KINETICS AND DYNAMIC EVALUATIONOF SPECIFIC IMMUNOTHERAPY

C. Pereira (1), F Botelho (2), B. Tavares (1), C . Lourenco (3),C. Baeta (3), A.-G . Palma-Carlos (4), J . Lima (3), C . Chieira (1)

SUMMARY: Specific immunotherapy (SIT) is frequently used in the treatment of allergic diseases . However, the mechanismsby which SIT achieves clinical improvement remained unclear. We decided to study the in vivo kinetics of this therapy, usinga nuclear medicine approach (leukocytes labelled with 99mTc-HMPAO) in patients on maintenance doses of specific immu-notherapy with confirmed clinical efficacy .

Material and methods : We studied 13 allergic patients grouped according to different treatment schedules: subcutaneousaqueous allergenic extract (3 latex and 2 hymenoptera venom), subcutaneous depot extract (2 house dust mite and 2 pollens),subcutaneous modified allergens (2 pollens), sublingual extract (2 house dust mites) . The control group included two allergicpatients submitted to subcutaneous injections of bacterial extract (1 patient - positive control), and aqueous solution (1 patient) .At the same time that the therapeutic allergen was administered subcutaneously, the autologous labelled white cells wereinjected intravenously in a peripheral vein in the contralateral arm. A thoracic dynamic acquisition of 60 mins, 64x64 matrix,2 frame/min, in anterior view was performed . Static acquisition for 256x256 matrix, during 5 mins each at 60, 90, 120, 180, 240,300 and 360 mins after the administration of the radiolabelled leukocytes, in thoracic (anterior and posterior), and abdominalview were performed . During the examination, the local erythema was monitored . A similar procedure was undertaken forsublingual administration of immunotherapy .

Results: The inflammatory activity at the site of SIT injection (aqueous depot extract) started in the first hour and the increa-se was time related . For modified allergen extract and sublingual SIT the activity was present since the beginning of the admi-nistration . The ascendant lymphatic drainage, which was directed to the homolateral axillary region, to the lymphoid tissue ofthe upper mediastinum and to the anterior region of the neck began earlier . Thoracic focalisations were present for all thepatients, whereas bowel focalisations were only observed for the subcutaneous route of administration . Sublingual SIT didnot induce axillary or intestinal' inflammatory focalisations, even though the patients had swallowed the allergenic extract . Theuptake coefficient in individualized areas corrected to the uptake coefficient background was also studied .

Conclusions : For the subcutaneous route of administration, except for gluteraldheyde modified allergen, the local inflam-matory activity at the allergenic injection site was significantly higher' in depth and was time dependent, maintaining activityeven after complete disappearance of the erythema and/or wheat These results express a prompt inflammatory involvementof the immune system with this allergenic therapy, which was unexpected until now . We also observed differences concer-ning allergic diseases, the type of allergenic extracts and routes of administration .Key-words: Specific immunotherapy - Subcutaneous immunotherapy - Sublingual immunotherapy - Allergic inflammation -Radio labelled cells - Scintigraphy, s mTc-HMPAO .

INTRODUCTION

Pllergen specific immunotherapy (SIT) and totalavoidance of exposure to allergens, to whichthe patient is sensitised, are the only interven-

tions that really influence in the long-term control ofthe allergic disease (1, 2) .

(1) Immunoallergology Department, Coimbra University Hospital ;Pneumology Center - Coimbra University (Portugal)(2) Biophysical Department, IBILI, Medicine Faculty - CoimbraUniversity.(3) Nuclear Medicine Department - Coimbra University Hospital .(4) Medicine Faculty of Lisbon University.

European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004

In fact, it is now obvious that SIT is the only curativetreatment for hymenoptera venom allergy or latexanaphylaxis, two examples of life-threatening IgEdependent mechanisms (1-4) . A lot of DBPC studies(double-blind, placebo-controlled) provide longitudi-nal data on the clinical efficacy of subcutaneous SITon mite, pollen, epithelium and some mould species .For other respiratory allergic diseases such as bron-chial asthma, allergic rhinitis and conjunctivitis, thereduction of symptom scores/need for medicationwere also observed (5) .Allergen vaccines are, in fact, the only treatment thatmay affect the natural course of allergic disease, pre-venting the development of asthma in patients withallergic rhinitis, reducing the severity of the disease

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and the need for anti-allergic drugs thereby impro-ving the patient's quality of life (1) . It is also now reco-gnized that immunotherapy is long-lasting effectiveand should be started earlier in the course of thedisease . It has been propose that SIT reduce the riskof development of new sensitisations (6, 7), but recentdata has not confirmed this protective effect onmonosensitised allergic patients (8) .There are several factors that are important to the cli-nical outcome of SIT concerning efficacy and toleran-ce: the use of premedication, the allergen source, thepurification, the standardization and the stability ofthe extract, the pharmaceutical presentation of theallergen (native or chemically modified ; aqueous ordepot) . The clinical manifestations before and duringthe treatment, the route of SIT administration, and theschedule followed, should also be evaluated (9) .The aqueous extracts are highly effective, but inducemore side-effects (local and systemic) than depot andmodified vaccines . These have been developed in anattempt to reduce or remove allergenicity, while pre-serving or increasing the immunogenicity. Aqueoussubcutaneous SIT is now the gold standard treatmentfor hymenoptera venom allergy and depot aeroaller-gen extracts for respiratory allergy. The use of chemi-cally modified allergens is not consensual . Althoughrecent papers demonstrate a clinical benefit with aller-goid therapy, DBPC studies comparing both extractsare still missing (10) .Local immunotherapy is another strategy for allergicdisease treatment . In spite of the therapeutical bene-fit observed in some studies, there is no consensus onthe use of the nasal, bronchial or oral route of admi-nistration of SIT (1, 2, 11) . On the other hand, the sub-lingual-swallowed administration of SIT in higherdoses, which is another type of local immunotherapy,has an A level evidence-based medicine for seasonaland perennial allergic rhinitis in adults and seasonalallergic rhinitis in children (1, 11, 12) .The clinical efficacy was demonstrated by DBPC studyand later, with the increment in the allergen doses,the immunological changes were confirmed (12, 13) .Recent papers have confirmed the long-lasting (14)clinical efficacy of sublingual administration in a ran-domised, placebo-controlled, double blind, double-dummy study (15) .In spite of the widespread knowledge in the scientificliterature, the mechanism by which SIT achieves cli-nical improvement remains unclear. For subcuta-neous immunotherapy the immunomodulation of theT-cell response is now accepted : immune deviation(increase in ThO/Th1), Tcell anergy (decrease inTh2/ThO), or both mechanisms (1) . However, this isnot so linear, because SIT did not induce Th1-typecytokine-associated pathology in treated respiratoryallergic patients to aeroallergens (16) . There are other

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immunological changes with subcutaneous SIT : therise in allergen-blocking IgG, the induction of IgE-modulating CD8+ T cells, the reduction of mast cellsand eosinophils on the target mucosa, the decrease ininflammatory mediators (1, 5, 17-21) and a modula-tion on the inflammatory trafficking cells by reducedexpression on VCAM-1 (22, 23) .Immune tolerance depends on different mechanisms,including Tcell anergy, Tcell depletion by apoptosis,and active immune suppression (24) . One of the targetmechanisms of SIT is the induction of tolerance to aller-gens to which the patient is sensitised (25) . IL-10 is pro-bably a relevant cytokine induced by this treatment andis related to regulatory T cells that actively control orsuppress the function of others cells, generally in aninhibitory pattern (5, 25) . The changes in microenviron-ment due to the decrease in histamine and PGE2 mastcell release and the IL-10 and TGF-_ release by dendriticcells could deviate the Tcell population pathway inorder to induce T regs and finally a therapeutic toleran-ce (26). This recent findings agree with what is current-ly known about the regulation induced by SIT . It is notcredible that it could promote a Th2 / Thl switch (25) .Much of the knowledge on SIT is based on studiesperformed in subcutaneous route of administration,but increasing data are now available on sublingualvaccines. The absorption of allergen through theintact mucosa and the interaction with local dendriticcells could induce the process and immune-tolerance .Besides the intrinsic local effect, the swallowed aller-gen could induce an additional GALT related mecha-nism (11, 12) . In fact, the first method of sublingualadministration of SIT in which the allergen was spatout is significantly less effective. Most of the mecha-nisms and immunological changes that occur withsublingual administration of high allergen dose havebeen recently demonstrated (1, 27-31) .There is lots of information available on immunomo-dulation with SIT, but studies on kinetics of SIT admi-nistration and its immune effects on allergic patientsare still lacking .The study of M Bagnasco et al. (32) was highly impor-tant in understanding much of the data . In this study,the administration of radiolabelled Par-j-1 by 123 1 isoto-pe to healthy individuals demonstrated the local persis-tence of the allergen at the sublingual region for a longperiod of time . The systemic radioactivity was only pre-sent after swallowing the allergen . For sublingual SIT itseems that the allergen is degraded in the gastroenteralenvironment and only peptides are absorbed (33) .There are no similar studies in the literature concerningthe subcutaneous route of administration .Radiolabelling of blood cells is a nuclear medicinetechnique used in the study of inflammatory diseasessuch as chronic bowel inflammatory disease (34-37) .We considered that this kind of methodology could be

European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004

interesting worth investigating further with regards toan in vivo approach to the study of SIT mechanism ofaction and immune response. We expected to studythe migration of autologous peripheral labelled-leu-kocytes that were reinjected to the site of the allergenextract administration in allergic patients, and theother possible resulting events. We think that the bio-logical response to the therapy can be easily obser-ved . The site or sites where the immune responsehappen and the different times of the response, canbe established. We decided to study the kinetic res-ponse to SIT in patients with different types of allergyconcerning the severity, the type of allergenic extractsadministered and different routes of administration .

MATERIAL AND METHODS

The Coimbra University Hospitals Ethics Committeeand Research Commission approved the design ofthis study protocol and all the volunteer patientssigned an informed consent form according to theinternational ethics consensus .

Subjects

13 adult allergic volunteer patients aged 20 to 51 yearold were enrolled for study . All of them were submit-ted to SIT for at least 2 years and in all of them the cli-nical efficacy was demonstrated by complete remis-sion of symptoms, the absence of medication withregular anti-allergic drugs, reduction of skin reactivityto allergens and reduction of serum specific IgE . Inpatients with previous anaphylaxis, the absence ofallergic reactivity after the beginning of treatmentwas confirmed by challenge tests or immunoblottingstudies .The patients were selected according to the differentallergenic extracts and routes of administration of SIT,including :a Aqueous subcutaneous allergenic extract (for ana-phylaxis) :•

2 patients - hymenoptera venom, honeybee venomextract ;•

3 patients - latex extract .- Depot subcutaneous allergenic extract (for respirato-

ry allergy) :•

2 patients - Dermatophagoides pteronyssinus extract ;•

2 patients - grass pollen extract .Modified subcutaneous allergenic extract (for respi-

ratory allergy) :•

1 patient - grass pollen extract;•

1 patient - Parietaria judaica extract .Sublingual allergenic extract (for respiratory allergy) :

•

2 patients - Dermatophagoides pteronyssinus extract .

European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004

Dermatophagoides pteronyssinus allergic patients(bronchial asthma and rhinitis) not submitted to SITwere studied as a control group .None of the patients had other clinical disorders,namely inflammatory diseases . All the femalepatients were submitted to a pregnancy test on theday of the study to exclude pregnancy .All the studies were performed under strict hospitalmedical care surveillance, in the day of SIT administra-tion, according to each individual schedule of treatment .

Methods

∎ Technetium -99m hexamethylpropyleneamineoximeleukocyte labelling (99mTC-HMPAO) : The white bloodcells labelling process with 99mTC-HMPAO was per-formed according to the standard University HospitalNuclear Medicine Department protocol, based on thetechnique described by Peters et al. (34) . 42 ml of thepatient's blood was collected from peripheral veinwith a 60 ml plastic syringe, containing 6 ml of acid-citrate-dextrose (ACD) plus 6 ml of hydroxy-ethil-star-ch (HES) at 6% .The ACD anticoagulant prevents cells from sticking tothe plastic wall of tubes and the HES accelerates ery-throcyte sedimentation by affecting the charge of thesialic acid groups on the outer membrane of the redblood cell . This blood in the syringe was maintainedin a vertical position for 30 mins in order to allow ery-throcyte sedimentation . A second 10 ml syringe ofblood was sampled with 1ml of ACD . This blood wascentrifuged at 2 000 g for 10 min in order to obtaincell-free plasma (CFP) .After 30 mins of blood syringe sedimentation, thesupernatant was centrifuged at 150 g for 5 min and thesupernatant, platelet rich plasma (PRP) was dischar-ged. The leukocyte pellet was resuspended in 0 .6 ml ofcell free plasma and labeled with 99mTC-HMPAO(Ceretec®, Amersham, UK) . For the leukocyte labelingprocedure, the HMPAO must be labeled first . For thispurpose, a 1110 MBq in 5 ml of saline of 99mTc fresh-ly eluted was added to a kit of HMPAO that contained0.5 mg of exametazime, 7.6 mg of SnCl 2 and 4.5 mg ofNaCl according to the manufacturer's indications. 3 mlof this 99mTc-HMPAO was added to the leukocytesresuspended in cell free plasma, and the mixture wasmaintained at room temperature for incubation, for 10min, in order to label the leukocytes . After this time, 10ml of PRP was added to the leukocytes in order to stopthe labeling procedure, and the mixture was centrifu-ged at 150g for 5 min .After centrifugation, the supernatant was removedand discharged and the labeled leukocyte pellet wasresuspended in 5 ml of CFP, counted and re-injectedinto the patients .

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The labeling yield ranged between 50% and 75%, asnormally described .The effective dose-equivalent for this examinationwas 0.021 mSv/MBq (38, 39) .

•

Allergenic therapeutic extract : The allergenic extra-cts administered were from ALK-Abe116 (Spain) for allthe patients except for modified subcutaneous aller-genic extract . These extracts were glutaraldehydemodified grass pollen (Bial/Aristegui- Spain) anddepigmented Parietaria judaica pollen (Leti- Spain) .All the patients were submitted to the maintenancedose according to the individual schedule .One allergic patient of the control group was submittedto a 0.5cc subcutaneous phenol aqueous injection sup-plied by ALK-Abe116 (Spain), and the other patient wassubmitted to a 0.5cc subcutaneous injection of a bacte-rial extract (RibomunylTM, Pierre Fabre Medicament,France: ribosomal fractions of Klebsiella pneumoniae,Streptococcus pneumoniae, Streptococcus pyogenes,Haemophilus influenzae and the membrane fraction ofKlebsiella pneumoniae), an nonspecific immunostimu-lant agent of IgG production .

• Administration procedures : Patients were lyingsupine under the gamma camera . A humeral vein wascatheterised to permit the reinjection of labeled auto-logous leukocytes. The allergen extract was adminis-tered subcutaneously, according to the usual method,in the contra lateral arm . Both injections were given atthe same time, simultaneously with the scintigraphicacquisitions .The 2 allergic patients under sublingual SIT were alsolying supine under the gamma camera at the begin-ning of the study . The drops of the allergenic extractwere administered at the same time as the reinjectionof the labeled autologous leukocytes in the humeralvein . Three minutes later, the-patients swallowed theallergen extract .For the control group the same procedure was adop-ted. The study was performed under the close super-vision of allergist and nuclear medicine specialists .

• Scintigraphic acquisitions (table 1) : Scintigraphicstudies were performed on a large field of view digi-tal gamma camera (GE XR, Milwaukee, USA, interfa-

maximum count

maximum count

Uptake Coefficient

Uptake Coefficient

average count

average count background

max count ROI /average count backgrou !

max count /average countUptake COWUCROI - UCBK

Table 1 .

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ced with a Camstar acquisition unit and an eNTEGRAprocessing unit), equipped with a parallel low-energygeneral purpose collimator .A dynamic acquisition was obtained in anterior viewat thoracic and neck level for 64x64 matrix for 60minutes (120 frames x 30 seconds) followed by staticimages at 60, 90, 120, 180, 240, 300 and 360 mins afteradministration of the radiolabeled leukocytes and theallergen for 256x256 matrix and for 5 mins each .These static images were obtained in anterior andposterior views at thoracic level and anterior views atabdominal projection .During the acquisitions, the visualized area was keptclose to the gamma-camera collimator, and the patientswere not allowed to move any part of the body, in orderto maintain the geometry of the projections .Throughout the acquisitions, the erythema and thewheal at the site of SIT administration were controlled .For quantitative results, the procedure was differentaccording to the type of image . For the dynamic tho-racic images, regions of interest (ROls) were drawnat SIT administration, background area (muscle),and for possible focalisations (cervical and axillaryareas and thorax) . For the thoracic static views, animage corresponding to the geometric mean bet-ween anterior and posterior views was calculated,and ROls were drawn at thoracic locations . For thestatic anterior abdominal views, ROls were drawnover each location .For each ROI the total counts, average counts perpixel and maximum counts were obtained, anddecayed was corrected. With these values, uptakecoefficients were calculated by the ratio between themaximum count rate at the target ROI and averagecounts at the background ROI . In order to normaliseand to help with the interpretation of the results anuptake coefficient of the background ROI was calcu-lated and subtracted from the uptake coefficient ofthe target ROls .

∎ Data analysis : The clinical pattern, allergen extra-ct and route of administration were analysed indivi-dually and the different groups of patients werecompared .The statistical analysis was performed by descripti-ve analysis .

European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004

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There were no adverse events caused by radiolabeledautologous cells reinjection or allergenic therapeuticextract administration .Clinical and allergenic therapeutic extract aspects areshown in table 2 and 3 .After scintigraphic processing, the ROI value wasobtained at the site of SIT administration, thoracic,axillary and cervical area, abdominal focalizationsand ROI background . We also calculated the correcteduptake coefficient (maximum count at the site of SITROI over average counts at the background ROI) atdifferent focalisations at different times .The uptake coefficients for the different focalisationson analysis are shown in Figs . 1-5 .We did not consider the scintigraphic acquisitions atthe site of the allergenic injection (patient 1, 2 and 9)and the axillary area (patient 1) due to technical pro-blems .The inflammatory activity started early in patientstreated with aqueous allergenic extract for anaphy-laxis than for in patients treated with depot extracts

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European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004

for other diseases (latex : 20-35 mins; hymenopteravenom : 15 mins; grass pollen : 25 and 30 mins; mites:25 and 45 mins) .For sublingual allergenic extracts the inflammatoryactivity started promptly (at 5 mins) with a rapidincrease that persisted during the study .In patients submitted to allergoid, the inflammatoryactivity at site of administration of the extract startedin the first minutes, although with a different pattern .In the allergic patient submitted to saline subcuta-neous injection, we observed low values of activity atthe site of administration, probably related with anirritative effect caused by phenol used as preservati-ve. This observation confirms the sensitivity of thelaboratory procedure. No other focalisations wereobserved during the study .The control patient submitted to bacterial injection sho-wed inflammatory activity at 10 minutes with an increa-se that was time related . We also observed activity inthe axillary, the cervical and the thoracic areas, but withno focalisations at the bowel region, Fig . 6.The inflammatory activity at the axillary and the cer-vical areas was present from the beginning of the

Figures 1: Uptake coefficient corrected oallergenic extract local administration .

Figures 2 : Uptake coefficient correctedaxillary area focalisations .

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Table 2 : Clinical aspects of patients sample .Disease: A= anaphylaxis ; BA+AR= bronchial asthma+allergic rhinitis .Allergen : L=latex ; HV= hymenoptera venom (honeybee) ; GP= grass pollen ; Dp= Dermatophagoides pteronyssinusjudaicaSIT route : sc= subcutaneous ; sl= sublingualSIT extract : depot= aluminium hydroxide-adsorbed ; alergoide=SIT (cc) : volume in ml administrated of the maintenance dose . Patients 12 and 13 are submitted to 5 dropsSIT dose: quantity of allergen in µg proteinAdministered activity : MBq

Table 3 : Control group . Clinical aspectsDisease: BA+AR= bronchial asthma+allergic rhinitis .Allergen : Dp= Dermatophagoides pteronyssinusAdministered activity : MBq

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European Annals of Allergy and Clinical immunology - volume 36 - n° 10 - 2004

Figures 3 : Uptake coefficient correctedcervical area focalisations .

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Figures 5: Uptake coefficient corrected ogut focalisations .

after 60 minutes after the injection, even though theywere observed earlier . There were no significant dif-ferences among the different groups .Focalisations at the abdomen acquisition views wereobserved in all the patients submitted to subcuta-neous SIT. We only considered ROls till 240 minutes .This time was coincident with the beginning of hepa-

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tic/gall bladder excretion of the product. No bowel acti-vity was observed in patients on SLIT treatment .There were other focalisations such as supraster-num and anterior mediastinum with high inflamma-tory activity, but we decided not to consider theseareas because they were sites of vascular confluen-ce. Bone marrow was another site of intense activi-

382

Figures 6 : Uptake coefficient corrected/timefor the ROls of the active control patient sub-mitted to bacterial extract .

Figures 7: Patient 1 study, dynamic and sta-tic acquiitions . Inflammatory activity at 25,90 120, 180, 240 min .) .

Figures 8 : Patient 12 study, dynamic andstatic acquisitions . Inflammatory activity at240 min .

ty, higher than that usually observed by this tech-nique .Distinct ROI count profiles were obtained with the dif-ferent routes of administration and with the differentallergenic extracts but all of them induced a time-dependent global inflammatory activation at sitesrelated with the immune system (Fig. 7-10) .

European Annals of Allergy and Clinical immunology - volume 36 - n° 10 - 2004

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European Annals of Allergy and Clinical immunology - volume 36 - n° 10 - 2004

DISCUSSION

In this study we decided not to label the allergen used,because it was only possible to make a linkage betweenan allergenic epitope and the radiolabel-drug, excludingthe other epitopes. The decision to use the commonadministration of SIT seemed to be the procedure thatminimised the error factors in the understanding of thistherapy dynamics. The technique is now a routine pro-cedure for analogous inflammatory pathologies . Therisks are minimal, the radioactive burden is low, and thebody-absorbed dose (effective dose) corresponds toabout 3mSv, which is similar to one year of naturalexposure in the continental area of Portugal .

383

Our first attempt was to study the local inflammationrelated to SIT administration . The activity observed inother areas and the speed of occurrence was a sur-prising result. Even in patients with excellent clinicalresponse to immunotherapy, the administration ofspecific allergen resulted in a local inflammatory res-ponse that ranged from minimal intensity to erythe-ma and/or wheal . The intensity of inflammation washigher for aqueous extract administrations in patientswith the most severe allergic diseases . However, itwas not related with clinical signs of inflammation atthe site of administration . The allergoids were relatedwith lower inflammatory activity when comparedwith depot extracts, as expected, according to the cli-nical knowledge (40) . In the 2 patients submitted tosublingual extracts the beginning of the radioactivitywas faster, with no signs of local effects . There was aminimal activity in the control patient who was sub-mitted to phenol-saline injection, which is usuallyused to dilute the extracts . We considered that thisreaction was related to phenol, which is a nonspecificirritant used as a preservative. This observationconfirmed the sensitivity of the technique . The othercontrol patient that was submitted to an injection ofbacterial derived fragments, also revealed activityaccording to the expected IgG dependent mechanismof action of this immune-stimulant (41) .In the first minutes ascending drainage directed to thehomolateral axillary area was induced by subcuta-neous SIT. Cervical and superior mediastinum lym-phoid tissues were the next structures involved . Thelate static acquisitions showed global activation onfocalisations related to the immune system : lymphoidtissue neck/cervical, suprasternum, mediastinum,lung, gut and bone marrow. The early involvement ofthe central lymphoid structures was an unexpectedfinding. The maximum ROI values were increasedeven when the radionuclide was eliminated throughurine and the half time was reached . This could reflectthe magnitude of the therapeutical allergen mecha-nism of restimulation and immune-tolerance .Concerning the sublingual immunotherapy the activi-ty was reached faster than for subcutaneous SIT.There was an obvious difference in the inflammatoryactivity profile comparing these two types of immu-notherapy. The most intense ROI values were achie-ved at the cervical lymphoid tissue, as expected .There was no activity in the axillar and the abdominalarea, compared with depot extract. This data couldexplain the need for daily or three times a week admi-nistration of sublingual SIT in order to promote fre-quent stimulation of the immune system .The allergoid extracts also induced inflammatory acti-vity when compared with depot pollen extracts. Therewere obvious differences in local activity betweenboth. Our results could deflect some criticism of lot of

384

authors who reject the use of polymerised vaccines orchemically modified allergens .This technique did not allow labelling specific circula-ting peripheral cells . The pellet resulting from a bloodsample contains all the lineages of white blood cells.At first, the lymphocytes seemed to be the only cellsresponsible for the local and global events in thestudy. However, we must reflect on all the knowledgeabout SIT, inflammation and immune response avai-lable till now .Dendritc cells (DC) have been a matter of exhaustivestudy and seem to be a nuclear target of inflammationand immune tolerance . The professional APC are divi-ded into two major systems : DC, including blood andtissue DC as well as epidermal Langerhans cells (LC),and the monocyte/macrophage system (42) .Concerning DC there are two different subsets : DC1 ormyeloid DC, and DC2 or lymphoid/plasmocytoid DC(42, 43) . DC2 are mainly found in thymic medulla, ton-sils and in T cell areas of secondary lymphoid organs,whereas DC1 are located outside the T cell areas . DC2are unable to internalise foreign antigen (42) . On theother hand, DC1 could traffic from the blood to theperipheral tissue to capture foreign antigens, maymigrate to the draining lymphoid organs in order toprime T cells towards Th1 or Th2 cytokine profile (26,43, 44). Under appropriate stimulus peripheral bloodmonocytes could differentiate to DC1 (42) .Langerhans cells belong to the DC1 system and arerelevant in the skin . After local inflammatory stimulusthe monocyte-chemoattractant protein (MCP) inducesrecruitment of LC progenitors from the bone marrow,migration to the skin and in the skin, and migration toperipheral lymph nodes (43) . It is now well establishedthat IL-10 DC release is an important factor of immunetolerance promoting Tregs lymphocytes (25, 26, 45) .The DC family of oral mucosa is different from that ofthe skin LC family. The oral microenvironment and TGF-

R are relevant as well as IL-10 (43) . They also presentedincreased expression of FccRl on their surface, whichwas partially occupied by IgE molecules (43) . The linka-ge of allergens to the FcERI receptor contributes to tole-rance by an IL-10 dependent mechanism (42, 43) .According to our results these data could be explai-ned by the following hypothesis : The subcutaneousallergenic extract induced activation of LC, recruit-ment of other circulating DC/monocytes ; migration tolymphoid areas ; cooperation with DC2 and finallyTregs induced by IL-10 and tolerance towards sensiti-sed allergens . The systemic activation of areas relatedwith the immune system could support the globalimunomodulation of SIT.Large amounts of allergen are administered by sub-lingual SIT route in an area with close proximity tolymphoid organs such as the lingual amygdale . It ispossible that DC could initiate the process as occurs

European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004

in the skin . However the presence of lymph nodeswith other populations of DC (DC2) could lead to asaturation of activity of DC1 and the inability to causean intense systemic inflammatory activity. The indu-ced tolerance and the efficacy of this therapy are,consequently less intense and require frequent stimu-lation and higher allergen doses. On the other hand,the swallowed allergen could interact with gastroin-testinal mucosa and promote additional tolerancemechanism, mediated by TGF-(3 and Th3 regulatorycells (43, 45) .Our results showed a real systemic effect of SIT, rela-ted to focalisations on immune system dependence .The inflammatory activity began quicker than weexpected and there were obvious differences concer-ning the different extracts. There seems to exist anobvious difference between the kinetic of sublingualand subcutaneous routes of administration . Aqueousallergenic extracts induce more intense and globalsystemic inflammatory activity than depot extractsand ultimately the allergoid extracts .In the future it will be important to study the kine-tic/dynamic of the built-phase of SIT in animal modelsin order to understand how the tolerance/immuno-modulation process occur throughout the treatment .

Acknowledgments: We thank Drs I . Carrapatoso andG. Loureiro for helping with the clinical study; NuclearMedicine technicians; and Jeremy Waterson for theEnglish revision . Research Grant from PneumologyCenter of Coimbra University (Dir . Prof . L.-C .Oliveira)/Portuguese Ministry of Education .

References1 . Bousquet and ARIA Workshop Group. - J Allergy Clinlmmunol2001;108 suppl 5: S147-S336 .2. Bousquet J, Lackey R.-F., Mailing H .-J. - WHO positionpaper. Allergen immunotherapy : therapeutic vaccines forallergic disease . Allergy 1998;53 suppl 44 : 1-42 .3 . Rueff F, Przybilla B, Muller U, Mosbech H . The sting chal-lenge test in Hymenoptera venom allergy . Position paper ofthe Subcommittee on Insect Venom Allergy of the EuropeanAcademy of Allergology and Clinical Immunology . Allergy1996;51 :216-254. Pereira C., Pedro E ., Tavares B . et al . Specific immunothe-rapy for severe latex allergy . Eur Annals of Allergy ClinImmunol 2003;35:217-25 .5. Akdis C.-A., Blaser K . - Mechanisms of allergen-specificimmunotherapy. Allergy 2000 ;55:522-30.6. Moller C ., Dreborg S ., Ferdousi H.-A . et al . - Pollen immu-notherapy reduces the development of asthma in childrenwith seasonal rhinoconjunctivitis (the PAT-study) . J AllergyClin Immunol. 2002;109 :251-6 .7 . Passalacqua G ., Canonica G.-W . - Long-lasting clinical effi-cacy of allergen specific immunotherapy . Allergy2002;57 :275-6.8 . Tella R ., Bartra J ., San Miguel M . et al . - Effects of specificimmunotherapy on the development of new sensitisationsin monosensitised patients. Allergol Immunopathol ( Madr)2003; 31 : 221-5 .

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9. Parmiani S., Fernandez-Tavora L ., Moreno C ., Guardia P.,Rico P - Clustered schedules in allergen-specific immuno-therapy. Allergol et lmmunopathol 2002;30:283-91 .10. Garcia-Selles J ., Pascual A ., Funes E ., Pagan J .-A ., NegroJ.-M . - Clinical efficacy of depigmented and glutaraldehydepolymerized therapeutic vaccines of Parietaria judaica .Allergol et Immunopathol 2003;31 :63-9 .11 . Kagi M .-K., Wuthrich B . - Different methods of local aller-gen-specific immunotherapy . Allergy2002 ;57:379-88.12. Lleonart R ., Monoz F., Eseverri J .-L . et al . - Inmunoterpaisublingual en ninos . Position Paper. Allergol etImmunopathol 2003;31:244-913. Passalacqua G., Canonica G.-W . - Sublingual or injectionimmunotherapy : the final answer? Allergy2004 ;59 :37-8 .14. Di Rienzo V., Marcucci F., Puccinelli P et al. - Long-lastingeffect of sublingual immunotherapy in children with asthmadue to house dust mites : a 10-year prospective study . ClinExp Allergy 2003;33:206-10 .15. Khinchi M.-S ., Paulsen L.-K ., Carat F., Andre C ., HansenA.-B ., Mailing H.-J . - Clinical efficacy of sublingual and sub-cutaneous birch pollen allergen-specific immunotherapy : arandomised, placebo-controlled, double-blind, double-dummy study. Allergy 2004;59 :45-53 .16. Durham S.-R ., Till S .-J . - Immunological changes asso-ciated with allergen immunotherapy? J Allergy ClinImmunol 1998;102:157-64 .17. Durham S.-R ., Ying S ., Varney V.-A . et al. - Grass pollenimmunotherapy inhibits allergen-induced infiltration ofCD4+ T lymphocytes and eosinophils in the nasal mucosaand increases number of cells expressing mesanger RNA forinterferon-gamma . J Allergy Clin Immunol 1996;97 :1356-65 .18. Hamid Q ., Schotman E ., Jacobson M ., Walker S ., DurhamS.-R . - Increases in interleukin-12 (IL-12) messenger RNA+(mRNA+) cells accompany inhibition of allergen induced lateskin responses following successful grass pollen immuno-therapy. J Allergy Clin Immunol 1997;99:254-60 .19. Ariano R ., Merendino R.-A., Minciullo P-L ., Salpietro C .-D., Gangemi S . - Specific immunotherapy effect on interleu-kin-18 and CD30 serum levels in monosensitized patientswith rhinitis . Allergy Asthma Proc 2003 ; 24: 179-83 .20. Lewis DB . Allergy immunotherapy and inhibition of Th2immune responses : a sufficient strategy? Curr OpinImmunol. 2002;14 :644-51 .21. Gardner L.-M ., O'Hehir R.-E ., Rolland J.-M . - High doseallergen stimulation of T cells from house dust mite-allergicsubjects induces expansion of IFN-gamma+ T cells, apopto-sis of CD4+IL-4+ cells and T cell anergy . Int Arch AllergyImmunol 2004 .22. Pereira C ., Paiva A ., Todo-Bom A . et al. - Expression ofVCAM-1 during allergic reaction in asthmatic patients .Effects of specific immunotherapy. In XVI World Congress ofAsthma . Ed HE Neffen, C Baena-Cagnani, A Yanez. MonduzziEd, Bologna, Italy; 113-8 .23. Palma Carlos A.-G ., Pregal A.-L ., Alonso E . et al . - Solubleadhesion molecule levels during immunotherapy for peren-nial allergic rhinitis and/or asthma . Actma-Asthma2001 ;2 :116-7 .24. Foussat A ., Cottrez F., Brun V., Fournier N ., BreittmayerJ .-P., Groux H . - A comparative study between T regulatorytype 1 and CD4+CD25+ T cells in the control of inflammation .J Immunol 2003 ; 17:5018-26 .25. Akbari 0 ., Stock R, Dekruyff R.-H ., Umetsu D.-T. - Role ofregulatory T cells in allergy and asthma . Current Opinion inImmunol 2003 ; 15 627-33 .26. Schmit-Weber C.-B ., Blaser K. - T-cell tolerance in allergicresponse . Allergy 2002;57 :762-8 .27. Passalacqua G ., Albano M ., Riccio A . et al . - Clinical andimmunological effects of a rush sublingual immunotherapyto Parietaria species: a double-blind, placebo controlledstudy. J Allergy Clin Immunol 1999;104 :964-8 .28. Guez S ., Vatrinet C., Fadel R ., Andre C . - House dust-mitesublingual-swallow immunotherapy (SLIT) in perenial rhini-tis : a double-blind, placebo-controlled studt . Allergy2002;55:369-75 .

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29 . Clavel R., Bousquet J ., Andre C. - Clinical efficacy of sub-lingual-swallow immunotherapy : a double-blind, placebocontrolled trial of standardized five-grass pollen extract inrhinitis . Allergy 1998; 53 :493-8 .30. Ippoliti F., De Santis W., Volterrani A . et al. -Immunomodulation during sublingual therapy in allergicchildren . PediatrAllergylmmunol2003;14:216-21 .31 . Reich M ., Zwacka G., Markert U.-R . - Nonspecific plasmaproteins during sublingual immunotherapy . Chem ImmunolAllergy 2003;82 :99-108 .32. Bagnasco M ., Mariani G., Passalacqua G . et al . -Absortion and distribution kinetics of the major Parietariajudaica (Par j 1) administered by noninjectable routes inhealthy human beings . J AllergyClin Immunol1997;100:122-9 .33. Passalacqua G ., Bagnasco M ., Mariani G ., Falagiani R,Canonica G .-W . - Local immunotherapy: pharmacokineticsand efficacy . Allergy 1998;53 :477-84 .34. Peters A.-M ., Danpure H .-J ., Osman S. et al . - Clinicalexperience with 99m Tc-hexamethyl propylene-amine oximefor labelling leucocytes and imaging inflammation . Lancet1986;2 :946-9 .35. de Labriolle-Vaylet C.-L ., Doloy M.-T., Salat-Trepas M .,Petiet A., Colas-Linhart N . - Radiobiology of 99m Tc-labelledlymphocytes . Eur J Nucl Med 1991 ;18 :556 .36. Frier M . - Leucocyte radiolabelling techniques : practicalaspects. Scand J Gastroenterol 1994;203 (Suppl) : 32-5 .

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37. Charron M., Di Lorenzo C ., Kocoshis S.-A . et al . - (99m)Tcantigranulocyte monoclonal antibody imaging for the detec-tion and assessment of inflammatory bowel disease newlydiagnosed by colonoscopy in children . Pediatr Radial.2001 ;31 :796-800 .38. Takur M ., Seifert C ., Madsen M. et al. - Neutrophil labelling :procedures and pitfalls . Semin Nucl Med 1984;14 :107-117 .39. International Comission on radiological protection . -Radiation dose to patients from radiopharmaceuticals . ICRpPublication 53, New York . Pergamon Press 1988 .40. Ariano R., Kroon AM, Augeri G., Canonica G.-W.,Passalacqua G . - Long-term treatment with allergoid immu-notherapy with Parietaria . Clinical and immunologic effectsin a randomized, controlled trial . Allergy 1999;54 :313-9 .41 . Clot J. Pharmacology of ribosomal immunotherapy .Drugs . 1997; 54 (Suppl 1) : 33-6 .42. von Bubnoff D ., de la Salle H ., We endorf J., Koch S .,Hanau D., Bieber T. - Antigen-presenting cells and toleranceinduction . Allergy 2002 ;57 :2-8 .43. Novak N ., Allam J .-R, Betten H., Haberstok J ., Bieber T. -The role of antigen presenting cells at distinct anatomicsites: they accelarate and they slow down allergies . Allergy2004;59 :5-14 .44. Lambrecht B.-N ., Hammad H . - Myeloid dendritic cellsmake it to the top . Clin Exp Allergy 2002;3 :805-10 .45. Jonuleit H., Schmitt E . - The regulatory T cell family : dis-tinct subsets and their interrelations . J Immunol2003;171 :6323-7 .

AVOIDING PEANUTS MAY RESULT IN RECURRENCE OF ALLERGY. FROM THEJOURNAL OF ALLERGY & CLINICAL IMMUNOLOGY

Children who keep avoiding peanuts after they outgrow their peanut allergy -h 3rat their peanut allergy will return, according to a study in the November 2004 Journal ;3f_Alle

I 'Immunology (JACI) . The JACI is the peer-reviewed journal of the American Academy of AI11*>k &'Immunology (AAAAI) .

,study,David M . Fliescher, MD, Johns Hopkins University School of Medicine, and colleagie .ants who had previously outgrown their peanut allergy . They sought to determine the percefdawho reacquire peanut allergy later; identify any risk factors for recurrent peanut allergy, and'de

c -+'eeE emendations for treating patients who have outgrown peanut allergy.

on dtlestionnaire results, repeat peanut-specific IgE levels, and repeat food challenges, the reseer6ti' that patients who have outgrown their peanut allergy have approximately an 8% chance of ft r'The study found this risk is significantly higher in patients who continue to avoid eating peanUtafter

-is resolved .

n the study's results, researchers recommend that patients eat concentrated forms of'penut of 1

cinch after outgrowing their peanut allergy in an attempt to maintain their tolerance to peanu

e ers: also recommended that patients and families who rarely eat peanuts or eat then~4n f"iti'4 mer outgrowing their allergy should continue to carry epinephrine at all times .

I is the largest professional medical specialty organization in the United States representing allergists, ast ' ;atti&ts,iclinical immunologists, allied health professionals and others with a special interest in the reseami and w_ ,of&Ifergicdisease . Established in 1943, the AAAAI has nearly 6,000 members in the United States, Canada ar+d `tries . The AAAAI serves as an advocate to the public by providing educational information through its Web l iatY .',saaai,org or the toll-free physician referral and information line at 1(800) 822-2762 .

ffIRIS NOTE: This study was published in the peer-reviewed, scientific journal of the American Academy: of Alte& immunology, but does not necessarily reflect the policies or the opinions of the Academy . To receive a yL

sleasl~ contact John Gardner ( jgardner@aaaai .org) at (414) 272-6071 . For more information and mess tiA

past JACI news releases, visit the Media Center of the AAAAI Web, site, www.aaaai .org .

European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004