results

ASTHMA

with

90

7

LU

-

70

-

10 -

o-

.

:. .. i. . . .ie 00.. B P III.

-----j

L

P

GROUPS

OF

PATIENTS

FIG. 2. Mean symptoms scores for asthma experienced by patients placed in the placebo (PJ, high-dose (HJ, or low-dose (LJ groups. Statistical analysis by Mann-Whitney U test.

III. Symptoms and medications presented by the patients during the period of survey

TABLE

Rhinitis Days with symptoms Mean SD

Symptom score Meall SD Medication score Mean SD Asthma Days with symptoms Mean SD Symptom score Mean SD

Low dosage

Placebo

22.9* 11.4

27.2*

40.2

63.6* 32.5

57.w 37.5

108.6 33.2

38.6 37.6

35.3$ 44.5

66.4 51.7

12.3* 13.4

12.6* 13.5

35.5 12.4

17.4* 20.2

12.8t 16.8

54.8 23.0

Statisticaldifference from placebo: ‘0.01 < p < 0.005. tp < 0.001. $0.05 < p < 0.02.

7.1

of skin test end point

grass-pollen

Immunotherapy Before After p Value

extract

High dosage

Low dosage

Placebo

6.3 2 1.6 4.2 e 1.1

5.8 + 1.4 4.5 ? 1.0 <0.02

6.1 -t 2.0 6.0 c 1.6 NS

-co.01

NS. Not significant. Statistical analysis between first and second values by Wilcoxon W test.

(860 + 1394 grains) than patients placed in the lowdose (21,932 + 51,104 grains; p < 0.04, MannWhitney U test) or in the high-dose groups (33,530 + 63,003 grains; p < 0.01, Mann-Whitney U test). There were no significant differences between the two allergoid-treated groups. The correlation between the threshold dose inducing a positive nasal challenge and the observed symptoms of the patients during the season (Fig. 3) was highly significant (p < 0.001, Spearman’s rank test). Most patients with a nasal challenge that was positive for a low number of pollen grains (50 to 250 grains) suffered from severe symptoms during the pollen season. In contrast, most patients who reacted during the challenge for a high number of pollen grains (>6250 grains) had fewer symptoms during the pollen season.

Quantitative

High dosage

15.5

orchard

CLIN. IMMUNOL. FEBRUARY 1990

skin tests

After immunotherapy, the mean end point titer of patients treated with placebo did not change by comparison to pretreatment and was significantly greater than end point titer of both treated groups (p < 0.05 and p < 0.02) (Table IV). The correlation between the mean end point titer after immunotherapy and the severity of symptoms during the pollen season was only minimally significant (p < 0.04, Spearman’s rank test).

Serum mixed grass pollen-specific and IgE

IgG

Mean serum-specific IgG level did not change after immunotherapy in the placebo-treated group, whereas it was significantly (p < 0.01 and p < 0.001, Wilcoxon W test) increased in both treated groups (Table V). After immunotherapy, mean serum-specific IgE levels increased nonsignificantly in the two allergoidtreated groups, and there was no change in the placebo-treated group (Table V). There was no significant correlation between nasal symptom-medication scores and IgG or IgE levels.

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Specific

85 7

immunotherapy

0.0

150

with allerc;oids

495

Fiho=0.71 p<0.0001

l

0 l

l

1 125 1

25 -

1

2

3

NASAL CHALLENGE FIG. 3. Correlation between the pollen season.

4

5t6

(log-5 grass pollen grains)

the results of nasal challenge

and the nasal symptom

scores during

TABLE V. Results of in vitro assays High dossge IgE (b binding) Before immunotherapy After immunotherapy p Value IgG (% binding) Beforr: immunotherapy After immunotherapy p Value

Low dowge

Ptoaebo

11.02 t 8.09 17.63 2 15.73 NS

16.80 t 13.25 26.13 ? 30.77 NS

15.56 -t 15.58 14.63 +- 13.70 NS

6.16 2 4.69 15.10 rt 11.41
6.28 !z 4.54 13.59 + II.55
6.06 5: 5.70 6.30 t- 5.71 NS

KS, Not significant. Statistical analysis between first and second values by Wilcoxon W test.

DISCUSSION

The resultsof the presentstudy indicate that specific immunotherapywith mixed grass-pollenHMW GOID is effective in decreasing symptoms of rhinitis and asthmaduring the pollen seasonand in inducing both a significant decreasein nasaland skin test reactivities to allergen and a significant increase in serum grass pollen-specific IgG. There was a highly significant correlation between the results of nasal challenge and symptoms during the season. Although allergoid

were increased by fivefold in the high-dose group, there was no difference in objective or subjective parameters of efficacy among both treated groups. The safety of the HMW GOID was confirmed in this study. since only one patient presentedan SR of moderateseverity. Specific immunotherapy was found to be effective in grass-pollen rhinitis and asthma, and the present study confirms previous studies with standardized ex1racts7.11. 17.3031or allergoids.‘.’ However, in those doses

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studies the incidence of SRs ranged between 5% and 45%. It has already been demonstrated that HMW allergen extracts are safe,3. 3z-.14 and this finding was confirmed in the present study in which one patient developed an SR of moderate severity that did not require adrenaline. Since specific immunotherapy may lead to serious side effects, including deaths,12.35-38 it is desirable to treat patients with safe extracts, and HMW preparations, which reduce the incidence of side effects, may therefore be of importance. The efficacy of HMW GOID was demonstrated by symptom-medication scores during the season and confirmed by nasal challenge with pollen grains, skin tests, and the evolution of serum-specific IgG. All the techniques used in this study were simple and previously validated in our clinic.‘-3, ” In particular, we only performed nasal challenge after the season, since we had previously controlled its reproducibility.2. 24 The results observed in the placebo-treated group are similar to results of other studies. When objective parameters were compared to symptom-medication scores during the season, there was no significant correlation with IgG antibody levels as previously observed in grass-pollen immunotherapy.‘-3, ‘. 39.4o In contrast, there was a highly significant correlation between the results of nasal challenge and symptommedication scores during the season. This finding confirms a previous study with allergoids’ and indicates that the severity of the pollen season may be predicted by this test. Moreover, this finding further validates the clinical relevance of nasal challenge with pollen grains. The correlation between the results of skin tests and symptom-medication scores was only minimally significant. Serum IgE increased during the early months of immunotherapy, as previously stated in other studies, but there was no clinical significance. The dose of allergen injected to patients is of importance. It has been demonstrated that low-dose regimens, such as the Rinkel method, are ineffective,‘” and it was decided that the largest tolerated dose be administered.6 However, large doses can lead to a high rate of SRs. Thus, it is desirable to reach an optimal dose leading to efficacy as well as reducing the incidence of SR.“, I6 In this study, when doses were increased fivefold, there was no improved efficacy in objective or subjective parameters, which suggests that the optimal dose is the smaller one, since it is likely to be better tolerated.

2.

3.

4.

5.

6. 7.

8.

9.

10.

I I.

12.

13.

14.

15.

We thank Mrs. M. Deltour for her technical assistance. 16. REFERENCES I. Bousquet J, Heijaoui A. Skassa-Brociek W, et al. Doubleblind, placebo controlled immunotherapy with mixed grasspollen allergoids. 1. Rush immunotherapy with allergoids and

17.

CLIN. IMMUNOL. FEBRUARY 1990

standardized orchard-grass pollen. J ALLERGY CLIN IYMUNOI 1987;80:591-8. Bousquet J, Maasch H. Martinot B, Hejjaoui A, Wahl R. Michel FB: Double-blind, placebo-controlled immunotherapy with mixed grass-pollen allergoids. II. Comparison between parameters assessing the efficacy of immunotherapy. J ALLKRGY CLIK hMUNOL 1988;82:439-46. Bousquet J, Maasch H, Hejjaoui A, et al. Double-blind, placebo-controlled immunotherapy with mixed grass-pollen allergoids. 111. Efficacy and safety of unfractionated and high-molecular-weight preparations in rhinoconjunctivitis and asthma. J AUERGY CLIN IMMUNOL 1989;84:546-56. Marsh DG, Lichtenstein LM. Campbell DH. Studies on “allergoids” prepared from naturally occurring allergens. I. Assay of allergenicity and antigenicity of formalinized rye group I component. Immunology 1970; 18:705-22. Marsh DC. Norman PS. Roebber M, Lichtenstein LM. Studies on allergoids from naturally occurring allergens. III. Preparation of ragweed-pollen allergoids by aldehyde modification in two steps. J ALLERGY CLIN IMMUNOL 1981;68:449-59. Norman PS. Immunotherapy. Prog Allergy 1981;32:318-55. Osterballe 0. Immunotherapy in hay fever with two major allergens 19.25 and partially purified extract of timothy-grass pollen. A controlled double blind study. In vivo variables, season I. Allergy 1980:35:473-89. Osterballe 0. Nasal and skin sensitivity during specific immunotherapy with hvo major allergens 19.25 and partially purified extract of timothy-grass pollen. Allergy 1982:37: 169-77. Nordvall SL, Berg T. Johansson SGO, Lanner A. Clinical studies of a purified timothy-pollen extract: desensitization therapy with a purified timothy pollen preparation compared to a crude timothy-pollen extract. II. Results of the tests in vitro and their relation to symptoms and tests in vivo. Int Arch Allergy Appl Immunol 1978:67: 132-8. Ortolani C, Pastorello E, Moss RB. et al. Grass-pollen immunotherapy: a single year, double-blind, placebo-controlled study in patients with grass pollen-induced asthma and rhinitis. J ALLERGY CLIN IMMCJNOL1984;73:283-90. Berg T, Nordvall SI, Lanner A. Clinical studies of a purified timothy-pollen extract: desensitization therapy with a purified timothy-pollen preparation compared lo a crude timothy-pollen extract. 1. Results of tests in vivo. Int Arch Allergy Appl lmmunol 1980;63:2fXi-74. Malling HJ. Specific immunotherapy [Position paper of the European Academy of Allergy and Clinical Immunology]. Allergy 1988;43(suppl 6): I-33. Van Metre TE, Adkinson NF, Lichtenstein LM, et al. A controlled study of the effectiveness of the Rinkel method of immunotherapy for ragweed-pollen hay fever. J ALLERGY CLIN IMMWOL 1980;65:288-97. Van Metre TE, Adkinson NF Jr, Amodio FJ, et al. A comparison of immunotherapy schedules for injection treatment of ragweed-pollen hay fever. J ALLERGY CLIN IMMUNOL 1982;69:181-93. Bousquet J, Gu&in B, Michel FB. Clinical trials with standardized extracts. In: Brede HD, Stevens E, eds. Proceedings of the Third Paul Ehrlich Seminar on Regulatory Control and Standardization of Allergenic Extracts. Stuttgart: Gustav Fischer Vedag. 1985:291-309. Turkeltaub PC. The importance of allergen dose on the safety ad efficacy of immunotherapy of ragweed hay fever with standardized short ragweed extracts. J ALLERGYCLIN IMMIJNOL 1986;77:211. Bousquet J. G&tin B, Dotte A, et al. Comparison of rush immunotherapywith a standardized grass-pollen extract and

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19

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21.

22. 23.

24.

25.

26.

27.

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