Paolo Falagiani

Absorption and distribution kinetics of the major Parietaria judaica allergen (Par j 1) administered by noninjectable routes in healthy human beings.

BACKGROUND AND OBJECTIVE The clinical effectiveness of noninjectable routes for specific immunotherapy has been demonstrated in many studies, but no data are available on the kinetics of allergens administered by these routes. Therefore we studied the kinetics of the radiolabeled purified major Parietaria judaica allergen (Par j 1) after sublingual, oral, and intranasal administration to healthy human beings. METHODS After tracer administration (10 to 12.5 microg of Par j 1 labeled with iodine 123) to nonallergic volunteers, scintigraphic images were recorded at various times. Blood samples were also obtained at serial intervals to evaluate the absorption and distribution of radioactivity in plasma and to identify circulating radioactive species by molecular exclusion gel chromatography. RESULTS When the sublingual route was used, no circulating radioactivity was detected until the tracer was kept under the tongue. The labeled allergen was rapidly degraded and absorbed in the gastrointestinal tract after swallowing. Plasma radioactivity peaked at about 1.5 to 3 hours and was mostly represented by free radioiodine and small radiolabeled peptides. Some activity not caused by free 123I remained associated with the oral mucosa up to 18 to 20 hours after administration. When the oral route was used, the results were similar to those observed after swallowing the sublingually administered allergen but without any persistence of the tracer in the mouth. When the intranasal route was used, the pattern of plasma radioactivity mimicked that of the sublingual and oral routes, with absorption of activity from the radiolabeled allergen occurring in the gastrointestinal tract after transport to the pharynx by mucociliary clearance. A relevant fraction of the tracer was retained on the nasal mucosa up to 48 hours after administration. CONCLUSION The data in this study provide the first experimental basis for exploring the in vivo kinetics of allergen administered through noninjectable routes for specific immunotherapy in human beings.

Safety of sublingual immunotherapy with a monomeric allergoid in very young children.

Sublingual immunotherapy (SLIT) is approved by official documents (1), nevertheless some concerns still remain about its use in children. The age below 5 years is considered a relative contraindication (1), as severe side effects are more difficult to treat. SLIT is devoid of relevant risks as testified by 20 years of use and of controlled trials, and in everyday clinical practice it seems to be safe in very young children also, but a systematic assessment is still to be carried out. We performed a safety survey of SLIT in children under the age of 5 years. They suffered from intermittent or mild persistent asthma or persistent rhinitis (2), and had positive skin-prick test for at least one of the following: dust mites, grasses, parietaria, olive, birch, cat and dog dander. The SLIT was a monomeric allergoid, standardized in Allergy Units (LAIS ; Lofarma S.p.A., Milan, Italy), prepared as aqueous drops. After a 3-week build-up, a maintenance of 4 drops/day of the maximum concentration (3000 AU/ml) was given continuously. The administration was by oral means. The first doses were given at the pediatrician’s office. Parents were carefully instructed on how to administer SLIT, and a physician was always available for telephonic contact. Parents had to fill a diary card at each dose. Side effects were subdivided into: eye symptoms, rhinitis, asthma, gastrointestinal (nausea, vomiting, abdominal pain), urticaria, angioedema, anaphylaxis. The severity was graded as: low (no treatment/dose adjusting), moderate (need for drugs/medical advice/ discontinuation), and severe (lifethreatening/hospitalization/emergency care) (3). Mild transient oral itching requiring no intervention was not considered. At the end of the treatment, the parents indicated if symptoms had worsened, improved or not changed. All parents gave their consent and the study was approved by the Ethical Committee. Thirty-six children (33 males), aged between 1 year 11 months and 3 years 10 months (mean 3 years 2 months) were enrolled: 17 had persistent asthma, 12 intermittent asthma and 33 rhinoconjunctivitis. Nineteen patients received mite-SLIT and 17 grass-SLIT. The mean follow-up was 22.2 months (1–3 years), and the number of doses was about 25 200. One episode of abdominal pain occurred in two children (5% of patients; 0.071 per 1000 doses) during the maintenance phase. One episode was mild and transient (<30 min) and one was moderate, requiring a temporary adjustment of the dose. In this latter case, the maximum maintenance dose was reached after few days without problems. In 30 of the 36 children, the omitted doses were <10/year (85%). The most frequent reasons for skipping doses wereaccidental omission and febrile episodes. The evaluation by parents was: highly improved in 21 children, moderately improved in nine, slightly improved in four and unchanged in two. Allergen immunotherapy is the only treatment capable of modifying the natural history of allergy (4, 5), and it is reasonable to expect that, when used as early as possible, the clinical outcome is more favorable. In our survey, the number of patients is small, and rare events could not be detected; nevertheless, the data were obtained by a rigorous followup in a real-life setting. Thus, the present observation suggests that SLIT could also be safely administered to very young children.

Monomeric chemically modified allergens : immunologic and physicochemical characterization

Allergenic extracts (Der p, grass, and Parietarin) or single allergens such as Par j I (the major allergen of Parietaria) and ovalbumin (OA), a food allergen widely used in animal models, were chemically modified by reaction with potassium cyanate (KCNO), which transforms the &‐amino group of the lysine of proteinaceous allergens into ureido groups. KCNO‐modified (carbamylated) allergens have low allergenic potency, as demonstrated in vitro (RAST inhibition) and in vivo (passive cutaneous anaphylaxis). When used to immunize rabbits, carbamylated allergens still induce IgG antibodies able to cross‐react with native allergens (immunoblotting experiments). An interesting feature distinguishing carbamylated allergens from other chemically modified allergens is the preservation of the native monomeric dimension as demonstrated by SDS‐PAGE analysis. Results are discussed from the perspective of clinical application of carbamylated allergens.

Pharmacokinetics of Der p 2 Allergen and Derived Monomeric Allergoid in Allergic Volunteers

Background: Presently, sublingual immunotherapy is widely used as an alternative to the injection route for respiratory allergy, but its pharmacokinetics in humans is poorly known, and data are available only for Par j 1 allergen. We aimed at assessing the biodistribution of iodine-123-radiolabelled Der p 2 in allergic volunteers. Methods: Purified Der p 2 and its monomeric allergoid were radiolabelled with iodine-123 and administered sublingually to 7 allergic volunteers. The subjects were allowed to swallow 6 min after administration. Dynamic (up to 10 min) and static scintigraphic images (30 min, 1, 2, 3 and 20 h) were recorded, and blood samples were obtained at different time points to measure the plasma radioactivity and to assess the presence of circulating radiolabelled species by gel chromatography. Results: The local pharmacokinetics did not differ between allergen and allergoid. Plasma radioactivity began to increase only after swallowing and peaked at 1–2 h. Both the allergen and the allergoid persisted in the mouth for several hours, and traces could be detectable up to 20 h. At radioactivity plasma peak, gel chromatography showed that a fraction of the allergoid, but not the allergen, was absorbed as an intact molecule. Conclusions: These results indicate that the pharmacokinetics of sublingual administration is independent of the allergen used and characterized by the long persistence in the mouth. The contribution of enteric absorption of the allergoid in the mechanism of action of sublingual immunotherapy remains to be defined.

Long-Lasting Effects of Sublingual Immunotherapy for House Dust Mites in Allergic Rhinitis with Bronchial Hyperreactivity: A Long-Term (13-Year) Retrospective Study in Real Life

Background: Subcutaneous immunotherapy for respiratory allergy has shown a long lasting efficacy after its discontinuation, whereas evidence in the case of sublingual immunotherapy (SLIT) is weak. This retrospective study evaluates whether SLIT exerts a long-lasting effect and whether it relates to its duration. Methods: Sixty-five patients allergic to mite and positive to methacholine challenge 13 years ago were studied. Twelve (control group, SLIT 0) were treated for 4 years only with standard pharmacological therapy (SPT), while 53 received SLIT and SPT. Among these, four groups were identified according to SLIT duration. Fifteen patients were treated for 1 year (SLIT 1), 10 for 2 (SLIT 2), 14 for 3 (SLIT 3) and 14 for 4 years (SLIT 4). Clinical parameters (symptom monthly score, SMS), bronchial reactivity and FEV1 were evaluated in 1992 (run-in), 1993 (baseline) and every 2 years from 1997 to 2005. Results: Two to 3 years after the treatment ended, a positive effect on SMS, but not methacholine challenge and FEV1, was seen in the SLIT groups versus SLIT 0. At this time interval an effect on methacholine challenge was also seen in SLIT 3. After 7–8 years a significant difference was seen for SMS, i.e., it was significantly better in SLIT 4 than in the other groups, while bronchial reactivity was still improved in SLIT 1, 3 and 4 only after 5–6 years. Conclusions:The effects of a 4-year SLIT on clinical parameters but not bronchial reactivity and FEV1 last 7–8 years after its discontinuation. SLIT shorter than 4 years yields proportionally less impressive results.

Sublingual swallow or spit

. THE sublingual route of administration of immunotherapy has been approved for clinical use on the basis of several controlled trials (1, 2). Usually, the vaccine is kept under the tongue for 1–2 min and then swallowed (sublingual/swallow). Nevertheless, in some of the earliest studies, the vaccine was kept in the mouth and then spat out (sublingual/spit route), and this modality also achieved a measurable clinical effectiveness. The rationale of the latter procedure implied the concept of direct and rapid absorption of the vaccine through the mucosa. In recent pharmacokinetic studies in man employing the radiolabeled major Parietaria judaica allergen (Par j 1), we showed that indeed no direct absorption could be detected (3, 4), but a longlasting persistence of the Par j 1 allergen on the mucosal surface was observed. By the procedure previously described (3), involving the administration of a radiolabeled allergen, we investigated the pharmacokinetics of the sublingual/spit route. Two healthy volunteers underwent this procedure after providing informed consent. Briefly, the purified Par j 1 (MW=12 500 kDa) was radiolabeled with carrier-free I to yield a specific radioactivity of about 100 mCi/mg (3.7 MBq/mg). About 15 mg of the radiolabeled Par j 1 allergen (corresponding to about 1.5 mCi [55 MBq]) was administered sublingually to two nonallergic volunteers. The allergen had to be kept sublingually for 3 min without swallowing and then spat into a proper container for subsequent radioactivity counting; afterward, the subjects were allowed to swallow and rinse their mouths. During the first 3 min, dynamic scintigraphy (1 frame/10 s) of the head and neck was recorded; static scans of the head, chest, and abdomen were also recorded 15, 30, and 60 min after spitting. Heparinized blood samples were collected at 5, 10, 15, 30, and 60 min for plasma radioactivity count. The results were compared with those obtained in three volunteers who had swallowed the entire dose after retaining it under the tongue for 20 min. About 30% and 26%, respectively, of the dose administered could be recovered in the saliva spat out by the two volunteers. After spitting, the subjects were permitted to swallow normally; therefore, the progression of the radiolabeled allergen along the gastrointestinal tract could be observed. Plasma radioactivity began to increase only after swallowing (thus confirming the absence of any direct sublingual absorption), and with a peak attained about 2 h after swallowing. The plasma radioactivity pattern following sublingual/spit administration did not differ from that of the volunteers who swallowed. These data suggest that the more appropriate and advantageous way to administer the allergen sublingually is the sublingual/swallow procedure. In fact, after spitting, most of the protein remains in the mouth; thus, the spitting method does not differ from the sublingual/swallow procedure. On the other hand, the sublingual/ spit procedure leads to a certain loss of the allergen administered. The observation that the sublingual/spit procedure also achieves favorable clinical results indirectly confirms that, although brief, the contact of the allergen with the oral mucosa is a crucial step in the mechanisms of action of this desensitizing treatment.

Effect of sublingual immunotherapy with grass monomeric allergoid on allergen-specific T-cell proliferation and interleukin 10 production

BACKGROUND Sublingual immunotherapy (SLIT) is safe and efficacious in the treatment of patients with allergic rhinitis. Although favorable clinical effects have been observed with controlled trials as early as a few months since the beginning of treatment, few biological changes induced by SLIT have been demonstrated. OBJECTIVE To investigate in grass-allergic patients the effect of a 2-month SLIT regimen, administered with a simplified protocol without up-dosing, on proliferation and production of cytokines characteristic of the regulatory T-cell phenotype (interleukin 10 [IL-10] and transforming growth factor beta [TGF-beta]) by allergen-specific T cells. METHODS Patients were recruited to the study in January 2006. SLIT was performed by self-administration and was continued for 60 days from February to April 2006. Eleven grass pollen-allergic patients with seasonal rhinitis were treated daily before the pollen season for 2 months with a modified allergen (monomeric allergoid) derived from a 3-grass pollen extract. Allergen-specific proliferation and production of IL-10 and TGF-beta were measured on peripheral blood mononuclear cells at baseline and treatment end. Tetanus toxoid served as the control antigen. RESULTS After SLIT, allergen-specific (P = .002) but not tetanus toxoid-specific proliferation decreased, whereas IL-10 transcription increased (P < .001). TGB-beta transcription was also increased after treatment, although not statistically significantly (P = .06). Changes in proliferation to allergen and in IL-10 transcription were correlated (r = -0.82, P = .003). CONCLUSIONS A short-term course of SLIT with modified allergen in grass-allergic patients is associated with the reduction of allergen-specific proliferation and with the up-regulation of the IL-10 regulatory cytokine.

Allergy to nonspecific lipid transfer proteins in Rosaceae: a comparative study of different in vivo diagnostic methods.

BACKGROUND Lipid transfer proteins (LTPs) are the major allergens in patients sensitive to Rosaceae (apple, peach, apricot, cherry, plum, and pear) who are not allergic to birch pollen. OBJECTIVE The purpose of this study was to find a sensitive, specific, and relatively easy method for detection of LTP-sensitive patients. METHODS We studied 36 persons who experienced oral allergy syndrome after the ingestion of fruits in the family Rosaceae. This study cohort was divided into two groups: 18 without allergy to birch pollen (patients) and 18 with birch pollen allergy (control subjects). All were tested by skin prick tests (SPTs) with fresh Golden Delicious apple, fresh peach, and extracts of peel and pulp from both fruits. Their specific IgE reactivities against peach peel extract were further investigated by immunoblot analysis. RESULTS All 18 subjects in the control group showed strongly positive skin reactions with both fresh apple and fresh peach, whereas no skin reactivity was found with extracts from peach peel, peach pulp, or apple pulp. Extract of apple peel produced positive skin reactions in 17 of 18 control subjects; however, the wheals were generally smaller than those induced by fresh fruits. Immunoblot analysis showed no reactivity for peach peel extract. In contrast, the SPTs with fresh fruits showed that some of the 18 patients had strongly positive reactions, but others had weak reactions or negative responses. Further, in a high proportion of the patients, consecutive SPT with fresh apple yielded inconsistent results. In all patients, SPTs with extracts from apple pulp and peach pulp were negative, whereas SPTs with peel extracts were strongly positive in all patients. In most patients, the wheal area induced by SPT with peel extracts was larger than that induced by SPTs with fresh fruits. Immunoblot analysis showed that serum specimens from all 18 patients reacted with a 10-kD protein in peach peel. This is the molecular mass of LTPs. CONCLUSIONS In birch pollen-allergic patients, the SPTs with fresh foods still remains the most reliable method of diagnosing vegetable food hypersensitivity. In contrast, in patients not allergic to birch pollen, the most reliable strategy for detection of patients sensitive to LTPs is skin prick testing with properly prepared fruit peel extracts. The loss of Bet v 1- and Bet v 2-like structures, which probably occurs during extraction, may facilitate immediate identification of the relevant allergen.

Environmental and clinical study of latex allergy in a textile factory.

Several reports have been published on occupational allergy to latex among health care personnel, and a few of them provide quantification of latex aeroallergens in the workplace.1, 2 Immediate hypersensibility reactions to latex have been described also in workers wearing natural rubber gloves to protect their hands,3 in glove manufacturing plants,4 and in a latex-doll manufacturing plant.5 To date, no other industrial environment has been identified as being at risk for latex hypersensitivity. This article reports the results of a medical and occupational hygiene survey in a textile factory, where latex threads (powdered with pure talc to reduce the stickiness of natural rubber) and nylon or polyamide fibers were braided to produce elasticized ribbon for underclothes. Ten workers were employed in the factory, and two of them had been referred to our hospital because of work-related cutaneous, ocular, and respiratory (nasal or bronchial) symptoms. This study set out to ascertain whether allergic manifestations to latex were present among workers and to quantify latex allergen exposure in the workplace.

Oral allergy syndrome (OAS) in pollinosis patients after eating pistachio nuts: Two cases with two different patterns of onset

We describe two uncommon cases of oral allergy syndrome (OAS) after eating pistachio nuts (p.n.) in subjects (a 54‐year‐old man and a 3‐year‐old girl) with exclusive skin prick test (SPT) positivity to Parietaria (P.) and pistachio nut (p.n.) allergens. Serologic P.‐ and p.n.‐specific IgE determinations were also carried out. A double‐blind, placebo‐controlled food challenge (DBPCFC) was performed, for ethical reasons, only in the adult patient, but we observed a positive intraoral reaction only after slight scratching of the oral mucosa. Since this patient had put three whole p.n. with their shells into his mouth, breaking them with his teeth, before the onset of symptoms, we suggest that slight injury of the oral mucosa may enhance the local response. Preliminary results with SDS‐PAGE and immunoblotting demonstrate the occurrence of a slight degree of cross‐reactivity between these allergens, hut further studies are necessary to obtain a definite conclusion.